Contributions of Primary Neuronal Cilia to the Normal and Abnormal Development of the Neocortex

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Contributions of Primary Neuronal Cilia to the Normal and Abnormal Development of the Neocortex
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Guadiana, Sarah M
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Doctorate ( Ph.D.)
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University of Florida
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Medical Sciences, Neuroscience (IDP)
Committee Chair:
Sarkisian, Matthew R
Committee Members:
Cohn, Martin J
Harfe, Brian
Semple-Rowland, Susan L
Boulton, Michael Edwin

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cilia -- neocortex -- neuronal -- primary
Neuroscience (IDP) -- Dissertations, Academic -- UF
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Medical Sciences thesis, Ph.D.
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Abstract:
Primary cilia are microtubule-based, sensory organelles found on most every cell in the mammalian body including the central nervous system (CNS)1-26. Primary neuronal cilia were long thought to be vestigial remnants until recent evidence has shed light on a number of specialized functions for these “extrasynaptic signaling” hubs. Seminal studies demonstrated these organelles are crucial for several early developmental processes in the CNS and primary cilia dysfunction has been implicated in a group of human diseases termed ciliopathies, many of which affect neurological function. However, their role on mature neurons during post-natal development and maturation is poorly understood. Although it was known the mammalian adult cortical neurons bear primary cilia, several key questions remained. We asked in our first group of experiments when cilia develop on neurons in the neocortex and examined if they persist throughout the lifespan. We found ciliogenesis is largely a postnatal process in the mouse and neurons assemble a cilium upon reaching their destined layer of the neocortex. The extension of neuronal cilia occurs over many weeks until plateauing around P90. We 16 also investigated if cilia still persist in the aged rat cortex. We found that ACIII positive cilia persist on aged neurons, however the levels of certain ciliary GPCRs suspected to be involved in cognition, learning and memory (e.g. SSTR3 and MCH1R) can fluctuate and/or are undetectable from aged neuronal cilia. In our second group of experiments, we examined how mutated neuronal cilia influence the development and maturation of excitatory pyramidal neurons in the neocortex. We found neurons with malformed cilia or absent cilia correlated with a hindered ability to extend full dendritic arbors. Remarkably, we found that restoring ciliary signaling molecules (e.g. ACIII) to the cilium returned dendritic arbors but this result was not achieved when cilia formation was blocked and ACIII was exogenously expressed within the neurons. Collectively, the results of the experiments in my thesis suggest that neuronal cilia defects may set the stage for abnormal cortical circuitry and may be important for the development of a fully functional neocortical network.
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In the series University of Florida Digital Collections.
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by Sarah M Guadiana.
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Thesis (Ph.D.)--University of Florida, 2013.
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Adviser: Sarkisian, Matthew R.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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1 CONTRIBUTIONS OF PRIMARY CILIA TO THE NORMAL AND ABNORMAL DEVELOPMENT OF THE NEOCORTEX By SARAH MARIE GUADIANA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

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2 2013 Sarah Marie Guadiana

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3 To my husband and my son for your love and support ; w o rds cannot express my gratitude

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4 ACKNOWLEDGMENTS First, I would like to thank my family and friends. My parents Debbie and Bob, and my siblings Kate, Kevin, Tom, and Bobby have given me so much encouragement and support throughout the entire process and I would like to sincerely thank them for their love and guidance. Thank you to Sofia, Tom, Nick, Dan and Cristina for our conversations, scientific or otherwise. I would also like to thank my best friend James for his ear and shoulder during the last four years. Thank you to my wonderful husband Juan for his unconditional love and support and his unwavering dedication to our family. Randy: Thank you son for reminding me why I did this in the first place. I would like to t hank several members of the UF scientific community for their mentorship and guidance both in life and scientific matters. Specifically, I would like to thank Habibeh Khoshbouie, Jada Lewis, Jennifer Bizon and Lucia Notterpek for being generous mentors and guiding my scientific endeavors. A special thanks to Dr. Sue SempleRowland for her role in sculpting my writing abilities as well as helping me become a better scientist. Thank you to our collaborators Dr(s). Jon Arellano, Joshua Breunig, Pasko Rakic, Ki rk Mykytyn, Tom Foster, Ashok Kumar, and Ron Mandel for their advice, suggestions, and scientific contributions. I would also like to thank my committee, Dr(s). Sue Semple Rowland, Brian Harfe, Michael Boulton, and Marty Cohn for their invaluable advice an d guidance. Thank you to Sarkisian lab members past and present including Ashton Seque ira, Gileno Fonseca Filho, Tyler Smith, Kathleen Park, Lan Hoang Min h and Dorit Siebzehnrbl. Finally, I would like to thank, Dr. Matthew Sarkisian for his mentorship an d advice in this entire process, without which none of this would have been possible. His generosity with his time and mind were unending, for which I am grateful.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURE S .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 15 CHAPTER 1 INTRODUCTION .................................................................................................... 17 Summary ................................................................................................................ 17 Primary Neuronal Cil ia Structure and Function ....................................................... 18 Primary Cilia Biology ........................................................................................ 18 Primary Cilia Function in the CNS: Early CNS Development and Disease ...... 23 Cilia function during embryogenesis and other early developmental processes ............................................................................................... 23 Roles prim ary cilia play in disease: models and insights ........................... 25 Hypothesis and Overview of Chapters 24 .............................................................. 26 2 DEVELOPMENT AND DISTRIBUTION OF NEURONAL CILIA IN THE MOUSE NEOCORTEX ......................................................................................................... 31 Background ............................................................................................................. 31 Results .................................................................................................................... 33 Expression of Adenylyl Cyclase III in Fetal and Postnatal Mouse Cortex ......... 33 Basal Body Docking Begins in Deeper Layer Cells of the Developing Cortical Plate ................................................................................................. 34 Neuronal Primary Cilia Axonemes Develop Postnatally Taking Several Weeks to Reach Maximal Lengths ................................................................ 36 Extension of Cilia from Ca2+ Binding ProteinContaining Interneurons ........... 40 Neuronal Cilia Do Not Appear Critical for Neuronal Polarity or Expression of Layer Specific Markers. ................................................................................. 41 Discussion .............................................................................................................. 41 Initiation of Ciliogenesis in the Developing Cortical Plate. ................................ 42 Appearance of Signaling Machinery in Neuronal Cilia ...................................... 48 Neuronal Cilia Do Not Seem Essential for Proper Neuronal Migration or for Acquisition of Neuronal Polarity. ................................................................... 50 Materials and Methods ............................................................................................ 51 Mice .................................................................................................................. 51 In Utero Electroporation ................................................................................... 52

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6 Immunohistochemistry ...................................................................................... 52 Antibody Characterization ................................................................................ 53 Analysis and Quantification of Cilia .................................................................. 57 Electron Microscopy ......................................................................................... 58 Western Blots ................................................................................................... 59 3 PROPERTIES OF NEURONAL CILIA IN RODENT AND HUMAN FOREBRAIN DURING SENESCENCE ........................................................................................ 77 Background ............................................................................................................. 77 Results .................................................................................................................... 79 Expression of ACIII, a Marker of Neuronal Cilia, Persists in the Aged Rat and Human Forebrain ................................................................................... 79 Expression of SSTR3 in Cilia of the Young and Aged Forebrain ...................... 80 P75NTR Expression in Young and Aged Forebrain ............................................ 82 Ciliary MCHR1 Expression in Young and Aged Forebrain. .............................. 83 Levels of Other Ciliary GPCRs, IFT, and Bardet Biedl Syndrome (BBsome)Associated Proteins in Young and Aged Human Forebrain ......... 84 Material and Methods ............................................................................................. 86 Animals ............................................................................................................. 86 Immunohistochemistry ...................................................................................... 86 Cilia Length Measurements .............................................................................. 87 Statistical Analysis ............................................................................................ 87 Western Blot ..................................................................................................... 88 Human Brain Transcriptome Data .................................................................... 88 4 ARBORIZATION OF DENDRITES BY DEVELOPING NEOCORTICAL NEURONS IS DEPENDENT ON PRIMARY CILIA AND TYPE 3 ADENYLYL CYCLASE ............................................................................................................... 97 Background ............................................................................................................. 97 Results .................................................................................................................... 99 Overexpression of Neuronal Cilia GPCRs in Developing Mouse Cortical Neurons Dramatically Increases Cilia Length and Disrupts Cilia Morphology ................................................................................................... 99 Aberrant Lengthening of Neuronal Cilia by GPCR Overexpression is Linked to Enhanced IFT Function ........................................................................... 101 5HT6 Overexpression is Associated with a Marked Decrease in Ciliary SSTR3 and ACIII Localization ..................................................................... 104 Neurons with Long, Malformed Cilia and Those with Blocked Cilia Formation Exhibit Abnormal Dendritic Outgrowth. ...................................... 105 Co Expression of 5HT6:EGFP with ACIII but Not Dnkif3a Reverses Dendrite Arbor Defects ................................................................................ 107 Discussion ............................................................................................................ 108 GPCRInduced Changes in Neuronal Cilia Length ......................................... 109 GPCR Overexpression May Compromise Neuronal Cilia Signaling ............... 111 Cilia Malformation, Dendrite Abnormalities, and ACIII .................................... 112

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7 Materials and Methods .......................................................................................... 114 Mice ................................................................................................................ 1 14 In Utero Electroporation ................................................................................. 115 Vectors ........................................................................................................... 115 Cell Culture ..................................................................................................... 116 Immunostaining .............................................................................................. 117 Analyses and Quantification of Cilia ............................................................... 118 Western Blots ................................................................................................. 118 Sholl Analysis ................................................................................................. 119 5 CONCLUSIONS ................................................................................................... 130 Summary of Findings and Implications ................................................................. 130 Cilia Roles on Different Neuronal Populations ...................................................... 132 Future Unanswered Questions and Challenges ................................................... 135 LIST OF REFERENCES ............................................................................................. 138 BIOGRAPHICAL SKETCH .......................................................................................... 157

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8 LIST OF TABLES Table page 2 1 Primary antibodies used in this study ................................................................. 60 2 2 Average length of cilia in the neocortical lamina at different ages ...................... 61 4 1 Plasmid cDNA vectors used in this study ......................................................... 121

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9 LIST OF FIGURE S Figure page 1 1 Primary neuronal cilia structure and IFT on pyramidal cells from the neocortex. .......................................................................................................... 29 1 2 Neuronal Cilia and ciliary ACIII localization throughout the Cortex. ................... 30 2 1 Expression of adenylyl cyclase III during fetal and postnatal cortical development. ..................................................................................................... 62 2 2 Basal bodies are more prevalent in deeper cortical plate at E16.5. .................... 63 2 3 Procilia in the fetal cortical plate at E16.5. ......................................................... 64 2 4 Elongation of neuronal cilia over several postnatal weeks. ................................ 65 2 5 Distribution of procilia in the neocortex at P0 and P4. ........................................ 66 2 6 Heterogeneity of procilia at P0 in the upper cortical plate. ................................. 67 2 7 Procilia in the deep cortical plate at P0. ............................................................. 68 2 8 Procilia in P4 neocortex. .................................................................................... 69 2 9 ACIII+ cilia extend from neurons in all lamina of neocortex at P7 and P14. ....... 71 2 10 Ultrastructur e of cilia at P8. ............................................................................... 72 2 11 Ultrastructure of cilia at P60. .............................................................................. 73 2 12 Different interneuron subtypes in neocortex extend cilia. .................................. 74 2 13 Mutants lacking cilia show normal gross cytoarchitecture. ................................ 75 2 14 Model of ciliogenesis stages in m ouse neocortical neurons. .............................. 76 3 1 ACIII+ cilia in young and aged rat cerebral cortices and hippocampi. ............... 90 3 2 SSTR3+ cilia of young and aged rat forebrain and SSTR3 expression in human cerebral cortex. ...................................................................................... 92 3 3 p75NTR expression in the young and aged cortex and hippocampus. .............. 93 3 4 Decrease in expression of MCHR1 in the adult aged forebrain. ........................ 94 3 5 Variablity in other GPCRs and IFT/ BBSome machinery during aging. ............ 95

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10 3 6 SSTR3 immunodetection in Fisher 344 rat hippocampus subfields CA1,DG, and CA3. ........................................................................................................... 96 3 7 MCHR1 peptide blocki ng to confirm antibody specificity. .................................. 96 4 1 Overexpression of 5HT6 and SSTR3 in mouse neocortical neurons induces abnormal growth of their primary cilia. ............................................................. 122 4 2 Cilia growth induced by GPCR overexpression is not significantly affected by l oss of GPCR function or the presence of protein tags. ................................... 123 4 3 GPCR overexpression induces upregulation of IFT proteins and premature cilia lengthening .............................................................................................. 124 4 4 Cilia of neurons overexpressing 5HT6:EGFP do not contain detectable levels of SSTR3. ......................................................................................................... 126 4 5 Cilia of neurons overexpressing 5HT6:EGFP do not contain detectable levels of ACIII.. ........................................................................................................... 127 4 6 5HT6, SSTR3 and dnKif3a overexpression reduce dendrite outgrowth of cultured cortical neurons. ................................................................................. 128 4 7 Co expression of ACIII:EGFP with 5HT6:EGFP, but not dnKif3a, restores ciliary ACIII, cilia structure and dendrite outgrowth. ......................................... 129 5 1 Model of cilia contributions to the n ormal and abnormal development of the neocortex. ......................................................................................................... 137

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11 LIST OF ABBREVIATIONS 5HT6 Serotonin receptor subtype 6 A1C Primary auditory cortex AB Antibody ACIII Type 3 Adenylyl Cyclase AD Adolescence AMY Amygdala ANOVA Analysis of Variance BB Basal Body BBS Bardet Biedl Syndrome BBSome Bardet Biedl Syndrome protein complex BN Brown Norway CA1 Cornu Ammonic region 1 CA3 Cornu Ammonic region 3 cAMP cyclic Adenosine Monophosphate CB Calbindin cDNA copy/complimentary DNA CLS Cilia localization signal CNS Central Nervous System CP Cortical P late CR Calretinin D1 Dopamine Receptor 1 DAPI 4',6 diamidino 2 phenylindole DC Daughter Centriole DCDC2 Doublecortin domain containing domain 2

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12 DFC Dorsolateral prefrontal cortex DG Dentate gyrus DNA Deoxyribonucleic Acid dnKif3a Dominant Negative KIF3A DRD1 Dopamine Receptor D1 (human) EGFP enhanced GFP EM Electron Microscopy FC Frontal Cortex GE Ganglionic E minence GFP Green Fluorescent Protein GPCRs G protein couple receptors HIP Hippocampus IFT Intraflagellar Transport IFT88 Intraflagellar Transport protein 88 IPC Posterior inferior parietal cortex ITC Inferior temporal cortex IZ Intermediate zone kDa kiloDalton KIF3A Kinesin Family Member 3A KO Knockout LA Late Adulthood LTP Long term potentiation M1C primary motor cortex MA Middle Adulthood MC Mother Centriole

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13 MCH1R Melanin Concentrating Hormone Receptor 1 MFC Medial prefrontal cortex MKS Meckel Grber Syndrome mRNA messenger RNA MT Microtubule MTOC Microtubule Organizing Center MW Molecular Weight NA Nucleus accumbens NES Nuclear Export Signal NeuN Neuronal Nuclei Marker NGFR Nerve Growth Factor Receptor (aka P75NTR) NLS Nuclear Localization Signal NPHP Nephonopthisis OC Occipital cortex OFC Orbital prefrontal cortex OFD1 Oral facial digital syndrome type 1 OT Olfactory Tubercle P75NTR p75 Neurotrophin receptor PC Parietal cortex PFA Paraformaldehyde PKAC Protein Kinase A Catalytic subunit PKD Polycystic Kidney Disease POMC Pro opiomelanocortin PV Parvalbumin RNA Ribonucleic A cid

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14 S1C Primary somatosensory cortex SEM Standard Error of the Mean Shh Sonic Hedgehog SMO Smoothened SSTR3 Somatostatin receptor subtype 3 STC Posterior superior temporal cortex ST R Striatum TC Temporal cortex TULP3 Tubby like 3 V1C Primary visual cortex VFC Ventrolateral prefrontal cortex VZ Ventricular zone YA Young Adulthood

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CONTRIBUTIONS OF PRIMARY CILIA TO THE NORMAL AND ABNORMAL DEVELOPMENT OF THE NEOCORTEX By Sarah Marie Guadiana August 2013 Chair: Matthew R. Sarkisian M ajor: Biomedical Sciences Major Neuroscience Primary cilia are microtubulebased sensory organelles found on most every cell in the mammalian body including the central nervous system (CNS) (Tucker et al., 1983, Mandl and Megele, 1989, Fuchs and Schwark, 2004, Whitfield, 2004, Corbit et al., 2005, Berbari et al., 2007, Bishop et al., 2007, Rohatgi et al., 2007, Berbari et al., 2008a, Berbari et al., 2008b, Han et al., 2008, Pedersen et al., 2008, Willaredt et al., 2008, Domire and Mykytyn, 2009, Stanic et al., 2009, Green and Mykytyn, 2010, Han and Alvarez Buylla, 2010, Lee and Gleeson, 2010, Domire et al., 2011a, Ezratty et al., 2011, Lee and Gleeson, 2011, Yoshimura et al., 2011, Arellano et al., 2012, GarciaGonzalo and Reiter, 2012, Guadiana et al., 2013, Sarkisian et al., 2013) Primary neuronal cilia were long thought to be vestigial remnants until recent evidence has shed light on a number of specialized functions for these extrasynaptic signaling hubs Seminal studies demonstrated these organelles are crucial for several early developmental processes in the CNS and primary cilia dysfunction has been implicated in a group of human diseases termed c iliopathies many of which affect neurological function However, thei r role on mature neurons during post natal development and maturation is poorly understood.

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16 Although it was known the mammalian adult cortical neurons bear primary cilia several key questions remained. We asked in our first group of experiments when cilia develop on neurons in the neocortex and examined if they persist throughout the l ifespan We found ciliogenesis is largely a postnatal process in the mouse and n eurons assemble a c ilium upon reaching their destined layer of the neocortex The extension of neuronal cilia occurs over many weeks until plateauing around P90. We also investigated if cilia still persist in the aged rat cortex. We found that ACIII positive cilia persist on aged neurons h owever the levels of certain ciliary GPCRs su spected to be involved in cognition, learning and memory (e.g. SSTR3 and MCH1R) can fluctuate and/ or are undetectable from aged neuronal cilia. In our second group of experiments we examined how mutated neuronal cilia influence the development and maturat ion of excitatory pyramidal neurons in the neocortex W e found neurons with malformed cilia or absent cilia correlated with a hindered ability to extend full dendritic arbor s. Remarkably, we found that restoring ciliary signaling molecules (e.g. ACIII) to the cilium returned dendritic arbors but this result was not achieved when cilia formation was blocked and ACIII was exogenously expressed within the neurons Collectively, the results of the experiments in my thesis suggest that neuronal cilia defects may set the stage for abnormal cortical circuitry and may be important for the development of a fully functional neocortical network.

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17 CHAPTER 1 INTRODUCTION Summary Primary cilia are microtubule ( MT ) based organelles found on nearly all cells in the body (Mandl and Megele, 1989, Fuchs and Schwark, 2004, Whitfield, 2004, Corbit et al., 2005, Berbari et al., 2007, Breunig et al., 2008, Pedersen et al., 2008, Willaredt et al., 2008, Domire and Mykytyn, 2009, Han and Alvarez Buylla, 2010, Lee and Gleeson, 2010, Ezratty et al., 2011, Louvi and Grove, 2011, Arellano et al., 2012, GarciaGonzalo and Reiter, 2012, Green et al. 2012, Guadiana et al., 2013) Their presence on most cells types has been known for over a century and these structures have been implicated in a number of diseases affecting a wide variety of organ systems. However, their function on many of these cell types is unclear. Moreover, their role on neurons is even less understood. Cilia presence on neurons was not fully appreciated until better techniques for identifying them became readily available. Initial stu dies using electron microscopy (EM) identified neuronal cilia on a number of cell types, including hippocampal and cerebellar neurons and in peripheral neurons and glia (Porter, 1955, De Robertis, 1956, Dahl, 1963, Del Cerro and Snider, 1969, Tucker et al., 1983, Mandl and Megele, 1989, Poole et al., 1997, Fuchs and Schwark, 2004, Louvi and Grove, 2011) Based on this technique, it was quite difficult to fully appreciate their ubiquity. In the last ten years new techniques and better tools have enabled us to get a better picture of how prevalent these specialized organelles are in the brain. The notion that primary cilia are found on most if not all neurons, at first being controversial, is now widely accepted as mounting evidence has shown their ubiquitous existence on neurons in most mammalian brain regions (Hamon et al., 1999, Handel et al., 1999,

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18 Brailov et al., 2000, Fuchs and Schwark, 2004, Berbari et al., 2007, Davenport et al., 2007, Domire and Mykytyn, 2009, Einstein et al., 2010, Green and Mykytyn, 2010, Domire et al., 2011a, Arellano et al., 2012, Green et al., 2012, Higginbotham et al., 2012, Guadiana et al., 2013, Heydet et al., 2013) Additionally, a barrage of recent reports suggest ing these ubiquitous organelles serve chemo sensory roles in both normal central nervous system (CNS) development and in CNS related disorders has led to a surge in efforts to better understand the functional roles of these organelles (Valente et al., 2006, Fliegauf et al., 2007, Gerdes et al., 2007, Adams et al., 2008, Bae and B arr, 2008, Leitch et al., 2008, Pedersen and Rosenbaum, 2008, Santos and Reiter, 2008, Sharma et al., 2008, Berbari et al., 2009, Lancaster and Gleeson, 2009, Nigg and Raff, 2009, Green and Mykytyn, 2010, Lee and Gleeson, 2010, Amador Arjona et al., 2011, Domire et al., 2011a, Lee and Gleeson, 2011, Logan et al., 2011, Novarino et al., 2011, Sattar and Gleeson, 2011, Wang et al., 2011, BachmannGagescu et al., 2012, GarciaGonzalo and Reiter, 2012, Green et al., 2012, McIntyre et al., 2012, Heydet et al., 2013) Primary Neuronal Cilia Structure and Function Primary Cilia Biology Most cilia are characterized by their MT arrangement and their capacity for mobility. Neuron al cilia have a 9+0 MT arrangement and lack of a central MT doublet, suggesting they are immotile structures, although there is evidence that some 9+0 MT based primary cilia are indeed motile (e.g. nodal cilia) The nine MT doublets, which comprise the bac kbone of the cilia axoneme, form from the older, mother centriole (MC) of the microtubuleorganizing center (MTOC). The MC along with the daughter centriole (DC) then go on to become the collective centrosome and eventually become the basal

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19 bodies after th e cell begins to build the cilium (Cohen et al., 1988, Doxsey et al., 1994, Brailov et al., 2000, Michaud and Yoder, 2006, Gerdes et al., 2007, Dave et al., 2009, Delaval and Doxsey, 2010, Ishikawa and Marshall, 2011, Arellano et al., 2012, Avasthi and Marshall, 2012, Baudoin et al., 2012) The ciliary plasma membrane is contiguous with the rest of the cellular plasma membrane and forms a specialized compartment, much like other organelles of the cell. The cilia axoneme is separated from the rest of the cellular space via a transition zone barrier, which prevents nonciliary proteins lacking a cilia localization signal (CLS) from entering the cilia cytoplasmic space (Berbari et al., 2008a, Pedersen and Rosenbaum, 2008, Ishikawa and Marshall, 2011, Li et al., 2011) The primary cilium also does not make protein de novo and t he proteins required for ciliogenesis and cilia maintenance must be made intracellularly in the Golgi complex and then transported to the cilium proper (Wheatley, 1967 a, b, Poole et al., 1997, Grissom et al., 2002, Rakic, 2003, Mykytyn and Sheffield, 2004, Nachury et al., 2007, Berbari et al., 2009, Green and Mykytyn, 2010, Green et al., 2012) These synthesized proteins are transported to and from the cilium via an elegant, evolutionary conserved trafficking mechanism known as intraflagellar transport (IFT) (Mandl and Megele, 1989, Slavotinek et al., 2000, Robert et al., 2007, Pedersen and Rosenbaum, 2008, Pedersen et al., 2008, Willaredt et al., 2008, Wong et al., 2009, Kim et al., 2010, Ishikawa and Marshall, 2011, Louvi and Grove, 2011, Sattar and Gleeson, 2011, Taschner et al., 2012) IFT is required for ciliogenesis and maintenance and involves molecular motors to transport cilia destined traffic towards the distal tip of the axoneme, known as anterograd e trafficking (or IFT B) using k inesin motors and retrograde trafficking (or IFT

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20 A) towards the basal body using dynein motors ( Figure 1 1) (Morris and Scholey, 1997, Pazour et al., 1998, Huangfu et al., 2003, Huangfu and Anderson, 2005, Jones et al., 2008, Spassky et al., 2008, Norman et al., 2009, Wong et al., 2009, Goetz and Anderson, 2010, Mukhopadhyay et al., 2010, Massinen et al., 2011, Qin et al., 2011, Zhao et al., 2012, Barakat et al., 2013) This mechanism also utilizes adaptor proteins such as Ift88 and Tulp3 to transport cargo such as G protein coupled receptors (GPCRs) along the motors. Although in other ciliated cell types, it is known that upwards of 2200 proteins localize to the their cil ia axonemes, also known as the c iliome, only a handful of proteins are known to localize to postnatal neuronal cilia. Many of which are neuromodulatory GPCRs and include se rotonin receptor subtype 6 (5ht6), somatostatin receptor subtype 3 (Sstr3), melanin concentrating hormone receptor subtype 1 (Mch1R), and dopamine receptor 1 (D1), along with a neurotrophin receptor (p75NTR) (Saito et al., 1999, Saito et al., 2004, Kang et al., 2005, Berbari et al., 2007, Bishop et al., 2007, Berbari et al., 2008a, Berbari et al., 2008b, MiyamotoMatsubara et al., 2008, Domire and Mykytyn, 2009, Chakravarthy et al ., 2010a, Chakravarthy et al., 2010b, Einstein et al., 2010, Marley and von Zastrow, 2010, Mukhopadhyay et al., 2010, Bittencourt, 2011, Domire et al., 2011a, Avasthi et al., 2012, Chakravarthy et al., 2012a, Chakravarthy et al., 2012b, Green et al., 2012, Armato et al., 2013, Guadiana et al., 2013, Mukhopadhyay and Jackson, 2013, Nagata et al., 2013) The axonemal localization of these receptors suggests t hat primary cilia are involved in signaling pathways associated with those receptors. Sstr3, 5ht6, and Mch1R subtype specificity for cilia axoneme localization has not only been shown to be highly specific to the neuronal cilia but also might imply a specialized function of neuronal cilia apart from

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21 other ciliated cell types that do not express these markers of cilia. Another neuronal cilia component found enriched in the axoneme, and one that is often used as the main cilia marker in the neocortex, is adenylyl cyclase subtype III (ACIII). Of the ten known adenylyl cyclases, ACIII is the only adenylyl cyclase identi fied through multiple sources to be neuronal cilia specific First identified by EM in rat olfactory neuron cilia (Menco et al., 1992) ACIII has been shown to functionally couple to GPCRs in those cilia (Wei et al., 1998, Wong et al., 2000, Bishop et al., 2007, Zou et al., 2007, Miyoshi et al., 2009, Ou et al., 2009, Wang et al., 2009, Green and Mykytyn, 2010, Wang et al., 2011, Arellano et al., 2012, Guadiana et al., 2013) This secondmessenger signaling molecule is found highly enriched within cilia across most of the cerebral cortex ( Figure 1 2). Localizing ACIII to the axoneme suggests cilia play a role in cAMP signaling and several groups have shown cAMP signaling defects in cilia mutants. Additionally, PKA C the active form of PKA, was found to localize to the base of the cilia near the basal body (Barzi et al., 2010, Tuson et al., 2011) Taken together, the findings that ACIII, PKAC, and GPCRs localize to cilia, s uggests that these organelles concentrate all the components necessary for receptor mediated extracellular/intracellular signaling. It is also generally thought that cilia are also unique depending on which protein(s) they localize to their axoneme, cell t ype they are harbored from, and from which region of the organism the cilia emanate. Evidence of cilia localization of these signaling proteins have, in part, sparked intense interest in understanding the exact function(s) neuronal cilia play in the cortex In addition to these molecules, another stable protein complex made up of 16 different proteins known as the BBSome (Bardet Biedl Syndrome) binds to cilia destined

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22 cargoes and helps transport them from the Golgi to the cilium and viceversa. BBSome prot eins have been shown to localize to the cilia axoneme, basal body and centrosomes. BBSome proteins are not only transported to the cilia through their CLSs but some also traffic in and out of the nucleus using nuclear export/localization signals (NESs/NL Ss). Interestingly both processes of cilia localization and nuclear localization are dramatically similar (Slavotinek et al. 2000, Mykytyn et al., 2001, Mykytyn et al., 2002, Riise et al., 2002, Mykytyn et al., 2003, Mykytyn et al., 2004, Mykytyn and Sheffield, 2004, Nishimura et al., 2004, Gerdes et al., 2007, Nachury et al., 2007, Rooryck et al., 2007, Berbari et al., 2008b, Domire et al., 2011a, Gascue et al., 2012, Heydet et al., 2013) Other cilia proteins aside from BBS proteins can also enter the nucleus using NLS/NES motifs. Whatsmore, recent evidence suggests the BBSome also play s a direct role in transcriptional regulation in the nucleus (Gascue et al., 2012) Th ese data provide the first evidence of a mechanism by which cilia proteins can signal to and from both the intracellular and extracellular space. This type of specialized signaling capacity has led to the coined phrase the cellular antennae or the extrasynaptic signaling hub to refer to primary cilia (Whitfield, 2004) However, the exact mechanisms by which these organelles provide this extrasynaptic signaling function on neurons are largely unknown as are the downstream targets and consequences of these s ignals Lastly, some ciliary functions on other ciliated cell types and even in the developing CNS have been well characterized for some time, but the purpose primary cilia serve on mature neurons in the cortex has yet to be e lucidated.

