An integrated cell signalling, cell aggregation, and mechanical process breaks symmetry to achieve periodic patterning of feathers

We first aimed to determine the mechanism underlying the local spatial patterning of feathers. Based on genetic evidence for their importance in feather patterning [29,33], we began by investigating the relationship between FGFs and BMPs, and their control of cell aggregation, in developing chicken dorsal skin. To define the role of FGF, we treated skin explant cultures with FGF9 (a member of the FGF9/16/20 subfamily with more stable activity than FGF20) [42,43] to assess its effect on cell behaviour and feather patterning. We used embryonic skin from the CAG-GFP transgenic line [44], which expresses green fluorescent protein (GFP) in all cells, to reveal feather primordia as sites of high cell density. Skin explants were cultured and imaged after perturbation of FGF signalling. Treatment of explants with FGF protein yielded differing results depending on the protein’s spatial availability. Local exposure via delivery from an FGF-soaked bead resulted in mesenchymal cell aggregation at the FGF source and disruption to the endogenous condensate pattern, whereas ubiquitous exposure through FGF incorporation into the culture medium inhibited all primordium formation (Fig 1C). Inhibition of FGF signalling, via treatment of explants with SU5402, further demonstrated the importance of FGF signalling in condensate formation (S1B Fig). These findings suggest that FGF protein acts as a chemoattractant inducing mesenchymal cell clustering at its local sources, but ubiquitous exposure blinds cells to endogenous sources. Consistent with a requirement for cell movement in feather condensate formation, Latrunculin A, an inhibitor of actin polymerisation [45] and thus cell movement, also suppresses feather pattern formation (Fig 1C).

Application of BMP4 or other BMP family members reduced the number of feather primordium rows formed in cultured explants in a dose-dependent manner (S1C Fig and S1D Fig). Pharmacological inhibition of BMP signalling using LDN193189 resulted in broad areas of skin assuming primordium identity, with loss of the interprimordium interval (S1E Fig and S1F Fig). Quantitative reverse transcription PCR (qRT-PCR) analysis detected suppression of FGF20 expression after 5 hours of BMP4 treatment, whereas inhibition of BMP signalling led to rapid elevation of FGF20 levels, showing that BMP rapidly negatively regulates FGF20 expression (Fig 1D and Fig 1E). Cotreatment of explants with FGF-soaked beads and ubiquitous BMP4 inhibited endogenous primordium formation but did not prevent cell aggregation at applied FGF protein sources (S1G Fig). Thus, BMP signalling inhibits FGF production but does not abolish cell coalescence at FGF sources.

At the core of periodic patterning systems based on intercellular signalling is the phenomenon of self-activation coupled to lateral inhibition [20,21]. If FGF and BMP interactions constitute such a system in feather patterning, then FGF signals should stimulate BMP expression. However, we were unable to detect activation of expression of BMP family members (BMP2/4/7) after FGF treatment (S2A Fig), suggesting that these genes are not regulated directly by FGF stimulation. Thus, considering FGF and BMP interactions, we are unable to construct a simple reaction-diffusion system capable of producing a periodic pattern de novo in chicken skin.

We next explored the potential for the process of local cell condensation itself to induce expression of patterning genes. We observed induction of BMP4 and FGF20 expression at sites of cell aggregation induced by applied FGF beads (Fig 1F and Fig 1G). Coadministration of Latrunculin A, to suppress cell movement and thus aggregation, prevented induction of gene expression, demonstrating that cell aggregation, rather than simple exposure to FGF, underlies induction of BMP4 and FGF20 (Fig 1F and Fig 1G). Thus, cell aggregation is both controlled by and stimulates production of these signals required for periodic pattern formation.

Shyer and colleagues recently reported that contraction of embryonic chicken skin, achieved by culturing the skin without a support, is driven by mesenchymal tension [16]. Such contraction causes rapid displacement of β-catenin protein (encoded by the CTNNB1 gene) from epithelial cell junctions and permits nuclear entry of this protein, mimicking the effect of canonical WNT signalling. We assessed whether tissue contraction as performed by Shyer and colleagues [16] might lead to activation of FGF20 expression by culturing chicken skin either unattached and thus free to contract or pinned to filters and thus under tension, and assessing subsequent changes in FGF20 expression. At both time points assessed (2 and 4 hours), FGF20 expression was markedly elevated in the contracted tissue, supporting a role for cell aggregation in rapid activation of FGF20 production (Fig 1H). No other genes that we analysed (AXIN2, BMP2, BMP4, EDA, and EDAR) were observed to alter expression in these conditions (S2B Fig).

