1 EVOLUTION OF VERTEBRATE CARTILAGE DEVELOPMENT By GUANGJUN ZHANG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007
2 2007 GuangJun Zhang
3 To my wife, Li Lei, who provided constant support from China to the US, and to my parents for their encouragement to pursue my goals and dreams.
4 ACKNOWLEDGMENTS I greatly acknowledge the he lp and support of my adviso r, Marty Cohn, who introduced me to a new field, evolution and developmen t, and expanded my ways of thinking about scientific questions. I especially thank him fo r his patience and tolera nce during the whole study period. I really feel privileged to be able to work with Marty. I am also grateful to my supervisory committee (Mike Miyamoto, Brian Harf e, Paul Oh and Steve Phelps) for inspiring discussions and constant support. I especially acknowledge Mike for the time that he spent helping me to conduct the phylogenetic studies. W ithout his help, I would not have gotten this far on the molecular analysis. I would like to tha nk James Albert for his great help when I first got into the program. I also want to thank Rob Robins and Griffin Sheehy for the help to adapt myself to Gainesville daily life. I would also thanks Renata Freitas for technical help and stimulating discussion, and Ashley Seifert for manuscript reading. Many thanks go to Larry Page, Rob Robins Simone Shuster, Ben Olaivar, Gordon Weddle, Gretchen Walker, Nick Holland and Linda Holland for assistance with specimen collection. I would also thank P hyllis Luvalle, Keith Choe, Kelley Hyndman, Justin Havird and David Evans for sharing materials and equipment. Lastly I thank my wife Li Lei for her understanding and unselfish suppor t. I also thank my parents for their encouragement and continuous support.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 GENERAL INTRODUCTION..............................................................................................12 Vertebrate Cartilage Structures...............................................................................................1 2 Tetrapods...................................................................................................................... ...12 Teleosts....................................................................................................................... .....13 Chondrichthyans..............................................................................................................14 Agnathans...................................................................................................................... ..14 Invertebrates.................................................................................................................. ..15 Compositions of Vertebra te Cartilage Matrix........................................................................16 Collagens...................................................................................................................... ...16 Proteoglycans.................................................................................................................. 17 Evolutionary History of Cartilage..........................................................................................18 Vertebrate Skeletal Development...........................................................................................21 Embryonic Origins and Mesenchymal Condensation.....................................................21 Skeletal Cell Lineage Determination...............................................................................22 Chondrogenesis...............................................................................................................25 Non-hypertrophic chondrocyte diffe rentiation and proliferation.............................26 Chondrocyte proliferation and hypertrophy.............................................................27 Blood vessel invasion and os teoclast development..................................................35 Complexity of Skeletal De velopment, Secondary Cart ilage and Influences of Epigenetic Factors........................................................................................................36 Summary........................................................................................................................ .........37 2 LAMPREY TYPE II COLLAGEN AND SOX9 REVEAL AN ANCIENT ORIGIN OF THE VERTEBRATE COLLA GENOUS SKEELTON.........................................................43 Introduction................................................................................................................... ..........43 Materials and Methods.......................................................................................................... .45 Gene Cloning and Sequence Analyses............................................................................45 In Situ Hybridization.......................................................................................................47 Immunohistochemistry....................................................................................................48 Results........................................................................................................................ .............48 Lampreys have Two Col2a1 Orthologues.......................................................................48 Col2a1a and Col2a1b are Expressed during Lamprey Chondrogenesis.........................48 Adult Lamprey Cartilage C ontains COL2A1 Protein.....................................................50 Upstream of Col2a1 : a Lamprey Orthologue of Sox9 .....................................................50
6 Sox9 Expression Co-Localizes with Col2a1a and Cola1b in the Developing Skeleton....................................................................................................................... .51 Discussion..................................................................................................................... ..........51 Molecular Evolution of Lamprey Collagens...................................................................52 Evolution of Collagen-Based Cartilage...........................................................................53 3 HAGFISH AND LANCELET FIBRILLAR CO LLAGENS REVEAL THAT TYPE II COLLAGEN-BASED CART ILAGE EVOLVED IN STEM VERTEBRATES...................64 Introduction................................................................................................................... ..........64 Materials and Methods.......................................................................................................... .65 Animals........................................................................................................................ ....65 Gene Cloning................................................................................................................... 65 Sequence Analysis and Mo lecular Phylogenetics...........................................................65 Histology, Immunohistochemistry and In Situ Hybridization.........................................67 Results........................................................................................................................ .............67 Identification of Col2a1 in Hagfishes.............................................................................67 COL2A1 Localizes to Hagfish Soft Cartilage.................................................................68 AmphiColA is Expressed in the Lancelet Notochord and Neural Tube...........................70 Discussion..................................................................................................................... ..........71 COL2A1-Based Cartilage is a Shared Character of Crowngroup Vertebrates..............71 Did vertebrate Chondrocytes E volve from the Notochord?............................................72 Clade A fibrillar Collagen Duplication F acilitated Evolution of the Vertebrate Skeleton....................................................................................................................... .72 4 MOLECULAR IDENTIFICATION OF A SCLEROTOME IN LAMPREYS AND SHARKS: IMPLICATIONS FOR THE OR GIN OF THE VERTEBRAL COLUMN.........81 Introduction................................................................................................................... ..........81 Materials and Methods.......................................................................................................... .84 Fish Embryos................................................................................................................... 84 Gene Cloning and Phylogenetic Analysis.......................................................................85 In Situ Hybridization and Cryosections...........................................................................86 Results........................................................................................................................ .............87 Isolation and Analysis of La mprey and Catshark Genes.................................................87 Expression of Sclerotomal Markers in th e Somites of Lampreys and Catsharks............87 Shark Pax1 and lamprey Pax1/9 ..............................................................................87 Bapx1 ........................................................................................................................88 Shark FoxC1 FoxC2 and lamprey FoxC1/2 ............................................................89 Twist .........................................................................................................................89 Shark Scleraxis Paraxis and lamprey Parascleraxis ..............................................90 Shark TBX18 and lamprey Tbx15/18 .......................................................................91 Shark Gli2 and lamprey Gli1/2/3 .............................................................................91 Discussion..................................................................................................................... ..........91 Molecular Evidence for a Lamprey Sclerotome..............................................................92 Bapx1 ........................................................................................................................93 FoxC1/2 ....................................................................................................................94
7 Twist .........................................................................................................................95 Paraxis and Scleraxis ...............................................................................................95 Tbx15/18 ...................................................................................................................96 Gli1/2/3 .....................................................................................................................97 Embryonic Origin of Lamprey Arcualia.........................................................................98 The Evolution of Sclerotome and Vertebral Columns....................................................99 5 GENERAL DISCUSSION...................................................................................................112 Evolution of Collagen Genes in Chordates..........................................................................112 Regulatory Relationships of SoxE and ColA Genes May Have Been Established Before the Origin of Vertebrates...................................................................................................114 Collagenous Skeletons verses Non-Collagenous Skeletons.................................................115 Vertebrates.................................................................................................................... .115 Invertebrates.................................................................................................................. 117 Roles of Gene Duplications in Vertebrate Novelties............................................................118 Origin of Vertebrate Chondrocytes...............................................................................119 Origin of Vertebrate Axial Skeletons............................................................................120 LIST OF REFERENCES............................................................................................................. 122 BIOGRAPHICAL SKETCH.......................................................................................................155
8 LIST OF FIGURES Figure page 1-1. Distribution of verteb rate cartilage structures.......................................................................39 1-2. Overview of extracellu lar composition of cartilage..............................................................40 1-3. Distribution of cartila ge tissue within metazoa.....................................................................41 1-4. Vertebrate skeletal cell lineage determination.......................................................................42 2-1. Minimum evolution phylogeny for fibril A collagen proteins as obtained with JTT+ distances ( = 0.906).........................................................................................................55 2-2. Extended majority-rule consensus tree fo r the BP analysis of the fibril A collagen proteins....................................................................................................................... ........56 2-3. Col2a1a and Col2a1b expression during lamprey development...........................................57 2-4. COL2A1 protein is abundant in adult lamprey cartilage.......................................................58 2-5. Alignment of inferred amino acid sequenc e for lamprey SOX9 with chordate SOXE proteins....................................................................................................................... ........61 2-6. Extended majority-rule consensus tree fo r the BP analysis of the chordate SoxE proteins....................................................................................................................... ........62 2-7. Sox9 expression during lamprey development......................................................................63 3-1. Extended majority-rule consensus tree for the Bayesian phylogeneti c analysis of clade A fibrillar collagen proteins...............................................................................................74 3-2. Maximum likelihood phylogeny of fibrillar collagen A proteins, as obtained with WAG plus gamma distances........................................................................................................75 3-3. Minimum evolution phylogeny of fibrillar collagen A proteins as obtained with WAG plus gamma distances (a=0.906)........................................................................................76 3-4. COL2A1 in cranial and tail fi n cartilages of A tlantic hagfish...............................................77 3-5. Unrooted extended majority-rule consensu s tree for the Bayesian phylogenetic analysis of fibrillar A, B and C collagen proteins............................................................................78 3-6. Expression of AmphiColA during lancelet development.......................................................79 3-7. Origin of type II collagen-based cartilage.............................................................................80 4-1. Evolution and development of axial morphological structures...........................................101
9 4-2. Minimum evolution phylogenetic analysis of the predicted protein sequences of catshark and lamprey clones............................................................................................102 4-3. Maximum likelihood phylogenetic analysis of the predicted protein sequences of catshark and lamprey clones, as obtaine d with JTT plus gamma distances....................103 4-4. Bayesian phylogenetic analysis of the pr edicted protein sequences of catshark and lamprey clones, as obtained with WAG plus gamma distances......................................104 4-5. Group I Pax genes are expressed in the sclerotome of catshark.........................................105 4-6. Expression of Nk3.2 genes in catshark and lamprey...........................................................106 4-7. FoxC1 and FoxC2 expression patterns in catshark and lamprey.........................................107 4-8. Expression of catshark and lamprey Twist genes................................................................108 4-9. Scleraxis and Parascleraxis expression in catshark and lamprey.......................................109 4-10. Expression of Tbx18 in catshark and lamprey...................................................................110 4-11. Gli gene expression in catshark and lamprey....................................................................111
10 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EVOLUTION OF VERTEBRATE CARTILAGE DEVELOPMENT By GuangJun Zhang December 2007 Chair: Martin J. Cohn Major: Zoology The phylogenetic relationships of the verteb rates were established largely based on anatomical characters, particularly those of th e skeleton. Skeletal development has been well studied in higher vertebrates (e.g., mouse and chicken), however, little is known about the developmental mechanisms responsible for evolutiona ry origin of the vertebrate skeleton. Here I investigate cartilage development in two jawles s vertebrates, hagfishes and lampreys, and in a sister group to the vertebrates, lancelets (amphioxus). I show that both lampreys and hagfishes have type II collagen based cartilage, suggesting that this type of car tilage was present in common ancestor of all crow n-group vertebrates. My analysis of lancelets revealed the presence of an ancestral clade A fibrillar collagen ( ColA ) gene that is expresse d in the notochord. The results suggest that, during the chordate-vertebrate transition, an ancestral Clade A fibrillar collagen gene underwent duplication and diversif ication, and this process may underlie the evolutionary origin of ve rtebrate skeletal tissues. By comparing the axial skeletal developmenta l genetic program in lamprey and catshark, I found that the network of scleroto mal genes is conserved in mediovental part of somites. These results suggested that lamprey po ssesses a sclerotome. The conserva tion of this sclerotomal gene network, the later chondrogenetic program, a nd the structure of lamprey arcualia and
11 gnathostome vertebrae suggest that the arcualia develop from scle rotome. Phylogenetic analysis showed all of the axial skeletal genes examin ed here arose from gene duplication events. I propose that the sclerotome, like other vertebra te novelties may have resulted from the same gene duplication events. These duplications al lowed differentiation of developmental control genes, which facilitated both subfunctionalization a nd acquisition of new expression domains.
12 CHAPTER 1 GENERAL INTRODUCTION Vertebrate Cartilage Structures Cartilage as a skeletal tissue t ype is found mainly in the endo skeleton system. Cartilage is diversely distributed across anim al taxa, suggesting it may potentially have a long evolutionary history (Hall, 2005). Although carti lage has some unique characters compared with bone, such as its low metabolic rate and avascular structure, cartilage is defined by its components. Person and MathewsÂ’ definition includes th ree criteria of true cartilage; chondrocytes suspended in rigid matrix; high content of collagen and acidic polys accharides (Person and Mathews, 1967). More recently, this definition was modi fied by replacing collagen with fibrous protein, in order to accommodate the non-collagenous proteins found in lamprey and hagfish (Cole and Hall, 2004a). Tetrapods Mammalian cartilage has been studied extens ively. In terapods, there are three major kinds of cartilages according the physical characters and matrix components. Hyaline cartilage is the most widespread type in mammals. Hya line cartilage is named by its semi-transparent and bluish-white color appearances. It forms th e scaffold for the endochondral bones in early embryos. During later development, most cart ilages are replaced by bone during development, although some may remain as cart ilage throughout the whole lifes pan. In the adults, hyaline cartilages are found mostly in epiphyses, which cap the long bones at thei r proximal and distal ends. Other less common types of cartilage are fi brocartilage and elastic cartilage, which contain numerous thick bundles of collage n fibers and elastic fibers, re spectively (Hall, 2005). Similar with hyaline cartilage, elastic cartilage is stiff but is also more elastic than hyaline cartilage since its matrix contains more elastin protein fibrils. Elastic cartilages usuall y are distributed in the
13 pinna of the ear, larynx and epig lottis (Naumann et al., 2002). The third type of cartilage is fibrocartilage, which contains type I collagen in its matrix. The type I collagen makes the fibrocartilage more tensile and tough. Fibrocartila ge is usually found in th e intervertebral disks and in the joints with tendons or ligaments attached 1(Benjamin and Evans, 1990; Benjamin and Ralphs, 2004; Eyre and Wu, 1983). I wish to note that this cartilage cl assification system does not necessarily include all the carti lages in the vertebrate lineage. Teleosts Whereas in mammals three major cartilage types are found, teleosts have many more diverse types of cartilages. Most of the histological work on te leost cartilage types comes from one laboratory, and Benjamin and co-workers divide d teleost cartilages into at least eight types, most of which are absent in mammals (1989, 199 0). Hyaline-cell cartilage is composed of compact chromophobic chondrocytes and hyaline cyt oplasm with little matr ix. It was found in the lips, rostral folds and other cranial cartilage s (Benjamin, 1989). Hyalin e-cell cartilage can be further classified into the follo wing sub-types: fibrohyaline-cell ca rtilage with greater quantities of collagen; elastic hyali ne-cell cartilage with mo re elastin matrix; and lipohyaline-cell cartilage which contains adipose cells as well as chondrocytes (Benjamin, 1990). Zellknorpel chondrocytes are more chromophilic than those of hyaline-cell cartilage and are shrunken within the large lacunae. Zellk norpel is usually found in the gill filaments, basa l plate, etc (Benjamin, 1990). Elastic/cell-rich cartilage is a kind of cartilage with highly cellular elastic fibers, and the cells are not hyaline. This type of cartilage can be distinguis hed from Zellknorpel and hyalinecell cartilage by elastic staining. Elastic/cell-rich cart ilage is usually found at the barbels and maxillary oral valves, and is surrounded by a thick fibrous perichondrium (Benjamin, 1990). Cell-rich hyaline car tilage is a hyaline-like cartilage with more cells and lacunae that occupy more than half of the total volume. Parts of ne urocranium and MeckelÂ’s cartilage belong in this
14 category (Benjamin, 1990). Matrix-rich hyalin e cartilage is the typical mammalian hyaline cartilage type. It is common in gill arches a nd part of neurocranium (Benjamin, 1990). Scleral cartilage is a special cartilage, which is loca ted in the sclera. A lthough a COL2A1 antibody can label most of the fish matrix-ric h cartilages, cell-rich cartilages is not stained (Benjamin, 1991). Chondrichthyans The skeletons of chondrichthyans are entirely cartilaginous, alt hough it was reported that shark may retain some bone, and primitive chondrichthyans possessed both exoskeleton endochondral bones (Coates et al., 1998; Kemp and Westrin, 1979; Moss, 1970; Moss, 1977; Peignoux-Deville et al., 1982). One obvious character of shar k cartilage is that it can be calcified, and biochemical studies showed that sh ark and skate cartilage cont ains type I collagen in addition to type II collagen (Mizuta et al., 2003; Moss, 1977; Rama and Chandrakasan, 1984). Agnathans Cartilage structures are also present in th e only two extant jawless fishes, lamprey and hagfish. In the head of larval lamprey, the mucocar tilage occurs as a kind of temporal cartilage without blood vessel invasion and is surr ounded by perichondrium (Hall, 2005). During metamorphosis, the mucocartilage is transformed into pistal and tongue ca rtilages (Hall, 2005). In the19th century, two kinds of cartila ges were identified, soft and hard in adult lampreys. The hard cartilage is kind of like mammalian hya line cartilage (Parker, 1883). The lamprey cartilages are mainly found in the cranial regi on except the axial cartil age nodes named arcualia and caudal fin rays (Morrison et al., 2000). Similar with lampre y, hagfish cartilage is also mainly present in the cranium and median fin ra ys. Cole described two types of cartilages in hagfishes; Â“softÂ” cartilage contains large hypert rophic chondrocytes that stain with hematoxylin (blue) and are surrounded by a thin extracellular matrix, whereas Â“hardÂ” cartilage contains smaller chondrocytes that are surrounded by an abundance of extr acellular matrix (Cole, 1905).
15 Biochemical analysis also supported the two types of hagfish cartilages, which were named Type I and Type II cartilage, with Type II being more similar to adult lamprey cartilage (Wright et al., 1984). Although they have similar morphologica l structures, adult lamprey and hagfish cartilages were reported to lack collagen. Inst ead, lamprey and hagfish cartilage was argued to be composed of elastin-like molecules which were named lamprin and myxinin, respectively (Wright et al., 2001). Like chondrichthyans, lamprey cartilage can be calcif ied in vitro (Langille and Hall, 1988). Calcified cartilage also was reported in the fossil agnathan Euphanerops (Janvier and Arsenault, 2002). Invertebrates True cartilage is usually assumed to be found only in the verteb rate lineage, although cartilage-like tissues (c hondoid or chondroid) were also iden tified in invertebrates (Wright, 2001 and Cole, 2004). Morphologically, some invert ebrate cartilages are i ndistinguishable from vertebrate cartilage. Thus far, cartilage tissues were identified in Cnidaria ( Metridium ); Molluska ( Sepia ), Arthropoda ( Limulus ), Brachiopoda ( Terebratulina ), Polychaeta ( Potamilla ), Hemichordata (Saccoglossus), Urochord ata (Styella), and Cephalochordata ( Branchiostoma ) (Cole and Hall, 2004a; Cole and Hall, 2004b; Wright et al., 2001). In general, there are three kinds of cartilages found in inverteb rates; central cell-rich cartilage ; vesicular cartilage with large vesicles or vacuoles; and acelluar cartilage. The cartilage-like tissues cross reacted with vertebrate type II and X collage n and proteoglycan antibodies (Cole and Hall, 2004a; Cole and Hall, 2004b). It was also reported that squid cartil age collagens were type V like instead of type II, although the details of inve rtebrate collagen types were not known (Sivakumar 1998 and Bairati 1999). Acelluar cartilage may have evolved on seve ral independent occasions, since it occurs in different lineages in metazoa (Junqueir a et al., 1983). Acelluar cartilage was also found in lancelets, however lan celet cartilage was thought to be non-collagenous due to its
16 incomplete digestion by cyanogen bromide, a chem ical that digests collag en protein (Wright et al., 2001). In general, cartilages show extensiv e variation across different taxa, and I summarize the major chordate cartilage structures in the Figure 1-1. Compositions of Vertebrate Cartilage Matrix Most of the supportive tissues of vertebrates are formed fr om extracellular fibers and matrix, both of which are produced by the cells of various connective tissues. Up to 90% of the dry weight of cartilage is extracel lular matrix, indicating that matrix proteins are particularly rich in cartilage (Hardingham and Fosang, 1992). In jawe d vertebrates, cartilage extracellular matrix is composed of mucopolysaccharides (proteogly cans) deposited on collagen fibers, mainly COL2A1 and the less abundant Â“minorÂ” collagens IX and XI (Bruckner and van der Rest, 1994). A diagram of cartilage matrix composition is illustrated in Figure 1-2, which is adapted from Knudson and Knudson (Knudson and Knudson, 2001). Collagens Collagens are the main components of animal extracellular matrix (Expositio JY, 2002). Thus far, 27 t collagen genes have been identifi ed (Pace et al., 2003). The collagens are divisible into two major groups, fibril and non -fibril collagens. The fibril collagens are further divisile into clade A, B and C (Aouacheria, A. et al. 2004 ). Clade A fibrillar collagens are the major fibril-forming collagen, including type, I, II, III and V (Aouacheria, A. et al. 2004). Clade A fibril procollagens are made of N-propeptide, N-telopeptide, triple helix, C-telopeptide, Cpropetide from N to C terminus. Triple helix is the collagenous domain consisting of Gly-XaaYaa triplet repeat. X and Y usually are prol ine and hydroproline. The propeptide will be removed during the maturation of collagen th rough posttranscriptional process by Nand Cproteinase (Ladler, KE, 1996; Expositio JY, 2002). Type II collagen is encoded by Col2a1 gene and was found to the major matrix protein in carti lage at the end of 1960s (Miller 1969). Each
17 type II collagen fibril is made of three identica l chains, conferring tensile strength and providing a scaffolding network for proteoglycans (van de r Rest and Garrone, 1991). As minor collagens in cartilage, type IX and XI colla gens belong to the clade B fibril lar collagen. They participate in the process of fibril formation (Eyre et al., 2004; Kadler et al., 1996; Li et al., 1995). The collagen types and quantities are different in di fferent types of cartila ge. The fibrocartilage contains certain amount of type I collagen (Benjamin and Evans, 1990) During the long bone development, there is also a collagen type tr ansition. In the prolifera tive cartilage, the major matrix is type II collagen, the hypertrophic cartil age major collagen type is type X collagen, and type I collagen is the major matr ix of bone (Olsen et al., 2000). Proteoglycans Proteoglycans are the second-most abundant pr oteins after the fibr illar collagens in cartilage matrix. Chondroitin sulfate ha s long been known to be the predominant glycosaminoglycan in cartilage, and aggrecan was found to be the most abundant proteoglycan in cartilage (Doege et al., 1991). Deposition of ag grecan is thought a hallmark of chondrogenesis, although it is also present in aort a, disks and tendons (Schwartz et al., 1999). Aggrecan not only contributes to the physical char acters of cartilage, it also prot ects cartilage collagen from degradation (Pratta et al., 2003). In addition to aggrecan, there are many small leucine-rich proteoglycans in cartilage, in cluding biglycan, decorin, fibr omodulin, lumican, epiphycan and perlecan. These small leucine-rich proteoglyc ans have a variety of functions in cartilage development and maintenance (Iozzo, 1998; Knudson and Knudson, 2001). Chondrocytes also express cell surface proteoglycans: syndecans an d glypican which may have the potential to interact with FGF and TGFsignals in cellÂ–cell and cellÂ–ma trix interactions (Iozzo, 1998; Song et al., 2007). In addition to th e proteoglycans and collagen protei ns, elastic cart ilage contains
18 elastin proteins. Recently, more minor proteins were identified in human articular cartilage by proteomic analyses, especially the posttranscript ional modification protei ns. (Belluoccio et al., 2006; Lammi et al., 2006). Evolutionary History of Cartilage As I described above, the cartilage structures ar e widely distributed in metazoan taxa and show great diversity. The dominan ce of cartilage in vertebrate sk eletons has led to the idea that cartilage is vertebrate-specific character. The existence of invertebrate cartilage suggested this specific tissue appeared before vertebrates and ha s a more ancient evolutionary history (Cole and Hall, 2004a). It has long been debated whether bone or cartilage occurred first, although the earliest vertebrate skeletons are believed to be unmineralized car tilaginous splanchnic noncranial endoskeletons (Donoghue and Sansom, 2002; Donoghue et al., 2006). Obviously, cartilage has a longer history than bone if we take into consideration of inverteb rate cartilage, whereas bone and dental skeletons are limited onl y to the vertebrate lineage. In higher vertebrates, there are some intermed iate skeletal types (H all, 2005). Chondroid tissues are cartilage-like and possess both characters of bone and cartilage (Cole and Hall, 2004a). Squid cartilage is very similar to mammalian hyaline cartilage morphologically, but there are some desmosomes and other direct cellcell connections in squid cartilage cells, while such cell-cell connections are not found among vertebrate chondrocytes (although they are in osteocytes) (Bairati et al., 1999) These combined characters suggest an ancestral state of vertebrate skeleton, and raise the possibility that the vertebrate skeleton may have evolved from invertebrate cartilage. In their comprehensive examination of invert ebrate cartilage distri butions, Cole and Hall proposed that all the metazoan animals may poten tially have the ability to make cartilage-like tissues, and some taxa may have lost this ab ility during evolution (Cole and Hall, 2004a).
19 Depending on relative amounts of cells and extracellular matr ix, there are generally four kinds of cartilage in vertebrates and inve rtebrates; matrix-rich cartilage, cell-rich cartilage, vesicular cartilage and acellular cartilage. Vesicular cartilage was proposed first by Benjamin as a special cell-rich cartilage with large ve sicles or vacuoles with chondr ocytes (Benjamin, 1990; Cole and Hall, 2004b). Polychaetes, horseshoe crabs and mollu sks have been reported to possess vesicular cartilage. To some degree, the vertebrate notoch ord is a kind of vesicu lar cell-rich cartilage, since notochordal cells are vacuolated and su rrounded by cartilage sp ectrum of extracellular matrix incuding type II collage, type I collagen, type X collage n, aggrecan, and polysaccharides (Domowicz et al., 1995; Eikenberr y et al., 1984; Linsenmayer et al., 1986; Welsch et al., 1991). Interestingly, the notochord is usually thought to be an epithelial structure instead of cartilaginous. It has been proposed that vertebrate cartila ge may has evolved from the notochord based on the evidence that matrix similarity and notoc hord predate the vertebrate cartilage (Stemple, 2005). Acellular cartilage is anothe r interesting type of cartilage with no chondrocytes. It is has been found in hemichordates, cephalochordates and vertebrates (stingray ) (Cole and Hall, 2004b; Rychel et al., 2006). Epithelial cells may secrete the matrix since there are no chondrocytes. Rychel has proposed that ect odermal acellular cartilage is an ancestral model in making pharyngeal cartilage in deuteros tomes (Rychel et al., 2006; Rychel and Swalla, 200 7). Cell-rich, matrix-rich or acellular cartilage may have evol ved independently or from a single type of connective tissue. The sequence of occurrence of these four cartilage types in a phylogenetic context is important for understanding the orig in and evolution of cartilage. The detailed distribution of cartilage in metazoa is illustrated in Figure 1-3. It seems that vertebrate cartilage may have two possible origins; notochord and/or ectoderm.