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23 Primary Cilia Funct ion in the CNS: Early CNS Development and Disease Cilia f unction d uring e mbryogenesis and o ther e arly d evelopmental p rocesses What is perhaps the most fascinating aspect of cilia function are their diverse roles on different cells types and in different t issue types during development. Cilia are utilized during mammalian embryogenesis for patterning of structures such as the limb buds, brain regions (i.e. cortex), and the neural tube, responding to cues from both growth factors and morphogens (Singla and Reiter, 2006, Caspary et al., 2007, Dubreuil et al., 2007, Sharma et al., 2008, Willaredt et al., 2008, Norman et al., 2009, Logan et al., 2011, Bay and Caspary, 2012, Pasek et al., 2012, Sun et al., 2012) Early approaches using transgenic mouse models and conditional knockouts (KOs) to induce cilia mutations resulted in defects of digit formation, dorsal telencephalic patterning, and neural tube closure. Cilia are also critical for neurogenesis in several CNS regions including the hippocampus, the neural tube, and cerebellum (Haydar et al., 1999, Higginbotham and Gleeson, 2007, Han et al., 2008, Lee and Gleeson, 2010, Amador Arjona et al., 2011, BennounaGreene et al., 2011, Louvi and Grove, 2011) These diverse organelles are also involved in mechano, photo, and chemotransduction in the renal, visual, and olfactory systems. One example fr om the renal system in which selective KOs of Ift88 (adaptor protein) and Kif3a (Kinesin2 motor), two proteins involved in IFT transport, were the first attempts at parsing out the underlying cilia contribution to disease pathology. Davenport et al show ed conditional systemic KO in mice of Ift88 and Kif3a during early development induces rapidonset of polycystic kidney disease (PKD) but when induced later in the postnatal period the phenotype was a slow onset of PKD. They further went on to show both conditional mutants are obese compared to their wildtype littermates and food intake was dramatically increased in

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24 these mice. They then conditionally deleted Kif3a from cilia specifically in hypothalamic neurons expressing pro opiomelanocortin (POMC) and f ound a robust hyperphagiainduced obesity attributed to cilia defects in the hypothalamus (Davenport et al., 2007) Disruptions of the same cilia molecule on the same cell population but at different times in development resulting in distinct phenot ypes indicates cilia may be performing distinct functions on the same cells throughout the lifespan. Another interesting finding from this work is the distinct phenotypes by deleting the same cilia molecule but in different cell types. This was the first evidence that cilia mouse models can not only recapitulate some of the patient pathology but also provided a direct link between cilia function, in this case a satiety role, and cilia mutations in the brain. Further evidence supporting the link between cognitive deficits and cilia mutations came from Einstein et al and Wang et al (Einstein et al., 2010, Wang et al., 2011) KO models of two key neuronal cilia proteins display deficits in learning and memory. More specifically, Einstein et al. demonstrated ciliary Sstr3 is critical for object recognition recall but not spatial memory while Wang et al showed ciliary ACIII is required for both novel object recognition and extinction of contextual memory. Einstein et al. further explored the functional consequences of Sstr 3 KOs and demonstrated these mice have impaired responses to Forskolininduced, longterm potentiation (LTP), indicating a cAMP signaling defect. LTP improvement was seen with a selective Sstr3 agonist while Sstr3 antagonist blocks familiar object recall in wildtype animals. These data indicate neuronal cilia, and some of the molecules that localize there, are required for specific types o f learning and memory and may be one factor in the neurological dysfunction seen in adult ciliopathy patients. Although it is clear that most cell types in

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25 the brain (including neuronal progenitor cells, postnatal neurons, glia, astrocytes and ependymal cells) harbor cilia and that cilia dysfunction is associated with neurological disorders such as ciliopathies, the onset of ciliogenesis on neurons, their presence and molecular profile into late adulthood, and their exact function on neurons in the mammalia n cerebral cortex postnatally remains elusive. Disruptions in cilia function and structure in all of the aforementioned regions above has been implicated in a group of multisystem diseases collectively termed ciliopathies (Valent e et al., 2006, Fliegauf et al., 2007, Gerdes et al., 2007, Adams et al., 2008, Bae and Barr, 2008, Leitch et al., 2008, Pedersen and Rosenbaum, 2008, Santos and Reiter, 2008, Sharma et al., 2008, Berbari et al., 2009, Lancaster and Gleeson, 2009, Nigg and Raff, 2009, Green and Mykytyn, 2010, Lee and Gleeson, 2010, Amador Arjona et al., 2011, Domire et al., 2011b, a, Lee and Gleeson, 2011, Logan et al., 2011, Novarino et al., 2011, Sattar and Gleeson, 2011, Wang et al., 2011, BachmannGagescu et al., 2012, Garcia Gonzalo and Reiter, 2012, Gascue et al., 2012, Green et al., 2012, McIntyre et al., 2012, Heydet et al., 2013) Roles primary cilia play in disease: models and insights Since almost every cell in the body ciliated, it is not surprising that ciliopathy patients present with pleiotropic pathologies including retinal degeneration, polydactyly, obesity, and renal cysts among many other symptoms. Also noteworthy is the prevalence of neurological symptoms in these patients including cognitive and behavioral deficits. Th e group of ciliopathies that affect neurological f unction includes Joubert Syndrome (JS), Meckel Grber Syndrome (MKS), Bardet Biedl Syndrome (BBS), Nephonopthisis (NPHP), and Oral facial digital syndrome type 1 (OFD1)(Slavotinek et al., 2000, Leitch et al., 2008, Williams et al., 2008, Logan et al.,

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26 2011, D'Angelo et al., 2012, Di Gioia et al., 2012, Thauv in Robinet et al., 2012) Although rare, these diseases are often lethal in fetal/embr yonic stages in both human and animal models, making it difficult to study postnatally. To circumvent these issues, many researchers have turned to conditional animal models to fully understand how cilia defects result in such a diverse group of diseases. Attempts to induce mutations in primary cilia often target the IFT and BBSome proteins as a strategy to create dysfuncti onal cilia to study the effects This strategy has proved fruitful in that many of these mutants have led to a better understanding of c iliopathy etiology and cilia function in the brain. However, little is known about cilia function in the post natal mammalian cortex and if these organelles are in fact contributing to the disease pathology in Ciliopathy patients postnatally. Hypothesis an d Overview of Chapters 24 The following contents of this thesis contain the original experiments that focus on the overarching hypothesis that primary neuronal cilia contribute to the normal and abnormal development of the neocortex. One key question that needed to be addressed was the timing of ciliogenesis on neurons in the cortex. Other remaining unanswered questions included whether cilia were maintained throughout the lifespan. Are cilia maintained in the aged brain and do they still localize the same proteins as they do during earlier stages of development? Also, what is the function(s) of cilia on neurons? Our data were the first to have identified and fully characterize the initial appearance of primary cilia on neurons in all six laminar layers of the neocortex as well as being the first to show these dynamic organelles are maintained throughout the mammalian lifespan. Furthermore, we show ed developing cortical neurons with cilia malformations

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27 that display a novel differentiation defect phenotype in two different cilia mutation strategies, which allowed for the testing of this hypothesis. In collaboration with investigators from Yale, I made significant contributions to the first full characterization of the process of primary neuronal ciliogenesis on neocortical excitatory neurons through immunohistochemical and electron microscopy techniques and this work is detailed in Chapter 2. We show ed that all neocortical neuron subtypes (e.g. inhibitory interneurons and excitatory pyramidal neurons ) harbor pr imary cilia and that cilia structure is maintained regardless sex and neuronal cell phenotype differences. This work was published in the Journal of Comparative Neurology (Arellano et al., 2012) E xperiments were also perfor med detailed in Chapter 3, which characterized primary cilia presence on neurons in the rat aged cortex and hippocampus. It is the first to have show n cilia and cilia localizing proteins such as Sstr3 and ACIII are maintained and localized to neuronal cil ia even into advanced aging (38 mos rat) although these findings may vary between strains and brain regions This work is also the first to show there are biciliated cells harboring the Mch1R receptor and ACIII in the nucleus accumbens and olfactory tuber cle of the ventral striatum. This work is currently being submitted for publication. In Chapter 4, I describe how two different types of cilia mutations result in abnormal differentiation phenotype in developing neocortical neurons in vitro These studies reveal ed that neurons harboring malformed cilia either absent or hyperelongated display ed severe dendritic arborization defects and that their cilia do not localize ACIII to the axoneme. This work was also the first to show Kif3a is required for 5ht6 induced lengthening of the cilium. Finally, this study reports one potential mechanism by which cilia affect dendritic arborization, since we found that

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28 r eturning ACIII expression to mutant cilia reversed both the cilia length and dendritic morphology defi cits This work was recently published in the Journal of Neuroscience (Guadiana et al., 2013)

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29 Figure 1 1 Primary neuronal cilia structure and IFT on pyramidal cells from the neocortex. A : Neurons in the neocortex extend primary cilia that localize GPCRs such as Sstr3 and second messenger molecules such as ACIII in the axoneme. The 9+0 microtubule axoneme begins to form from the mother centriole (MC) while the daughter centriole is tethered to the MC. IFT or intraflagellar transport is the bidirectional trafficking mechanism used to transport carg o to and from the cilium base. B : The cilium is most often located near the apical dendrite of the excitatory pyramidal layer II/III neuron (green ) and is enriched with ACIII (red) while the basal body is enriched with the pericentrin protein (blue).

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30 Figure 1 2 Neuronal Cilia and ciliary ACIII localization throughout the Cortex ACIII is found enriched within the cilia axoneme throughout all of the neocortical layers (depicted here in Layers I V). Confocal Z stack of adult rat brain immunostained for cilia marker ACIII and mature neuronal marker NeuN. Bar =10m. ACIII / NeuN

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31 CHAPTER 2 DEVELOPMENT AND DISTRIBUTION OF NEURONAL CILIA IN THE MOUSE NEOCORTEX Background The primary cilium of neurons has been mostly overlooked for the last 50 years. Although its presence was occasionally documented by ultrastructural studies using electron m icroscopy (EM) (Dahl, 1963, LeVay, 1973, White and Rock, 1980) the lack of a specific marker for this organelle and intrinsic difficulties with random sampling for cilia with EM did not allow for more extensive studies on their morphology or distribution. More recently, cilia were shown to be enriched with type III adenylyl cyclase (ACIII), a mediator of cAMP signaling typically associated with G protein coupled membrane receptors (Bishop et al., 2007) The development of antibodies against ACIII has allowed describing the ubiquitous presence of cilia in neurons in different regions of the brain (Bishop et al., 2007) although confirmation of its presence in specific neuronal cell types is lacking. In addition, little is known about their development and function (for review see: (Fuchs and Schwark, 2004, Whitfield, 2004, Green and Mykytyn, 2010) ). During embryonic development, primary cilia extend from interphase radial glial cells into the lateral ventricles (Cohen et al., 1988, Dubreuil et al., 2007, Li et al., 2011) Loss or reduction in the expression of genes that are important for cilia growth and function (e.g. Ift88, stumpy and Bardet Biedl Syndrome proteins (BBS)) can lead to cortical morphogenesis defects in mice and human brain (Rooryck et al., 2007, Breunig et al., 2008, Willaredt et al., 2008, BennounaGreene et al., 2011) In the postnatal brain, primary cilia are found also on astrocyte like neural precursors in the hippocampus that express sonic hedgehog (Shh) signaling machinery and respond to

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32 secreted Shh to regulate neurogenesis and differentiation (Breunig et al., 2008, Han et al., 2008) In addition to ACIII, a few neuronal cilia specific receptors have been identified including somatostatin receptor subtype 3 (SSTR3), melaninconcentrating hormone receptor 1 (MchR1), dopamine receptor 1 (D1) and serotonin receptor subtype 6 (5hydroxytryptamine, 5HT6) ((Handel et al., 1999, Brailov et al., 2000, Berbari et al., 2008b, Domire and Mykytyn, 2009, Domire et al., 2011b) ). SSTR3 receptors localize on neuronal cilia in the neocortex, hippocampal CA fields, olfactory bulb, and dentate gyrus neuronal populations (Handel et al., 1999, Berbari et al., 2008b) In the hippocampus, SSTR3 appears between P03 in rat (Stanic et al., 2009) ; however the neocortical developmental appearance of SSTR3 and other neuronal cilia receptor subtypes is unclear. Recent studies have shown that disruption of these molecules or impairment of ciliogenesis in general can lead to abnormalities in object recognition memory, synaptic plasticity, an d obesity (Davenport et al., 2007, Wang et al., 2009, Einstein et al., 2010, Amador Arjona et al., 2011) Cilia are composed of a microtubular backbone (the axoneme) surrounded by a specialized membrane that is continuous with the plasma membrane. The axoneme extends from the basal body, a structure derived from the mother centriole (the older of the two centrioles) which can form the cilium in two different ways. In one case, a vesicle from the Golgi apparatus attaches to the mother centriole. The centriole starts growing the cilium inside the vesicle, which eventually fuses with the cell membrane and the cilium continues growing into the extracellular space. Alternatively, the mother Reprinted with permission from Arellano, J.I., et al., Development and distribution of neuronal cilia in mouse neocortex. J Comp Neurol, 2012. 520(4): p. 84873.

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33 centriole can migrate and anchor to the cell membrane to form the basal body from which the cilium grows into the extracellular space ( (Sorokin, 1962, Sorokin, 1968) ; for review see (Pedersen et al., 2008, Nigg and Raff, 2009) ). The pr ocess of cilia growth can be rapid upon serum starvation (Ou et al., 2009) whereby the axoneme can quickly disassemble if cells r e enter the cell cycle and subsequently reassemble upon reaching G1/G0 (Tucker et al., 1979a, Tucker et al., 1979b) To our knowledge, there are no studies on the development of cortical neuronal cilia, although it has been shown that failure of neuroblasts to properly segregate centrioles during neurogenesis can result in cortical neurons with multiple centrioles and cilia (Anastas et al., 2011) During neuronal migration, the centrioles are a core component of the centrosome, which aids in guiding neuronal migration (Higginbotham and Gleeson, 2007) Therefore, it would be expected that axoneme extension would occur after cells have completed migration. In this study, we attempt to address these questions, characterizing the development of cilia in neocortical neurons, their presence in specific neuronal types and describing their morphological features using different techniques such as western blot, immunohistochemistry and ultrastructural analysis. Results Expression o f Adenylyl Cyclase III in Fetal a nd Postnatal Mouse Cortex Cilia throughout the mature cortex are enriched in ACIII (Bishop et al., 2007) which thus serves as a surrogate cilial marker ( Figure 2 1A). To study the expression of ACIII in the developing cortex, we used western bl ot analysis to examine protein lysates from mouse cortex at ages E11.5, E13.5, E16, E18.5, P0, P7, P14, P21 and young adult (P60; Figure 2 1B). We found low levels of ACIII could be detected at ~E13.5

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34 (approximating early mid cortical neurogenesis), but levels were higher at birth and noticeably increased over the first several postnatal weeks ( Figure 2 1B). ACIII persisted at P60, but the signal intensity was lower compared to P21 lysates ( Figure 2 1B). Quantification of ACIII relative to actin from the same blot confirmed these observations (data not shown). We found that the signal for ACIII is ~125130 kDa, suggesting that cortical ACIII may predominantly be in a less glycosylated form (Wei et al., 1996, Murthy and Makhlouf, 1997) To confirm this we compared lysates isolated from P90 olfactory epithelium and frontal cortex from the same brain. As rep orted previously (Wong et al., 2000) we observed a very strong signal for glycosylated ACIII between ~190200kDa in olfactory epithelium which was not observed in cortex ( Figure 2 1C). Taken together, these data suggest that ACIII, a protein enriched in neuronal cilia, is expressed mostly in a lesser glycosylat ed form in the neocortex which increases in the late embryonic stages becoming more robust in postnatal cortex. Basal Body Docking Begins in Deeper Layer Cells of t he Developing Cortical Plate To study the possibility of late embryonic growth of neuronal cilia, we analyzed sections of mouse cortex that had been electroporated at embryonic day (E) 13.5 with a plasmid expressing green fluorescent protein (GFP) and subsequently fixed at E16.5 and prepared for immunoelectron microscopy (immunoEM). We first s creened cells localized throughout the cortical plate (CP) ( Figure 2 2A,B) for the presence and location of cilia and centrioles/basal bodies (quantified in Figure 2 2C). We found only a few cilia precursors at a very early stage of development ( Figure 2 2 D F and Figure 2 3), and most cells exhibited centrioles either free in the cytoplasm (undocked centrioles, Figure 2 1 G I, 2 3A) or located adjacent to the cell membrane with one centriole

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35 attached to the plasma membrane (docked centrioles/basal bodies, Fi gure 2 2D F, Figure 2 3B, C). The centrioles appeared surrounded by or in close proximity to Golgi apparatus, and in some cases multivesicular bodies were also observed in their vicinity ( Figure 2 3A, C). Results of our analysis are summarized in Figure 2 C. Out of 36 centriole encounters in the deeper CP, 25/36 were docked with the membrane while 11/36 were undocked ( Figure 2 2 C). Electroporation of a cohort of neural progenitors from the ventricular zone with GFP cDNA allowed us to track the neural cells derived from them. Cells generated at E13.5 and later are mostly directed to upper layers of the neocortex, and therefore, GFP+ cells located in the deep CP at E16.5 would most likely be migrating cells. In fact, docked centrioles were frequently within non GFP+ cells that typically displayed ultrastructural features of nonmigrating cells (e.g. more rounded soma, less elongated nuclei) both in the upper and deep CP (e.g. Figure 2 2 D F). In contrast, undocked centrioles in the deeper CP were in GFP+ cells t hat also typically exhibited leading processes and elongated nuclear morphology, suggesting they were in fact cells migrating to upper layers (e.g. Figure 2 2G). Moreover, deep CP cells with docked centrioles sometimes exhibited short (0.10.5 m) protrusi ons consisting of a membranous bud lacking microtubular organization that we called procilia (e.g. Figure 2 2E, F and Figure 2 3). We found only rare cases of undocked centrioles with a vesicle attached ( Figure 2 2G and Figure 2 3A) that could represent th e previous step before docking of the centriole to the cell membrane (Pedersen et al., 2008) The relative rarity of these findings suggests that vesicle attachment to the mother centriole could be quickly followed by centriole docking to the cell membrane. According to this model,

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36 ciliation of immature neurons would occur at the cell membrane an d not inside the vesicle attached to the mother centriole (Sorokin, 1968) In upper CP, 27/31 centrioles were undocked ( Figure 2 2C, H, I). Thus ~70% of deeper layer cells possess a docked centriole whereas only ~10% of upper CP cells contained similarly docked centrioles. These data suggest that cells initiate docking of the mother centriole in the CP once cells have completed migration and/or reached their appropriate lamina (Experiments for this section were performed by M.R.S. and J.I.A). Neuronal Primary Cilia Axonemes Develop Postnatally Taki ng Several Weeks t o Reach Maximal Lengths In the ro dent cortex, the cilia and basal bodies are immunodetectable with ACIII and pericentrin, respectively (Bishop et al., 2007, Anastas et al., 2011) ( Figure 2 4A E). To s tudy the development of neocortical cilia, ACIII immunostaining and EM was used at different ages. Cilia length was estimated by tracing ACIII signal on collapsed confocal z stack images from tissue sections at E18.5, P0, P4, P7, P14, P60, P90, P170 and P365. Average and maximum cilia length was recorded for each age and cortical layer ( Table 22 Figure 2 4F). ACIII staining revealed regional and laminar differences in the length of cilia in the neocortex. To avoid regional bias we consistently measured ci lia in the dorsal neocortex overlying the hippocampus (see methods for details). To distinguish laminar differences we used DAPI and NeuN to delineate layers 1 6 when possible ( Figure 2 5). These methods do not allow for the establishment of absolute values of cilia length, but provide information about cilia elongation trends in different cortical compartments over time. A total of 3756 cilia were measured, averaging ~85 per layer and age studied. To correspond with our analysis of ACIII, EM

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37 studies were p erformed at P0, P4, P8 and P60 to assess structural developmental characteristics. At E18.5 and P0, ACIII staining revealed a punctate pattern suggesting slightly elongated growth of cilia compared to E16.5 ( Figure 2 4A, B, Figure 2 5A D and Table 2 2 ). Ne uN is not fully expressed by all neurons in the region analyzed at P0 and is almost absent in layer 2, upper layer 3 and deep layer 6, although many neurons in the subplate are intensely immunostained. Thus, we only made a distinction between superficial ( layers 24) and deep (layers 56) strata based on cytoarchitecture and NeuN expression ( Figure 2 5A D). ACIII was found in small specks in all layers, with rare rodlike cilia. Layer 1 had short cilia compared to the CP, in which both superficial and deep strata showed similar lengths ( Table 22 Figure 2 4F, and Figure 2 5A D). Ultrastructural analysis at P0 supported those findings and showed the presence of procilia in all cells analyzed (n=21), although with large heterogeneity in their length ( Figure 2 6 and 2 7). These procilia were similar to some observed at E16.5 ( Figure 2 6 and Figure 2 2D I and Figure 2 3), but they had different ciliar content. Sometimes they exhibited a short axoneme with doublets of outer microtubules poorly developed and only distinguishable for about 100200 nm into the procilium. The presence of vesicles was a common finding inside the procilia (see for example Figure 2 6B, C) and some procilia appeared filled with a number of vesicles of different size ( Figure 2 7B, C). Interestingly, we found one cell located in the subplate region bearing a short cilium, but with a well organized axoneme ( Figure 2 7D). At P4, NeuN expression is not fully mature and is very weak in layer 2 and deep layer 6 ( Figure 2 5E, G, J). Layer 1 at P4 showed relatively long cilia compared to upper

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38 layers 24, where most ACIII staining appeared as thick punctae (0.7 1.5 m) with few intermingling longer cilia (2 3 m) ( Figure 2 5F H). In deeper layers 5 and 6, the heterogeneity was more dramatic, and two clear populations could be distinguished: a majority of short punctae (0.52 m), and a population of longer cilia especially in some cells in layer 5 (up to 6.6 m) and in the subplate (up to 5.4 m) ( Table 22 Figure 2 4F, G, Figure 2 5I K). These results suggest most cilia in upper and deep layers remained in a procilia stage, while some specific subpopulatio ns in deeper layers start elongating earlier. Consistent with this, in the EM analysis at P4 in supragranular layers 14 we found only procilia in the cells studied (n=11; Figure 2 8). The procilia at P4 were slightly longer but similar to those described at P0. They showed occasional vesiclelike structures and diffuse electrondense material, and sometimes the membrane was expanded in a mushroom like shape ( Figure 2 8A C). Similar to P0, procilia at P4 had short and poorly organized axonemes, extending about 200 nm into the procilium, and in deeper layer cells, microtubules were observed but lacked clear axonemal organization ( Figure 2 8D, F). From P4 to P7, upper layer cilia showed an increase in length to match deeper layers lengths ( Figure 2 4F, G, and F igure 2 5 and Figure 29), and all layers had similar average cilia length between 3 and 4.4 m, with maximum lengths of about 7 microns in layers 2, 3 and 5 ( Table 22 Figure 2 4F, G and Figure 2 9). From this age on, ACIII staining of cilia showed a pat tern of intense staining in the proximal segment that tapered towards the tip ( Figure s 2 1A and 2 4C E). Consistent with immunohistochemistry results, EM analysis at P8 revealed longer, straighter cilia with a developed axoneme that can be followed several microns into the parenchyma ( Figure 2 10). Unfortunately, the lengthening of cilia creates technical difficulties to

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39 follow the entire cilium length across a series of EM sections. Thus from this age on, we do not have information about the full structure of cilia. At P14 and P60 there is a progressive elongation of cilia in all layers ( Table 22 Figure 2 4F, G and Figure 2 9H N) and ultrastructural analysis at P60 showed well developed axonemes and rounded shaped cilia that could only be partially reconstructed ( Figure 2 11). At P90 ACIII staining showed similar or only slightly longer cilia lengths in all layers compared with P60, suggesting that cilia stop growing by ~P60P90. Cilia exhibited maximum lengths at these ages, with averages of ~5 m i n layer 2, 3, 4 and 6, and ~6 m in layer 5. Maximum values in each layer varied from ~8 m in layers 2, 4 and 6; ~9 m in layer 3, and 10.8 m in layer 5 ( Table 22 Figure 2 4F, G). Surprisingly, we found a notable shortening of the estimated cilia leng th at P170 and P365 as compared to P60P90 ( Table 22 Figure 2 4F, G). This could be due to an actual shortening of cilia, although an alternative possibility is that in aging animals ACIII protein does not fill the cilia completely and is absent from the distal tip. In fact, there were also slight decreases in estimated cilia length in layer 1 and 2 between P60 and P90 that could be due to the same phenomenon (see discussion). To determine whether or not sex played any significant influence in cilia leng th, we examined the average length in upper and deeper layers of the neocortex of males (n=2) and females (n=2) at P14. This analysis did not reveal significant differences in cilia length between sexes (3.060.19 m and 3.030.19 m (mean SEM) for males and females respectively ( Figure 2 4H)). Taken together, our results suggest cilia elongation in neocortex is a slow process that starts postnatally with slightly different dynamics depending on cortical layer but is not significantly affected by gender.

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40 Maximal cilia lengths are different depending on cortical layer, and overall it can take up to 3 months to be reached. Extension of Cilia f rom Ca2 + Binding ProteinContaining Interneurons Although ACIII+ cilia have been described in cultured hippocampal c ells expressing glutamate acid decarboxylase (Berbari et al., 2007) their presence in inhibitory neurons in the neocortex has not been described. Inhibitory neurons migrate tangentially into the neocortex during development to invade the cortical plate (Wonde rs and Anderson, 2006) Inhibitory interneurons can be identified by the expression of several markers. From those, calcium binding proteins parvalbumin (PV), calbindin (CB) and Calretinin (CR) distinguish three large subpopulations of inhibitory interneurons with little overlap (Defelipe et al., 1999) In the mature rodent cortex, parvalbumin (PV) expressing neurons comprise ~50% of GABAergic inhibitory interneurons (Wonders and Anderson, 2006). We examined confocal z stack images of P60 tissue sections immunostained for PV, ACIII and NeuN and measured cilia from these cells compared to cells that did not express PV ( Figure 2 12A, B). Cells PV+ appeared to extend a cilium from the soma ( Figure 2 12A). We found that the lengths of cilia from PV+ neurons appeared comparable with those of neighbouring PV neurons (putatively excitatory neurons) (Mean: PV+ = 5.12 m 0.42 SEM; PV = 5.06 0.25) ( Figure 2 12B). In addition, ACIII+ cilia were found in other interneuron subtypes expressing Calbindin and Calretinin ( Figure 2 12C, D). These results suggest that neocortical interneurons also extend ACIII+ cilia that reach lengths similar to neighbouring excitatory pyramidal neurons.

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41 Neuronal Cilia Do Not Appear Critical for Neuronal Polarity or Expression of Layer Specific Markers. On pyramidal shaped neurons, cilia generally extend from the base of the apical dendrite (Anastas et al., 2011) This suggests that cilia could be related to the orientation/polarity of neocortical neurons, and particularly to the extension/orientation of the apical dendrite. To analyze this, we examined neocortical neurons in Stumpy (including neurons) either lack or extend a poorly formed, stunted cilia (Town et al., 2008, Anastas et al., 2011) Though the lack of cilia leads to hydrocephalus and noticeable compression of the neocortex starting at approx imately P4 ( Figure 2 13), we Figure 2 13A C). In addition, laminar mice ( Figure 2 13D,E). These data suggest that failure o f neurons to extend normal cilia does not dramatically alter polarity or gross laminar pattern of neurons in the neocortex (Experiments for this section were performed by M.R.S. and J.I.A). Discussion Previous studies in vitro and in vivo have gone a long way towards characterizing neuronal cilia and the expression of specific proteins within these structures (for review see: (Green and Mykytyn, 2010, Lee and Gleeson, 2010) ). Indeed, there is growing evidence that dysgenesis of the primary cilium can be associated with neurological disorders (Sharma et al., 2008) In addition, during the past decade much has been learned about the cell biology of ciliogenesis (Goetz and Anderson, 2010) However, the genesis of neuronal cilia during brain development has not been well characterized.

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42 Our results suggest that ciliogenesis in mouse cortical neurons initiates after cells complete migration by docking of the mother centriole to the cell membrane and growing a procilium: a rudiment of the cilium lacking axonemal organization that will largely develop and elongate postnatally. The elongation of neocortical cilia is a surprisingly protracted process lasting several weeks that follows different temporal patterns and reaches different final lengths depending on laminar position. In addition to pyramidal neuron cilia, we also describe the presence of cilia in interneurons, with lengths comparable to those in pyr amidal cells. Collectively, our study provides a comprehensive initial characterization of neuronal ciliogenesis and cilia distribution in the developing and mature mouse neocortex. Initiation o f Ciliogenesis in t he Developing Cortical Plate EM analysis o f the cortical plate at E16.5 showed frequent undocked centrioles in cells with migratory morphology in the upper layer of the cortical plate, whilst cells with nonmigratory profile in deeper layer showed predominant basal body docking and in some cases b udding procilia. These observations suggested that cessation of migration is a necessary step before docking of the mother centriole to the membrane and the subsequent ciliogenesis. Furthermore, because centrioles are an integral part of the centrosome dur ing neuronal migration (Higginbotham and Gleeson, 2007) th is conclusion is not unexpected. It is also not surprising that the laminar positions of neocortical neurons are not dramatically altered in brains of mice that have mutations in genes that disrupt neuronal ciliogenesis because the centrioles have already performed their role in migration prior to docking. Detailed analysis of the centrioles at E16.5 showed the presence of procilia, or short membranous protrusions (0.10.8 m long) growing in the distal aspect of the

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43 mother centriole from cortical cells. T hese procilia were present in cells with docked centrioles analyzed in serial sections, and in some cases also in undocked centrioles in a vesicular stage, most likely immediately before docking to the membrane ( (Sorokin, 1962, Sorokin, 1968) ; for review see: (Pedersen et al., 2008) ). We cannot discard the possibility that some form of cilia could be present at earlier stages of development or in other pallial compartments, for example in early migrating immature neurons or in the population of immature neurons with multipolar morphology in the intermediate zone. However, if there is any previous form of cilia, it is very likely transient and probably reabsorbed before final migration of the neurons to their destination in the cortex, as our data suggest that cilia are not present in migrating neurons in the cortical plate. More extensive studies are needed to assess this possibility. With our data we propose the following model ( Figure 2 14): when a migrating neuro n reaches its appropriate lamina, the mother centriole migrates and docks with the nearby plasma membrane, either directly, or attaching a vesicle, to their distal tip prior to docking (Sorokin, 1968) Membrane docking generally occurs close to the proximal portion of the leading process that will become the apical dendrite (present results; (Anastas et al., 2011) Once the mother centriole docks to the membrane, the procilium grows slightly (~ 0.2 0.5 m) and reaches about 0.52 m by P0 and P4. We differentiate procilia from cilia based on the lack of axonemal organization. We define the procilia as membranous protrusions characterized by the lack of axoneme, that instead have only an axonemal rudiment extending about 100300 nm int o the procilia. The procilia cytoplasm is filled with diffuse material, frequently contains vesicles and rod like structures and occasional short and disorganized microtubules. The term

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44 procilia has been used by other investigators (Morris and Scholey, 1997, Sfakianos et al., 2007) to describe short budding cilia in different contexts, sometimes including a well developed axoneme. Our description of procilia matches previous observations of the budding primary cilia in diverse avian and mammalian tissues (Sorokin, 1962, Sorokin, 1968) and in the chick developing neural tube (Sotelo and TrujilloCenoz, 1958) However, this latter study reported various cycles of complete ciliogenesis in a period of 4.5 days, indicating a quick assembly of cilia (with fully elongated axoneme), in sharp contrast with the very slow development of procilia from E16.5 to P0 and P4 recorded in our study. We cannot entirely rule out the possibility that the developing axoneme in budding cilia of young neurons is susceptible to rapid microtubule breakdown before the specimen is fully fixe d, a possibility that would explain the lack of axoneme at early ages. However, this alternative is in doubt given our observations of cilia with well organized axonemes in P0 tissues. In fact, it should be emphasized that cilia development is not a synchr onous and homogeneus process in the neocortex, and differences in length and axonemal content are present particularly during early postnatal timepoints. For example, in the same brain where we detected (rare) neuronal cilia in the deepest cortical plate w ith well developed axonemes, we also observed (rare) neurons in the upper cortical plate with docked centrioles lacking a clear procilium. This difference in cilia development would seem to follow the insideout pattern of corticogenesis, and correlate wit h the length differences observed with ACIII staining. Further, at P0, ACIII staining revealed occasional neurons in the subplate exhibiting relatively longer procilia than other layers, suggesting that those cells could display more mature axonemes as the ones found at

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45 EM level. Similarly, at P4, ACIII staining revealed some relatively long procilia located in the subplate and in a subpopulation of neurons in layer 5. EM analysis at the same age revealed lack of axonemes in all procilia analyzed, but proci lia in neurons in the deep cortical plate were frequently enriched with microtubular content, suggesting the beginning of axonemal development. Based on this, we propose that axoneme elongation begins ~P0 only in some cells in the subplate, but it is an ongoing process that develops further by P4 in the deep cortical plate and extends subsequently to all layers, and probably parallels the cilia elongation pattern reported by ACIII staining. In fact, the axoneme was first observed in all layers at P8, coinci dent with significant ACIII immunodetection by western blot and sprout in length of cilia. This coincidence suggests that axoneme development is probably responsible for the secondary elongation of the cilia. Although we focused our study on neurons, at P0 and P4 the high packing density of cortical cells and the incomplete expression of NeuN (Lyck et al., 2007) precludes the selective study of neurons. Therefore it is possible that some ACIII+ particles would belong to nonneuronal cells. In animals older than P4, we could analyze neuronal cilia more reliably due to lower neuronal density and mature NeuN expression. However, some cilia could be incomplete due to sectioning trimming, and we are measuring the planar projection of cilia, and therefore underestimating their length. Therefore, our quantification study is not intended to provide precise values of neuronal cilia length, but aimed to give a general picture of cilia distribution and elongation in different cortical layers along development. Similarly, we focused on cells

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46 with neuronal features in our EM analysis, but we cannot discard the possibility that some non neuronal cilia were included in the study. From P7 onwards, a more or less steady elongation of cilia is observed, first and faster in deep layers, which is followed later by upper layers. Length values stabilize between P60P90 with average lengths of about 5 microns in all layers, except layer 5 that has longer cilia (~6 m on average and up to ~11 m), far from the maxima in other layers. Layer I is unlike layers 26 due to its scarce neuronal content, but as expected, the few neurons detectable with NeuN typically exhibited a long cilium enriched with ACIII. Initially, layer 1 neurons displayed long cilia compared to upper l aminas, but after P7 their values were similar to other layers. These laminar differences in length and rate of growth could be related to differences in neuron/soma size and maturation rate in different layers (Lund et al., 1977, Van Eden and Uylings, 1985) but we did not study those relationships in the present work. On the other hand, we did not find significant differences in estimated cilia length between sexes. Surprisingly, we found that the lengths of cilia at P170 and P365 were decreased compared to P60P90. The explanation for this decrease is currently unclear. It is possible that cilia slightly shorten with aging, but another possibility is a reduced or incomplete localization of ACIII in older animals. ACIII staining tapers towards the tip of the cilium (Poole et al., 1997) ; present results) and therefore reduced levels of ACIII could lead to incomplete filling of the cilia length. Separate studies on aged rats (>3years) did not show significant cilia shortening (data not shown), suggesting that cilia do not continue to sh orten with advanced aging. Further studies are needed to clarify these questions.