These results point to the operation of an integrated reaction-diffusion-taxis system in feather patterning. If such a system underlies patterning, then the appearance of cell aggregates should neither precede nor follow local changes in gene expression, but both features should emerge synchronously. To assess the relative timing of formation of mesenchymal cell aggregates and the activation of feather primordium gene expression, we assessed both cell distribution and gene expression in the same cultured skin samples (Fig 1I and S1H Fig). Detection of marker genes that are specific for or excluded from feather primordia (CTNNB1, AXIN2, EDAR, EDA, FGF20, and BMP4) in CAG-GFP embryonic skin revealed that changes in gene expression occurred simultaneously with changes in mesenchymal cell density. Time-lapse imaging of feather pattern formation in the CAG-GFP skin (S1 Video) shows the appearance and resolution of unstable intermediates. In fixed skin specimens, shapes consistent with such unstable intermediate condensations also show overlapping gene expression (S1H Fig) highlighting the tight temporal coincidence of these two phenomena. This indicates that the formation of the feather periodic pattern does not arise from a strict reaction-diffusion prepatterning system or a pure mesenchymal cell motility-led system but has key features of both. These observations contrast with observations made during mouse primary hair follicle formation, in which periodic foci of gene expression are observed prior to mesenchymal cell condensation [46].

From these experimental results, we developed a model of the molecular and cellular interactions involved in feather pattern formation (Fig 1J). In this model, local epithelial FGF stimulates mesenchymal cell aggregation. The localised aggregation of mesenchymal cells activates both the expression of BMP4 and amplifies epithelial FGF20 production. BMP proteins diffuse and inhibit FGF production through transcriptional suppression of FGF20. The regulation of cell aggregation by BMP’s suppression of FGF production, together with the local depletion of mesenchymal cells from the regions surrounding the aggregates, results in the formation of discrete and characteristically spaced feather primordia. Such a structure of interactions, incorporating aspects of both reaction-diffusion and mesenchymal condensation into a hybrid reaction-diffusion-taxis model, is capable of breaking symmetry to produce a periodic pattern de novo (Fig 2A). We have recently reported a detailed mathematical analysis of this network structure and its properties [47].

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Fig 2. A travelling wave of primordium induction is required for high-fidelity pattern formation.

(A) Time course output of a simulation of the patterning network shown in Fig 1J. A 2-dimensional patterning (bottom) yields an irregular array, whereas the same network deployed in a wave (top) yields a highly regular hexagonal pattern. (B) E6.5 skin explants cultured for a total period of 48 hours. Control explants display a hexagonal periodic pattern of high regularity. Explants treated for 24 hours with 150 ng/ml Latrunculin A to suppress feather primordium formation, followed by 24-hour incubation in normal medium to permit patterning, produces a pattern of low regularity. Scale bar: 1 mm. (C) Schematic of pattern fidelity analysis. CTNNB1 expression was detected and imaged, and a mask of the image was generated. Voronoi tessellation was constructed with primordium centroids as seeds. Boundary Voronoi cells were removed. (D) Plot of the distribution of shapes of Voronoi cells (i.e., number of first neighbours) in control and Latrunculin-paused skin patterns, as in panel B. The proportion of each n-sided shape for each skin sample is shown as a single data point. The numerical values for D can be found in S2 Data. E, embryonic day.

https://doi.org/10.1371/journal.pbio.3000132.g002

High feather-pattern fidelity is achieved by patterning in a wave

At the ideal packing density, periodic patterns tend to form a hexagonal lattice. In silico modelling using only the interactions described in Fig 1J [47] did not replicate the chicken skin pattern but instead produced a dappled pattern rather than an evenly hexagonal array. Only when this patterning system is driven by a travelling wave of activation are the spatiotemporal progression of sequential row insertion and a uniformly hexagonal patterning outcome observed (Fig 2A).

We tested in embryonic skin the importance of a wave of patterning in producing a hexagonal arrangement by imposing a transient block on patterning. We noted that inhibition of cell movement, and thus primordium formation, in developing explants via Latrunculin A treatment is reversed once the chemical is removed. E6.5 skin explants cultured with a 24-hour Latrunculin A treatment followed by another 24 hours under control conditions displayed irregular primordium arrangements (Fig 2B), consistent with the absence of a patterning wave resulting from the temporary block on cell movement.

To quantify pattern fidelity, defined as the tendency of the lattice of feather primordia to attain a hexagonal arrangement, images of cultured explants were masked and individual primordia were used as seeds to construct a Voronoi diagram (Fig 2C). The distribution of number of neighbours of the Voronoi cells gives a measure of the hexagonality of the experimentally observed patterns. Approximately 75% and 50% of the Voronoi cells in control skin samples and Latrunculin A–paused explants, respectively, exhibited six neighbours, demonstrating a decrease in pattern fidelity in the latter (Fig 2D). These results are consistent with the presence of a pattern-triggering wave spreading across the skin, such that when the Latrunculin block is removed, patterning does not reinitiate at the midline but occurs essentially simultaneously across the entire tract.

An expanding wave of EDA interacts with a receding wave of β-catenin/EDAR to trigger FGF20 expression

The nature of the wave that triggers feather patterning has been elusive since the first descriptions of sequential feather row appearance [11,48–50]. Initiation of feather primordia was reported by Shyer and colleagues [16] to rely on local tissue compression, which was mimicked experimentally by permitting the skin to contract by culturing without a supporting substrate. We assessed whether such local compaction might serve to initiate patterning of the dorsal tract by preparing skin cultures in which a strip of skin parallel to and displaced from the midline was free of the underlying support. This strip was compressed, while the remainder of the skin, including the midline, was pinned taut to a filter to prevent generalised compression. In this condition, we observed an unaltered initiation site and timing of patterning, starting as normal at the midline and expanding symmetrically across the control and experimentally compressed sides of skin at the same rate (S3 Fig). This indicates that feather pattern initiation and its spread from the midline does not rely on heterogeneities in tissue compression.

