Author response: Molecular shifts in limb identity underlie development of feathered feet in two domestic avian species

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Birds display remarkable diversity in the distribution and morphology of scales and feathers on their feet, yet the genetic and developmental mechanisms governing this diversity remain unknown. Domestic pigeons have striking variation in foot feathering within a single species, providing a tractable model to investigate the molecular basis of skin appendage differences. We found that feathered feet in pigeons result from a partial transformation from hindlimb to forelimb identity mediated by cis-regulatory changes in the genes encoding the hindlimb-specific transcription factor Pitx1 and forelimb-specific transcription factor Tbx5. We also found that ectopic expression of Tbx5 is associated with foot feathers in chickens, suggesting similar molecular pathways underlie phenotypic convergence between these two species. These results show how changes in expression of regional patterning genes can generate localized changes in organ fate and morphology, and provide viable molecular mechanisms for diversity in hindlimb scale and feather distribution. https://doi.org/10.7554/eLife.12115.001 eLife digest Animals ranging from fish to birds display dramatic diversity within and among species; yet remarkably little is known about the genetic and developmental mechanisms that underlie this variation. In birds and their extinct dinosaur relatives, the distribution of scales and feathers on the feet is a highly variable trait. Different breeds of domestic pigeon all belong to the same species but have feet that can be feathery or scaly to different extents. Classical genetics experiments suggested that only a few genes are involved in the transition from scaled to feathered skin on the feet of pigeons. However, the molecular basis for this transition was unknown. Domyan et al. set out to identify the genes involved in the transition from scaled to feathered feet by mating different breeds of pigeon in the laboratory and then sequencing the birds’ DNA. They also surveyed the entire DNA sequences of many additional pigeon breeds with and without feathered feet. This combined approach showed that two regions of the pigeon genome have a profound effect on the number and size of feathers on the feet of domestic pigeons. These regions contain genes that are known to play key roles in controlling the growth of a limb and whether it develops into a leg or a wing. In developing pigeon embryos, Domyan et al. found that a gene called Pitx1, which is typically considered a hindlimb gene, is expressed at lower levels in the developing legs of breeds with feathered feet than in a breed with scaled feet. The experiments also showed that Tbx5, a gene that is expressed in the forelimbs of many animals, is expressed abnormally in the embryonic hindlimbs of breeds of pigeon and chicken with feathery feet. Together, these findings suggest that the hindlimbs of domestic birds with feathery feet are more like wings at the molecular level, which results in them being covered in feathers rather than scales. Future work will now aim to discover the specific DNA sequences that alter the expression of Pitx1 and Tbx5 in feather-footed breeds, and whether the same genes control the foot feathers of other species of birds. https://doi.org/10.7554/eLife.12115.002 Introduction In birds, the genetic and developmental mechanisms that control the decision between scale and feather development remain poorly understood. Most birds possess scales on the foot (tarsometatarsus and toes) and feathers elsewhere. Exceptions to this pattern can provide insights into the evolutionary and developmental basis of skin appendage diversity. Some raptors and boreal birds evolved foot feathers instead of scales ('ptilopody'; Danforth, 1919; Lucas and Stettenheim, 1972), but the lack of appendage variation within these species precludes their use as genetic models. Likewise, paravians (birds and their close non-avian theropod dinosaur relatives) and other dinosaurs evolved diverse feather coverings on their legs and feet that sometimes resemble flight-like feathers (Xu et al., 2003; Hu et al., 2009; Turner et al., 2012; Zheng et al., 