In this study, I first defined two types of natal down formation in the dorsal skin of zebra finch, in contrast to only one type in chicken. The absence of natal down in Type I feather formation signifies the altricial phenotype in zebra finch. Previous studies found that the naked skins in the sc/sc and naked neck chicken were due to the abolishment of feather bud formation (Mou, et al. 2011; Wells, et al. 2012). However, in zebra finch hatchlings I found typical feather buds in some regions of the skin (Figure 4),
suggesting that a different regulatory mechanism suppresses feather growth. Moreover, according to the expression patterns of SHH (Figure 12), the difference between AD and PD skin regions at the developmental stages studied is not due to heterochrony because AD skin never grows natal down. I utilized the comparative transcriptomics approach to infer that molecules in the FGF/MAPK pathway are involved in the natal down growth suppression and epithelial thickening, leading to naked AD skin regions in zebra finch hatchlings.
FGFs are key players in the processes of proliferation and differentiation of a wide variety of animal cells and tissues (Ornitz and Itoh 2001). In feather elongation, FGFs may play two opposite functions. Some, such as FGF2 and FGF4 (Widelitz, et al. 1996;
Song, et al. 2004), may induce or promote feather growth, while others, such as FGF10 and FGF16, may play a suppressor role (Tao, et al. 2002; Yue, et al. 2012).
Overexpression of FGF10 thickens the epithelium, up-regulates NCAM and
down-regulates SHH. FGF10 suppresses the chicken natal down growth through the epithelium/mesenchyme signaling interaction (Tao, et al. 2002), leading to a phenotype similar to that in zebra finch AD skin in which periodic feather germs are formed, but feather elongation is suppressed.
The natal down growth suppressors showed functional conservation between different skin regions and between avian species. In chicken, FGF10 was shown to suppress the natal down growth in the leg skin previously (Tao, et al. 2002) and in dorsal skin in this study (Figure 16). In my transcriptome analysis, FGF10 expressed higher in AD skin than in PD skin of zebra finch embryos (Table A4), suggesting a role in natal down growth suppression. On the other hand, FGF16 expressed higher in AD skin than in PD skin of zebra finch embryos (Table A4), and suppressed the natal down elongation in the leg skin of chicken embryo, suggesting a role in natal down growth suppression in altricial hatchlings. However, due to experimental limitations in zebra finch, I was unable to overexpress or knock down FGF16 in zebra finch. Furthermore, due to the low expression of FGF16 in zebra finch (FPKM value 1~8), I found it difficult to distinguish noise from the true signal by in situ hybridization. Therefore, I cannot rule out the possibility that the natal down growth was indirectly suppressed due to the wide range of FGF16 overexpression by the RCAS system. More experimental innovations are needed to address these issues.
TWIST2 is known to be a feather growth initiator, but overexpression of TWIST2 induced thickened dermis with normally shaped ectopic feather buds (Hornik, et al.
2005). There are two possible explanations for its role in natal down suppression. First, other molecules such as SNAI1 that showed coexpression with TWIST2 may work in a
It should also be pointed out that the developmental process of natal down is diverse among altricial birds. For example, in most finches, the natal down development is finished at hatchlings, but in the parrots, the natal down growth continues after
hatching (data not shown). Furthermore, when I mapped the altricial and precocial phenotypes onto the recently published avian phylogeny (Zhang, et al. 2014), I found that the altricial-precocial transition occurred multiple times in the past 70 million years, as previously proposed (Starck and Ricklefs 1998). Although the precocial phenotype is considered ancestral to the altricial phenotype, some precocial orders, such as
ciconiiformes and gruiformes, are clustered with altricial lineages, while some altricial orders, such as cuculiformes and apodiformes, are clustered with precocial lineages (Starck and Ricklefs 1998; Zhang, et al. 2014). Thus, different mechanisms may act in the natal down growth regulation in birds. Whether the FGF/MAPK signaling pathway is utilized as the natal down growth suppressor in all altricial birds needs to be
investigated.
The feather bud elongation in AD skin of zebra finch embryos stopped at around E9, and the phenotype of the suppressed feather bud is similar to that in FGF16 overexpressed chicken skins (Figure 4J and Figure 14C). However, the epithelium invagination and feather follicle formation still proceed in the AD skin of E12 zebra finch embryos (Figure 4O), but not in FGF16 overexpressed chicken skins (Figure 14E).
This difference suggests that overexpression FGF16 may suppress invagination and follicle formation or the FGF/MAPK pathway is not the only factor for natal down growth suppression. More works remain to be investigated to identify the whole regulatory network of altricial feather suppression.