Based on the mechanistic model defined above, the first step of feather primordium formation is a mild chemotaxis to a source of FGF. We thus reasoned that the key activity of the pattern-triggering wave should be capable of stimulating FGF20 production, which is often detected in a faint streak ahead of and parallel to the most recently formed feather rows (S4A Fig). The expression or activity of the triggering factor should also expand across the skin coincident with the spread of the feather patterning process. Thus, we began by identifying molecules able to induce FGF20 expression. Canonical WNT/β-catenin activation and EDA/EDAR signalling have been reported to induce FGF20 expression in mouse skin [51]. We found that stimulation of each of these pathways, with CHIR99021 or recombinant chicken EDA protein (Fc-chEDA1) [52] respectively, rapidly induced FGF20 expression in embryonic chicken skin (S4B Fig). We also found that stimulation of WNT/β-catenin activity induced EDAR expression in cultured skin (S4C Fig). In situ hybridisation revealed that EDA is expressed as an expanding wave in dorsal skin (Fig 3A). In contrast, CTNNB1 and EDAR expression are first present in a block of skin defining the presumptive tract, and their expression is then maintained in the feather primordia but lost from the interprimordium space as feather rows are laid down. Thus, in the skin, there is a moving front of loss of CTNNB1 and EDAR expression, starting at the midline and receding towards both sides [32,39], as the EDA expression wave advances (Fig 3A). Spreading of EDA expression coincident with patterning is also observed in the other anatomical tracts (S4D Fig). Expansion of EDA expression and regression of CTNNB1 expression across the skin is observed in FGF20^sc/sc mutant embryos, which define tracts at the molecular level but fail to undergo subsequent specification of feather primordia (S5 Fig) [29,39]. This demonstrates that neither FGF20 function nor primordium formation is required for the propagation of these waves. Simultaneous detection of EDA and CTNNB1 by double whole-mount in situ hybridisation shows that primordium formation is closely associated with the leading edge of EDA expression and the lagging edge of the regressing CTNNB1 wave (Fig 3B). EDA is a secreted protein [32,53], and its availability may extend beyond the mRNA location detected by in situ hybridisation.

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Fig 3. An EDA travelling wave triggers feather patterning.

(A) E6.5 skin after 24 hours and 48 hours in culture showing expression of CTNNB1, EDAR, and EDA transcripts. Scale bar: 1 mm. (B) Whole-mount double in situ detection of CTNNB1 (orange) and EDA (purple) at E7.5. Scale bar: 200 μm. (C) qRT-PCR analysis of FGF20 expression levels in E6.5 skin after 5 hours in 20 μM CHIR99021 (an activator of WNT/β-catenin signalling), 50 ng/ml Fc-chEDA1 (an activator of EDAR signalling), or a combination of both. Filled circles indicate individual data points; red bars indicate the mean. Statistical significance was calculated using a Student t test (*p < 0.05, **p < 0.01, ***p < 0.001). (D) Comparison of the periodically patterned region, defined by FGF20 expression, in E6.5 skin cultured for 24 hours in the presence of either 15 μM LDN193189 (an inhibitor of BMP signalling) or 2 μg/ml Fc-chEDA1. Scale bar: 1 mm. (E) Quantification of patterned region width in control, LDN193189-treated, and Fc-chEDA1-treated explants. Statistical significance was calculated using a Student t test (**p < 0.01, ***p < 0.001). (F) Comparison of patterned region width in E6.5 skin cultured for 48 hours in either control or 10 μg/ml Ecto-D2 (an inhibitor of EDA/EDAR signalling) supplemented medium. Scale bar: 1 mm. (G) Quantification of patterned region width in control and Ecto-D2-treated explants. Statistical significance was calculated using a Student t test (*p < 0.05). (H) Detection of FGF20 expression in in ovo control antibody (Aprily2) and Ecto-D2-treated embryos for comparison of feather patterned region width. Scale bar: 2 mm. (I) Quantification of width of primordium carrying dorsal region in Ecto-D2 in ovo injected E8.5 embryos compared to controls. Statistical significance was calculated using a Student t test (***p < 0.001). The numerical values for C, E, G, and I can be found in S3 Data. BMP, bone morphogenetic protein; E, embryonic day; EDA, Ectodysplasin A; EDAR, EDA receptor; qRT-PCR, quantitative reverse transcription PCR.

https://doi.org/10.1371/journal.pbio.3000132.g003

As primordium induction occurs where the EDA wave’s leading edge meets the CTNNB1 receding edge (Fig 3A and Fig 3B), and because of the ability of both canonical WNT and EDA/EDAR signalling to induce FGF20 expression (S4B Fig), we reasoned that these two signalling pathways may function synergistically to spark primordium formation at their junction. Indeed, we found that when both signalling pathways were stimulated simultaneously via cotreatment with low doses of CHIR99021 and Fc-chEDA1, FGF20 expression is induced to a greater degree than in individual treatments, demonstrating a synergistic regulatory effect (Fig 3C). This explains the induction of FGF20 expression along the stripe where the leading edge of the EDA wave meets the receding edge of the CTNNB1/EDAR wave.

