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Detection Of Tfap2 Family Proteins In Developing Mouse Teeth

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Title:
Detection Of Tfap2 Family Proteins In Developing Mouse Teeth
Series Title:
19th Annual Undergraduate Research Symposium
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Gutierrez, Galaxy
Language:
English
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Undetermined

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Center for Undergraduate Research
Center for Undergraduate Research
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Conference papers and proceedings
Poster

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Abstract:
Mammalian teeth develop from two tissues, dental mesenchyme and dental epithelium. The dental mesenchyme is composed of cells derived from the neural crest, a cell population which forms early in development and goes on to form many structures in the developing embryo, including teeth. Many genes are necessary for proper tooth development. AP-2 transcription factors are involved in cell proliferation and apoptosis during neural crest development and it is known that transcription factor AP-2 alpha (Tfap2a) is necessary for craniofacial development in mice. The research question we are addressing is: are AP-2a, AP-2b, and AP-2c proteins present in neural crest-derived cells during tooth development? We have already ascertained the presence of Tfap2a and Tfap2b mRNA in developing mouse teeth. Subsequently, we need to confirm the location of these protein products, AP-2a and AP-2b, as well as a related protein, AP-2c. We employ immunohistochemistry (IHC) to detect proteins on histological sections from wild-type and transgenic embryonic mouse heads. In these transgenic mice, neural crest-derived cells fluoresce green and IHC reveals red fluorescent AP-2 proteins. In wild type mice, we have documented the presence of AP-2a protein in the molar epithelium and we are currently examining AP-2 proteins in transgenic mice. ( en )
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Research authors: Galaxy C. Gutierrez, Emily D. Woodruff, Martin J. Cohn - University of Florida
General Note:
University Scholars Program
General Note:
Faculty Mentor: Mammalian teeth develop from two tissues, dental mesenchyme and dental epithelium. The dental mesenchyme is composed of cells derived from the neural crest, a cell population which forms early in development and goes on to form many structures in the developing embryo, including teeth. Many genes are necessary for proper tooth development. AP-2 transcription factors are involved in cell proliferation and apoptosis during neural crest development and it is known that transcription factor AP-2 alpha (Tfap2a) is necessary for craniofacial development in mice. The research question we are addressing is: are AP-2a, AP-2b, and AP-2c proteins present in neural crest-derived cells during tooth development? We have already ascertained the presence of Tfap2a and Tfap2b mRNA in developing mouse teeth. Subsequently, we need to confirm the location of these protein products, AP-2a and AP-2b, as well as a related protein, AP-2c. We employ immunohistochemistry (IHC) to detect proteins on histological sections from wild-type and transgenic embryonic mouse heads. In these transgenic mice, neural crest-derived cells fluoresce green and IHC reveals red fluorescent AP-2 proteins. In wild type mice, we have documented the presence of AP-2a protein in the molar epithelium and we are currently examining AP-2 proteins in transgenic mice. - Center for Undergraduate Research, University Scholars Program

