鳥類腳部由肉質鱗片和羽毛這兩種皮膚衍生器官構成其豐富的型態多樣性。
多數鳥類在發育過程中會表現肉質鱗片，在高緯度或高海拔地區的鳥類及少數人
為培育種則於腳趾外側有羽毛分布。目前相信調控毛腳性狀的基因座位於雞的第
13 對染色體上，然而至今並沒有明確且詳細的研究說明此外觀差異是在胚胎發
育中受哪些訊號分子的調控。從先前文獻中我們推測一種同源異形轉錄因子—
PITX1 基因—和毛腳性狀有極大的關聯性。不僅因為 PITX1 在脊椎動物的後肢發
育扮演重要角色，更有學者發現 PITX1 基因在高程度表現毛腳性狀的鴿子的表
現量越低、在肉質鱗片性狀中的鴿子則表現量高。在雞種 (Gallus gallus
domesticus)中一樣能觀察到毛腳性狀(ptilopody)的型態多樣性，但是在先前的研究中並未提及 PITX1 基因和雞種的毛腳性狀的關聯性，這則是此研究中致力的
重點。我們選擇三種表現不同程度毛腳性狀的雞種，並觀察在七個不同發育階段
中胚胎的 PITX1 基因表現量變化，發現有毛腳性狀的雞胚胎中 PITX1 基因在不
同胚胎發育階段的表現量相對較少。更進一步利用反轉錄病毒系統 (RCAS)感染
雞胚胎來抑制 PITX1 基因在後肢發育的表現，卻未發現明顯型態改變。我們比較
不同雞種中 PITX1 強化子 Pen 的序列後並未發現單一鹼基對差異(SNPs)，卻發
現在有毛腳性狀的雞種基因組中、於 PITX1 基因上游處有約 55kb 的基因缺失。

Birds show remarkable diversity in the morphology of their feather traits. Most birds have scales on their feet; in some wild birds or some domestic bird mutants, feathers grow on the feet instead. However, the genetic and developmental mechanisms, which determine whether scales or feathers should develop on avian feet, still remain unknown. The homeodomain transcription factor PITX1 loss-of-function mutation has been demonstrated to be responsible for feathered feet in pigeons, but not in the chicken which are ptilopody (feathered shank). However, the PITX1 gene located in the Pti-1 locus, which was responsible for chicken foot feathering with a dominant allele from previous studies.
To investigate if the differential expression of PIXT1 leads to transformation of skin appendages, I compared PITX1 expression in three domestic chicken breeds at seven development stages and the effects of PITX1 inhibition on hindlimbs by RCAS, the retrovirus transfection system. Decreased PITX1 expression was found in feathered foot chicken rather than scaled ones, but no morphology changes were observed after RCAS treatments. Sequences of PITX1 enhancer, Pen, among chicken breeds were also under investigation but no single nucleotide polymorphisms were detected. However, this study found a 55-kb Pen deletion upstream of PITX1 gene in ptilopody chicken.

Abstracts………………………………………………………………………………iv
Introduction……………………………………………………………………………1
Materials and Methods………………………………………………………………6
Results…………………………………………………………………………12
Discussion……………………………………………………………………………22
Figures and Tables……………………………………………………………………34
Appendix……………………………………………………………………………...48
References……………………………………………………………………………49

Al-Qattan, M. M., A. Al-Thunayan, I. Alabdulkareem and M. Al Balwi (2013). "Liebenberg syndrome is caused by a deletion upstream to the PITX1 gene resulting in transformation of the upper limbs to reflect lower limb characteristics." Gene 524(1): 65-71.
Alibardi, L. (2004). "Dermo-epidermal interactions in reptilian scales: speculations on the evolution of scales, feathers, and hairs." J Exp Zool B Mol Dev Evol 302(4): 365-383.
Alibardi, L. and R. H. Sawyer (2002). "Immunocytochemical analysis of beta keratins in the epidermis of chelonians, lepidosaurians, and archosaurians." J Exp Zool 293(1): 27-38.
Benton, M. J., D. Dhouailly, B. Jiang and M. McNamara (2019). "The Early Origin of Feathers." Trends Ecol Evol.
Bhattacharjee, M. J., C. P. Yu, J. J. Lin, C. S. Ng, T. Y. Wang, H. H. Lin and W. H. Li (2016). "Regulatory Divergence among Beta-Keratin Genes during Bird Evolution." Mol Biol Evol 33(11): 2769-2780.