The EDA wave triggers periodic pattern formation and controls tract expansion

Having shown that a wave of EDA induces FGF20 expression and is coincident with the onset of periodic patterning, we assessed the functional effects of EDA on feather primordium formation. Specifically, we predicted that if EDA is the limiting factor for triggering pattern formation, then generalised application of EDA should expand the periodically patterned region. Conversely, suppression of endogenous EDA function should restrict pattern expansion. We tested this by treating skin explants with modulators of EDA/EDAR signalling and measuring the width of the periodically patterned region. Ubiquitous stimulation of EDA/EDAR signalling, using recombinant EDA-A1 for 24 hours, almost doubled the width of FGF20 patterned skin (Fig 3D and Fig 3E), supporting the hypothesis that limited EDA availability normally restricts pattern formation. The converse experiment, that of inhibiting EDA/EDAR signalling with the anti-EDA function–blocking antibody Ecto-D2 [52], narrowed the patterned area compared to control cultures (Fig 3F and Fig 3G), further supporting a key role for endogenous EDA in defining patterning wave expansion. Suppression of BMP signalling, which is required for periodic patterning, did not produce such an effect (Fig 3D and Fig 3E), demonstrating that activation of feather patterning potential—rather than simple suppression of BMP activity—is required to drive the advance of the primordium wave.

To test the role of the EDA wave on feather wave progression in an intact and growing embryo, we introduced the blocking antibody Ecto-D2 into the circulation of E5.5 chicken embryos and continued incubation to day E8.5. Such systemic treatment with Ecto-D2 is capable in mouse of inducing an ectodermal dysplasia phenotype that matches that of EDA gene mutation in exposed embryos [52]. In chicken embryos, this blockade of EDA/EDAR signalling permitted feather patterning but restricted the width of the feather pattern in all tracts as compared to controls (Fig 3H and Fig 3I; S6 Fig), agreeing with the results from skin culture experiments. This treatment also abolished the formation of scleral papillae of the eye, similar to but more severe than the effect of the FGF20^sc mutation on these structures (S7 Fig). The feather primordia generated under conditions of EDA suppression failed to develop fully, yielding largely naked skin by incubation days E11.5 and E13.5 (S8A Fig and S8B Fig) and indicating an ongoing need for EDA/EDAR signalling during feather outgrowth. Although placodes and condensates are formed in conditions of EDA suppression, at E8.5 their expression of FGF20 is not polarised normally (S8C Fig and S8D Fig), and this may lead to a failure of their morphogenesis and disappearance over the following days. The converse experiment, stimulating EDA/EDAR signalling in ovo using the EDAR-activating antibody mAb-EDAR3 [54] at E5.5 to E7.5, expanded the feather pattern in the dorsal tracts, also agreeing with the ex vivo explant culture experiments (S9 Fig).

A cell density wave precedes the EDA wave to produce the feather patterning field

These findings show that EDA availability controls the spatiotemporal wave of periodic patterning in avian skin but that this signal is neither absolutely necessary nor absolutely sufficient for this process. Thus, a second factor, independent of EDA, must be both limiting for and sufficient to initiate periodic patterning. Based on the long-recognised importance of cell density in feather development [8,10], we considered mesenchymal cell density as a candidate for this second factor limiting competence to undergo periodic patterning. To characterise mesenchymal cell density at different stages of skin development, we counted cells on sectioned embryos, finding a medial-lateral decreasing gradient of cell density (Fig 4A and Fig 4B). Quantification of cell density across the presumptive tracts of developing FGF20^sc/sc embryos lacking mesenchymal condensates, and so used to avoid the distortion of mesenchymal distribution by cell recruitment to forming condensates, shows that this medial-high cell density gradient is maintained throughout the period of patterning, even as total mesenchymal cell density increases (Fig 4B). Thus, the EDA wave travels across a dynamic gradient of mesenchymal cell density as it triggers feather formation.

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Fig 4. Mesenchymal cell density dynamics in dorsal skin.

(A) DAPI-stained sections of E6.5 skin from the lateral and medial regions of the dorsal tract. Medial regions have a higher density of dermal mesenchymal cells than lateral regions. Scale bar: 50 μm. (B) Mesenchymal cell density quantification across the dorsal tract of E6.5, E7.5, and E8.5 chicken embryos. Arrowheads denote areas of tracts displayed in A. (C) A series of stills from a 70-hour time-lapse imaging of feather patterning during dorsal tract development in CAG-GFP skin, starting at E6.5. Intense GFP signal corresponds to regions of high cell density. Anterior is to the top of each panel. (D) Density kymograph shows high cell density medially and in lateral zones at early stages, followed by sequential feather row formation. (E) X-velocity kymograph depicting displacement of cell density (red: rightward; blue: leftward). A wave of generalised cell density displacement towards the midline occurs at early stages, followed by bilateral feather row formation from 30 hours. (F) Streamlines computed for average displacement fields of 10 consecutive frames showing different phases of primordium formation: (i) cell displacement towards the midline, (ii) formation of individual feather primordia of the midline row, and (iii) formation of the primordia in newly induced rows. Scale bar side: 500 μm. The numerical values for B can be found in S4 Data. a.u., arbitrary units; E, embryonic day; GFP, green fluorescent protein.

