5.5 Beaks, feathers and silk Birds and reptiles have their own keartins , very different from the -keratins in mammals. Like keratins, bird and reptile keratinsare composites made up from both fibrous and matrix components. The fibrous feather keratin can only stretch some 6% before breaking, unlike hair akeratin that can stretch to twice its length. Protein chemists think that the main secondary structure in bird and reptile keratin is the -sheet. The -sheet does not lie flat but twists gradually (Figure 13). Each polypeptide chain in these -keratinshas a central helical section with less regular regions at each end. These regions contribute to the matrix component and have some -S-Scross-links. http://resources.schoolscience.co.uk/unilever/16-18/proteins/Protch5pg6.html THE LOOM Your Inner Feather POSTED THU, 11/20/2014 Feathers are like eyes or or hands. They’re so complex, so impressive in their adaptations, so good at getting a job done, that it can be hard at first to believe they evolved. Feathers today are only found on birds, which use them to do things like fly, control their body temperature, and show off for potential mates. The closest living relatives of birds– alligators and crocodiles–are not exactly known for their plumage. At least among living things, the glory of feathers is an all-or-nothing affair. But the more we get to know feathers, the more we can appreciate how they evolved. The general rule is that complex things–be they feathers, hands, or eyes–take a very long time to evolve. As I wrote in National Geographic in 2011, the fossil record has gone a very long way in helping us to understand how feathers took on the form we see today. Birds evolved from dinosaur ancestors, and those ancestors already had feathers. Feathers started out as simple filaments, turning to fuzz, and then diversifying into a lot of different forms– including the ones that eventually let birds take to the air. A sampling of feathered dinosaurs and early birds. Xing Lida/National Geographic Now a new study in the journal Molecular Biology and Evolution offers an even deeper look into the history of feathers. Instead of looking at fossils, the scientists look at the genetic recipe for feathers written in the DNA of birds. It turns out that a lot of that recipe already existed hundreds of millions of years before anything vaguely resembling a feather existed on Earth. In fact, you, my fine unfeathered friend, have most of the genetic information required for making feathers, too. Scott Edwards, a Harvard ornithologist, and his colleagues couldn’t have carried out this study even a few years ago, because scientists have only recently figured out a lot of the details of how feathers develop. Bird embryos starts out featherless. But in their skin, they develop lots of tiny blobs of cells known as placodes in which cells are switching on genes in a distinctive pattern. The reason that certain genes switch on in the placodes and others don’t is that genes have little on-off switches near them. If a particular combination of proteins lands on a gene’s switch, the gene will start making a protein of its own. The development of a feather. The middle figure is a cross-section of the primordial feather shown on the left. Source: Ng et al 2012. PLoS Genet 8(7): e1002748 At first, the cells in the placodes multiply quickly. Then they start grow into shafts, which then split open to form feathers. Depending on the bird, and on the spot on the bird’s body where it grows, the feather may split into a downy plume, or into a paddle-shaped flight feather, or into an ornamental tail feather. Along the way, the cells differentiate, producing different combinations of proteins. The cells that make up the central shaft of the feather are stiffened with certain types of keratin, for example, while cells that are in the more delicate regions of the feather produce more flexible forms of the protein from different genes. Clusters of cells produce pigment molecules to give the feather colors and patterns. Every cell has an entire genome, which means it has all the genes for making any part of the feather. But its switches ensure that it only uses a certain combination of those genes. Frizzled chicken. Photo by Alisha Vargas via Creative Commons https://flic.kr/p/7XmQqu Edwards and his colleagues combed the scientific literature for genes that are important for feather development. Scientists have studied frizzled chickens, for example, identified the mutation for the breed’s frizzles, and thereby identified a gene essential for developing feathers. All told, Edwards and his colleagues found 193 feather genes this way. Their list included 67 that encode variations of keratin, and 126 that help establish the pattern of feathers. Next, the scientists searched for the switches that control those genes. This isn’t so easy. The switches are short stretches of DNA, often nestled deep inside much longer stretches of DNA that are just gibberish. What’s more, genes have distinctive segments that let you know you’re looking at a gene. The switches are much harder to distinguish from the gibberish. The scientists used several strategies to zero in on the switches. They took advantage of the fact that most switches for a gene are close to the gene itself. So they only searched in the neighborhood of the 167 feather genes. They also took advantage of the fact that switches evolve relatively little, because most mutations will be harmful to them. So the scientists compared the DNA around feather genes in several different species and sought out the stretches that were noticeably similar from one species to the next. Using these two strategies, the scientists discovered a staggering 13,307 feather gene switches (technically known as conserved nonexonic elements, or CNEEs for short). Next, the scientists asked when each part of this feather cookbook evolved. If they found a gene or a switch only in the DNA of birds, then they could be confident that it had evolved after the ancestors of birds split off from the ancestors of alligators and crocodiles. But if they found a gene or a switch in birds and alligators and crocodiles, then it must have evolved earlier, in their common ancestor. (You may be asking, how did these new genes evolve? The short answer is that they can evolve through the duplication of old genes, or the transformation of genetic gibberish–a k a noncoding DNA. If you want to get more details, watch this TED-Ed video I wrote the script for.) To see how far back the evolution of feather genes went, the scientists compared birds to a wide range of vertebrates, including humans, turtles, and pufferfish. They found that the instructions for making feathers got their start a long, long time before feathers themselves (see the tree at the bottom of the post or embiggen it here). The genes that establish the basic pattern of placodes already existed in the common ancestor of living fish and birds (and us)–in other words, about half a billion years ago. Even more feather genes evolved as our common ancestors climbed ashore and walked around on land 350 million years ago. Many switches for feather genes also emerged during this period, too. About 300 million years ago, our ancestors began to lay hard-shelled eggs. Those early animals would give rise to mammals, reptiles, and birds (collectively known as amniotes, named for the amniotic egg). Edwards