20 Cartilage is a kind of connective tissue. C onnective tissues are widely distributed in metazoan animals. At the most basic level, there are two groups of extracellular molecules, ground substances (glycosaminoglycans, proteoglycan s) and fibrous proteins (collagen fibrils) in all kinds of connective tissues. In histological sections, the verteb rate connective tissues can be distinguished by relative quantities of ground and fi bril molecules and orientation of fibrils in addition cell types (Cole and Hall, 2004a). Like the hyaline cartilage matrix is dominated by type II collage fibril, while the fi brocartilage contains more type I collagen. The elastic cartilage contains elastin fibrils in addition to type II co llagen fibril. The hypertr ophic cartilage mainly contains type X collagen. Bone and tendon contain ma inly type I collagen, but the former is also composed of osteonectin, osteopotin and other uni que proteins. Although none of the collagen types are expressed exclusively in certain connective tissues, they are still widely used as molecular indicators. Due to th e uniqueness of extrac ellular matrix types, the evolutionary history of each connectiv e tissue will be well reflected by the natural history matrix molecules. The current view on the evolution of vertebrate cartilage matrix contrasts with the idea of an invertebrate origin. The gnathostomes, fr om sharks to mammals, express the same major matrix, COL2A1, in their chondrocytes (Wright et al., 2001). The agnathans, hagfishes and lampreys have been reported to possess the noncollagenous cartilage. Th eir cartilage matrix was said to be composed by elastin-like molecu les, named lamprin and myxinin respectively. The lancelets were thought to be non-collagenous due to the insolubility of their cartilage in cyanogen bromide. These differences in matrix structure suggested th at the ancestor of vertebrates is non-collagenous and that agnath ans and gnathostomes evolved their cartilages independently (Cole and Hall, 2004a; Wright et al., 2001). The similar non-collagenous biochemical matrixes of their skeleton provi de some unifying char acters, since putative
21 homologies between the endoskeleton elements re main dubious and made it difficult to ascertain the gross structure of vertebrate skeletons (Donoghue and Sansom, 2002; Donoghue et al., 2006). The controversy of agnathan and gnathostome carti lage matrix composition raised the possibility that vertebrate cartilage has a dual origin, e ither non-collagen-based or collagen-based. Although it is difficult to sort out the vertebrate cartilage matrix origin s, there is still some evidence that squid and cuttlefis h cartilage contain some collage n, although they are different than type II collagen (Bairati et al., 1999; Bairati and Gioria, 2004; Kimura and Karasawa, 1985; Kimura and Matsuura, 1974). In addition, horseshoe crab cartilage was shown to contain chondroitin sulphate (Sugahara et al., 1996). The cartilage matrix genes in invertebrates suggested that the chondrogeneti c genetic program might be deeply conserved. So, further investigation of agnathan cartila ge matrix will shed light on the origin of vertebrate cartilage. Vertebrate Skeletal Development Embryonic Origins and Mesenchymal Condensation Vertebrate skeletons mainly came from diffe rent three embryonic lineages. The cranial neural crest cells contribute mo st of the head skeleton, which is also derived form cranial mesoderm and the first five somites (Couly et al ., 1993); axial skeletons are made from paraxial mesoderm; appendicular skeletons are formed by lateral plate mesoderm. However, not all the vertebrate skeletons are composed by these three lineages. Especially in teleosts, and early fossil vertebrates, even in some tetrapod, like arma dillo, the dermal skeletons are derived from dermomyotome or ectoderm (Vickaryous and Hall, 2006). No matter what the cell lineages, mesenchymal cells undergo condensati on as a characteristic early step towards skeletogenesis. Skeletogenesis occurs in four steps; cells migrate to the right sites, epithelialmesenchymal tissue interactions occur, mesenchy mal condensations form and cells differentiate into chondroblasts and osteoblas ts (Hall and Miyake, 2000). Cells committed to skeletal fate
22 have no obvious morphological characters prior to condensation, when the cells begin to aggregate together, reduce their sizes and appe ar rounded with higher density once condensation begins (Wezeman, 1998). By regulating prolifera tive cell rate through grow th factor signalling, extracellular matrix is necessary for the condensation (Streuli et al ., 1993). It was reported Type I collagen is expressed in these mesenchymal cells, but is downregulated once condensation begins. At this stage, type II collagen became the major fibrillar protein and type I collagen persists only in the perichondrium (Dessau et al., 1980; Wezeman, 1998). Mesenchymal condensation is also characterized by cell surface adhesion molecules, like N-cadherin N-CAM, Hyaladherin Versican Tenascin Sydecan Heparin Sulphate and Chondroitin Sulphate Proteoglycans (Hall and Miyake, 2000). The sequence of matrix molecule expression is well summarized by Shum at al (Shum et al., 2003). These adhesive molecules have important functions in addition to mediating the condensation process. For example, N-Cadherin has been shown to recruit cells into c ondensations and disruption of N-cadherin led to condensation inhibition (DeLise and Tuan, 2002; Haas and Tuan, 1999). Syndecan-3 and Tenascin-C were shown to set boundaries and are involved in pe riosteum development (Koyama et al., 1995). Most of these adhesive markers are downregulat ed at the commencement of overt differentiation (Wezeman, 1998). Skeletal Cell Line age Determination Vertebrate cartilage and bone are primarily composed of th ree cell lineages, chondrocytes, osteoblasts and osteoclasts. The former two cell types are derived from the common mesenchymal progenitor cell condensation, wh ereas the osteoclasts are of blood-borne hematopoietic origin. After condensation, mese nchymal cells start to differentate into chondrocytes. These chondrocytes may remain as cartilage throughout life, or the cartilage templates may undergo hypertrophy and eventually be replaced by bone. This process is called
23 endochondral ossification. Alternatively, the mese nchymal cells may form bone directly in a process named intramembranous ossification, which is usually seen in cran ial skeletons. In both ossification types -intram embranous and endochondral -osteoblasts aggregate as mesenchymal condensations. (Karsenty and Wagn er, 2002; Yang and Karsenty, 2002; Zelzer and Olsen, 2003). Several lines of evidence have suggested th at the condensed mesenc hymal cells have both chondrogenic and osteogenic pot ential, since they express both the chondrogenesis and osteogenesis master genes, Sox9 and Runx2 (Bi et al., 1999; Ducy et al., 1997; Otto et al., 1997; Yamashiro et al., 2004). Moreover, cultured em bryonic calvarial cells may form both bone and cartilage (Fang and Hall, 1997; Toma et al., 1997; Wong and Tuan, 1995). Inactivation of Sox9 in the cranial neural crest-derived mesenchymal cells block the cartilage differentiation, but this also leads to ectopic expression of osteoblast specific genes such as Runx2 Osterix and Col1a1 (Mori-Akiyama et al., 2003). Conversely, it was reported that in Oste rix mutants, ectopic chondrocytes formed at the expense of the bone collar in long bones and in some intramembrane bones (Nakashima et al., 2002). These data s upport the idea that the common mesenchymal progenitors have three possible differentia tion fates, chondrogenesis, intramembranous ossification or endochon dral ossification. The mesenchymal cells also can take other cell fates like ad ipose tissues (Karsenty, 2003; Karsenty and Wagner, 2002). Only recently have the molecular mechanisms underlying these different pathways become clear. The canonical WNT pathway was shown to be a key regulator for this mesenchymal cell lineage determination. Wnt genes are the vertebrate orthologs of fruit flyÂ’s wingless gene, and there are at least 19 Wnt genes in humans (Logan and Nusse, 2004; Miller, 2002). This group of s ecreted molecules is highly conser ved in metazoan animals from
24 cnidarians to humans, and play major roles in normal development and tumorigenesis (Kusserow et al., 2005; Lee et al., 2006; Logan and Nusse, 2004; Prud'homme et al., 2002). WNT proteins bind to their receptor, Frizzed, and transduce the input into the cell with the co-receptor, named LDL Receptor related Protein 5/6 (LRP5/6). There are at least three intracellular pathways, the canonical pathway, which is mediated by -Cate nin, Ca-PKC pathway and planar cell polarity pathway (Miller, 2002). Canonical Wn t signals previously were s hown to be involved in the skeletal development (Bodine et al., 2004; Boyden et al., 2002; Gong et al., 2001; Hartmann and Tabin, 2001; Kato et al., 2002; Lit tle et al., 2002; Rawadi et al., 2003). Recently several critical lines of evidence revealed that the WNT canonica l pathway regulates cell fate determination through a cell-autonomous mechanism to indu ce osteoblast differentiation and repress chondrocyte differentiation (Day et al., 2005; Glass et al., 2005; Hill et al., 2005; Hu et al., 2005; Rodda and McMahon, 2006). When -Catenin is conditionally removed in early mesenchyme (skeletal progenitors) using Prx-Cre mouse line, osteoblast differe ntiation was arrested and no cortical and intramembrane bones were formed, however this could be rescued by IHH and BMP2. In the mutants, periosteal cells turned into chondroc ytes instead of osteoblasts, and some ectopic cartilages were found in the membranous bone region (Hill et al., 2005). Similar phenotypes were found when -Catenin was deleted from skel etal primordium (mesenchymal condensation) using Dermo1 -Cre and Col2a1 -Cre mouse lines (Day et al., 2005). The ectopic chondrocytes formed at the expense of osteoblasts (Day et al., 2005; Hu et al., 2005) Moreover, micromass cell culture experiments showed that -Catenin levels can control the Sox9 and Runx2 expression in vitro (Day et al., 2005). Collectively, Catenin controls early osteo-chondroprogenitor differentiation into chondrocytic or osteoblasti c lineages. High level of -Catenin lead to
25 osteogenic differentiation and low level lead to chondrogenic differentiation (Day et al., 2005; Hill et al., 2005). The process is summarized in the Figure 1-4. This mechanism is not only important developmentally to designate skeletal types, but also is important for skeletal evolution. It seems that vertebra te skeletal variation can be ac hieved through tinkering with the temporal and spatial expression of canonical WNT signals. Both Sox9 and Runx2 are expressed in mesenchymal osteo-chondrogenic progenitors; removal of Sox9 will abolish bone and cartilage formation. This suggested that Sox9 is required for their overt skeletal differentiation (Akiyama et al., 2005). Very recently, Sox9 was found to be dominant to Runx2 (Zhou et al., 2006), this finding leads us to assume that skeletal progenitor cells will automatically differentiate into cartilage if there are no other transcriptional factors to upregulate Runx2 In chicken embryos, it was shown that high levels of Sox9 and Runx2 commit the cells to chondrogenesis and osteogenesis, resp ectively (Eames et al., 2004). The cross talk between Sox9 and -Catenin was proven recently. Sox9 can inhibit -Catenin-dependent promoter activation through the interaction between HMG-box and Armadillo repeats. Sox9 also promotes degradation of -Catenin by ubiquitatio n or the proteasome pathway (Akiyama et al., 2004). Naturally, we expect more evidence to en sure about the interac tions between -Catenin and RUNX2. Although Wnt and Sox9 were clearly shown to play major roles in the cell fate determination process, it does not exclude other possibilities that may be found in the future. Chondrogenesis Embryonic cartilage has at least two potential fa tes. A part of cartilage remains permanent cartilage, like the articular cart ilage. Most cartilages eventu ally are replaced by bone through endochondral ossification process. Although there are three types of cartilage, as mentioned at the beginning of this disserta tion, only hyaline cartilage was we ll characterized, especially in limb development. Chondrocytes are not simp ly a single cell population, but as suggested by
26 Karsenty and Wagner, there are three subpopul ation cells; non-hypertrophic chondrocytes, hypertrophic chondrocytes and articular chond rocytes, and each cell type has a unique morphology, gene expression profile and appears se quentially at certain developmental stages (Karsenty and Wagner, 2002). Non-hypertrophic chondrocyte di fferentiation and proliferation After overt differentiation, th e cells in mesenchymal condensation then develop into chondrocytes, which is marked by Type II collagen and aggrecan as major matrix proteins and type IX and XI collagens as minor matrix proteins. The master tr anscription factor that regulates this process is Sox9, one of the vertebrate S oxE family members, which contains a highmobility-group (HMG)-b ox DNA binding domain. Sox9 was the earliest marker of chondrogenesis as it starts to express during the mesenchymal condensation (Healy et al., 1996; Wright et al., 1995). Sox9 is required for cartilage-specific extracellular matrix component such as COL2A1, COL9, COL11 and AGGRECAN, and wa s proved to be the di rect regulator of Col2a1 and aggrecan in mice and its major role s was chondrocyte differentiation (Lefebvre and de Crombrugghe, 1998; Lefebvre et al., 1997; Liu et al., 2000; Ng et al., 1997; Zhang et al., 2003; Zhou et al., 1998). Haploinsufficiency of Sox9 in human and mice will cause campomelic dysplasia (CD) (Foster et al., 1994; Wagner et al., 1994). Sox9 homozygous knockout mice died prior to birth and Sox9 heterozygous mutants exhibited se vere cartilage hypoplasia. The cartilage matrix maker genes were downregulated in the mutants too. Furthermore, the Sox9 null embryonic stem cells could not form cartilage in teratomas (Bi et al., 1999; Bi et al., 2001). Conversely, ectopically expressed Sox9 in chick using RCAS retrovir al vector induced cartilage formation in dermomyotome (Healy et al., 1999 ). Detailed analys is revealed that Sox9 did not work by itself, and that its normal function duri ng overt chondrocyte diffe rentiation requires two important cofactors, Sox5 and Sox6 both members of the SoxD group (Lefebvre et al., 1998).
27 These two SoxD genes modulate the function of Sox9 (Stolt et al., 2006). Collectively, the Sox trio ( Sox9 Sox5 and Sox6 ) is a very important regulator fo r chondrocyte differentiation (Ikeda et al., 2004; Lefebvre et al., 2001; Lefebvre and de Crombrugghe, 1998; Lefebvre et al., 1998; Smits and Lefebvre, 2003; Stolt et al., 2006). The targets of Sox trio was unclear until recently, when two targets, Sox100A1 and Sox100B were discovered (Saito et al., 2007). In addition to Sox5 and Sox6 there are some other Sox9 cofactors, like CBP/P300 TRAP230 and PGC-1a, which are important for the functions of Sox9 (Kawakami et al., 2005; Liu et al., 2007; Rau et al., 2006; Zhou et al., 2002). Given the importance of Sox9 its temporal and spatial regulation is very important. Unfortunately, few gene s have been identified that regulate Sox9, although there are some genome-wide analyses targeted this direction (Bagheri-Fam et al., 2006; Bagheri-Fam et al., 2001). BMP, TGF- and -Catenin are imp licated in this process (Akiyama et al., 2004; Bagheri-Fam et al., 2006; Chimal-Monroy et al ., 2003; Yoon et al., 2005; Zou et al., 1997). Chondrocyte proliferation and hypertrophy Cartilage anlagens have two pot ential pathways for differentiation; they can remain as permanent cartilage, in which case the cells ne ver express IHH and BMP6, or they can undergo endochondral ossification (Eames and Helms, 2004) During the endochondral ossification, the specified chondrocytes in the cente r of skeletal anlagen proliferat e and develop successively into prehypertrophic, hypertrophic and terminal hypertr ophic chondrocytes. The chondrocytes were surrounded by perichondrium, which contains flatte ned fibroblastic cells. The perichondrium is also influenced by hypertrophy of the underlying chondrocytes, a nd the fibroblastic cells will differentiate into osteoblasts, wh ich are involved in bone collar formation (Olsen et al., 2000; Provot and Schipani, 2005). The prehypert rophic and hypertrophic cell subpopulations are marked by Ihh and BMP6, or type X collagen and VEGF, respectively. Interestingly, the terminal hypertrophic chondrocytes do express bone markers, like Bsp Osteocalcin Osteonectin
28 Osteopotin and AlkalinePhosphatase (Eames and Helms, 2004; Go ldring et al., 2006; Karsenty and Wagner, 2002). Runx2 is a Runx domain containing gene that is also named PEBP2A (polyoma enhancer binding protein), Osf2 (osteoblast spec ific factor 2), AML3 (acute myelogenous leukemia 3) and Bcfa1 (core-binding factor) (van Wijnen et al., 2004). It was shown to control chondrocyte maturation in addition to osteoblast differentiatio n (Ducy et al., 1999; Ducy et al., 1997; Komori et al., 1997; Otto et al., 1997). Cleidocranial dysplasia (CCD) is a ra re skeletal dysplasia characterized by short stature, distinctive facial features and narrow, sloping shoulders caused by defective or absent collarbones (cla vicles). Haploinsufficiency of Runx2 causes CCD in human and mice (Mundlos et al., 1996; Mundlos a nd Olsen, 1997a; Mundlos and Olsen, 1997b), In Runx2 null mice, the entire skeleton is cartilaginous due to the matu rational arrest of osteoblasts, and there is a complete loss of chondrocyte hypertrophy in most of these skeletons (Inada et al., 1999; Kim et al., 1999; Takeda et al., 2001). More interestingly, either Runx2 was conditionally over-expressed or knocked out fr om chondrocytes using transgenic mice, the chondrocytes maturation rates were changed (Ueta et al ., 2001). There are th ree closely related Run x genes in mouse, Runx1 Runx2 and Runx3 and all the three genes ar e expressed in chondrocytes, suggesting that Runx1 and Runx3 are also involved in the hype rtrophy and maturation (Levanon et al., 2001; Lian et al., 2003; Sm ith et al., 2005; Stricker et al., 2002; Wang et al., 2005). In fact, this was proved by Runx1 and Runx2 double knockout experiments, in which the chondrocyte hypertrophy was inhibited more comple tely (Yoshida et al., 2004). Collectively, these observations suggested that Runx2 is an essential transcription factor for chondrocyte maturation, and that Runx1 and Runx3 are also important and may have distinct roles. In contrast to the function of Runx2 in chondrocytes, Runx2 in perichondrium was shown to inhibit
29 chondrocyte proliferation and hype rtrophy during later stages of chondrogenesis (Hinoi et al., 2006). These results suggest that Runx2 has complex functions during chondrogenesis. It will be very interesting to know the ta rgets, cofactors and regulators of Runx2 One good example is histone deacetylase 4 ( HDAC4 ), which regulate chondrocyte hype rtrophy through its interaction with Runx2 as a corepressor (Vega et al., 2004). HDAC4 normally is expressed in the prehypertrophic zone. In the null mutant mi ce, ectopic and prematur e chondrocyte hypertrophy was observed. Over expression of HDAC4 led the opposite phenotypes (Vega et al., 2004). The chondrocyte proliferation and differentia tion is tightly regulated by IHH and PTHrP negative loop (Karp et al., 2000; Lanske et al., 19 96; St-Jacques et al., 1999; Vortkamp et al., 1996). The PTHrP is a peptide hormone that is s ecreted by the most distal perichondrium. Its Gprotein coupled receptors are located at the preh ypertrophic zone, which the cells proliferate. Normally, PTHrP prevents the c hondrocytes from exiting the prol iferative cell cycle, then inhibits hypertrophy (St-Jacques et al., 1999). Ectopic expressi on of PTHrP or its receptor severely inhibits cartilage maturation (Schip ani et al., 1997; Weir et al., 1996). Moreover, mutations of PTHrP and its receptor cause JansenÂ’s chondrodysplasia, in which the patients have reduced skeletal growth and abnormal metaphases (Schipani et al., 1995). The mutant mice had phenotypes similar to the human disease, and exhi bit long bone dwarfism (Karaplis et al., 1994; Lanske et al., 1996). Ihh is a member of verteb rate hedgehog family genes, and localizes at the prehypertrophic zone, along with the PTHrP recep tor. Mice without Ihh in their skeletons exhibited severe dwarfism due to proliferation reducti on and osteoblast absence. This suggested that Ihh plays a major role in chondrocyte maturation (Vortkamp A, et al 1996). In the IHH mutants, PTHrP expression was lost (Razza que et al., 2005; St-Jacques et al., 1999). Conversely, Ihh over-expression data showed that IHH can induce expression of PTHrP in distal
30 chondrocytes, thereby promoting the proliferatio n and antagonizing hypertrophy. As a result of the sustained proliferati on and delayed maturation, Ihh expression is reduced. Collectively, the IHH-PTHrP feedback loop is necessary fo r proliferation and differentiation. Since Runx2 and the IHH-PTHrP loop are both involved in the regu lation of chondrocyte hypertrophy, it is not a surprise that Runx2 and Runx3 was shown to induce Ihh expression (St-Jacques et al., 1999; Yoshida et al., 2004). It is interesting that Ihh was also reported to inhibit Runx2 expression through the PKA signaling pathway (Iwamoto et al., 2003; Li et al., 2004). Very recently, it was reported that Bapx1 ( Nk3.2 ) is a downstream target of IHHPTHrP loop and, at least in part, mediates chondrocyte hypertr ophy (Provot et al., 2006). Fgfs and their receptors are also critical regulators of chondrocyte proliferation and differentiation. In humans and mice there are 22 Fgf genes and 4 FGF receptors ( Fgfr ) many of which are involved in the sk eletal genesis, includi ng those that signal through Fgfr1 Fgfr2 and Fgfr3 (Ornitz and Marie, 2002). Fgf9 has been show n to regulate differentiation of hypertrophic chondrocytes and to direct vascularizati on of the limb skeleton (Hung et al, 2007). Fgf18 is expressed in the perichondrium, and it si gnals to the chondroc ytes through Fgfr3. Fgfr1 is found in prehypertrophic and hypertrophic zone, Fgfr2 and Fgfr3 are expressed in perichondrial cells and the proliferating zone, respectively. Each of the three receptors has a unique function. Fgfr3 mutation will cause achondropl asia, hypochondroplasia and thanat ophoric dysplasia in humans (Olsen et al., 2000). The chondrocyte column length was increased in Fgfr3 null mice and this is caused by an increase in the pr oliferative rate (Colvin et al ., 1996; Deng et al., 1996). This suggested that FGFR3 is a negati ve regulator of proliferation in the growth plate, and this process is mediated through STAT1-P21 pathwa y (Sahni et al., 1999). Moreover, if FGFR3 signaling is increased, there will be more apoptosis and a reduc tion of chondrocyte proliferation
31 (Sahni et al., 1999). As with Fgfr3 conditional removal of Fgfr1 using Col2 -Cre and Col1 -Cre in mice results in the hypertr ophic chondrocyte zone being expa nded (Jacob et al., 2006). This suggested Fgfr1 is also a negative regul ator of proliferation. In contrast to Fgfr1 and Fgfr3 Fgfr2 and Fgfr4 were positive regulators of skeletal development (Eswarakumar et al., 2002; Weinstein et al., 1998; Yu et al., 2003). Another group of important chondrocyte regul ators are the BMPs and their receptors, which play multiple roles in chondrocyte diffe rentiation and prolifer ation (Zou et al., 1997). Bmp7 is mainly found in the prolif erating chondrocytes, whereas BMP2 3 4 and 5 are primarily expressed in the perichondrium (Lyons et al., 1995; Minina et al., 2001). Bmp2 and Bmp6 are expressed in hypertrophic chondr ocytes (Solloway et al., 1998) These distinctive expression patterns suggest that each of these Bmps has a unique function. Manipulating the BMP antagonist Noggin showed that BMP2 has anti-prolif eration roles in the gr owth plate. In Noggin over-expressing experiment, the BMP signal was attenuated and chondroc yte proliferation was reduced (De Luca et al., 2001; Mi nina et al., 2001). Conversel y, skeletons overgrew in the Noggin null mice (Brunet et al., 1998). Bmp also was proven to be able to increase Collagen X promoter activity (Volk et al., 1998), thus Bm ps may also be invol ved in the chondrocyte differentiation. Bmp receptor IA was shown to work downstream of IHH and regulate chondrocyte differentiation, while Bmp receptor IB was related to cell death (Zou et al., 1997). Recently, there is further evidence that Bmps promote chondrocyte differentiation in vivo. Kobayashi et. al. investigated the roles of Bmp receptor IA during chondrocyte development by overexpressing it using Col2a1 -Promoter driven and UAS-Gal binary systems. In the overexpression mice, long bones were shortened and, in the growth plate, the proliferating chondrocytes in the columnar layer we re reduced (Kobayashi et al., 2005).
32 The BMP signal, FGF signal, IHH-PTHrP signa ls and the transcri ption factors Runx2, Runx3 and Sox trio do not function independently. In fact, they have to work as a network. There is increasing evidence that links the functions of these genes together. Runx2 was upregulated and Sox9 was downregulated in hypertrophic chondrocytes, suggesting that Sox9 plays a negative role in hypert rophic maturation (Eames, BF 2003) Bmps can interact with IHH-PTHrP loop by promoting Ihh expression, and in turn, Ihh ma intains Bmp expression level (Minina et al., 2001; Pathi et al., 1999). In ge neral, Fgf pathways have opposite functions to Bmp pathways (Minina et al., 2002). Thus, the balance of BMP and FGF signals are important regulators of chondrocyte proliferation (Ornitz 2005). Osteogenesis As discussed above, mesenchymal cells are committed to the osteoblast when there are high levels of -Catenin and Runx2 expression. In tetrapods, th ese osteoblasts continue to mature and eventually become osteocytes regardless of ossification mode, be it intramembranous, perichondrial or endochondral The osteoblasts do not always become osteocytes, especially in advanced teleosts (F ranz-Odendaal et al., 2006). The last step of endochondral bone formation is blood vessel invasi on, in which the osteoclasts are introduced. Here, I will mainly focus on e ndochondral bone formation since it was most extensively studied and is most relevant to the topic of this dissertation. The roles of -catenin have been discussed as they re late to the skeletal cell lineage determination. Another master gene that controls the osteoblast is Runx2 Runx2 is involved multiple roles in both chondrogenesis and osteogenesis. In the Runx2 null mice, no intramembranous or endochondral ossification was observed, and the calvar ial cells transformed into chondrocytes or adipocytes (Kobayashi et al., 2000; Komori et al., 1997; Otto et al., 1997).