2013; Foth et al., 2014; Godefroit et al., 2014), but the absence of living specimens preclude mechanistic molecular studies. In contrast, domestic pigeons (Columba livia) exhibit stunning variation within a single extant species (Shapiro and Domyan, 2013). Most breeds have feet covered by scaled epidermis (wild-type), but scales are replaced by small feathers in grouse (gr) mutants, and by larger feather 'muffs' in birds that also carry mutant alleles at the Slipper (Sl) locus (Doncaster, 1912; Wexelsen, 1934; Hollander, 1937; Levi, 1986) (Figure 1A). In muffed breeds, scutellate scales are generally absent or poorly developed on the feathered epidermis covering the tarsometatarsus and toes, and feathers are surrounded by soft integument. The molecular identities of both gr and Sl are unknown, and additional loci probably control quantitative variation in the muff phenotype. Because both scale-footed and feather-footed pigeon breeds belong to the same species, we can use traditional genetic crosses and whole-genome resequencing to map the genes that control this striking variation (Shapiro et al., 2013; Domyan et al., 2014). Therefore, we can study diversity of the magnitude usually observed among different species without the roadblock of hybrid incompatibility that often eliminates the possibility of genetic mapping studies. Figure 1 with 1 supplement see all Download asset Open asset Two QTL differentiate scale- and feather-footed domestic pigeons. (A) Common phenotypes of domestic rock pigeon, in order of increasing foot feathering (left to right): scaled, groused, small- and large-muffed. (B-F) QTL scans and effect plots: proportion of tarsus feathered (B,C), number of toe feathers (D,E), and length of toe feathers (F,G). Mean phenotypes ± S.E. are plotted in (C,E,G). For (E) and (G), genotypes at the QTL with the higher LOD score are on the x-axis, and genotypes at the other QTL are inset. See Tables 1 and 2 for detailed QTL statistics. S, allele from scale-footed grandparent; F, allele from feather-footed grandparent. https://doi.org/10.7554/eLife.12115.003 During development in vertebrates, skin appendages form through interactions between the ectoderm-derived epidermis and the mesoderm-derived dermis, and signals from the dermis determine epidermal appendage fate (Dhouailly, 2009; Hughes et al., 2011). Previous analyses of mutants and gene misexpression in chickens suggest candidates for feathered feet in the Hedgehog (Harris et al., 2002), BMP (Zou and Niswander, 1996; Harris et al., 2002; 2004), Delta-Notch (Crowe et al., 1998), and Wnt (Chang et al., 2004) pathways. Our study of genetic variation and embryonic development in pigeons, however, reveals a surprisingly different mechanism with broad implications for limb identity and patterning. Results Two loci of major effect control foot feathering in pigeons To identify chromosome regions that contribute to feathered feet, we generated an F2 intercross between muffed (Pomeranian pouter) and scaled (Scandaroon) breeds (Figure 1—figure supplement 1). F1 hybrids displayed intermediate foot feathering, demonstrating a semi-dominant inheritance pattern. Scaled, muffed, and intermediate phenotypes were recovered in the F2 population, confirming that a small number of major-effect loci contribute to this trait. Among F2 offspring, digit 3 bore the largest and greatest number of feathers (digit 1, 7.04 ± 5.08 feathers; digit 2, 7.64 ± 7.54; digit 3, 15.46 ± 10.43; digit 4, 6.13 ± 5.70). Using quantitative trait locus (QTL) mapping with 130 F2 offspring genotyped at 3803 polymorphic markers (Broman et al., 2003), we identified two linkage groups (LG11 and LG20) that had significant effects on three different aspects of foot feathering (log10 odds ratio (LOD) > 4.6; Figure 1B–G, Table 1). LG11 had the largest effects on the proportion of the tarsometatarsal epidermis that was transformed from scaled to feathered (LOD = 7.69, percent variance explained (PVE) = 28.4%) and toe feather number (LOD = 6.72, PVE = 21.3%), and a smaller effect on toe feather length (LOD = 8.51, PVE = 15.8%). LG20 had the largest effect on toe feather length (LOD = 20.9, PVE = 52.2%), and a smaller effect on toe feather number (LOD = 5.36, PVE = 16.5%). When toe feather number was analyzed for each digit individually, the