and his colleagues found that the first amniotes already had the entire complement of feather patterning genes. That means you, as an amniote, have them too. Later, the early amniotes split away into their major lineages. The lineage that includes alligators, birds, and extinct dinosaurs–called archosaurs–originated about 250 million years ago. Edwards and his colleagues detected many new keratin genes evolving during the origin of archosaurs, along with 86 percent of their 13,000 or so feather gene switches. It would not be another 100 million years or so before the oldest known birds flew. And yet just about everything you need to make a feather, genetically speaking, was already in place. It may seem strange to consider the fact that you, as a mammal, have all the known genes required to pattern a feather, and yet you do not look like Big Bird. The reason for this discrepancy is that genes can do different jobs. Depending on where and when they make their proteins, they can build different kinds of anatomy. But it didn’t take much rewiring of genetic switches to turn the scaly skin of early reptiles into feathers. Indeed, the deep history of feathers could explain why paleontologists are finding so much evidence of simple feather-like filaments not just in dinosaurs, but in their close relatives like pterosaurs. Evolution was tinkering with the same toolkit. Edwards and his colleagues noticed something else intriguing in the genomes of birds. They found a lot of switches that were not near feather genes, but were unique to birds. When the scientists looked for the nearest genes, they noticed that many of the genes help birds grow. They control the size of bird bodies, for example, or the size of their limbs. This is an intriguing finding, because the fossil record reveals that as dinosaurs evolved into birds, their bodies shrank, while their arms got long for their size. The shift made it possible for birds to generate a lot of lift with big wings, which only had to keep a small body aloft. Edwards and his colleagues may have found the molecular signature of that change. If they’re right, the cookbook for feathers is very old, but it took the evolution of a new kind of body for birds to use their feathers to fly. From Lowe et al 2014 (Click to embiggen) With apologies to Neil Shubin for riffing on his title. ABOUT CARL ZIMMER Carl Zimmer is a columnist for the New York Times. His award-winning articles also appear frequently in National Geographic and other publications. He is the author of 13 books, including Parasite Rex and The Tangled Bank: An Introduction to Evolution. You can find him on Twitter, Facebook, Pinterest, and Google+. http://phenomena.nationalgeographic.com/2014/11/20/your-inner-feather/ BMC Evol Biol. 2015; 15: 82. Published online 2015 May 7. doi: 10.1186/s12862-015-0360-y PMCID: PMC4423139 PMID: 25947341 Convergent evolution of cysteine-rich proteins in feathers and hair Bettina Strasser, Veronika Mlitz, Marcela Hermann, Erwin Tschachler, and Leopold Eckhart Author information ► Article notes ► Copyright and License information ► Disclaimer This article has been cited by other articles in PMC. Abstract Go to: Background Feathers and hair consist of cornified epidermal keratinocytes in which proteins are crosslinked via disulfide bonds between cysteine residues of structural proteins to establish mechanical resilience. Cysteine-rich keratinassociated proteins (KRTAPs) are important components of hair whereas the molecular components of feathers have remained incompletely known. Recently, we have identified a chicken gene, named epidermal differentiation cysteine-rich protein (EDCRP), that encodes a protein with a cysteine content of 36%. Here we have investigated the putative role of EDCRP in the molecular architecture and evolution of feathers. Go to: Results Comparative genomics showed that the presence of an EDCRP gene and the high cysteine content of the encoded proteins are conserved among birds. Avian EDCRPs contain a species-specific number of sequence repeats with the consensus sequence CCDPCQ(K/Q)(S/P)V, thus resembling mammalian cysteine-rich KRTAPs which also contain sequence repeats of similar sequence. However, differences in gene loci and exonintron structures suggest that EDCRP and KRTAPs have not evolved from a common gene ancestor but represent the products of convergent sequence evolution. mRNA in situ hybridization demonstrated that chicken EDCRP is expressed in the subperiderm layer of the embryonic epidermis and in the barbule cells of growing feathers. This expression pattern supports the hypothesis that feathers are evolutionarily derived from the subperiderm. Conclusions Electronic supplementary material Go to: Background The evolution of genes that facilitate the cornification of keratinocytes was crucial for the evolution of skin appendages such as hair and feathers. Mature skin appendages consist of dead keratinocytes which are interconnected by stable junctions and filled with highly cross-linked proteins. The process of intracellular protein cross-linking involves either transglutamination, the covalent connection of glutamine and lysine residues, or disulfide bonding, that is, the covalent connection of cysteine residues. Mammals have distinct sets of proteins that have evolved as efficient substrates for cornification-associated cross-linking [1]. These cornification substrates include cysteine-rich keratins, also known as hair keratins [2,3], and keratin-associated proteins (KRTAPs) [4,5] as well as proteins encoded by genes of the so-called epidermal differentiation complex (EDC) [6,7]. The latter is a cluster of genes that are expressed during terminal differentiation of epidermal keratinocytes. Many proteins encoded by EDC genes contain glutamine and lysine-rich sequence motifs and some of them also have a high cysteine content around 15% [6]. Recently, we have reported that sauropsids (reptiles and birds) have genes homologous to hair keratin genes [3] as well as a gene cluster homologous to the mammalian EDC [8]. However, homologs of KRTAPs have not been identified in sauropsids [3]. The EDC of the chicken contains a gene coding for a protein with an extremely high content of cysteine residues, named epidermal differentiation cysteine-rich protein (EDCRP) [8]. Cysteine makes up 140 of the 385 amino acid residues of chicken EDCRP. The EDCRP gene and its neighboring genes have 2 exons, of which the second one contains the entire coding region [8]. Thus, EDCRP has the same gene structure as the genes encoding the so-called beta-keratins (also known as corneous beta-proteins) [8], which are the most abundant proteins of sauropsidian scales and claws as well as avian feathers [9,10]. Expression of EDCRP was detected by RT-PCR screening in embryonic skin from various body sites of the chicken [8]. However, its expression pattern at the cellular level has remained elusive. Here we report the investigation of the evolutionary history and the expression pattern of EDCRP in the skin and feathers of the chicken. Our data suggest an important role of EDCRP in the molecular architecture and in the evolution of feathers. Go to: Results EDCRP is expressed in subperiderm and feathers of the chicken Based on our previous analysis of the gene structure of chicken EDCRP [8], we designed primers and probes suitable for the specific detection of EDCRP mRNA by RT-PCR and in