33 Osterix ( Sp7 ), the downstream gene of Runx2 is also important for osteoblast determination, since its null mutant phenotype s are similar to those of Runx2 mutants (Nakashima et al., 2002). In -catenin conditional knockout mice, Osterix is absence although the Runx2 was intact, suggesting Osterix is a target gene of catenin. Collectively these three genes collaborate to determine the osteoblast lineage and inhibit chondroge nesis and adipogenesis. IHH is required for perichondrial mesenchyme to differentiate into osteoblasts in the perichondrium. Instead, the pe richondrial mesenchyme cells adopt chondrogenic fates in the absence of IHH (Long et al., 2004). In the Ihh mutants, Runx2 was present in chondrocytes, but not in the perichondriu m, indicating that Ihh works upstream of Runx2 and is required for Runx2 expression in the perichondrium. Similar to Runx2 -catenin was absent in the perichondrium in Ihh mutants (Hu et al., 2005). Taken toge ther, spatial and te mporal signaling by Ihh and Wnt/catenin promote the osteogenesis and inhibit cho ndrogenesis through thei r interaction with Runx2 Osterix and Sox9 Osteoblastic lineage determination Unlike Ihh both Runx2 and -Catenin signaling are required for osteoblast differentiation after the osteogenic fate is de termined. As discussed above, Runx2 promotes osteoblast formation from the mesenchymal skelet al progenitors. Consistently, many in vitro experiments revealed that Runx2 upregulates the bone matrix gene promoters, like Col1A1, Col1A2, osteopontin, bone sialoprotein, osteocalcin, fibr onectin, MMP13 and OPG (Komori, 2002). This was further supported by the finding that conditional deletion of Runx2 from mature osteoblasts resulted in down-regulation of Col1a1 Col1a2 osteopontin osteocalcin and Bsp (Ducy et al., 1999). It is interesting that th e same gene was shown to have some negative regulatory functions at later stages. When Runx2 was over-expressed under the Col1a1 promoter, multiple fractures and osteopenia were observed in mice, and most of the osteoblasts were immature (Geoffroy et
34 al., 2002; Liu et al., 2001). Taken together, Runx2 is important for osteoblast differentiation; it promotes differentiation at early stages a nd inhibits maturation at late stages. Another important osteoblast differentiation regulator is ATF2, a basic leucine-zipper transcription factor, which bel ongs to CREB family. It is re quired for the late osteoblast development, since in the Atf2 deficient mice only the late markers of osteoblast differentiation, Bsp and osteocalcin, were down regulated and Runx2 Osterix and Col1a1 were not affected (Yang and Karsenty, 2004). These data also suggested that Atf2 works independently of Runx2 Atf2 is capable to interact with Ap 1 ( Fos and Jun ), other Creb gene and Bcl-2 to control cell cycle and proliferation (Luvalle et al., 2003). It is not a surprise that the mutation of Ribosomal serine/threonine kinase 2 ( RSK2 ), a growth factor regulated kinase cause Coffin-Lowry Syndrome, an X-linked mental retardati on with skeletal abnormalities because Atf2 is the direct target of RSK2 (Yang and Karsenty, 2004). RSK2 also is required for the phosphorylation of Ap1 which is also important for bone formation. A p1 transcription factor complex is composed of Fos ( c-Fos FosB Fra-1 Fra-2 ), Jun ( c-Jun, JunB, JunD ) and ATF family (Jochum et al., 2001). Conditional knockout Fra-1 leads to bone matrix reduc tion and overexpression of Fra-1 or Delta-FosB in mice results in the opposite phenotype, osteopetr osis due to increased osteoblast number and bone formation (Eferl et al., 2004; Karreth et al., 2004; Kenner et al., 2004; Kveiborg et al., 2004). Very recently, a new regulator of Atf2 and Runx2 Satb2 was reported (Dobreva et al., 2006). Satb2 not only regulates osteoblas t differentiation through its interaction with Runx2 and Atf2 but also patterns the cran iofacial skeleton by repressing Hoxa2 (Dobreva et al., 2006). Other gene s were also reported to regula te osteoblast differentiation through interaction with Runx2 like Msx1 Msx2 Dlx5 and Dlx6 etc (Ichida et al., 2004; Ishii et al., 2005; Lee et al., 2005; Robledo et al., 2002; Sa tokata et al., 2000). However, most of these
35 genesÂ’ functions remain unclear, with the exception of Twist1 and Twist2 Twist genes are transcription factors containi ng the basic helix-loop-helix ( b-HLH ). Both Twist1 and Twist2 are co-expressed with Runx2 in the osteoprogenitors in the he ad and trunk, respectively, during early developmental stages. And osteoblasts start to differentiate after Twist expression is downregulated (Bialek et al., 2004). In the Twist1 or Twist 2 null mice, osteoblasts differentiated prematurely, and overexpression of Twist1 resulted in delayed oste oblast differentiation. Further analysis showed twist genes directly interact with Runx2 through their twist box domain (Bialek et al., 2004). Not all the osteoblast regulators need the interaction with Runx2 and Atf2 Recently, Calcineurin/Nfat was identified to be able to re gulate osteoblast proliferation and osteoclastogenesis, probably th rough the interaction with Wnt pathway (Winslow et al., 2006). Blood vessel invasion and osteoclast development The final step of endochondral osteogenesis is replacement of hypertrophic cartilage by bone (Erlebacher et al., 1995; Olse n et al., 2000). At l east four major events feature in this process: blood vessel invasion (a ngiogenesis), osteoclast differe ntiation, osteoblast development into osteocytes and bone marrow formation. Hypert rophic chondrocytes secr ete agiogenic factors like vascular endothelial growth factor (VEGF) and specific me talloproteinase (MMP) 9 and 13 to facilitate the vascular inva sion into the skeleton (Gerber an d Ferrara, 2000; Inada et al., 1999; Stickens et al., 2004). Then hypertrophic chondr ocytes die through apoptosis, the perichondrial osteoblasts and blood-borne osteoc lasts are introduced through blood vessels, and finally the osteoblasts further develop into osteocytes, whic h are buried by bone specific matrices such as type I collagen. Runx2 is a potential regulator of Vegf and Mmp13 therefore, Runx2 is also important for bone angiogenesis (Inada et al., 1999) (Otto et al., 1997; Zelzer et al., 2001; Zelzer et al., 2004). Since these major ev ents were already reviewed in detail, and my review mainly focuses on the cartilage development, I will not discuss these processes further. For further
36 details, the reader is referred to the followi ng references: angiogenesis (Gerber and Ferrara, 2000), osteoclasts (Boyle et al., 2003; Karsenty and Wagner, 2002; Kobayashi and Kronenberg, 2005; Shinohara and Takayanagi, 2007; Wagner and Karsenty, 2001) and osteocytes (FranzOdendaal et al., 2006). Complexity of Skeletal Development, Secondary Cartilage and Influences of Epigenetic Factors Our current knowledge of skeletal developmen tal is mainly based on work in mouse and chicken. The development of many intermediate sk eletal tissues, especially in fishes, remains mysterious. Even in mice, development of fi bril cartilage and elastic cartilage is not well studied. For bony tissues, not all bones ar e formed through either endochondral and intramembranous ossification; the clavicle, for example, has a complex development that probably involves a mixed mechanism (Huang et al., 1997). Secondary cartilages are named after thei r position, which is at the margin of intramembranous bones. Phylogenetically, second ary cartilages are found in birds, mammals and teleosts, suggesting that th ey are not homologous tissues (H all, 2005). They are usually found in several human sutures, where epigen etic factors, such as oxygen saturation and mechanical loading, influence this kind of carti lage development (Hall, 1967; Hall, 1970). The developmental process of secondary cartilage is like the reverse pro cess of endochondral bone formation: cartilages are formed from the perios teum. This was further confirmed by the finding that upregulation of Sox9 and downregulation of Runx2 lead to the formation of secondary cartilages. This induced the chondrocytes to rapidly exit the cell cy cle and increase the expression of Ihh (Buxton et al., 2003). Ihh expression in prehypertrophic chondrocytes works like a bone formation center without a cartilage template. The mech anical forces stimulate the formation of secondary cartilages (Buxton et al., 2003). There are some reports that physical
37 strain is able to change gene expression prof iles of skeletal cells. After application of mechanical force, osteoblasts increased cyclooxygenase-2 and c-fos transcripts in an hour and osteopontin and osteocalcin level in one day (Pavalko et al., 1998; Walker et al., 2000). Recently it was proved that mechanical for ce across the midpalatal suture promotes osteoblast maturation in the perios teum and induces osteoclast ac tivity in vivo (Hou et al., 2007). In the same mice, secondary cartilage formati on was inhibited and new cartilage formation was promoted (Hou et al., 2007). Anot her well-characterized epigenet ic factor is hypoxia, which is mediated through the transcription fa ctor hypoxia inducible factor 1 ( Hif1 ). Hif1 is required for the chondrocyte survival th rough its interaction with Vegf (Schipani et al., 2001). Recently evidence was provided that the growth plat e is hypoxic during fetal development and Hif1 was turned on in the hypoxic environment to regulate chondrocyte differentiation, joint development, autophagy and apoptosis (Bohensky et al., 2007; Provot et al., 2007). Hypoxia and Hif-1 are also involved in tumor progression, su rvival of tissues with low blood supply, and many other pathological events (Schipani, 2006). Summary In this dissertation, I investig ate the evolutionary origin of cartilage development in vertebrates. Based on the extens ive model system work describe d above, I take a comparative approach and ask when the key components of th e skeletogenic gene netw ork were recruited for chondrogenesis. In particular, I focus on the or igin of collagen2-based cartilage, which has long been thought to be a unique charac ter of the jawed vertebrates. My results challenge this view and uncover an unexpectedly ancient origin of a c onserved skeletogenic program in vertebrates. I also examine the evolutionary origin of the axial skeleton, by determining when the embryonic precursor of vertebrae, the sclerotome, first a ppeared in vertebrate evolution. The results presented below show that the collagenous skelet on evolved at an earlier node than previously
38 expected, and rather than being a unique feature of gnathostomes, is a character that unites the extant jawed and jawless vertebra tes. Moreover, I provide molecular evidence for the presence of a sclerotome in lampreys, wh ich supports the century -old hypothesis that lamprey arcualia and gnathostome vertebrae may have a common developm ental origin. I discuss the implications of these results for the question of homology of th ese structures, and propose a new model for the role of gene duplication in the evol utionary origin of the skeleton.
39 Figure 1-1. Distribution of verteb rate cartilage structures. Th e phylogenetic relationships are based on Bourlat and Neidert (Bourlat et al., 2006; Mallatt and Winchell, 2007; Neidert et al., 2001). The bars indicate th e emergence of the cartilage structures.
40 Figure 1-2. Overview of extra cellular composition of cartilage. Depicted is a cartilage chondroc yte. Cartilage collagen fibril s (collagens types II, IX and XI) have been indicated. The proteoglycans and th eir positions are marked respectively. Modified from Knudson and Knudson (Knudson and Knudson, 2001).
41 Figure 1-3. Distribution of cartila ge tissue within metazoa. Th e phylogenetic relationships were based on Gerlach, D et. al. (G erlach et al., 2007). The ta xa with cartilage were highlighted with black line underneath the taxa name. The taxa with cartilage were based on the previous reports (Cole and Hall, 2004a; Wright et al., 2001)
42 Figure 1-4. Vertebrate skeletal cell lineage determination. The early skeletal cells co-express Sox9 and Runx2 and Sox9 dominantely inhibits Runx2 Dependings on the expression level of -catenin the skeletal precursor cells go to the chondrogenic or osteogenic pathway. Adapted from Hartmann C. 2006.
43 CHAPTER 2 LAMPREY TYPE II COLLAGEN AND SOX9 RE VEAL AN ANCIENT ORIGIN OF THE VERTEBRATE COLLAGENOUS SKEELTON Introduction The earliest known vertebrates ar e jawless fishes that date to the Lower Cambrian (~520 Ma) (Shu et al., 1999). The remarkab le preservation of these skelet ons has revealed the patterns of early vertebrate skeletal e volution, however little is known about the developmental processes and molecular mechanisms that gave rise to these patterns. In the cartil age of jawed vertebrates (gnathostomes), the major extracellular matrix molecule is type II collagen (COL2A1). By contrast, the extant jawless fishes (lampreys and hagfishes) have been reported to lack collagenbased cartilage (Janvier and Arsenault, 2002; Wr ight et al., 2001; Wright and Youson, 1983). In lampreys and hagfishes, some skeletal elements are composed of elastin-like molecules, such as lamprin and myxinin (Robson et al., 2000; Robson et al., 1993). This difference in skeletal structure has raised the hypothe sis that the collagenous skelet on is a gnathostome synapmorphy, and the earliest vertebrates have been presumed to have non-collagen based cartilage (Wright et al., 2001). Collagens are estimated to have appeared in the late Proterozoic (about 800 ma) and then diversified into two major groups the fibril-forming and non fibr il-forming collagens (MorvanDubois et al., 2003). The fibril-forming group, cons isting of collagen types I, II, III, V and XI, share a high degree of sequence and structural similarity and provi de stiffness to a variety of tissues, whereas the non-fibril-forming group is structurally and functionally heterogeneous (Exposito et al., 2002; Morvan-Dubois et al., 2003; van der Rest and Garrone, 1991). The former then split into two phyloge netic groups that are designated (A) for types I, II, III, and V 2 versus (B) for types V 1, V 3, V 4, V 5, and XI (Morvan-Dubois et al., 2003)
44 During chondrocyte differentiation in gnathostomes, expression of the Col2a1 gene leads to secretion of Col2a1 protein into the extracellular matrix, wher e it mineralizes. Expression of Col2a1 is regulated directly by Sox9 which binds to a chondrocyte-s pecific enhancer to activate Col2a1 transcription (Bell et al., 1997; Lefebvre et al., 1997). The Sox family of transcription factors consists of at least te n subgroups (A-H) that are charac terized by a specific 79-amino acid DNA-binding region, termed the high-mobility-g roup (HMG) box (Bowles et al., 2000). Sox9 a member of the SoxE subgroup, is required fo r COL2A1 expression and for chondrogenesis in jawed vertebrates (Bi et al., 1999; Yan et al., 2005). Mutation of Sox9 in zebrafish disrupts the stacking of chondrocytes and the separation and sh aping of individual cart ilage elements, and in humans causes campomelic dysplasia (W agner et al., 1994; Yan et al., 2002). Despite the different compositions of lamp rey and gnathostome cartilage matrix, the genetic pathway that regulates early developm ent of the cranial skeleton is well conserved (Cohn, 2002; McCauley and Bronner-Fr aser, 2003; Neidert et al., 2001; Shigetani et al., 2002). This raises the question of how the same cascade of gene expre ssion can lead to activation of different cartilage matrix gene targets in th ese two lineages. Alt hough studies of lamprey cartilage matrix have identified non-collagenous proteins in particular skeletal elements (McBurney et al., 1996a; McBurn ey et al., 1996b; Robson et al., 1993), it is not clear whether these elastin-related proteins exist in place of or in addition to collagen. Indeed, comparative anatomical studies from the 19th century identified true hyaline cartilage in lampreys and noted striking structural similarities to gnathostome cartilage (Parker, 1883), suggesting that there may be hitherto undiscovered molecular similarities in lamprey and gnathostome cartilage matrix. Here we revisit the evolutionary origin of co llagenous cartilage from a molecular developmental perspective, and we report that development of the lamprey skeleton involves type II collagen.
45 Our experiments show that lampreys have tw o type II collagen genes, and that both are expressed in the developing skelet on. We also find COL2A1 prot ein in cranial and postcranial cartilages. We go on to show that lampreys have a true orthologue of Sox9 that is co-expressed with both Col2a1 genes during development of the lampre y skeleton. Thus, we conclude that lampreys have collagen-based cartilage, and that the genetic pathway for chondrogenesis is conserved all the way to cartilage matrix gene activation in lampreys and gnathostomes. The results indicate that a collage nous skeleton evolved prior to th e divergence of the lamprey and gnathostome lineages and suggest that collagen-based cartilage may be a unifying character of crown vertebrates. Materials and Methods Gene Cloning and Sequence Analyses Degenerate RT-PCR was performed to amplify fragments of lamprey Col2a1 and Sox9 orthologues from a Petromyzon marinus cDNA library. PCR products were cloned into pDrive vector (Qiagen) and sequenced in both directions. These sequences have been submitted to GenBank (Accession numbers: DQ136023-DQ136025). The inferred protein sequences for the new lamprey cDNAs were initially assigned to the Col2a1 and Sox9 families on the basis of BLAST searches and conserved domains (Bowles et al., 2000; Koopman et al., 2004; Valkkila et al., 2001). These initial assignments were followe d by estimates of their amino acid identities and phylogenetic relationships. Multiple sequence alignments for availabl e fibrillar A collagens and SoxE proteins, including the new lamprey sequences, were generated with CLUSTAL X and then refined according to their known tertiary structur es (Bowles et al., 2000; Koopman et al., 2004; Valkkila et al., 2001). GenBank accession numbers for all sequences used in these phylogenetic analyses: human COL2a 1, NP_001006952; mouse COL2A1, B41182; rat COL2A1, NP_037061; chicken COL2A1, NP_98 9757; frog COL2A1, B40333; salamander
46 COL2A1, BAA82043; Zebrafish Col2a1, XP_692723; Lamprey COL2A1a, DQ136024; Lamprey COL2A1b, DQ136025; human COL1A 1, BAD92834; mouse COL1A1, CAI25880; zebrafish COL1A1, BC063249; Human COL1 A2, AAH42586; mouse COL1A2, NP_031769; zebrafish Col1a2, NP_892013; human COL3 A1, AAL13167; mouse COL3A1, NP_034060; human COL5A2, NP_000384; mouse COL5A 2, NP_031763; sea uchin ColP2a, NP_999675; human Sox8, AAH31797; mouse SOX8, BAC2 8299; rat SOX8, XP_220283; chicken SOX8; NP_990062; frog SOX8, AAQ67212; zebrafish Sox8, AAX73357; pufferfish, SOX8, AAT42231; human SOX9, AAP35521; mouse SOX9, NP_035578; pig SOX9, AAB81431; chicken SOX9, NP_989612; frog SOX9, BAA95427; alligator SOX9, AAD17974; zebrafish Sox9a, AAM13696; zebrafish Sox9b, AAH67133; pu fferfish SOX9, CAG00200; medaka SOX9, BAC06353; trout SOX9, AB006448; stickleback SOX9a, AAQ62978; stickleback SOX9b, AAQ62979; sturgeon SOX9, AAW78521; lamprey SOX9, DQ136023; human SOX10, NP_008872; mouse SOX10; XP_128139; rat S OX10; AAH62067; chicken SOX10, NP_990123; frog SOX10; AAO13216; zebrafish Sox10, NP_571950; pufferfish Sox10a, AAQ18509; pufferfish SOX10b, AAQ18510; lamprey SOXE1, AAW34332; sea squirt SOXE, CAD58841. Phylogenetic analyses of these multiple prot ein alignments were conducted with maximum parsimony (MP), minimum evolution (ME), maximum likelihood (ML), and Bayesian phylogenetics (BP) methods (Felsenstein, 2004). Th e MP analyses included both the equal and unequal weighting of amino acid replacements, with th e latter relying on th e Â“ProtParsÂ” cost matrix. The former relied on branch-and-bound searches, whereas the latter was based on heuristic ones with tree-bisec tion-and-reconnection branch swa pping and 1,000 starting trees that were generated from different random sequence additions. The pa irwise distances in ME were corrected for multiple replacements with the JTT rate matrix and the gamma ( ) distribution for
47 site-to-site heterogeneity in rates. The ME an alyses relied on heuristic searches with closeneighbor-interchanges starting from neighbor joining trees. The ML and BP analyses also relied on but in combination with the improved WAG rate matrix available in their computer programs (but not in that for ME; see below). In ML and BP, the parameter for was estimated, whereas it was fixed to its ML estimates in ME. The ML analyses relied on a fast heuristic procedure that simultaneously searched for both optimal branch lengths and topologies. The BP analyses were based on three indepe ndent runs of 2,000,000 generations apiece, with each run consisting of one cold and three heated chains ( T =0.2) and with samples taken from the former every 100 generations. The reliability of groups was evaluated in MP, ME, and ML with 1,000 bootstrap replicates apiece and in BP with poste rior probabilities that were calculated after discarding the first 1,000 samples of each run as burnin. The MP, ME, ML, and BP analyses were conducted with PAUP*4.0b10 (Swoffor d, 2002), MEGA3 (Kumar et al., 2004), PHYML2.4.4 (Guindon and Gascuel, 2003), a nd MRBAYES3.1.1 (Ronquist and Huelsenbeck, 2003) respectively. In Situ Hybridization Whole mount in situ hybridization was performed as desc ribed for chick embryos (Nieto et al., 1996), with the following modifications: embryos were treated with prot einase K (10 mg/ml) for 15-30 min at room temperature, and 10% di methylformamide was added to color reaction solution. For histological analys is, specimens were equilibrated in 15% sucrose then 30% sucrose in 20% gelatin, after which they we re embedded in 20% gelatin for cryosectioning (10mm).
48 Immunohistochemistry Lamprey specimens were fixed in 70% ethano l and processed for paraffin sectioning using standard methods. Sections were cut at 6 mm and antigen retrie val was performed by autoclaving slides in 0.01 M ci trate buffer (pH6). Antibody st aining was performed using the Vectastain ABC kit according to manufacturerÂ’s instructions. Primary antibodies against human COL2A1 (Santa Cruz Biotechnology Inc.) were used at concentra tions of 1:500-1:1000. Results Lampreys have Two Col2a1 Orthologues As a first step towards resolving whether co llagen genes are involved in lamprey skeletal development, we searched fo r expressed orthologues of Col2a1 using a degenerate PCR screen of a P. marinus embryonic cDNA library. We isolated two 1.74 kb clones w ith 75% nucleotide sequence identity to one another, and their deduced amino acid se quences were 80% identical to mouse Col2a1. Absence of gaps in the nucleotid e alignments suggested th at the two transcripts were not splice variants from a single gene. Mo lecular phylogenetic analyses with MP, ME, ML and BP methods consistently placed the lamprey se quences within the vertebrate Col2a1 family, and supported their position as a sister group to the gnathostome COL2A1 clade (Figure 2-1. and Figure 2-2.). These results indicate the presence of two Col2a1 genes in lamprey, which we designate Col2a1a and Col2a1b This represents the first demons tration of true type II collagen orthologues outside of jawed ve rtebrates, and reveals that an independent duplication of the ancestral Col2a1 gene occurred within the lamprey lineage. Col2a1a and Col2a1b are Expressed during Lamprey Chondrogenesis If the ancestral Col2a1 gene was involved in skeletoge nesis in the common ancestor of lampreys and gnathostomes, then an evolutiona ry signature may be detectable during lamprey embryonic development. We therefore examined whether Col2a1a and Col2a1b are expressed
49 in tissues that give rise to the lamprey skeleton. Using whole mount in situ hybridization, we found that Col2a1a is expressed during somitogenesis in a segmental pattern along the lamprey trunk (Figure 2-3.A). Histologi cal sections showed that Col2a1a transcripts in the somites were localized ventromedially to the sclerotome, which gives rise to the axial skeleton, and dorsolaterally to the dermatome, wh ich gives rise to the dermis (Figure 2-3. B). In the midline, lamprey Col2a1a expression was observed in the floor pl ate of the neural tube and in the hypochord, but not in the notochord (F igure 2-3. B). This represents a subset of the zebrafish Col2a1 expression pattern, which occu rs along the midline in thr ee domains, the floor plate, notochord, and hypochord (Yan et al., 1995). The second lamprey orthologue, Col2a1b was expressed in a pattern similar to Col2a1a along the anteroposterior axis and in the midline; however Col2a1b transcripts also localiz ed to the notochord and to endoderm immediately ventral to the hypochord (F igure 2-3. C and D). During differentiation of the lamprey skeleton, both Col2a1a and Col2a1b were expressed in chondrogenic cells. The axial sk eleton of lampreys consists of a series of paired cartilaginous vertebrae or arcualia, wh ich are serially repeated on either side of the notochord and have long been thought to be homologous to the neural ar ches of gnathostome vertebrae (Gadow, 1933). Col2a1a expression persisted in sclerotomal cells (F igure 2-3. E) and could be seen in the prevertebral condensations by stage 30 (Figure 2-3. F). Col2a1b expression appeared in the sclerotome by stage 26, but expression in the mid line was downregulated anteriorly (Figure 2-3. G-I). In the developing head skeleton, Col2a1a expression was detected throughout the cartilaginous branchial basket and posterior to the oral cavity (Fig. 2J). Transcripts were most abundant in the chondrocyte stacks that make up each gill bar (Figure 2-3. K). By contrast, Col2a1b expression could not be detected in the pharyngeal arches (Fig ure 2-3. L). During
50 development of the median fin skeleton, Col2a1a and Col2a1b were expressed respectively in the posterior and anterior parts of the finfold (data not shown). Thus, both lampreys and jawed vertebrates exhibit widespread expression of Col2a1 genes during skeletal development. Adult Lamprey Cartilage Co ntains COL2A1 Protein The discovery that two Col2a1 genes are transcribed during lamprey skeletal development raised the possibility that la mpreys may possess a collagenous sk eleton. As a direct test of whether lamprey differentiated cartilage contai ns type II collagen protein, we performed immunohistochemical analysis of an adult using an antibody specific to COL2A1. We detected COL2A1 protein in the extracel lular matrix of pharyngeal ca rtilages, notochord, notochordal sheath and arcualia (Figure 2-4.) confirming that COL2A1 protei n is abundant in the lamprey skeleton. These results demonstrat e that a collagenous skeleton e volved prior to the divergence of the lamprey and gnathostome lineages, and su ggest that COL2A1 was involved in skeletal development at least as early as the co mmon ancestor of crown vertebrates. Upstream of Col2a1 : a Lamprey Orthologue of Sox9 In gnathostomes, transcription of Col2a1 is regulated directly by SOX9, and these two genes are co-expressed during c hondrogenesis (Ng et al., 1997; Yan et al., 1995; Yan et al., 2002; Yan et al., 2005). Given the critical role of SOX9 in the development of cartilages derived from both neural crest and mesoderm, we next asked whether lampreys have a Sox9 orthologue. Using degenerate RT-PCR, we isolated a 1.3KB clone whose inferred amino acid sequence most closely matched those of the gnathostome SOX9 pr oteins. For example, this inferred protein sequence was 96% identical to human SOX9 with in the HMG-box (Figure 2-5.). Furthermore, this new amino acid sequence included the SOX9-s pecific signature motif (Bowles et al., 2000; Koopman et al., 2004) that occurs immediately 3Â’ to the HMG-domain. Molecular phylogenetic analyses consistently joined this new lamprey pr otein to the base of the SOX9 clade, with BP
51 providing a posterior probability of 98% for this assignment (Figure. 2-6.). In concert with its overlapping gene expression patter n with that of SOX9 in jawe d vertebrates (see below), these results collectively provide clear evidence fo r the designation of th is clone as lamprey Sox9 Sox9 Expression Co-Localizes with Col2a1a and Cola1b in the Developing Skeleton We next investigated whether lamprey Sox9 is expressed in a pattern consistent with a role in regulation of Col2a1. Lamprey Sox9 expression along the primary body axis resembled the lamprey Col2a1b pattern at stage 23 (compare Figure 27.A with Figure 2-3. C). Transcripts were localized to the ventral neural tube, notoc hord and hypochord along the midline, as well as the sclerotome and dorsal endode rm at stage 24 (Figure 2-7. C). Neural expression of Sox9 extended from the spinal cord to the forebrai n (Figure 2-7. B). At stage 23, the pharyngeal arches were negative for Sox9 however by stage 24, expression was detected in streams of neural crest cells extending from the hindbrain towards the arches (Figure 2-7. B). At stage 26, Sox9 was expressed throughout the developing bran chial basket, and in the otic and optic placodes (Figure 2-7. D). Like Col2a1b, Sox9 expression in the notocho rd later retracted from anterior to posterior, but transcripts remained in sclerotome and neural tube (Figure 2-7. E-G). Thus, lamprey Sox9 expression co-localizes with Col2a1 transcripts during chondrogenesis, and closely follows the pattern described for gna thostomes (Ng et al., 1997; Yan et al., 2005). Discussion The data presented here demonstrate th at lampreys have two orthologues of Col2a1 that are expressed during development of the cartil agenous skeleton. Our discovery of type II collagen protein throughout the adult lamprey skel eton challenges the view that collagen-based cartilage is a gnathostome character and indicate s that a collagenous skle leton evolved prior to the divergence of lampreys and gnathostomes. Expression of lamprey SOX9 in chondrogenic cells suggests that a common suite of genes is ta rgeted during skeletal di fferentiation in jawed
52 and jawless vertebrates. We conc lude that a collagenous skeleton is a shared, derived feature of all vertebrates and not of jawed vertebrates only. Molecular Evolution of Lamprey Collagens Our molecular phylogenetic analyses of fibril A collagens leads us to conclude that Col1 to Col5 duplicated before the speciation of lampreys and jawed vertebra tes. Thus, one prediction of our phylogenetic results is that future st udies will recover lamp rey orthologues of Col1 Col3 and/or Col5 which should join at the bases of their re spective gnathostome cl ades in the fibril A collagen tree (Fig. 1). An independent duplication of the Col2a1 gene then occurred in the lamprey lineage. The expression patterns of Col2a1a and Col2a1b suggest that subfunctionalization followed this duplication of the ancestral Col2 a1 gene, with the ancestral expression pattern being partitioned between Col2a1a and Col2a1b Force and colleagues proposed a mechanism by which duplicated genes are preserved during evolution by subfunctionalization, in which both members of th e pair undergo reduction of their activity and expression patterns such that together they equal that of their single ancestral gene (Force et al., 1999; Lynch et al., 2001). Our findi ng that the expression domains of Col2a1a and Col2a1b in lampreys correspond to that of the single Col2a 1 gene in jawed vertebrates supports their duplication-degeneration-complement ation (DDC) model. It is also noteworthy that collagen genes are physically linked to the Hox clusters in gnathostomes (Bailey et al., 1997; MorvanDubois et al., 2003). In lampreys, there are at least three Hox clusters (Force et al., 2002; Fried et al., 2003; Irvine et al., 2002). Our identification of two Col2a1 genes supports the suggestion that one of their three Hox clusters may have arisen by an in dependent duplication in the lamprey lineage (Force et al., 2002; Fried et al., 2003; Irvine et al., 2002). If the fibril A collagens are linked with Hox clusters in all chordates, then the presence of a single Hox cluster in amphioxus
53 makes it tempting to speculate that, barring ta ndem duplications, cephalo chordates may possess a single fibril A collagen gene. Evolution of Collagen-Based Cartilage Localization of type II collagen mRNA and prot ein in the lamprey skeleton reveals that a collagenous skeleton is not restri cted to the gnathostome lineage but instead is a character shared by the crown vertebrates. This may provide a molecular explanation for ParkerÂ’s observation in 1883 that lampreys ha ve hard hyaline cartilage (Par ker, 1883). We suggest that the additional cartilage matrix molecules (e.g., la mprin and myxinin) of agnathans may represent derived character states that that were added onto the more ancient collagenous skeleton. Expression of elastin-related molecule s in a subset of lamprey crania l cartilages that also express COL2A1 may underlie the different structural and mechanical properties within the lamprey skeleton. Expression of Col2a1a in the lamprey dermatome was unexpected, as vertebrate dermis is generally characterized by type I co llagen (van der Rest and Garrone, 1991). While this may be a derived feature of lampreys, it coul d also reflect their descent from agnathans with dermal armor (Forey and Janvier, 1993). Five independent molecular phylogenetic analyses joined lamprey SOX9 to the gnathostome Sox9 clade. Thus, the duplication of the ancestral SOXE gene that gave rise to Sox9 occurred before the divergence of the lamprey and gnathostome lineages. Sox9 is expressed in strikingly similar patterns in lamprey and gnathos tome embryos. The co-expression of Sox9 with Col2a 1 during skeletogenesis in both lineages raises the possibility that the regulatory relationship between th ese two genes had already been established in their common ancestor. In gnathostomes, Sox9 is a target of parathyroid hormone-related protein (PTHrP), which regulates chondrocyte di fferentiation through a negative feedback loop with Indian hedgehog (Huang et al., 2001; Vortkamp et al., 1996). Interestingly, PTHrP expression has
54 recently been detected in lamprey cartilage (Tri vett et al., 2005). Our discovery of conserved expression of Sox9 and Col2a1 taken together with the extensive conservation of upstream regulatory genes such as AP2, Dlx, Msx, Id and PTHrP (Cohn, 2002; McCauley and BronnerFraser, 2003; Neidert et al., 2001; Shigetani et al., 2002; Trivett et al., 2005), suggests that the genetic program for chondrogenesis -from the in itial induction of chondrogenic mesenchyme to the synthesis of collagen matrix -was assemble d surprisingly early in vertebrate evolution. Comparative analyses of fibrillar collagen and SOXE genes in hagfishes, amphioxus, ascidians and hemichordates will further refine the evol utionary history of the collagenous skeleton.