same two QTL were identified and had the most pronounced effects on digits 3 and 4 (Table 2). In summary, two major QTL have marked and separable effects on qualitative and quantitative variation in epidermal appendages. Table 1 Summary of foot feathering QTL. https://doi.org/10.7554/eLife.12115.005 TraitLGLoc (cM)ScaffoldPositionLODPVEMean ± S.D.SSSFFFProportion tarsus feathered11117799,205,2867.6928.40.46 ± 0.040.58 ± 0.03**0.80 ± 0.04***Number of toe feathers111247912,325,9776.7321.343.6 ± 8.967.3 ± 6.5*105.3 ± 8.1***Number of toe feathers2015951,451,1275.3616.545.3 ± 8.178.2 ± 6.5**100.3 ± 9.9***Toe feather length (mm)20070136,74620.8952.25.2 ± 2.319.9 ± 2.0***37.3 ± 2.5***Toe feather length (mm)111247912,325,9778.5115.811.4 ± 3.318.7 ± 2.4*28.5 ± 3.0** LG, linkage group; Loc, genetic location of peak LOD score in centimorgans; S, allele from scaled parent; PVE, percent variance explained; F, allele from feathered parent; LOD, log10 odds ratio. (Welch two sample t-test of means compared to SS homozygote; *p≤ 0.05, **p≤0.005, ***p≤0.0005.) Table 2 Summary of QTL for numbers of feathers on individual toes. https://doi.org/10.7554/eLife.12115.006 DigitLGLoc (cM)ScaffoldPositionLODPVEMean ± S.D.SSSFFFDigit 2, left foot111487911,624,7015.2020.243.75 ± 1.136.95 ± 1.12*12.19 ± 1.16***Digit 3, right foot20070136,7467.7124.19.06 ± 1.7017.35 ± 1.48**19.86 ± 1.83***Digit 3, right foot111487911,624,7016.4719.59.98 ± 1.6015.60 ± 1.62*21.02 ± 1.65***Digit 3, left foot20070136,74610.7929.848.66 ± 1.5917.06 ± 1.41**21.09 ± 1.86***Digit 3, left foot111487911,624,7019.4825.529.37 ± 1.5216.26 ± 1.52**21.30 ± 1.57***Digit 4, right foot2032952,464,7887.3321.362.44 ± 0.907.24 ± 0.75**10.14 ± 1.10***Digit 4, right foot11118795,475,4746.7819.53.17 ± 0.896.91 ± 0.92*9.95 ± 1.00***Digit 4, left foot2030952,464,7889.3726.832.44 ± 0.796.63 ± 0.66**9.65 ± 0.96***Digit 4, left foot111487911,624,7017.5820.833.75 ± 1.146.95 ± 1.13*12.19 ± 1.17*** LG, linkage group; Loc, genetic location of peak LOD score in centimorgans; S, allele from scaled parent; PVE, percent variance explained; F, allele from feathered parent; LOD, log10 odds ratio. (Welch two sample t-test of means compared to SS homozygote; *p≤0.05, **p≤0.005, ***p≤0.0005.) In parallel to our experimental cross, we performed probabilistic whole-genome scans of allele-frequency differentiation (pFst; see Kronenberg et al., 2014) across a genetically and phenotypically diverse panel of breeds by comparing 15 feather-footed birds (4 groused and 11 muffed) to 28 scale-footed birds (Shapiro et al., 2013). Using this independent approach across breeds, the two most-highly differentiated pFst signals implicate the same genomic regions as the QTL study: genomic scaffold 79 is located on LG11 (p=4.44 x 10–16, genome-wide significance threshold = 2.11 x 10–9), and scaffolds 70 and 95 are adjacent to one another on LG20 (p=9.81 x 10–13) (Figure 2A, Figure 2—figure supplement 1A,B) Figure 2 with 3 supplements see all Download asset Open asset Two regions of genomic differentiation and H3K27ac enrichment distinguish scale- and feather-footed pigeons. (A) Whole-genome pFst comparisons between genomes of feather-footed and scale-footed pigeons. Scaffolds are ordered by genetic position in a linkage map from an F2 cross (see Figure 1). Dashed line, genome-wide significance threshold. (B) pFst and extended haplotype homozygosity (EHH) plots for region of high differentiation on scaffold 79. Feather-footed birds (n=10, red in EHH plot) homozygous for a 44-kb deletion are differentiated from scale-footed birds (n=28, black) and show extended haplotype homozygosity in this region. Smoothed lines follow a generalized additive model (Wickham, 2009). (C) H3K27ac ChIP-seq enrichment differed significantly between embryonic wing and leg buds of the scale-footed racing homer (RH) in several regions (blue shading), including within a 44-kb interval that is deleted in the muffed Indian fantail (IF; blue arrowheads). This deleted region is orthologous to a known human limb enhancer (hs1473). (D) Selection scans show similar patterns of differentiation on scaffold 70 between muffed (n=11, red in EHH plot) and scale-footed birds (n=28, black). (E) H3K27ac ChIP-seq enrichment differed significantly between leg buds of racing homer and Indian