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47 Overall, our study shows that ciliogenesis is a protracted process in mouse neocortex: procilia seem to remain undifferentiated from late fetal to early postnatal stages and the subsequent elongation of axonemes and cilia takes several weeks to complete, till P60P90 ( Figure 2 14). This delayed time course of cilia maturation is very surprising considering cilia growth can be very rapid (on the order of hours) in many nonneu ral cells in culture (Tucker et al., 1979a, Tucker et al., 1979b) and in the order of days in the chick neural tube (Sotelo and TrujilloCenoz, 1958) Further, cilia are observed within days on cultured neurons (derived from fetal/perinatal tissues) from a variety of brain regions including hippocampus, striatum, amygdala, cerebellum and spinal cord (Berbari et al., 2007, Miyoshi et al., 2009, Barzi et al., 2010, Belgacem and Borodinsky, 2011, Domi re et al., 2011b) It is difficult to compare the growth that we see in vivo to these newly maturing neurons until studies are performed that describe the in vivo development of these structures in each brain region. It is noteworthy that neurons from different brain regions grow cilia of different lengths (Fuchs and Schwark, 2004) but whether neurons from different brain regions exploit different mechanisms to control the onset and duration of ciliogenesis needs further analysis. With respect to neocortex (and possibly other regions), prolonged maturation of cilia overlaps with neocortical neuron maturation that comprises key developmental processes such as dendrite growth and synaptogenesis: processes that are defined by actin and microtubule rearrangements and extensive vesicle trafficking, similar to ciliogenesis (Kim et al.) Whether the coincidental developmental timeframes of cilia and other neuronal processes are interrelated also requires further study.

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48 Appearance of Signaling Machinery in Neuronal Cilia ACIII can exist at different molecular weights (e.g. ~125 and ~200 kDa) based on glycosylation (Wei et al., 1996) We found that ACIII from cortex appeared to be in the less glycosylated ~125 kDa form as reported by others (Murthy and Makhlouf, 1997) We also found that this differed from the more glycosylated form that is observed in lysates of olfactory epithelium ( (Wong et al., 2000) present study). The significance of these different forms of ACIII between different brain regions is unclear. Our analysis of ACIII protein in fetal cortical lysates showed early detection at very low levels at E13.5, E16 and E18.5. These results could correspond with the formation of procilia starting at E13.5, although we cannot rule out the possibility that our blots are detecting nonneuronal or potentially nonciliar y ACIII expression. At E18.5, we observed short ACIII+ procilia coinciding with per icentrin+ basal bodies. Later, as development continues postnatally, ACIII expression levels increase dramatically, which directly correlated with the elongation of ACIII positive cilia throughout the neocortex. These levels may also correlate with the cel lular production or available levels of cytosolic tubulin, which recently were reported to correspond with cilia length (Sharma et al., 2011) It was unclear why the levels of ACIII protein at P60 appear to be lower than earlier in development. Whether this reflects a role for ACIII earlier in development during cilia elongation (Ou et al., 2009) a change in ACIII concentration within cilia over time, or some other mechanism requires further study. From E16.5 throug h P0 until ~P4, our EM analysis showed a slight growth of the prociliar length without apparent axonemal development (except for some subplate cells). The elongation of the cilia axoneme is coordinated by intraflagellar transport (IFT), a bidirectional tra fficking mechanism to shuttle cilia components to and from the

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49 cilium tip (Pedersen and Rosenbaum, 2008) Mutations in IFT molecules are often associated with defective assembly of primary cilia. For example, Pazour and colleagues (Pazour et al., 2000) showed that impairment of IFT in mice resulted in short cilia resembling the procilia shown here. Thus, it is tempting to speculate that IFT might not be fully active at these early stages of ciliogenesis, and later development of this mechanism could be responsible for axoneme and cilia elongation. This is one potential hypothesis for future studies on neuronal cilia. The EM micrographs typically reveal ed Golgi apparatus and different types of vesicles in close apposition to the basal body and centriole, and also inside the procilia, suggesting that trafficking to the newly forming cilium might be significant at these ages. Considering the presence of AC III in procilia from E18.5 to P4, when the axoneme is poorly developed, suggests that ACIII localization to the procilium is, or at least can be, independent of the presence of a fully developed axoneme. This is notable in light of the fact that downregulation of BBS2 and BBS4 (Bardet Biedl syndrome (BBS) proteins, which regulate vesicular transport to the cilium) impedes SSTR3 but not ACIII localization in cilia and do not notably alter cilia elongation (Berbari et al., 2008b) In cell lines, it has been suggested that ACIII can contribute to the elongation of cilia (Ou et al., 2009) However, ACIIIdeficient mice extend cilia of comparable lengths and remain able to traffic neuronal cilia receptors such as SSTR3 (Wang et al., 2009) Taken together, these data suggest that independent routes of protein trafficking t o the cilium may underlie the developmental time course of receptor expression and cilium maturation.

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50 In hippocampus, SSTR3 appears in cilia between ~P0P3 (Stanic et al., 2009) and in neocortex we previously observed SSTR3 in neonatal rat neocortex (Anastas et al., 2011) However, no studies are available about the development of SSTR3 localization in cilia in the neocortex. The field awaits further characterization of content and developmental appearance of other neuronal cilia receptors. Neuronal Cilia Do Not Seem Essential for Proper Neuronal Migration or for Acquisition of Neuronal Polarity. We observed that in pyramidal neurons ( (Anastas et al., 2011) present data), the docking of the basal body generally forms on the pial side of the cell. However, in the major interneuron subtypes, PV+ and CB+ cells, the cilia/basal body seemed to be more randomly located on the soma. CR+ cells, however of ten exhibit a highly polarized morphology, and we found the cilia could be in the apical dendrite. Overall, these data suggest that the positioning of the cilium might be related to the polarity of the neural cell type in that there is correlation between the docking location of the basal body and the relative position of the mother centriole as migration ceases. However it seems that cilia formation is not essential for neuronal polarity or expression of layer specific markers in neocortex. Despite a sev ere defect in ciliogenesis, analysis of mutant Stumpy mice revealed normal polarity of pyramidal neurons with a well developed apical dendrite. The preserved expression of laminar markers suggests loss of cilia does not grossly disrupt timing of neuronal m igration, which is consistent with our findings that cilia initiate growth post migration. We cannot rule out that loss of cilia alters the fine positioning or morphology of cortical neurons. For example we have observed subtle abnormalities in Purkinje cell position and morphology in the

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51 granule cell dysgenesis or altered Sonic hedgehog signalling in the region (Chizhikov et al., 2007, Breunig et al., 2008, Spassky et al., 2008) Duplication of a neurons cilium number also does not seem to alter the general ori entation of pyramidal neurons (Sarkisian et al., 2001, Anastas et al., 2011) Thus, dramatic changes to a neurons cilium do not grossly affect its position or morphology. How or whether such mutations ultimately influence intracellular signalling pathways or connections between neurons requires further investigation. Despite the increased attention given to this organelle, more precise genetic manipulations will be needed to definitively assess the role of primary cilia in neurons. Moreover, a proper understanding of t he dynamics of ciliogenesis will be needed to harness these technologies and to create informed hypotheses about these organelles. Our detailed description of the process of neuronal ciliogenesis will be useful for these endeavours and for future studies examining the significance and functional role of neuronal cilia. Materials and Methods Mice Mice of the CD1 strain (24 animals per age) were collected on embryonic (E) days 11.5, 13.5, 16, 18.5 and post natal (P) days 0, 1, 3, 4, 7, 8, 14, 21, 60, 90, 170 and 365. P0 and older mice were intracardially perfused with saline followed by 4% paraformaldehyde (PFA) in 0.1M phosphate buffer solution (PBS). After dissection and fixation, brains were either sectioned (60 m coronal) in a vibratome or cryoprotected, frozen over liquid N2 and sectioned (20, 50 or 60 m coronal) on a cryostat. Stumpy mutant brain tissue was generated as previously described (Town et al., 2008) Briefly, a floxed stumpy allele was deleted in the presence of Cre under the control of the

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52 Nestin promoter. Floxed stumpy mice were originally generated on a B6/129 back ground and then backcrossed into a B6 background for 10 generations. Floxed mice were then crossed with NestinCre deleter mice on a pure B6 background (The Jackson Laboratory). Animal care procedures were performed in accordance with the Laboratory Anim al Welfare Act, the Guidelines for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD) and the approval of both the University of Florida and Yale University Institutional Animal Care and Use Committee. In Utero Electropora tion We used in utero electroporation to deliver plasmid DNA, pCAGGS GFP into fetal cerebral cortices as previously described (Sarkisian et al., 2006, Rasin et al., 2007) Briefly, at 13.5 days into gestation (E13.5), female CD1 mice were anestheti zed by an intraperitoneal injection of ketamine (100mg/kg) and xylazine (10mg/kg) diluted in sterile saline. The uterine horns were exposed and ~1L of DNA [0.5g/L] mixed with 0.025% Fast Green) was microinjected through the uterine wall into the lateral ventricles of the cerebral cortices of the mouse embryos using pulled glass capillaries. Electroporation was achieved by discharging 40V across the cortex in five 50msec pulse series spaced 950msec apart using a BTX ECM 830 Square Wave Electroporator. F ollowing injections, the dams were sutured and allowed to recover on heating pads. Electroporated embryos were harvested at E16.5 and brains were dissected and processed for immunoEM as described below. Immunohistochemistry Tissue sections were probed 2448 hours at 4C using the following primary antibodies (dilutions listed in Table 1): rabbit anti adenylyl cyclase (ACIII), mouse anti NeuN, mouse anti parvalbumin, goat anti Foxp2, rabbit anti CDP (aka Cux1), rabbit

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53 anti pericentrin; mouse monoclonal anti Calretinin, mouse anti pericentrin, chicken anti green fluorescent protein (GFP). After rinse in PBS, appropriate species specific, fluorescent conjugated secondary antibodies were used (1:200; Jackson ImmunoResearch) for each antibody. After rinse in PBS immunostained sections were coverslipped using ProLong Gold Antifade media containing 4', 6diamidino 2 phenylindole dihydrochloride (DAPI) (Invitrogen). For combination of ACIII and pericentrin using both rabbit antibodies, tissue was incubated first i n ACIII and developed with Fitc conjugated monovalent Fab secondary antibodies (1:200, Jackson Immunoresearch), followed by incubation in pericentrin and development in conventional Cy3conjugated secondary antibodies. No overlapping domains were observed in the cortex as shown in Figure 2 1A. Antibody Characterization The antibodies used in this study were tested by immunostaining of mouse brain sections or by western blot analyses of mouse brain lysates. The data we collected for each antibody was consist ent with known information about each protein. Antibody information is detailed in Table 2 1 with further specificity details listed below. Rabbit anti ACIII was raised against the C terminal 20 amino acids of mouse type III adenylyl cyclase (ACIII). By w estern blot, this antibody can detect bands between ~125 and ~200 kDa depending on the level of glycosylation (Wei et al., 1996, Murthy and Makhlouf, 1997, Wei et al., 1998, Wong et al., 2000) Immunostaining in the brain reveals specific enrichment in neuronal cilia which was confirmed by the abs ence of ACIII detection in cilia from ACIII knockout mice (Bishop et al., 2007, Wang et al., 2009) The pattern of staining in our study is consistent with the above citations.

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54 Mouse monoclonal antibody against actin was raised against a modified cytoplasmic actin N ter minal peptide (Ac Asp Asp Asp Ile Ala Ala Leu Val Ile Asp Asn Gly Ser Gly Lys) conjugated to KLH. By western blot, the antibody detects a 42 kDa (predicted MW of actin) from lysates of cultured mouse, human or chicken fibroblast extracts result in a band. Results below reveal an identical pattern and molecular weight for actin (see Figure 2 1). Mouse monoclonal antibody against calbindin D 28K was produced by hybridization of mouse myeloma cells with spleen cells isolated from mice immunized with CB D28k purified from chicken gut. This antibody specifically stains the 45Cabinding domain of calbindin D 28k (MW 28kDa, IEP 4.8) in a twodimensional gel. By radioimmunoassay it detects calbindin D 28k with a sensitivity of 10 ng/assay and an affinity of 1. 6 x 1012 L/M. Immunoblots of tissue originating from several species including rodents and primates show a band of 28 kDa, and the antibody does not crossreact with other known calcium binding proteins (Celio et al., 1990) Antibody CB300 immunolabels a subpopulation of neurons in the normal brain with high efficiency but does not stain in the brain of calbindin D 28k knockout mice (Airaksinen et al., 1997) Mouse anti calretinin was produced in mice by immunization with recom binant human calretinin22k (Zimmermann and Schwaller, 2002) an alternative splice product o f the calretinin gene and identical with calretinin up to Arg178. The antibody 6B3 stains a 29kD band (calretinin MW = 29 kD) on immunoblots of brain extracts from mouse, rat and macaque. Immunohistochemistry with the antibody stains a subpopulation of nonpyramidal cells in the cortex of mice that is absent in CR KO mice (Bearzatto et al.,

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55 2006) The antibody does not cross react with CalbindinD 28K or other calcium binding proteins as shown by immunoblots or immunostaining in brain tissue. Chicken anti GFP antibody was raised against the recombinant full length synthetic peptide of jellyfish Aequorea victoria. B y western blot, analysis of transgenic mouse spinal cord or mouse carcinoma cell lines express GFP, this antibody recognizes a distinct band between 27 and 30kDa (predicted MW of GFP = 27kDa). Immunostaining of untreated wildtype brain tissue with this ant ibody shows no detectable staining. Mouse monoclonal against neuronspecific nuclear antigen NeuN was originally made against cell nuclei purified from mouse brain, and western blotting with this antibody shows three bands in the 46 48 kDa range (Mullen et al., 1992) The anti NeuN antibody showed a pattern of neuronal nuclei in the developing mouse brain as previously described (Mullen et al., 1992) We also observed NeuN in the neuronal cell body which is not only consistent with previous reports (Lyck et al., 2007, Kao et al., 2008, Lorenzo et al., 2008, SainoSaito et al., 2011) but also recent identification of NeuN as splicing regulator Fox 3 whi ch appears to have isoforms that localize to the cytoplasm (Kim et al., 2009, Dredge and Jensen, 2011) Mouse monoclonal anti parvalbumin reacts specifically with parvalbumin (PV) in cultured nerve cells and in mouse tissue, and specifically stains the 45 Cabinding spot of PV (MW=12 kDa and IEF 4.9) in a two dimensional immunoblot. (Celio and Heizmann, 1981) Staining was located in a subpopulation of nonpyramidal cells in the neocortex as previously described (Cho et al., 2011)

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56 Rabbit polyclonal antibody anti pericentrin was raised against a fusion protein containing 60 kDa of pericentrin (amino acids 870 1370 of the mouse protein) and T7Gene10 (Doxsey et al., 1994) This antibody recognizes a single 220 kDa band by immunoblotting and has been extensively characterized in a previous study (Doxsey et al., 1994) Staining in the developing neocortex showed a pattern of pericentrin immunoreactivity that was identical to previous descriptions (Westra et al., 2008) Mouse monoclonal anti pericentrin was raised against amino acids 16921814 of mouse pericentrin and purified by affinity chromatography. As described in an original characterization of pericentrin (Doxsey et al., 1994) this antibody detects a band at 220kDa (MW of pericentrin). The pattern of immunostaining with this antibody is also comparable to other studies that show enrichment in centrioles (Jurczyk et al., 2010) We additionally found that this antibody colocalizes with rabbit pericentrin where we found it to be localized to the basal body at the cilium base (data not shown). Rabbit polyclonal anti Cux1 (anti CDP) was raised against amino acids 11111332 of CDP (CCAAT displacement protein) of mouse origin. By western blot of mouse liver extracts, this antibody detects a band at ~200kDa which is knocked down by specific microRNAs (Xu et al.) This antibody has also been shown by multiple investigators to effectively label neurons in upper layers 2/3 of neocortex (Feng and Cooper, 2009, Tury et al., 2011) The pattern of staining in our study is consistent with these citations. Goat anti Foxp2 was r aised against a synthetic peptide REIEEEPLSEDLE corresponding to C terminal amino acids 703 715 of human FOXP2 protein. By western blot analysis of human cerebellum lyastes, this antibody detected a single band at

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57 ~80kDa (predicted MW = ~79.9kDa) (manufact urers technical information). Staining for Foxp2 reveals strong expression in neuronal nuclei of deep layers of neocortex (Ferland et al., 2003, Stillman et al., 2009, Waclaw et al., 2010) The patterns of antibody staining in our study resemble that reported in the cited studies above. Analy sis and Quantification of Cilia For quantification of cilia length, mice at P0, P4, P7, P14, P60, P90, P170 and P365 were analyzed (2 brains/age and 2 sections/brain) in area S1Tr from the mouse brain atlas (Paxinos and Franklin, 2004) located dorsal to the hippocampus and medial to the barrel field cortex. Vibratome sections (60 m) were immunostained for ACIII and NeuN, and co unterstained with DAPI. Mosaic stacks (918 images separated ~ 1 m in the z axis) were taken from the pial surface to the white matter using a Zeiss Apotome system attached to a Zeiss Axioplan2 microscope with a 20x objective using Zeiss Axiovision softwa re. Stacks were collapsed to the maximum intensity projection and the resulting images were adjusted for grey levels for each channel using the same software. Images were imported into Reconstruct software (Fiala, 2005) where they were aligned, layers were delimitated, and cilia were traced and measured. A total of 3756 cilia were measured, averaging ~85 per layer and age studied in this analysis performed by JIA. In addition, a separate analysis was performed by SMG at ages E18.5, P0, P3, P7, P14, P21, P60, P90, using an Olympus IX81DSU spinning disc confocal microscope. Z stack of images (at 0.75 m steps) of sections immunostained for ACIII (Santa Cruz) and pericentrin (BD Biosciences) were collected and subsequently collapsed to the maximum intensity projection. Resulting images were analyzed using Image J64 (http://rsbweb.nih.gov/ij/) whereby cilia lengths were measured from the pericentrin puncta side to the end of the continuous ACIII+ signal in

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58 at least 2 brains and 68 fields/section for each age. Although not displayed, similar results were obtained in both analyses. For analysis of ACIII+ cilia extending from PV+ cells, Z stack images of sections i mmunostained for ACIII and PV were converted to maximum intensity projection images, and ACIII+ cilia lengths were analyzed in Image J64 as described above. Analyses of cilia length between sexes were compared using Students t tests (two tailed). In all t ests, a p value < 0.05 was considered significant. Electron Microscopy (All EM analysis was done by JIA) For the immunoEM analysis, electroporated brains were fixed at E16.5 by immersion in 4% PFA and 0.3% glutaraldehyde in PBS for 24 hours at 4C. Brai ns were dissected, embedded in 4% agarose and sectioned (100 m thick) using a vibratome (Leica). Sections were collected in PBS, cryoprotected with 30% sucrose and freezethawed over liquid nitrogen to permeabilize the tissue. After rinse in PBS, sections were incubated with antibodies against GFP for 24 hours, rinsed in PBS, incubated with biotinylated secondaries (Jackson ImmunoResearch) for 2 hours, rinsed in PBS, incubated in ABC elite kit (Vector), rinsed in PBS and developed using DAB (Vector) as chr omogen. From this point, sections were postfixed and processed for EM in the same way as described below. For the conventional EM analysis, animals were intracardially perfused with saline followed by 4% PFA in PBS for P60 brains and 1% PFA and 1.25% glut araldehyde in PBS for the rest of ages. Brains were dissected and postfixed in the same fixative overnight. After rinse in PBS, brains were sectioned (60100 m thick) coronally with a vibratome. Animals P8 or younger were embedded in 4% agarose before sec tioning. P60 sections were collected in PBS and postfixed in 2% glutaraldehyde for 1 hour. All sections were postfixed in 1% osmium tetroxide for 40

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59 min, and then rinsed, dehydrated, embedded in Durcupan (Fluka, Buchs, Switzerland) and cured in an oven for 48 hours at 60C. Neocortical regions of interest were sectioned at 70 nm in a Reichert ultracut ultramicrotome. Serial sections were collected in slot grids covered with Formvar, counterstained with uranyl acetate and lead citrate and analyzed in a Jeol JEM 1010. Pictures were taken with a Gatan MSC600W digital camera and adjusted for bright and contrast using Adobe Photoshop. Western Blots Protein lysates from mouse cortex were prepared by homogenizing tissue in 1X RIPA buffer [containing 20 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% vol/vol Triton X 100, 2.5 mM sodium pyrophosphate, 1 mM glycer ophosphate, 1 mM Na3VO4, 1 mg/mL leupeptin, and 1 mM PMSF] (Cell Signaling Technology). For developmental timepoints, similar total amounts ( 30g/lane) were loaded onto 412% NuPAGE gel (Invitrogen) and separated by SDS PAGE. Proteins were transferred onto PVDF membranes using an iBlot (Invitrogen). Blots were blocked in 5% BSA in Tris Buffered Saline containing 0.1% Tween (TBST) for 1 h at RT. The following primary antibodies were diluted in 2.5%BSA in TBST incubated overnight at 4C: rabbit anti ACIII, or mouse anti actin. Membranes were rinsed in TBST, and incubated with appropriate HRP conjugated secondary antibodies (1;10,000; BioRad). Bl ots were developed using chemiluminescence (ECLPlus Kit (GE HealthCare), and images were captured using an Alpha Innotech FluorChemQ Imaging System (Cell Biosciences).

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60 Table 21 Primary antibodies used in this study Antigen Immunizing antigen Manufacturer Details Working Dilution Adenylyl Cyclase III (ACIII) Synthetic peptide with the C terminal 20 amino acids of mouse ACIII (PAAFPNGSSVTLPHQVVDNP) Santa Cruz Biotechnologies; cat #: sc 588; rabbit polyclonal 1:1000 (WB) 1:1000(IHC) actin A modified cytoplasmic actin N terminal peptide (DDDIAALVIDNGSGK, conjugated to KLH) Sigma; cat #: A5316; mouse monoclonal 1:10,000 (WB) Calbindin D28K Calbindin D 28k purified from chicken gut Swant; cat #: 300; mouse monoclonal 1:4000 (IHC) Calretinin Recombinant human calretinin 22k Swant; cat #: 6B3; mouse monoclonal 1:2000 (IHC) Enhanced green fluorescent protein (eGFP) Recombinant full length eGFP Abcam; cat # ab13970; mouse monoclonal 1:5000 (IHC) Microtubule associated protein 2 (MAP2) Rat brain microtubule associated proteins Sigma; cat #: M4403; mouse monoclonal 1:1,000 (IHC) Neuronal nuclear protein (NeuN) Cell nuclei purified from mouse brain Chemicon; cat #: MAB377; mouse monoclonal 1:1000 (IHC) Parvalbumin Purified parva lbumin from carp muscle Swant; cat # 235; mouse monoclonal 1:4000 (IHC) Class III tubulin (Tuj1) Purified microtubules from rat brain Covance; cat #: MMS 435P mouse monoclonal 1:1250 (IHC) Pericentrin A fusion protein containing ~60kD of pericentrin ( amino acids 870 1370 of the mouse protein). Covance; cat #: PRB 432C; rabbit polyclonal 1:500 (IHC) Pericentrin Amino acids 1692 1814 of mouse pericentrin BD Biosciences; cat # 611815; mouse monoclonal 1:200 (IHC) Cux 1 Amino acids 1111 1332 of mouse CDP (CCAAT displacement protein) Santa Cruz Biotechnologies; cat #: sc 13024; rabbit polyclonal 1:100 (IHC) FOXP2 Synthetic peptide: REIEEEPLSEDLE, corresponding to amino acids 703 715 of human FOXP2 Abcam; cat #: ab1307; goat polyclonal 1:100 (IHC) Arellano, J.I., et al. Development and distribution of neuronal cilia in mouse neocortex. J Comp Neurol, 2012. 520(4): p. 84873.

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61 Table 22 Average l ength of cilia in the n eocortical lamina at d ifferent a ges L1 L2 L3 L4 L5 L6 P0 0.90.1 (2) 1.30.1 (3.3) 1.30.1 (3.3) 1.30.1 (3.3) 1.20.1 (3.7) 1.20.1 (3.7) P4 2.50.2 (4.6) 1.30.1 (2.2) 1.80.1 (3.1) 1.80.1 (3.1) 3.40.1 (6.6) 2.90.1 (4.5) P7 2.70.3 (4.2) 3.80.1 (7.1) 4.40.1 (7) 3.40.1 (5.3) 3.80.1 (6.9) 30.1 (5.3) P14 40.4 (6.5) 4.50.1 (6.9) 4.80.1 (8.3) 3.50.1 (5.7) 4.60.2 (8.4) 3.50.1 (6.9) P60 4.60.2 (7) 50.1 (7.7) 5.20.1 (9.2) 4.50.1 (7.5) 6.30.2 (9.7) 4.40.1 (7.5) P90 4.10.2 (6.5) 4.30.1 (6.1) 5.20.1 (7.9) 5.10.1 (7.7) 6.20.1 (10.8) 5.10.1 (8.1) P170 31 (4.9) 3.90.3 (6.6) 3.60.2 (6) 3.70.4 (5.9) 4.80.2 (8.3) 4.40.1 (8.1) P365 4.90.5 (6.6) 4.40.2 (7.3) 4.30.1 (6.2) 3.90.2 (4.8) 50.3 (9.9) 4.60.1 (6.5) Average length SEM and maximum length noted (in parentheses) of cilia in the cortical layers at different ages. At P0 upper layers were indistinguishable, and the same value from upper layers is used for layers 2, 3 and 4. The same applies to layers 3 and 4 at P4. The s ubplate at P0 was included in layer 6, but at P4 showed marked differenc es, with a mean cilia length of 3.8 0.1 and a maximum of 5.4 m. Arellano, J.I., et al ., Development and distribution of neuronal cilia in mouse neocortex. J Comp Neurol, 2012. 520(4): p. 84873.

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62 Figure 2 1. Expression of adenylyl cyclase III during fetal and postnatal cortical development. A: Maximum intensity projection of a z stack of layer 3 pyramidal neurons in the neocortex of a P90 mouse. Cilia were immunostained with ACIII (green), basal bodies with pericentrin (red), neuronal somata with NeuN (purple) and nuclei stained with DAPI (blue). Scale bar = 10m. B: Western blot detection of ACIII from mouse cortical lysates from embryonic day 11.5 (E11.5) to young adult (~P60). The upper blot for ACIII revealed a band close to the predicted MW of ung lycosylated ACIII (molecular weight (MW) at ~125kDA). Generally, ACIII expression significantly increases between P0 and P21. At P60, there is a decrease in actin (lower blot) was used as a loading control. C: Protein l ysates of P90 olfactory epithelium (OE) or frontal cortex (FC) were separated by western blot and probed for ACIII. Very strong expression of ACIII is detected at ~190200kDa which has been shown to reflect high levels of glycosylated ACIII (bracket with ). This higher MW signal was absent in FC sample that revealed only a lower MW signal for ACIII (lower bracket).

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63 Figure 2 2. Basal bodies are more prevalent in deeper cortical plate at E16.5. E13.5 embryos electroporated with cDNA encoding GFP, sacrif iced and fixed for immunoEM at E16.5. A: DAB immunostaining with anti GFP antibodies shows GFP+ cells (arrows) with leading and trailing processes in both deeper layer and upper layer of the cortical plate (CP). B: Higher magnification of the upper CP showing a cell (arrow) with a typical migrating profile. C: Bar graph shows a summary of the position of centrioles/basal bodies that were identified in deeper and upper layers of the CP docked (to the plasma membrane) or undocked. In deep CP ~70% were docked (mostly in cells that were GFP ). In upper CP, most centrioles (~90%) were undocked. D I: Electron micrographs examples of centrioles and basal bodies in deep (D G) or upper (H,I) CP. D1: A docked basal body in a GFP cell (boxed area shown in D2). GFP im munoprecipitate is visible in the upper left cell (arrow). E: A docked basal body with a small axoneme extension (arrowhead). F1: Another example of a docked basal body in a GFP cell. The boxed region is shown in F2 and an adjacent section (AS) in F3. G1 and G3: Adjacent sections of a GFP+ cell (arrows point to GFP precipitate) with a leading process. Boxed regions in are magnified in G2 and G4, respectively. H1: A GFP+ cell in the upper CP with undocked centrioles (boxed area is magnified in H2 which sho ws two centrioles (arrowheads). I1: Another example of a GFP+ cell in the upper CP. The boxed area of I1 is enlarged in I2 with an adjacent section shown in I3. Scale bars in (A) =50m; (D1, E, F1, G1, H1, I1)= 2m; (D2, F2, G2, H2) =0.5m.

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64 Figure 2 3. Procilia in the fetal cortical plate at E16.5. Cells of interest and procilia are pseudocolored in red to help identification. A: Serial sections (70 nm thick) illustrating an undocked mother centriole with a vesicle attached. located adjacent to the c ell membrane (asterisks). Inset in A1 shows the daughter centriole found in adjacent sections and (G) indicates Golgi cisterns around the centrosome (G). A2: A membranous centriolar protrusion, consistent with a procilium budding into the vesicle. This ves icular attachment is predictably the step before docking of the mother centriole to the cell membrane. Scale bar = 0.25 m in A1A3. B: Docked basal body with a protruding small procilium. B1: panoramic view of the cell studied. Square indicates the magnif ied region in B2. B2: detail of the mother centriole (arrow) and the daughter centriole (arrowhead) located between the nucleus and the pial oriented process. B3B6: serial sections (70 nm thick) illustrating the short procilium (~0.1 m long) protruding f rom the attached mother centriole (arrow), Scale bar = 3.6 m in B1; 0.5 m in B2 and 0.25 m in B3B6. C. Docked basal body with developed procilium. C1: panoramic of the cell analyzed showing the procilium (~0.3 m long; arrow) protruding outside the cel l. Inset shows the centriole surrounded by Golgi cisterns, located in the position of the asterisk in adjacent sections. C2C5: serial sections (70 nm thick) illustrating the extent and mushroom shape of the procilium (red and arrow). Scale bar = 0.54 m i n C1 and 0.25 m in C2 C5.