situ hybridization, respectively. RTPCR was performed on RNAs from skin and skin appendages of chicken embryos and adult chicken (Figure 1). In the skin of the legs, EDCRP was detected on embryonic day E18 but not, at significant amounts, on days E10 and E14 nor in adult leg skin. By contrast, feather follicles and feathers were positive for EDCRP from E10 to adults. To more precisely determine the expression pattern of EDCRP, we performed mRNA in situ hybridization. In the embryonic epidermis, EDCRP mRNA was absent from the basal and suprabasal epidermal layers that correspond to those of adult chicken skin and in the superficial embryonic skin layer, the periderm. By contrast, strong staining was present in the subperiderm (Figure 2A), a layer of embryonic periderm-related cells that is specific to archosaurs (Crocodilia and Aves) [11]. The negative control experiment in which the mRNA antisense probe was replaced by a labeled probe in sense orientation yielded no staining (Figure 2B), thereby confirming the specificity of the assay. In some regions of the embryonic skin, the subperiderm showed little or no labeling, which was likely caused by masking or degradation of EDCRP mRNA in advanced cornification of the subperiderm. In situ hybridization also revealed prominent expression of EDCRP in barbule cells of the feathers. Positive labeling was observed in samples of E15 (not shown) and on E18 (Figure 2C, E, G). The labeling was strongest in the developmentally youngest barbule cells in the lateral part of the feather (Figure 2C, E, G) whereas cornified barbs were not stained. Negative control experiments with sense probes confirmed the specificity of the signals (Figure 2D, F, H). The feather sheath and feather pulp were consistently negative (Figure 2C-H). The expression pattern of EDCRP is consistent with the hypothesis that the cyclical growth and shedding of feathers is a modified replication of a series of steps in embryonic skin development (Figure 3). In this model, the feather sheath is the equivalent of the embryonic periderm, as suggested by the common expression of scaffoldin and presence of periderm granules [12] (blue layers in Figure 3); the permanent components of the feathers are equivalent to the embryonic subperiderm with both expressing EDCRP (red in Figure 3); and the epithelial cell layer, that borders on the dermis (grey layers in Figure 3) during early feather development and later degenerates [13], is equivalent to the epidermis proper of the embryo (yellow layers in Figure 3). EDCRP appears to function both in the subperiderm and in the feathers, presumably by facilitating intermolecular crosslinking via its many cysteine residues. The cysteine-rich sequence of EDCRP is conserved among birds To test which features of chicken EDCRP are conserved among birds, we characterized EDCRP genes in a panel of genome sequences from phylogenetically diverse avian species and compared the sequences. Indeed, we identified partial or complete coding sequences of EDCRP in all birds investigated (Additional file 1: Table S1, Additional file 2: Figure S1). Gaps in the genome sequence assemblies and presumable artefacts of genome sequencing or sequence assembly caused gaps or premature ends in the coding sequence of EDCRP orthologs of several species (Additional file 2: Figure S1, and data not shown). A frameshift within the coding sequence of EDCRP was present in the genome sequence of the zebra finch deposited in the GenBank (Accession number NC_011489.1). However, amplification and sequencing of the zebra finch EDCRP gene revealed a contiguous open reading frame encoding all the protein domains present in chicken EDCRP (Additional file 2: Figure S1). The genome sequence of ostrich (Struthio camelus australis) contained a gap within the EDCRP gene. Amplification and sequencing of this region suggested the presence of 2 EDCRP forms, perhaps corresponding to 2 alleles, which differed by the absence or presence of a 27 bp stretch of nucleotides within a repetitive sequence region (Additional file 2: Figure S1). Thus, our data indicate that EDCRP is conserved among birds. All available complete avian EDCRP genes contained a single coding exon that was preceded by a sequence highly similar to the experimentally verified non-coding exon 1 of chicken EDCRP [8]. Sequence comparison of exon 1 and the proximal promoter of phylogenetically diverse species of birds, including ostrich and tinamou from the basal clade Palaeognathae, showed high degrees of nucleotide sequence conservation (Additional file 3: Figure S2). A canonical TATA box, that is conserved in other avian and non-avian EDC genes [8] (Additional file 4: Figure S3), is replaced by the TATA-like element AATAAAA [14,15] in all avian EDCRP genes except for that of the loon (Additional file 3: Figure S2). This suggests that the evolution of the promoter of avian EDCRP might have involved a specific mutation replacing the ancestral TATA box with the TATA-like element and the reversion of this mutation in the loon. The proteins encoded by EDCRP genes of different species vary in length (Additional file 1: Table S1) but have essentially the same basic organization in which a central segment containing multiple sequence repeats is flanked by amino-terminal and carboxy-terminal segments with unique sequences (Figure 4 and Additional file 2: Figure S1). The amino-terminal segment differs significantly between species of the basal avian clade Palaeognathae (ratites, e.g. ostrich, and tinamous) and Neognathae (all other birds), indicating an early evolutionary divergence in the structure of EDCRP. The carboxy-terminal segment shows a widely conserved basic organization which, however, appears to tolerate insertions and deletions of residues at several positions (Figure 4). The central region of EDCRP contains 6–56 repeats of 7–9 (and in exceptional cases 10) residues with the core sequence CCDPCQ. Each species has 2–4 types of repeat units that are defined by the residues on the carboxy-terminal side of the repeat core. The main repeat types are CCDPCQKP, CCDPCQK(T/S)V, CCDPCQ(T/S), and CCDPCQQS(V). In many, but not all, species the different repeat types are arranged in regular patterns. For example, the repeat units CCDPCQKP and CCDPCQQSV alternate 14 times in EDCRP of the saker falcon (Falco cherrug) (Additional file 2: Figure S1). The number of repeats shows high variability even among closely related species such as the penguins (Additional file 5: Figure S4). The amino-terminal and carboxy-terminal segments of EDCRP comprise 8–20 and 52–75 residues, respectively, and contain sequence motifs that are conserved among all birds investigated (Figure 4). Despite the local sequence variabilities described above, all avian EDCRPs are characterized by a high content of cysteine (29-31% in Palaeognathae and 32-38% in Neognathae), suggesting that the capability of EDCRP to form disulfide bonds is important across birds. Of note, two consecutive cysteine residues (CC) are found at a periodicity of 8–11 residues (with exceptions) along the entire length of EDCRP proteins. In addition, glutamine and lysine residues, i.e. the target sites of transglutamination, are also abundant at conserved positions within EDCRP (Figure 4). The conserved presence of amino acid residues capable of protein crosslinking makes EDCRP highly competent to participate in the formation of