https://doi.org/10.1371/journal.pbio.3000132.g004

To trace the cell density dynamics involved in the emergence of periodic pattern on the dorsal tract, we performed time-lapse imaging of cultured embryonic skin from the CAG-GFP transgenic line. Time-lapse imaging of skin begun with tissue collected at E6.5 shows a stripe of high cell density along the dorsal midline. Over the following 70 hours, cells rearrange to produce an ordered periodic pattern with the breaking up of this stripe into a row of spots, followed by bilateral additions of pairs of rows of feather primordia (Fig 4C). During this process, extensive changes in cell density and dynamic relocation of cell aggregates are apparent (S1 Video).

To represent cell density across the skin over time in a 2-dimensional form, we plotted GFP signal intensity as a kymograph (Fig 4D). Each frame from the time-lapse series is depicted as a single line, with red indicating high and blue indicating low cell density. This depiction compresses the 2-dimensional information of each original frame into a line that depicts cell density relative to the left-right axis of the original image, with the compression removing anterior-posterior information. The y-axis on the kymograph is produced by stacking these frames to show the progression of cell density over time. This plot shows the initial midline stripe of high cell density, soon followed by the emergence of two broad lateral regions of high cell density between 10 and 25 hours, after which the addition of the first lateral feather primordium rows occurs from 25 hours. Progressive addition of rows is apparent as the emergence of additional vertical red stripes, though ongoing cell depletion from interprimordium intervals leads to a reduction in overall fluorescence for the most mature (i.e., central) rows.

To track general cell motility, we plotted a velocity kymograph (Fig 4E) showing for each pair of frames compressed along their y-axes the change in cell density (i.e., the change in GFP signal intensity) occurring across the time course of imaging, with blue indicating leftward movement and red indicating rightward movement. Thus, meeting points of red and blue, where red is to the left of blue, show the sites of cell accumulation. This plot shows a generalised movement of cells towards the midline at early stages of patterning, even before the first periodic condensates become apparent there. This widespread midline movement is followed by progressively more lateral local clustering at later stages to form the distinct feather rows. We used streamlines calculated from a particle image velocimetry (PIV)-based velocity field averaged over 10 consecutive frames to better represent cell behaviours during specific phases of dorsal tract patterning (Fig 4F), here maintaining the x-axis (left-right) and y-axis (anterior-posterior) of the original skin images. This shows the initial generalised flow of cells towards the midline (time 5:10–6:50), followed by periodic patterning of condensates on the midline (11:50–13:30) and then cell condensation laterally to form the next rows of feather condensates (36:50–38:30).

To assess the extent to which these observations reflect cellular events specifically in the mesenchymal component of the skin, we visualised mesenchymal cells by transplanting somites from the newly developed tdTomato (TPZ) transgenic line (S1 Supporting Methods) into a CAG-GFP host embryo. Four days after somite transplantation, we collected the chimeric skin at day E6.5 for time-lapse imaging (S10A Fig and S10B Fig). In this condition, the somite derivatives, which are entirely mesenchymal in the skin [55], will on one side of the embryo produce red fluorescent signal, whereas the host cells, including the overlying ectoderm, will be green. Computational analyses of time-lapse imaging of skin explants prepared from host-GFP/mesenchyme graft–tdTomato chimeras show the same cell behaviours as observed in entirely CAG-GFP skin, demonstrating that the key behaviours observed arise from mesenchymal cell dynamics (S10C Fig and S2 Video). Together, these investigations show that the process of dorsal tract patterning involves an initial movement of mesenchymal cells towards the midline, followed by laterally expanding dense dermis and rapid cell density changes to generate a periodic condensate array.

EDA sets the mesenchymal density threshold required for periodic patterning

To assess the functional impact of mesenchymal cell density on feather patterning, we reduced cell density by suppressing proliferation using methotrexate (MTX). This treatment in culture reduced cell density in the dermis (S11 Fig) and limited the area of the feather patterned region, causing it to arrest at five normally spaced rows. This reduction in feather row number occurred without any change in the gene expression waves of CTNNB1 and of EDA, which continued to extend to the normal edge of the presumptive tract (Fig 5A). Thus, the molecular wave propagates independently of either the cell density wave or of feather primordium formation.

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Fig 5. Mesenchymal cell density and EDA signalling interact during skin patterning.