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65 Figure 2 4. Elongation of neuronal cilia over several postnatal weeks. A E: Examples of basal bodies (pericentrin+; red; example indicated by red arrowhead) and cilia (ACIII+; green; example indicated by green arrowhead) for the indicated ages. Bar (in A) for A E = 2.5m. F: Bar graph shows the average SEM of cilia length (m) in the neocortical layers at the indicated ages. Because it was difficult to differentiate neocortical lamina at early ages, average lengths for laye rs 2, 3 and 4 were pooled for P0, while at P4 layers 3 and 4 were also pooled. G: Graphic representation of cilia elongation across age in the cortical layers. Mean values of length are used (SEM values are omitted for clarity but can be found in Table 22 ). H: Comparison of cilia length in upper layers (24; UL) and deep layers (5 6; DL) of the neocortex in male and female P14 mice did not show significant effect of gender.

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66 Figure 2 5. Distribution of procilia in the neocortex at P0 and P4. A: Low magnification view of the P0 neocortex immunostained for ACIII (green) and NeuN (red) and counterstained with DAPI (blue). NeuN is expressed in some cells of layer 25, scarce cells in layer 6 and intensely by some cells in the subplate (Sp). B D: Details of t he neocortex illustrating approximate layers 14 (L1, L24) in panel B, layers 5 and 6 (L56) in C and the subplate (Sp) in D. ACIII+ specks were found in all layers, with rare longer, rodshaped cilia. Scale bar (in K) = 15 m in A and 8 m in B D. E: Low magnification view of the P4 neocortex immunostained for ACIII and NeuN and counterstained with Dapi. F K: High magnification details of the cortical layers from layer 1 to the subplate. NeuN is still not fully expressed by all neurons, and layer 2 (G) an d deep layer 6 (J) have few stained neurons. In contrast, the subplate (K) shows large neurons intensely stained with NeuN. Overall, intense ACIII+ puncta are predominant, although scattered longer cilia can be found in all layers (arrows) and particularly in some neurons in layer 5 (I) and in the subplate (K). Scale bar = 30 m in E and 10 m in F K.

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67 Figure 2 6. Heterogeneity of procilia at P0 in the upper cortical plate. Cells and procilia of interest are pseudocolored in red to help with identificati on. A1: Low magnification view of a cell with neuronal morphology in layer 2; arrow indicates the position of the docked centriole (basal body). A2 shows higher magnification of the basal body (Bb) and part of the adjacent daughter centriole (C). Note the lack of procilium (arrow) that was also absent in adjacent serial sections. Scale bar is 4 microns in A1, 0.18 microns in A2. B1B7: A procilium in a cell with neuronal morphology in layer 2. B1: Low magnification view of the cell with an arrow indicating the location of the procilium. B2: Cross section of the basal body showing microtubular doublets (arrows) in the transitioning basal body/axonemal rudiment. B3B6: Serial sections (70 nm) through the procilium show the lack of axoneme but the presence of a tubular/vesicular network (arrows) inside the procilium. B7: 3D reconstruction of the procilium (red) and basal body (blue). Scale bar is 3 microns in B1, 70 nm in B2B6, 0.15 microns in B7. C1 C6: Procilium in a cell with neuronal morphology. C1: Low magnification view of a cell with an arrow indicating the location of the procilium. C2: Transition between the basal body (arrowhead) and procilium. C3C5: Serial sections illustrate a short procilium lacking microtubules and containing some vesicular struct ures (arrows). C6: 3D reconstruction of the basal body (blue) and the procilium (red) indicating the levels of C2C5 sections. Scale bar is 2 microns in C1, 70 nm in C2C5, 0.1 microns in C6. D1D6: Serial sections showing a procilium growing inside the cy toplasm in a cell in the upper cortical plate. D1: The basal body (arrowhead) and an adjacent vesicle (small arrow). D2: The basal body (arrowhead) and the budding procilium with a vesicle attached (large arrows). D3D4: The procilium is surrounded by the cell membrane and reaching the extracellular space in contact with an adjacent cell (discontinuous white line). Vesicles in and around the procilium are indicated with arrows. D5: final section containing the procilium (asterisk). D6: Schematic of the proc ilia (Pc) growing inside the cytoplasm. Bb is basal body, V are vesicles and P are polyribosomes. Scale bar is 90 nm in D1D5.

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68 Figure 2 7. Procilia in the deep cortical plate at P0. Cells and procilia of interest are pseudocolored in red to help with identification. A1: Low magnification view of a cell with neuronal morphology bearing a procilium (arrow) in its pial aspect. A2A6: Serial sections of the procilium from the cell in A1. A2: The transition between the basal body (arrowhead) to the procilium. The procilium lacks an axoneme, but some microtubule like structures are detectable (arrows in A2A6). A7: 3D reconstruction of the basal body (blue) and the (incomplete) procilium (red) indicating the relative position of sections A2 and A6. Scale bar is 2 microns in A1, 90 nm in A2 A6, 0.26 microns in A7. B1B3: Procilium in the deep cortical plate with cabbage like morphology. B1: Low magnification image of the cell with neuronal morphology bearing the procilium. B2 B3: serial micrographs showing the basal body (arrowhead) and the procilium with membrane foldings and multiple vesicles (arrows). Scale bar is 2 microns in B1, 0.18 microns in B2B3. C1 C2: Another example of a procilium containing multiple vesicles (arrows) and occasional tubular struct ures (small arrow). Vesicles (V) were also frequent around the basal body (arrowhead in C1). Scale is 0.24 microns in C1C2. D1 D5: A cilium with axoneme in a cell with neuronal morphology in the subplate. D1: Low magnification image of the cell bearing the cilium (arrow). D2 D5: Selected serial oblique sections of the cilium illustrating the presence of parallel microtubules (arrows) forming the axoneme. The distance (nm) between sections is indicated in the upper right corner. Surprisingly, the microtubul es are more visible in distal sections (D4, D5) than in proximal ones (D2, D3). D6: A 3D reconstruction of the cilium (basal body is not illustrated) showing the levels of sections D3D5. Scale bar is 1.8 microns in D1, 0.2 microns in D2D3; 0.16 microns i n D4 D5, 0.25 microns in D6.

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69 Figure 2 8. Procilia in P4 neocortex. Cells of interest and procilia are pseudocolored in red to help identification. Illustrated are examples of procilia lacking axonemes (A C), and procilia located in the deep cortical plate with some microtubular structures but lacking a well formed axoneme (D F). A C: Enlarged, apparently immature procilia, present both in the deep (A) and superficial (B, C) cortical plate. A1: Low magnification view of a cell with neuronal morphology in the deep cortical plate; the arrow indicates the location of the procilium. A2A3: Serial longitudinal sections along the basal body (arrowhead) and procilium containing some microtubules (arrow). A multivesicular body (MB) and vesicles (V) could be found in the vicinity. Scale bar is 2 microns in A1, 0.18 in A2A3. B1 B2: serial longitudinal sections along the basal body (arrowhead in B1) and stubby procilium containing a dark structure (arrowhead in B2). Vesicles (arrows in B2) were common close to the basal body. Scale bar is 0.18 in B1B2. C1: Low magnification view of a cell with neuronal morphology located in the deep cortical plate; the arrow indicates the location of the procilium. C2C6: Serial oblique sections through the basal body (arrowhead) and procilium (C3 C6) containing vesiclelike structures (arrow in C5). C7: 3D reconstruction of the basal body (blue) and the cilium (red). Scale bar is 2 microns in C1, 0.18 microns in C2C6 and 0.25 in C7. DF: Examples of procilia containing microtubul e like structures. D1: Low magnification view of a cell with neuronal morphology located in the deep cortical plate; the arrow indicates the location of the procilium. D2D5: serial sections (70 nm thick) through the procilium. D2: The basal body (arrowhead) and the adjacent Golgi apparatus (G). D3D5: Oblique sections through the procilium showing lack of axoneme with presence of scattered tubular structures (arrows). D6: 3D reconstruction of the basal body (blue) and the cilium (red). Scale bar is 3 micro ns in D1, 0.16 microns in D2D5, 0.18 microns in D6. E1 E6: Serial longitudinal sections along the basal body (arrowhead in E1) and the procilium containing disorganized microtubules (arrows). Golgi apparatus (G) vesicles were located close to the basal bo dy. E6: 3D reconstruction of the basal body (blue) and the cilium (red). Scale bar is 0.18 microns in E1E5, 0.22 in E6. F1: Low magnification view of the cell (red) with neuronal morphology; the arrow indicates the location of the procilium. F2 F5: Serial longitudinal sections along the basal body (arrowhead in F2) and the procilium that contains a few microtubules in a parallel arrangement (arrows), compatible with a developing axoneme. F5: 3D reconstruction of the basal body (blue) and the cilium (red). Scale bar is 2 microns in F1, 0.2 microns in F2F5.

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71 Figure 2 9. ACIII+ cilia extend from neurons in all lamina of neocortex at P7 and P14. A: Low magnification view of P7 neocortex immunostained for ACIII (green) and NeuN (red) and counterstained w ith Dapi (blue). B G: High magnification views of layers 1 (B), 2 (C), 3 (D), 4 (E), 5 (F) and 6 (G). Compared to P4 ( Figure 2 5), layers are more developed, neuropil is expanding, and cilia are longer and growing at similar rates in all layers (see Table 2 2 ). Scale bar (located in N) = 70 m in A and 24 m in B G.H N: Low magnification view of P14 neocortex immunostained for ACIII and NeuN and counterstained with DAPI. High magnification views of layers 1 (I), 2 (J), 3 (K), 4 (L), 5 (M), and 6 (N) are sh own to the right. Note the expansion of the neuropil and more elongated appearance of cilia in all layers. At this age, cilia lengths reach ~7090% of maximal lengths (see Table 22 ). Scale bar = 70 m in H and 20 m in I N.

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72 Figure 2 10. Ultrastructur e of cilia at P8. Cells of interest and procilia are pseudocolored in red to help with identification. A: Serial micrographs of a cilium at P8. A1: panoramic view of the cytoplasm and initial apical dendrite of a pyramidal neuron with the basal body attached to the membrane (arrow). G: Golgi apparatus. A2A7: Serial sections of the cilium (arrow) protruding outside of the cell. Well formed and structured microtubules in the proximal segment (inset in A6 and A7) appear disorganized distally (inset in A7). Sc ale bar = 0.5 m in A1; 0.4 m in A2A7; numbers in upper right corner indicate the Z distance between sections in nm. B: Serial sections of a cilium from a pyramidal neuron at P8. B1: panoramic view of the pyramidal cell (red). Cilium location is indicate d (arrow). B2 B4: serial micrographs of the cilia from neuron in B1. B2 illustrates the transition between the basal body and the cilium, and B3 and B4 are transverse and oblique sections though the cilium showing a straight morphology and a well developed axoneme along the available length of 0.56 m. Vesicles were less frequent at this age. Scale bar = 5 m in B1 and 0.12 m in B2B4.

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73 Figure 2 11. Ultrastructure of cilia at P60. Cells of interest and procilia are pseudocolored in red to help with identification. A1 A5: Serial sections along the basal body (arrowhead) and proximal segment (arrow) of a neuronal cilium. Notice the welldeveloped microtubules. Scale bar is 0.17 m. B: Serial transverse sections of a pyramidal cell cilium (incomplete). B1: Panoramic view of a pyramidal neuron (red). Location of the cilium is indicated (arrow). B2B4: Details of transverse sections through the cilium of the pyramidal neuron in A1. (B2) Transition basal body cilium; (B3) mid distance of the series; (B4) final section at about 1.2 m from the origin. Note the rounded shape of t he ciliar membrane and the well developed axoneme along the available length. B5: 3D reconstruction of the basal body (blue) and the (incomplete) cilium (red). Positions of images B2B4 a re indicated. Scale bar =1.5 m in B1; 0.1 in B2B4 and 0.18 m in B5; numbers in upper right corner indicate the Z distance to the cilium origin in nm.

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74 Figure 2 12. Different interneuron subtypes in neocortex extend cilia. Examples of ciliated interne urons in the neocortex of P60 mice. (A F) An ACIII+ (arrow) cilium extending out of a parvalbumin (PV) positive cell. (G) Bar graph shows cilia length between PV+ and neighbouring PV cells were comparable (~5m). (H M) Example of a Calbindin (CB) positive interneuron with an ACIII+ cilium (arrow). (N R) A Calretinin (CR) positive interneuron extending an ACIII+ cilium (arrow). Interestingly, some bipolar CR+ cells extended their cilia from the proximal part of the ascending (pial oriented) dendrite, as illustrated. Scale bar = 12 m in A F; 10 m in H M and 7.5 m in N R; numbers in the right upper corner of images indicate z step increments in microns.

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75 Figure 2 13. Mutants lacking cilia show normal gross cytoarchitecture. A: Panoramic view of the (green) and counterstained with DAPI (blue). The boxed areas in A represent higher magnification views of layers 23 (B) and 5 (C). In spite of the cortical ice lacking cilia exhibit normal polarization in pyramidal neurons, with well formed and oriented apical dendrites (arrows in B and C). D and E: Panoramic view of the cortical plate immunostained for Cux1 (upper layer neurons marker; green) and Foxp2 (deeper layer neurons marker; red). Stratification of cortical layers was also grossly preserved in mice lacking cilia. D2 and E2 have the DAPI channel removed.

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76 Figure 2 14. Model of ciliogenesis stages in mouse neocortical neurons. Based on our observations, we propose the following model: migrating neurons do not bear cilia but r ather their mother centriole (MC) and daughter centriole (DC ) are free in the cytoplasm. Once cells terminate migration and reach their appropriate lamina, the mother centriole attach a vesicle, likely from the Golgi apparatus, buds a very short procilium and docks to the plasma membrane (It is also possible that the mother centriole docks directly to the plasma membrane w ithout vesicle attachment as indicated by the discontinuous arrow. Docking to the membrane involves developing specific structures such as transition filaments and the mother centri ole will become a basal body (BB ), frequently surrounded by vesicles (aster isk). This basal body will grow the procilium: a membranous expansion about 0.5 to 2 m in length, whose main feature is the lack of proper axoneme, and typically contains vesicles, short and disorganized tubular structures and electrondense diffuse content. This procilium does not display typical axonemal characteristics until ~ P8 although axonemal growth seem to start ~P0 in some subplate cells and could start ~P4 in some populations of neurons that showed early elongation of cilia (e.g. some layer 5 neurons). Overall, cilia will take weeks to fully elongate towards a peak ~P60P90, with some differences between layers

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77 CHAPTER 3 PROPERTIES OF NEURONAL CILIA IN RODENT AND HUMAN FOREBRAIN DURING SENESCENCE Background Primary cilia are nonmotile, microtubulebased organelles that extend from nearly every cell type in the body They sens e and respond to a wide variety of chemical changes in the local extracellular/intracellular environment like small cellular antennae (Green and Mykytyn, 2010, Lee and Gleeson, 2010, Louvi and Grove, 2011) Throughout the cerebral cortex, neurons extend a single primary cilium (Mandl and Megele, 1989, Fuchs and Schwark, 2004, Bishop et al., 2007, Anastas et al., 2011, Arellano et al., 2012, Guadiana et al., 2013) In m ouse cortex the formation and elongation of neuronal cilia begins perinatally and continues for several months reaching their mature length around postnatal day 90 (P90) (Arellano et al., 2012) Primary cilia contain several signaling mol ecules, including type III adenylyl cyclase (ACIII) and the p75 neurotrophin factor receptor ( p75NTR) (Chakravarthy et al., 2010b) ,as well as several G proteincoupled receptors (GPCRs ) including melanin concentrating hormone receptor 1 (MCHR1), serotonin receptor subtype 6 (5HT6), somatostatin receptor 3 (SSTR3) and dopamine receptor 1 (D1) (Handel et al., 1999, Berbari et al., 2008b, Marley and von Zastrow, 2010, Domire et al., 2011a, Guadiana et al., 2013) Recent studies have linked alt ered neuronal cilia structure and signaling capacity to defects in neuronal connectivity and reduced intellectual function (Einstein et al., 2010, Amador Arjona et al., 2011, Wang et al., 2011, Kumamoto et al., 2012, Guadiana et al., 2013) Specifically, w e found that neocortical pyramidal neurons, in which ciliogenesis was disrupted, also exhibited defects in dendrit e growth and arborization. In cases where disruption of ciliogenesi s produced abnormally long cilia,

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78 we observed reduced levels of ACIII in these cilia. Importantly, we found that increasing ACIII expression in these cells reversed the dendritic morphology defect s, a result that suggests that ciliary ACIII levels influenc e normal dendrite formation and arborization(Guadiana et al., 2013) Recent studies of adult born dentate granule neurons have produced similar results. In these studies, ablation of ACIII positive cilia by expressing a dominant negative form of Kif3a (dnKif3a) in these cells was shown to disrupt the development of dendrites and the ability of these newly born neurons to integrat e in to the adult brain (Kumamoto et al., 2012) The results of these studies suggest that dysfunction or loss of primary cilia may induce cellular changes that in turn lea d to altered neuronal and brain function. This scenario is supported by the observations that loss of both ACIII and SSTR3 disrupt synaptic plasticity and novel object recognition memory in mice (Einstein et al., 2010, Wang et al., 2011) and that loss of MCHR1 leads to impaired synaptic plasticity and deficits in cognition (Adamantidis et al., 2005, Pachoud et al., 2010) Additionally, many ciliopathy patients present with cognitive impairments and other CNS related dysfunction (Fuchs and Schwark, 2004, Lee and Gleeson, 2010, Louvi and Grove, 2011, Arellano et al., 2012, Guadiana et al., 2013) Learning and memory impairments often accompany human aging (Roberson et al., 2012, SinghManoux et al., 2012) Since disruption of ciliogenesis has been linked to learning and memory impairment (Einstein et al., 2010, Green and Mykytyn, 2010, Wang et al., 2011, Guadiana et al., 2013) it is possible that the learning and memory impairments that accompany aging are in part due to changes in neuronal cilia signal ing and maintenance. T he goal of this study w as to determine if the neurons in the forebrain

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79 of aged rats are ciliated and whether the levels of key receptors and signaling proteins in the primary cilia of these cells mirror those observed in the forebrain of younger rats. We also compared some of our findings to published aging human forebrain transcriptome data to determine if changes observed in rat cortex resembled human forebrain expression patterns. Results Expression o f ACIII, a Marker of Neuronal Cilia, Persists in the Aged Rat and Human Forebrain ACIII is enriched in the axonemes of the primary cilia of neurons in mouse cortex from birth to 1 year of age [5, 6]. To determine whether ACIII+ cilia are present in the neocortices and hippocampi of aged rats, we stained tissue sections of Fisher 344 and Fisher 344xBrown Norway (F344xBN) rats for ACIII to identify cilia and NeuN to label the neuronal cell bodies. Comparisons of the ACIII stained neocortices and hippocampi of 6 month (mos) and 2224 mos Fisher 344 rats revealed that no significant differences were observed in the numbers of neurons possessing cilia in these brain regions ( Figure 3 1A L). Interestingly, we found that the average length of the cilia increased as a function of age in neocor tex ( Figure 3 1J) but not in hippocampus ( Figure 3 1L). Next, we asked whether neurons in the neocortices and the CA1 region of the hippocampi of 38 mosF344xBN rats are ciliated. We found that ACIII+ cilia were present in the neocortices and hippocampi of these older rats ( Figure 3 1M P) and that the increase in the average length of neocortical cilia that was observed at 24 mos ( Figure 3 1J) was still apparent at 38 mos ( Figure 3 1Q). No changes in the lengths of the cilia in CA1 were observed at 38 mos ( F igure 3 1R). We also observed a significant reduction in NeuN staining in 38 mos neocortex ( Figure 3 1N) and

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80 hippocampus ( Figure 3 1P), an observation consistent with a previous report in spinal cord neurons [27]. Together these results show that neurons i n the aged mammalian forebrain retain the ability to elaborate ACIII+ primary cilia. Recently, a spatiotemporal transcriptome of the human brain was generated from the results of deep sequence analyses of mRNA that was isolated from embryonic (4 post con ception week (PCW) <8 PCW) through aged brain (up to 82 years old) ([26] and http://hbatlas.org). We used this resource to examine agerelated changes in the expression of ACIII (ADCY3) and other ciliaassociated genes in human forebrain. A heat map of ACI II expression suggests that expression increases from birth and persists into late adulthood in most cortical regions ( Figure 3 1S). Although there was a statistically significant increase in expression levels of ACIII (ADCY3) when comparing young adulthood (YA) and late adult (LA) cortex, no significant changes were observed when comparing ACIII mRNA expression levels in middle (MA) to late (LA) adult cortex. The average cortical expression level of ACIII over the course of aging in adult brain was found t o be 8.37. The persistence of ACIII expression in human neocortex is consistent with our findings in aged rat cortex. Together, these results suggest that both ciliary ACIII localization and human ACIII mRNA expression does not dramatically decline with advanced age in the forebrain. Expression o f SSTR3 in Cilia of the Young a nd Aged Forebrain The majority of ACIII+ cortical neuronal cilia also stain positively for SSTR3, a localization pattern that appears as early as embryonic day 13.5 (E13.5) within the budding vesicle of the procilium in rodents [5, 11, 13, 16, 28, 29]. SSTR3+ cilia have also been shown to be present in the hippocampi of up to 5 monthold rats [30]. To determine whether neuronal cilia in the cortices and hippocampi are SSTR3+ of aged

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81 rats, we co stained brain sections for SSTR3 and ACIII to label cilia, and NeuN to label neuronal cell bodies. We first examined the primary cilia in primary somatosensory cortex (S1C), motor cortex (M1C), and the subfields of the hippocampus of young (6 mos) and aged (24 mos) Fisher 344 rats by immunohistochemistry ( Figure 3 2). We found that the majority of the ACIII+ primary cilia in the somatosensory and motor cortices of young and aged rats ACIII+ were also SSTR3+ ( Figure 3 2A D, white arrows). A sma ll number of ACIII+ cilia in both young and aged brain di d not stain for SSTR3 ( Figure 3 2A B ). We did not find significant differences between the percentages of SSTR3+ cilia relative to NeuN+ neurons in young and aged SIC and M1C cortices ( Figure 3 2E ) s uggesting that aging does not significantly alter the levels of ciliary SSTR3 in these regions of cortex. We also asked whether the primary cilia of neurons in the CA1, DG and CA3 subfields of the hippocampi of aging Fisher 344 rats were SSTR3+. We found that the numbers of ACIII+/SSTR3+ cilia these regions of the hippocampi of 6 mos and 24 mos F344 rats were not different ( Figure 3 6 A F ). We also did not find significant differences between the percentages of SSTR3+ cilia relative to NeuN+ neurons in any of t he hippocampal subfields ( Figure 3 2F ). In addition to localization to the primary cilia, SSTR3 immunostaining was also present in the somas of the cortical (M1C) and hippocampal neurons, a finding also reported by others [2, 10, 31]. In contrast to F isher 344 rats, SSTR3+ primary cilia in the neocortices of 38 mos F344BN were not detected using either SSTR3 antibody ( Figure 3 2I L ) despite the presence of ACIII+ cilia in these brains ( Figure 3 2L Figure 3 1N,P ). These results suggest that transport of SSTR3 to the primary cilia in neocortex may be influenced by either strain or significantly advanced age in rat.

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82 Analyses of SSTR3 gene expression across regions of human forebrain revealed that SSTR3 expression levels were relatively steady as a function of age. Significant decreases in SSTR3 expression were identified in select cortical regions from the AD period to YA period ( Figure 3 2M), and there was a trending but nonsignificant decrease from YA to MA. However, there were no significant changes into LA. These results show that SSTR3+ cilia are still present on neurons in aged rat cortex, and suggest that aging can alter the level of this receptor localized to the cilia of these neurons. Our analyses also suggest that the changes were see in levels of SSTR3 in aged rat cortex mirror those observed in aged human forebrain. P75NTR Expression in Young a nd Aged Forebrain The nerve growth factor receptor p75NTR has been shown to localize to the axonemes of neur onal cilia in both the frontal cortices and the hippocampi of rodents [10, 32]. Activation of this receptor has been implicated in the inhibition of both adult neurogenesis in the hippocampus and memory formation [33]. We asked whether the levels of this r eceptor change during normal aging in the rat frontal cortex and hippocampus. Western blot analyses revealed that there were no significant changes in the protein levels of p75NTR in lysates derived from the frontal cortices ( Figure 3 3A, top panel) or hi ppocampi of young (6 mos) and aged (2224mos) F344 rats ( Figure 3 3A, bottom panel). Analyses of the expression of NGFR in human cortex and hippocampus as a function of age revealed that levels of NGFR remained steady over the course of aging in all forebrain areas across all time points analyzed ( Figure 3 3B). These results suggest p75NTR expression levels in aging rat and huma n forebrain do not dramatically change.

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83 Ciliary MCHR1 Expression i n Young a nd Aged Forebrain. The melaninconcentrating hormone receptor 1 (MCHR1) has been detected in the primary cilia of neurons in the hypothalamus, nucleus accumbens (NA), and olfactory tubercle (OT) [29, 3440]. We performed immunohistochemistry using an antibody raised against MCHR1 to determine whether ciliary MCHR1 in the NA and OT is altered by aging. We observed MCHR1+ cilia in both the NA and OT (white arrows, Figure 3 4A D), a result confirming previous studies. Interestingly, during our confocal imaging analyses, we observed single cells in both regions of young and aged NA and OT that possessed two MCHR1+ cilia ( Figure 3 4E) that were intermingled with cells possessing one MCHR1+ cilium. In support of this observation, we found that the ratio of MCH1R+ cilia to NeuN+ cells in these regions was greater than 1 (100%) in the 6 mos group ( Figure 3 4G). Examination of the ratios of bi ciliated, MCHR1+ cilia to NeuN+ cells in both the OT and NA as a function of age revealed the numbers of MCHR1+ cilia associated with these doubly ciliated neurons significantly decreased in aged rats ( Figure 3 4H). In a separate analysis, we confirmed the presence of bi ciliated and singly ciliated cells in these same regions, using ACIII immunostaining ( Figure 3 4F). We did not observe a change in the percentage of ACIII+ singl y ciliated cells with age in either the NA or the OT (data not shown), nor was there significant decrease in the numbers of ACIII+ cilia on bi ciliated neurons in the OT. We did, however, find a dramatic decrease in the number of ACIII+ cilia on bi ciliate d neurons in the NA. This result suggests that while there is not obvious cilia loss in these regions, there is a reduction in levels of MCH1R and ACIII associated with the cilia on the bi ciliated cells in these regions. The reduction in ciliary MCHR1 in aged rat mirrored reductions that we found in our analyses of the human transcriptome data for MCHR1

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84 mRNA expression in cortex as a function of age ( Figure 3 4J). The transcriptome data suggest that MCHR1 expression in cortex decreases with age. Additional ly, the transcriptome analyses revealed that levels of MCHR1 were reduced in human hippocampus and striatum. Together, the results of these experiments and our transcriptome analyses [34] are consistent with reduced expression of MCHR1 mRNA in aging human forebrain and rat brain (data not shown) and reduced localization of MCHR1 to the primary cilia of neurons in these regions. Levels o f Other Ciliary GPCRs IFT, a nd Bardet Biedl Syndrome (BBsome) Associated Proteins in Young a nd Aged Human Forebrain Mutations in genes encoding key ciliary proteins have been shown to be detrimental to cilia formation, trafficking, and function in the developing and adult human brain [2, 3, 15, 18, 36, 4159]. In these analyses we wanted to determine whether the human transcriptome could point to potential changes in the expression of other ciliaassociated proteins that accompanies aging. We analyzed heat map matrixes of mRNAs of genes encoding ciliary GPCRs and proteins associated with IFT and the BBSome machinery. Two neuronal cilia GPCRs were examined, 5HT6 (HTR6) and D1 (DRD1) ( Figure 3 5). Our analyses revealed that the levels of mRNAs encoding HTR6 and DRD1 in human forebrain were reduced in cortex and hippocampus with aging. These analyses and our MCHR1 data suggest that there may be a selective, agerelated decline in the levels of certain GPCRs that are normally enriched in neuronal primary cilia, such as MCHR1, 5HT6, and D1, in the cortex and hippocampus. The levels of other cilia GPCRs, such as p75NTR, app ear to be unaffected by aging. BBS proteins (e.g., BBS2 and BBS4 [11, 45, 6067] and tubby or tubby like protein 3 (e.g., TULP3) [6871] are in part responsible for shuttling proteins such as

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85 GPCRs to the cilia axoneme. We asked whether levels of expres sion of BBS2 and BBS4, two proteins associated with neuronal cilia that are components of the BBSome complex, change in aging human forebrain. These analyses revealed no change in expression in BBS2 from AD to MA or from AD to LA, while a significant decrease in expression occurred between AD and YA. In contrast, a significant increase in expression was seen in BBS2 expression between YA and LA. Human BBS4 expression was found to significantly increase from AD, YA and MA in all cortical regions examined into the LA period. GPCR trafficking requires core IFT proteins that include anterograde molecular motors such as kinesin (e.g., KIF3A) and IFT cargo proteins such as IFT88 and TULP3, proteins whose loss severely disrupts ciliogenesis [1, 56, 7282]. In th ese analyses, we looked the expression of IFT88, KIF3A, and TULP3 in the human brain transcriptome as a function of age ( Figure 3 5). We found that KIF3A appears to increase significantly between the adolescent and young periods and from young to middle ad ulthood. However, in all periods assessed (AD, YA, and MA), KIF3A was expressed significantly higher than in the late adulthood (LA) period showing a general decreasing trend over the course of aging in the human forebrain. The expression profile for IFT88 showed significant increases over the course of aging with adolescent period being the lowest in expression among all brain regions assessed. There was a significant increase in IFT88 expression from YA to MA and level in both YA and MA cortex were significantly lower than in LA cortex. TULP3 expression was significantly lower in the MA and LA periods than in AD and YA time points. Taken together, these data indicate that there may be a reduction in the levels of some ciliary components such as certain G PCRs,

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86 motors, and adaptor proteins in the aged brain while others, such as IFT transport proteins, are upregulated. Whether these changes in expression affect ciliary function in the normal, aged brain has yet to be elucidated. Material and Methods Anima ls Brain tissues analyzed in this study were derived from two strains of behaviorally characterized, [2325] memory unimpaired rats: Fisher 344 x Brown Norway rats (young 1 mos, n=2; aged38 mos, n=2) and Fisher 344 (young 6 mos, n=8; aged2224 mos, n=8) For histological analyses, brains were immersion fixed in or perfused with 4% paraformaldehyde in 0.1M phosphate buffer (PFA), cryoprotected in 30% sucrose, and sectioned coronally at 50m. For hippocampal measurements and immunohistochemistry, hippocampi were subdissected from fresh tissue and fixed, frozen, and sectioned coronally at 50m. Procedures involving animal subjects were reviewed and approved by the UF Institutional Animal Care and Use Committee and were in accordance with guidelines establi shed by the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals. Immunohistochemistry Immunohistochemical analyses were performed using a previously published protocol [8] and the following primary antibodies: rabbit anti ACIII (1:10,000; Encor Biotechnology), rabbit anti MCHR1 (1:10,000, Encor Biotechnology), mouse anti NeuN (1:2000; Chemi con), goat anti SSTR3 (1:200; Santa Cruz), and rabbit anti SSTR3 (1:500; Gramsch). Appropriate species specific, fluorophoreconjugated, secondary antibodies were used to visualize binding of the primary antibodies (1:400; Jackson ImmunoResearch). Stained sections were coverslipped with Prolong Antifade Gold