mechanically resilient and hard epidermis-derived structures such as feathers. Phylogenetic analysis suggests independent evolution of EDCRP-like features in mammalian KRTAPs The sequences of chicken, pigeon, and ostrich EDCRP were used as queries in tBLASTn searches for EDCRPlike genes in the genomes of non-avian vertebrates. Genes encoding proteins with both high cysteine content and sequences similar to that avian EDCRP were identified in the green anole lizard (A. carolinensis) and in mammals (Figure 5) but not in crocodilians (the sister group of birds), snakes (the sister group of the anole lizard), turtles, and anamniotes (fish and frogs). The EDCRP-like protein of the lizard was previously also termed EDCRP [8]. The mammalian proteins with EDCRP-like sequence motifs belong to the protein family of the KRTAPs [16]. The sequences of lizard EDCRPs and mammalian cysteine-rich KRTAPs are mostly similar to the central repetitive region of avian EDCRPs (Figure 5). However, some terminal sequence elements of avian EDCRP were also found in lizard EDCRP and KRTAPs (Figure 5). It is important to note that the sequences of all these proteins are dominated by a few amino acid residues, i.e. cysteine, proline, lysine, glutamine, and serine, so that complex sequence motifs are rare. To further evaluate the likelihood that avian EDCRPs, lizard EDCRP and mammalian KRTAPs have a common ancestor, we compared their gene structures and flanking genes (synteny). Lizard EDCRP has the same gene structure (1 non-coding, 1 coding exon) as avian EDCRPs [8]. In contrast to the promoters of avian EDCRPs, the promoter of lizard EDCRP contains a canonical TATA box (Additional file 4: Figure S3). The lizard EDCRP gene is located at a similar position within the EDC as chicken EDCRP, i.e. between the conserved genes EDWM and the loricrin genes. Moreover, the orientation of the EDCRP genes relative to these genes is identical in birds and lizards [8]. These similarities are compatible with the hypothesis that EDCRPs of birds and lizards are orthologous. However, when we screened the genomes of other sauropsids (crocodilians, turtles, and snakes) for genes encoding EDCRP-like proteins, we did not find orthologs (our unpublished data). This suggests that this ancestral EDCRP gene, if it existed in a common ancestor of modern sauropsids, was lost in some of its descendants. Alternatively, genes of similar sequence may have emerged by convergent evolution in birds and lizards. Mammalian KRTAP genes differ from avian EDCRP with regard to the exon-intron structure because KRTAPs have only a single exon. This exon is preceded by a promoter in which a TATA box is present. Unlike EDCRP genes, KRTAPs do not have a non-coding exon 1 [17]. The chromosomal locations of mammalian KRTAP genes are not syntenic with the sauropsidian EDCRP gene locus. In humans, more than 100 human KRTAP genes are distributed in 6 clusters on 4 different chromosomes (chromosome 2, 11, 17, and 21) with no KRTAP cluster being present in the EDC (chromosome 1). Actually, the human EDC does not have any gene in the region that corresponds to the locus of EDCRP genes in birds and the anole lizard, i.e. between PGLYRP3 and LOR [8]. Similar KRTAP gene distributions are present in other mammals [18]. It is also important to note that KRTAPs diversified to encode proteins with high cysteine contents (similar to EDCRP) and proteins with low cysteine (but high glycine and tyrosine) contents [17]. Together, the analyses of exon-intron structures and gene locus syntenies suggest homology of avian and lizard EDCRP but non-homology of these proteins to mammalian KRTAPs. The parsimonious evolutionary pathways leading to avian and lizard EDCRP as well as of mammalian KRTAPs are schematically depicted in Figure 6. Accordingly, sequence similarities between avian EDCRP and mammalian KRTAPs are likely to be the products of convergent evolution. Thus, the evolution of feathers and hair was associated with and perhaps facilitated by the independent origins of cysteine-rich structural proteins (Figure 7). Discussion This study shows that birds have a protein with sequence similarity to cysteine-rich proteins of mammalian hair. The avian cysteine-rich protein is expressed in an archosaur-specific embryonic skin layer, the subperiderm, and in the feathers. Together, these findings shed new light into the cornification of cells that become the building blocks of feathers and allow to refine the hypotheses about the evolution and development of feathers [19-22]. Our genome screening has identified EDCRP homologs in all birds investigated and revealed conservation of sequence elements as well as considerable tolerance for insertions and deletions of amino acid residues at many positions. The central region of EDCRP consists of sequence repeats in which the amino-terminal part of the repeat unit is highly conserved whereas other residues are variable. In some species 2 types of repeats are arranged in an alternating pattern (Figure 4) whereas other species do not have this regular arrangement of different types of repeat units. The number of repeat units varied even among closely related species, such as the emperor penguin and the Adélie penguin (Additional file 5: Figure S4) [23]. Together, these data indicate that neither repeat regularity nor the length of the central region are critical for the function of EDCRP. The most striking and most conserved feature of the EDCRP amino acid sequence is the highly biased abundance of individual amino acids. The relative cysteine content is among the highest of all proteins reported so far [24]. Only some mammalian KRTAPs have higher percentages of cysteine residues [18]. Moreover, EDCRP is rich in lysine and glutamine residues. While cysteine residues allow protein cross-linking via disulfide bonds, lysine and glutamine residues do so by undergoing transglutamination. Thus, this amino acid sequence qualifies EDCRP as an ideal cross-linking substrate during the cornification of keratinocytes. Cysteine residues are typically cross-linked during the formation of hard skin appendages such as claws/nails, hair and feathers, but to a much lower extent in the cornification of interfollicular epidermis or epidermal regions devoid of skin appendages, such as the soles [25]. Interestingly, a large portion of cysteine residues of EDCRP is present in the form of consecutive cysteine residues (CC) and notably, CCs are arranged in a regular pattern not only in the central repetitive region but also in the terminal segments. A similar pattern is present in many mammalian KRTAPs and has been proposed to facilitate protein cross-linking [16]. The results of this study suggest that the evolutionary origin of EDCRP occurred during the diversification of so-called simple EDC genes (SEDCs), which are genes comprising a 5′-terminal non-coding exon, one intron and a second exon in which the entire coding region resides [[8]. An ancestral gene with this structure was likely already present in the last common ancestor of birds, reptiles and mammals. According to our hypothesis, duplications and sequence modifications of this primordial SEDC gene have led to the evolution of more than 20 SEDC genes in each chicken, lizard and humans [8]. Our data indicate that the evolution of EDCRP involved the replacement of an ancestral canonical TATA box by a TATA-like element, the loss of amino acid