(A) E6.5 skin cultured for 48 hours in 5 μM MTX, an inhibitor of cell proliferation. Primordium formation is detected by CTNNB1 expression. Inhibition of cell proliferation reduces the width of the patterned region, but the extent of the EDA expression wave is unaffected. Scale bar: 2 mm. (B) Schematic of assessment of the critical cell density at which patterning occurs. (C) and (D) Mesenchymal cell density within 100 μm of the edge of the patterned region of skin explants in the presence of (C) an activator (Fc-chEDA1) or (D) an inhibitor (Ecto-D2) of EDA/EDAR signalling. Filled circles indicate individual data points; red bars indicate the mean. Statistical significance was calculated using a Student t test (***p < 0.001). (E) E6.5 CAG-GFP skin explants cultured for 48 hours with 5 μM MTX, 10 μg/ml Ecto-D2, or both. Scale bar: 2 mm. (F) Quantification of patterned region widths, based on FGF20 expression, shows reduction in patterned region of cotreated samples compared to each single treatment. Statistical significance was calculated using a Student t test (***p < 0.001). (G) One-dimensional representation of a simulation of the network shown in Fig 1J, with peaks representing high cell density (condensates). Reduction of the wave or of cell growth restricts the spread of pattern, whereas reduction of both has a stronger effect. The numerical values for C, D, F, and G can be found in S5 Data. E, embryonic day; EDA, Ectodysplasin A; EDAR, EDA receptor; GFP, green fluorescent protein; MTX, methotrexate.

https://doi.org/10.1371/journal.pbio.3000132.g005

We assessed the mesenchymal cell density at which patterning occurs in different EDA signalling regimens by making incisions directly adjacent to the FGF20-defined margin of the patterned region of EDA modulated explants and quantifying the mesenchymal cell density within the first 100 μm lateral to the pattern margin (Fig 5B). Application of soluble EDA, which led to expansion of the patterned region (Fig 3D and Fig 3E), permitted patterning at a lower mesenchymal cell density than normal (at the lateral pattern margins 66 cells/100 μm versus 49 cells/100 μm after 24 hours in culture) (Fig 5C). Conversely, suppression of EDA function permitted patterning after 48 hours in culture only where a density of 98 cells/100 μm was attained (Fig 5D), restricting patterning to a narrow strip at the midline (Fig 3F and Fig 3G). Thus, EDA signalling lowers the mesenchymal cell density at which the dermis becomes permissive to periodic pattern formation.

To test whether EDA signalling sets the critical mesenchymal cell density quorum independently of skin location, we assessed the effects of combined modulation of both cell density and EDA function. As before, suppression of either cell proliferation or of EDA function individually reduced the width of the patterned region of the skin. Combined suppression of cell density and EDA function yielded a still greater reduction of the periodically patterned region (Fig 5E and Fig 5F), showing that a high cell density, rather than any other means of circumventing EDA activity, is required for medial skin patterning in conditions of EDA suppression. Simulations of the patterning process involving a priming wave, representing EDA, and including a mesenchymal cell density gradient show that such behaviour is expected of the network upon suppression of cell density, suppression of EDA, or the enhanced effect of suppressing both (Fig 5G) (see S2 Supporting Methods). Thus, mesenchymal cell density has a defining effect on determining whether periodic patterning occurs or not, with EDA setting the minimum density of mesenchymal cells required to undergo pattern formation.

Continual movement of individual mesenchymal cells occurs in loose and dense dermis prior to and during condensate formation

Having defined a requirement for a mesenchymal quorum in feather patterning and its relationship to EDA, we assessed cell behaviour in dense and loose dermis. We aimed to determine the mode of cell accumulation at condensates and whether differences in cell behaviour might account for the requirement for a minimal mesenchymal density to initiate periodic patterning. To track single cells and quantify their behaviour, we employed embryos of the Chameleon transgenic chicken line. This line carries an array of fluorescent protein encoding genes, one of which is selected for expression upon Cre-mediated recombination at the transgene locus ([56] and Saunders and colleagues in preparation). We treated cultured Chameleon transgenic skin with a cell-permeable TAT-Cre to sparsely label cells, applying the reagent to the mesenchymal side of the dissected skin. This results in stochastic labelling of scattered cells with different colours (Fig 6A and Fig 6B). Detection of TWIST1/2 (also referred to as Dermo-1), a nuclear marker of mesenchymal cells contributing to pattern formation and dermal condensates [57–59], shows Cre-mediated recombination and resulting cytoplasmic fluorescence in this cell population at all levels in the dermis (Fig 6C).

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Fig 6. Individual mesenchymal cell movement and morphology are unaffected by cell density.

(A) Cells of the Chameleon line express Nuclear Blue until exposure to Cre recombinase, which causes cells to express one of three colours of cytoplasmic fluorescent proteins: red, cyan, or yellow. (B) E6 Chameleon skin treated with TAT-Cre and cultured for 24 hours, demonstrating sparse labelling of cells. Two feather condensates are present at the midline (vertical centre). Scale bar: 200 μm. (C) Frozen transverse section of TAT-Cre labelled Chameleon mesenchyme. Colocalisation of nuclear TWIST1/2 staining (a marker of mesenchymal cells, white) and cytoplasmic tdTomato or eYFP (shown as green) confirms that labelled cells have dermal mesenchymal identity. Scale bar: 20 μm. (D) Mesenchymal cell speed and track straightness plotted for each region of skin, from medial (i) and intermediate (ii, iii) to lateral (iv). Analysis derived from confocal time-lapse imaging of labelled E6.5 Chameleon skin explants cultured for 24 hours. Error bars indicate S.E.M. (E) CAG-GFP explants cultured for 24 hours carrying either BSA- or FGF9-coated beads across the presumptive dorsal tract (high density) and intertract loose mesenchyme (low density). Scale bar: 1 mm. (F) Angle plots showing direction of individual mesenchymal cell movement in the 10 hours before and the 10 hours during the formation of a feather primordium row (Row) or the same time period in loose mesenchyme that does not form feather primordia (Loose). Line length indicates cell movement track length. Midline: 180°, Anterior: 90°. The numerical values for D and F can be found in S6 Data. BSA, bovine serum albumin; E, embryonic day; eYFP, enhanced yellow fluorescent protein; FGF, fibroblast growth factor.