55 Figure 2-1. Minimum evolution phylogeny for fibril A collagen pr oteins as obtained with JTT+ distances ( = 0.906). Numbers indicate bootstr ap scores for each node, based on 1,000 replicates, whereas branch lengths are proportional to expected replacements per site. This tree is rooted by sea urch in COLP2. Equally and unequally weighted MP, ML, and BP also place the two lamprey sequences together at the base of the COL2A1 clade, with bootstrap scores or a posterior probability of 93%, 77%, 71%, and 87%, respectively. In contrast to this consistent support for a Col2A1 grouping, this ME tree is unique among the different phylogenies in its placement of the root along the Col5 clade. This discrepancy ove r the root is illust rated with the BP phylogeny and discussed further in Figure. 2-2.
56 Figure 2-2. Extended majority-rule consensus tree for the BP analysis of the fibril A collagen proteins. Numbers indicate posterior probabilities that are given for each group with >50% credibility and for those clades that ar e combinable with this first set. Branch lengths are proportional to the means of th e posterior probability densities for their expected replacements per site. This tree is rooted by sea urchin COLP2. This root along the COL3 clade illustrates an a lternative placement for MP, ML, and BP relative to the COL5 rooting fo r ME (Figure. 2-1.). In contrast to this new position, the latter is consistent with available phyl ogenetic and linkage data for the physically linked Hox gene clusters (Bailey et al., 1997; Morvan-Dubois et al., 2003). Nevertheless, this discrepancy over the r oot does not diminish the fact that all phylogenetic methods converge onto the same placement of the two lamprey collagens at the base of the COL2A1 cl ade. Thus, no ambiguity exists over the identification of the two new lamprey sequences as orthologues of COL2A1.
57 Figure 2-3. Col2a1a and Col2a1b expression during lamprey development. Whole mount in situ hybridization of lamprey embryos at stag es 23 (A-D), 25 (E), 26 (G-I) and 30 (F, JL). Anterior is to right in (A, C, J and L) ; All sections are transverse with dorsal to the top. A, B, Col2a1a expression is evident in the somites, within the dermatome and sclerotome. Expression in the midline is restricted to floor plate and hypochord (B). C, D, Col2a1b is expressed in the floor pl ate, notochord, hypochord and dorsal endoderm (asterisk). E. Col2a1a in the dermatome, sclerotome, floor plate and hypochord. F. Col2a1a expression in a prevertebral condensation (arrows). G-I, Sections through the hindbrain (G), mid-trunk (H) and tail (I) show an anterior to posterior retraction of the Col2a1b domain in the notoc hord, hypochord and floor plate. J, Col2a1a is expressed throughout the branchial skeleton (boxed) and posterior to the oral cavity. K, Col2a1a expression in a stack of chondrocytes in a branchial bar. (L). Col2a1b is not expressed in the bran chial skeleton. Abbreviations: bb, branchial basket; d, dermatome; fp, fl oor plate; h, hypochord; n, notochord; oc, oral cavity; s, sclerotome; so, somite.
58 Figure 2-4. COL2A1 protein is abundant in adult lamprey cartilage. Immunohistochemical staining of lamprey cartilage with COL2 A1 antibody. A. Sagittal section through pharyngeal cartilage bars. B. Transverse section through notochord. Note staining of notochord cells and notochorda l sheath. C. Transverse section through arcualia on ventrolateral side of notochord. D. Contro l section through pharynge al cartilage, after omission of primary antibody.
59 10 20 30 40 50 60 70 80 90 100 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Lamprey-Sox9 -------------------------------------------------------------------------------S Q VL K G Y D W T LV P M P V R V N G A lligator-Sox9 LL D P F M K M T EE Q E K C I S G A P S P T M S DD S A G S P C P S G S G S D T E N T R P Q E N T F P K G D --P D L KK D S DED K F P V C I R E A V S Q VL K G Y D W T LV P M P V R V N G Chicken-Sox9 LL D P F M KM T EE Q D K G L S G A P S P T M S DD S A G S P C P S G S G S D T E N T R PP Q E N T F P K G D --P D L KK E S DED K F P V C I R E A V S Q VL K G Y D W T LV P M P V R V N G Human-Sox9 LL D P F M K M T DE Q E K G L SG A P S P T M S ED S A G S P C P S G S G S D T E N T R P Q E N T F P K G E --P D L KK E S EED K F P V C I R E A V S Q VL K G Y D W T LV P M P V R V N G Pig-Sox9 LL D P F M K M T DE Q E K G L S G A P S P T M SE G S R G S P C P S G S G S D T E N T R P Q E N T F P K G E --P D L KK E S EED K F P V C I R E A V S Q VL K G Y D W T LV P M P V R V N G M ouse-Sox9 LL D P F M K M T DE Q E K G L S G A P S P T M S ED S A G S P C P S G S G S D T E N T R P Q E N T F P K G E --P D L KK E S EED K F P V C I R E A V S Q VL K G Y D W T LV P M P V R V N G Frog-Sox9 LL D P F L KM T EE Q E K C L S G A P S P S M S ED S A G S P C P S G S G S D T E N T R P Q E N T F T K G D --Q D L KK E T EDE K F P V C I R E A V S Q VL K G Y D W T LV P M P V R V N G Sturgeon-Sox9 -------------C L S D A P S P S M S ED SA G S P C P S G S G S D A E N T R P S E N S LL G P D S Q M P D F KK E G DDD K F P V C I R D A V S Q VL K G Y D W T LV P M P V R V N G Trout-Sox9 LL D P F L K M T DE Q D K C L S D A P S P S M S ED S V G S P C PS G S G S D T E N T R P S E N G LLM G P D G P LV E F KK D DDD K F P V C I R D A V S Q VL K G Y D W T LV P M P V R M N G M edaka-Sox9 LL D P Y L K M T EE Q D K C L S D A P S P S M S ED S A G S P A S P S G S G S D T E N T R P R E N G LM R A D G A L S D F KK D EDD K F P A C I R E A V S Q VL K G Y D W T LV P M P V R V N G Stickleback-Sox9A LL D P Y LK M T EE Q D K C L S D A P S P S M S ED S A G S P C P S G S G S D T E N T R P S E N G LL G L D G --E F KK D EDD K F P A C I R E A V S Q VL K G Y D W T LV P M P V R V N G Zebrafish-Sox9A LL D P Y L K M T DE Q E K C LS D A P S P S M S ED S A G S P C P S A S G S D T E N T R P A E N S LL AA D G T L G D F KK D EED K F P V C I R E A V S Q VL K G Y D W T LV P M P V R V N G Pufferfish-Sox9 LL D P Y L K M T EE Q E K C H S D A P S P S MS ED S A G S P C P S G S G S D T E N T R P S D N H LLL G P D ----Y KK E N EEE K F P V C I R D A V S Q VL K G Y D W T LV P M P V R V N G Stickleback-Sox9B LL D P Y L K M T EE Q E K C H S D A P S P S M S ED S A G S P C -S VS G S D T E N T R P S D N H LLL G A D ----Y KK E G EEE K F P V C I R D A V S Q VL K G Y D W T LV P M P V R V N G Zebrafish-Sox9B LL Q R G L K M S -----V S G A P S P S L S ED S A G S P C A S A G S G S D S E T P R A -E PP L H RDE ----------Q E K F P V C I R D A V S Q VL K G Y D W S LV P M P V R V S G Frog-Sox8 M SS D Q ----E PP C S P T G T A SS M S H V S D S D S D S P L S P A G S E G R G ----S H R PP G I S --K R D G EE P M DE R F P A C I R D A V S QVL K G Y D W S LV P M P V R G S G Chicken-Sox8 M T EE H D K A L E A P C S P A G TTSS M S H V D S D S D S P L S P A G S E G L G C A P A P A P R PP G AA P L G A K V D AA E V DE R F P A C I R D A V S Q VL K G Y D W S LVP M P V R G N G Rat-Sox8 M S E A R A ---Q PP C S P S G T A SS M S H V ED S D S D A PP S P A G S E G L G ------R A G --GGG R G D T A E AA DE R F P A C I R D A V S Q VL K G Y D W S LV P M P V R GGG M ouse-Sox8 M S E A R A ---Q PP C S P S G T A SS M S H V ED S D S D A PP S P A G S E G L G ------R A G --GGG R G D T A E AA DE R F P A C I R D A V S Q VL K G Y D W S LV P M P V R GGG Human-Sox8 M S E A R S ---Q PP C S P S G T A SS M SH V ED S D S D A PP S P A G S E G L G ------R A G V A V GG A R G D P A E AA DE R F P A C I R D A V S Q VL K G Y D W S LV P M P V R GGG Pufferfish-Sox8 M T EE H D K C V N D Q P C S P S G T N SS M S Q D E S D S D A P SS P T G SD G Q G --------S LL TS L G R K V D S E DDE R F P A C I R D A V S Q VL K G Y D W S LV P M P V R G N G Zebrafish-Sox8 M S EE R E ----K C SS P T G S C SS E C P D E C D S D P S C S P A G P AA L R ---------------M G QQ A EDED G R F P V C I R D AV S Q VL K G Y D W S LV P M P V R V S G Rat-Sox10 M A EE Q D L S E V E L S P V G S EE P R C L S P SS A P S L G P D GG ---GGG S G L R A S P G P G E L G K V KK E QQ D G E A DDD K F P V C I R E A V S Q VL S G Y D W T LV P MP V R V N G M ouse-Sox10 M A EE Q D L S E V E L S P V G S EE P R C L S P G S A P S L G P D GG ---GGG S G L R A S P G P G E L G K V KK E QQ D G E A DDD K F P V C I R E A V S Q VL S G Y D W T LV P M P V R V N G Human-Sox10 M A EE Q D L S E V EL S P V G S EE P R C L S P G S A P S L G P D GG ---GGG S G L R A S P G P G E L G K V KK E QQ D G E A DDD K F P V C I R E A V S Q VL S G Y D W T LV P M P V R V N G Chicken-Sox10 M A DD Q D L S E V E M S P V G S ED HH C L S PG -P S M A S D -------N SS H L A SS G N G E M G K V KK E QQ D S E A DDD K F P V C I R E A V S Q VL S G Y D W T LV P M P V R V N G Frog-Sox10 M S DD Q S L S E V E M S P V G S ED P S L T P D P L PP H A H SS -----P DDDDDDDEEEEEE T K V KK EQ --D S EDE R F P V C I R E A V S Q VL N G Y D W T LV P M P V R V N G Pufferfish-Sox10B S R EE Q S F S E A D L S P G M S DD S R S L S P G H SS G A T GGG D S P LL G S QQ P H L A G M D N TT A S C S --S A K S DDEDE R F P V E I R D AV S Q VL N C Y D W T IV P M P V R V N S Zebrafish-Sox10 S A EE H S M S E V E M S P G V S DD G H S M S P G H SS G A P GG A D S P L P G QQ S Q M S G I G DD G A G V S GG V S V K S DEEDD R F P I G I R E A V S Q VL N G Y D W TLV P M P V R V N S Pufferfish-Sox10A S A EE H S L S E A E M S P GG S DD G H S L S P S Q P GG PP S Q G S P L S V T P QQ L S A L C V G D G S G DD GG R S G A K S EEEDD R F P I G I R E A V S Q VL D G Y D W T LV P M P V R V NN Lamprey-SoxE1 H V P S P D V S D V E S E P S L H GG S L S G A S D S DE S G L G S Y G A G M S GGGGGG L A G N V GG V GG H G V A G S R K S G C DDE K F P D S I R E A V S Q VL K G Y D W T LV P M P V R V N G Sea squirt-SoxE TS E I F S M SS P E SL S N F C N V K S A V A V A R AAA T LL E S K SS EEE N Y DE S LM N T G SS A R S A S P G T N DD L S DE K DD M S K D I K D A V S Q VL K G Y D W T LV P M P V R M N G 110 120 130 140 150 160 170 180 190 200 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Lamprey-Sox9 SS K S K P H V K R P M N A F MV W A Q A T RR K L A D Q Y P H L H N A E L S K T L G K L W R LL S E N E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S G K N G Q S E S D SS G E Q T H A lligator-Sox9 SS K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E S E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K N G Q S E Q EE G S E Q T H Chicken-Sox9 SS K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E S E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K T V K N G Q S E Q EE G S E Q T H Human-Sox9 SS K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E S E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K N G Q A E A EE A T E Q T H Pig-Sox9 SS K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E S E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K N G Q A E A EE A T E Q T H M ouse-Sox9 SS K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E S E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K N G Q A E A EE A T E Q T H Frog-Sox9 SS K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E T E K R P F V EE A E R L R I Q H KK D H P D Y K Y Q P RRR K S V K N G Q S E Q ED G S D Q T H Sturgeon-Sox9 SS K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E G E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K N G Q N E A ED G P E Q T H Trout-Sox9 SS K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E G E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S M K N G Q S E S DD G S E Q T H M edaka-Sox9 ST K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E G E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K S GG S E A ED GG E -H Stickleback-Sox9A SS K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E G E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K N G Q S E S ED G S E Q T H Zebrafish-Sox9A SS K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E V E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K N G Q S E S ED G S E Q T H Pufferfish-Sox9 S N K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E V E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K N G Q N D P ED G E Q T H Stickleback-Sox9B SS K S K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E A E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K N G Q N D P ED G E Q T H Zebrafish-Sox9B S G K S K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E G E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K S G S A E S ED G E Q T Q Frog-Sox8 G K A K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL S E N E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K A G Q S D S D S G A E L G H Chicken-Sox8 S K A K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL S E N E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K A G Q S D S D S G A E L S H Rat-Sox8 GG K A K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL S E S E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K T G R S D S D S G T E L G H M ouse-Sox8 GG K A K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL S E S E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K T G R S D S D S G T E L G H Human-Sox8 GG K A K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL S E S E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S A K A G H S D S D S G A E L G P Pufferfish-Sox8 S K N K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL S E S E K R P F V DE A E R L R I Q H KK D H P D Y K Y Q P RRR K N A K P G Q S D S D S G A E L A H Zebrafish-Sox8 S G K S K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL T E S E K R P F V EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K P G H A E S E A G S E LM Q Rat-Sox10 A S K S K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E S D K R P F I EE A E R L R M Q H KK D H P D Y K Y Q P RRR K N G K AA Q G E A E C P D Q GG A M ouse-Sox10 A S K S K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E S D K R P F I EE A E R L R M Q H KK D H P D Y K Y Q P RRR K N G K AA Q G E A E C P E Q GG A Human-Sox10 A S K S K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E S D K R P F I EE A E R L R M Q H KK D H P D Y K Y Q P RRR K N G K AA Q G E A E C P E Q GG T Chicken-Sox10 S N K S K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E S D K R P F I EE A E R L R M Q H KK D H P D Y K Y Q P RRR K N G K A T Q G E G E G Q E A GG A Frog-Sox10 G S K S K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E N D K R P F I EE A E R L R M Q H KK D H P D Y K Y Q P RRR K N G K P S P G E G D G S A E GG A Pufferfish-Sox10B G S K N K P H V K R P M N A F MV W A Q AA RR K L A D Q H P H L H N A E L S K T L G K L W R LL N E S D K R P F I EE A E R L R K Q H KK D Y P D Y K Y Q P RRR K N G K P G S G E A D G H S E G E I Zebrafish-Sox10 G S K S K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E T D K R P F I EE A E R L R K Q H KK D Y P E Y K Y Q P RRR K N G K P G SS E A D A H S E G E V Pufferfish-Sox10A G N K A K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E T D K R P F I EE A E R L R K Q H KK D Y P D Y K Y Q P RRR K N G K LM AA E S D G Q G E G E A Lamprey-SoxE1 SS K C K P H V K R P M N A F MV W A Q AA RR K L A D Q Y P H L H N A E L S K T L G K L W R LL N E N E K R P F I EE A E R L R V Q H KK D H P D Y K Y Q P RRR K S V K G S G D G D AA S P C G Sea squirt-SoxE S Q K T K P H V K R P M N A F MV W A Q A A R R K L A D Q Y P H L H N A E L S K T L G K L W R LL S E T E KK P F V DE A E R L R I K H KK D H P D Y K Y Q P RRR K SS K T A S GG T Q G A L K QQ S
60 210 220 230 240 250 260 270 280 290 300 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Lamprey-Sox9 I TT N A I Y K A L Q A D S P ------S A D V H S P G E H S G Q S Q G PP T PP TT P K T D V Q S L D I K R E G -P L Q E GG -R QQ I D F S N V D I R E L S R E VI S N M E S F D V A lligator-Sox9 I S P N A I F K A L Q A D S P Q -SSSS M S E V H S P G E H S G Q S Q G PP T PP TT P K T D V Q P Q D L K R E G -P L Q E GG R Q PP H I D F R D V D I G E L SS D VI S N I E T F D V Chicken-Sox9 I S P N A I F K A L Q A D S P Q -SSSS I S E V H S P G E H S G Q S Q G PP T PP TT P K T D A QQ P Q D L K R E G -P L A E GG R Q PP H I D F R D V D I G E L SS D VI S N I E T F D V Human-Sox9 I S P N A I F K A L Q A D S P H -SSS G M S E V H S P G E H S G Q S Q G PP T PP TT P K T D V Q P A D L K R E G -P L P E GG R Q PP I D F R D V D I G E L SS D VI S N I E T F D V Pig-Sox9 I S P N A I F K A L Q A D S P H -SSS G M S E V H S P G E H S G Q S Q G PP T PP TT P K T D V Q P A D L K R E G -P L P E GG R Q PP I D F R D V D I G E L SS D VI S N I E T F D V M ouse-Sox9 I S P N A I F K A L Q A D S P H -SSS G M S E V H S P G E H S G Q S Q G PP T PP TT P K T D V Q A V D L K R E G -P L A E GG R Q PP I D F R D V D I G E L SS D VI S N I E T F D V Frog-Sox9 I S P N A I F K A L Q A D S P H -S A SS M S E V H S P G E H S G Q S Q G PP T PP TT P K T D V Q PP D L K R E G -P L Q E S G R Q PP H I D F R D V D I G E L SS E VI S N I E T F D V Sturgeon-Sox9 I S P T A I F K A L QQ A D S P H -S A SS M S E V H S P G E H S G Q S Q G PP T PP TT P K T D V Q AA D L K R E G -P L Q E GGG R Q P H I D F R D V D I G E L SS D VI S N I E T F D V Trout-Sox9 I S P G A I F K VL QQ A D S P ---A SS M G E M H S P G E H S G Q S Q G PP T PP TT P K T D I Q VV D L K R E G -P L H E G T G R Q L N I D F R D V D I G E L SS D VI S H I E T F D V M edaka-Sox9 I ST N A I F K A L QQ A D S P ---A SS M G E V H S P A E H S G S Q A PP T PP TT P K T D C S -M D L K R E G R P L P D G A G R Q L N I D F R D V D I G E L SS D VI S H I E T F D V Stickleback-Sox9A I S P N A I F K A L QQ A D S P ---A SS M G E V H S P G E H S G S Q G PP T PP TT P K T D V S S M D L K R E G R S L S D G T G R Q L N I D F R D V D I G E L SS D VI S H I E T F D V Zebrafish-Sox9A I S P N A I F K A L QQ A D S P ---A SS M G E V H S P S E H S G Q S Q G PP T PP TT P K T D T Q P A D L K R E A R P L Q E N T G R P L S I N F Q D V D I G E L SS D VI --E T F D V Pufferfish-Sox9 I S P N A I F K A L QQ A D S P ---A SS L G E V H S P G D H S G Q S Q G PP T PP TT P K T D LV S A D L K R E G -P M Q E G TS R Q L N I D F G A V D I G E L SS E VI S N M G S F D V Stickleback-Sox9B I S P N A I F K A L QQ V D S P ---A SSS G E V H S P G E H S G P S Q G PP T PP TT P K T D L P S A D L K R E G -P M Q E G TS R Q L N I D F G A V D I G E L SS E VI S N M G S F D V Zebrafish-Sox9B I ST N A L F R A L Q R A E T P ---D SST G E L H S P G E H S G Q S Q G PP T PP TT P K T D L P V C A D L K R E R ---R D R E P L Q D G I D F G A V D I G E L SS D VI S N I E A F D V Frog-Sox8 H P G S Q M Y K S D S G M G S M G -------E N H L H S E H A G Q N H G PP T PP TT P K T D L HH G QE L K H E G R MM D N G --R Q N I D F S N V D I N E L SS E VI S N I E A F D V Chicken-Sox8 H A G T Q I Y K A D S G L GG M A -------D G HHH G E H A G Q P H G PP T PP TT P K T D L HH G Q E L K H E G R LV E S G --R Q N ID F S N V D I S E L SS E VI NN M E T F D V Rat-Sox8 H P GG P M Y K T D T VL G ----------D A H R H S D H T G Q T H G PP T PP TT P K T D L H Q A Q E L R L E G R LV D S G --R Q N I D F S N V D I S E L SS E VI S N M D T FD V M ouse-Sox8 H P GG P M Y K A D A VL G ----------E A HHH S D HH T G Q T H G PP T PP TT P K T D L H Q A Q E L R L E G R LV D S G --R Q N I D F S N V D I S E L SS E VI S N M D T F D V Human-Sox8 H P GGG A V Y K A E A G L G ----------D G HHH G DH T G Q T H G PP T PP TT P K T E L QQ A P E L K L E G R P V D S G --R Q N I D F S N V D I S E L SS E VM G T M D A F D V Pufferfish-Sox8 H ----M Y K A E P G M GG L A G ---L T D A HHH A E H A G Q P H G PP T PP TT P K T DL HH G Q D L K H E G R LV D S G --R Q N I D F S N V D I S E L ST D VI S N M E T F D V Zebrafish-Sox8 H ----M Y K A E P G M G R L T ------G S P D H I T D H T G H T H G PP T PP TT P K T E H P Q A ----------------K Q N I D F S N V D I SE L ST D VI G N L T F D L Rat-Sox10 AA I Q A H Y K S A H L D H R H P EE G S P M S D G N P E H P S G Q S H G PP T PP TT P K T E L Q S G A D P K R D G R S L G E G -G K P H I D F G N V D I G E I S H E VMS N M E T F D V M ouse-Sox10 AA I Q A H Y K S A H L D H R H P EE G S P M S D G N P E H P S G Q S H G PP T PP TT P K T E L Q S G A D P K R D G R S L G E G -G K P H I D F G N V D I G E I S H E VM S N M E T F D V Human-Sox10 AA I QA H Y K S A H L D H R H P G E G S P M S D G N P E H P S G Q S H G PP T PP TT P K T E L Q S G A D P K R D G R S M G E G -G K P H I D F G N V D I G E I S H E VM S N M E T F D V Chicken-Sox10 A S I Q A HY K N A H L D H R H P G E G S P M S D G H P E H SS G Q S H G PP T PP TT P K T E L Q A G A D S K R E G R S L G E G -G K P H I D F G N V D I G E I S H E VM S N M E T F D V Frog-Sox10 A S I Q A H Y K N SH L D H R H ---G S P M S D G N S E H ST G Q S H G PP T PP TT P K T E L Q A G S D G K R D G H A L R E G -G K P Q I D F G N V D I G E I S H D VM S N M E T F D V Pufferfish-Sox10B S H S Q S H Y K G F H L D VV H S GGA G S P L A D G HH P H AA G Q S H S PP T PP TT P K T E P Q S GG D G K R E GGG S R ST V E S G K P H I D F G N V D I G E M S H E VMV N M E P F D V Zebrafish-Sox10 S H S Q S H Y K S L H L E V A H GG AA G S P L G D G HH PH A T G Q S H S PP T PP TT P K T E L Q GGG E G K R E G R S G L G V G A S G K P H I D F G N V D I G E I S H D VM A N M E P F D V Pufferfish-Sox10A S H S Q S H Y K T L H L E H N --G A G S P L DD L HHHHH P A G Q G H S PP TPP TT P K T E L Q S G S D P K R D G R G A L G V GG N A K P H I D F G A M D I G E I S H E VM S N I E P F D V Lamprey-SoxE1 A D P H GG I F K G V H G E GG S L ------G D P I S L S A H T G Q A Q S PP T PP ST P K T E Q G A KS QQQQQ H N Q L HHHH Q P A R Q H I D F S N V D M G E L SS E VI S N M E P F D V Sea squirt-SoxE G K V R K Q D S Q SS DE C Q G V QQ A LV A N P I S G K L SS H H S P Q S V C H S P S N Q S P Q G S I T N IM DE S KK P G S K QQ R G S LTS E C SS H S M H G VM D T N T Q D IM A K P G F D V 310 320 330 340 350 360 370 380 390 400 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Lamprey-Sox9 N E F D Q Y L PP N ------G H P G H G Q S ------------V AA S Y GG T G Y S I N ------G H A W L S K Q QQQQQQQQ H T L SS P R A H V K T E Q L S P S H Y S D QQQ A lligator-Sox9 N E F D Q Y L PP N ------G H P G V P A T ----H G Q P G Q V T Y T G S Y G I SST AA T P T --G A G H V W M S K Q P Q P Q AA H T M T P L S G R P H I K T E Q L S P S H Y S E QQ Chicken-Sox9 N E F D Q Y L PP N ------G H P GV P A T ----H G Q -V TT Y S G T Y G I SSS A SS P A --G A G H A W M A K -Q PP A Q H T L P ST E R R P H I K T E Q L S P S H N S E QQ Human-Sox9 N E F D Q Y L PP N ------G H P G V P A T ----H G Q --V T Y T G S Y G I SST AAT P A --S A G H V W M S K QQQQ P Q A H T L TT L SS R T H I K T E Q L S P S H Y S E QQ Pig-Sox9 N E F D Q Y L PP N ------G H P G V P A T ----H G Q --V T Y T G S Y G I SST AA T P A --G A G H V W M S K QQQQ P Q A H T L TT LSS R T H I K T E Q L S P S H Y S E QQ M ouse-Sox9 N E F D Q Y L PP N ------G H P G V P A T ----H G Q --V T Y T G S Y G I SST A P T P A --T A G H V W M S K QQQQQQ A H T L TT L SS R T H I K T E Q L S P S H Y S E QQ Frog-Sox9 N E F D Q Y L PP N ------G H P G V A ST ----Q V T----Y T G S Y G I SS AA GG P A --G A G H A W M P K Q -QQQQ H G L P T L S N R T H I K T E Q L S P S H Y S D QQQ Sturgeon-Sox9 N E F D Q Y L PP N ------G H P G V P A T N -S A H G Q S G Q V T Y T G S Y G I SSTS V P Q AA N V AG H A W M A K Q -QQQQ H S L P T L SS TT H I K T E Q L S P S H Y N E QQ Trout-Sox9 N E F D Q Y L PP N ------G H P G A P G A ---A T G Q --V S Y T G T Y G I SSS VV S Q AA GG A T G H S W M T K S -QQQQ H S L TT L G S G P T H IK T E Q L S P S H Y N D QQ S M edaka-Sox9 N E F D Q Y L PP N ------G H P G AA P G ------ST A P V S Y S G N Y S I S G A PP L S P Q A GGG P A W M A K A Q -QQ H S L T P L G T R T Q I K T E Q L S P S H Y S E QQ Stickleback-Sox9A N E F D Q Y L PP N ------G H P G --------------------------------L AG AAAA W L A K S Q G QQQ H T L T P L GG R T Q I K T E Q L S P S H Y T E QQ Zebrafish-Sox9A N E F D Q Y L PP N ------G H Q N ------------------------------A P Y A GG Y AA W M T K P S P Q SS Q L T P L N P TT H I K T E Q L S P S H Y N E QQ Pufferfish-Sox9 DE F D Q Y L PP H ------S H A G V S G A --P Q A G -----Y T G S Y G I SSSS V S Q AA G V G A Q A W M S K Q QQQQQ H S L T A L S GG P A Q I K T E Q L S P S H Y S E QQ Stickleback-Sox9B DE F D Q Y L PP H ------S H I G V TS ----SS G R -----Y A S G Y G I GG SS V G H AA N V G A H A W M S K Q QQ --H S L TT L GGG TT Q I K T E Q L S P S H Y S E QQ Zebrafish-Sox9B N E F D Q Y L PP H ------G A P G P A G A G -----------F SS G Y G -------------S AA W M H K P -----L A SSS M A N A A Q I K TE Q L S P G H Y S QQ P Frog-Sox8 H E F D Q Y L P L N G H G ---A I P A D H G Q N TT ----------AA P Y G P S Y P H AA G -A T P A P V W S H K SSSTSSS SS I E S G QQ R P H I K T E Q L S P S H Y N D Q S Q Chicken-Sox8 H E F D Q Y L PL N G H T ---A M P A D H G P G ------------A G F Y STS Y S H S AA G A GG A G Q V W T H K S P A S A S P SS A D S G QQ R P H I K T E Q L S P S H Y S D Q S H Rat-Sox8 H E F D Q Y L P L N G H S ---A L A T E P S Q A T A ----------SG S Y GG A S Y S H S G A T G I G A S P V W A H K G A P S A S A S P T E A G P L R P H I K T E Q L S P S H Y N D Q S H M ouse-Sox8 H E F D Q Y L P L N G H S ---A L P T E P S Q A T A ----------S G S Y GG A S Y S H S G A T G I G A S P V W A H K G A P S A S A S P T E A G P L R P Q I K T E Q L S P S H Y N D Q S H Human-Sox8 H E F D Q Y L P L GG-----P A PP E P G Q A ---------------Y GG A Y F H A G A S ----P V W A H K S A P S A S A S P T E T G PP R P H I K T E Q P S P G H Y G D Q P R Pufferfish-Sox8 H E F D Q Y L P L N G H TSSSS G L P S D Q PP A P -----------V G S Y A SS Y G H A GI N ---G S A W S R K G A M SSSS P SS G E V G Q H R L Q I K T E Q L S P S H Y S E H S H Zebrafish-Sox8 Q E F D Q Y L P L T P D Q ---------------------------------------------G A C S RR A PP A G A H ----L H P Q R V H I K T E Q R S P Q H Y S E H S Rat-Sox10 T E L DQ Y L PP N ------G H P G H V G S Y S -AA G ---------Y G L SS A L A V A S G H --S A W I S K PP V A L P T V S PP A V D A K A Q V K T E TT G P Q G PP H Y T D M ouse-Sox10 T E L D Q Y L PP N ------G H P G H V G S Y S -AA G ---------Y G L G S A L A V A S G H --S A W I S K PP V A L P T V S PP G V D A K A Q V K T E TT G P Q G PP H Y T D Human-Sox10 A E L D Q Y L PP N ------G H P G H V SS Y S --AA G ---------Y G L G S A L A V A S G H --S A W I S K PP V A L P T V S PP G V D A K A Q V K T E T A G P Q G PP H Y T D Chicken-Sox10 N E F D Q Y L PP N G H A ---G H P G H V GG Y A -AAA G --------Y G L G S A L AAA S G H --S A WI S K Q H V S L S A TTS P VV D S K A Q V K T E G S A P GG -H Y T D Frog-Sox10 N E F D Q Y L PP N G H A ---G H P S H I GG Y T -SS Y ----------G L T G A L AA G P ----S A W A L A K ----Q H S Q T V A D S K A Q V K T E SS --STSH Y T E Pufferfish-Sox10B N E F D Q Y L PP N ------G H P G V G Q T A G -AAAA V A G N P A S Y T Y G I SS A L AAA S G H --S AA W L S K QQ H Q HH G T P L G S D A S K A Q I K S E A GG T GG H F A E S A Zebrafish-Sox10 N E F D Q Y L PP N ------G HP Q A S A T A S -A G S AA P ---S Y T Y G I SS A L AAA S G H --ST A W L S K QQ P S QQ -H L G A D GG K T Q I K S E T H F P G ---D T A Pufferfish-Sox10A N E F D Q Y L PP N ------G H P Q S A T V A S -AA G SSTS --S Y A Y ---A L AAA S GH --S A W L S K QQ P Q A S -P SST D P S K A Q I K S E S A S G S H Y A E A S Lamprey-SoxE1 N E F D Q Y L P H S ----Q Y G Y T AAA V AA G ---W T A K L Q E Q E R P T H I K T E Q L S P S H Y S QQQQ AA V QQQQQQQQQQ P T I S Y G S FL Q N A R S QQQQ H Y E Y A E HH S Sea squirt-SoxE T E F E Q Y M P G A C N P A C N F T D TS Q N M P S P --------V D C S V QQ N P M F S P N K Q G T P Q Y R P SS W V E G Y E S P L Q SSS V TS Q S G EE L P V KK E Q I S P I QQ H FF P H