Boer, E. F., H. F. Van Hollebeke and M. D. Shapiro (2017). "Genomic determinants of epidermal appendage patterning and structure in domestic birds." Dev Biol 429(2): 409-419.
Brito, J. M., M. A. Teillet and N. M. Le Douarin (2006). "An early role for sonic hedgehog from foregut endoderm in jaw development: ensuring neural crest cell survival." Proc Natl Acad Sci U S A 103(31): 11607-11612.
Bustin, S. A., V. Benes, J. A. Garson, J. Hellemans, J. Huggett, M. Kubista, R. Mueller, T. Nolan, M. W. Pfaffl, G. L. Shipley, J. Vandesompele and C. T. Wittwer (2009). "The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments." Clin Chem 55(4): 611-622.
Chan, Y. F., M. E. Marks, F. C. Jones, G. Villarreal, Jr., M. D. Shapiro, S. D. Brady, A. M. Southwick, D. M. Absher, J. Grimwood, J. Schmutz, R. M. Myers, D. Petrov, B. Jonsson, D. Schluter, M. A. Bell and D. M. Kingsley (2010). "Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer." Science 327(5963): 302-305.
Chen, C. F., J. Foley, P. C. Tang, A. Li, T. X. Jiang, P. Wu, R. B. Widelitz and C. M. Chuong (2015). "Development, regeneration, and evolution of feathers." Annu Rev Anim Biosci 3: 169-195.
Chen, C. K., C. S. Ng, S. M. Wu, J. J. Chen, P. L. Cheng, P. Wu, M. Y. Lu, D. R. Chen, C. M. Chuong, H. C. Cheng, C. T. Ting and W. H. Li (2016). "Regulatory Differences in Natal Down Development between Altricial Zebra Finch and Precocial Chicken." Mol Biol Evol 33(8): 2030-2043.
Chen, C. W. and C. M. Chuong (1999). "Avian integument provides multiple possibilities to analyse different phases of skin appendage morphogenesis." J Investig Dermatol Symp Proc 4(3): 333-337.
Chuong, C. M., R. B. Widelitz, S. Ting-Berreth and T. X. Jiang (1996). "Early events during avian skin appendage regeneration: dependence on epithelial-mesenchymal interaction and order of molecular reappearance." J Invest Dermatol 107(4): 639-646.
Conlon, F. L., L. Fairclough, B. M. Price, E. S. Casey and J. C. Smith (2001). "Determinants of T box protein specificity." Development 128(19): 3749-3758.
DeLaurier, A., R. Schweitzer and M. Logan (2006). "Pitx1 determines the morphology of muscle, tendon, and bones of the hindlimb." Dev Biol 299(1): 22-34.
Dhouailly, D. (1973). "Dermo-epidermal interactions between birds and mammals: differentiation of cutaneous appendages." J Embryol Exp Morphol 30(3): 587-603.
Dhouailly, D. (1975). "Formation of cutaneous appendages in dermo-epidermal recombinations between reptiles, birds and mammals." Wilehm Roux Arch Dev Biol 177(4): 323-340.
Dhouailly, D. (2009). "A new scenario for the evolutionary origin of hair, feather, and avian scales." J Anat 214(4): 587-606.
Domyan, E. T., Z. Kronenberg, C. R. Infante, A. I. Vickrey, S. A. Stringham, R. Bruders, M. W. Guernsey, S. Park, J. Payne, R. B. Beckstead, G. Kardon, D. B. Menke, M. Yandell and M. D. Shapiro (2016). "Molecular shifts in limb identity underlie development of feathered feet in two domestic avian species." Elife 5: e12115.
Domyan, E. T. and M. D. Shapiro (2016). "Pigeonetics takes flight: Evolution, development, and genetics of intraspecific variation." Dev Biol.
Feng, C., Y. Gao, B. Dorshorst, C. Song, X. Gu, Q. Li, J. Li, T. Liu, C. J. Rubin, Y. Zhao, Y. Wang, J. Fei, H. Li, K. Chen, H. Qu, D. Shu, C. Ashwell, Y. Da, L. Andersson, X. Hu and N. Li (2014). "A cis-regulatory mutation of PDSS2 causes silky-feather in chickens." PLoS Genet 10(8): e1004576.
Gordon, C. T., F. A. Rodda and P. G. Farlie (2009). "The RCAS retroviral expression system in the study of skeletal development." Dev Dyn 238(4): 797-811.