The natal down divergence between altricial and precocial hatchlings is thought to
be associated with heat transfer and conservation (Starck and Ricklefs 1998; Bicudo 2010). In altricial hatchlings, most of their body heat is conferred by the parents, and the naked dorsal skin is thought to be associated with heat transduction (Starck and Ricklefs 1998). The cornified epidermal keratinocytes, such as the feathers of birds and the hairs of mammals, are essential for the adaptation of the terrestrial animals (Strasser, et al.
2015). I found that epithelial thickening is a phenotype in featherless AD skin of zebra finch hatchlings. In the naked mole-rat, lack of fur is compensated by a thicker
epidermal layer and a marked reduction in sweat glands (Daly and Buffenstein 1998).
Similar mechanisms might be shared between these naked organisms for environmental adaptation.
The evolution of feathers was so successful as to enable the birds to become the most diverse amniotes. However, like the recurrent losses of limb or eye in animal evolution (Lande 1978; Protas, et al. 2011), feather evolution is not unidirectional.
Fossil records showed that most ancestral birds had flight feathers on their legs, but this phenotype is rare in modern birds (Dhouailly 2009; Zheng, et al. 2013). The loss of the leg flight feather might have enhanced flight ability (Dial, et al. 2008). This study provided another case of feather growth suppression. My view is that the feather growth suppression during Type I feather formation is due to the overexpression of specific suppressors, but not due to the functional loss of the feather growth promoters. The
SUMMARY AND PROSPECTIONS
My major research interest is feather development and evolution. Feather is a unique evolutionary innovation. Feathers are skin appendages but have highly ordered and hierarchically branched structures. Feathers evolved in dinosaurs but underwent dramatic diversification in birds, allowing birds to adapt to various ecological niches.
Natal down is the plumage of avian hatchlings and is used to classify birds into altricial and precocial. Signaling molecules involved in natal down development may be
associated with the natal down divergence and my study showed that the FGF (fibroblast growth factor)/MAPK (mitogen-activated protein kinase) pathway is
involved in natal down growth suppression in zebra finch hatchling (Chen, et al. 2016).
My study provides insights into the regulatory divergence in natal down formation between precocial chicken and altricial zebra finch, but raises questions about bird and feather evolution. The first important question is whether the FGF /MAPK pathway is used by all the naked altricial hatchlings. To answer this question, one needs to understand the evolution of the precocial to altricial continuum. Generally, one avian order only shows one type of developmental mode. I mapped the altricial and precocial phenotypes onto the recently published avian phylogeny (Zhang, et al. 2014) and found that the precocial to altricial transition occurred multiple times in the past 70 million years, as previously proposed (Figure 17) (Starck and Ricklefs 1998; Deeming and Reynolds 2015). Although the precocial phenotype is considered ancestral to the
altricial phenotype, some precocial orders, such as Eurypygiformes and Cariamiformes, are clustered with altricial lineages. Some altricial orders, such as Phoenicopteriformes and Mesitornithiformes, are clustered with precocial lineages (Figure 17) (Starck and Ricklefs 1998; Zhang, et al. 2014; Deeming and Reynolds 2015). Thus, the evolution of
the precocial to altricial continuum should include multiple independent events and different mechanisms may act in the natal down growth regulation in birds. More investigations of the molecular mechanisms of natal down development in different kinds of birds are necessary to resolve this question.
Second, although the FGF/MAPK pathway had been proposed in natal down suppression in this study (Chen, et al. 2016), I cannot identify the evolutionary change in sequence of or close to FGF16 between zebra finch and chicken, showing that some regulators may work upstream to FGF16. A recent bioinformatics pipeline developed in our lab that was used to predict the transcription factors of the specific genes depend on gene co-expression and sequence conservation could be used to predict the upstream transcription factors of FGF16 (Bhattacharjee, et al. 2016).
Third, although the hatchlings of altricial birds are almost naked and those of precocial birds are covered with natal down, most feather follicles (both downy and naked follicles) are replaced by contour feathers when birds are ready to leave the nest in their juveniles (Figure 18) (Podulka, et al. 2004). After several times of moulting, more functional feathers develop from the juvenile feather follicles to achieve specific function in adult birds (Terres and National Audubon Society. 1991), including feathers used in camouflage, migration, overwintering, or courtship (Dunn, et al. 2011). The duration and frequency of juvenile to adult plumage transitions vary among birds, and
regulatory transition from natal down to contour feathers in altricial zebra finch and precocial chicken to understand the similarities and differences between the two types of birds.
Ultimately, by using feather development as the model, I want to understand how changes in gene regulation affect development and cell differentiation to produce new phenotypes.