sequence motifs encoded by ancestral EDC genes [8], the accumulation of mutations that increased the cysteine content and the increase in the number of sequence repeat units in the central region by a mechanism such as inequal cross-over. From the presence of EDCRP in the avian species investigated it can be inferred that the time of origin of EDCRP has preceded the diversification of modern birds. Due to the common ancestry of all SEDC genes, avian EDCRP and lizard EDCRP have also evolved from a common ancestral gene which was present in the amniote cenancestor (see above) [8]. However, the question remains whether the sequence similarity between avian EDCRP and lizard EDCRP and their high cysteine content have been derived from a common ancestor or whether they appeared by convergent evolution. The assumption that a gene coding for a cysteinerich EDCRP ancestor has been present in the ancestor of all modern sauropsids would imply that this gene (or its major sequence features) has been conserved only in the lineages leading to birds and lizards whereas it has been lost independently in the evolutionary lineages leading to 3 different clades of reptiles, namely crocodilians, turtles, and snakes, because none of the latter has an EDCRP homolog of comparable cysteine content. A more parsimonious explanation for the observed distribution of EDCRP among modern sauropsids is convergent evolution of the similar repeat units of avian and lizard EDCRPs from a common ancestor with lower cysteine content (Figure 6). Convergent evolution is also the most likely mechanism for generating the sequence similarity between avian EDCRP and mammalian KRTAPs because the genes encoding EDCRP and KRTAPs are very likely to have different evolutionary origins. This notion is suggested by the difference in exon-intron structures (EDCRP has 2 exons whereas KRTAPs have 1 exon) and by the lack of gene locus synteny (EDCRP is located within the EDC whereas none of the KRTAP genes is located there) (Figure 6). A possible scenario, similar to a previously published hypothesis [26], for the evolutionary origin of KRTAP genes is the mutation of a keratin gene. KRTAP genes have the same organization as exon 1 of keratins, and both KRTAPs and the exon 1 of socalled “hair keratins” encode cysteine-rich amino acid sequences. Notably, there is strong evidence that the exon-intron structure of keratins and the increased cysteine content of “hair keratins” (which originally might have been claw keratins) have evolved prior to the divergence of the lineages leading to modern mammals and sauropsids [3]. As the genes encoding type 1 cysteine-rich keratins are the neighbors of a cluster of KRTAP genes in mammalian but not sauropsidian genomes [3], it is conceivable that the 3′-terminal truncation of a cysteine-rich keratin gene has generated the first KRTAPgene in mammals. Subsequently, this gene might have undergone duplications, mutations and translocations to generate the various subtypes of modern KRTAPs. The results of our mRNA in situ hybridization experiments show that EDCRP is expressed in the subperiderm layer of embryonic epidermis prior to its cornification and shedding [27] as well as in the barbule cells of growing feathers prior to their cornification. Like KRTAP-mediated cornification of hair keratinocytes [5], EDCRP-mediated cornification of feather keratinocytes must be expected to abrogate the detectability of mRNAs in situ. Indeed, fully cornified subperidermal cells and feather cells that have already passed the EDCRP-positive differentiation stages do not yield in situ hybridization signals (Figure 2). The expression and cross-linking of EDCRP may contribute to the apparent stiffness of the subperiderm that allows its desquamation (together with the periderm) “in the form of extended epithelial sheets” [27]. The observed in situ hybridization pattern in feather follicles is compatible with the hypothesis that EDCRP is involved in the cysteine-dependent protein crosslinking and hardening of cells that become the building blocks of feathers. Based on the detection of EDCRP expression in the subperiderm, a temporary embryonic skin layer, and in the feathers, a cyclically shed skin appendage, we put forward a model of feather development that emphasizes the constant topology of epidermal layers during the growth of feathers (Figure 3). This model integrates prior hypotheses about the link between the subperiderm and feather barbs and barbules [11,20], the key role of the tubular shape of the feather follicle in establishing the complex branching of feathers [19,28] and the role of cell death in removing cells that separate the branches of growing feathers [13,29,30]. In essence, a series of steps in embryonic skin development are replicated, in modified form, during feather growth and shedding in adult birds (Figure 3). Notably, the timing of EDCRP expression in feather follicles is decoupled from that in extrafollicular epidermis already in the embryo. To achieve completion of the complex morphogenesis of feathers before hatching, cell differentiation and expression of EDCRP (Figure 1B) are started in feather follicles at much earlier time points than in apteric (featherless) skin. EDCRP is the second protein type, besides beta-keratins, which is expressed both in the subperiderm and in feathers [31]. The properties of feathers are likely to depend on both EDCRP and beta-keratins, which may interact via disulfide bonding or other mechanisms. However, EDCRP has a uniquely high content of cysteine and, different from beta-keratins, is present in birds but not in the phylogenetically closely related crocodilians. Therefore, EDCRP might have played a particularly important role in the evolution of feathers. The significance of our findings is further underscored by the finding of similarity of avian EDCRP to mammalian cysteine-rich KRTAPs, which indicates that the origin of highly cysteine-rich proteins was a key step in the evolution of both feathers and hair (Figure 7). Taken together, EDCRP appears to represent one of the critical innovations during the evolution of feathers which may be summarized as follows. (1) The evolutionary origin of the subperiderm in a common ancestor of archosaurs (crocodiles and birds as well as extinct dinosaurs) provided the cellular ancestors of cornified feather keratinocytes [11]. (2) The evolution of a feather follicle with tubular shape was an essential evolutionary innovation in the lineage leading to modern birds after its divergence from the crocodilian lineage [19]. (3) The origin of the EDCRP gene by duplication of an ancestral EDC gene [8] and/or the modification of its sequence to increase the cysteine content of EDCRP contributed to the ability of subperidermal keratinocytes to establish durable protein cross-links. It is likely that other cysteine-rich proteins evolved in parallel in birds. The extensive disulfide bonding facilitated the formation of delicate, yet stable structures of feathers. (4) The co-option of signaling and cell differentiation pathways facilitated the formation of the branching pattern of feathers. Dermo-epidermal interactions and differential cell growth and cell death processes in the adjacent layers of the feather follicle established the first feathers which gained complexity by the fine-tuning of epithelial growth and fusion processes during evolution [29,32]. Thus, it