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87 mounting media containing DAPI (Life Technologies). Peptide blocking was used to confirm the specificity of MCHR1 staining. Tissue sections were incubated overnight with MCHR1 antibody alone or with ant ibody that had been incubated with blocking peptide (AB: peptide ratios 1:0, 1:1, 1:5, 1:20, CGIRLPNPDTDLYWFTLYQF, Peptide 2.0, Figure 3 7 ). Images of stained sections were captured using an Olympus IX81DSU spinning disc confocal microscope and are displayed as collapsed z stacks that were collected in 0.51m steps. Cilia Length Measurements Each rat brain was immunostained for ACIII and NeuN and cilia length (in m) was measured as previously described [8]. Confocal z stack images were opened and analyzed in Image J64 (http:// rsbweb.nih.gov/ij/), such that the continuous ACIII ciliary signal could be traced. We analyzed cilia in the neocortex and hippocampal subfields (CA1, DG, CA3) of 4 brains, 68 fields were examined per for each rat age (1 mos, 6 mos 22 24 mos) and 3 brains, six to eight fields/section for 38 mos. Statistical Analysis Statistical analysis was performed using Statview software (SAS Institute Inc.). T tests were performed in instances of only two groups and analysis of variance (ANO VA) was performed where appropriate. For multiple groups, differences were evaluated using twoway ANOVA. If there was a significant interaction between the main effects or a main effect in a oneway ANOVA, individual contrasts were carried out using a Sch eff post hoc analysis (Scheff 1953) Significance was determined if p < 0.05. Most, if not all human samples consisted of raw data from both l eft and right hemispheres; however, in some cases, samples consisted of data from only one

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88 hemisphere. Furthermore, in order to reduce false positives, we determined if there was an effect of hemisphere. If no effect of hemisphere was found, the data were averaged and counted as one subject for analysis. Western Blot Freshly dissected samples of rat frontal cortex or hippocampus were solubilized in ice cold 1X lysis buffer (Cell Signaling Technology) that was supplemented with protease and phosphatase inhibitor cocktails 1 and 2 (1:100; Sigma) and 1mM (phenylmethanesulfonylfluoride) PMSF. Protein lysates (30 g/lane for frontal cortex and 10 g/lane for hippocampus) were boiled in 2X sample buffer (NuPAGE), separated on Bis Tris gels (NuPAGE, Life Technologi es), and transferred to PVDF membranes using an iBlot Dry Blotting System (Life Technologies). Blots were blocked for 1h in 1X Tris buffered saline containing 0.1% Tween20 (TBST) and 5% Nonfat dry milk (NFDM) and were then incubated in Tris Buffered Saline with Tween (TBST) containing 2.5% NFDM and either mouse anti actin (1:10,000; Sigma) or rabbit anti p75NTR (1:1000; Alamone Labs) overnight at 4C. The blots were then washed and incubated for 1 hr at RT in species appropriate secondary antibodies that were conjugated to horseradish peroxidase (1:10,000; Jackson Immuno). Antibody binding was visualized using chemiluminescence (Pierce) and images of the blots were captured and analyzed using an Alpha Innotech FluorChem Q imager (Proteinsimple). Huma n Brain Transcriptome Data The heat maps of human gene expression used in this study were generated from the human brain atlas (http://hbatlas.org) and are similar to previously published maps [26]. We were interested in the following genes that encode known ciliary proteins: ACIII (ADCY3), SSTR3, MCHR1, p75NTR (NGFR), IFT88, KIF3A, TULP3,

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89 5HT6 (HTR6), D1R (DRD1), BBS2, BBS4. Briefly, genome wide, exon level transcriptome data from disease free, postmortem brains were analyzed from the following brain regi ons: amygdala (AMY), hippocampus (HIP), and striatum (STR) and 11 neocortical areas and subregions: Frontal cortex (FC) Orbital prefrontal cortex OFC, Dorsolateral prefrontal cortex (DFC), Ventrolateral prefrontal cortex (VFC), Medial prefrontal cortex ( MFC), and primary motor cortex (M1C); Parietal cortex (PC) Primary somatosensory cortex (S1C) and Posterior inferior parietal cortex (IPC); Temporal cortex (TC) Primary auditory cortex (A1C), Posterior superior temporal cortex (STC), and Inferior temporal cortex (ITC); Occipital cortex (OC) Primary visual cortex (V1C). The adenylyl cyclase III (ADCY3) heat map includes the entire temporal periods sampled beginning in early embryonic period 1(48 weeks post conception) and ending in late adulthood peri od 15 (~60+ years of age up to 82 years of age). All other heat maps begin in adolescence (AD, 1220 years of age period 12) and transition to young adulthood (YA, 2135 years of age, period 13), middle adulthood (MA, 3655 years of age, period 14) and end in late adulthood (LA, 5582 years of age, period 15). Each heat map matrix shows gene expression displayed as log2transformed signal intensity across analyzed regions and time periods using a color scale from low (blue) to high (red). Microarray analyses were performed with R system software (http://www.R project.org, V2.3.0). Additional information and detailed descriptions of the milestones/events occurring in each period during human development can be found at http://hbatlas.org or [26].

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90 Figure 3 1 ACIII+ cilia in young and aged rat c erebral cortices and hippocampi. I mmunostaining of 6 mos (left panels) and 24 mos (right panels) Fisher 344 rat neocortex and hippocampal subregions for ACIII (green, cilia marker) and NeuN (red; a neuronal marker) (A H). ACIIIpositive cilia were detected extending from neurons in layer II/III of the cerebral cortex (A, B) and from neurons in hippocampal subregions CA1(C, D), DG (E, F) and CA3 (G, H) in the brains of both 6 and 24 mos rats. Nuclei are labeled with DAPI. Bars = 10m. Percent of neocortical neurons with cilia in 6 mos (grey bars) and 24 mos (black bars) F344 rats (ttest, p> 0.05, I). Mean lengths of cilia extending from neurons in layer II/III of the neocortex of 6 and 24 mos F344 rats (ttest, p <0. 05, J). Percent of ciliated neurons in hippocampal subfields CA1, DG, and CA3 of 6 mos (grey bars) and 24 mos (black bars) F344 rats (ttest, p >0.05 K). Mean lengths of cilia extending from hippocampal neurons in CA1, DG, and CA3 of 6 and 24 mos F344 rats (ttest, p >0.05 L). Immunostaining of 1 mos (left panel) and 38 mos (right panel) Fisher 344 x Brown Norway rat neocortex (M, N) and CA1 (O, P) for ACIII (green) and NeuN (red). Despite the loss of neuronal marker NeuN in aged Fisher 344 x BN rat brain, ACIII+ cilia (arrows) were still detected. Nuclei are counterstained with DAPI. Asterisk in panel N = lipofuscin granules. Bars = 10m. Mean lengths of cilia extending from neurons in layer II/III of the neocortex and from CA neurons in 1 mos (black) and 38 mos (grey) Fisher 344 x Brown Norway brains (ttest, p< 0.05 Q, ttest, p >0.05 R respectively). Heat map matrix showing temporal and spatial expression changes in adenylyl cyclase III (ADCY3) mRNA in human brain as a function of brain region and age (S ). Time points and regions are denoted by their abbreviations: AD adolescence, YA young adulthood, MA middle adulthood, LA late adulthood, OFC orbital prefrontal cortex, DFC dorsolateral prefrontal cortex, VFC ventrolateral prefrontal cortex, MFC medial prefrontal cortex, M1C primary motor cortex, S1C primary somatosensory cortex, IPC posterior inferior parietal cortex, A1C primary auditory cortex, STC posterior superior temporal cortex, V1C primary visual cortex, HIP hippocampus. The relative level of expression in each region is displayed and ranges from dark blue (low)=6.75 to bright red (high)= 9. An effect of age was seen from YA to LA time point (F(3,32) = 5.045, p= 0.0038). Above the horizontal black line indicates neocortical regions and below the hippocampus. Left y axis: Forebrain regions analyzed. Right Y axis: The average ADCY3 signal intensity plotted as a function of time from adolescence (AD) into late adulthood (LA) denoted by a black line with black diamonds, average signal intensity for all cortical regions over time is 8.37. Average signal intensity for hippocampus is 8.24.

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91

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92 Figure 3 2. SSTR3+ cilia of young and aged rat forebrain and SSTR3 expression in human cerebral cortex. Immunostaining from young (6mos, A) and aged (24mos, B) in rat primary somatosensory cortex (S1C; A, B) and primary motor cortex (M1C; C, D)co stained for the cilia axoneme marker, ACIII (green) and SSTR3 (red) and the neuronal marker NeuN (blue)(A D). Arrows indicate cilia positively stained for both SSTR and ACIII and asterisks indicate ACIII +/SSTR3 cilia. Bars = 10m. Comparison of the mean percent of NeuN+ neurons that possess SSTR3+ cilia in S1C and M1C of young (6 mos; grey bar) and aged (24 mos; b lack bar) rats (ttest, p>0.05, E ). Comparison of the mean percent of NeuN+ neurons that possess SSTR3+ cilia in the hippocampal subfields CA3, DG, and CA1 of young (6 mos; grey bar) and aged (24 mos; black bar) rats (ttest, p>0.05, F ). Immunostai ning of R b SSTR3 (green, Gramsch) in the 1 (A) and 38 month (mos, B) Fisher/Brown Norway rat neocortex (G J). Nuclei (red) are counterstained using DAPI. Bar=10m. Arrowheads indicated SSTR3+ cilia, asterisks indicate lipofuscin granules. Immunostaining of 38 mos ( C) of Gt SSTR3 (red, Santa Cruz) and Rb ACIII (green, EnCor) in rat neocortex (K L). Nuclei are counterstained with DAPI (blue). Arrowheads indicated ACIII+/SSTR3cilia. Asterisks indicate lipofuscin granules. Heat map showing changes in SSTR3 mRNA levels in human brain as a function of brain region and age (M). Above the horizontal black line indicates neocortical regions while below indicates hippocampus. Left y axis: Forebrain regions analyzed are abbreviated using their three letter acronym (detailed i n Figure 3 1). Black line with diamond symbols indicates the average signal intensity across all cortical areas in adolescence (AD) through late adulthood (LA) cortex. An effect of age was seen in SSTR3 expression from AD to YA (F(3,32) = 4.602 p=0.0068) a nd a trending but not significant change in intensity from YA to MA (p=0.0762). The relative level of expression in each region is color coded from low (dark blue) 3.75 to high (bright red) 6.75. Average cortical signal intensity for SSTR3 is 5.15 and aver age hippocampal intensity is 4.81.

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93 Figure 3 3 p75NTR expression in the young and aged cortex and hippocampus. Western Blots of p75NTR (75kDa) levels in 6 mos versus 24 mos in frontal cortex and hippocampal lysates as compared t actin levels (45kD) A:. Student t test analysis indicates no significant changes in protein levels in either the cortex (P= 0.8732) or the hippocampus (P= 0.9135). Heat map matrix of human mRNA P75NTR expression in various cortical regions across agin g B: Horizontal bla ck line separates cortical region expression (above) from hippocampal region expression (below). The relative expression levels from low (dark blue) 5 to high (dark red) 8 are color coded. The average cortical level across aging is 5.89 and the hippocampal average intensity is 5.85. Left y axis: Forebrain regions analyzed abbreviated by three letters (See Figure 3 1 for reference). Right Y axis: average signal intensity from all cortical areas from adolescence (AD) into late adulthood (LA) shown by a black line with diamonds (F(3,32)=6.794, p =0.543).

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94 Figure 3 4 Decrease in expression of MCHR1 in the adult aged forebrain. Immunostaining images from young (6 mos; A, C) and aged (22 mos; B, D) rat olfactory tubercle (OT, top panel) and nucleus accumbens ( NA, bottom panel) immunostained for MCHR1. Bar = 10m. Arr ows indicate MCHR1+ cilia. E,F: Co immunostaining for MCHR1 (red) and ACIII (green) in NA of 38 mos F344xBN brain. Doubly labeled cilia were not detected. G: Confocal images of NA cells displaying t wo MCHR1+ cilia in 22 mos F344. Confocal z stacks following two cells (upper and lower panels) reveal separate cilia (arrows) whose axonemes enter and exit the plane of view and appear to stem fr om the same cell. Bar = 5m. H: Confocal z stack (0.5 m ste ps) examples of NeuN+ cells that appear to harbor two ACIII+ cilia in the 22 mos NA. Bar = 5m. I: The percent ciliated (total MCHR1+ cilia/number of NeuN+ cells) in the NA and OT of aged brains of F344 sections (t test, NA: p< 0.001, O T: p<0.01). J: The percent of bi ciliated MCHR1+ neurons (MCHR1+ double cilia/number of NeuN+) in the NA and OT between young and aged (t test, NA and OT: p<0.001). K : The percent of bi ciliated ACIII+ neurons (ACIII+ double cilia/number of NeuN+) in the OT ( p= 0.8374) and NA (p<0.001). L: Heat map of MCHR1 mRNA expression from all forebrain regions analyzed (see Figure 3 1 or Methods for abbreviations). All periods (AD, YA, and MA) had significantly higher expression levels than in the LA period (F(3,32)= 5.422, AD LA, p value= 0.0069, YA LA, p value = 0.0001, MA LA p value= 0.0005). The average expression level of MCHR1 across all regions of cortex is indicated by a trend line (black line +diamonds).

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95 Figure 3 5 Variablity in other GPCRs and IFT/ BBSome machinery during aging.Heat map matrixes of human mRNA expression from genes from cilia known ciliary GPCRs ( DRD1 and 5HT6) and other cilia molecules known to be involved in intraflagellar transport (IFT) ( KIF3A,IFT88, TULP3 ) and BBSome machinery (BBSome, BBS2 and BBS4 )of neuronal cilia. Left y axis: Forebrain regions analyzed (see Figure 3 1 for region abbreviations). Right Y axis: average signal intensity from all cortical areas from adolescence (AD) into l ate adulthood (LA). Black horizontal line indicates cortical areas above the line and other forebrain regions analyzed below the line. The average expression level of each gene assessed across all regions of cortex are individually plotted on their respec tive heat maps denoted by a black line with diamonds. 5HT6 expression appears to be dramatically reduced as a function of age (F(3,32)=5.045, AD YA p value=0.0016, AD MA p value= 0.0020, AD LA F p value= 0.0001, YALA p value= 0.002). Average cortical int ensity for 5HT6 is 6.22 while the average signal intensity for hippocampus and striatum are 5.73 and 8.32 respectively. DRD1 also shows a significant decrease in expression levels into aging (F(3,32)=1.190, AD LA p value=0.0014, MA LA p value=0.0035). Aver age cortical intensity for DRD1 is 5.30 while the average signal intensity for hippocampus and striatum are 4.69 and 7.94 respectively. From AD to YA, BBS2 shows significant reduction in expression levels (F(3,32)= 8.231, p=0.0008) while from YA to LA ther e is a significant increase in expression (p value < 0.001). Average cortical intensity for BBS2 is 9.35 while the average signal intensity for hippocampus and striatum are 9.44 and 10.05 respectively. There is a robust decrease in BBS4 expression with agi ng from all time point into LA (F(3,32)=14.102, p value< 0.001). Average cortical intensity for BBS4 is 6.94 while the average signal intensity for hippocampus and striatum are 7.40 and 6.74 respectively. A decrease in the expression of KIF3A from all time points into LA was also observed (F(3,32)=12.380, AD LA p= 0.0045, YA LA p=0.0001, MA LA p=0.0141). Average cortical intensity for KIF3A is 10.34 while the average signal intensity for hippocampus and striatum are 10.10 and 9.39 respectively IFT88 expression increases over the course of aging (F(3,32)=36.418, AD YA, AD MA, and AD LA, p value <0.001). Average cortical intensity for IFT88 is 6.02 while the average signal intensity for hippocampus and striatum are 6.38 and 6.78 respectively. T ULP3 significantly decreases with aging into the MA and LA periods (F(3,32)=3.962, AD MA p value= 0.0035; YA MA p value = 0.0076; AD LA p value = 0.0425). Average cortical intensity for TULP3 is 6.99 while the average signal intensity for hippocampus and striatum are 7.16 and 7.55 respectively.

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96 Figure 3 6 SSTR3 immunodetection in Fisher 344 rat hippocampus subfields CA1,DG, and CA3. Confocal z stack images of young (6 mos; A C) and aged (24 mos; D F ) rat hippocampal subfields CA1 (A,D), DG (B,E), and CA3 (C,F ) immunostained for SSTR3 (red), ACIII (green) and NeuN (blue)( far right panel, merge). Arrows indicate cilia positively stained for both SSTR and ACIII. Arrowheads indicate cilia examples in higher mag insets in 24 mos (H J). Figure 3 7 M CHR1 peptide blocking to confirm antibody specificity Immunostaining of MCHR1(green) in the OT demonstrating MCHR1 specificity including a counterstain with DAPI (blue). Increased ratio of antibody to peptide concentration reveals loss of specificity and a complete loss of staining by 1:5 MCHR1 antibody: Blocking Peptide (middle, right panels) as compared to antibody alone (left panel) enriched within the cilia axoneme. Bar=10m.

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97 CHAPTER 4 ARBORIZATION OF DENDRITES BY DEVELOPING NEOCORTICAL NEURONS IS DEP ENDENT ON PRIMARY CILIA AND TYPE 3 ADENYLYL CYCLASE Background Interest in the biological functions of the primary cilia of cortical neurons and their influence on neuronal maturation and cortical development has dramatically increased in recent years (Louvi and Grove, 2011) in part because several developmental and neurological disorders have now been linked to defects in ciliogenesis (Green and Mykytyn, 2010, BennounaGreene et al., 2011, Lee and Gleeson, 2011, Novarino et al., 2 011, Sattar and Gleeson, 2011) As a first step toward understanding the impact that primary cilia have on neuron function and maturation, we characterized the time course of neuronal ciliogenesis in developing mouse neocortex (Arellano et al., 2012) Neuronal ciliogenesis progresses through several stages in post migratory neurons, the first of wh ich is distinguished by the appearance of the procilium that is formed as a result of outgrowth of the early stage ciliary plasma membrane (Arellano et al., 2012) Shortly after birth, the elongation stage begins during which time microtubules within the procilium assemble and organize to form the axoneme that continues to elongate over the first 8 12 weeks of life. During the procilium and elongation stages of ciliogenesis, the cilia become enriched with signaling enzymes (e.g. type 3 adenylyl cyclase (ACIII) (Berbari et al., 2007, Bishop et al., 2007, Anastas et al., 2011, Arellano et al., 2012) nerve growth factor receptors (e.g. p75NTR (Chakravarthy et al., 2010b) ), and specific GPCRs (e.g. 5HT6, SSTR3, MCHR1, and D1 (Brailov et al., 2000, Miyoshi et al., 2006, Berbari et al., 2008a, Berbari et al., 2008b, Stanic et al., 2009, Marley and von Zastrow, 2010) ) that enable the cilia to respond to ligands in the extracellular environment. The

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98 process of intraflagellar transport (IFT) not only promotes neuronal ciliogenesis but also supports the bidirectional trafficking of these ciliary signaling molecules within the cilium. There is growing evidence that disruption of the formation or function of neural cilia adversely affects neuronal differentiation. For example, in neurons born in the adult hippocampus, blocking the function of Kif3a, an anterograde motor protein that i s required for CNS ciliogenesis (Chizhikov et al., 2007, Davenport et al., 2007, Han et al., 2008, Spassky et al., 2008) has been reported to disrupt dendritic arborization of these neurons and their synaptic integration into the hippocampus (Kumamoto et al., 2012) In addition, overexpression of the doublecortin domaincontaining protein 2 (DCDC2) in cultured rat hippocampal neurons, a protein that interacts with Kif3a, has been reported to induce cilia elongation and alter the dendritic branching of these neurons (Massinen et al., 2011) These findings raise the question as to whether the growth and differentiation of other cortical neuron subtypes are altered by disruption of their cilia. For example, while targeted ablation of cilia hinder ed dendritic outgrowth in adult born hippocampal granule neurons (Massinen et al., 2011) ablating cilia in post migratory cortical interneurons did not significantly alter differentiation (Higginbotham et al., 2012) Given the range of these observations, we wondered whether cilia regulate the differentiation and maturation of projection neurons in developing neocortex. The purpose of this study was to investigate the relationship between ciliogenesis and the differentiation of neocortical neurons in developing cortex. We hypothesized that disruption of ciliogenesis in developing neocortical neurons would Reprinted with permission from Guadiana, S.M., et al., Arborization of dendrites by developing neocortical neurons is dependent on primary cilia and type 3 adenylyl cyclase. J Neurosci, 2013. 33(6): p. 262638.

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99 induce abnormal dendritic outgrowth. In this study, we overexpressed neuronal cilia GPCRs in developing neocortical neurons to disrupt ciliogenesis; a strategy that we found induces significant lengthening of primary cilia of immortalized cells. We found that neurons overexpressing cilia GPCRs developed exceedingly long, malformed cilia, and that dendrite outgrowth from these neurons was severely stunted in a manner similar to that observed for neurons lacking cilia. Moreover, our findings suggest that changes in the complement of signaling proteins present in the cilia that were induced by overexpression of the GPCRs contributed to the abnormal dendritic phenotype exhibited by these neurons. Results Overexpression o f Neuronal Cilia G PCRs i n Developing Mouse Cortical Neurons Drama tically Increases Cilia Length and Disrupts Cilia Morphology In this study we targeted neocortical neurons and their cilia by delivering ciliary genes to developing E15.5 neural progenitors using in utero electroporation. The effects of expression of these genes by the pyramidal neurons were examined in culture or in situ at different postnatal stages of neocortical development ( Figure 4 1A). We found that expression of control plasmids encoding cytoplasmic EGFP by electroporated neuro ns did not induce any gross morphological changes in the cilia of these cells or alter the normal position of the cilia on the soma, which emerges near the base of the apical dendrite ( Figure 4 1B). In addition, the staining patterns for ACIII and pericent rin in EGFP positive, control neurons and neighboring nonelectroporated pyramidal neurons in layers 2/3 were similar; ACIII was enriched in the axoneme and pericentrin was localized to the basal body (Anastas et al., 2011, Arellano et al., 2012) ( Figure 4

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100 1B). We also observed that cytoplasmic EGFP was not detected in ACIII positive cilia ( Figure 4 1B). 5HT6 and SSTR3 receptors are normally expressed in the neocortex of e arly postnatal brain (Breunig et al., 2008, Riccio et al., 2009, Stanic et al., 2009, Anastas et al., 2011) and localize to the plasma membranes of neuronal pr imary cilia (Hamon et al., 1999, Handel et al., 1999, Brailov et al., 2000) We previously noted that overexpression of GPCRs in immortalized cell lines induced abnormal growth of the cilia of these cells. In this experiment, we used in utero electroporation to deliver bicistronic 2A vectors encoding mCherry and either SSTR3:EGFP or 5HT6:EGFP to the brains of E15.5 mice to determine if similar changes in the cilia of developing neur ons would occur if we overexpressed GPCRs in these cells. Examination of electroporated neurons in layers 2/3 of P14 neocortex revealed that both 5HT6:EGFP and SSTR3:EGFP were trafficked into the cilia ( Figure 4 1 C, D); however, unlike 5HT6:EGFP, SSTR3:EGFP also appeared to be diffusely distributed throughout the somas and neurites of the electroporated cells ( Figure 4 1D). Notably, the cilia elaborated by neurons expressing 5HT6:EGFP were not only significantly longer than those of neurons express ing SSTR3:EGFP, but were also abnormally branched ( Figure 4 1 E, F). The robust primary and secondary order branching of 5HT6:EGFP+ cilia occurred at varicosities that were distributed along the lengths of the cilia ( Figure 4 1C, G). Branching of the cilia of neurons expressing SSTR3:EGFP, while not common, was occasionally observed ( Figure 4 1H). We confirmed that the cilialike processes elaborated by the electroporated cells were in fact cilia by staining the cells for pericentrin. Examination of the stained cells showed that pericentrin was localized at the

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101 base of all hyper elongated EGFP+ organelles ( Figure 4 1I). In sum, our data show that overexpression of ciliary GPCRs in developing neocortical neurons dramatically alters ciliogenesis, inducing grow th of cilia that are abnormally long and branched. Aberrant Lengthening of Neuronal Cilia b y GPCR Overexpression is Linked t o Enhanced IFT Function While conducting our cortical experiments, we found that overexpression of 5HT6:EGFP and SSTR3:EGFP in NIH3 T3 cells induced changes in the structures and lengths of their cilia that were similar to those observed in neurons ( Figure 4 2A, B, G). We took advantage of this observation and used these cells as a model system to examine possible factors that could explain the effects of overexpression of these receptor proteins on ciliogenesis. To determine whether the function of the overexpressed receptor was critical to obtaining the cilia phenotype induced by overexpression of 5HT6, we examined the effects of overexpression of two signaling defective 5HT6 receptors, K265A and D72A, on ciliogenesis. We also determined if fusion of 5HT6 to protein tags contributed to the cilia phenotype. Finally, we examined the specificity of this effect by overexpressing nonciliary fusion proteins targeted to the cilia by ciliary targeting sequences; the two proteins examined were EGFP fused to the fibrocystin ciliary targeting sequence (PKD 1C1 68:EGFP), a protein that is normally excluded from neuronal cilia (e.g. Figure 4 1B), and the transferrin receptor fused to EGFP, a single transmembranespanning protein not normally found in cilia (PC2TRFR:EGFP). The results of these experiments suggest that the abnormal cilia growth that accompanies overexpression of 5HT6:EGFP is not dependent on the activity of this receptor ( Figure 4 2C, D, G) and is only modestly influenced by the size or the position of the EGFP or HA protein tags fused to the receptor ( Figure 4 2G). More importantly,

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102 we found that abnormal cilia growth could n ot be induced in cells overexpressing nonciliary transmembrane (PC2TRFR:EGFP) or soluble proteins (PKD 1C1 68:EGFP) targeted to the cilia ( Figure 4 2E, F, G). Trafficking of GPCRs within the cilium is believed to depend on IFT, a process that is mediated by proteins that are also critical for promoting ciliogenesis (Nachury et al., 2007, Berbari et al., 2008b, Mukhopadhyay et al., 2010, GarciaGonzalo and Reiter, 2012) We next asked if overexpression of GPCRs in neurons alters IFT in a manner that would support the increased growth of the primary cilia of these cells. Western blot analyses of proteins extracted from electroporated cortex revealed that the levels of Kif3a, an anterograde motor subunit required for neuronal ciliogenesis (Chizhikov et al., 2007, Davenport et al., 2007, Han et al., 2008, Kumamoto et al., 2012) were significantly elevated within subdissected regions of P14 cortex overexpressing either SSTR3:EGFP or 5HT6:EGFP (n=3 4 pooled hemispheres/group) compared to extracts from electroporated and nonelectroporated control tissues ( Figure 4 3A D). We also found that the levels of other IFT associated proteins were elev ated in these cortical regions: the retrograde transport protein cytoplasmic dynein, D1 IC74 (Pazour et al., 1998, Tai et al., 1999, Grissom et al., 2002, Makokha et al., 2002) ; the IFT complex B protein, IFT88 (Kozminski et al., 1993, Willaredt et al., 2008, Goetz and Anderson, 2010, Satir et al., 2010, Taschner et al., 2012) ; and the GPCR ciliary trafficking protein, Tubby like protein 3 (TULP3) (Mukhopadhyay et al., 2010) Examination of neurons overexpressing 5HT6:EGFP that were stained with IFT88 antibodies showed that this transport protein was localized to the cilium and was distributed along its entire length ( Figure 4 3E, F). Together, our Western and immunohistochemical data show that

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103 o verexpression of 5HT6:EGFP induces an increased levels of several proteins associated with IFT and suggest that these proteins are distributed to the cilia and would support increased IFT and cilia growth. Interestingly, the levels of TULP3, which were bar ely detectable in control cortex and cortex overexpressing 5HT6:EGFP, were increased in cortices overexpressing SSTR3:EGFP ( Figure 4 3D). We next asked whether overexpressing GPCRs in neurons not yet elaborating cilia could trigger IFT and premature ciliogenesis. In normal developing neocortex, neurons begin to elaborate procilia at P1 that resemble puncta when visualized by ACIII immunostaining (Arellano et al., 2012) To address this question, we electroporated E15.5 neocortex with a vector encoding mCherry and 5HT6:EGFP (mCherry(AU1)2A 5HT6:EGFP) and examined the cilia of the electroporated neurons at P1. We found that many of the 5HT6:EGFP positive cilia that were elaborated by mCherry positive P1 neurons were significantly longer than the ACIII positive procilia associated with neighboring, nonelectroporated neurons ( Figure 4 3G, H), a result which showed overexpression of 5HT6 was able to induce premature ciliogenesis. To determine if IFT was essential to support the early onset of ciliogenesis in developing layer 2/3 neurons, we electroporated neurons at E15.5 with either a plasmid encoding 5HT6:EGFP (mCherry(AU1)2A 5HT6:EGFP) to visualize cilia or a mixture of this plasmid and one encoding dominant negative Kif 3a (mCherry(AU1) 2A dnKif3a). Dominant negative Kif3a (dnKif3a) is a truncated, nonfunctional form of Kif3a that does not support IFT and has recently been reported to block ciliogenesis in adult born granule neurons (Kumamoto et al., 2012) As expected, examination of neurons in P1 brains expressi ng 5HT6:EGFP alone revealed numerous cells bearing long, EGFP -

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104 positive cilia ( Figure 4 3I). In contrast, neurons coexpressing 5HT6:EGFP and dnKif3a, failed to localize 5HT6:EGFP to the developing cilia; in these cells 5HT6:EGFP was largely restricted to the neurons cell bodies ( Figure 4 3J). This result indicates that 5HT6 induced ciliogenesis in P1 neocortical neurons requires IFT. Collectively, these data show that the premature cilia growth in developing neocortical neurons that is induced by overex pression of ciliary GPCRs is accompanied by increased levels of IFT proteins, which may reflect a demand for increased transport of the GPCRs into or within the cilia. 5HT6 Overexpression is Associated w ith a Marked Decrease i n Ciliary SSTR3 a nd ACIII Loca lization Next, we asked whether the abnormal length and branching of 5HT6:EGFP+ cilia could compromise trafficking of other ciliatargeted molecules to the cilia that are required for normal cilia function. In normal neocortex, SSTR3 is trafficked into the majority (>90%) of ACIII+ neuronal cilia ( Figure 4 4A, C) (Stanic et al ., 2009; Einstein et al ., 2010). Thus, in our first experiment, we examined the cilia of neurons overexpressing 5HT6:EGFP for the presence of SSTR3. Strikingly, we found that the cilia elaborated by ~95% of the neurons overexpressing 5HT6:EGFP failed to stain for SSTR3 ( Figure 4 4B, C), an observation that suggests that overexpression of 5HT6:EGFP disrupts trafficking of SSTR3 into the cilium. In view of this result, we next asked if overexpression of 5HT6:EGFP alters the levels of other cilia targeted signaling molecules in the cilia. One key signaling molecule associated with primary cilia is ACIII. ACIII, which is localized to the primary cilia of most neurons, is believed to partic ipate in the signal transduction cascades triggered by many receptors in the ciliary membrane (Berbari et al., 2007, Bishop et al., 2007, Ou et