fantail embryos in regions immediately 5’ of Tbx5 (blue shading). Foot and wing drawings modified after Levi (1986). https://doi.org/10.7554/eLife.12115.007 The peak pFst region on scaffold 79 contained a 44-kb deletion (from 6.719–6.763 Mb) that was homozygous in 10, and heterozygous in 2 of the 15 feather-footed birds (Figure 2B,C; Figure 2—figure supplement 2A). Birds homozygous for the deletion showed elevated levels of haplotype homozygosity relative to scaled birds, a signature of positive selection on this region (Figure 2B). This deletion spans an element orthologous to a known human limb enhancer, hs1473 (Spielmann et al., 2012), which contains active chromatin marks (Cotney et al., 2012) and is bound by the hindlimb-specific transcription factor Pitx1 in the developing mouse hindlimb (Infante et al., 2013) (Figure 2C). The locus was homozygous for the deletion in 35 of 54 additional feather-footed birds from 21 breeds, but was never homozygous in 96 scale-footed birds from 56 breeds (Chi-square, p<0.0001; Figure 2—figure supplement 2B). The 3 feather-footed birds from our whole-genome panel that did not have this deletion (including the male founder of the aforementioned genetic cross implicating this same region) also showed allelic differentiation from scale-footed birds over this interval, suggesting that an additional feathered-foot allele may also exist at this locus (Figure 2—figure supplement 2A). In contrast to the differentiation signal we observed between scale-footed and all feather-footed birds on scaffold 79, only the muffed birds (more heavily feathered) showed signatures of selection and shared similar haplotypes on scaffold 70 (higher pFst signal than the adjacent scaffold 95) (Figure 2D, Figure 2—figure supplements 2C, 3). Thus, both QTL analyses and whole-genome scans show that mutation on scaffold 79 alone is sufficient for the grouse phenotype (gr locus), and point to scaffold 70 as the major-effect locus for longer toe feathers in birds with muffs (Sl locus) (Figure 1F,G). Expression of limb outgrowth and identity genes differs between scale-footed and feather-footed breeds Next, we examined scaffolds 79 and 70 for candidate genes that might control the scale-to-feather transition. The highest pFst peak on scaffold 79 – corresponding to the major-effect QTL on LG 11 for the proportion of tarsometatarsal feathering – was approximately 200 kb upstream of Pitx1, a gene that encodes a homeobox-containing transcription factor that is normally expressed in the vertebrate hindlimb but not the forelimb (Figure 2B). The highest pFst peak on scaffold 70 – corresponding to the major-effect QTL on LG 20 for toe feather length – was 40 kb upstream of Tbx5, a gene that encodes a T-box transcription factor that is normally expressed in the vertebrate forelimb but not the hindlimb (Figure 2D). These regions were especially intriguing because these two genes encode key transcriptional regulators of forelimb (Tbx5) and hindlimb (Pitx1) identity and development (Logan et al., 1998; Logan and Tabin, 1999; Rodriguez-Esteban et al., 1999; Szeto et al., 1999; Takeuchi et al., 1999). For example, misexpression of Pitx1 in the embryonic chick forelimb blocks feather development (Logan and Tabin, 1999), while misexpression of Tbx5 in the early hindlimb field of embryonic chickens is sufficient to induce a partial wing-like transformation, including feather formation on the feet (Takeuchi et al., 1999). In mouse, Pitx1 (but not Tbx5) plays a role in determining limb-type identity (Szeto et al., 1999; Minguillon et al., 2005; DeLaurier et al., 2006), suggesting that the roles of Tbx5 in limb outgrowth and identity have diversified during amniote evolution (Horton et al., 2008). We did not identify any fixed non-synonymous coding changes in Pitx1 or Tbx5 between scale-footed and feather-footed breeds of pigeon. However, we found striking differences in embryonic hindlimb expression of these genes among three different breeds – racing homer (scale-footed), Indian fantail (small-muffed), and English trumpeter (large-muffed) – at Hamburger-Hamilton stage 25 (HH25; Hamburger and Hamilton, 1951). Pitx1 expression was reduced in both muffed breeds (expression relative to racing homer: Indian fantail 0.75 ± 0.06, p=0.0007; English trumpeter 0.40 ± 0.05, p=0.0007; n = 6 each) and was more severely reduced in the large-muffed English trumpeter (p=0.002) (Figure 3A). Conversely, Tbx5, the forelimb-specific transcription factor, was ectopically expressed in the hindlimbs of both muffed breeds (hindlimb expression relative to racing homer forelimb: racing homer 0.001 ± 0.0004; Indian fantail 0.01 ± 0.008, p=0.0007; English trumpeter 0.14 ± 0.05, p=0.0007; n = 6 each), and was higher in the large-muffed English trumpeter (p=0.002) (Figure 3B). Forelimb expression of Tbx5 was indistinguishable among the three breeds, indicating that upregulation of Tbx5 in feather-footed breeds is restricted to the hindlimb (Figure 3—figure supplement 1A). Figure 3 with 3 supplements see all Download asset Open asset Limb-type gene expression varies among feathered and scaled pigeons and chickens. (A,B,F,G) qRT-PCR analyses of Pitx1 and Tbx5 expression in HH25 hindlimbs of pigeon (A,B) and chicken (F,G). Boxes span 1st to 3rd quartiles, bars extend to minimum and maximum observed values if within 1.5 times the interquartile range of the box, circles indicate values outside of this range, black line indicates median. **=p<0.01, ***=p<0.001. (C-E, H-J) RNA in situ hybridization for Tbx5 expression in HH25 embryos of racing homer (C), Indian fantail (D), and English trumpeter (E) pigeons; and white leghorn (H), Cochin (I), and silkie (J) chickens. Arrowheads indicate sites of ectopic Tbx5 expression. Scale bar = 1 mm. https://doi.org/10.7554/eLife.12115.011 Figure 3—source data 1 Source data from quantitative RT-PCR experiments. Abbreviations: fl, embryonic forelimb; hl, embryonic hindlimb. https://doi.org/10.7554/eLife.12115.012 Download elife-12115-fig3-data1-v1.xlsx We examined expression of additional genes within the two candidate regions at HH25, and found that the Tbx5 paralog Tbx3 was also differentially expressed in both feather-footed pigeon breeds relative to racing homer (Figure 3—figure supplement 1B). This could be due to the fact that Tbx3 is a target of Tbx5 (Mori et al., 2006; Postma et al., 2008), and additional experiments confirm that cis-regulatory changes do not drive this expression difference (see below). The hindlimb-specific transcription factor Tbx4 is not contained in the candidate regions defined by our QTL mapping and genome-wide association studies, but this gene is a downstream transcriptional target of Pitx1 (Logan and Tabin, 1999; Takeuchi et al., 1999; Duboc and Logan, 2011). We compared expression levels of Tbx4 among scaled and feathered breeds at HH25, but found significant differences at this stage homer ± Indian fantail ± English trumpeter ± n = 6 each) (Figure 3—figure supplement Thus, the embryonic hindlimbs of muffed pigeons show quantitative expression changes in transcription with limb expression including the striking of a key hindlimb identity gene and the expression of a key forelimb-specific gene We analyzed the patterns of Tbx5 and Pitx1 expression in scaled and muffed pigeon embryos at ectopic hindlimb expression of Tbx5 in muffed embryos was different than forelimb pattern in pigeons and other et al., Logan et al., 1998; et al., 1999; et al., Tbx5 is typically expressed the of the but ectopic Tbx5 expression was localized to the of the and hindlimb of the Indian fantail (Figure Figure 3—figure supplement 3B). This was in the large-muffed English trumpeter (Figure Figure 3—figure supplement with the quantitative differences in expression between the two breeds (Figure 3B). This a striking with regions of epidermal transformation, as foot feathers are usually and most on the digits Levi, 1986) (Figure 3—figure supplement In contrast, and quantitative expression differences among breeds, Pitx1 had a similar expression in embryos of scale-footed and feather-footed breeds at this stage (Figure 3—figure supplement 2). Therefore, with the role of in determining fate et al., ectopic expression of Tbx5 is with transformation of epidermal appendages. changes contribute to expression differences in Pitx1 and Tbx5 in muffed pigeons are for the differences in Pitx1, Tbx5, Tbx3 expression between embryos of scale-footed and feather-footed pigeons, then expression of and feathered-foot alleles in a To this we generated F1 hybrid pigeon embryos by an to a and allele expression