Figure 18. The juvenile (contour) feather is growing and carries the old natal down on its tip. (Podulka, et al. 2004).
Figure 19. The plumages in hatchling and posthatch day 7 zebra finch. The sections of the hatchling were stained with H&E. AD: anterior dorsal; PD: posterior dorsal; D7:
posthatch day 7. ep: epithelium; me: mesenchyme; ff: feather follicle; MND: mature natal down; Scale bar: 100 μm.
REFERENCES
Abzhanov A, Protas M, Grant BR, Grant PR, Tabin CJ. 2004. Bmp4 and morphological variation of beaks in Darwin's finches. Science 305:1462-1465.
Adkins-Regan E. 2009. Hormones and sexual differentiation of avian social behavior.
Dev Neurosci 31:342-350.
Alev C, Shinmyozu K, McIntyre BA, Sheng G. 2009. Genomic organization of zebra finch alpha and beta globin genes and their expression in primitive and definitive blood in comparison with globins in chicken. Dev Genes Evol 219:353-360.
Basu M, Mukhopadhyay S, Chatterjee U, Roy SS. 2014. FGF16 promotes invasive behavior of SKOV-3 ovarian cancer cells through activation of mitogen-activated protein kinase (MAPK) signaling pathway. J Biol Chem 289:1415-1428.
Bhattacharjee MJ, Yu CP, Lin JJ, Ng CS, Wang TY, Lin HH, Li WH. 2016. Regulatory Divergence among Beta-Keratin Genes during Bird Evolution. Mol Biol Evol.
Bicudo JEPW. 2010. Ecological and environmental physiology of birds. Oxford ; New York: Oxford University Press.
Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114-2120.
Brown WRA, Hubbard SJ, Tickle C, Wilson SA. 2003. The chicken as a model for large-scale analysis of vertebrate gene function. Nature Reviews Genetics
3:169-195.
Chen CK, Ng CS, Wu SM, Chen JJ, Cheng PL, Wu P, Lu MJ, Chen DR, Chuong CM, Cheng HC, et al. 2016. Regulatory Differences in Natal Down Development between Altricial Zebra Finch and Precocial Chicken. Mol Biol Evol.
Chuong C-M. 1998. Molecular basis of epithelial appendage morphogenesis. Austin, TX:
R.G. Landes.
Chuong CM, Widelitz RB, Ting-Berreth S, Jiang TX. 1996. Early events during avian skin appendage regeneration: dependence on epithelial-mesenchymal interaction and order of molecular reappearance. J Invest Dermatol 107:639-646.
Clayton DF, Balakrishnan CN, London SE. 2009. Integrating genomes, brain and behavior in the study of songbirds. Curr Biol 19:R865-873.
Clayton DF, George JM, Mello CV, Siepka SM. 2009. Conservation and expression of IQ-domain-containing calpacitin gene products (neuromodulin/GAP-43, neurogranin/RC3) in the adult and developing oscine song control system. Dev Neurobiol 69:124-140.
Consortium ICGS. 2004. Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432:695-716.
Daly TJ, Buffenstein R. 1998. Skin morphology and its role in thermoregulation in mole-rats, Heterocephalus glaber and Cryptomys hottentotus. J Anat 193 ( Pt 4):495-502.
Deeming DC, Reynolds SJ. 2015. Nests, eggs, and incubation : new ideas about avian reproduction. In.
Dhouailly D. 2009. A new scenario for the evolutionary origin of hair, feather, and avian scales. J Anat 214:587-606.
Dial KP, Jackson BE, Segre P. 2008. A fundamental avian wing-stroke provides a new perspective on the evolution of flight. Nature 451:985-989.
Dunn JL, Alderfer JK, National Geographic Society (U.S.). 2011. National Geographic field guide to the birds of North America. Washington, D.C.: National Geographic Society.
Ewing B, Green P. 1998. Base-calling of automated sequencer traces using phred. II.
Error probabilities. Genome Res 8:186-194.
Geiss GK, Bumgarner RE, Birditt B, Dahl T, Dowidar N, Dunaway DL, Fell HP, Ferree S, George RD, Grogan T, et al. 2008. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 26:317-325.
Greenwold MJ, Sawyer RH. 2010. Genomic organization and molecular phylogenies of the beta (beta) keratin multigene family in the chicken (Gallus gallus) and zebra finch (Taeniopygia guttata): implications for feather evolution. BMC Evol Biol 10:148.
Hamburger V, Hamilton HL. 1992a. A Series of Normal Stages in the Development of the Chick-Embryo, (Reprinted from Journal of Morphology, Vol 88, 1951).
Developmental Dynamics 195:231-&.