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105 al., 2009, Arellano et al., 2012) Given its central role in cilia function, we asked whether levels of ACIII are altered in the cilia of cortical neurons overexpressing either 5HT6:EGFP or SSTR3:EGFP. Intriguingly, ACIII was not detected in the cilia of layer 2/3 neurons overexpressing 5HT6:EGF P ( Figure 4 5A), but was present in the cilia of neurons overexpressing SSTR3:EGFP ( Figure 4 5B). Quantification of ACIII staining revealed that >90% of the cilia produced by neurons overexpressing SSTR3:EGFP were ACIII+ whereas <10% of the cilia elaborated by neurons overexpressing 5HT6:EGFP were ACIII+ ( Figure 4 5C). Collectively, these data suggest that the growth and structural changes observed in the primary cilia of neurons overexpressing 5HT6:EGFP are accompanied by a dramatic reduction in ciliary ACIII levels that we predict would compromise the signaling capabilities of these organelles. Neurons with Long, Malformed Cilia and Those w ith Blocked Cilia Formation Exhibit Abnormal Dendritic Outgrowth. Recent studies suggest that abnormal neuronal ciliogenesis is associated with dendrite outgrowth defects (Massinen et al., 2011, Kumamoto et al., 2012) To determine whether the abnormal ciliogenesis induced by overexpression of GPCRs in neocortical neurons is accompanied by changes in dendrite outgrowth, we compared the dendritic arbors of cortical neurons that had been electroporated at E15.5 with vectors encoding either EGFP (control) ( Figure 4 6A), SSTR3:EGFP ( Figure 4 6B), or 5HT6:EGFP ( Figure 4 6C) and then cultured for 12 days. Visual comparisons of the confocal images of these neurons revealed that overexpression of 5HT6:EGFP dramatically reduced dendrite outgrowth while overexpression of SSTR3:EGFP produced moderate reductions in dendrite outgrowth compared to controls. Using Sholl analyses to quantify these observations, we foun d that dendritic outgrowth from

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106 5HT6:EGFP and SSTR3:EGFP neurons was significantly less than from control neurons (p<0.001; twoway ANOVA) ( Figure 4 6E). The complexity of the dendritic arbors elaborated by neurons overexpressing SSTR3:EGFP resembled that of control neurons within a radius of ~20 m of the soma center, but then abruptly decreased between ~30 to 150 m of the soma center. Within this later region, the complexity of the dendritic arbors was greater than that observed for 5HT6:EGFP neurons but less than that of control neurons. Within 100 m of the soma, the complexity of the dendritic arbors elaborated by SSTR3:EGFP+ neurons was significantly greater than that exhibited by 5HT6:EGFP+ neurons. We also found that overexpression of 5HT6:EGFP ( n= 80 cells) compared to EGFP alone (n= 80 cells) hindered formation of dendritic arbors by neurons electroporated at E13.5 that were destined to form the deeper layers of neocortex (data not shown). This observation shows that the effects of overexpression of 5HT6:EGFP on dendrite formation are not limited to neurons that populate layers 2/3 but are also observed in other neocortical neuron subtypes. To determine if the absence of a cilium would induce similar effects on the dendrite arbor formation, we el ectroporated developing cortical neurons with a vector encoding dnKif3a fused to EGFP (EGFP:dnKif3a). We found that overexpression of EGFP:dnKif3a in NIH3T3 cells and cultured neurons blocks ciliogenesis as evidenced by the absence of acetylated alpha tubulin and ACIII staining, respectively (data not shown). The dendritic arbors of neurons expressing EGFP:dnKif3a were significantly less complex than those of neurons overexpressing SSTR3:EGFP and control neurons ( Figure 4 6D, E). It is noteworthy that the effects of overexpression of 5HT6: EGFP on dendritic complexity, while the most severe, closely resembled those induced by

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107 overexpression of EGFP:dnKif3a. Collectively, our data suggest that developing neurons that either elongate malformed cilia or lack cilia fail to elaborate normal dendritic arbors. Co Expression o f 5HT6 : EGFP w ith ACIII b ut Not Dnkif3a Reverses Dendrite Arbor Defects We observed that the inability of neurons to arborize dendrites was most pronounced when their cilia were either both very long and branched or were absent. In either case, ACIII could not be detected in the cilia of these cells ( Figure s 4 5 and 6). In contrast, the cilia of neurons overexpressing SSTR3:EGFP, although longer than control cilia, were ACIII+ and their dendritic arbors were less severely attenuated than those of neurons expressing either 5HT6:EGFP or dnKif3a. Thus, we asked whether overexpression of ACIII in neurons overexpressing 5HT6:EGFP could reverse the dendritic arbor defect s exhibited by these neurons. First, we generated an EGFP tagged ACIII construct which, unlike EGFP ( Figure 4 7A), was able to localize to cilia in cultured electroporated neurons ( Figure 4 7B). We then electroporated E15.5 cortex with a mixture of vectors encoding ACIII:EGFP, 5HT6:EGFP and mRFP, harvested the electroporated neurons at E16.5, and examined them after 12 days in culture. Examination of mRFP+ neurons revealed that >90% of EGFP+ cilia also stained positively for ACIII ( Figure 4 7C, D). Compared to neurons overexpres sing ACIII:EGFP alone, whose cilia lengths were similar to control, coexpression of 5HT6:EGFP and ACIII:EGFP in neurons significantly reduced the lengths of the cilia elaborated by these cells compared to neurons expressing 5HT6:EGFP alone ( Figure 4 7E). As expected, in neurons coexpressing ACIII:EGFP and dnKif3a, we were unable to detect ACIII+ cilia ( Figure 4 7E). These results indicate that coexpression of ACIII:EGFP with

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108 5HT6:EGFP in neurons reduced the ciliary defects induced by overexpression of 5H T6:EGFP alone and increased the levels of ACIII in the cilium. We next asked whether there were any differences in the dendritic arbors of neurons expressing either ACIII:EGFP alone, 5HT6:EGFP alone, or ACIII:EGFP and 5HT6:EGFP. As shown above ( Figure 4 6C,E), we found that the dendritic arbors of neurons overexpressing 5HT6:EGFP alone were the most poorly arborized ( Figure 4 7F). In contrast, the dendritic arbors of neurons overexpressing ACIII:EGFP were significantly more elaborate than neurons expressi ng 5HT6:EGFP alone ( Figure 4 7F). Remarkably, the dendritic arbors of neurons coexpressing ACIII:EGFP and 5HT6:EGFP were significantly more arborized than those of neurons expressing 5HT6:EGFP alone, resembling those of neurons expressing ACIII:EGFP alone ( Figure 4 7F). To determine whether the effects of overexpression of ACIII on dendritic arborization were dependent on localization of the ACIII to the cilium, we examined the dendritic arbors of cultured neurons that had been coelectroporated with vec tors encoding dnKif3a and ACIII:EGFP. Strikingly, we found that the dendritic processes elaborated by these neurons were not significantly different from those elaborated by neurons overexpressing 5HT6:EGFP alone ( Figure 4 7F). Collectively, these result s strongly suggest that the formation of normal dendritic arbors by developing cortical neurons requires ACIII to be localized to their cilia. Whether the regulation of arborization is mediated directly by ACIII or whether ACIII works in concert with other ciliary molecules to regulate dendrite arborization requires further investigation. Discussion The results of our study show that disruption of ciliogenesis in developing cortical neurons by either overexpressing ciliaassociated GPCRs or by blocking IFT inhibits the

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109 ability of these neurons to form normal dendritic arbors. Overexpression of ciliary GPCRs in these neurons induces an upregulation of IFT associated molecules and premature elongation of primary cilia by developing neurons. The most striking changes in cilia morphology were induced by overexpression of 5HT6:EGFP, which were accompanied by a lack of SSTR3 and ACIII in the cilia. Coexpression of ACIII:EGFP and 5HT6:EGFP reversed the abnormal dendrite phenotype associated with overexpression of 5HT6:EGFP alone. However, overexpression of ACIII:EGFP in neurons lacking cilia due to coexpression with dnKif3a was unable to reverse this dendritic phenotype. Collectively, our data suggest that the process of neuronal dendritogenesis is dependent on the ability of neurons to generate ACIII enriched primary cilia. Mutations that disrupt ciliogenesis or the ability of signaling proteins to localize to the cilium are thus likely to alter dendritogenesis and therefore the ability of neurons to form normal network connections and brain circuitry. GPCRInduced Changes i n Neuronal Cilia Length The primary cilia of cortical neurons in the postnatal brain elongate over a period of many weeks (Arellano et al., 2012, Kumamoto et al., 2012) While many factors are believed to affect cilia length (Ou et al., 2009, Miyoshi et al., 2011, Sharma et al., 2011, Avasthi and Marshall, 2012) our data show that cilia length homeostasis is dramatically disrupted by overexpression of GPCRs. Our observation is consistent with and extends recent results showing that class A GPCRs (e.g. dopamine 1) can induce elongation of NIH3T3 primary cilia (Avasthi and Marshall, 2012) and that the primary cilia of neurons in the amygdalae of BBS4/ mice become elongated due to increased ciliary accumulation of dopamine 1 receptors (Domire et al., 2011a) ( K. Mykytyn unpublished observation). In contrast, the absence o f neuronal cilia GPCRs does not appear to

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110 have an appreciable effect on cilia length homoeostasis. For example, the cilia of hippocampal or nucleus accumbens neurons in mice lacking either BBS2 or BBS4 appear normal even though they do not contain detectable levels of SSTR3 or MCHR1 (Berbari et al., 2008b) Similarly, the primary cilia of neurons in SST R3 knockout mice also appear to be structurally normal (Einstein et al., 2010) We found that overexpression of either SSTR3:EGFP or 5HT6:EGFP in neurons not only induced premature growth of their primary cilia but also increased expression levels of Kif3A, IFT88, and cytoplasmic dynein in these cells, all of which support IFT. Interestingly, we found increased levels of TULP3 in neocortical regions overexpressing SSTR3:EGFP, but not in those overexpressing 5HT6:EGFP. TULP3 binds to the IFT A complex and promotes ciliary trafficking of SSTR3 and MCHR1 into neural cilia but not Smoothened (Smo) (Mukhopadhyay et al., 2010) The increase in TULP3 in neurons overexpressing SSTR3 could reflect differences between 5HT6 and SSTR3 transport within and/or to neuronal cilia, and suggests that the neuronal response induced by overexpression of specific G PCRs may be receptor specific. Over the course of our experiments we noticed that the cilia of neurons overexpressing 5HT6:EGFP were typically longer and more irregular than neurons overexpressing SSTR3:EGFP. The underlying cause(s) for this dif ference is not clear. It is possible that 5HT6 is trafficked into the cilia more efficiently or that neurons regulate the levels of expression of these GPCRs differently. An additional possibility is ubulin and mobilize microtubule plus ends (Dave et al., 2009, Yu et al., 2009, Dave et al., 2012) The 5HT6 receptor is coupled to Gs, whereas the SSTR3 receptor is coupled to Gi (Law et al., 1994) Thus, it

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111 is tempting to speculate that the dramatic effects of overexpression of 5HT6 on cilia formation is, at least in part, due to an increase in the amount and activity of G s within the cilium that leads to increased axoneme growth and demand for IFT proteins. GPCR Overexpression May Compromise Neuronal Cilia Signaling It is likely that increased trafficking of ciliary GPCRs (especially 5HT6) into developing cilia alters the signaling properties of the cilia. In addition to ciliary branching and varicosities, we found that native SSTR3 was rarely detected in the cilia of neurons overexpressing 5HT6:EGFP. The reduced levels of SSTR3 in these cilia could reflect impaired traffi cking of SSTR3 into the cilium in the presence of the more abundant 5HT6:EGFP receptor which would be expected to alter ciliary signaling. A recent study suggests that the various GPCRs present in cilia physically interact with each other (Green and Mykytyn, 2010) Since heteromerization of ciliary GPCRs has been proposed to increase the complexity of ciliary signaling, overexpression of specific receptors would cause an imbalance in the ratios of these receptors and may compromise ciliary signaling. Moreover, the forced exclusion of receptors such as SSTR3 from the cilia of neurons overexpressing 5HT6 could contribute to learning and memory deficits as observed in SSTR3 knockout mice (Einstein et al., 2010) Normally, ACIII is trafficked into cilia during the earliest stages of neuronal ciliogenesis and persists throughout adulthood (Bishop et al., 2007, Arellano et al., 2012) We were unable to detect ACIII in the cilia of neurons overexpressing 5HT6:EGFP and did not detect an accumulation of ACIII around the bases of the elongated cilia or in the soma from these cells. Surprisingly, overexpression of SSTR3:EGFP, which lengthened neuronal ci lia, was not accompanied by a loss of ciliary ACIII staining. In addition, the ciliary phenotype associated with overexpression

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112 of SSTR3:EGFP was not as severe as that observed in cells overexpressing 5HT6:EGFP. Together, these results suggest that the unique transport demands created by overexpression of 5HT6:EGFP reduced the trafficking of native ACIII, SSTR3, and perhaps other important signaling molecules into the cilium. The reduced levels of these molecules in these cilia would likely impair the ability of the cilia to detect and respond to cues in the local extracellular milieu. Cilia Malform ation, Dendrite Abnormalities, a nd ACIII We found that neocortical neurons overexpressing cilia GPCRs grow excessively long/malformed cilia and defective dendr itic arbors. Intriguingly, the severity of the dendritic defect was positively correlated with the severity of the ciliary structural defect. The dendritic defects that we observed in our study were more severe than those reported in a recent study of the adult dentate gyrus. In that study, blocking the formation of cilia by expressing dnKif3a in neurons born in the adult dentate gyrus shortened the dendrites elaborated by these cells and delayed their arborization, but did not alter their complexit y as a ssessed by Sholl analysis (Kumamoto et al., 2012) Although we did not measure the lengths of the dendrites elaborated by our cultured neocortical neurons expressing dnKif3a, we did find that overexpression of either dnKif3a or ciliary GPCRs in these cells reduced the complexity of the perisomal den drites elaborated by these cells. The differences observed in the results of these two studies could indicate that different populations of neurons, in this case adult born dentate granule versus developing neocortical pyramidal neurons, respond differentl y to disruption of Kif3a function. It is noteworthy that overexpression of DCDC2, a molecule that interacts with KIF3A, has recently been reported to induce abnormal cilia growth in

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113 hippocampal neuron cultures and in ciliated C. elegans neurons that was as sociated with abnormal dendritic outgrowth (Massinen et al., 2011) To what extent are ciliogenesis and dendrite formation in neocortical neurons linked? Our data suggest these two processes are interdependent. We observed that overexpression of 5HT6:EGFP induced abnormal grow th of the cilia, significantly reduced ciliary ACIII levels, and disrupted dendrite growth. We also found that overexpression of ACIII in these cells reversed these effects. Importantly, overexpression of ACIII and dnKif3a did not alter the dendritic defects exhibited by these cells. Together, these results suggest that ciliary ACIII is required for normal dendritogenesis. Whether ACIII itself directly reg ulates dendrite formation, or functions in concert with other signaling molecules requires further st udy. Interestingly, a recent study of ACIII deficient mice shows that these mice exhibit deficits in learning and memory (Wang et al., 2011) Our results suggest these deficits may, in part, reflect defects in growth of neuronal dendrites. At this time, it is unclear whether the reduction in the lengths of the cilia produced by neurons coexpressing 5HT6:EGFP and ACIII:EGFP was due to a reduction in the amount of 5HT6:EGFP trafficked into the cilia or restoration of ACIII signaling from the cilia. In sum, our data and those of recent studies support a link between cilia and dendrite growth. We observed that disruption of ciliogenesis in layer 2/3 cortical neurons, as well as in cells destined for the deeper layers of neocortex, reduced dendritogenesis, suggesting that the effects of cilia on cytoarchitecture are not limited to one neuronal cell type. It is possible that this effect does not generalize to all neocortical neuron subtypes. For example, a recent study of developing neocortical

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114 inhibitory interneurons shows that targeted ablation of the cilia of these cells induced aberrant migration of these cells and their final position in neocortex, but did not dramatically alter their post migratory differentiation (Higginbotham et al., 2012) Interestingly, these authors also found that targeted ablation of Arl13b in projection neurons did not alter their migration, but did induce axonal outgrowth and connectivity defects. Taken together, these results suggest that the impact that ciliogenesis has on neuronal development and maturation may depend on the developmental age and subtype of the neuron. Unraveling these phenotypes may represent a key to understanding the various neurological symptoms exhibited by patients diagnosed with ciliopathies, including cognitive deficits, autism spectrum disorders, seizures, schizophrenia, and developmental dyslexia (Marley and von Zastrow, 2010, Lee and Gleeson, 2011, Louvi and Grove, 2011, Massinen et al., 2011) The specific changes induced in the neural network or in signaling pathways by cilia defects m ay depend on the nature of the defect and the type of neuron affected. Materials and Methods Mice All animal protocols were approved by and carried out in accordance with the Institutional Animal Care and Use Committee at the University of Florida. CD1 mouse brains were collected on embryonic (E) day 14.5 (n=9), E16.5 (n = 92), postnatal (P) day 1 (n=5), P10 (n=10), and P14 (n= 37). Postnatal mice of either sex were intracardially perfused with saline followed by 4% paraformaldehyde in 0.1M phosphate buffe r solution (4% PFA). All brain tissues were post fixed overnight in 4% PFA at 4C. Following fixation, the brains were rinsed, cryoprotected in sucrose, frozen over liquid N2, and sectioned (4050m coronal) using a cryostat.

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115 In Utero Electroporation Vectors were delivered to the developing cortices of E15.5 mice using in utero electroporation (IUE) as previously described (Sarkisian et al., 2006) Briefly, at 15.5 days into gestation, female mice were anesthesized by intraperitoneal injections of ketamine (100mg/kg) and xylazine (10mg/kg) diluted in sterile saline. Mice received Meloxicam (1mg/kg) as an analgesic. The uterine horns were exposed and approximately 1L of DNA ([0.52g/L ] mixed with 0.025% Fast Green) was microinjected through the uterine wall into the cerebral lateral ventricles of the mouse embryos using pulled glass capillaries. Electroporati on was achieved by discharging 50V across the cortex in 5pulse series spaced 50msec apart (pulse duration =950msec) using a BTX ECM 830 Square Wave Electroporator. Following injections, the dams were sutured and allowed to recover on heating pads. Vectors Table 2 1 describes the vectors used in this study and their promoter/protein tag information if applicable. pEGFP N3 vectors were constructed that encoded either mouse SSTR3 or mouse 5HT6 that were fused to EGFP on the C termini. The expression of SSTR3:EGFP and 5HT6:EGFP in these vectors was controlled by a human CMV immediate early promoter. Lentiviral vectors were generated that encoded mCherry tagged with AU1 upstream of dnKif3a, or either SSTR3 or 5HT6 that were fused to EGFP on the C termini. mCh erry(AU1) and dnKif3a, SSTR3:EGFP or 5HT6:EGFP in these vectors were fused to each other using the pTV1 2A cleavage peptide. The expression of all lentiviral transgenes was controlled by an elongation et al ., 2011). Additional 5HT6 vectors used included pcDNA3.1HA:5HT6 encoding 5HT6 with a HA tag fused to the N terminus,

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116 and two signaling defective 5HT6 receptor constructs 5HT6 (D72A):EGFP and 5HT6 (K265A):EGFP, which have been previously described (Kang et al., 200 5; Zhang et al., 2006). The K265A point mutation completely abolishes the signaling capabilities of the 5HT6 receptor and reduces cAMP levels to 5% of those found in wild type cells. The D72A point mutation reduces the binding affinity of the 5HT6 receptor for 5HT and reduces downstream activation of adenylyl cyclase III by 60%. The PKHD 1C1 68 vector encodes 68 amino acids immediately downstream of the transmembrane domain of fibrocystin, the human autosomal recessive polycystic kidney disease protein. T hese 68 amino acids, which include the 18residues near the N terminus that contains the cilia targeting sequence, were fused to the N terminus of EGFP (Follit et al., 2010) The PC 2 TRFN vector encodes the cilia targeting sequence of polycystin2 (PC2) (Geng et al., 2006) fused to the first 61 amino acids of the N terminus of the human transferrin receptor (hTFR), all of which were fused to the N terminus of EGFP. The transferrin receptor (hTFR) imports iron into cells and is not normally trafficked to the cilium (Geng et al., 2006, Avasthi et al., 2012) The vector encoding full length mouse type 3 adenylyl cyclase (ACIII) was fused to the C terminus (ACIII:EGFP) and under control of t he ubiquitin C (UbiC)promoter. All control experiments were performed using pCAGGS EGFP, pCAGGS EGFP 2A mCherry expressing vectors. Cell Culture NIH3T3 cells were seeded onto glass coverslips in DMEM supplemented with 10% fetal bovine serum (FBS) and 1X antibiotic antimycotic solution (ABX, Life Technologies) (cDMEM). Cells were seeded into 24well plates at a density of 3.6 x 106 cells/well and were transfected 24 hours later. The cells were transfected with the indicated cDNAs (0.8g/well) using LipofectamineTM2000 (Life Technologies) in serum

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117 free DMEM (Mediatech Cellgro # 10013 CV). After 4 6 hours, the transfection media was replaced with cDMEM and the cells were allowed to grow for 48 hrs at 37oC in 5% CO2. After 48 hrs of growth, the c ells were fixed in 4% PFA for 15 min at room temperature (RT) and washed 3X with PBS. Electroporated mRFP or EGFP+ neurons located in the dorsal telencephalon were dissected from the brains of E16.5 mice (68 fetal cortices/group) and were placed into ice cold Hanks Balanced Salt Solution (HBSS) containing 25mM HEPES buffer and 0.5% (w/v) glucose. The tissues were transferred into prewarmed Trypsin LE solution (Life Technologies) supplemented with 10mM HEPES and were then dissociated by trituration with a fire polished glass pipette. The dissociated cells were re suspended in Neurobasal medium (Life Technologies) supplemented with 2M sodium pyruvate, 4M Lglutamine, 1X antibiotic anti mycotic liquid (Life Technologies), 5% FBS, and 2% (v/v) B27 (Life Technologies) and were seeded at a density of 1.5 x 105 cells/well in 24well plates containing sterile glass coverslips that had been coated with poly ornithine (0.001%) and laminin (5g/mL). To promote neuron differentiation, 50% of the culture media was r eplaced with media lacking serum 24 hr after seeding followed by fifty percent of the media being replaced every other day. Throughout the experiment the cultures were maintained at 37C in 5% CO2. Neurons were cultured for up to 12 days in vitro (DIV) and were then fixed in 4% PFA for 15 min at RT and washed 3X with PBS. Immunostaining Cultured cells or brain cryosections were incubated overnight at 4C with the following primary antibodies: mouse anti tubulin (1:2000; Sigma), rabbit anti adenylyl cyclase (ACIII) (1:10,000; EnCor BioTechnology, 1:1000; Santa Cruz),

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118 mouse anti pericentrin (1:500; BD Biosciences), rabbit anti IFT88 (1:500; Covance), chicken anti GFP (1:5,000; Abcam), and goat anti SSTR3 (1:200; Santa Cruz). Appropriate, species specific, secondary antibodies conjugated to fluorescent tags were used to visualize the primary antibodies (1:200; JacksonImmunoResearch). Immunostained sections were cover slipped using ProLong Gold Antifade media containing 4',6diamidino2 phenylindole dihydrochloride (DAPI) (Life Technologies). Analyses and Quantification of Cilia Immunostained cell cultures and brain sections were examined using an Olympus IX81DSU spinning disc confocal microscope. Z stack images (0.5 0.75 m per step) of ACIII, ac tubulin or EGFP positive cilia were collected and then collapsed to create maximum projection images that were saved as tiff files and subsequently analyzed using Image J64 (http://rsbweb.nih.gov/ij/). We analyzed the brains of P1 and P14 elect roporated mice (68 collapsed z stack images/brain). Cilia tubulin+ or EGFP+ structures. The pixel values were converted to microns and were plotted as the mean SEM for each group. A OneWay ANOVA (wi th Fishers PLSD post hoc analysis) was used to compare groups. A pvalue <0.05 was considered significant. Western Blots Protein lysates were prepared from mouse cortex by homogenizing tissue in 1X RIPA buffer (Cell Signaling Technology). Lysate samples (10 g total protein/lane) were separated on 412% NuPAGE gels (Life Technologies) and transferred onto PVDF membranes using an iBlot (Life Technologies). Blots were blocked in Tris buffered saline containing 0.1% Tween (TBST) and 5.0% BSA (w/v) for 1h at RT and were then incubated overnight at 4C with one of the following primary antibodies diluted in TBST

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119 containing 2.5% BSA: rabbit anti Kif3a (1: 1000; Protein Tech); rabbit anti TULP3 (1: 500; gift from J Eggenschwiler); rabbit anti cytoplasmic dynein ( 74.1) (1:5000; Covance); rabbit anti actin (1:10,000; Sigma). The next day, the blots were washed with TBST, incubated for 1hr at RT with appropriate HRP conjugated secondary antibodies (1;10,000; BioRad), and were developed using an ECLPlus chemiluminescence kit according to the manufacturers instructions (GE HealthCare). Images of the blots were captured and analyzed using an Alpha Innotech FluorChemQ Imaging System (ProteinSimple). The signal intensities of the protein actin on the same blot which served as a loading control. Each protein sample was analyzed a minimum of three times. Sholl Analysis To visualize cell morphology, cells were elect roporated in utero with vectors encoding eit her EGFP or mRFP alone [0.5g/L], or one of these vectors was paired with a vector encoding either ACIII:EGFP, 5HT6:EGFP, SSTR3:EGFP, EGFP:dnKif3a, or mCher(AU1)2A dnKif3a [2g/L]. Sholl analyses were carried out as previously described (Sarkisian and Siebzehnrubl, 2012) Briefly, from several experiments, fixed EGFP+ or mRFP+ cultured neurons selected from 48 coverslips (~1015 neurons/coverslip) were photographed using an Olympus IX81 spinning disc confocal microscope fitted with a 40x dry objective. All raw images of EGFP+ or mRFP+ cells were resampled to obtain smaller, higher resolution grayscale images (e.g. 18.667x14.222 at 72 pixels/in to 3x2.286 at 300 pixels/in). The grayscale images were thresholded to create highcontrast, binary images using Adobe Photoshop (Version 11.01) and were saved as tiff files. Images were then opened in Image J64

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120 (http://rsbweb.nih.gov/ij/) and analyzed using the Sholl Analysis plugin. The intersections of each cells processes with concentric rings placed every 10 m up to 200 m from a point positioned in the center of each soma was counted and the means of the various treatment groups were compared using TwoWay ANOVA (with Fishers PLSD posthoc analysis), with p <0.05 considered significant.

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121 Table 41. Plasmid cDNA vectors used in this s tudy Plasmid Vector backbone Promoter Source Species (if applicable) Fusion Tag Location Obtained From 5HT6:EGFP pEGFPN3 CMV mouse CT K Mykytyn SSTR3:EGFP pEGFPN3 CMV mouse CT K Mykytyn mCherry(AU1) 2A 5HT6:EGFP pFIN mouse CT S Rowland mCherry(AU1) 2A SSTR3:EGFP pFIN mouse CT S Rowland EGFP2amCherry(AU1) pFIN n/a CT S Rowland PC2 TRFR:EGFP pEGFPN CMV human (TRFR) CT K Mykytyn PKHD 1C168:EGFP pEGFPN CMV mouse CT K Mykytyn 5HT6 (D72A):EGFP pEGFPN3 CMV mouse CT K Mykytyn 5HT6 (K265A):EGFP pEGFPN3 CMV mouse CT K Mykytyn HA:5HT6 pcDNA3.1 CMV mouse NT K Mykytyn mRFP pCAGGS CAG n/a none J LoTurco EGFP pCAGGS CAG n/a none P Rakic EGFP:dnKif3a pEGFPC1 CMV bp1252 2255 of mouse Kif3a NT S Ge mCherry(AU1) 2A dnKif3a pFIN bp1252 2255 of mouse Kif3a none S Rowland A CIII:EGFP pUB UbiC mouse CT J Breunig Guadiana, S.M., et al ., Arborization of dendrites by developing neocortical neurons is dependent on primary cilia and type 3 adenylyl cyclase. J Neurosci, 2013. 33(6): p. 262638.

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122 Figure 4 1. Overexpression of 5HT6 and SSTR3 in mouse neocortical neurons induces abnormal growth of their primary cilia A: Flow diagram of electroporation and experimental procedures used to induce and analyze the effects of overexpression of ciliary GPCRs on ciliogenesis in cultured and in situ neocortical neurons. Neurons analyzed in culture were dissected from electroporated cortex at E16.5, dissociated, and cultured up to 12 days (12DIV). Neurons in situ were analyzed in sections of brains between postnatal day (P) 1 and P40 electropor ated mice. B: Examples of layer 2/3 neurons electroporated at E15.5 with a vector encoding EGFP and labeled at P14. EGFP+ pyramidal neurons show normal positioning and morphology of ACIII+ cilia (red, arrows) with pericentrin+ basal bodies (blue, arrowhea ds) near the base of the apical dendrite, and are comparable to neighboring nonelectroporated neurons. EGFP does not colocalize with ACIII in t he EGFP+ neurons. C, D : E15.5 cortices were electroporated with vectors encoding either 5HT6:EGFP (C) or SSTR3:E GFP (D) under ere analyzed in situ at P14. C: Example of layer 2/3 neurons overexpressing 5HT6:EGFP which is enriched in cilia (arrows). D: Example of layer 2/3 neurons overexpressing SSTR3:EGFP which is detected in cilia (arrows) and throughout the cell bodies (asterisk). E: Tracings of EGFP+ cilia elongated by neurons expressing either 5HT6:EGFP and SSTR3:EG FP. Bar = 10m. F: Comparison of cilia lengths from control (ACIII+), SSTR3:EGFP+ and 5HT6:EGFP+ neurons in layers 2/3 of P14 neocortex. **p< 0.01, ***p<0.001 (ANOVA). G, H: Higher magnification confocal z stacks of branching cilia with varicosities (arrows) synthesized by mCherry+ pyramidal neurons expressing either 5HT6:EGFP (E) o r SSTR3:EGFP (F). Bar= 10m. I: Sections of P14 neocortex containing mCherry+ neurons (red) with abnormally long and malformed 5HT6:EGFP+ cilia (green) were immunostained for pericentrin(blue). Five EGFP+ cilia with basal bodies were numbered and their magnified images are displayed as separate channels (3 images) grouped vertically by cilium on the right. Pericentrin+ basal bodies are present at the base of each EGFP+ cilium (I, arrows). Bar = 10m.

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123 Figure 4 2 Cilia growth induced by GPCR overexpression is not significantly affected by l oss of GPCR function or the presence of protein tags. (A F) Representative confocal images of NIH3T3 cells expressing the proteins indicated above each panel whose expression was driven by the CMV promoter. All cells were immunostained with antibodies agai nst the axonemeenriched protein, acetylated alpha tubulin (AAtub) (red) and GFP (green). Nuclei we re labeled with DAPI (blue). A: Cilium of a cell transfected with a vector encoding SSTR3:EGFP (arrow). B: Cilium of cell overexpressing 5HT6:EGFP (arrow) ad jacent to a cilium of a nontransfected control cell (arrowhead). The insets show single channel EGFP and AAtub staining of the cilium of the transfected cell. (C and D) Cilia elaborated by cells overexpressing the signaling defective 5HT6 receptors, 5HT6( K265A):EGFP (C) or 5HT6(D7 2A):EGFP (D) (arrows). E, F: Overexpression of EGFP fused to fibrocystin (PKHD 1C1 68:EGFP) (E) or human transferrin receptor (PC2TRFR:EGFP) (F), two non cilia proteins fused to a cilia localization signal. G: Mean axoneme length s of cilia produced by cells expressing the experimental vectors shown in panels A F and HA:5HT6. From left to right, n = 50, 34, 44, 56, 38, 22, 26, and 30 cilia analyzed/group, respectively. Each bar represents the mean SEM. *** = p<0.001, ns = not sig nificant (One way ANOVA).