https://doi.org/10.1371/journal.pbio.3000132.g006

We collected time-lapse confocal images of cells in such sparsely labelled skin during patterning in culture, permitting the tracking of individual cells. We observed that cells in all parts of the skin move, with cells in the epidermis moving in a highly coherent manner, as predicted based on their physical attachment to one another in a sheet (S3 Video, S12A Fig). We analysed cell behaviour by confocal layer, finding that the more superficial layers, which are epithelial, undergo coordinated cell movement, whereas the deeper layers of mesenchyme have cells undergoing random and uncoordinated movement (S12A Fig). However, we detected very little movement upward or downward at any skin depth, with most cells remaining in the same confocal layer throughout and thus moving in parallel with the plane of the epidermis (S12B Fig). The net movement for individual mesenchymal cells over the course of imaging occurs almost entirely towards the midline (S12C Fig).

By measuring individual mesenchymal cell behaviours over the entire time course of imaging, we found that cells in dense and loose dermis move, though somewhat more rapidly in the lateral, loose dermis (Fig 6D and S4 Video). This may result from freer movement of cells where there are fewer cell–cell interactions. Thus, the failure of patterning in loose dermis is not a result of impaired cell movement, nor is it due to an inability to respond to FGF sources, as FGF beads are readily capable of inducing cell aggregation in both dense and loose dermis (Fig 6E). By tracking cells specifically prior to and during the formation of condensates, we observe that before the formation of a new primordium row, cells move medially; but to form the condensates themselves, movement is predominantly along the anterior-posterior axis (Fig 6F). Throughout this process, there is remarkably constant migration speed and straightness (persistence), though the midline-directed cell movement occurring before condensate formation displays the greatest persistence of all classes (S13A and S13B Fig). Individual condensates are thus formed by the FGF-guided accretion of cells that move constantly with approximately the same velocity and persistence.

The skin’s mesenchymal cells display extensive and dynamic projections when they are both outside and within condensates, and this morphology does not differ between low- and high- cell-density mesenchyme (S13C Fig). We used the MacGreen reporter line, which expresses GFP specifically in macrophages under control of CSF1R regulatory elements [60], to detect macrophages in embryonic chicken skin. These cells are distinct from other mesenchymal cells in size of cell body and characteristics of the projections, confirming that we are not inadvertently tracking this cell population (S13D Fig). Together, this approach shows that in morphology and movement, mesenchymal cells in dense and loose regions display very similar observable behaviours. Thus, the high-cell-density dependence for feather development more likely rests on a need for rapid mesenchymal cell condensation and stabilisation of FGF20 expression than on distinct individual cell phenotypes in dense and loose mesenchyme.

Loss of feather patterning waves in flightless birds

We find that the skin of chicken, duck, ostrich, and emu embryos have pronounced differences in their feather arrangements. The two flighted species, chicken (of which wild ancestors and light domestic breeds can fly, though this ability is lost in some breeds because of increased body weight [61]) and duck, display a highly regular feather arrangement, whereas the two flightless species have a much less regular pattern (Fig 7A). Voronoi tessellation analysis shows that flightless species have a pattern of lower hexagonality than the flighted species (Fig 7B). Whereas the flighted species form their feather pattern through a medial-lateral travelling wave, in the flightless ostrich and emu sequential feather primordium formation is absent (S14 Fig).

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Fig 7. Low-fidelity feather pattern in flightless birds.

(A) Visualisation of feather primordia (CTNNB1 gene expression) during and after dorsal patterning in flighted (chicken and duck) and nonflighted (ostrich and emu) birds. Both flighted species display a high-fidelity hexagonal arrangement of feather primordia, whereas nonflighted species display a low-fidelity arrangement. Inset: high-magnification image of the corresponding panel showing low-fidelity arrangement of the small emu primordia. Scale bar: 1 mm (Inset: 500 μm). (B) Measurement of the fidelity of primordium arrangements of chicken, duck, ostrich, and emu dorsal regions. Each data point represents the proportion of the specified n-sided shape from a single piece of skin. (C) In situ hybridisation detecting CTNNB1 expression in emu embryos prior to and during feather patterning. Dorsal and lateral views of embryos from the ages indicated are shown. Scale bar: 5 mm. The numerical values for B can be found in S7 Data. E, embryonic day.