61 410 420 430 440 450 460 470 480 490 500 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| Lamprey-Sox9 QQQ P QQQQQ S P F S I Q H Y G AA VV P A I S R S Q Y S Y A D HHH AAA YY S G H S A Q T A G L Y S G F S Y M G P S Q R P S Y T P I A D A T G V P S I P Q P -H S P -P S W E Q P V A lligator-Sox9 Q H S P QQ I N Y SS F N L Q H Y SSS Y P T I T R S Q Y D Y T D H Q SS N S YY S H AA G Q STS L Y ST F T Y M N P T Q R P M Y T P I A D TS G V P S I P Q T -H S P -Q H W E Q P V Chicken-Sox9 Q H P QQQ L G Y G SF N L Q H Y G F S Y PP I T R S E Y D Y T E H Q N S G S YY S H AA G Q S G S L Y ST F T Y M N P T Q R P M Y T P I A D TS G V P T I P Q T -H S P Q Q H W E Q P V Human-Sox9 Q H S P QQ I A Y S P F N L P HY S P S Y PP I T R S Q Y D Y T D H Q N SSS YY S H AA G Q G T G L Y ST F T Y M N P A Q R P M Y T P I A D TS G V P S I P Q T -H S P -Q H W E Q P V Pig-Sox9 Q H S P QQ I A Y S P F N L P H Y S P S Y PP IT R S Q Y D Y T D H Q N S G S YY S H A R S Q G S VL Y ST F T Y M N P A H G P M Y T P I A D TS G V P S I P Q T -H S P -Q H W E Q P V M ouse-Sox9 Q H S P QQ I S Y S P F N L P H Y S P S Y PP I T R S Q Y D Y A D H Q N S G S YY S H AA G Q G S G L Y ST F T Y M N P A Q R P M Y T P I A D TS G V P S I P Q T -H S P -Q H W E Q P V Frog-Sox9 Q H S PQQ L N Y TS F N L Q H Y G ST Y P T I T R S Q Y D Y T E H Q G S N S YY S H AA G Q SSS L Y ST F S Y M N P S Q R P M Y T P I A D TT G V P S I P Q T -H S P -Q H W E Q P V Sturgeon-Sox9 H S P Q S I N Y G S F NL Q H Y SS A Y P T I T R S Q Y D Y S E H Q G A N S YY S H AA S Q G S G L Y ST F T Y M S R A -----------------------------------Trout-Sox9 SS PP Q H V N Y G S F N L Q H F SSS Y P S I T R G Q Y D F S D H Q G T N S YY S H T A G Q G S G L Y SF SS Y M S P S Q R P M Y T P I A D TT G V P S V P Q T -H S P Q HH W D QQ P V M edaka-Sox9 G S P Q N A P Y S P F N L Q H Y SSS Y PP I S R A QQ Y D Y P D P Q G -G F Y S P A G A Q G S G L Y ST F S Y M SS P S Q R P M Y T P I A D N A G V P S I P Q G --S P -Q H W E Q A P V Stickleback-Sox9A G S P Q HV A Y S P F N L Q H Y SS A Y P AA I S R A QQ Y D Y S D H Q G AA G YY S H A G A Q G S G L Y ST F S Y M SS P S Q R P M Y T P I A D TT G V P S I P Q S --S P -Q H W E Q A P V Zebrafish-Sox9A G S P Q H I S Y G S F N V QH L Q TS F P S I T R A Q Y D Y S D S H Q A SS YY T H A G Q SS G L Y ST F S Y M SS S Q R P M Y T P I A D ST G V P S I P Q S N H S P -Q H W D QQ P V Pufferfish-Sox9 G S P Q H V T Y G S F N L Q H Y SSS Y P S -M T R A Q Y D Y S D H Q G A N S YY S H AA G Q G S G L Y ST F S Y M N P S Q R P M Y T P I A D N A G V P S V P Q T -H S P -Q H W E QQ P I Stickleback-Sox9B G S P Q H V T Y G S F N L Q H Y SSS Y P S I T R A Q -Y D Y S D H Q G A N S YY S Q S -Q G S G L Y ST S A T C P S Q R P M Y T P I A D TT G V P S V P Q T -Q S P -Q H W E QQ P I Zebrafish-Sox9B --P QQQ F Y -------S A P Y ---S R A Q Y T E Y S E Q H S -A YY S P Y P -------T FS Y S R P ----P Y T P AAAA D T A H T ----------HH W D P Q P V Frog-Sox8 G S P S D Y N T Y A Q A C A TT V SS A T V P T A F P SS Q C D Y T D L P S S N YY N P Y S Y P SS L Y Q Y P Y F H SS -RR P Y A T P IL N S L ----S I PP S -H S P T S N W D Q P V Chicken-Sox8 G S P S D Y G S Y T Q A C A TT A ST A T AAA S F SSS Q C D Y T D L Q S S N YY N P Y P Y P SS I Y Q Y P Y F H SS -RR P Y A T P IL N G L ----S I PP A H S P T A N W D Q P V Rat-Sox8 G S P A D Y G S Y A Q A S V TT AA S A T AA SS F A S A Q C D Y T D L Q A S N YY S P Y P Y PP S L Y Q Y P Y F H SS -RR P Y A SS LL N G L ----S M PP A H S P S S N W D Q P V M ouse-Sox8 G S P A D Y G S Y A Q A S V TT AA S A T AA SS F A S A Q C D Y T D L Q A S N YY S P Y P Y PP S L Y Q Y P Y F H SS -RR P Y A S P LL N G L ----S M PP A H S P S S N W D Q P V Human-Sox8 G S P D Y G S C G QSS A T P AA P --A G P F A G S Q G D Y G D L Q A SS YY G A Y P Y A P G L Y Q Y P C F H S P -RR P Y A S P LL N G L ----A L PP A H S P T S H W D Q P V Pufferfish-Sox8 R S P S D Y G S Y S P A C V TS A TS AA S V P FS G S Q C D Y S D I Q S T N YY N P Y S Y SS G L Y Q Y P Y F H SS -RR P Y G S P IL N S L ----S M A P A H S P T G S G W D Q P V Zebrafish-Sox8 ----ST L Y SSS -------------SS A Q C E Y T E H S ---F Y S P Y S Y P --Y P Y P Y L HR --------P IL N -------I P A P H SSS A H W D P P V Rat-Sox10 Q P TS Q I A Y TS L S L P H Y G S A F P S I S R P Q F D Y S D H Q P S G P YY G H A G Q A S G L Y S A F S Y M G P S Q R P L Y T A I S D P S --P SG P Q S -H S P -T H W E Q P V M ouse-Sox10 Q P TS Q I A Y TS L S L P H Y G S A F P S I S R P Q F D Y S D H Q P S G P YY G H A G Q A S G L Y S A F S Y M G P S Q R P L Y T A I S D P S --P S G P Q S -H S P -T H W E Q P V Human-Sox10 Q P -TS Q I A Y TS L S L P H Y G S A F P S I S R P Q F D Y S D H Q P S G P YY G H S G Q A S G L Y S A F S Y M G P S Q R P L Y T A I S D P S --P S G P Q S -H S P -T H W E Q P V Chicken-Sox10 Q P TS Q I A YTS L S L P H Y G S A F P S I S R P Q F D Y P D H Q P S G P YY S H SS Q A S G L Y S A F S Y M G P S Q R P L Y T A I S D P A --P S V P Q S -H S P -T H W E Q P V Frog-Sox10 Q P TS Q L T Y TS L G L P HY G S A F P S I S R P Q F D Y A D H Q P SSS YY S H S A Q A SS L Y S A F S Y M G P P Q R P L Y T A I S D P ---P S V A Q S -H S P -T H W E Q P V Pufferfish-Sox10B S A G A H V T Y T P L S L P H Y SS A F P S FA S R A Q F A D Y A D H Q A S G S YY A H SS Q A S G L Y S A F S Y M G P S Q R P L Y T A I T D P ---A N V P Q S -H S P -T H W E Q P V Zebrafish-Sox10 S G S H V T Y T P L T L P H Y SS A F P S L A S R A Q FA E Y A E H Q A S G S YY A H SS Q TS G L Y S A F S Y M G P S Q R P L Y T A I P D P ---G S V P Q S -H S P -T H W E Q P V Pufferfish-Sox10A SS P G T H V T Y T P L S L P H Y G S A F P S L A S R P Q F D Y G D H QA P G A YY A H SS Q A P G L Y S A F S Y M G P T Q R P L Y TT I G D P ---SS V G P S -H S P -T H W E Q P V Lamprey-SoxE1 G A HH QQ L Q HH Q H ST AA VM SSSSSSSSSSSSSSSS P S AAAAA YY S Q M S G Q A S G L Y S F SS Y A G AA G Q C S L Y A P GGE AA P L H A S V A P AAA H S P -Q H W E Q P I Sea squirt-SoxE V PPP Y Q A Y F HH N A Y D H S GG R F D A C S A K QQ E M N L D S P T R H A A Y D S N I T N F S L P LI P L A S H Y P K P N E R QQ L Y S A P E G F S Y P H N Q H Y N -M A QQQQ N W P L P ST Figure 2-5. Alignment of inferred amino acid se quence for lamprey SOX9 with chordate SOXE proteins. The inferred amino acid sequen ce of the new lamprey Sox clone most closely matched those of the gnathostome SOX9 family. For example, this new lamprey sequence was 96% identical to human SOX9 within the HMG-box domain (highlighted here in yellow). Outside of this domain, the available SOXE proteins often showed considerable le ngth variation, thereby maki ng these regions difficult to align. Such problematic regions were removed prior to phylogenetic analyses (Swofford et al., 1996), resulting in the mu ltiple sequence alignment shown here with 495 aligned positions. In ME, gapped positions were subjected to complete deletion (i.e., the removal of their entire column in the multiple sequence alignment) to ensure uniform comparisons among all pairwise distances (Nei and Kumar, 2004). However, this approach also reduced the number of aligned positions for ME to 219 sites, thereby resulting in even less inform ation for the placement of the new lamprey sequence at the base of the SOX9 clade (as indicated by its low bootstrap score in Fig. 4). In the end, we based our current assignment of the new lamprey sequence on three different lines of evidence (Bowles et al., 2000; Koopman et al., 2004): (a) on the presence of a SOX9-signa ture motif (which is loca ted immediately 3Â’ to the HMGbox and is underlined in the figur e); (b) on the consistent phylogenetic placement of the lamprey protein to the base of the SOX9 clade (Figure. 2-6.); and (c) on its overlapping gene expression pattern w ith that of SOX9 in jawed vertebrates (see text).
62 Figure 2-6. Extended majority-rule consensus tr ee for the BP analysis of the chordate SOXE proteins. Numbers indicate pos terior probabilities for gr oups with >50% credibility and for those clades that are combinable with this first set. Branch lengths are proportional to the means of the posterior probability densities for their expected replacements per site. This tree is rooted by sea squirt SOXE. Equally and unequally weighted MP, ME, and ML also place the new lamprey sequence at the base of the SOX9 clade, with bootstrap scores of 86% 74%, 28%, and 75%, respectively. The surprisingly low bootstrap score for ME is re lated to our complete deletion of gapped positions in the pairwise distance calculations (Figure 2-5.). Despite this low score, the optimal ME phylogeny still supports as best a SOX9 assignment for the new lamprey protein and is therefore consiste nt with the other phylogenetic results.
63 Figure 2-7. Sox9 expression during lamprey devel opment. Expression of lamprey Sox9 in embryos at stages 23 (A) and 24 (B). Inset in (b) shows sagittal sec tion through hindbrain and pharyngeal arches. Arrows indicate Sox9 expression in streams of ne ural crest cells invading pharyngeal arches. C. Transverse section th rough trunk of embryo shown in (B). Note Sox9 expression in notochord, hypochord, ne ural tube, sclerotome and dor sal endoderm (asterisk). D. Sox9 expression in stage 26 lamprey head. E-G. Sections through the hindbrain (E), mid-trunk (F) and tail (G) at stage 26 show an an terior to posterior retraction of the Sox9 domain in the notochord. Note persistant expr ession in neural tube, pharyngeal arch mesenchyme, sclerotome and hypochord. Abbreviations: fb, forebrain; hb, hindbrain; h, hypochor d; mb, midbrain; n, notochord; nt, neural tube; op, optic placode; ot, otic placode ; pa, pharyngeal arches; s, sclerotome.
64 CHAPTER 3 HAGFISH AND LANCELET FIBRILLAR COLLA GENS REVEAL THAT TYPE II COLLAGEN-BASED CARTILAGE EVOLVED IN STEM VERTEBRATES Introduction The phylogenetic relationships of the vertebra tes were set up largely based on anatomical characters, particularly those of the skeleton. Th e skeletons of jawed vert ebrates (gnathostomes) are composed of cartilage and bone, which contai n high levels of COL2A1 and COL1A1 protein, respectively. By contrast, the ca rtilaginous skeletons of lampreys and hagfishes, the only extant jawless fishes (agnathans), have been reported to be non-collagenous and to contain instead the elastin-like proteins lamprin and myxinin (Wri ght et al., 2001). This difference in the histological matrices of vertebrate skeletons ha s led to the idea that type II collagen became the major structural component of gnathostome cartila ge after the divergence of these two lineages (Wright et al., 2001). However, this view was challenged by our recent report that lampreys have two Col2a1 orthologues and that both genes are ex pressed during chondrogenesis. Adult lamprey cartilage was also shown to be rich in COL2A1 protein (Chapter 2). Furthermore, we identified a lamprey orthologue of Sox9 a direct transcript ional regulator of Col2a1 in gnathostomes and showed that it is co-expressed with Col2a1 (Chapter 2). This revealed that the genetic pathway for vertebrate chondrogenesi s predated the divergen ce of lampreys and gnathostomes, although precisely how ear ly this characte r arose is unknown. Here we investigate the evol utionary origin of COL2A1-b ased cartilage by expanding our analysis to the most inclusive cl ade of vertebrates, which includes the hagfishes, and to a sister group to the vertebrates, the lancelets (Donoghue et al., 2000; Ze ng and Swalla, 2005). We report that hagfishes also have COL2A1-based cartilage suggesting that this type of cartilage was present in common ancestor of all crown vertebrate s. Our analysis of lancelets revealed the presence of an ancestral clade A fibrillar coll agen (COLA) gene that is expressed in the
65 notochord. Thus, during the chordate-vertebrat e transition, an ancestral Clade A fibrillar collagen gene underwent duplication and diversif ication, and this process may underlie the evolutionary origin of ve rtebrate skeletal tissues. Materials and Methods Animals Adult lancelets (Branchiostoma floridae) were collected in Tampa Bay, FL. and spawning was induced in the lab, according to published methods (Stokes and Holland, 1995). Atlantic hagfishes ( Myxine glutinosa ) were purchased from Wards Na tural Science (Rochester). Gene Cloning RNA was extracted using TR Izol Reagent (Invitrogen). cDNA was made by reverse transcription reactions using S uperScript II first-strand cDNA synt hesis kit (Invitrogen). Fibrillar collagen genes were amplified with degenera te primers designed using CODEHOP program ( http://bioinformatics.weiz mann.ac.il/ blocks/ codehop.html ) (Rose et al., 2003). PCR reactions were carried out using BD Advantage 2 PCR enzyme system (Clontech) from cDNA. In 50l reactions containing 1 l forward (5Â’-GGCCCTCCCGGC CTGcarggnatgcc-3Â’) and 1 l reverse (5Â’GGGGCCGATGTCCACGccraaytcyt g-3Â’) primers (20pmol/ l), 5 l 10 buffer, 1 l cDNA template, 1 l dNTP at 10mM (each), 1 l Taq polymerase mixture and 40 l of double distilled water. Reactions were amplified as follows: 94 -1 min followed by 35 cycles of 94 -45 sec, 65 -45 sec, 68 -3min, and a final 10-min elongation at 68 PCR products were purified using QIAGEX II gel extraction kit (Qiagen) then cloned into pCRII-TOPO (Invitrogen) for sequencing. Hagfish COL2A1 and lancelet Am phiCOLA sequences have been submitted to GenBank, under accession numbers DQ 647926 and DQ647925, respectively. Sequence Analysis and Molecular Phylogenetics Inferred protein sequences for hagfish and am phioxus cDNAs were initially assigned to the clade A fibrillar collagen families on the basis of BLAST searches and conserved domains. These preliminary assignments were followed by estimates of their amino acid identities and
66 phylogenetic relationships. Multiple sequence al ignments for available fibrillar A, B and C collagen proteins, including the new hagfish and amphioxus sequences, were generated with CLUSTAL X. Phylogenetic analyses of these mu ltiple protein alignments were conducted with Bayesian phylogenetics (BP), maximum lik elihood (ML) and mini mum evolution (ME) methods, as described pr eviously (Chapter 2). The following amino acid sequences were retr ieved from GenBank for inclusion in our phylogenetic analyses: Mouse Col2a1: B 41182; Rat COL2A1: NP_037061; Dog COL2A1: NP_001006952; Chick COL2A1: NP_989757; Horse COL2A1: T45467; Salamander COL2A1: BAA82043; Frog COL2A1: B40333; Zebrafish Col2a1:XP_692723; Lamprey COL2A1a: DQ136024; lamprey COL2A1b: DQ136025; Huma n COL1A1: BAD92834; Mouse COL1A1: CAI25880; Dog COL1A1: NP_001003090; Cow COL1A1: AAI05185; Salamander COL1A1: BAA36973; Frog COL1A1: BAA29028; Halibut COL1A1: BAD77968; Zebrafish Col1a1: AAH63249; Rainbow trout Col1a1: BAB 55661; Human COL1A2: AAH42586; Mouse COL1A2: NP_034060; Dog COL1A2: NP_001 003187; Chick COL1A2: XP_418665; Frog COL1A2:AAH49287; Salmon COL1A2: BAB7 9229; Halibut COL1A2: BAD77969; Fugu COL1A2: CAG11117; Zebrafish Col1a2: NP_892013; Human COL3A1: AAL13167; Mouse COL3A1: NP_031763; Dog COL3A1: XP_851009; Cow COL3A1: XP_588040; Frog COL3A1: AAH60753; Human COL5A2: NP_000384; Mouse COL5A2: NP_031763; Rat COL5A2: XP_343565; Dog COL5A2: XP_535998; Co w COL5A2: XP_581318; Pig COL5A2: BAD91584; Chick COL5A2: XP_421846; Sea urchin: NP_999675. acorn worm DQ233249; Human COL5A1: NP_000084; Mouse COL5 A1: NP_056549; Chick COL5A1: NP_990121; Human COL5A3: NP_056534; Mouse COL5A3 : P25940; Human COL11A1: NP_001845; Mouse COL11A1: NP_031755; Chick COL11A 1: XP_422303; Human COL11A2: CAA20240;
67 Mouse COL11A2: NP_034056; Human CO L24A1: NP_690850; Mouse COL24A1: NP_082046; Chick COL24A1: XP _422363; Human COL27A1: NP_116277; Mouse COL27A1: NP_079961; Chick COL27A1: XP_415514. The tunica te fibrillar collage n genes (clade A fibrillar collagen: ci0100150759; clade B fibrilla r collagen: ci0100154301) were retrieved from the JGI Ciona intestinalis genome website (http: //genome.jgi-psf.org/ciona4/ciona4. home.html). Histology, Immunohistochemistry and In Situ Hybridization Formalin-fixed hagfish specimens were washed in 70% ethanol, embedded in paraffin and sectioned (6 m). Sections were either used for imm unohistochemistry or were stained with hematoxylin, eosin and fast green (MalloryÂ’s trichrome) using standard staining methods. Immunohistochemical staining for COL2A1 was carried out using an antibody against human COL2A1 protein, as described prev iously (Chapter 2) Whole-mount in situ hybridization of amphioxus embryos was performed following published methods (Shimeld, 1997). Results Identification of Col2a1 in Hagfishes We searched for an expressed Col2a1 orthologue in hagfish using degenerate RT-PCR, and recovered a 2214bp cDNA fragment with a seque nce that corresponds to the region between the major triple helix and the C-propeptide dom ains of gnathostome COL2A1. The deduced amino acid sequence of the hagfish clone was 76% identical to mouse COL2A1, and comparison with the two lamprey COL2A1 orthologues showed 80% identity to lamprey COL2A1a and 77% identity to lamprey COL2A1b. We next c onducted molecular phylogenetic analyses using Bayesian phylogenetics (BP) minimum evolution (ME) and maximum likelihood (ML) methods. All three analyses plac ed the hagfish sequence within the vertebrate Col2a1 clade, supporting the designation of this gene as hagfish Col2a1 (Figure 3-1, 3-2 and 3-3). Interestingly, each tree further positioned hagfis h COL2A1 as a sister to lamprey COL2A1a,
68 with lamprey COL2A1b falling out as the sister branch (Figure 3-1, 3-2 and 3-3). The topology of these trees suggests that Col2a1 was present in the common ancestor of agnathans and gnathostomes, and that an addi tional duplication gave rise to Col2a1a and Col2a1b in the cyclostome (lampreys + hagfishes) lineage. COL2A1 Localizes to Hagfish Soft Cartilage We next asked whether Col2a1 is expressed in hagfish cartil age. Little is known about hagfish embryonic development because of difficulti es in obtaining fertilized embryos (Ota and Kuratani, 2006; Powell, 2005; Wicht and Northcutt, 1995). Eptatretus embryos were last collected in 1930 (Wicht and Northcu tt, 1995) and only three embryos of Myxine have ever been found (Ota and Kuratani, 2006; Powell, 2005). The una vailability of embryos therefore precludes analysis of hagfish Col2a1 expression during development, however COL2A1 protein is known to be detectable in adult cartilage of gnathostomes and lampreys (Hamerman, 1989). We therefore investigated whether COL2A1 is present in hagfish cartilage using a human antibody against the highly specific N-terminal region of COL2A1 (Cotrufo et al., 2005; Guo et al., 2004; Yang et al., 2003). Immunohistoche mical analysis revealed the presence of COL2A1 in the extracellular matrix of several cartilage elements in the hagfish head and tail (Figure 3-4). COL2A1 protein was also detected in the notocho rd (data not shown), which is known to contain collagen fibers (Welch et al., 1998 ). Although a number of cartila ge elements in hagfish were rich in COL2A1 protein (Figure 3-4 AÂ’ and BÂ’), others showed a mosaic distribution (Figure 3-4 CÂ’) or lacked COL2A1 altogether (Figure 3-4 DÂ’). Hagfishes ha ve been described as having two types of cartilage; Â“softÂ” cart ilage contains large hypertrophic chondrocytes that stain with hematoxylin (blue) and are surrounded by a thin ex tracellular matrix, whereas Â“hardÂ” cartilage contains smaller chondrocytes th at are surrounded by an abundance of extracellular matrix (Cole, 1905a; Robson et al., 2000). Cartilage elements th at were positive for COL2A1 had the cellular
69 characteristics of Â“softÂ” cartilage (Figure 3-4 A, AÂ’, B and BÂ’), whereas cartilages that lacked COL2A1 exhibited the features of Â“hardÂ” cartilage (Figure 3-4 D and DÂ’). Elements that showed a mosaic distribution of COL2A1 also showed a mosaicism of Â“hardÂ” and Â“softÂ” features, and COL2A1 was restricted to the Â“softÂ” regions of these structures (Figure 3-4 C and CÂ’). Hagfish Â“softÂ” cartilage has been proposed to be struct urally similar to lampre y cartilage (Robson et al., 2000), and our finding that each is composed of COL2A1 protein supports this idea. The presence of COL2A1 in the cartil ages of hagfishes, lampreys (C hapter 2) and gnathostomes (Yan et al., 1995) strongly suggests that their last common an cestor had a COL2A1-based endoskeleton. Duplication and Divergence of Clade A Fibr illar Collagen Genes Occurred in Stem Vertebrates COL2A1 belongs to the clade A fibrillar collag en family, which includes collagen types I, II, III and Va2 (Aouacheria et al., 2004). In order to investigate the relationship between evolution of the clade A collagens and the origin of the vertebrate sk eleton, we extended our analysis to lancelets, a sister group to the vertebrates (Zeng and Sw alla, 2005). Comparative studies of a multitude of genes in lancelets a nd vertebrates show that the lancelet lineage diverged prior to the duplication events that occurred in the vertebra te genome (Panopoulou et al., 2003). We screened for lancelet fibrillar collagen cDNAs using degenerate RT-PCR, and isolated a 2196bp clone with a deducted amino aci d sequence that is 54% identical to mouse COL2A1 and 53% identical to mouse COL1A1 a nd COL3A1. To determine the relationship of the lancelet clone to the vertebrate fibrilla r collagen proteins, we carried out molecular phylogenetic analyses using C-propeptide of clad e A, B and C fibrillar collagens. BP, ML and ME methods each supported its designa tion as a clade A fibrillar collagen ( AmphiColA ; Figure 35). We then used the C-propeptide and triple helix domains to further refine the position of
70 AmphiColA within the chordate clade A family, and all three methods pl aced it as the sister clade to the vertebrate (incl uding cyclostome) clade A collagens (F igure 3-1). These results suggest that duplication of the ancestral ColA, the precursor of the clade A fibrillar collagen multi-gene family, occurred in the vertebrate lineag e after the divergence of lancelets. AmphiColA is Expressed in the Lancelet Notochord and Neural Tube To gain insight into the expr ession pattern of the ancestral ColA gene, we investigated the developmental expression of AmphiColA by RNA in situ hybridization on lancelet embryos. During neurulation, AmphiColA was expressed in the notochord (Figure 3-6 A). At 30 hr postfertilization, AmphiColA remained in the notochord, and a new domain of expression was detected in the neural tube (Figure 3-6 B). By 36 hr, AmphiColA expression was being downregulated in the middle third of the ne ural tube and notochord, but remained strong anteriorly and posteriorly (Figur e 3-6 C). In 5-day old larvae, AmphiColA was detected in the tail bud and in the anterior region of the neural tube, up to the base of the cerebral vesicle (Figure 3-6 D). Expression of AmphiColA in the lancelet notochord and neur al tube is strikingly similar that of clade A fibrillar collagens in gnathostomes and lampreys. We did not detect AmphiColA in the embryonic and larval pharynx by in situ hybridization, although Rychel et al showed that the gill bars of adult cephalochordates can be stained with a chicken COL2A1 antibody (Rychel et al., 2006). Our phylogenetic analyses indicate that AmphiColA is not a strict orthologue of COL2A1, but rather is a sister to the entire vertebrate COLA family, and whether the COL2A1 antibody can recognize AmphiColA protein is unclea r. Nonetheless, our results cannot exclude the possibility that AmphiColA is activated in the pharynx dur ing metamorphosis, when the gill region undergoes extensive remodeling.