Hamburger, V. (1938). "Morphogenetic and axial self-differentiation of transplanted limb primordia of 2-day chick embryos." Journal of Experimental Zoology 77(3): 379-399.
Hamburger, V. and H. L. Hamilton (1992). "A series of normal stages in the development of the chick embryo. 1951." Dev Dyn 195(4): 231-272.
Hardy, M. H., D. Dhouailly and P. Sengel (1981). "Scales into feathers--an effect of retinoic acid on tissue interactions in the developing chick." Ann N Y Acad Sci 359: 394-395.
Hartley, J. L., G. F. Temple and M. A. Brasch (2000). "DNA cloning using in vitro site-specific recombination." Genome Research 10(11): 1788-1795.
Himly, M., D. N. Foster, I. Bottoli, J. S. Iacovoni and P. K. Vogt (1998). "The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses." Virology 248(2): 295-304.
Hu, D., J. A. Clarke, C. M. Eliason, R. Qiu, Q. Li, M. D. Shawkey, C. Zhao, L. D'Alba, J. Jiang and X. Xu (2018). "A bony-crested Jurassic dinosaur with evidence of iridescent plumage highlights complexity in early paravian evolution." Nat Commun 9(1): 217.
Hughes, S. H. (2004). "The RCAS vector system." Folia Biol (Praha) 50(3-4): 107-119.
Hughes, S. H., J. J. Greenhouse, C. J. Petropoulos and P. Sutrave (1987). "Adapter Plasmids Simplify the Insertion of Foreign DNA into Helper-Independent Retroviral Vectors." Journal of Virology 61(10): 3004-3012.
Ishii, Y. and T. Mikawa (2005). "Somatic transgenesis in the avian model system." Birth Defects Res C Embryo Today 75(1): 19-27.
Ishii, Y., D. E. Reese and T. Mikawa (2004). "Somatic transgenesis using retroviral vectors in the chicken embryo." Dev Dyn 229(3): 630-642.
Klopocki, E., C. Kahler, N. Foulds, H. Shah, B. Joseph, H. Vogel, S. Luttgen, R. Bald, R. Besoke, K. Held, S. Mundlos and I. Kurth (2012). "Deletions in PITX1 cause a spectrum of lower-limb malformations including mirror-image polydactyly." Eur J Hum Genet 20(6): 705-708.
Kragesteen, B. K., F. Brancati, M. C. Digilio, S. Mundlos and M. Spielmann (2019). "H2AFY promoter deletion causes PITX1 endoactivation and Liebenberg syndrome." J Med Genet.
Kragesteen, B. K., M. Spielmann, C. Paliou, V. Heinrich, R. Schopflin, A. Esposito, C. Annunziatella, S. Bianco, A. M. Chiariello, I. Jerkovic, I. Harabula, P. Guckelberger, M. Pechstein, L. Wittler, W. L. Chan, M. Franke, D. G. Lupianez, K. Kraft, B. Timmermann, M. Vingron, A. Visel, M. Nicodemi, S. Mundlos and G. Andrey (2018). "Dynamic 3D chromatin architecture contributes to enhancer specificity and limb morphogenesis." Nat Genet 50(10): 1463-1473.
Lanctot, C., A. Moreau, M. Chamberland, M. L. Tremblay and J. Drouin (1999). "Hindlimb patterning and mandible development require the Ptx1 gene." Development 126(9): 1805-1810.
Logan, M. and C. Tabin (1998). "Targeted gene misexpression in chick limb buds using avian replication-competent retroviruses." Methods 14(4): 407-420.
Logan, M. and C. J. Tabin (1999). "Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity." Science 283(5408): 1736-1739.
Marcil, A., E. Dumontier, M. Chamberland, S. A. Camper and J. Drouin (2003). "Pitx1 and Pitx2 are required for development of hindlimb buds." Development 130(1): 45-55.
Musser, J. M., G. P. Wagner and R. O. Prum (2015). "Nuclear beta-catenin localization supports homology of feathers, avian scutate scales, and alligator scales in early development." Evol Dev 17(3): 185-194.
Naiche, L. A. and V. E. Papaioannou (2007). "Tbx4 is not required for hindlimb identity or post-bud hindlimb outgrowth." Development 134(1): 93-103.
Ng, C. S. and W. H. Li (2018). "Genetic and Molecular Basis of Feather Diversity in Birds." Genome Biol Evol 10(10): 2572-2586.