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124 Figure 4 3. GPCR overexpression induces upregulation of IFT proteins and premature cilia lengthening. A D: Comparisons were made between protein expression in nonelectroporated control cortex (A), fetal cortex that was electroporated at E15.5 with either a vector encoding EGFP and mCherry:AU1 (IUE control) (B), or mCherry:AU1 and either SSTR3:EGFP or 5HT6:EGF P (C). Expression indicate cortical regions of P14 brains that were used to prepare the protein lysat es analyzed by Western blot. D: were probed for proteins associated with either anterograde (Kif3a) or retrograde (cytoplasmic dynein, D1 IC74) IFT complex B protein (IFT88), or actin wa s used as a loading control. E: Cultured, nonelectroporated control cortical neuron immunostained for pericentrin (basal body, red), IFT88 (green), and the neuronal marker, MAP2 (blue). The arrow in the middle panel points to an IFT88+ cilium extending from a pericentrin+ ba sal body (arrow left panel). F: Example of an abnormally long, branched 5HT6:EGFP+ cilium synthesized by a cultured neuron expressing 5HT6:EGFP(green) under the control of the CMV promoter and mRFP (pseudocolored blue). Ift88(red) and EGFP were colocalized along the length and branches of the cilium (w hite arrows). Bar = E15.5 brains were electroporated with a vector encoding mCherry(AU1)2a5HT6:EGFP. At P1, electroporated brains were sectioned and stained with an antibody against ACIII. Examination of the upper layers of the cortical plate rev ealed mCherry+ neurons (red) that possessed longer 5HT6:EGFP+ cilia (arrowheads) than their neighboring nonelectroporated cells whose ACIII stained cilia appear puncta Comparison of the lengths of the cilia of neurons overex pressing 5HT6:EGFP and control ne urons. (***Students t test) I: Section of brain electroporated and processed as described for (G) but not including the red channel used to visualize mCherry. Numerous, often long cilia (arrows) were present in the upper l ayers of the cortical plate. J: P1 neurons in the upper cortical plate that were coelectroporated at E15.5 with vectors encoding mCherry and 5HT6:EGFP (mCherry(AU1)2a5HT6:EGFP) and mCherry and dnKif3a (mCherry(AU1)2adnKif3a). The elongated 5HT6:EGFP+ cilia of neurons expressing 5HT6:EGFP alone (I) are not observed in cells coexpressing 5HT6:EGFP and dnKif3a.

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125

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126 Figure 4 4 Cilia of neurons overexpressing 5HT6:EGFP do not contain detectable levels of SSTR3. A: Control P14 neocortex stained with antibodies against ACIII (green) and SSTR3 (red). The majority of ACIII+ cilia are also S STR3+ (arrows). Bar = 10m. B: Cilia synthesized by neurons expressing 5HT6:EGFP (arrows) are not correspondingly SSTR3+. Bar = 1 0m. C: The percentage of ACIII+ control (n= 1056) or 5HT6:EGFP+ (n=688) cilia that are also SSTR3+.

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127 Figure 4 5 Cilia of neurons overexpressing 5HT6:EGFP do not contain detectable levels of ACIII.The brains of E15.5 embryos were electroporated with vectors promoter and were imm unostained for ACIII at P14. A: Pyramidal neurons in layers 2/3 of neocortex expressing mCherry:AU1 (blue) and 5HT6:EGFP (green). Sections were immunostained for ACIII (red), which normally is enriched in cilia of virtually all neocortical neurons. White arrows point to 5HT6:EGFP+ cilia projecting from mCherry:AU1+ neurons that lack detectable ACIII staining. Bar = 10m. B: Pyramidal neurons in layers 2/3 of neocortex expressing mCherry:AU1 (blue) and SSTR3:EGFP (green). SSTR3:EGFP+ cilia also stain fo r ACIII (yellow arrowheads). C: The percentage of SSTR3:EGFP+ (n=123) or 5HT6:EGFP+ (n=89) cilia that are also ACIII+.

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128 Figure 4 6 5HT6, SSTR3 and dnKif3a overexpression reduce dendrite outgrowth of cultured cortical neurons. Neurons were electroporated at E15.5 with a vector encoding EGFP (Control) or the vector encoding EGFP plus vectors encoding either SSTR3:EGFP, 5HT6:EGFP or EGFP:dnKif3a under the CMV promot er. Typical cilia phenotypes associated with each group is indicated. At E16.5, cells were harvested, dissociated and fixed after 12DIV. Confocal images of EGFP+ cells were converted to black and white images. A C: Examples of neurons at 12DIV expressing (A) EGFP alone, (B) SSTR3:EGFP, (C) 5HT6:EGFP, and (D) EGFP:dnKif3a. (D) Sholl analyses reveal the extent of arborization of EGFP control (green line), SSTR3:EGFP (blue), dnKif3a (red) and 5HT6:EGFP (grey) neurons. N= total number of cells analyzed. Statistical comparisons were against EGFP using Twoway ANOVA (***p < 0.001, **p<0.01, *p<0.05)

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129 Figure 4 7. Coexpression of ACIII:EGFP with 5HT6:EGFP, but not dnKif3a, restores ciliary ACIII, cilia structure and dendrite outgrowth. A, B: Neurons electroporated at E15.5 with vectors encoding either (A) mRFP and EGFP or (B) mRFP and ACIII:EGFP. Electroporated neurons were cultured for 6DIV, fixed, and immunostained for ACIII (red). Analyses of mRFP+ neurons (blue) showed that EGFP did not t raffic into the cilia as evidenced by an absence of co localization with ACIII (A; arrow in inset). When fused to ACIII, the cilia were positive for both ACIII staining and EGFP fluorescence (B; arrow in inset). C: Example of a cultured neuron coelectroporated with 5HT6:EGFP and ACIII:EGFP possessing a cilium that is positive for both ACIII staining and EGFP (inset shows zoom of cilia in indi vidual channels and merge). D: Quantification of the number of cells coelectroporated with 5HT6:EGFP and ACIII:EGFP whose cilia were both ACIII and EGFP positive (N= 139/153). E: Comparisons of the lengths of the cilia elaborated by neighboring nonelectroporated (control) neurons (n=142) or neurons transfected with vectors encoding mRFP plus either ACIII:EGFP (n=60), ACIII:EGFP and 5HT6:EGFP (n=120), 5HT6:EGFP (n=120) or dnKif3a (n=118) and cultured for 12DIV (**p<0.01, ***p<0.001, ns=not significant, N.D.= not determinable). F: Sholl analyses of the complexity of the dendritic arbors elaborated by neurons transfected with mRFP plus either 5HT6:EGFP (grey), ACIII:EGFP (green), EGFP:dnKif3a + ACIII:EGFP (red), or ACIII:EGFP + 5HT6:EGFP (blue) and maintained in culture for 12 days (n= number of cells analyzed). The complexity of the arbors of neurons expressing 5HT6:EG FP were statistically compared to those of the other groups using twoway ANOVA (***p < 0.001, ns=not significant).

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130 CHAPTER 5 CONCLUSIONS Summary of Findings and Implications The work presented here supports the hypot hesis that neuronal cilia play a role in the development of the neocortex and that cilia dysfunction contribute to abnormal development in this brain region. Although it has been proposed for some time that cilia dysfunction may be perturbing delicate earl y developmental processes in the cortex, the exact role neuronal cilia have in the post natal neocortex remains unclear. Many functional roles have been attributed to cilia in the other parts of the body but the role primary cilia play on post mitotic neur ons was largely unknown. Now that there is a more complete picture of cilia development and maintenance throughout the mammalian lifespan on excitatory neurons, more informed hypotheses about the function of neuronal cilia can be made We now know how neur onal ciliogenesis occurs and some of the proteins that are localized within the axoneme during the earliest stages (procilium) of that process. We also know cilia are still found on aged cortical neurons even up to the most advanced stages of aging in the mammalian brain. It is intriguing to speculate that these structures are still active and functional throughout the lifespan They may still be performing signaling functions into and throughout senescence as they also still retain most of their molecular profile with aging However, there is a possibility that aged cilia are not able to signal to their full capacity as evidenced by loss of immunodetectability with some GPCR in specific brain regions. Another interesting aspect of the work in Chapter 3 includes the data that showed neurons in the NA and OT of the ventral striatum do not retain their localization of MCH1R in their double cilia and that in the nucleus accumbens, cells may have shed

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131 their cilia prematurely due to advanced age. The loss of cilia in the ventral striatum may be a link to loss of cilia function in this region of the brain as it has been heavily implicated in the reward pathway and this cilia receptor has been shown to be involved in feeding and satiety behaviors. Interestingly, a predominant pathology seen in ciliopathy patients is obesity. Chapter 4 describes a novel link between neocortical excitatory pyramidal neuron development and cilia malformations. Due to this work, the cilia malformation approaches and the cell culture methods for analysis used in that study may be beneficial for future studies dissecting out exact mechanism(s) of neuronal cilia signaling contributions to dendritogenesis during development of the neocortex. Although this finding will need to be repeated in vivo it suggests cilia are providing a service in the formation of cortical circuitry. This work also provides a link between the possibility that cilia mutations may be underlying the learning/memory and cognitive dysfunction seen in ciliopathies. Although we partially attributed ACIII localization within the axoneme as one important mechanism of action by which cilia may be contributing to the differentiation of neurons, there are likely many others to e xplore. The data we show in C hapter 4 also reveals an interesting aspect of cilia manipulations. Although two different cilia mutations were used ( blunt cilia vs. elongated cilia) both were able to produce the same differentiation defect in neocortical dendrites. One of the most intriguing aspects of this study was the fact that in dnKif3a group, returning ACIII back to those cells did not rescue the differentiation defects. This suggests the ciliary localization of ACIII is crucial to nor mal dendritogenes is. However it is not yet clear whether mere localization of the ACIII protein itself is important for both proper neuronal cilia function and dendritic arbor extensions or if direct signaling from

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132 ciliary ACIII is the main contributing factor to normal dendritic development. Whatsmore, each of the GPCR overexpression approaches (SSTR3 versus 5HT6) showed distinct defects. For example, the majority of SSTR3 induced hyperelongated cilia were ACIII+, indicating either this receptor uses another AC or it is a function of Gprotein coupling as this receptor is Gi coupled to AC in contrast to the more severe cilia malformation using 5HT6 w h ere it is Gs coupled to AC. The fact that the 5HT6 overexpression group included many examples where cilia axonemes were forked and branched suggests the microtubule dynamics were strained or unstable, although this result will certainly need ultrastructure characterization to better define the axonemal defects. These aspects of the work will need to be fully explored in order to better understand these results. Future work is needed to examine these questions and identify both up and downstream targets in this pathway Cilia Roles on Different Neuronal Populations All cilia are not created equal. During the course of my the sis several groups have investigated the functions of cilia on both excitatory and inhibitory cortical neurons. Now emerging are two strikingly different roles for these two cortical neuron subtypes. First, we reported excitatory projection neurons in the neocortex do not extend a cilium from their basal body unti l the neuron has completed its migration up radial glia fibers into its proper laminar layer. Once there, the post migratory, excitatory neuron begins to build its cilium, expressing ACIII and other signaling molecules, during the first several weeks of development while other important developmental processes are occurring simultaneously such as dendritogenesis, synaptogenesis and neuronal maturation. We found that the earlier processes such as laminar patterning were not perturbed when cilia were absent (Stumpy) suggesting cilia do not have a functional role

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133 on excitatory projection neurons in layer positioning. This is not surprising since the centros ome is the key cellular component required for this migration patterning and the cilia have yet to be formed until this process has completed (Arellano et al., 2012) Kumamoto et al then reported the timecourse of ciliogen esis on hippocampal excitatory adult born dentate granule cells (DGCs) also being a protracted process which lasts several weeks, initiated after migration of those cells has been finalized (Kumamoto et al., 2012) Recently, Higginbotham et al ablated cilia on excitatory projection neurons and they too found no obvious migration defects when cilia were disrupted (Higginbotham et al., 2012) Thus, it appears that both neocortical and hippocampal excitatory neurons do not use their cilia for migration processes. Kumamoto and colleagues then showed that by blocking ciliogenesis on these excitatory, adult born DGCs using a dominant negative version of Kif3a (dnKif3a), synaptic integration and dendritic refinement defects were observed. This group also showed that 5 days after the initial cilia formation, blocking ciliogenesis at this timepoint revealed a less severe phenotype (Kumamoto et al., 2012) We showed in the neocortical excitatory neurons that by inducing cilia malformations (either absent using dnKif3a or hyperelongated and malfor m ed using an overexpression of receptors 5ht6 or Sstr3) in primary neuronal cultures, dendritic morphology defects were observed. These defec ts were progressively worse as the neurons matured in vitro (Guadiana et al., 2013) These data suggest excitatory neurons use their cilia to help differentiate the cell and potentially integrate into their proper circuit in the cortex, although more s tudies are needed to confirm this idea.

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134 On the contrary to the role cilia play on excitatory neurons, the function on interneurons appears to be important during earlier developmental processes. Higginbotham et al. ablated interneuron cilia by deleting Arl13b using a floxed allele mouse line crossed to a Dlx5/6Cre (expressed in interneurons) In contrast to excitatory neurons, they found disruption of inhibitory interneuron cilia did not induce post migratory differentiation defects; however these mice displayed severe migration patterning defects, abnormal interneuron accumulation in the subpallial region, and fewer GABAergic cells reaching their final cortical destination. A fascinating aspect of their work was seen in the timelapse imaging of interne uronal tangential migration C ilia from th ese cells extend out into the extracellular milieu en route to the cortical plate. The cilium elongates once the cells have paused in their migration process and seem to sense and respond to Shh. The cells then ret ract their cilia just prior to proceeding on with migration into the tangential stream (Higginbotham et al., 2012) Another group, Baudoin et al showed similar findings in their study, which too ablated inhibitory interneuron cilia during cortical migration by interrupting Kif3a and Ift88. They showed cells that did not bear cilia were l ess able to leave the tangential migratory stream, turn properly into and invade the cortical plate (Baudoin et al., 2012) These two studies provide a link between cilia dysfunction and the cortical malformation seen in cili opathy patients such as Joubert syndrome. Together, these data suggest an entirely different role for cilia on inhibitory interneurons, which appear to be utilizing their cilia for migration and proper entry into the cortex. The model ( Figure 5 1) illustrates the identified roles thus far of cilia on excitatory and inhibitory neurons in the cortex. Cilia on inhibitory interneurons (red), born in the

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135 GE, are utilized for migration through the tangential stream and invasion of the cortical plate. Pri mary cilia on excitatory neurons (green) (either adult born DGCs born in the proliferative zone near the SVZ (hippocampus) or projection neurons born in the proliferative zones of the VZ in the cortex) ) however, do not use cilia for this purpose. Instead cilia on these excitatory neurons are utilized for differentiation, synaptic integration and refinement of dendritic processes. The work presented in my thesis outlines the important role cilia play on the different iation of excitatory neurons in one aspec t of this model and how this process may go askew when cilia are malformed. This work from my thesis has led to a better understanding of how primary cilia on projection neurons influence cortical development and may be contributing the neurological diseas e pathology seen in ciliopathic conditions. Future Unanswered Questions and Challenges Although a clearer picture of primary neuronal cilia function in the development and maturation of the cortex is beginning to emerge, there are still numerous unanswer ed questions. Future investigation into their roles will likely include studies on cellular mechanisms of differentiation and migration and how cilium signaling is involved. Although several have been elucidated in the last few years, there are likely to b e many more mechanisms by which these sensory organelles function. Other questions that remain after this work and currently in field include whether cilia have other functional roles in the cortex on neuronal populations than the ones discussed here. Are there distinct signaling properties from these organelles on different cell populations? What is the c iliome of neuronal versus nonneuronal populations? Future questions will need to address the distinction between cilia specific function and the possibil ity that the other noncilia function is the root cause for the cellular abnormalities

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136 observed. This is a valid question often brought up among the researchers in the field and an important one to address. Additionally, better tools are essential to separ ating out the cilia vs. noncilia functional roles. As more cilia proteins are pathways are identified, a more complete story of primary neuronal cilia may start to take shape.

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137 Figure 5 1. Model of c ilia contributions to the n ormal and a bnormal d evelopment of the n eocortex ( Left ) In the normal developing cortex, primary cilia are present on radial glial (grey) and progenitor cells (grey) in the VZ (roles not discussed here), migrating and differentiating inhibitory interneurons (red), and post mitotic pyramidal cells (green). Migrating interneurons use cilia to coordinate migration from the GE and to depart from their tangential streams into the CP. Excitatory pyramidal neurons assemble their cilia after radial migration, which then take many weeks to fully elongate. In the hippocampus, cilia on adult born, dentate granule neurons (blue cells) also develop primary cilia, which also take several weeks to mature. ( Right ) When cilia structure and function is conditionally targeted in different animal models, interneurons develop a variety of deficits in migration (e.g. accumulating at the pallial/subpallial boundary, abnormal migratory morphologies, failed turning behavior towards the CP), which ultimately result in abnormal positioning within different lamina of the cortex and hippocampus. Post migratory differentiation of interneurons is unaffected. Excitatory pyramidal neurons and adult born dentate granule neurons in vivo migrate appropriately upon cilia deficits but subsequentl y develop deficits in dendritic arbors and synaptic integration. Abbreviations: VZ ventricular zone, IZ intermediate zone, CP cortical plate, GE ganglionic eminence

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138 LIST OF REFERENCES Adamantidis A, Thomas E, Foidart A, Tyhon A, Coumans B Minet A, Tirelli E, Seutin V, Grisar T, Lakaye B (Disrupting the melaninconcentrating hormone receptor 1 in mice leads to cognitive deficits and alterations of NMDA receptor function. The European journal of neuroscience 21:28372844.2005). Adams M, Sm ith UM, Logan CV, Johnson CA (Recent advances in the molecular pathology, cell biology and genetics of ciliopathies. J Med Genet 45:257267.2008). Airaksinen MS, Eilers J, Garaschuk O, Thoenen H, Konnerth A, Meyer M (Ataxia and altered dendritic calcium s ignaling in mice carrying a targeted null mutation of the calbindin D28k gene. Proceedings of the National Academy of Sciences of the United States of America 94:14881493.1997). Amador Arjona A, Elliott J, Miller A, Ginbey A, Pazour GJ, Enikolopov G, Roberts AJ, Terskikh AV (Primary cilia regulate proliferation of amplifying progenitors in adult hippocampus: implications for learning and memory. The Journal of neuroscience : the official journal of the Society for Neuroscience 31:99339944.2011). Anastas SB, Mueller D, SempleRowland SL, Breunig JJ, Sarkisian MR (Failed cytokinesis of neural progenitors in citron kinasedeficient rats leads to multiciliated neurons. Cereb Cortex 21:338344.2011). Arellano JI, Guadiana SM, Breunig JJ, Rakic P, Sarkisian M R (Development and distribution of neuronal cilia in mouse neocortex. The Journal of comparative neurology 520:848873.2012). Armato U, Chakravarthy B, Pacchiana R, Whitfield JF (Alzheimer's disease: an update of the roles of receptors, astrocytes and pri mary cilia (review). International journal of molecular medicine 31:310.2013). Avasthi P, Marley A, Lin H, Gregori Puigjane E, Shoichet BK, von Zastrow M, Marshall WF (A chemical screen identifies class a g protein coupled receptors as regulators of cili a. ACS Chem Biol 7:911919.2012). Avasthi P, Marshall WF (Stages of ciliogenesis and regulation of ciliary length. Differentiation 83:S3042.2012). Bachmann Gagescu R, Ishak GE, Dempsey JC, Adkins J, O'Day D, Phelps IG, Gunay Aygun M, Kline AD, Szczaluba K, Martorell L, Alswaid A, Alrasheed S, Pai S, Izatt L, Ronan A, Parisi MA, Mefford H, Glass I, Doherty D (Genotype phenotype correlation in CC2D2A related Joubert syndrome reveals an association with ventriculomegaly and seizures. J Med Genet 49:126137. 2012).

PAGE 139

139 Bae YK, Barr MM (Sensory roles of neuronal cilia: cilia development, morphogenesis, and function in C. elegans. Front Biosci 13:59595974.2008). Barakat MT, Humke EW, Scott MP (Kif3a is necessary for initiation and maintenance of medulloblastoma. Carcinogenesis.2013). Barzi M, Berenguer J, Menendez A, Alvarez Rodriguez R, Pons S (Sonic hedgehog mediated proliferation requires the localization of PKA to the cilium base. Journal of cell science 123:6269.2010). Baudoin JP, Viou L, Launay PS, Luccar dini C, Espeso Gil S, Kiyasova V, Irinopoulou T, Alvarez C, Rio JP, Boudier T, Lechaire JP, Kessaris N, Spassky N, Metin C (Tangentially Migrating Neurons Assemble a Primary Cilium that Promotes Their Reorientation to the Cortical Plate. Neuron 76:1108112 2.2012). Bay SN, Caspary T (What are those cilia doing in the neural tube? Cilia 1:19.2012). Bearzatto B, Servais L, Roussel C, Gall D, BabaAissa F, Schurmans S, de Kerchove d'Exaerde A, Cheron G, Schiffmann SN (Targeted calretinin expression in granule cells of calretinin null mice restores normal cerebellar functions. FASEB J 20:380382.2006). Belgacem YH, Borodinsky LN (Sonic hedgehog signaling is decoded by calcium spike activity in the developing spinal cord. Proceedings of the National Academy of Sciences of the United States of America 108:44824487.2011). BennounaGreene V, Kremer S, Stoetzel C, Christmann D, Schuster C, Durand M, Verloes A, Sigaudy S, Holder Espinasse M, Godet J, Brandt C, Marion V, Danion A, Dietemann JL, Dollfus H (Hippocampal dysgenesis and variable neuropsychiatric phenotypes in patients with Bardet Biedl syndrome underline complex CNS impact of primary cilia. Clinical genetics (epub Apr 25, 2011).2011). Berbari NF, Bishop GA, Askwith CC, Lewis JS, Mykytyn K (Hippocampal neurons possess primary cilia in culture. Journal of neuroscience research 85:10951100.2007). Berbari NF, Johnson AD, Lewis JS, Askwith CC, Mykytyn K (Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Proteincoupl ed Receptors. Molecular biology of the cell 19:15401547.2008a). Berbari NF, Lewis JS, Bishop GA, Askwith CC, Mykytyn K (Bardet Biedl syndrome proteins are required for the localization of G proteincoupled receptors to primary cilia. Proceedings of the N ational Academy of Sciences of the United States of America 105:42424246.2008b).

PAGE 140

140 Berbari NF, O'Connor AK, Haycraft CJ, Yoder BK (The primary cilium as a complex signaling center. Curr Biol 19:R526535.2009). Bishop GA, Berbari NF, Lewis J, Mykytyn K (Type III adenylyl cyclase localizes to primary cilia throughout the adult mouse brain. The Journal of comparative neurology 505:562571.2007). Bittencourt JC (Anatomical organization of the melaninconcentrating hormone peptide family in the mammalian brain. General and comparative endocrinology 172:185197.2011). Brailov I, Bancila M, Brisorgueil MJ, Miquel MC, Hamon M, Verge D (Localization of 5HT(6) receptors at the plasma membrane of neuronal cilia in the rat brain. Brain research 872:271275.2000). Br eunig JJ, Sarkisian MR, Arellano JI, Morozov YM, Ayoub AE, Sojitra S, Wang B, Flavell RA, Rakic P, Town T (Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling. Proceedings of the National Academy of Sciences of the United States of America 105:1312713132.2008). Caspary T, Larkins CE, Anderson KV (The graded response to Sonic Hedgehog depends on cilia architecture. Dev Cell 12:767778.2007). Celio MR, Baier W, Scharer L, Gregersen HJ, de Viragh PA, Norman AW (Monoclonal a ntibodies directed against the calcium binding protein Calbindin D 28k. Cell Calcium 11:599602.1990). Celio MR, Heizmann CW (Calcium binding protein parvalbumin as a neuronal marker. Nature 293:300302.1981). Chakravarthy B, Gaudet C, Menard M, Atkinson T, Brown L, Laferla FM, Armato U, Whitfield J (Amyloid beta peptides stimulate the expression of the p75(NTR) neurotrophin receptor in SHSY5Y human neuroblastoma cells and AD transgenic mice. Journal of Alzheimer's disease : JAD 19:915925.2010a). Chakravarthy B, Gaudet C, Menard M, Atkinson T, Chiarini A, Dal Pra I, Whitfield J (The p75 neurotrophin receptor is localized to primary cilia in adult murine hippocampal dentate gyrus granule cells. Biochemical and biophysical research communications 401:4584 62.2010b). Chakravarthy B, Gaudet C, Menard M, Brown L, Atkinson T, Laferla FM, Ito S, Armato U, Dal Pra I, Whitfield J (Reduction of the immunostainable length of the hippocampal dentate granule cells' primary cilia in 3xAD transgenic mice producing human Abeta(142) and tau. Biochemical and biophysical research communications 427:218222.2012a).

PAGE 141

141 Chakravarthy B, Menard M, Ito S, Gaudet C, Dal Pra I, Armato U, Whitfield J (Hippocampal membraneassociated p75NTR levels are increased in Alzheimer's disease. Journal of Alzheimer's disease : JAD 30:675684.2012b). Chizhikov VV, Davenport J, Zhang Q, Shih EK, Cabello OA, Fuchs JL, Yoder BK, Millen KJ (Cilia proteins control cerebellar morphogenesis by promoting expansion of the granule progenitor pool. The Journal of neuroscience : the official journal of the Society for Neuroscience 27:97809789.2007). Cho YJ, Lee JC, Kang BG, An J, Song HS, Son O, Nam DH, Cha CI, Joo KM (Immunohistochemical study on the expression of calcium binding proteins (calbindinD2 8k, calretinin, and parvalbumin) in the cerebral cortex and in the hippocampal region of nNOS knock out( / ) mice. Anat Cell Biol 44:106115.2011). Cohen E, Binet S, Meininger V (Ciliogenesis and centriole formation in the mouse embryonic nervous system. An ultrastructural analysis. Biol Cell 62:165169.1988). Corbit KC, Aanstad P, Singla V, Norman AR, Stainier DY, Reiter JF (Vertebrate Smoothened functions at the primary cilium. Nature 437:10181021.2005). D'Angelo A, De Angelis A, Avallone B, Piscopo I Tammaro R, Studer M, Franco B (Ofd1 controls dorsoventral patterning and axoneme elongation during embryonic brain development. PloS one 7:e52937.2012). Dahl HA (Fine structure of cilia in rat cerebral cortex. Z Zellforsch Mikrosk Anat 60:369386.1963) Dave RH, Saengsawang W, Lopus M, Dave S, Wilson L, Rasenick MM (A molecular and structural mechanism for G proteinmediated microtubule destabilization. The Journal of biological chemistry 286:43194328.2012). Dave RH, Saengsawang W, Yu JZ, Donati R, R asenick MM (Heterotrimeric G proteins interact directly with cytoskeletal components to modify microtubuledependent cellular processes. Neurosignals 17:100108.2009). Davenport JR, Watts AJ, Roper VC, Croyle MJ, van Groen T, Wyss JM, Nagy TR, Kesterson R A, Yoder BK (Disruption of intraflagellar transport in adult mice leads to obesity and slow onset cystic kidney disease. Curr Biol 17:15861594.2007). De Robertis E (Electron microscope observations on the submicroscopic organization of the retinal rods. J Biophys Biochem Cytol 2:319330.1956).

PAGE 142

142 Defelipe J, Gonzalez Albo MC, Del Rio MR, Elston GN (Distribution and patterns of connectivity of interneurons containing calbindin, calretinin, and parvalbumin in visual areas of the occipital and temporal lobes of the macaque monkey. The Journal of comparative neurology 412:515526.1999). Del Cerro MP, Snider RS (The Purkinje cell cilium. Anat Rec 165:127130.1969). Delaval B, Doxsey SJ (Pericentrin in cellular function and disease. J Cell Biol 188:181190.2010). Di Gioia SA, Letteboer SJ, Kostic C, BandahRozenfeld D, Hetterschijt L, Sharon D, Arsenijevic Y, Roepman R, Rivolta C (FAM161A, associated with retinitis pigmentosa, is a component of the ciliabasal body complex and interacts with proteins involved in ciliopathies. Human molecular genetics 21:5174 5184.2012). Domire JS, Green JA, Lee KG, Johnson AD, Askwith CC, Mykytyn K (Dopamine receptor 1 localizes to neuronal cilia in a dynamic process that requires the Bardet Biedl syndrome proteins. Cellular a nd molecular life sciences : CMLS 68:29512960.2011a). Domire JS, Green JA, Lee KG, Johnson AD, Askwith CC, Mykytyn K (Dopamine receptor 1 localizes to neuronal cilia in a dynamic process that requires the Bardet Biedl syndrome proteins. Cellular and molecular life sciences : CMLS.2011b). Domire JS, Mykytyn K (Markers for neuronal cilia. Methods in cell biology 91:111121.2009). Doxsey SJ, Stein P, Evans L, Calarco PD, Kirschner M (Pericentrin, a highly conserved centrosome protein involved in microtubul e organization. Cell 76:639 650.1994). Dredge BK, Jensen KB (NeuN/Rbfox3 Nuclear and Cytoplasmic Isoforms Differentially Regulate Alternative Splicing and NonsenseMediated Decay of Rbfox2. PloS one 6:e21585.2011). Dubreuil V, Marzesco AM, Corbeil D, Hut tner WB, Wilsch Brauninger M (Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin1. J Cell Biol 176:483495.2007). Einstein EB, Patterson CA, Hon BJ, Regan KA, Reddi J, Melni koff DE, Mateer MJ, Schulz S, Johnson BN, Tallent MK (Somatostatin signaling in neuronal cilia is critical for object recognition memory. The Journal of neuroscience : the official journal of the Society for Neuroscience 30:43064314.2010).

PAGE 143

143 Ezratty EJ, Stokes N, Chai S, Shah AS, Williams SE, Fuchs E (A role for the primary cilium in Notch signaling and epidermal differentiation during skin development. Cell 145:11291141.2011). Feng L, Cooper JA (Dual functions of Dab1 during brain development. Mol Cell B iol 29:324332.2009). Ferland RJ, Cherry TJ, Preware PO, Morrisey EE, Walsh CA (Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain. The Journal of comparative neurology 460:266279.2003). Fiala JC (Reconstruct: a free editor for serial section microscopy. J Microsc 218:5261.2005). Fliegauf M, Benzing T, Omran H (When cilia go bad: cilia defects and ciliopathies. Nat Rev Mol Cell Biol 8:880 893.2007). Follit JA, Li L, Vucica Y, Pazour GJ (The cytoplasmic tail of fibr ocystin contains a ciliary targeting sequence. J Cell Biol 188:2128.2010). Fuchs JL, Schwark HD (Neuronal primary cilia: a review. Cell Biol Int 28:111118.2004). Garcia Gonzalo FR, Reiter JF (Scoring a backstage pass: mechanisms of ciliogenesis and ciliary access. J Cell Biol 197:697709.2012). Gascue C, Tan PL, Cardenas Rodriguez M, Libisch G, Fernandez Calero T, Liu YP, Astrada S, Robello C, Naya H, Katsanis N, Badano JL (Direct role of Bardet Biedl syndrome proteins in transcriptional regulation. Journal of cell science 125:362375.2012). Geng L, Okuhara D, Yu Z, Tian X, Cai Y, Shibazaki S, Somlo S (Polycystin2 traffics to cilia independently of polycystin1 by using an N terminal RVxP motif. Journal of cell science 119:13831395.2006). Gerdes JM, Liu Y, Zaghloul NA, Leitch CC, Lawson SS, Kato M, Beachy PA, Beales PL, DeMartino GN, Fisher S, Badano JL, Katsanis N (Disruption of the basal body compromises proteasomal function and perturbs intracellular Wnt response. Nature genetics 39:13501360.2007). Goetz SC, Anderson KV (The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet 11:331344.2010). Green JA, Gu C, Mykytyn K (Heteromerization of ciliary g proteincoupled receptors in the mouse brain. PloS one 7:e46304.2012).