71 Discussion COL2A1-Based Cartilage is a Shared Ch aracter of Crown-group Vertebrates Type II collagen based cartilage has long been considered a unifying character of gnathostomes that separates them from lampreys and hagfishes (Wright et al., 2001; Wright and Youson, 1983). Recently, we reporte d that lampreys possess two Col2a1 orthologues, and that adult lamprey cartilage is composed of COL2A1 protein (Chapter 2). He re we have gone on to show that hagfishes, the sister group to lampreys, also possess a Col2a1 orthologue, and we demonstrate that COL2A1 protein is localized to their soft ca rtilage. Taken together, these results suggest that the common ancestor of all crown-group (the livi ng jawed and jawless) vertebrates had COL2A1-based cartilage. Pres ence of an undifferentiated clade A fibrillar collagen in lancelets and tunicates (Wada et al ., 2006) suggests that the expansion of the COLA gene family occurred in stem ve rtebrates after the divergence of lancelets and tunicates. Thus, COL2A1-based cartilage is a synapomor phy of all crown-group vertebrates. Our finding that hagfish Â“hardÂ” cartilage l acks COL2A1 indicates that hagfishes also possess some nonCOL2A1 based cartilage. This finding highlights a relationship between the profile of collagen expressi on and the structure of skeletal tissue s in hagfishes. In gnathostomes, cartilage matrix is composed predominantly of COL2A1 whereas bone matrix is mostly COL1A1. During endochondral o ssification, which involves the transition from cartilage to bone, COL2A1 is replaced by COL1A1 in the skel etal matrix. Our observation that a single skeletal element in the hagfish can have a mosaic structure (both Â“softÂ” a nd Â“hardÂ” cartilage) that corresponds to mosaic distribution of COL2A1 raises the question of whether one type of cartilage may develop from the other, perhaps by altering the proportion of COL2A1 relative to other types of collagen or non-collagen matrix proteins. Alternativel y, Â“softÂ” and Â“hardÂ” cartilage elements may arise from distinct chondrocyte lineages (COL2A1-positive and
72 negative), with mosaic cartilages having a mixed lineage. Further charac terization of hagfish hard cartilage will clarify whether it is composed of other types of collagen or whether this subset of the skeleton is entirely non-collagenous. Did vertebrate Chondrocytes Evolve from the Notochord? It has been suggested that the notochord may represent a primitive form of cartilage, based on their many shared structural, cellular and molecular properties, and that vertebrate chondrocytes may have evolved from notochordal cells (Cole a nd Hall, 2004a; Stemple, 2005). In gnathostomes, the notochord and/or notochordal sh eath expresses most of the vertebrate clade A fibrillar collagen genes (Dubois et al., 2002; Gh anem, 1996; Yan et al., 1995; Zhao et al., 1997). Our finding that AmphiColA is expressed in the notoc hord and notochordal sheath of Branchiostoma floridae taken together with the recently reported data on Ciona intestinalis CiFCol1 and Branchiostoma belcheri BbFCol1 (both clade A fibrillar collagens), supports the idea that an ancestral ColA gene was expressed in the not ochord of stem-group chordates (Robson et al., 2000; Satou et al., 2001; Wada et al., 2006). We suggest that duplication and divergence of the clade A collagen genes in st em-group vertebrates may have facilitated the evolutionary origin of chondrocytes from notochor dal cells. This hypothesis deals specifically with the origin of vertebrate c hondrocytes, and it is important to note that cartilage is also found in several invertebrates, incl uding cephalopods, snails, and ho rseshoe crabs (Cole and Hall, 2004a; Wright et al., 2001). Future work on the molecular basis of invertebrate chondrogenesis should reveal whether fibrillar collagens also we re utilized in these i ndependent evolutionary events. Clade A fibrillar Collagen Duplication Facilitat ed Evolution of the Vertebrate Skeleton The Identification of an undifferentiated, ancestral ColA gene in lancelets indicates that the duplication event that gave rise to Col2a1 occurred after the divergence of lancelets and
73 vertebrates. We have focused our analysis on hagfishes and la ncelets, however it has recently been suggested that tunicates are the closest sister group to verteb rates (Delsuc et al., 2006). As noted above, tunicates also have an undifferentiated ColA gene (Wada et al., 2006), indicating that the duplication also post-da tes their divergence from the lin eage leading to vertebrates. Taken together, the data show that the origin of Col2a1 from the ancestral ColA gene must have occurred in stem vertebrates. We propose that this duplication event may have been critical for the origin of the vertebra te skeleton (Figure 3-7). Our data fit with the hypothe sis that at least one round of genome duplication occurred between the origin of chordate s and the origin of vertebrate s (Boot-Handford and Tuckwell, 2003; Panopoulou and Poustka, 2005). The expansi on of the clade A fibr illar collagen gene family, particularly the origin of Col2a1 and Col1a1 may account for the unique skeletal matrices of vertebrate cartilage and bone. A lthough cartilage has evolved multiple times in metazoa, it is unclear from the fossil record whether cartilage or bone evolved first in the vertebrates (Donoghue et al., 2006a). Our results raise the possibility that Col1a1 and Col2a1 arose from the same duplication event, and thus, the major matrix components of bone and cartilage may have evolved at the same time. Finally, it is noteworthy that clade A fibrillar collagens are physically linked with Hox gene clusters in verteb rates and in echinoderms (Cameron et al., 2006). Duplicati on of the clade A fibrillar colla gen genes therefore may have coincided with the Hox cluster duplications (Bailey et al ., 1997; Morvan-Dubois et al., 2003). Coordinated expansion of the ColA and Hox gene families, respectively, may have facilitated the diversification of verteb rate connective tissue types and provided a mechanism for differentially patterning them.
74 Figure 3-1. Extended majority-rul e consensus tree for the Bayesi an phylogenetic analysis of clade A fibrillar collagen proteins. Nu mbers at each node indicate posterior probability values based on 1,000,000 replicates Branch lengths are proportional to means of the posterior probability (pp) de nsities for their expected replacements per site. The tree is rooted by tunicate ( Ciona intestinalis) clade B fibrillar collagen (COLB) and sea urchin fibrillar collagen (COLP2a). Hagfish COL2A1 (boxed) is grouped with lamprey COL2A1a and COL2A1b with a pp of 0.99. This cyclostome COL2A1 clade joins to the base of the gna thostome COL2A1 clad e with a pp of 0.96. Lancelet clade A fibrillar collagen (Amphi ColA, boxed) is joined to the vertebrate clade A collagen family with a pp of 0.99. ML and ME methods confirm these positions for hagfish COL2A1 and AmphiColA (see also Figure 3-2. and 3-3).
75 Figure 3-2. Maximum likelihood ph ylogeny of fibrillar collagen A proteins, as obtained with WAG plus gamma distances. Numbers indicate bootstrap values for each node, based on 1000 replicates. Branch lengths are propor tional to expected replacements per site. The tree is rooted by tunicate ( Ciona intestinalis) clade B fibrillar collagen (COLB). Consistent with BP analysis, the hagfish sequence is placed within the COL2A1 clade, as a sister of lamprey CO L2A1a. The lancelet clade A fibrillar collagen (AmphiColA) joins to the root of the vertebrate clade A fibrillar collagens.
76 Figure 3-3. Minimum evolution phy logeny of fibrillar collagen A proteins as obtained with WAG plus gamma distances (a=0.906). Nu mbers indicate bootstrap values for each node, based on 10,000 replicates. Branch le ngths are proportional to expected replacements per site. Th e tree is rooted by tunicate ( Ciona intestinalis) clade B fibrillar collagen (COLB). The phyloge netic positions of hagfish COL2A1 and AmphiCOLA are consistent with the BP a nd ML analyses shown in Figure 3-1. and 3-2..
77 Figure 3-4. COL2A1 in cranial and tail fin cartilages of Atlantic hagfish. Transverse s ections through adult hagfish stained with hematoxylin, eosin and fast green (A-D) or with COL2A1 antibody (AÂ’-DÂ’). (A, AÂ’) Â“SoftÂ” cranial cart ilage stains with hematoxylin, contains large hypertrophic c hondrocytes surrounded by thin layer extracellula r matrix (A), and is positive for COL2A1 (AÂ’). (B, BÂ’) Section through caudal fin shows cartilaginous fin rays with Â“softÂ” cartilage characteristics (B) that are rich in COL2A1 (BÂ’). (C, CÂ’) Cranial cartilage element exhibiting a mosaic distribution of Â“softÂ” (blue-stained chondrocytes) and Â“hardÂ” (red-stained chondroc ytes) cartilage (C). Note that COL2A1 protein is restricted to the Â“softÂ” cartilage region (CÂ’). (D, DÂ’) Â“HardÂ” cranial cartilage stains with eosin but not hematoxyli n, contains small chondrocytes surrounded by thick layer of extracellular matr ix (D), and is negative for COL2A1 (DÂ’).
78 Figure 3-5. Unrooted extended majority-rule consensus tree for the Bayesian phylogenetic analysis of fibrillar A, B and C collagen proteins. Numbers at each node indicate posterior probability values based on 1,000,000 replicates. Branch lengths are proportional to means of the posterior probabi lity (pp) densities for their expected replacements per site. AmphiCOLA joins the fibrillar collagen A clade, which is separated from the B and C clades with a pp of 1.00. ML and ME methods support this grouping of the clad e A proteins, with bootstrap values of 97% and 85%, respectively.
79 Figure 3-6. Expression of AmphiColA during lancelet development. Whole-mount in situ hybridizations of lancelet embryos with an AmphiColA antisense riboprobe. Anterior is to left in A-C and to the right in D. Stages shown are 18 hr (A), 30 hr (B), 36 hr (C) and 5 days (D) post-fertili zation. Inset in A shows tr ansverse section of 18 hr embryo. Arrowhead indicates notochord and arrows indica te neural tube.
80 Figure 3-7. Origin of type II co llagen-based cartilage. Phylogenet ic relationships of tunicates, lancelets, hagfishes, lampreys and gn athostomes (Blair and Hedges, 2005; Donoghue et al., 2006b). Dashed lines indicate alte rnative branch positions (Delsuc et al., 2006). Letters indicate major steps in Co l2a1-based cartilage evolution. Solid rectangle indicat es origin of Col2a1 gene by ColA gene duplication(s). Hollow rectangle indicat es origin of Col2a1a and Col2a1b by Col2a1 gene duplication A. Ancestral clade A collagen gene expression in notochord of stem chordates. B. Origin of COL2A1 based cartilage in stem vertebrates. C. Subfunctionalization of Col2a1, in which expression domain of ancestral gene is partitioned between the duplicate genes (Col2a1a and Col2a1b) ; arrows indicate possible positions of this event.
81 CHAPTER 4 MOLECULAR IDENTIFICATION OF A SCLEROTOME IN LAMPREYS AND SHARKS: IMPLICATIONS FOR THE ORGIN OF THE VERTEBRAL COLUMN Introduction The vertebral column is one of the key charac ters that define vert ebrates. A conserved feature of gnathostome vertebrae is the general design of a centrum, the central of the vetrebrae, a dorsal pair of neural arches surrounding th e spinal cord, and a dorsal neural spine. Actinopterygians and chondrichthyans also possess a ventral pair of hemal arches and a hemal spine. In tetrapods, the vertebra l column is differentiated into fi ve regions: cervical, thoracic, lumbar, sacral and caudal. The structure and num ber of vertebrae shows a great diversity across taxa (Figure 4-1A). The phylogene tic transitions of axial struct ures are summarized in Figure 41A. This diversity is not only of interest for taxonomic purposes but also fascinating for understanding vertebrate morphological novelties from perspective of the phylogenetic change. The notochord serves as the primary functiona l axial supporting struct ure in all chordate embryos, although its fate is strikingly different in adults of different taxa. In chondrichthyans and bony fishes, the notochord pers ists, giving rise to the centra, whereas in amniotes does not contribute to vertebrae but instead forms a component of the intervertebral disk. Embryonically, vertebrae develop from the sclerotome, the ventromedial part of somites. Most of our knowledge of vert ebral developmental mechanisms derives from studies in amniotes, especially in chicken and mice (Chris t et al., 2004; Christ et al., 2007; Scaal and Christ, 2004; Scaal and Wiegreffe, 2006). The para xial mesoderm is segmented into somites, and somites are further divided into two part s, ventromedial sclerotome, which forms the vertebrae, and dorsolatera l dermomyotome which give rises to the dermis and skeletal muscle of the trunk and limbs (Figure 4-1B). Ventromedi al cells lose their epithelial characters aggregating as loose mesenchyme of the sclerotome. These sclerotomal cells migrate toward and
82 envelop the notochord, where they develop into the vertebral bodies and the connective tissue. The lateral sclerotomal cells contribute to the ne ural arches and ribs (Brand-Saberi and Christ, 2000). This specific subdivision of the somites depends on the relative positions to notochord and neural tube (Dockter and Or dahl, 2000; Pourquie et al., 1993). Based on grafting experiments in chicken, it was reported that there are two midline signaling centers which are importa nt in designating vertebral cell fates. The ventral signaling center consists of the floor plate of neural tube and notochord, whic h secrete the morphogen protein sonic hedgehog (Shh) and Noggin. The dorsal signaling center is located in the dorsal neural tube and surface ectoderm, which both secrete bone morphogen proteins (BMPs) and Wnts (Dietrich et al., 1997; Dockter and Orda hl, 2000; Monsoro-Burq and Le Douarin, 2000). Shh and Noggin signals promote sclerotome cell fate, whreas BMPs and Wnts have the opposite functions, thus the dorsalizing and ventralizing si gnals work antagonistical ly (Dietrich et al., 1997). The responses of the somite cells to th e two signal centers are dose dependent, such as the cells close to notochord are induced to scle rotomal fate. The dorsal domain of Bmp also functions to induce differentiation of the sp inous processes (Monsor o-Burq et al., 1994; Monsoro-Burq et al., 1996; Watanabe et al., 1998). Sclerotome is defined both by its mesenchymal organization and expression of a network of sclerotomal genes (Chris t et al., 2007). Two group I Pax genes, Pax1 and Pax9 are expressed in similar, though not identical patterns. The Pax1 first is detectable just before the deepithelialization in the early developing ventral sclerotomes. This makes Pax1 an identical sclerotome marker. Pax9 expression begins a little bit later than Pax1 As the sclerotome differentiates, Pax1 and Pax9 are limited to the ventromedial and ventrolateral compartments of sclerotome. Both genes are downregulated once the sclerotomal cells undertake the
83 chondrogenenic process (Peters et al., 1995; Peters et al., 1999) Knockout mouse experiments have shown that Pax1 is required for vertebral development, but Pax9 is not necessary. Mice null for both Pax1 and Pax9 showed more severe vertebral de fects than the individual knockouts (Peters et al., 1998; Peters et al., 1999). Similarly, another pair of homeobox transcription factors, NK3.1 and NK3.2 / Bapx1 have indistinguishable expr ession patterns in the early sclerotome (Herbrand et al., 2002). Nk3.2 is first detected at similar stages as Pax1 in the mouse. Bapx1 knockout mice show a similar phenotype to Pax1 and Pax9 double knockout mice (Lettice et al., 1999; Tribioli and Lufk in, 1999). Later, it was shown that Bapx1 is the direct target of Pax1/9 (Rodrigo et al., 2003). The expression group I Pax genes and Nk3 genes in sclerotome have been shown to be Shh -dependent (Dietrich et al., 1993; Kos et al., 1998; Koseki et al., 1993; Murtaugh et al., 2001; Neubuser et al., 1995). Bapx1 is a key transcription factor that links both the development of the sclerotome and its later chondrogenesis, and it plays major roles in regulating Sox9 and Runx2 both themselves regulators of chondrogenesis (Lengner et al., 2005; Zeng et al., 2002). The transcription factor Sox9 is expressed in a ll chondroprogenitors and has an essential role in chondr ogenesis, as forced expression of Sox9 can induce chondrogenic commitment in mouse. Sox9 can bind the promoter of Col2a1, aggrecan and other cartilage matrix genes (Bi et al., 1999; Lefebvre et al., 1997). In addition to the Group I Pax genes, Nk3 genes and chondrogenic genes, there are also some other widely used molecular markers of these sclerotome, including the bHLH transcription factors FoxC1 and FoxC2 and the T-box gene Tbx18 (Brand-Saberi and Christ, 2000; Dockter, 2000; Lettice et al., 2001). The FoxC1 and FoxC2 compound null mice have been shown to lack somites, as the paraxial markers Paraxis, Pax1 and Meox1 cannot be detected (Kume et al., 2001). Twist, Scleraxis and Paraxis are closed basic helix-loop-helix
84 (bHLH) transcription factors expressed in the developing somites. In chickens and mice, Scleraxis is expressed in a subset of the sclerotome, which was recently given the name syndetome because it gives rise to tendon cells (Brent et al., 2003). The detailed regulatory relationships between these sclerotome ma rkers are summarized in Figure 4-1C. Lampreys, one of only two extent jawless fi shes, possess paired ca rtilaginous nodes known as arcualia on either side of the notochord a nd the have been proposed to be homologs of the primitive axial skeleton (Gadow and Abbott, 1895; Gadow, 1933). No vertebral centra have been reported in lampreys, although the primitive gnathostome vertebral column is usually thought to consist of a persistent notochord and cartilaginous centra (Goodrich, 1930; Romer, 1985). The embryonic origin of these arcualia re mains unclear. It was reported that Japanese lamprey somitic mesoderm does not express the group I Pax gene LjPax9, and this result suggested that lampreys may lack a sclerotome (Ogasawara et al., 2000). We recently found the lamprey cartilage matrix genes ( Col2a1a and Col2a1b ) and the chondrogenic regulatory gene ( Sox9 ) expressed in the lamprey paraxial mesode rm (Zhang et al., 2006). This conserved chondrogenic genetic pathway in somites raises th e possibility that the arcualia may develop from a sclerotome in lamprey. In order to te st our hypothesis, here we cloned several members of the sclerotome gene network from both lamp rey and catshark. By comparing the expression patterns of these scletome markers in both sp ecies, we propose lampreys indeed possess a sclerotome, and that acquisition of sclerotome may have coinci ded with the origin of the primitive cartilaginous axial skeleton. Materials and Methods Fish Embryos Lesser spotted catshark ( Scyliorhinus canicula ) eggs were collected from Menai Strait (North Wales). Embryos were isolated from th e eggshells, dissected from the yolk sac in ice-
85 cold phosphate buffered salin e solution (PBS) and staged accordi ng to Ballard et al (Ballard et al., 1993) Adult mountain brook lamprey ( Ichthymyzon greeleyi ) were colleted from Ohio River branches (Kentucky, USA). The eggs were artific ially fertilized and rais ed in filtered river water. European brook lamprey ( Lampetra planeri ) eggs were colleted from New Forest, England. The lamprey embryos were staged according to Tahara et al. (Tahara, 1988) Gene Cloning and Phylogenetic Analysis Degenerate RT-PCR was performed to amplif y catshark gene fragments using cDNA from a mixed stage. RNA was extracted using TR Izol Reagent (Invitrog en). cDNA was made by reverse transcription reactions us ing SuperScript II first-strand cDNA synthesis kit (Invitrogen). Degenerate primers for the scleromte marker gene s used in these chapte r were designed using CODEHOP program ( http://bioinformatics.weizmann.ac.il/ blocks/ codehop.html ) (Rose et al., 2003). Lamprey genes were isolated by RT-PCR from a Petromyzon marinus cDNA library (a gift from J. Langeland) using Advantage GC-P CR Kit (Clontech). The amplified fragments were cloned into PGEM-T Easy Vector (Promega ) or pDrive Cloning Vect or (Qiagen). Some gene fragments were expanded to full-length se quences by rapid amplification of cDNA ends (RACE). For gene diagnosis, each gene was identified primarily by BLAST searches. The sequence alignments were generated with Clusta l X. Phylogenetic anal ysis were done by using both minimum evolution, maximum likelihood and Ba yesian analysis methods (Zhang et al., 2006). All three methods resulted in similar topologies and supported the robustness of our results. The sequences reported in this paper were deposited in the Genbank (accession number: EU196399-EU196410). The following amino acid sequenc es were retrieved from GenBank for inclusion in our phylogenetic analyses: human PAX1, NP_006183; mouse PAX1, NP_032806; rat PAX1, XP_001056369.1; cow PAX1, XP_617873; dog PAX1, XP_542866; horse PAX1, XP_001492632; fugu PAX1, CAG09888, frog P AX1, NP_001090451; Japanese lamprey
86 PAX1/9, BAB12396; lancelet PAX1/9, AAA8 1364; human PAX9, NP_006185; dog PAX9, XP_547776; mouse PAX9, EDL36706; rat PAX9, NP_001034628; fugu PAX9, AAG44703; frog PAX9, AAH84222; platypus PAX9, XP_001511262; zebrafish Pax9a, NP_571373; zebrafish Pax9b, AAC60035; human FOXC 1, NP_001444; mouse F OXC1, NP_032618; frog FOXC1, NP_001007864; zebrafish FOXC1a, NP_571803; zebrafish Foxc1b, NP_571804; human FOXC2, NP_005242; cow FOXC2, XP_872296; mouse FOXC 2, Q61850; horse FOXC2, NP_001077066; frog, FOXC2, NP _998857; lancelet FOXC, CAH69694; human NK3.1, NP_006158; cow NK3.1, XP_001250882; dog NK 3.1, XP_543240; rat NK3.1, NP_001029316; frog NK3.1, AAH47968; see urchin NK3, XP _784735; cnidarian NK3, AAP88431; cow Nk3.2, XP_874137; dog Nk3.2, XP_545940; mouse NK3 .2, AAI45875; fugu NK3.2, CAG08309; chicken Nk3.2, BAB40713; salamander NK3.2, AAC08704; zebrafish Nk3.2, NP_835233; human TWIST, CAA62850; mouse TWIST, AAA40514; rat TWIST, NP_445982; frog TWIST, NP_001079352; chicken TWIST, AAF00072; zebrafi sh Twist, NP_571059; lancelet TWIST, O96642; human SCLERAXIS, NP_001073983; mouse SCLERAXIS, NP_942588; chicken SCLERAXIS, NP_989584; human PARAXIS, CAC00470; fugu PARAXIS, CAG03890; chicken PARAXIS, AAC60208; zebrafish Pa raxis, NP_571047; mouse MYOD, AAA39798; frog MYOD, CAE18108;chicken MYOD, AAA 74374; zebrafish MyoD, NP_571337; lancelet MYOD, AAR12639. In Situ Hybridization and Cryosections The catshark and lamprey whole mount in situ hybridization was performed as described for chicken method (Nieto et al., 1996) with th e following modifications. Both catshark and lamprey embryos were treated with proteinase K (10ug/ml) for 15-30min at room temperature. Levamisol (2 mM) was added to both KTBT an d NTMT washes. The pre-hybridization and hybridization was performed at 70C. For the color reaction, 10% dimethyl formamide (DMF)
87 was added to maintain NBT and BCIP in soluti on. For histological anal ysis, specimens were equilibrated in 15% sucrose then 30% sucrose in 20% gelatin, after which they were embedded in 20% gelatin for cryosectioning (10um). Results Isolation and Analysis of Lamprey and Catshark Genes In order to identify molecular compartments of the lamprey and shark somites, we set out to isolate orthologs of genes that both mark th e sclerotome and regulate the skeletogenic pathway in jawed vertebrate embryos. Using dege nerate RT-PCR on cDNA pool s and cDNA libraries from the catshark S. canicula and the lampre y P. marinus to isolate cDNA fragments, followed by RACE to obtain full-length cDNAs, we isolat ed 11 clones from catshark and 10 clones from lamprey with putative orthology. Phylogenetic analyses using Minimum Evolution, Maximum Likelihood, and Baysian Phylogenetic analysis resolved the id entities of catshark PAX1, BAPX11, FOXC1, FOXC2, TWIST, SCLERAXIS, PARAXIS, TBX18, GLI2, and lamprey PAX1/9, BAPX1, FOXC1/2, Twist, Parascleraxis, TBX15/18, GLI1/2/3 (Figure 4-2, 4-3 and 44). We then conducted whole mount and section in situ hybridizations to map the expression patterns of these genes during somite developmen t in catshark and lamprey embryos. Below we describe the results of these experiments. Expression of Sclerotomal Markers in the Somites of Lampreys and Catsharks Shark Pax1 and lamprey Pax1/9 Group I Pax genes ( Pax1 and Pax9 ) define the sclerotomal compartment of the somites in osteichthyans. We examined the expression of Pax1 in catshark embryos at stage 26 and observed segmental domains of expression in th e somites, along with strong expression in the pharyngeal pouches (Figure 4-5A). Analysis of Pax1 expression in transverse sections confirmed the somitic expression and revealed that it was restricted to the ventral-medial regions