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University of Florida
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Copyright Galaxy Gutierrez. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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Detection of Tfap2 Family Proteins in Developing Mouse Teeth Galaxy C. Gutierrez 1 Emily D. Woodruff 1 Martin J. Cohn 1,2 1 Department of Biology, 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Discussion Background Information Research Question We have documented the expression of AP-2a protein in upper and lower incisors in the dental epithelium at day 14 in wild type and transgenic mice. We have documented AP-2a protein expression in wild-type mice at a late stage of tooth development (mouse embryonic day 16.5). AP-2a is localized to the molar epithelium with intense staining at the tips of the cusps (where the enamel and dentin form). The results of the immunouorescence performed for AP-2b and AP-2c on wild-type mice were unclear due to high background staining. We are currently working on optimizing the protocol for AP-2b and c Hypothesis Are AP-2a, AP-2b, and AP-2c proteins present in neural crest-derived cells during tooth development? Methods Adult mice have both incisor and molar teeth (Fig 1). We are studying developmental genetic differences between incisor and molar teeth in laboratory mice to identify genes that help dictate differences in tooth shape. Immunouorescence Visual-spatial detection of proteins on histological sections. 1. A specic primary antibody attaches to the target AP-2 protein under investigation in the tissue. 2. A secondary antibody with a uorescent molecular tag attaches to the primary antibody. 3. Proteins tagged with a uorescent dye are visualized using confocal microscopy. AP-2 Transcription Factors AP-2 transcription factors are involved in cell proliferation and apoptosis during neural crest development it is known that transcription factor AP-2 alpha ( Tfap2a ) is necessary for craniofacial development in mice. I predict the locations of AP-2a and AP-2b proteins at the bud and cap stages (embryonic days 13.5-14.0) will correspond with the location of the mRNA expression that we demonstrated in previous experiments. Specically, I expect that AP-2a will be in the dental epithelium and dental mesenchyme, and AP-2b will be expressed solely in the dental mesenchyme. I hypothesize the location of AP-2c protein at the at the bud and cap stages of developing mouse teeth will be in the dental epithelium alone, the dental mesenchyme alone, in both the dental epithelium and mesenchyme, or neither tissue. References Results Mammalian teeth develop from two tissues, the dental epithelium and the dental mesenchyme. Dental mesenchyme is derived predominantly form the cranial neural crest Mouse Tooth Development Figure 1. Lower dentition of a mouse, viewed from the top. A. Ever-growing incisors. B. Diastema (space), C. Molars. Photo: EDW. Embryonic Day 11 Embryonic Day 13.5 Embryonic Day 14.5 Embryonic Day 16.5 Oral epithelium thickens and invaginates into the mesenchyme ( Nanci 2013) Inltration of epithelium into the underlying mesenchyme of the jaw ( Nanci 2013) The tooth continues to grow, and molecular signals from clusters of nondividing epithelial cells orchestrate the arrangement of cusps on the tooth surface ( Nanci 2013) Continued growth of the enamel organ resembles a "bell" shape and the tooth crown undergoes morpho differentiation into its nal shape ( Nanci 2013) Figure 4. A. H&E stained anterior section through the face of a mouse at embryonic day (E) 13.5; B. H&E stained upper mouse incisor at E13.5; C. Upper incisor of Sox10icre;mTmG transgenic mouse at E13.5; D. In situ hybridization of upper mouse incisor at E13.5; E. AP-2a immunouorescence on upper mouse incisor at E13.5. AP-2a is present in the dental epithelium. Blue is a nuclear stain (Hoechst), magenta is AP-2a proteins. Figure 5. A. H&E stained anterior section through the face of a mouse at embryonic day (E) 13.5; B. H&E stained lower mouse incisor at E13.5; C. Lower incisor of Sox10icre;mTmG transgenic mouse at E13.5; D. In situ hybridization of lower mouse incisor at E13.5; E. AP-2a immunouorescence on lower mouse incisor at E13.5. Blue is a nuclear stain, magenta is AP-2a proteins. Figure 6. A. H&E stained anterior section through the face of a mouse at embryonic day (E) 13.5; B. H&E stained upper and lower mouse molars at E13.5; C. Upper and lower mouse molars of Sox10icre/ mTmG transgenic mouse at E13.5; D. In situ hybridization of upper and lower mouse molars at E13.5; E. AP-2a immunouorescence on upper and lower mouse molars at E13.5. Blue is a nuclear stain, magenta is AP-2a proteins. Figure 3. Two views of mouse embryonic head at embryonic day 13.5; A. Arrow denotes direction head was cut. Planes denote location of sections in head. Green shows location of Fig. 4, Red shows Fig. 5, and blue shows Fig. 6; B. Front view Figure 2. Mouse Tooth Developmental Stages. Drawings by EDW, after Tucker and Sharpe 2004. Nanci Antonio, and Ten Cate A. R." Ten Cates oral histology: development, structure, and function 8th ed., St. Louis, MO, Elsevier, 2013. Tucker, A. and Sharpe, P. The Cutting-Edge of Mammalian Development; How the Embryo Makes Teeth. Nature Reviews. 2004 Jul; 5: 499-508. A B C D E A A B B C C D D E E A B