PAGE 144

144 Green JA, Mykytyn K (Neuronal ciliary signaling in homeostasis and disease. Cellular and molecular life sciences : CMLS 67:32873297.2010). Grissom PM, Vaisberg EA, McIntosh JR (Identification of a novel light intermediate chain (D2LIC) for mammali an cytoplasmic dynein 2. Molecular biology of the cell 13:817829.2002). Guadiana SM, Semple Rowland S, Daroszewski D, Madorsky I, Breunig JJ, Mykytyn K, Sarkisian MR (Arborization of dendrites by developing neocortical neurons is dependent on primary cil ia and type 3 adenylyl cyclase. The Journal of neuroscience : the official journal of the Society for Neuroscience 33:26262638.2013). Hamon M, Doucet E, Lefevre K, Miquel MC, Lanfumey L, Insausti R, Frechilla D, Del Rio J, Verge D (Antibodies and antisense oligonucleotide for probing the distribution and putative functions of central 5 HT6 receptors. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 21:68S 76S.1999). Han YG, Alvarez Buylla A (Role of primar y cilia in brain development and cancer. Curr Opin Neurobiol 20:5867.2010). Han YG, Spassky N, RomagueraRos M, Garcia Verdugo JM, Aguilar A, Schneider Maunoury S, Alvarez Buylla A (Hedgehog signaling and primary cilia are required for the formation of a dult neural stem cells. Nature neuroscience 11:277284.2008). Handel M, Schulz S, Stanarius A, Schreff M, ErdtmannVourliotis M, Schmidt H, Wolf G, Hollt V (Selective targeting of somatostatin receptor 3 to neuronal cilia. Neuroscience 89:909926.1999). Haydar TF, Bambrick LL, Krueger BK, Rakic P (Organotypic slice cultures for analysis of proliferation, cell death, and migration in the embryonic neocortex. Brain Res Brain Res Protoc 4:425437.1999). Heydet D, Chen LX, Larter CZ, Inglis C, Silverman MA, Farrell GC, Leroux MR (A truncating mutation of Alms1 reduces the number of hypothalamic neuronal cilia in obese mice. Developmental neurobiology 73:113.2013). Higginbotham H, Eom TY, Mariani LE, Bachleda A, Hirt J, Gukassyan V, Cusack CL, Lai C, Caspary T, Anton ES (Arl13b in primary cilia regulates the migration and placement of interneurons in the developing cerebral cortex. Dev Cell 23:925938.2012). Higginbotham HR, Gleeson JG (The centrosome in neuronal development. Trends in neurosciences 30:2762 83.2007).

PAGE 145

145 Huangfu D, Anderson KV (Cilia and Hedgehog responsiveness in the mouse. Proceedings of the National Academy of Sciences of the United States of America 102:1132511330.2005). Huangfu D, Liu A, Rakeman AS, Murcia NS, Niswander L, Anderson KV (Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426:8387.2003). Ishikawa H, Marshall WF (Ciliogenesis: building the cell's antenna. Nat Rev Mol Cell Biol 12:222234.2011). Jones C, Roper VC, Foucher I, Qian D, Banizs B, Petit C, Yoder BK, Chen P (Ciliary proteins link basal body polarization to planar cell polarity regulation. Nature genetics 40:6977.2008). Jurczyk A, Pino SC, O'Sullivan Murphy B, Addorio M, Lidstone EA, Diiorio P, Lipson KL, Standley C, Fogarty K, Lifs hitz L, Urano F, Mordes JP, Greiner DL, Rossini AA, Bortell R (A novel role for the centrosomal protein, pericentrin, in regulation of insulin secretory vesicle docking in mouse pancreatic betacells. PloS one 5:e11812.2010). Kang H, Lee WK, Choi YH, Vukoti KM, Bang WG, Yu YG (Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5 HT6) and the alpha subunit of GS protein. Biochemical and biophysical research communications 329:684692.2005). Kao HT, Li P, Chao HM, Janoschka S, Pham K, Feng J, McEwen BS, Greengard P, Pieribone VA, Porton B (Early involvement of synapsin III in neural progenitor cell development in the adult hippocampus. The Journal of comparative neurology 507:18601870.2008). Kim J, Lee JE, Heynen Genel S, Suyama E, Ono K, Lee K, Ideker T, AzaBlanc P, Gleeson JG (Functional genomic screen for modulators of ciliogenesis and cilium length. Nature 464:10481051.2010). Kim KK, Adelstein RS, Kawamoto S (Identification of neuronal nuclei (NeuN) as Fox 3, a new member of the Fox 1 gene family of splicing factors. The Journal of biological chemistry 284:3105231061.2009). Kozminski KG, Johnson KA, Forscher P, Rosenbaum JL (A motility in the eukaryotic flagellum unrelated to flagellar beati ng. Proceedings of the National Academy of Sciences of the United States of America 90:55195523.1993). Kumamoto N, Gu Y, Wang J, Janoschka S, Takemaru K, Levine J, Ge S (A role for primary cilia in glutamatergic synaptic integration of adult born neurons Nature neuroscience 15:399405, S391.2012).

PAGE 146

146 Lancaster MA, Gleeson JG (The primary cilium as a cellular signaling center: lessons from disease. Curr Opin Genet Dev 19:220229.2009). Law SF, Zaina S, Sweet R, Yasuda K, Bell GI, Stadel J, Reisine T (Gi alpha 1 selectively couples somatostatin receptor subtype 3 to adenylyl cyclase: identification of the functional domains of this alpha subunit necessary for mediating the inhibition by somatostatin of cAMP formation. Mol Pharmacol 45:587590.1994). Lee J E, Gleeson JG (Cilia in the nervous system: linking cilia function and neurodevelopmental disorders. Curr Opin Neurol 24:98105.2011). Lee JH, Gleeson JG (The role of primary cilia in neuronal function. Neurobiol Dis 38:167172.2010). Leitch CC, Zaghloul NA, Davis EE, Stoetzel C, Diaz Font A, Rix S, Alfadhel M, Lewis RA, Eyaid W, Banin E, Dollfus H, Beales PL, Badano JL, Katsanis N (Hypomorphic mutations in syndromic encephalocele genes are associated with Bardet Biedl syndrome. Nature genetics 40:443448 .2008). LeVay S (Synaptic patterns in the visual cortex of the cat and monkey. Electron microscopy of Golgi preparations. The Journal of comparative neurology 150:5385.1973). Li A, Saito M, Chuang JZ, Tseng YY, Dedesma C, Tomizawa K, Kaitsuka T, Sung CH (Ciliary transition zone activation of phosphorylated Tctex 1 controls ciliary resorption, S phase entry and fate of neural progenitors. Nat Cell Biol 13:402411.2011). Logan CV, Abdel Hamed Z, Johnson CA (Molecular genetics and pathogenic mechanisms for the severe ciliopathies: insights into neurodevelopment and pathogenesis of neural tube defects. Mol Neurobiol 43:1226.2011). Lorenzo LE, Ramien M, St Louis M, De Koninck Y, RibeirodaSilva A (Postnatal changes in the Rexed lamination and markers of nociceptive afferents in the superficial dorsal horn of the rat. The Journal of comparative neurology 508:592604.2008). Louvi A, Grove EA (Cilia in the CNS: the quiet organelle claims center stage. Neuron 69:10461060.2011). Lund JS, Boothe RG, Lund RD (D evelopment of neurons in the visual cortex (area 17) of the monkey (Macaca nemestrina): a Golgi study from fetal day 127 to postnatal maturity. The Journal of comparative neurology 176:149188.1977).

PAGE 147

147 Lyck L, Kroigard T, Finsen B (Unbiased cell quantificati on reveals a continued increase in the number of neocortical neurones during early post natal development in mice. The European journal of neuroscience 26:17491764.2007). Makokha M, Hare M, Li M, Hays T, Barbar E (Interactions of cytoplasmic dynein light chains Tctex 1 and LC8 with the intermediate chain IC74. Biochemistry 41:43024311.2002). Mandl L, Megele R (Primary cilia in normal human neocortical neurons. Z Mikrosk Anat Forsch 103:425430.1989). Marley A, von Zastrow M (DISC1 regulates primary cil ia that display specific dopamine receptors. PloS one 5:e10902.2010). Massinen S, Hokkanen ME, Matsson H, Tammimies K, TapiaPaez I, Dahlstrom Heuser V, Kuja Panula J, Burghoorn J, Jeppsson KE, Swoboda P, PeyrardJanvid M, Toftgard R, Castren E, Kere J (Increased expression of the dyslexia candidate gene DCDC2 affects length and signaling of primary cilia in neurons. PloS one 6:e20580.2011). McIntyre JC, Davis EE, Joiner A, Williams CL, Tsai IC, Jenkins PM, McEwen DP, Zhang L, Escobado J, Thomas S, Szym anska K, Johnson CA, Beales PL, Green ED, Mullikin JC, Sabo A, Muzny DM, Gibbs RA, Attie Bitach T, Yoder BK, Reed RR, Katsanis N, Martens JR (Gene therapy rescues cilia defects and restores olfactory function in a mammalian ciliopathy model. Nat Med 18:1423 1428.2012). Menco BP, Bruch RC, Dau B, Danho W (Ultrastructural localization of olfactory transduction components: the G protein subunit Golf alpha and type III adenylyl cyclase. Neuron 8:441 453.1992). Michaud EJ, Yoder BK (The primary cilium in cell signaling and cancer. Cancer Res 66:64636467.2006). MiyamotoMatsubara M, Saitoh O, Maruyama K, Aizaki Y, Saito Y (Regulation of melanin concentrating hormone receptor 1 signaling by RGS8 with the receptor third intracellular loop. Cellular signalling 20:20842094.2008). Miyoshi K, Kasahara K, Miyazaki I, Asanuma M (Lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Biochemical and biophysical research communications 388:757762.2009). Miyoshi K, Kasahara K, Miyazaki I, Asanuma M (Factors that influence primary cilium length. Acta Med Okayama 65:279285.2011).

PAGE 148

148 Miyoshi K, Onishi K, Asanuma M, Miyazaki I, Diaz Corrales FJ, Ogawa N (Embryonic expression of pericentrin suggests universal roles in ciliogenesis. Dev Genes Evol 216:537542.2006). Morris RL, Scholey JM (Heterotrimeric kinesinII is required for the assembly of motile 9+2 ciliary axonemes on sea urchin embryos. J Cell Biol 138:10091022.1997). Mukhopadhyay S, Jackson PK (Cilia, tubby mice, and obesity. Cilia 2:1. 2013). Mukhopadhyay S, Wen X, Chih B, Nelson CD, Lane WS, Scales SJ, Jackson PK (TULP3 bridges the IFT A complex and membrane phosphoinositides to promote trafficking of G proteincoupled receptors into primary cilia. Genes Dev 24:21802193.2010). Mullen RJ, Buck CR, Smith AM (NeuN, a neuronal specific nuclear protein in vertebrates. Development 116:201211.1992). Murthy KS, Makhlouf GM (Differential coupling of muscarinic m2 and m3 receptors to adenylyl cyclases V/VI in smooth muscle. Concurrent M2medi ated inhibition via Galphai3 and m3mediated stimulation via Gbetagammaq. The Journal of biological chemistry 272:2131721324.1997). Mykytyn K, Braun T, Carmi R, Haider NB, Searby CC, Shastri M, Beck G, Wright AF, Iannaccone A, Elbedour K, Riise R, Baldi A, Raas Rothschild A, Gorman SW, Duhl DM, Jacobson SG, Casavant T, Stone EM, Sheffield VC (Identification of the gene that, when mutated, causes the human obesity syndrome BBS4. Nature genetics 28:188191.2001). Mykytyn K, Mullins RF, Andrews M, Chiang AP, Swiderski RE, Yang B, Braun T, Casavant T, Stone EM, Sheffield VC (Bardet Biedl syndrome type 4 (BBS4) null mice implicate Bbs4 in flagella formation but not global cilia assembly. Proceedings of the National Academy of Sciences of the United States of America 101:86648669.2004). Mykytyn K, Nishimura DY, Searby CC, Beck G, Bugge K, Haines HL, Cornier AS, Cox GF, Fulton AB, Carmi R, Iannaccone A, Jacobson SG, Weleber RG, Wright AF, Riise R, Hennekam RC, Luleci G, Berker Karauzum S, Biesecker LG, Stone EM, Sheffield VC (Evaluation of complex inheritance involving the most common Bardet Biedl syndrome locus (BBS1). American journal of human genetics 72:429437.2003).

PAGE 149

149 Mykytyn K, Nishimura DY, Searby CC, Shastri M, Yen HJ, Beck JS, Braun T, Streb LM, C ornier AS, Cox GF, Fulton AB, Carmi R, Luleci G, Chandrasekharappa SC, Collins FS, Jacobson SG, Heckenlively JR, Weleber RG, Stone EM, Sheffield VC (Identification of the gene (BBS1) most commonly involved in Bardet Biedl syndrome, a complex human obesity syndrome. Nature genetics 31:435438.2002). Mykytyn K, Sheffield VC (Establishing a connection between cilia and Bardet Biedl Syndrome. Trends in molecular medicine 10:106109.2004). Nachury MV, Loktev AV, Zhang Q, Westlake CJ, Peranen J, Merdes A, Slusarski DC, Scheller RH, Bazan JF, Sheffield VC, Jackson PK (A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell 129:12011213.2007). Nagata A, Hamamoto A, Horikawa M, Yoshimura K, Takeda S, Saito Y (Ch aracterization of ciliary targeting sequence of rat melaninconcentrating hormone receptor 1. General and comparative endocrinology.2013). Nigg EA, Raff JW (Centrioles, centrosomes, and cilia in health and disease. Cell 139:663678.2009). Nishimura DY, F ath M, Mullins RF, Searby C, Andrews M, Davis R, Andorf JL, Mykytyn K, Swiderski RE, Yang B, Carmi R, Stone EM, Sheffield VC (Bbs2null mice have neurosensory deficits, a defect in social dominance, and retinopathy associated with mislocalization of rhodopsin. Proceedings of the National Academy of Sciences of the United States of America 101:1658816593.2004). Norman RX, Ko HW, Huang V, Eun CM, Abler LL, Zhang Z, Sun X, Eggenschwiler JT (Tubby like protein 3 (TULP3) regulates patterning in the mouse embry o through inhibition of Hedgehog signaling. Human molecular genetics 18:1740 1754.2009). Novarino G, Akizu N, Gleeson JG (Modeling human disease in humans: the ciliopathies. Cell 147:7079.2011). Ou Y, Ruan Y, Cheng M, Moser JJ, Rattner JB, van der Hoorn FA (Adenylate cyclase regulates elongation of mammalian primary cilia. Exp Cell Res 315:28022817.2009). Pachoud B, Adamantidis A, Ravassard P, Luppi PH, Grisar T, Lakaye B, Salin PA (Major impairments of glutamatergic transmission and long term synaptic plasticity in the hippocampus of mice lacking the melaninconcentrating hormone receptor 1. Journal of neurophysiology 104:14171425.2010).

PAGE 150

150 Pasek RC, Berbari NF, Lewis WR, Kesterson RA, Yoder BK (Mammalian Clusterin associated protein 1 is an evolutionarily conserved protein required for ciliogenesis. Cilia 1:20.2012). Paxinos G, Franklin KBJ (2004) The Mouse Brain in Stereotaxic Coordinates. London: Academic Press. Pazour GJ, Dickert BL, Vucica Y, Seeley ES, Rosenbaum JL, Witman GB, Cole DG (Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J Cell Biol 151:709718.2000). Pazour GJ, Wilkerson CG, Witman GB (A dynein light chain is essential for the retrograde particle movement of intraflagellar transport (IFT). J Cell Biol 141:979992.1998). Pedersen LB, Rosenbaum JL (Intraflagellar transport (IFT) role in ciliary assembly, resorption and signalling. Curr Top Dev Biol 85:2361.2008). Pedersen LB, Veland IR, Schroder JM, Christensen ST (Assembly of primary cilia. Dev Dyn 237:19932006.2008). Poole CA, Jensen CG, Snyder JA, Gray CG, Hermanutz VL, Wheatley DN (Confocal analysis of primary cilia structure and colocalization with the Golgi apparatus in chondrocytes and aortic smooth muscle cells. Cell Biol Int 21:483494.1997). Porter KR (The submicroscopic morphology of protoplasm. Harvey lectures 51:175228.1955). Qin J, Lin Y, Norman RX, Ko HW, Eggenschwiler JT (Intraflagellar transport protein 122 antagonizes S onic Hedgehog signaling and controls ciliary localization of pathway components. Proceedings of the National Academy of Sciences of the United States of America 108:14561461.2011). Rakic P (Developmental and evolutionary adaptations of cortical radial gl ia. Cereb Cortex 13:541549.2003). Rasin MR, Gazula VR, Breunig JJ, Kwan KY, Johnson MB, LiuChen S, Li HS, Jan LY, Jan YN, Rakic P, Sestan N (Numb and Numbl are required for maintenance of cadherinbased adhesion and polarity of neural progenitors. Natur e neuroscience 10:819827.2007). Riccio O, Potter G, Walzer C, Vallet P, Szabo G, Vutskits L, Kiss JZ, Dayer AG (Excess of serotonin affects embryonic interneuron migration through activation of the serotonin receptor 6. Mol Psychiatry 14:280290.2009).

PAGE 151

151 Riise R, Tornqvist K, Wright AF, Mykytyn K, Sheffield VC (The phenotype in Norwegian patients with Bardet Biedl syndrome with mutations in the BBS4 gene. Archives of ophthalmology 120:13641367.2002). Roberson ED, Defazio RA, Barnes CA, Alexander GE, Bizon JL, Bowers D, Foster TC, Glisky EL, Levin BE, Ryan L, Wright CB, Geldmacher DS (Challenges and opportunities for characterizing cognitive aging across species. Frontiers in aging neuroscience 4:6.2012). Robert A, Margall Ducos G, Guidotti JE, Bregerie O, Celati C, Brechot C, Desdouets C (The intraflagellar transport component IFT88/polaris is a centrosomal protein regulating G1S transition in nonciliated cells. Journal of cell science 120:628637.2007). Rohatgi R, Milenkovic L, Scott MP (Patched1 reg ulates hedgehog signaling at the primary cilium. Science 317:372376.2007). Rooryck C, Pelras S, Chateil JF, Cances C, Arveiler B, Verloes A, Lacombe D, Goizet C (Bardet biedl syndrome and brain abnormalities. Neuropediatrics 38:59.2007). SainoSaito S, Hozumi Y, Goto K (Excitotoxicity by kainateinduced seizure causes diacylglycerol kinase zeta to shuttle from the nucleus to the cytoplasm in hippocampal neurons. Neuroscience letters 494:185189.2011). Saito Y, Nothacker HP, Wang Z, Lin SH, Leslie F, Ci velli O (Molecular characterization of the melanin concentrating hormone receptor. Nature 400:265269.1999). Saito Y, Tetsuka M, Li Y, Kurose H, Maruyama K (Properties of rat melaninconcentrating hormone receptor 1 internalization. Peptides 25:15971604. 2004). Santos N, Reiter JF (Building it up and taking it down: the regulation of vertebrate ciliogenesis. Dev Dyn 237:19721981.2008). Sarkisian MR, Arellano JI, Breunig JJ (2013) Primary cilia in cerebral cortex: growth and functions on neuronal and nonneuronal cells. In: Cilia and Nervous System Development and Function(Tucker, K. L. and Caspary, T., eds), pp 105129 Dordrecht: Springer. Sarkisian MR, Bartley CM, Chi H, Nakamura F, HashimotoTorii K, Torii M, Flavell RA, Rakic P (MEKK4 signaling regul ates filamin expression and neuronal migration. Neuron 52:789801.2006). Sarkisian MR, Frenkel M, Li W, Oborski JA, LoTurco JJ (Altered interneuron development in the cerebral cortex of the flathead mutant. Cereb Cortex 11:734743.2001).

PAGE 152

152 Sarkisian MR, Siebzehnrubl D (Abnormal levels of Gadd45alpha in developing neocortex impair neurite outgrowth. PloS one 7:e44207.2012). Satir P, Pedersen LB, Christensen ST (The primary cilium at a glance. Journal of cell science 123:499503.2010). Sattar S, Gleeson JG (The ciliopathies in neuronal development: a clinical approach to investigation of Joubert syndrome and Joubert syndromerelated disorders. Dev Med Child Neurol 53:793798.2011). Scheff HA (1953) A method for judging all contrasts in analysis of variance. Sfakianos J, Togawa A, Maday S, Hull M, Pypaert M, Cantley L, Toomre D, Mellman I (Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells. J Cell Biol 179:11331140.2007). Sharma N, Berbari NF, Yoder BK (Ciliary dy sfunction in developmental abnormalities and diseases. Curr Top Dev Biol 85:371427.2008). Sharma N, Kosan ZA, Stallworth JE, Berbari NF, Yoder BK (Soluble levels of cytosolic tubulin regulate ciliary length control. Molecular biology of the cell 22:8088 16.2011). Singh Manoux A, Czernichow S, Elbaz A, Dugravot A, Sabia S, Hagger Johnson G, Kaffashian S, Zins M, Brunner EJ, Nabi H, Kivimaki M (Obesity phenotypes in midlife and cognition in early old age: the Whitehall II cohort study. Neurology 79:755762 .2012). Singla V, Reiter JF (The primary cilium as the cell's antenna: signaling at a sensory organelle. Science 313:629633.2006). Slavotinek AM, Stone EM, Mykytyn K, Heckenlively JR, Green JS, Heon E, Musarella MA, Parfrey PS, Sheffield VC, Biesecker LG (Mutations in MKKS cause Bardet Biedl syndrome. Nature genetics 26:1516.2000). Sorokin S (Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J Cell Biol 15:363 377.1962). Sorokin SP (Centriole formation and ciliogenesis. Aspen Emphysema Conf 11:213216.1968). Sotelo JR, Trujillo Cenoz O (Electron microscope study on the development of ciliary components of the neural epithelium of the chick embryo. Z Zellforsch Mikrosk Anat 49:112.1958).

PAGE 153

153 Spassky N, Han YG, Agui lar A, Strehl L, Besse L, Laclef C, Ros MR, GarciaVerdugo JM, Alvarez Buylla A (Primary cilia are required for cerebellar development and Shh dependent expansion of progenitor pool. Dev Biol 317:246259.2008). Stanic D, Malmgren H, He H, Scott L, Aperia A, Hokfelt T (Developmental changes in frequency of the ciliary somatostatin receptor 3 protein. Brain research 1249:101112.2009). Stillman AA, Krsnik Z, Sun J, Rasin MR, State MW, Sestan N, Louvi A (Developmentally regulated and evolutionarily conserved expression of SLITRK1 in brain circuits implicated in Tourette syndrome. The Journal of comparative neurology 513:2137.2009). Sun X, Haley J, Bulgakov OV, Cai X, McGinnis J, Li T (Tubby is required for trafficking G proteincoupled receptors to neuronal cilia. Cilia 1:21.2012). Tai AW, Chuang JZ, Bode C, Wolfrum U, Sung CH (Rhodopsin's carboxy terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex 1. Cell 97:877887.1999). Taschner M, Bhogaraju S, Lorentzen E (Architecture and function of IFT complex proteins in ciliogenesis. Differentiation 83:S12 22.2012). ThauvinRobinet C, Thomas S, Sinico M, Aral B, Burglen L, Gigot N, Dollfus H, Rossignol S, Raynaud M, Philippe C, Badens C, Touraine R, Gomes C, Franco B, Lopez E, Elkhartoufi N, Faivre L, Munnich A, Boddaert N, Maldergem LV, EnchaRazavi F, Lyonnet S, Vekemans M, Escudier E, AttieBitach T (OFD1 mutations in males: phenotypic spectrum and ciliary basal body docking impairment. Clinica l genetics.2012). Town T, Breunig JJ, Sarkisian MR, Spilianakis C, Ayoub AE, Liu X, Ferrandino AF, Gallagher AR, Li MO, Rakic P, Flavell RA (The stumpy gene is required for mammalian ciliogenesis. Proceedings of the National Academy of Sciences of the Uni ted States of America 105:28532858.2008). Tucker RW, MeadeCobun KS, Jayaraman S, More NS (Centrioles, primary cilia and calcium in the growth of Balb/c 3T3 cells. J Submicrosc Cytol 15:139143.1983). Tucker RW, Pardee AB, Fujiwara K (Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells. Cell 17:527535.1979a). Tucker RW, Scher CD, Stiles CD (Centriole deciliation associated with the early response of 3T3 cells to growth factors but not to SV40. Cell 18:10651072.1979b).

PAGE 154

154 Tury A, Mairet Coello G, Dicicco Bloom E (The Cyclin Dependent Kinase Inhibitor p57Kip2 Regulates Cell Cycle Exit, Differentiation, and Migration of Embryonic Cerebral Cortical Precursors. Cereb Cortex.2011). Tuson M, He M, Anderson KV (Protein kinase A acts at t he basal body of the primary cilium to prevent Gli2 activation and ventralization of the mouse neural tube. Development 138:49214930.2011). Valente EM, Silhavy JL, Brancati F, Barrano G, Krishnaswami SR, Castori M, Lancaster MA, Boltshauser E, Boccone L, Al Gazali L, Fazzi E, Signorini S, Louie CM, Bellacchio E, Bertini E, Dallapiccola B, Gleeson JG (Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of Joubert syndrome. Nature genetics 38:623625.2006). Van Eden CG, Uyling s HB (Cytoarchitectonic development of the prefrontal cortex in the rat. The Journal of comparative neurology 241:253267.1985). Waclaw RR, Ehrman LA, Pierani A, Campbell K (Developmental origin of the neuronal subtypes that comprise the amygdalar fear ci rcuit in the mouse. The Journal of neuroscience : the official journal of the Society for Neuroscience 30:69446953.2010). Wang Z, Li V, Chan GC, Phan T, Nudelman AS, Xia Z, Storm DR (Adult type 3 adenylyl cyclase deficient mice are obese. PloS one 4:e6979.2009). Wang Z, Phan T, Storm DR (The type 3 adenylyl cyclase is required for novel object learning and extinction of contextual memory: role of cAMP signaling in primary cilia. The Journal of neuroscience : the official journal of the Society for Neuroscience 31:5557 5561.2011). Wei J, Wayman G, Storm DR (Phosphorylation and inhibition of type III adenylyl cyclase by calmodulindependent protein kinase II in vivo. The Journal of biological chemistry 271:2423124235.1996). Wei J, Zhao AZ, Chan GC, Baker LP, Impey S, Beavo JA, Storm DR (Phosphorylation and inhibition of olfactory adenylyl cyclase by CaM kinase II in Neurons: a mechanism for attenuation of olfactory signals. Neuron 21:495504.1998). Westra JW, Peterson SE, Yung YC, Mutoh T, Barral S Chun J (Aneuploid mosaicism in the developing and adult cerebellar cortex. The Journal of comparative neurology 507:19441951.2008). Wheatley DN (Cells with two cilia in the rat adenohypophysis. Journal of anatomy 101:479485.1967a).

PAGE 155

155 Wheatley DN (Cilia and centrioles of the rat adrenal cortex. Journal of anatomy 101:223237.1967b). White EL, Rock MP (Threedimensional aspects and synaptic relationships of a Golgi impregnated spiny stellate cell reconstructed from serial thin sections. J Neurocytol 9:615 636.1980). Whitfield JF (The neuronal primary cilium --an extrasynaptic signaling device. Cellular signalling 16:763767.2004). Willaredt MA, HasenpuschTheil K, Gardner HA, Kitanovic I, Hirschfeld Warneken VC, Gojak CP, Gorgas K, Bradford CL, Spatz J, Wolfl S, Theil T, Tucker KL (A Crucial Role for Primary Cilia in Cortical Morphogenesis. The Journal of neuroscience : the official journal of the Society for Neuroscience 28:1288712900.2008). Williams CL, Winkelbauer ME, Schafer JC, Michaud EJ, Yoder BK (Functional redundancy of the B9 proteins and nephrocystins in Caenorhabditis elegans ciliogenesis. Molecular biology of the cell 19:21542168.2008). Wonders CP, Anderson SA (The origin and specification of cortical interneurons. Nat Rev Neurosci 7:6876 96.2006). Wong ST, Trinh K, Hacker B, Chan GC, Lowe G, Gaggar A, Xia Z, Gold GH, Storm DR (Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice. Neuron 27:487497.2000). Wong SY, Seol AD, So PL, E rmilov AN, Bichakjian CK, Epstein EH, Jr., Dlugosz AA, Reiter JF (Primary cilia can both mediate and suppress Hedgehog pathway dependent tumorigenesis. Nat Med 15:10551061.2009). Xu H, He JH, Xiao ZD, Zhang QQ, Chen YQ, Zhou H, Qu LH (Liver enriched tran scription factors regulate microRNA 122 that targets CUTL1 during liver development. Hepatology 52:14311442.2010). Yoshimura K, Kawate T, Takeda S (Signaling through the primary cilium affects glial cell survival under a stressed environment. Glia 59:333 344.2011). Yu JZ, Dave RH, Allen JA, Sarma T, Rasenick MM (Cytosolic G{alpha}s acts as an intracellular messenger to increase microtubule dynamics and promote neurite outgrowth. The Journal of biological chemistry 284:1046210472.2009). Zhao C, Om ori Y, Brodowska K, Kovach P, Malicki J (Kinesin2 family in vertebrate ciliogenesis. Proceedings of the National Academy of Sciences of the United States of America 109:23882393.2012).

PAGE 156

156 Zimmermann L, Schwaller B (Monoclonal antibodies recognizing epitopes of calretinins: dependence on Ca2+ binding status and differences in antigen accessibility in colon cancer cells. Cell Calcium 31:1325.2002). Zou DJ, Chesler AT, Le Pichon CE, Kuznetsov A, Pei X, Hwang EL, Firestein S (Absence of adenylyl cyclase 3 per turbs peripheral olfactory projections in mice. The Journal of neuroscience : the official journal of the Society for Neuroscience 27:66756683.2007).

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157 BIOGRAPHICAL SKETCH Sarah Marie Guadiana was born Sarah Marie Parrish in 1983 to Parents Debbie and Michael Parrish. She was the youngest sibling on three girls and grew up in Indianapolis, Indiana for the first decade of her life until the family moved to the Chicagoland Area. Sarah graduated from Donald E. Gavit High School in 2001 and then began a sales job in San Antonio Texas from 2001 to 2005. She then relocated back to the Chicagoland area and began attending Purdue University. Sarah was the President and charter member of the Purdue Honors Program, assisting in the formation of the program during its f irst year. She graduated Summa c um Laude in 2008 with a Bac helor of Science degree in biology with a concentration in biotechnology. She was also named Student of the Year and Outstanding Student of the Year in Biology for 2008. In the fall of 2009, Sarah began attending University of Florida Interdisciplinary Graduate Program in biomedical s ciences with a concentration in Neuroscience and graduated in the summer of 2013. Sarah will continue her research training as a postdoctoral fellow at the Univer sity of Chicago in the lab of Dr. Elizabeth Grove.