Hamburger V, Hamilton HL. 1992b. A series of normal stages in the development of the chick embryo. 1951. Dev Dyn 195:231-272.
activators in feather formation: implications for periodic patterning. Dev Biol 196:11-23.
Lande R. 1978. Evolutionary Mechanisms of Limb Loss in Tetrapods. Evolution 32:73-92.
Langmead B, Trapnell C, Pop M, Salzberg SL. 2009. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25.
Laurell T, Nilsson D, Hofmeister W, Lindstrand A, Ahituv N, Vandermeer J, Amilon A, Anneren G, Arner M, Pettersson M, et al. 2014. Identification of three novel FGF16 mutations in X-linked recessive fusion of the fourth and fifth metacarpals and possible correlation with heart disease. Mol Genet Genomic Med 2:402-411.
Lin CM, Jiang TX, Baker RE, Maini PK, Widelitz RB, Chuong CM. 2009. Spots and stripes: pleomorphic patterning of stem cells via p-ERK-dependent cell
chemotaxis shown by feather morphogenesis and mathematical simulation. Dev Biol 334:369-382.
Liu WY, Chang YM, Chen SC, Lu CH, Wu YH, Lu MY, Chen DR, Shih AC, Sheue CR, Huang HC, et al. 2013. Anatomical and transcriptional dynamics of maize embryonic leaves during seed germination. Proc Natl Acad Sci U S A 110:3979-3984.
Loftus SK, Larson DM, Watkins-Chow D, Church DM, Pavan WJ. 2001. Generation of RCAS vectors useful for functional genomic analyses. DNA Res 8:221-226.
Mandler M, Neubuser A. 2004. FGF signaling is required for initiation of feather placode development. Development 131:3333-3343.
Mardis ER. 2008a. The impact of next-generation sequencing technology on genetics.
Trends Genet 24:133-141.
Mardis ER. 2008b. Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet 9:387-402.
McDevitt D, Rosenberg M. 2001. Exploiting genomics to discover new antibiotics.
Trends Microbiol 9:611-617.
McKinnell IW, Turmaine M, Patel K. 2004. Sonic Hedgehog functions by localizing the region of proliferation in early developing feather buds. Dev Biol 272:76-88.
Meinhardt H, Gierer A. 2000. Pattern formation by local self-activation and lateral inhibition. BioEssays : news and reviews in molecular, cellular and
developmental biology 22:753-760.
Metzker ML. 2005. Emerging technologies in DNA sequencing. Genome Res 15:1767-1776.
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621-628.
Mou C, Pitel F, Gourichon D, Vignoles F, Tzika A, Tato P, Yu L, Burt DW, Bed'hom B, Tixier-Boichard M, et al. 2011. Cryptic patterning of avian skin confers a developmental facility for loss of neck feathering. PLoS Biol 9:e1001028.
Murray JR, Varian-Ramos CW, Welch ZS, Saha MS. 2013. Embryological staging of the Zebra Finch, Taeniopygia guttata. J Morphol 274:1090-1110.
Ng CS, Chen CK, Fan WL, Wu P, Wu SM, Chen JJ, Lai YT, Mao CT, Lu MY, Chen DR,
SJ, et al. 2004. Combined action of extracellular signal-regulated kinase and p38 kinase rescues Molt4 T cells from nitric oxide-induced apoptotic and necrotic cell death. Free Radic Biol Med 37:463-479.
Olivera-Martinez I, Viallet JP, Michon F, Pearton DJ, Dhouailly D. 2004. The different steps of skin formation in vertebrates. Int J Dev Biol 48:107-115.
Ornitz DM, Itoh N. 2001. Fibroblast growth factors. Genome Biol 2:REVIEWS3005.
Paznekas WA, Okajima K, Schertzer M, Wood S, Jabs EW. 1999. Genomic organization, expression, and chromosome location of the human SNAIL gene (SNAI1) and a related processed pseudogene (SNAI1P). Genomics 62:42-49.
Pinaud R. 2010. Genome of a songbird unveiled. J Biol 9:19.
Podulka S, Rohrbaugh RW, Bonney R. 2004. Handbook of bird biology. Ithaca, NY:
Cornell Lab of Ornithology in association with Princeton University Press.
Protas ME, Trontelj P, Patel NH. 2011. Genetic basis of eye and pigment loss in the cave crustacean, Asellus aquaticus. Proc Natl Acad Sci U S A 108:5702-5707.
Prum RO. 2005. Evolution of the morphological innovations of feathers. J Exp Zool B Mol Dev Evol 304:570-579.
Prum RO, Brush AH. 2002. The evolutionary origin and diversification of feathers. Q Rev Biol 77:261-295.