88 of the somites, in a domain that surrounded the notochord and extended dorsally around the neural tube, consistent with a sclerotomal pattern of expression (Figure 4-5B). Surprisingly, Lampetra Pax1/9 was also expressed in the somites at stages 23 and 25 as well as in the pharyngeal pouches at stage 25 (Figure 4-5 C and E). Transverse sections show that the somitic domains are restricted to the ve ntral-medial margins at stage 23 (Figure 4-5 D), and at stage 25 the domains surround the notochord and extend to the ventral aspect of the neural tube (Figure 45 F). Expression was also observed in the ventral neural tube (Figure 4-5 F). Thus, in sharks and lampreys, Pax1/9 expression marks the region of th e somite that co rresponds to the sclerotome of osteichthyans. Analysis of a purported Pax9 gene in the Japanese lamprey, Lethteron japonicum revealed expression in the pharyngeal pouches, however Og asawara and co-workers reported it to be absent from the scleroto me (Ogasawara et al., 2000). We re-examined the L. japonica sequence in our phylogenetic analyses and fi nd that it is a sister gene to Pax1 and Pax9 clades and, as such, should be designated Pax1/9 To determine whether expressi on in the sclerotome is unique to Lampetra planeri we examined Pax1/9 expression in another lamprey genus, Ichthyomyzon greelei We find that in both lamprey genera examined, Pax1/9 is expressed in the ventromedial region of the somites (Figure 4-5 G, H). It is therefore unclear whet her the previous report simply missed this expression domain, or whether Lethenteron japonica is a particularly derived genus, which has lost the sclerotomal expression of Pax1/9 Bapx1 Bapx1 ( Nkx3.2 ) was reported to be a direct downstream target gene of Pax1/9 in the mouse (Rodrigo et al., 2003). We therefore asked whether Bapx1 is expressed in a pattern similar to Pax1/9 in catshark and lamprey. Whole mount in situ hybridization of stage 25 catshark embryos showed clear expression in the somite s along the entire trunk and in the pharyngeal
89 pouches (Figure 4-6 A). Sections through the trunk indicated that expression in the somites was confined to the sclerotome, in domains that extended ventrally around th e notochord and dorsally adjacent to the ventral neural t ube (Figure 4-6 B). The catshark Bapx1 is limited around the midpoints of pharyngeal arches (F igure 4-6 C). In lamprey embryos at stages 23 and 25, whole mount in situ hybridization revealed Bapx1 expression in the regions of the pharynx and brain, and in two post-cranial domains in the regions of the neural tube and ventral somites (Figure 4-6 D, F). We also found lamprey Bapx1 broadly expressed in the phar yngeal arches (Figure 4-6 H). Transverse sections through the lamp rey trunk at stage 23 confirmed that Bapx1 transcripts were localized to the ventral-medial as pect of the somites and to the ve ntral neural tube (Figure 4-6 E, G). As in the shark, lamprey Bapx1 expression was detected in the pharyngeal pouches Shark FoxC1 FoxC2 and lamprey FoxC1/2 At stage 26, the expression of FoxC1 and FoxC2 in catshark embryos was indistinguishable from the patterns reported for chickens and mice (Figure 4-7 A-D) (Sasaki and Hogan, 1993). Transcripts for both genes were also detected in the gut epithelium and in the pharyngeal arches. FoxC1 and FoxC2 expression persisted as the sclerotomal region surrounding the notochord. (Figur e 4-7 B, D). The lamprey FoxC1/2 was expressed in the paraxial mesoderm as in mice and chickens (Fig ure 4-7 E-H). The stronge st expression in the trunk occurred in the ventral-medi al aspects of the somites at stage 25 (Figure 4-7 H). Analysis of the single FoxC1/2 gene that we isolated from lampreys showed that it is expressed in the ventral-medial regions of the somites at stages 23 and 25, similar to the sclerotomal expression observed in catsharks and tetr apods (Figure 4-7 F, H). Twist Twist is anther important somite marker in ve rtebrates and it has been shown to play important roles during axial sk eletal development in medaka (Yasutake et al., 2004). In
90 catsharks, Twist was detected in head mesoderm, somites and fin folds (Figure 4-8 A). Somitic expression was confined to the sclerotome, which persisted as these cells surrounded the notochord (Figure 4-8 B). In lampreys, Twist was also found in the head region and somites (Figure 4-8 C, E), and transver se sections through the trunk at stage 23 showed that it was expressed in the lateral somite in the neural tube (Figur e 4-8 D). Lamprey Twist is expressed in the neural tube and ventromedial region of the somites in the sections through both the stage 23 and stage 25 (Figure 4-8 C-F). Shark Scleraxis Paraxis and lamprey Parascleraxis We previously reported median fin expression of catshark Scleraxis and lamprey Parascleraxis the sister gene to the gnathostome Scleraxis and Paraxis genes (Freitas et al., 2006). We expanded that analysis to th e entire embryo and found that catshark Scleraxis was expressed in the head, the somites a nd neural tube (Figure 4-9 A-D). Scleraxis expression in the somites was confined to the sclerotomal region (Figure 4-9 B, D), in a broader domain than reported for chick and mouse, where it marks the s yndetome, which gives rise to tendon (Brent et al., 2003). Catshark Paraxis was found in newly developed som ites (Figure 4-9 E), as reported in tetrapods. Both Scleraxis and Paraxis were expressed in the catsh ark neural tube (Figure 4-9 B, D and F), which was not reported in chicken and mice. In lampreys, we failed to isolate strict orthologs of the Scleraxis and Paraxis genes, but we previously identified an undifferentiated gene, which we named Parascleraxis, that is a sister to the Scleraxis and Paraxis clade (Freitas et al., 2006). Lamprey Parascleraxis was expressed throughout the somites and did not show compartmentally restricted expres sion (Figure 4-9 G-J). Lamprey Parascleraxis like Paraxis and Scleraxis of the shark, also was detected in the neural tube (Figure 4-9 H and J).
91 Shark TBX18 and lamprey Tbx15/18 Tbx18 is expressed in the anterior halves of developing somites in mouse and zebrafish (Begemann et al., 2002; Kraus et al., 2001), a nd we reported previously that catshark Tbx18 and lamprey Tbx15/18 (a sister to the Tbx15/18 clade) are expressed in the median fins of both organisms (Freitas et al., 2006). Analysis of their expressi on patterns during somitogenesis shows that both genes are expressed in the vent ral-medial region of the somites, immediately adjacent to the notochord in lamprey and catshark (Figure 4-10 B and D). In catsharks, we also noted Tbx18 expression in the midbrain, hindbrain, hear t, pectoral fin and around the eye and ear (Figure 4-10 A). Lamprey Tbx15/18 was observed in the head and in the ventral neural tube (Figure 4-10 C and D). Anterior -posterior polarity of the somite s was clearly seen in catshark although it was not apparent in lamprey. Shark Gli2 and lamprey Gli1/2/3 Gli1 and Gli2 are expressed in the sclerotome and Gli3 is expressed throughout the somite in mouse embryos (Buttitta et al., 2003). Gli2 and Gli3 mediate Shh induction of sclerotome, and are required for activation of Pax1 and Pax9 (Sasaki et al., 1999). Since expression of Pax1/9 is conserved in lamprey and catshark (Figure 4-5), we investigated Gli2 expression in both species. We found that both catshark Gli2 and lamprey Gli1/2/3 were expressed in the ventral-medial region of the somites, consistent with the sclerotomal pattern described for jawed vertebrates (Figure 4-11). Catshark Gli2 and lamprey Gli1/2/3 were also detected, ex cluded from floor plate in the neural tube, which is consistent with th e pattern reported from tetropods. (Figure 4-11 B and D). Discussion The vertebral column is a key defining characte r for all vertebrates. In higher vertebrates, the vertebral columns are derived mainly from sc lerotome. In lampreys, arcualia were purported
92 to be homologs of vertebral cartilages by comparat ive anatomists of the last century, who also suggested that lampreys might have a small scle rotome (Brand-Saberi an d Christ, 2000; Maurer, 1906; Scott, 1882). However a recen t study of the Japanese lamprey LjPax9 gene showed that this sclerotome marker is abse nt in paraxial mesoderm (Ogasa wara et al., 2000) This finding suggested that lampreys might lack a sclerotome. In addition, the major matrix components of lamprey cartilage were reported to be elastin-like molecules, named lamprin, instead of the Col2a1 protein found in jawed vertebrates (Wri ght et al., 2001). This chondrogenic difference suggested that lamprey cartilage development mi ght involve a different genetic program. Thus, the embryonic origin of arcualia remained puz zling. Recently, we found the lamprey cartilage matrix contains Col2a1 proteins and Sox9 which suggested that th is chondrogenic genetic pathway is conserved in all vertebrates (lampr eys and gnathostomes). More importantly, the Col2a1 and Sox9 genes in lamprey are also expressed in the positions of the arcualia (Zhang et al., 2006). Thus, the conserved chondrogenic path way, the anatomical structure of these axial cartilages, and evidence from the early compar ative embryology lead us to hypothesize that lamprey arcualia develop from sclerotome like the tetrapods. Molecular Evidence for a Lamprey Sclerotome Pax genes play important roles duri ng animal development and all the Pax genes shared a conserved 128 amino acid paired domain. To date, in higher vertebrates, nine Pax genes have been classified into four subgroups based on thes e motifs (Dahl et al., 1997; Strachan and Read, 1994). Group I Pax genes include Pax1 and Pax9 which possess an octape ptide in addition to the paired domain, but not a homeobox domain. Pax1 and Pax9 were all expressed in the pharyngeal slits in the living deuterostomes (Holland and Holland, 1996; Ogasawara et al., 2000; Ogasawara et al., 1999). In mouse, Pax1 and Pax9 are all expressed at the developing somites and play critical roles for the vertebral column s (Peters et al., 1999). So these two genes are
93 widely used as sclerotome markers (Balling et al., 1996). Previous studies on invertebrate deuterostomes and lampreys have sugg ested that axial expression of group I Pax genes are a novel event for the gnathostome lineage (Holland and Holland 1996; Ogasawara et al., 2000; Ogasawara et al., 1999). Due to th e conflict between the absence of Pax1/9 in lamprey and presence of conserved chondroge nic program (Ogasawara et al ., 2000) (Zhang et al., 2006), we investigated the Pax1/9 gene in another two lamprey species, Lampetra and Ichthymyzon In both Lampetra and Ichthymyzon we detected their expression in the ventromedial somite, which was sustained as these cells surrounded the notochord. The Pax1/9 pharyngeal expression was same as in Lethenteron (Ogasawara et al., 2000). Our catshark Pax1 data showed pharyngeal and sclerotomal domains are typica l of gnathostomes. The lamprey Pax1/9 data suggests that this axial expression domain is a shared char acter of lampreys and gnathostomes and evolved coincident with the morphologica l sclerotome (Maurer, 1906). The difference between our results and those of previous publications may be caused by species differences, as proposed for lamprey HoxL6 (Cohn, 2002; Takio et al., 2004), or simply the result the sensitivity of in situ hybridization methods, as suggested by Ogas awara et. al. (Ogasawara et al., 2000). Since Pax1/9 marks the sclerotome in amniotes, this suggests that lamprey possess a sclerotome. To confirm this, we investigated more sclerotome markers; consistently these makers were expressed in the ve ntromedial part of somites. Bapx1 Bapx1 ( Nk3.2 ) is one member of NK3 genes that is homologous to the fruitfly bagpipe gene. There are two Nk3 genes in human and mouse, NK3.1 and Nk3.2 both genes are homeobox containing transcripti on factors and expressed in the sclerotome. Although the Nk3.1 mutant does not show sclerotomal defects, the Bapx1 mutant phenotypes were similar with Pax1 and Pax9 double mutants (Akazawa et al., 2000; Bha tia-Gaur et al., 1999; Lettice et al., 1999;
94 Schneider et al., 2000; Tribioli and Lufkin, 1999). Bapx1 was shown to be a direct target of group I Pax genes in mouse (Rodrigo et al., 2003). Moreover, Bapx1 was demonstrated to promote chondrogenesis in the somatic lineage (M urtaugh et al., 2001; Zeng et al., 2002). So the Baxp1 is the key gene, which determines the scle rotomal cell fate to the chondrogenic lineage. Bapx1 has been proposed to play important roles in the axial skeleton evolu tion after the split of jawless and jawed vertebrates (Let tice et al., 2001). Our lamprey Nk3 expression in the somites suggests the co-option of Nk3 from gut-associated mesenchyme to paraxial mesoderm happened before the split of jawed and jawless vertebrates. The Bapx1 results also have implications for the origin of hinged jaws. The co-option of Bapx1 expression in the first arch was proposed to be critical for the origin of a jaw since its e xpression resides at the ma ndibular joint of jawed vertebrates (Miller et al., 2003). Our finding that the lamprey Nk3 gene is expressed in all the pharyngeal arches suggests ther e may have been a reduction of NK3 gene expression from all gill arches of jawless vertebrates to first gill arch of gnathostomes. Modulation of the spatial extent of gene expression domains within the pharyngeal arches may have occurred with other genes during the agnathan-gnathostome transitions, as a similar reduction appears to have occurred with the pharyngeal Hox code (Cohn, 2002). FoxC1/2 FoxC genes encode transcription factors with a winged helix/forkhead domain and are known to be required for proper sclerotome developm ent (Iida et al., 1997; Winnier et al., 1999). Lamprey and shark FoxC1 / 2 showed similar expression patterns to mouse, chicken and frog sclerotomes (Buchberger et al., 1998; El-Hodiri et al., 2001; Kume et al., 2001). Both genes were shown to be important in specifying the paraxial mesoderm (Wilm et al., 2004). The FoxC2 null mutant axial skeletal defects are similar with Bapx1 knockout mice, and the chondrogenic markers like Sox9 and Col2a1 are severely reduced (Iid a et al., 1997; Winnier et
95 al., 1999). Double mutants of FoxC2 and Pax1 showed more severe defects of vertebral columns than single mutants, and bot h genes are required for sclero tomal cell proliferation in a Shh dependent manner (Furumoto et al., 1999) The conserved expression patter of FoxC1/2 in catshark and lamprey suggests that they played a ro le in formation of the earliest sclerotomes. Twist Twist a basic helix-loop-helix tran scription factor, is expressed in the sclerotome and was shown to play critical roles in mesoderm diff erentiation in a variety of species (Castanon and Baylies, 2002; O'Rourke and Tam, 2002; Stickn ey et al., 2000) and dur ing the development of vertebral columns, especially the neural arch es in medaka (Yasutake et al., 2004). The expression of catshark Twist is similar with mouse, suggesting its conserved function in sclerotome development, pharyngeal arch es and limb or fin formation. Lamprey Twist is not strictly homologous to the gnathos tome twist gene; instead it is a paralogue to the higher vertebrate Twist1 and Twist2 genes. It was cloned very recently as Twist (Sauka-Spengler et al., 2007). Our finding is consistent with their data that lamprey Twist is expressed in the notochord and developing somites, especially in the sclerotome. Lamprey Twist is also found in the dermomyotome, suggesting it may play part of the roles of Twist2 ( Dermo1 ), or it may be related to the mesenchymal-epithelial transition. Taken t ogether with lancelet twist expression in the differentiating notochordal and paraxial mesode rm, lamprey showed an intermedial state between lancelet and gnathostomes. Paraxis and Scleraxis Paraxis and Scleraxis are two closely-related bHLH tran scription factors expressed in the early paraxial mesoderm and later in the sclerotome, but Paraxis declines after sclerotome formation whereas Scleraxis persists in a subset of the sclero tome (Burgess et al., 1995). In the absence of Paraxis the axial skeleton and muscle formed but with abnormal patterns due to the
96 disruption of somite A/P polarity (Bur gess et al., 1996; Johnson et al., 2001). Scleraxis -null mice are unable to form mesoderm and Scleraxis Â–null cells are excluded from the sclerotome in chimeric mouse analysis, suggesting ScleraxisÂ’ role in sclerotome de velopment (Brown et al., 1999). Recently, Scleraxis was shown to be a early marker of tendons and ligaments, and the Scleraxis positive somite compartment of somitic tendon population was termed the syndetome (Brent et al., 2003; Schweitz er et al., 2001). Catshark Paraxis and Scleraxis are both expressed in the sclerotome. Interestingly both catshark Paraxis and Scleraxis are found in the neural tube. Xenopus Paraxis expression has been reported in the ne ural tube (Carpio et al., 2004). The lamprey Paraxis and Scleraxis orthologue, Parascleraxis, is also expressed in the sclerotome and neural tube. Taken together, Scleraxis and Paraxis sclerotome expression domains are conserved in all the living vert ebrates, while the neural tube expression domain is lost in amniotes (chicken and mouse). The S cleraxis sclerotomal domain in catshark is similar to the Pax1/9 expression domain, suggesting that differen tiation of syndetome may be a more recent event in amniotes. Indeed, th at syndetome forms a further molecular subdivision of the ventromedial somite within the Pax1 expression domain. The roles of Scleraxis in shark myoseptum are very interesting since it has b een proposed that tendon may have evolved from the myoseptum in gnathostomes (Gemballa et al., 2003; Summer s and Koob, 2002). Tbx15/18 Tbx18 or Tbx15 are closely related T-box containing genes that are e xpressed in the pharyngeal arches, sclerotomes and limb/fin buds in mouse, chicken and zebrafish (Agulnik et al., 1998; Begemann et al., 2002; Haenig and Kisp ert, 2004; Kraus et al., 2001; Singh et al., 2005; Tanaka and Tickle, 2004). In chicken and mouse, Tbx18 is important to maintain the somite boundary; in addition it marks the sclero tome (Bussen et al., 2004 ; Tanaka and Tickle, 2004). The Tbx18 deficient mouse exhibits posterior lateral sclerotome derivative defects,
97 pedicles, and the proximal ribs are severely expanded (Busse n et al., 2004). The catshark Tbx18 shows a typical gnathostome expression patt ern and is found in the pharyngeal arches, sclerotome and fin buds. Lamprey Tbx15/18 is found in the pharyngeal arches and perinotochordal region of the somites. Interestingly, the Ciona Tbx15/18/22 was reported to be expressed in the paraxial mesoderm although Ciona do not have overt morphological segmentation (Takatori et al., 2004 ). In addition, lancelet Tbx15/18/22 is also evident in the anterior part of the newly formed somites (Beas ter-Jones et al., 2006). The current data suggests that Tbx15/18/22 gene was originally deployed br oadly in the mesoderm (as in Ciona) then became restricted to paraxial mesoderm (as in lancelets), and finally af ter the gene duplication events, Tbx18 and Tbx15 genes became confined to so mite boundaries and used during sclerotome development. Gli1/2/3 The Ci vertebrate homologues, Gli1 Gli2 and Gli3 are zinc finger transcription factors that mediate hedgehog signaling (Ingham and McMahon, 2001). In mouse, all three genes are expressed in the sclerotome, and Gli2 and Gli3 were demonstrated to be required for Shh dependent sclerotome induction (Buttitta et al., 2003; Mo et al., 1997). In the Gli2 and Gli3 double mutants, axial skeletal defects are similar to Pax1/Pax9 double mutants, whereas Gli1 mutant mice exhibit no developmental defect s (Buttitta et al., 2003). The catshark Gli2 and the lamprey paralogous gene, Gli1/2/3, are all expressed at the ne ural tube and sclerotome, suggesting their ancien t roles in mediating Shh signals for inducing sclerotome and perhaps motor neuron development. Recently, la ncelet was reported to have a single Gli gene expressed in the neural tube and somites (Shimeld et al., 2007), suggesting that the vertebrate Gli gene duplication may have coincided with subdivision of the somites.
98 Embryonic Origin of Lamprey Arcualia Vertebrate cartilage is characterized by th e presence of chondrocytes and extracellular matrix molecules such as type II collagen fibril s and proteoglycans. The type II collagen protein is encoded by the Col2a1 gene, and predominantly localize to cartilage as the major matrix protein. Sox9 is a member of the Sox family of transcription factor that contains a high-mobilitygroup (HMG)-box DNA binding domai n. Haploinsufficiency of Sox9 in humans and mice causes the skeletal disease, campomelic dysplasia (CD) (Foster et al ., 1994; Wagner et al., 1994). Sox9 was proved to be the direct regu lator of Col2a1 and aggrecan in mice and its major roles during chondrocyte differentiation through direct regulating on the Col2a1 promoter (Bi et al., 1999; Bi et al., 2001; Lefebvre and de Crombrugghe, 1998; Le febvre et al., 1997). (Bi et al., 1999; Bowles et al., 2000; Lefebvre and de Crombrugghe, 1998). Recently we demonstrated that lamprey and hagfish have conserved this chondrogenic gene tic program to build ca rtilage (Zhang and Cohn, 2006; Zhang et al., 2006). Arcualia are paired cartilaginous nodules sitting on either si de of the notochord. They are irregularly-shaped and variably distributed in the tail of ammocoetes and hagfish, and the trunk region of adult lampreys (Gadow and Abbott, 1895). If they were homologous to vertebrae, then they would be expected to have the same devel opmental origin. If lampreys lack a sclerotome, as suggested, this would cast doub t on the common origin of vert ebrae and arcualia. Our eight molecular markers of the sclerotomal populat ion strongly suggest th at lamprey do possess sclerotome. This finding is consistent with the observations that arcualia develop from paraxial mesodermal cells (Gadow and Abbott, 1895). Hence, the genetic programs for sclerotome formation during chondrogenesis are conserved in th e lampreys, strongly s uggesting that arcualia derive embryonically from the sclerotome as axia l endoskeletons do in gnathostomes. Recently, it has become possible to get live hagfish embryo s (Ota et al., 2007), and thus it would be very
99 interesting to look at the sclerotomal molecu lar markers in hagfish embryos since some cartilaginous elements were reported in the hagf ish tail region (Gadow and Abbott, 1895). This work could resolve questions over the phylogenetic positions of the two ex tent jawless fishes, since it is still controversial wh ether hagfish belong to vertebrata or should be separated as craniata. The Evolution of Sclerotome and Vertebral Columns As pointed out by Scaal and Wiegreffe, modern embryologists have focused mostly on the amniote somites, contrasting to the late 19th and early 20th century embryologists who worked extensively on anamniotes. The basal vertebra tes, like the cyclostomes, chondrychthians and chondroistei generally have been neglected (S caal and Wiegreffe, 2006). Our experiments in lamprey and catshark demonstrated that sclero tome is a vertebrate character that unites gnathostomes and lampreys, suggesting its presen ce in their common ancestor. Analysis of hagfish somites may reveal whet her the origin of sclerotome dates to the common ancestor of modern agnathans and gnathostomes, which is co ngruent with the largescale gene duplication (Steinke et al., 2006). The size of sclerotome is hi ghly variable in different taxa. In the amniotes, the sclerotome is comparatively bigger than th e situation in fishes (B rand-Saberi and Christ, 2000). Clearly there is a trend of increasing the size of sclerotome from fish to tetrapods. The sclerotome size was determinated by the balance between the dorsal and ve ntral signals. The dorsal signals from the surface ectoderm and the dorsal neural tube promote the development of dermomyotome, but inhibit the sclerotome forma tion in amniotes. While the ventral signals promote sclerotome development and inhibit dermomyotome formation (Christ et al., 2004; Dockter, 2000). Currently, th e dorsal signals are mediated by the WNT and BMP family members, while the ventral signals are mediated by Shh and Noggin secreted by notochord (Dockter, 2000; Lee et al., 2001; Lee et al., 2000; McMahon et al., 1998; Yusuf and Brand-
100 Saberi, 2006). The evolution of vertebrate scle rotome size may be a reflection the evolution of the interaction of these tw o signaling centers. Furthe r functional st udies on the Wnt and Shh gene interaction in fishes will shed light on this intriguing question. Although composed mainly by arches and centr a, vertebral columns are highly variable structures in different taxa. The major compone nts of vertebrae appear ed at different times, which are summarized in Figure 4-1A. The developmental mechanisms of centra and arches were shown to be different, which may reflect th eir modular evolutionary history. For example, Pax1 Pax9 and Bapx1 null mice mainly showed centra defect s (Lettice et al., 1999; Peters et al., 1999), while in Twist FoxC2 and Zic1 knockout mice, the arch structures were mainly affected (Aruga et al., 1999; Furumoto et al., 1999; Yasutake et al., 2004). Vertebral centra are absent and only some arch structures are present in cyclostomes and chondrosteis (Gadow and Abbott, 1895). Hans Gadow classified the vertebral centra into two categor ies, according to the participation of notochordal shea th and sclerotomal cells, which he termed chorda-centra and arch-centra (Gadow and Abbott, 1895). He sugge sted that the former centra mainly develop from notochordal sheath, with some participation of sclerotomal cells, li ke the chondrychthians, whereas the latter centra are en tirely made by sclerotomal cells and all the amniotes belong to this category (Gadow and Abbott, 1895). So, the diversity of vertebrae c ould be interpreted as the results of phylogenetic differences in the inte ractions between the no tochord and sclerotome. Indeed, there is some functional evidence that s upport this idea. The no tochord is required to induce Pax1 expression (Brand-Saberi et al., 1993; Muller et al., 1996), and in Pax1 mutant mice, the notochord is enlarged due to more notoc hordal cell proliferation, suggesting reciprocal interaction between sclerotome and notochord (Wal lin et al., 1994). Furthe r functional studies in different animal taxa are necessa ry to test this hypothesis.