appears that the evolution of a structural protein complemented the evolution non-structural genes and regulatory elements [33,34]. Paleontological findings have unraveled a series of steps in the evolution of feather morphology [35]. Molecular biological studies, including the present characterization of EDCRP, should now help to elucidate the evolution of the feather architecture at the molecular level. Go to: Conclusion In conclusion, this study suggests that the evolution of avian EDCRP has been instrumental in the evolution of feathers and that EDCRP contributes to the structural integrity of feathers in modern birds. Go to: Methods Comparative genomics and sequence analysis The genome sequences from the bird species listed in Additional file 1: Table S1 [36] and from the following non-avian species were investigated: Chinese alligator (Alligator sinensis), American alligator (American alligator), saltwater crocodile (Crocodylus porosus), gharial (Gavalis gangeticus) [37], green sea turtle (Chelonia mydas), Chinese softshell turtle (Pelodiscus sinensis) [38], green anole lizard (Anolis carolinensis) [39], king cobra (Ophiophagus hannah) [40], Burmese python (Python bivittatus) [41], African clawed frog (Xenopus laevis), fugu (Takifugu rubripes), platypus (Ornithorhynchus anatinus), opossum (Monodelphis domesticus) and human (Homo sapiens). All genome sequences are available in the GenBank. The tBLASTn algorithm (http://www.ncbi.nlm.nih.gov/) was used to screen for homologs of chicken EDCRP [8]. Amino acid sequences of both complete EDCRP and distinct motifs of EDCRP were used as queries. Sequences were aligned using the programs MUSCLE and Multalin [42]. Genomic DNA of the ostrich was prepared from commercially marketed ostrich meat, amplified with the primers 5′-AGAAGTCCAGCCGCTGTGTCA-3′ and 5′-GGTATGCAGTACTTTCTCATGG-3′ and sequenced. Genomic DNA from a zebra finch (kindly provided by Dr. Lorenzo Alibardi, University of Bologna, Italy) was amplified with the primers 5′- TGCTCTGTCGTGAAGAGCAAG-3′ and 5′-CGGGCTTCTTCACCACGTAG-3′. The PCR product was purified and sequenced [GenBank: KP224277]. RT-PCR and sequencing RNA was prepared from chicken embryos on embryonic days 10, 14, 18 and from adult chicken as described previously [12]. All animal procedures were approved by the Animal Care and Use Committee of the Medical University of Vienna (66.016/0014-II/3b/2011). The RNAs were reverse-transcribed and subjected to PCRs with intron-spanning primer pairs specific for EDCRP (5′-CTCAACTGAACCCCTCAGTTAG-3′ and 5′CAGCACACTGTCTTGCTCTTC-3′) and caspase-3, as a control (5′-TGGCGATGAAGGACTCTTCT-3′ and 5′-CTGGTCCACTGTCTGCTTCA-3′). mRNA in situ hybridization A probe annealing to the 3′-untranslated region of chicken EDCRP mRNA (nucleotides 11–286 downstream of the stop codon) was cloned in sense and antisense orientation into pCR®2.1-TOPO® plasmids (Life Technologies, Paisley, UK) and transcribed in vitro using the DIG RNA labeling kit (Roche Applied Science). The in situ hybridizations with antisense and sense probes were performed at a hybridization temperature of 45°C (incubation time 1 h) on sections of formaldehyde-fixed and paraffin-embedded chicken tissues according to a published protocol [12]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4423139/ Published online 27 January 2010 | Nature | doi:10.1038/news.2010.39 News Fossil feathers reveal dinosaurs' true colours Pigment-storage sacs found in fossils give hints about hue. Matt Kaplan Sinosauropteryx may have had orange feathers and a stripey tail.Jim Robbins Pristine fossils of dinosaur feathers from China have yielded the first clues about their colour. A team of palaeontologists led by Michael Benton of the University of Bristol, UK, and Zhonghe Zhou of the Institute of Vertebrate Paleontology and Paleoanthropology in Beijing, has discovered ancient colour-producing sacs in fossilized feathers from the Jehol site in northeastern China that are more than 100 million years old. These pigment-packed organelles, called melanosomes, have only been found in fossilized bird feathers before now. The team discovered the melanosomes in fossils of the suborder Theropoda, the branch of the dinosaur family tree to which the flesh-eating species Velociraptor and Tyrannosaurusbelong. However, it was not in these two iconic dinosaurs that the organelles were found, but in smaller species that ran around low to the ground with tiny feathers or bristles distributed across their bodies. The team discovered two types of melanosome buried within the structure of the fossil feathers: sausageshaped organelles called eumelanosomes that are seen today in the black stripes of zebras and the black masks of cardinal birds, and spherical organelles called phaeomelanosomes, which make and store the pigment that creates the rusty reds of red-tailed hawks and red human hair. The team didn't find cell structures or machinery responsible for other colours, such as yellows, purples and blues. They suggest that dinosaur cells might have produced coloured pigments such as carotenoids and porphyrins, but that the proteins that make them degrade more rapidly than organelles, so do not leave a trace in fossils. Melanosomes, by contrast, are an integral part of a feather's tough protein structure so can survive for longer. The team found pigment-storing eumelanosomes (left) and phaeomelanosomes (right) in the fossil feathers.University of Bristol. "We always tell introductory palaeontology students that things like sound and colour are never going to be detected in the fossil record," says Benton. "Obviously that message needs to be reconsidered." Fossils of one theropod dinosaur, Sinosauropteryx, reveal that it had light and dark feathered stripes along the length of its tail. The team found that feathers from the darker regions of the tail were packed with phaeomelanosomes, indicating they were russet-orange in colour. The lighter stripes could have been white, says Benton, but because some pigments degrade and don't leave a fossil signature, it is difficult to be sure. Sinosauropteryx was not the only colourful feathered species. Another small theropod species, Sinornithosaurus, had feathery filaments that were dominated by either eumelanosomes or phaeomelanosomes, hinting that its individual feathers varied in colour between black and russet-orange. Birds of a feather A lot of questions have been raised about the structures that are found on the earliest of the feathered dinosaurs, such as Sinosauropteryx. Some palaeontologists argue that these bristle-like structures are actually fossilized connective tissue rather than early feathers. However, says Benton, in modern birds — which evolved from the theropod dinosaurs — melanosomes are found only in the developing feathers and not in the connective tissue. The fact that the melanosomes in the dinosaur feathers are also found inside the bristles themselves resolves the debate, he says. Samples were taken from a dark stripe near the base of Sinosauropteryx's tail.The Nanjing Institute "This paper puts the nails in the coffin of arguments countering the feather nature of these structures," agrees Luis Chiappe, a palaeontologist at the Natural History Museum of Los Angeles County, California. "It is deeply gratifying that this colour discovery is allowing us to finally agree that the structures on Sinosauropteryxwere actually early feathers," says evolutionary ornithologist Richard Prum of Yale University in New Haven, Connecticut. "Now we can get on with studying their evolution." In addition, the discovery of colour in the earliest feathers may also sway the biggest dinosaur debate of them all — what the feathers were actually being used for. The tiny bristles on early feather-bearers could not have been used for flight, as some have suggested, because they would have provided no lift. But they could have served as insulation, or for display. "It is looking increasingly likely to me that these dinosaurs were making a visual statement," says Benton. "What that statement was, we don't know, but you don't have a orange-and-white striped tail for nothing." 