Ortolani-Machado, C., P. De Freitas, M. E. Borges and C. Faraco (2008). "Special features of dermal melanocytes in white silky chicken embryos." Anat Rec (Hoboken) 291(1): 55-64.
Proszkowiec-Weglarz, M., S. E. Higgins and T. E. Porter (2011). "Changes in gene expression during pituitary morphogenesis and organogenesis in the chick embryo." Endocrinology 152(3): 989-1000.
Rodriguez-Esteban, C., T. Tsukui, S. Yonei, J. Magallon, K. Tamura and J. C. Izpisua Belmonte (1999). "The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity." Nature 398(6730): 814-818.
Ruvinsky, I. and J. J. Gibson-Brown (2000). "Genetic and developmental bases of serial homology in vertebrate limb evolution." Development 127(24): 5233-5244.
Sauka-Spengler, T. and M. Barembaum (2008). "Gain- and loss-of-function approaches in the chick embryo." Methods Cell Biol 87: 237-256.
Seki, R., C. Li, Q. Fang, S. Hayashi, S. Egawa, J. Hu, L. Xu, H. Pan, M. Kondo, T. Sato, H. Matsubara, N. Kamiyama, K. Kitajima, D. Saito, Y. Liu, M. T. Gilbert, Q. Zhou, X. Xu, T. Shiroishi, N. Irie, K. Tamura and G. Zhang (2017). "Functional roles of Aves class-specific cis-regulatory elements on macroevolution of bird-specific features." Nat Commun 8: 14229.
Shames, R. B., A. G. Jennings and R. H. Sawyer (1991). "Expression of the cell adhesion molecules, L-CAM and N-CAM during avian scale development." J Exp Zool 257(2): 195-207.
Shames, R. B. and R. H. Sawyer (1987). "Expression of beta-keratin genes during development of avian skin appendages." Curr Top Dev Biol 22: 235-253.
Shapiro, M. D. and E. T. Domyan (2013). "Domestic pigeons." Curr Biol 23(8): R302-303.
Sharpe, P. T. (2001). "Fish scale development: Hair today, teeth and scales yesterday?" Curr Biol 11(18): R751-752.
Somes, R. G., Jr. (1992). "Identifying the ptilopody (feathered shank) loci of the chicken." J Hered 83(3): 230-234.
Szeto, D. P., C. Rodriguez-Esteban, A. K. Ryan, S. M. O'Connell, F. Liu, C. Kioussi, A. S. Gleiberman, J. C. Izpisua-Belmonte and M. G. Rosenfeld (1999). "Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development." Genes Dev 13(4): 484-494.
Takeuchi, J. K., K. Koshiba-Takeuchi, K. Matsumoto, A. Vogel-Hopker, M. Naitoh-Matsuo, K. Ogura, N. Takahashi, K. Yasuda and T. Ogura (1999). "Tbx5 and Tbx4 genes determine the wing/leg identity of limb buds." Nature 398(6730): 810-814.
Takeuchi, J. K., K. Koshiba-Takeuchi, T. Suzuki, M. Kamimura, K. Ogura and T. Ogura (2003). "Tbx5 and Tbx4 trigger limb initiation through activation of the Wnt/Fgf signaling cascade." Development 130(12): 2729-2739.
Thompson, A. C., T. D. Capellini, C. A. Guenther, Y. F. Chan, C. R. Infante, D. B. Menke and D. M. Kingsley (2018). "A novel enhancer near the Pitx1 gene influences development and evolution of pelvic appendages in vertebrates." Elife 7.
Wu, P., J. Yan, Y. C. Lai, C. S. Ng, A. Li, X. Jiang, R. M. Elsey, R. Widelitz, R. Bajpai, W. H. Li and C. M. Chuong (2018). "Multiple Regulatory Modules Are Required for Scale-to-Feather Conversion." Mol Biol Evol 35(2): 417-430.
Xie, K. T., G. Wang, A. C. Thompson, J. I. Wucherpfennig, T. E. Reimchen, A. D. C. MacColl, D. Schluter, M. A. Bell, K. M. Vasquez and D. M. Kingsley (2019). "DNA fragility in the parallel evolution of pelvic reduction in stickleback fish." Science 363(6422): 81-84.
Xing, L. D., J. K. O'Connor, R. C. McKellar, L. M. Chiappe, K. Tseng, G. Li and M. Bai (2017). "A mid-Cretaceous enantiornithine (Aves) hatchling preserved in Burmese amber with unusual plumage." Gondwana Research 49: 264-277.