101 Figure 4-1. Evolution and development of axial morphological structures. (A) The e volution of axial st ructures in living deuterostomes. The phylogenetic relationships are base d on Koob and Neidert (Koob a nd Long, 2000; Neidert et al., 2001). The bars on the tree indicate the emergence of the mor phological structures. The dashed lines show the alternative possibility. The tree structures are based on Koob and Bourlat (Bourla t et al., 2006; Koob and Long, 2000). (B) Subdivisions of a somite, modified from Williams (Williams, 1959). Sclerotome markers are listed at the right bottom corner. (C) The genetic regulatory circui t of axial skeleton development. The relationships are based on several published studies (Bialek et al., 2004; Furumoto et al., 1999; Hornik et al., 2004; Kume et al., 2000; Lengner et al., 2005; Rodrigo et al., 2003; Zeng et al., 2002; Zhou et al., 2006).
102 Figure 4-2. Minimum evolution phy logenetic analysis of the pr edicted protein sequences of catshark and lamprey clones. The numbers indicate bootstrap scores for each node, based on 10,000 replicates, whereas branch lengths are proportional to expected replacements per site. Each tree is rooted by a lancelet sequence. The phylogenetic relationships are consistent with maximum likelihood and Bayesian analyses that are provided as supplementary information. (A) Pax1 and Pax9. (B) Nk3.1 and Nk3.2. (C) FoxC1 and FoxC2. (D) Twis t (E) Gli1, Gli2 and Gli3.
103 Figure 4-3. Maximum likelihood ph ylogenetic analysis of the pr edicted protein sequences of catshark and lamprey clones, as obtained with JTT plus gamma distances. The numbers indicate bootstrap scores for each node, based on 1,000 replicates, whereas branch lengths are proportional to expected replacements per site. Each tree is rooted by the lancelet sequence. The phylogenetic relationships are consistent with minimum evolution (Fig. 2) and Bayesian an alysis (Fig. S2). (A) Pax1 and Pax9. (B) Nk3.1 and Nk3.2. (C) FoxC1 and FoxC2. (D ) Twist (E) Gli1, Gli2 and Gli3.
104 Figure 4-4. Bayesian phylogeneti c analysis of the predicted pr otein sequences of catshark and lamprey clones, as obtained with WAG plus gamma distances. Numbers indicate posterior probabilities for groups with >50% credibility and for those clades that are combinable with this first set. Branch lengths are proportional to the means of the posterior probability densities for their exp ected replacements per site. Each tree is rooted by the lancelet sequen ce. The phylogenetic relations hips are consistent with minimum evolution (Fig. 2) and maximu m likelihood (Fig. S2). (A) Pax1 and Pax9. (B) Nk3.1 and Nk3.2. (C) FoxC1 and FoxC2. (D) Twist (E) Gli1, Gli2 and Gli3.
105 Figure 4-5. Group I Pax genes are expressed in the sc lerotome of catshark (A, B) an d lamprey (C-H). Catshark Pax1 gene is expressed in the somites and th e pharyngeal pouches at stage 26 (A ). Transverse section shows Pax1 is mainly located at the sclerotome (B). Lampetra Pax1/9 expression patterns at stages 23 and 25, wh ich is evident at th e pharyngeal pouches and somites (C, E). In transverse sections, Pax1/9 is seen at the ventromedial part of somite, sclerotome, and ventral neural tube (D, F). Ichthymyzon Pax1/9 shows similar expression patterns with Lampetra (G, H). All the sections are with dorsal to the top. d dermomyotome; gp pharyngeal pouches; h hypochord; n notochord; nt neural tube; s sclerotome; so somites.
106 Figure 4-6. Expression of Nk3.2 genes in catshark and lamprey. Catshark Nk3.2 gene expression is evident in the somites and pharyngeal pouches at stage 26 (A), and a transverse section shows it is located at the sclerotome (B). It is not only evident at the mandible joint, but also in the midpoint of other pharyngeal arches in catshark (C). The lamprey Nk3.2 gene is broadly expressed in the pharyngeal pouches and somites at stages 23 (D) and 25 (F, H). Transverse sections show lamprey Nk3.2 is evident in the neural tube and sclerotome (E G). All the sections are with dorsal to the top. d dermomyotome; gp pharyngeal pouches; n notochord; nt neural tube; s sclerotome.
107 Figure 4-7. FoxC1 and FoxC2 expression patterns in catshark and lamprey. FoxC1 in strongly expressed at the newly formed somites and pharyngeal arches at stage 26 (A). Transverse section shows catshark FoxC1 is located at the sclerotome (B). Catshark FoxC2 is evident in the somites and pharyngeal arches at stage 26 (C). Catshark FoxC2 is located at the sclerotome in the somite (D). Lamprey FoxC1/2 is evident at the somites at stages 23 (E) and 25 (G). It is also expressed at the pharyngeal arches at stage 25 (G ). Transverse sections show lamprey FoxC1/2 and catshark FoxC2 expression in the sclerotome (F, H) and gut region (H). All the sections are with dorsal to the top. d dermomyotome; ga pharyngeal arches; gu gut; n notochord; nt neural tube; s sclerotome.
108 Figure 4-8. Expression of catshark and lamprey Twist genes. At stage 26, catshark Twist is expressed in the somites, fin fold and pharyngeal arches (A). Tran sverse section shows that Twist is limited to the sclerotome (B). The lamprey Twist is also expressed in the pharyngeal arches and so mites at stage 23 (C) and stage 25 (E). Transverse section shows that lamprey Twist is evident at the neural tube, dermomyotome and sclerotome at st age 23 (D). While at stage 25, lamprey Twist is limited in the sclerotome and neural tube (F). All the sections are with dorsal to the top. d dermomyotome; fb fin bud; ga pharyngeal arches; h hypochord; n notochord; nt neural tube; s sclerotome.
109 Figure 4-9. Scleraxis and Parascleraxis expression in catshark and lamprey. Catshark Scleraxis is expressed in the neural tube and head paraxial mesoderm at stag e 21 (A, B). At stage 26, catshark Scleraxis is found in the craniofaci al region, neural tube and sclerotome (C, D). Catshark Paraxis is expressed in the newly formed somite s at stage 26 (C) and transverse section shows it is evident at the neural t ube and sclerotome (D). Lamprey Parascleraxis is evident in the pharyngeal region and somites at stages 23 and 25 (D, E). Tr ansverse sections show that lamprey Parascleraxis is expressed in the neural tube and sclerotome (I, J). All the sect ions are with dorsal to the top. d dermomyotome; n notochord; nt neural tube; s sclerotome.
110 Figure 4-10. Expression of Tbx18 in catshark and lamprey. The catshark Tbx18 is evident in the somites at stage 26 (A). Transverse sections show its expression in the sclerotome (B). Lamprey Tbx15/18 is expressed at the somite regi on (C, D). All the sections are with dorsal to the top. d dermomyotome; n notochord; nt neural tube; s sclerotome.
111 Figure 4-11. Gli gene expression in catshark and lamprey. Catshark Gli2 is evident in the pharyngeal arches and somites at stage 26 (A). The transv erse section showing that catshark Gli2 is expressed in the neural tube and somite, but is excluded from the floor plate (B). The lamprey Gli1/2/3 is found in the pharyngeal arches and somites at stage 23 (C). It is evident at the neural tube and sclerotome (D ). All the sections are with dorsal to the top. d dermomyotome; n notochord; nt neural tube; s sclerotome.
112 CHAPTER 5 GENERAL DISCUSSION Evolution of Collagen Genes in Chordates Collagens are the main components of animal extracellular matrix (Expositio JY, 2002). Thus far, 27 types collagen genes have been id entified (Pace et al., 2003 ). The collagens are divisible into two major groups: fi bril and non-fibril collagens. Th e fibril collagens are further divisible into clades A, B and C (Aouacheria, A. et al. 2004). Cl ade A fibrillar collagens are the major fibril-forming collagen, including types I, II, III and V (Aouacheria, A. et al. 2004). The identification of type II collagen in lamprey and hagfish suggests that the differentiation of clade A fibrillar collagen ge nes predated the split of agnathans and gnathostomes. An independent duplication of the Col2a1 gene then occurred in the lamprey lineage. The expression patterns of Col2a1a and Col2a1b suggest that subfunctionalization followed this duplication of the ancestral Col2 a1 gene, with the ance stral expression pattern being partitioned between Col2a1a and Col2a1b Force and colleagues proposed a mechanism by which duplicated genes are preserved during evolution by subfunctionalization. The proposal states that members of the pair undergo reduction of their activity and expression patterns until together the pair of genes equal that of their single ancestral ge ne (Force et al., 1999; Lynch et al., 2001). Our finding that the expression domains of Col2a1a and Col2a1b in lampreys correspond to that of the single Col2a1 gene in jawed vertebrate s supports their duplicationdegeneration-complementation (DDC) model. The hagfish Col2a1 was clustered with lamprey Col2a1a with lamprey Col2a1b falling out as a sister branch. This topology suggests that Col2a1 was present in the common ancestor of agnathans and gnathostomes, and that an additional duplication gave rise to Col2a1a and Col2a1b in the cyclostome (lampreys + hagfishes) lin eage. The results predict that there should
113 be another Col2a1 gene in hagfish. Efforts to locate the hagfish Col2a1 were unsuccessful. Either we did not isolate it due to the limitation of degenerative PCR method, or it was lost in the hagfish lineage during evol ution. The isolation of Col2a1 genes also suggests that other clade A fibrillar collagen genes may be present in the two agnathans due to the duplications of clade A fibrillar collagen genes. Further studies are needed to prove these conjectures. The presence of an ancestral state of clade A fibrillar collagen gene, AmphiColA in lancets suggests that duplication of the ancestral ColA, the precursor of the clade A fibrillar collagen multi-gene family, occurred in the vertebrate lineage after the divergence of lancelets. The five types of clade A fibrillar collagen are limited to vertebrates. Our findings on lancelet AmphiColA gene were further supported by the recen t report of several de uterostome collagen genes, including the similar genes in Chines e lancelet (Wada et al ., 2006). Wada and his collagues also isolated one copy of cl ade a fibrillar collagen gene named BbCol1 Whether AmphiColA and BbCol1 is the same gene or they are paralogous genes, requires further investigation. Ascidians also possess one copy of clade fibrillar collagen gene and this gene also can not be clustered with the five type s of vertebrate collagens (Wada et al., 2006), and this finding further supports my hypothesis th at the duplication event happened in the vertebrate stem group, before the split of the agnathans and gnathostomes. The sea urchin was reported to have three different undifferentiated clade A fibrillar collagen genes (Aouach eria et al., 2004; Wada et al., 2006), which may suggest that they evolved inde pendently in the echinoderm lineage. Very recently, one copy of clade a fibrillar collagen wa s isolated from hemichordate, specifically the acorn worm (Rychel and Swalla, 2007). Their analysis of hemichordate ColA gene and other invertebrate sequences proved that all the invertebrate ColA genes do not cluster with the
114 vertebrate collagen genes and verified that ColA gene underwent the exte nsive duplications in vertebrate lineage. After the dup lication events, the type, I, II, III and V differentiated from the ancestral ColA gene. Regulatory Relationships of SoxE and ColA Genes May Have Been Established Before the Origin of Vertebrates The Sox family of transcription factors consis ts of at least ten s ubgroups (A-H) that are characterized by a specific 79-amino acid DNA-bi nding region, termed the high-mobility-group (HMG) box (Bowles et al., 2000). SoxE subgroup includes three genes, Sox8 Sox9 and Sox10 It was well established that Sox9 is required for Col2a1 expression and for chondrogenesis in jawed vertebrates (Bi et al., 1999; Yan et al., 2005). Expression of Col2a1 is regulated directly by Sox9 which binds to a chondrocyte-sp ecific enhancer to activate Col2a1 transcription (Bell et al., 1997; Lefebvre et al., 1997). Mutation of Sox9 in zebrafish disrupts the stacking of chondrocytes and the separation and shaping of individual cartilage elem ents, and in humans causes campomelic dysplasia (Wagne r et al., 1994; Yan et al., 2002). Although it was reported that the compositions of lamprey and gnathostome cartilage matrices are different (Wright et al., 2001), the genetic pathway th at regulates early development of the cranial skeleton (e.g., Bmp2/4 Dlx Msx etc. ) is well conserved (Cohn, 2002; McCauley and Bronner-Fraser, 2003; Neidert et al., 2001; Sh igetani et al., 2002). Our finding of presence of type II collagen in lamprey cartilage led us to hypothesize that the genetic program to make cartilage in agnathans and gnathos tomes is conserved. To test th is hypothesis, we isolated one lamprey Sox9 orthologous genes by degenerate RT-PCR. In situ hybridization using the amplicon as a probe revealed that lamprey Sox9 co-localized with two Col2a1 genes during catilage development. Therefore, this conser vation of regulatory rela tionship between Sox9 and Col2a1 was established before the split of agnathan s and gnathostomes. Moreover, our isolation
115 of Col2a1 genes and localization of Col2a1 protein in hagfish head cartilage suggested that hagfish might use the same conserved chondroge nic program. Very recently, the isolation and expression of hagfish Sox9 gene confirmed this conservation (Ota et al., 2007). Thus, it seems likely that in all the vertebrates, Sox9 can regulate Col2a1 In invertebrate chordates, ther e are no strict orthologues to Sox9 and Col2a1 genes, instead they have SoxE and ColA genes. The relationship of the SoxE and ColA is very interesting since Sox9 is able to regulate Col2a1 in vertebrates. In the lancelet, we isolated the ColA gene and found it is expressed in the floor plate of neural tube and in the notochord. This finding was also supported by BbCol1 (Wada et al., 2006). Very recently, the lancelet SoxE was isolated and it was found in the notochord, and thus it co-localizes with ColA (Meulemans and Bronner-Fraser, 2007 ) Interstingly, the two genes were also found to co-expressed in the lancelet pharyngeal cartilages (Meulemans and Bronner-Fraser, 2007; Rychel et al., 2006; Rychel and Swalla, 2007). The similar co-expression of SoxE and ColA was also reported in hemichordate cartilages (Rychel et al., 2006; Rychel and Swalla, 2007). This evidence suggests that the relationship between SoxE and ColA had been established at least in the ancestor of deuterostomes, long before the origin of vertebrates. Consideri ng this conservation, it is not surprising that, in addition to Sox9 Sox10 can also directly activate Col2a1 gene expression (Suzuki et al., 2006). Collagenous Skeletons verses Non-Collagenous Skeletons Vertebrates The phylogenetic relationships of the vertebra tes were set up largely based on anatomical characters, particularly those of the skeleton. Th e skeletons of jawed vert ebrates (gnathostomes) are composed of cartilage and bone, which c ontain high levels of COL2A1 and COL1A1 proteins, respectively, in their ex tracellular matrices. By contra st, the cartilaginous skeletons of lampreys and hagfishes, the only extant jawless fi shes (agnathans), have been reported to lack
116 collagen-based cartilage and to contain instead the elastin-like proteins lamprin and myxinin (Janvier and Arsenault, 2002; Wr ight et al., 2001; Wright and Youson, 1983). This difference in the histological matrices of vert ebrate skeletons has led to three ideas: that type II collagen became the major structural component of gnathos tome cartilage after th e divergence of these two lineages. The collagenous skeleton is a gnathostome synapmorphy, and the earliest vertebrates were presumed to have non-coll agen based cartilage (Wright et al., 2001). Localization of type II collagen mRNA and prot ein in the lamprey skeleton reveals that a collagenous skeleton is not restri cted to the gnathostome lineage but instead is a character shared by the crown vertebrates. This may provide a molecular explanation for ParkerÂ’s observation in 1883 that lampreys ha ve hard hyaline cartilage (Parker, 1883). I suggest that the additional cartilage matrix molecules (e.g., lamp rin and myxinin) of agnathans may represent derived character states that that were added onto the more ancient collagenous skeleton. Expression of elastin-related molecule s in a subset of lamprey crania l cartilages that also express Col2a1 may underlie the different structural and mechanical properties within the lamprey skeleton. Furthermore, Sox9 is expressed in strikingly similar patterns in lamprey and gnathostome embryos. The co-expression of Sox9 with Col2a 1 during skeletogenesis in both lineages raises the possibility that the regulat ory relationship between these two genes had already been established in their common ances tor. In gnathostomes, SOX9 is a target of parathyroid hormone-related protein (PTHrP), which regu lates chondrocyte differentiation through a negative feedback loop with Indian hedgehog (H uang et al., 2001; Vortkamp et al., 1996). Interestingly, PTHrP expression has recently been detected in lamprey cartilage (Trivett et al., 2005). Our discovery of conserved expression of Sox9 and Col2a1 taken together with the
117 extensive conservation of upstream regulatory genes such as AP2, Dlx, Msx, Id and PTHrP (Cohn, 2002; McCauley and BronnerFraser, 2003; Neidert et al ., 2001; Shigetani et al., 2002; Trivett et al., 2005), suggests that the genetic program for chondrogenesis from the initial induction of chondrogenic mesenchyme to the s ynthesis of collagen matrix was assembled surprisingly early in vertebrate evolution. We have also found that hagfishes, the sister group to lampreys, also possess a Col2a1 orthologue, and that COL2A1 protein is localize d to their soft cartilage (Chapter 3). Taken together with lamprey COL2A1 data, these resu lts suggest that the common ancestor of all crown-group (the living jawed a nd jawless) vertebrates had COL2 A1-based cartilage. Thus, COL2A1-based cartilage is a synapomor phy of all crown-group vertebrates. Invertebrates Presence of an undifferentiated clade A fibrilla r collagen in lancelet s and tunicates (Wada et al., 2006) suggests that the expansion of the ColA gene family occurred in stem vertebrates after the divergence of lancelets an d tunicates. The expression of ColA in notochord and pharyngeal arches suggested that the lancelet skelet on is collagenous (Meulemans and BronnerFraser, 2007; Rychel et al., 2006; Rychel and Sw alla, 2007), not non-collag enous as previously proposed (Wright et al., 2001). The expression of SoxE and ColA genes in hemichordates further supports the idea that the collagenous skeleton evolved in the deuter ostomes before the origin of vertebrates. Based on biochemical analysis, th e cartilage of cephalopod mollusks was shown to contain two collagen chains, a1 and a2 (Kimura and Karasawa, 1985; Sivakumar and Chandrakasan, 1998; Sivakumar et al., 2003). Th e evolutionary depth of the origin of collagenous skeletons needs further investiga tion. Although the completed arthropod genomes indicate that fibrillar collagen was lost, car tilage tissue was report ed in the bookgills of horseshoe crab (Libbin et al., 1992; Libbin et al., 1976; Wright et al., 2001). Furthermore,
118 horseshoe crab cartilage was shown to contain chondroitin sulphate (Sugahara et al., 1996). Whether horseshoe crab cartilage is collagenous or non-collagenous is very interesting due to its pivotal phylogentic position. Furthe r investigations would be usef ul in probing the evolutionary depth of collagenous skeletons. Roles of Gene Duplications in Vertebrate Novelties Ohno first raised the idea that genome duplication played a majo r role in the evolution of vertebrate complexities (Ohno, 1970). It is now clear that, in fact vertebrates tend to have more copies of genes than do invertebra tes, and that there is extensive variation in gene number among different clades of vertebrates (Dehal a nd Boore, 2005; Meyer and Schartl, 1999; Panopoulou and Poustka, 2005). Recent studies of 14 differe nt genomes confirmed that the whole genome duplication occurred at the dawn of vertebrate evolution (Blomm e et al., 2006). One of the most intensively studied cases of gene family expansion is that of Hox genes (Holland et al., 1994; Martinez and Amemiya, 2002; Ruddle et al., 1994a; Ruddle et al., 1994b). Hox genes code for homeodomain-containing transcripti on factors, which tend to occur in tightly linked clusters in the vertebrate genomes. They are involved in pa tterning the vertebrate bo dy plan (Shashikant et al., 1991). Now it is widely accepted that the Hox cluster number reflect s the genome duplication events (Panopoulou and Poustka, 2005). These four Hox clusters are thought to have evolved in two rounds of whole geno me duplication, and the Hox cluster duplications gave the vertebrates the opportunity to generate morphological novel ties (Holland et al., 1994; Wagner et al., 2003). Based on the 2R (two rounds of duplication) hypothesis, one genom e duplication happened at the stem of all vertebrates and the other in the stem lineage of jawe d vertebrates, the gnathostomes (Panopoulou and Poustka, 2005). This scenario is also supported by the evolutionary history of nonHox genes, like Engrailed and Otx (Escriva et al., 2002; Garcia-Fernandez, 2005; Germot et al., 2001; Holland and Williams, 1990).
119 It is also noteworthy that collage n genes are physically linked to the Hox clusters in gnathostomes (Bailey et al., 1997; Morvan-Dubois et al., 2003). In lampreys there are at least three Hox clusters (Force et al., 2002; Fried et al., 2003; Irvine et al., 2002). Our identification of two Col2a1 genes supports the suggesti on that one of their three Hox clusters may have arisen by an independent duplication in the lamprey lin eage (Force et al., 2002; Fried et al., 2003; Irvine et al., 2002). The presence of Col2a1 gene in hagfish and ColA gene in lancelets further support this idea since lancelet has one Hox gene cluster and hagfish has at least 7 Hox clusters (Stadler et al., 2004). The skeleton has been prop osed to be one of the vertebrate innovations that resulted from the gene number increas e in genome (Shimeld and Holland, 2000). The duplications of Hox clusters and the linked clade A fibrillar collagen genes may have facilitated the evolution of vertebrate morphological novelties. The Hox clusters play major roles in animal body patterning, and the clade A fi brillar collagen genes lay down the connective tissues, including the skeletons. Origin of Vertebrate Chondrocytes It has been suggested that the notochord may represent a primitive form of cartilage, based on their many shared structural, cellular and molecular properties, and that vertebrate chondrocytes may have evolved from notochordal cells (Cole a nd Hall, 2004; Stemple, 2005). In gnathostomes, the notochord and/or notochordal sh eath expresses most of the vertebrate clade A fibrillar collagen genes (Dubois et al., 2002; Gh anem, 1996; Yan et al., 1995; Zhao et al., 1997). My finding that AmphiColA is expressed in the notoc hord and notochordal sheath of Branchiostoma floridae taken together with the recently reported data on Ciona intestinalis CiFCol1 and Branchiostoma belcheri BbFCol1 (both clade A fibrillar collagens), supports the idea that an ancestral ColA gene was expressed in the not ochord of stem-group chordates (Robson et al., 2000; Satou et al., 2001; Wada et al., 2006). We suggest that duplication and
120 divergence of the clade A collagen genes in st em-group vertebrates may have facilitated the evolutionary origin of chondrocytes from notochor dal cells. This hypothesis deals specifically with the origin of vertebrate chondrocytes, and, as mentioned a bove, it is important to note that cartilage is also found in several invertebra tes, including cephalopods, snails, and horseshoe crabs (Cole and Hall, 2004; Wright et al., 2001 ). Future work on the molecular basis of invertebrate chondrogenesis should reveal whether fibrillar collagens also were utilized in these independent evolutionary events. Origin of Vertebrate Axial Skeletons The vertebral column is a key defining characte r of all vertebrates. Lampreys possess some axial skeletal structures named arcualia, which ha ve been suggested to be homologs of vertebral elements. In higher vertebrates, the vertebral columns derive ma inly from sclerotome. Almost a century ago, comparative anatomists suggested that lampreys may have a small sclerotome (Brand-Saberi and Christ, 2000; Maurer, 1906; Sc ott, 1882). However, a recent study on the Japanese lamprey LjPax9 gene showed that this sclerotome marker is absent in paraxial mesoderm (Ogasawara et al., 2000). This finding suggested that la mpreys may lack a sclerotome. In addition, the major matrix of lamprey cartilage was reported to be an elastin-like molecule, named lamprin, instead of COL2A1 protein (Wri ght et al., 2001). Th e chondrogenic difference suggested that lamprey cartilage development may use a different genetic program. So, the embryonic origin of arcualia remains puzzling. Here I reported that th e lamprey and hagfish cartilage matrix contains CO L2A1 proteins and that lamp rey chondrocytes also express Sox9 a chondrogenic master gene in jawed-vertebrates, which suggests that a chondrogenetic pathway is conserved in all extant verteb rates (cyclostomes and gnathos tomes). More importantly, the Col2a1 and Sox9 genes in lamprey are also expressed in the arcualia (Zhang et al., 2006).
121 According to my finding on the conserved c hondrogenetic pathway in lamprey and the previously described anatomical structure of axial cartilages from comparative embryology, I hypothesized that lamprey arcualia develop from the sclerotome, as in tetrapods. By studying eight sclerotomal marker gene expression patter ns in lamprey and catshark, I confirmed that lamprey and catshark possess a sclerotome at the molecular level. Given the conservation of chondrogenetic pathways, it is very likely the la mprey arcualia develop from the sclerotome. More interestingly, the sclerotomal genes are members of multigene families, making it likely that the origin of the vertebral column was one of the vertebrate novelties that resu lted from the large gene or whole genome duplication events that occurred during chordate evolution. Taken together, the results presented in this thesis pu sh back, to an unexpectedly early evolutionary node, the genomic and developmental events th at gave rise to th e vertebrate skeleton.
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155 BIOGRAPHICAL SKETCH GuangJun Zhang was born in northeast of Ch ina. He entered the Medical College of TongJi University in 1993 and finished clinical medicine training and earned his medical degree in 1998. Immediately after medical school, he star ted a masters program at the Chinese Academy of Preventive Medicine in late 1998. He lear ned from Dr. MingYi Xia during the masters training, where his work focused mainly on the phylogeography of human blood fluke parasites, schistosomes, by using ribosomal and mitochondrial gene markers. After that he worked as research associate for two years at the Institute of Neuroscience, Chinese Academy of Science. During this time, he worked predominantly on the signal transduction pa thways in cortical neurons. In August of 2003, GuangJun Zhang be gan his Ph.D. training at the Zoology Department, University of Florida. With Dr Martin Cohn as his advisor, GuangJun Zhang accomplished his dissertation work on the vertebra te skeletal development and evolution. During his Ph.D. program, he was awarded Grinter Fellow ship and Winstar Dissertation Fellowship at the University of Florida.