1. References Zhang , F. et al. Nature advance online publication doi:10.1038/nature08740 (2010). https://www.nature.com/news/2010/100127/full/news.2010.39.html SHARE Share on facebook11K Share on twitter Share on reddit105 Share on linkedin Dark, iron-rich hematite particles may have preserved protein fragments in this 195-million-year-old dinosaur rib, according to one of two independent studies of dinosaur proteins. ROBERT REISZ Scientists retrieve 80-million-year-old dinosaur protein in ‘milestone’ paper By Robert F. ServiceJan. 31, 2017 , 11:00 AM It’s not quite Jurassic Park: No one has revived long-extinct dinosaurs. But two new studies suggest that it is possible to isolate protein fragments from dinosaurs much further back in time than ever thought possible. One study, led by Mary Schweitzer, a paleontologist from North Carolina State University in Raleigh who has chased dinosaur proteins for decades, confirms her highly controversial claim to have recovered 80-millionyear-old dinosaur collagen. The other paper suggests that protein may even have survived in a 195-million-yearold dino fossil. The Schweitzer paper is a “milestone,” says ancient protein expert Enrico Cappellini of the University of Copenhagen’s Natural History Museum of Denmark, who was skeptical of some of Schweitzer’s earlier work. “I’m fully convinced beyond a reasonable doubt the evidence is authentic.” He calls the second study “a long shot that is suggestive.” But together, Cappellini and others argue, the papers have the potential to transform dinosaur paleontology into a molecular science, much as analyzing ancient DNA has revolutionized the study of human evolution. Back in 2007 and 2009, Schweitzer reported in Science that she and her colleagues had isolated intact protein fragments from 65-million- and 80-million-year-old dinosaur fossils. But the claims were met with howls of skepticism from biochemists and paleontologists who saw no way that fragile organic molecules could survive for tens of millions of years, and wondered whether her samples were contaminated with modern proteins. SIGN UP FOR OUR DAILY NEWSLETTER Get more great content like this delivered right to you! 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Required fields are indicated by an asterisk (*) Then last year Cappellini and Matthew Collins, a paleoproteomics expert at the University of York in the United Kingdom, and colleagues managed to identify protein fragments from 3.8-million-year-old ostrich egg shells, a claim that most of their colleagues found convincing. Now, the case for dramatically older proteins seems to be firming up, too. Last week in the Journal of Proteome Research, Schweitzer, her postdoct Elena Schroeter, and colleagues report that they did a complete makeover of their 2009 experiment to rule out any possible contamination. They took new samples from the same 80-million-year-old fossil, of a duck-billed dinosaur called Brachylophosaurus canadensis. They reworked procedures for extracting would-be proteins from the bone, identified protein fragments with a more sensitive mass spectrometer, and compared the recovered protein sequences to those from many more living animals. Schroeter even went so far as to break down the mass spectrometer piece by piece, soak the whole thing in methanol to remove any possible contaminants, and reassemble the machine. “About the only thing that is the same [as the 2009 experiments] is the dinosaur,” Schweitzer says. In their 2009 paper Schweitzer’s team had identified three fragments of a protein called collagen 1 from their fossil. Collagen is the main protein in connective tissue and is abundant in bone. Each fragment contained about 15 amino acids strung together, which the mass spectrometer was able to identify. In their current study, Schweitzer’s team identified eight protein fragments, two of which matched those identified originally. “If [both sets] are from contamination, that’s almost impossible,” Schweitzer says. The three protein fragments originally recovered most closely resembled the collagen found in living alligators and other reptiles. But the new data show that B. canadensis collagen was a better match to that of birds. That’s just what paleontologists, who consider birds to be descendants of extinct dinosaurs, would predict. Just how those collagen sequences survived tens of millions of years is not clear. Schweitzer suggests that as red blood cells decay after an animal dies, iron liberated from their hemoglobin may react with nearby proteins, linking them together. This crosslinking, she says, causes proteins to precipitate out of solution, drying them out in a way that helps preserve them. That’s possible, Collins says. But he doesn’t think the process could arrest protein degradation for tens of millions of years, so he, for one, remains skeptical of Schweitzer’s claim. “Proteins decay in an orderly fashion. We can slow it down, but not by a lot,” Collins says. Researchers claim this scanning electron microscope image captures collagen fibrous material from an 80million-year-old dinosaur fossil. MARY SCHWEITZER NORTH CAROLINA STATE UNIVERSITY, AND ICAL FACILITY, MONTANA STATE UNIVERSITY The second paper, published this week in Nature Communications, goes back even further in time but offers weaker evidence, Cappellini says. In this work, researchers led by paleontologist Robert Reisz at the University of Toronto in Canada reported finding what they believe is collagen in a 195-million-year-old fossil rib from a large plant-eating dinosaur called Lufengosaurus that lived in what is now southwestern China. Reisz says his team’s methods, called Raman spectroscopy and synchrotron radiation Fourier transform infrared microspectroscopy (SR-FTIR), can probe the chemical makeup of a sample without the need to purify it first, which lowers the risk of contamination. The rib, he and his colleagues report, absorbed infrared light in wavelengths that match those of collagen from modern animals. Schweitzer and Cappellini caution that while SR-FTIR is good at spotting the so-called amide chemical bonds that link successive amino acids in proteins, it can’t pin down exactly which protein is present, or the protein's sequence. Thus it isn't useful for evolutionary studies. This method also can’t rule out that the amide bonds are in other compounds, such as the epoxy used to assemble microscope slides. “Synchrotron data is very powerful, but it’s limited,” Schweitzer says. “I would like to have seen confirmatory evidence,” such as exposing the fossilized material to an antibody that binds solely to collagen to see whether it targeted the fossilized material. Reisz agrees “that