in the hybrid embryonic hindlimb at HH25 (Figure et al., 2014). with expression differences we observed among breeds (Figure expression of the feathered-foot allele of Pitx1 was approximately lower than the allele (expression of feathered-foot relative to ± n = 6 and expression of the feathered-foot allele of Tbx5 was higher than the allele expression of feathered-foot ± n = (Figure In contrast, expression levels of feathered-foot and alleles of Tbx3 were indistinguishable expression of feathered-foot ± 0.05, n = (Figure These results show that genetic changes alter expression of feathered-foot alleles of Pitx1 and Tbx5, but not in the embryonic pigeon hindlimb. Figure 4 Download asset Open asset expression cis-regulatory changes in Pitx1 and Tbx5. (A) of Tbx5 expression as an in Tbx5 expression between scale-footed and feather-footed breeds could be due to expression differences between breeds are due to changes only then expression of the two Tbx5 alleles in hybrid embryos will be the same In contrast, if cis-regulatory changes underlie differences in Tbx5 expression between the breeds, then expression of the two Tbx5 alleles in hybrid embryos will be different in hybrid hindlimb buds indicate cis-regulatory between scale-footed and muffed pigeon breeds in Pitx1 (B) and Tbx5 (C), but not in Tbx3 Dashed blue line indicates of expression of (E) in hybrid hindlimb buds indicate cis-regulatory in Tbx5 between feather-footed and scale-footed chicken Boxes in span 1st to 3rd quartiles, bars extend to minimum and maximum observed values if within 1.5 times the interquartile range of the box, circles indicate values outside of this range, black line indicates median. Figure data 1 Source data from experiments. Abbreviations: fl, embryonic forelimb; hl, embryonic hindlimb. Download of chromatin between scale-footed and feather-footed pigeon breeds We performed genome-wide comparisons of enhancer in embryonic of racing and Indian H3K27ac as a for chromatin et al., two regions of significantly different enrichment in racing homer hindlimbs relative to forelimbs were within the 44-kb genomic region that is deleted in the Indian and one of these regions (log10 ratio = is adjacent to the hs1473 limb enhancer (Figure 2C). the most significant differentially region in Indian fantail hindlimbs relative to racing homer hindlimbs was upstream of Tbx5 (log10 ratio = (Figure The patterns of enrichment in Indian fantail hindlimbs and forelimbs suggest that ectopic hindlimb expression of Tbx5 could be due to of forelimb-specific In summary, the expression of Pitx1 and Tbx5 among pigeon breeds (Figure and between alleles (Figure is also by chromatin at these pigeon breeds patterning changes In mouse and chicken embryos, experimental of Pitx1 and Tbx5 expression can result in and in both chick and mouse that Pitx1 plays a (but not role in determining morphology of the and (Logan and Tabin, 1999; Takeuchi et al., 1999; Minguillon et al., 2005; DeLaurier et al., 2006; Duboc and Logan, 2011). ectopic expression of Tbx5 in the hindlimbs of chick embryos can also induce and Tbx5 not control limb patterning or determine morphology in et al., 1999; Takeuchi et al., 1999; Minguillon et al., 2005; et al., However, patterning of limb and is on Tbx5 and Tbx4 in et al., These between and point to differences in limb development in different (Horton et al., 2008). the dramatic observed in other with Pitx1 and Tbx5 we compared the hindlimb morphology of pigeons to that of the English trumpeter and the We found patterning in both feather-footed pigeon the on the rather than of the the et a rather than and a of the with the (Figure These changes are of are not to a more We also found that the which is normally and than the in pigeons, was (Figure and two of digit 4 were in feather-footed breeds These are not limb-type However, experimental ectopic expression of Tbx5 in the hindlimbs of chick embryos an of the of pigeon and Takeuchi et al., compared this morphology to a a at like a corresponding of the all of the modified of pigeons in the in the and which are the sites of ectopic Tbx5 expression. Thus, the changes to the hindlimbs of feather-footed pigeon breeds are more than skin Figure Download asset Open asset pigeons have hindlimb and wing-like foot morphology of scale-footed