Reimand J, Arak T, Vilo J. 2011. g:Profiler--a web server for functional interpretation of gene lists (2011 update). Nucleic Acids Res 39:W307-315.
Reimand J, Kull M, Peterson H, Hansen J, Vilo J. 2007. g:Profiler--a web-based toolset for functional profiling of gene lists from large-scale experiments. Nucleic Acids Res 35:W193-200.
Sang H. 2004. Prospects for transgenesis in the chick. Mech Dev 121:1179-1186.
Scholl FA, Dumesic PA, Barragan DI, Harada K, Bissonauth V, Charron J, Khavari PA.
2007. Mek1/2 MAPK kinases are essential for Mammalian development, homeostasis, and Raf-induced hyperplasia. Dev Cell 12:615-629.
Schuster SC. 2008. Next-generation sequencing transforms today's biology. Nat Methods 5:16-18.
Song HK, Lee SH, Goetinck PF. 2004. FGF-2 signaling is sufficient to induce dermal condensations during feather development. Dev Dyn 231:741-749.
Starck JM, Ricklefs RE. 1998. Avian growth and development : evolution within the altricial-precocial spectrum. New York: Oxford University Press.
Stern CD. 2004. The chick embryo--past, present and future as a model system in developmental biology. Mech Dev 121:1011-1013.
Stern CD. 2005. The chick: A great model system becomes even greater. Developmental Cell 8:9-17.
Strasser B, Mlitz V, Hermann M, Tschachler E, Eckhart L. 2015. Convergent evolution of cysteine-rich proteins in feathers and hair. BMC Evol Biol 15:82.
Tao H, Yoshimoto Y, Yoshioka H, Nohno T, Noji S, Ohuchi H. 2002. FGF10 is a mesenchymally derived stimulator for epidermal development in the chick embryonic skin. Mech Dev 116:39-49.
Tarazona S, Garcia-Alcalde F, Dopazo J, Ferrer A, Conesa A. 2011. Differential
Biotechnol 31:46-53.
Trapnell C, Pachter L, Salzberg SL. 2009. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105-1111.
Vleck CM, Vleck D. 1987. Metabolism and energetics of avian embryos. J Exp Zool Suppl 1:111-125.
Warnes GR, Bolker B, Bonebakker L, Gentleman R, Huber W, Liaw A, Lumley T, Maechler M, Magnusson A, Moeller S. 2009. gplots: Various R programming tools for plotting data. R package version 2.
Warren WC, Clayton DF, Ellegren H, Arnold AP, Hillier LW, Kunstner A, Searle S, White S, Vilella AJ, Fairley S, et al. 2010. The genome of a songbird. Nature 464:757-762.
Wells KL, Hadad Y, Ben-Avraham D, Hillel J, Cahaner A, Headon DJ. 2012.
Genome-wide SNP scan of pooled DNA reveals nonsense mutation in FGF20 in the scaleless line of featherless chickens. BMC Genomics 13:257.
Widelitz RB, Jiang TX, Lu J, Chuong CM. 2000. beta-catenin in epithelial
morphogenesis: conversion of part of avian foot scales into feather buds with a mutated beta-catenin. Dev Biol 219:98-114.
Widelitz RB, Jiang TX, Noveen A, Chen CW, Chuong CM. 1996. FGF induces new feather buds from developing avian skin. J Invest Dermatol 107:797-803.
Wright MT. 2006. Birds of the world : recommended English names. London:
Christopher Helm.
Yue Z, Jiang TX, Wu P, Widelitz RB, Chuong CM. 2012. Sprouty/FGF signaling
regulates the proximal-distal feather morphology and the size of dermal papillae.
Dev Biol 372:45-54.
Zann RA. 1996. The zebra finch : a synthesis of field and laboratory studies. Oxford ; New York: Oxford University Press.
Zhang GJ, Li C, Li QY, Li B, Larkin DM, Lee C, Storz JF, Antunes A, Greenwold MJ, Meredith RW, et al. 2014. Comparative genomics reveals insights into avian genome evolution and adaptation. Science 346:1311-1320.
Zheng X, Zhou Z, Wang X, Zhang F, Zhang X, Wang Y, Wei G, Wang S, Xu X. 2013.
Hind wings in Basal birds and the evolution of leg feathers. Science 339:1309-1312.
Zhou Z, Zhang F. 2004. A precocial avian embryo from the Lower Cretaceous of China.
Science 306:653.
APPENDIX Appendix Figures
Figure A1. The cluster dendrogram of differential expressed genes. The cut-off for
Figure A1. The cluster dendrogram of differential expressed genes. The cut-off for