certainly would be the next step.” But he’ll have to team up with other specialists to carry that out. Still, his work, too, suggests that collagen fragments can survive for astonishing periods of time. Meanwhile, Schweitzer’s team is going beyond collagen. In a 2015 paper in Analytical Chemistry, her group reported isolating fragments of eight other proteins from fossils of dinosaurs and extinct birds, including hemoglobin in blood, the cytoskeletal protein actin, and histones that help package DNA. Comparing those sequences from many different species could reveal evolution’s handiwork over geological time, much as studies of ancient DNA do today. Her team won’t be the only ones exploring these methods. Now that it seems that very ancient protein fragments can, in fact, be isolated and examined, it’s a safe bet that many new collaborations will soon take shape to pin down the evolutionary relationships among different dinosaurs, as well as among ancient mammals and other extinct creatures. Says Schweitzer: “The door is now open.” Posted in: Paleontology doi:10.1126/science.aal0679 Robert F. Service Bob is a news reporter for Science in Portland, Oregon, covering chemistry, materials science, and energy stories. http://www.sciencemag.org/news/2017/01/scientists-retrieve-80-million-year-old-dinosaur-protein-milestonepaper Click and Clone Mimi the Mouse http://learn.genetics.utah.edu/content/cloning/clickandclone/ Complete Dinosaur Cladogram http://www.gavinrymill.com/dinosaurs/cladogram.html Building a Dinosaur from a Chicken, Jack Horner TEDTalk https://www.ted.com/talks/jack_horner_building_a_dinosaur_from_a_chicken?language=en NATURE | NEWS Sharing 'Dino-chickens' reveal how the beak was born Chicken embryos have been altered so that the birds grow dinosaur-like snouts. Ewen Callaway 12 May 2015 Article tools Rights & Permissions Bhart-Anjan Bhullar The skull of a chicken embryo ready to hatch usually has a beak (left), but when certain proteins are blocked (middle) develops a reptilian 'snout' from two bones, rather like a modern-day alligator (right). Biologists have created chicken embryos with dinosaur-like faces by tinkering with the molecules that build the birds' beaks. The research, details of which are published today in Evolution1, does not aim to engineer flocks of hybrid ‘dino-chickens’ or to resurrect dinosaurs, says Bhart-Anjan Bhullar, a palaeontologist now at the University of Chicago in Illinois, who co-led the work. “We’re never going back to the actual dino-chicken or whatever it is.” Rather, he says, the team wants to determine how snouts might have turned into beaks as dinosaurs evolved into birds more than 150 million years ago. The transition from dinosaur to bird was messy — no specific anatomical features distinguished the first birds from their meat-eating dinosaur ancestors. But in the early stages of bird evolution, the twin bones that formed the snout in dinosaurs and reptiles — called the premaxilla — grew longer and joined together to produce what is now the beak. “Instead of two little bones on the sides of snout, like all other vertebrates, it was fused into a single structure,” Bhullar says. Facial reconstruction Related stories Darwin’s iconic finches join genome club Flock of geneticists redraws bird family tree Rival species recast significance of ‘first bird’ More related stories To better understand how these bones might have become fused, a team led by Bhullar and Arhat Abzhanov, an evolutionary biologist at Harvard University in Cambridge, Massachusetts, analysed the embryonic development of beaks in chickens and emus, and of snouts in alligators, lizards and turtles. They reasoned that reptile and dinosaur snouts develop from premaxilla in a similar way, and that the developmental pathways that form the snout were altered in the course of bird evolution. The team found that two proteins known to orchestrate the development of the face, FGF and Wnt, were expressed differently in bird and reptile embryos. In reptiles, the proteins were active in two small areas in the part of the embryo that turns into the face. In birds, by contrast, both proteins were expressed in a large band across the same region in the embryo. Bhullar sees the result as tentative evidence that altered FGF and Wnt activity contributed to the evolution of the beak. To test this idea, the team added biochemicals to block the activity of both proteins in dozens of developing chicken eggs. The researchers did not actually hatch the eggs, says Bhullar, because they did not write that step into their approved research protocol. Instead, they discerned differences in the faces of ready-to-hatch chicks, which looked subtly different from chicks without their proteins inhibited. The altered chicks still had a flap of skin over their would-be beaks, so the difference is not obvious, says Bhullar. “Looking at these animals externally, you would still think it’s a beak. But if you saw the skeleton, you’d just be very confused," he says. "I would not say we gave birds snouts.” In some embryos, the premaxillae were partly fused, whereas in others the two bones were distinct and much shorter; some of the altered embryos did not look all that different from those of regular chickens. The team created digital models of their skulls with a computed tomography scanner and found that some of these more closely resembled the bones of early birds such as Archaeopteryx and dinosaurs such as Velociraptor, than unmodified chickens. “Very cool,” says Clifford Tabin, a developmental biologist at Harvard Medical School in Boston, Massachusetts. He thinks that Bhullar’s team makes a strong case that altered expression of FGF and Wnt shaped the bird's beak. Identifying the genetic changes responsible, however, will prove much more difficult. They could lie in the genes coding for FGF and Wnt, or to genes in related biochemical pathways, or in ‘regulatory’ DNA that influences gene expression. If these changes could be identified, it might be possible to modify chicken genomes to include them (and, conversely, to make reptiles more bird-like through genome editing). Jack Horner, a palaeontologist at Montana State University in Bozeman, hopes to take a genetic approach to imbuing chickens with dinosaur-like tails. In a paper published last year2, his team identified mutations potentially involved in the disappearance of the tail in modern birds. But applying these insights to engineering ‘dino-chickens’ has proved difficult, he says. “We’re having a little more trouble with the tail. There are so many components.” Other anatomical features could be altered by tinkering with development proteins, Horner adds. “It gives us a lot of opportunities to think about making new kinds of animals.” Bhullar says that he admires Horner’s vision, but he is more interested in replaying evolution to reveal how it creates new forms. His lab plans to study the expansion of the mammalian skull and the unusual lower limbs of crocodiles by resurrecting ancient anatomy. “I think it will open as big a window as you could possibly get into the deep past without having a time machine,” he says. Nature doi:10.1038/nature.2015.17507 References 1. Bhullar, B.-A. S. et al. Evolution http://dx.doi.org/10.1111/evo.12684 (2015). Show context 2. Rashid, D. J. et al. EvoDevo 5, 25 (2014). https://www.nature.com/news/dino-chickens-reveal-how-the-beak-was-born-1.17507