https://doi.org/10.1371/journal.pbio.3000132.g007

Prior to patterning, both flighted and flightless (S14 Fig and S15 Fig) species define the presumptive dorsal tracts, denoted by CTNNB1 and EDA expression, but the flightless species lack the spreading wave of EDA expression during the period of feather patterning. Rather than patterning in a wave, the ostrich embryonic skin undergoes sporadic patterning within the CTNNB1-defined dorsal tract in regions where mesenchymal cells attain a high density (S14A Fig). This ultimately produces a set of tracts that can be mapped onto those present in the chicken anatomy, though with some fusions (S14A Fig and S16 Fig). Indeed, the flighted Chilean tinamou (Nothoprocta perdicaria), a ratite related to ostrich and more closely to emu [62,63], has tracts in the same arrangement as chicken, indicating that this arrangement (S17 Fig) is ancestral to all extant birds. In the emu, in contrast, the first signs of feather formation occur late in development, at E17, after loss of CTNNB1 expression from the presumptive tract. These feather primordia appear outside the characteristic tract locations, beginning instead on the flanks (Fig 7C), which are prominent intertract regions in chicken and ostrich. Sequential double in situ hybridisation to detect CTNNB1 and EDA in emu embryos shows the feather primordia forming specifically in EDA-negative skin regions, lateral to the presumptive dorsal tract (S18A Fig).

To assess whether the absence of feather formation in the emu presumptive dorsal tract is of mesenchymal or epithelial origin, we performed reciprocal epidermal-dermal recombinations between chicken and emu embryonic skin. A combination of chicken epidermis with emu dermis did not produce feather primordia. In the reciprocal recombination, however, emu epidermis can undergo rapid primordium formation in conjunction with chicken dermis (Fig 8A). Use of CAG-GFP transgenic chicken skin allowed tracking of the chicken and emu components of the skin composites in sectioned tissue, confirming the emu origin of epidermis undergoing feather formation when combined with chicken tract dermis (Fig 8B). Therefore, the evolutionary loss of emu feather tract patterning in the dorsal skin is a result of a mesenchymal defect, with the emu epidermis being competent to participate in feather development.

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Fig 8. Emu dermis has low density and is not permissive to tract patterning.

(A) Epidermal-dermal recombinations between CAG-GFP chicken and emu skin. E6.5 chicken and E14 emu dorsal skins were recombined and cultured for a period of 96 hours. Chicken epidermis and emu dermis do not form feather primordia, whereas emu epidermis recombined with chicken dermis does. Scale bar: 500 μm. (B) GFP detection of chicken cells in sections of recombined skin, stained with DAPI. Scale bar: 50 μm. (C) and (D) Immunodetection of TWIST1/2 protein (a nuclear marker of mesenchymal cells, brown) in the lateral and medial regions of the dorsal skin of embryonic stage HH23, HH26, and HH29 (C) chicken and (D) emu embryos. Scale bars: 50 μm. (E) Quantification of mesenchymal cell densities across the dorsal region of E4.5 (HH23), E5.5 (HH26), and E6.5 (HH29) chicken embryos. (F) Mesenchymal cell density quantification across the dorsal region of emu embryos from E9 to E20. Density increases markedly after E15. The numerical values for E and F can be found in S8 Data. E, embryonic day; GFP, green fluorescent protein; HH, Hamburger Hamilton stage.

https://doi.org/10.1371/journal.pbio.3000132.g008

To determine the nature of the mesenchymal defect that abolishes tract patterning in emu, we compared the status of the developing dorsal mesenchyme in chicken and emu embryos. TWIST1/2 staining in developing chicken and emu dorsal skin revealed a profound delay in the densification of the dermis in the presumptive emu tract compared to chicken (Fig 8C, Fig 8D and S18B Fig). Quantification of mesenchymal cell density at these stages shows that the population of the dorsal skin with mesenchyme is limited in emu embryos, which develop a dermis displaying a uniformly low mesenchymal cell density when compared to chicken embryos (Fig 8E and Fig 8F). In earlier-stage embryos, cell proliferation was observed in the dermomyotomal lip of both Hamburger Hamilton stage (HH)21 chicken and emu embryos, as determined by immunodetection of phospho-Histone H3 (Ser10) (S18C Fig), suggesting that delayed dermis densification in emu is not due to the lack of production of mesenchymal cell progenitors but rather due to a failure of cell ingression towards the dorsal midline during emu development. These observations suggest that in emu, the failure to develop a sufficient mesenchymal density at the appropriate time in development underlies the failure of tract patterning by desynchronising the cellular and molecular influences required to act coordinately in the generation of a patterned feather array.

Ethics statement

All animal work was approved by the Roslin Institute AWERB and the United Kingdom Home Office.

Eggs and incubation

Dekalb White embryos from Henry Stewart & Co (Norfolk, UK) were used unless stated otherwise. Scaleless eggs were obtained from the University of California Davis Meyer Hall Avian Facility, USA, and incubated at the Roslin Institute. Transgenic green fluorescent (CAG-GFP) [44], TPZ, Chameleon, and MacGreen reporter chicken eggs were obtained from the Roslin Institute National Avian Research Facility (NARF); Khaki Campbell/Indian runner duck eggs were obtained from a small supplier. Ostrich (Struthio camelus) and emu (Dromaius novaehollandiae) eggs were supplied by Woodbine Farms (Northamptonshire, UK). Chilean tinamou (N. perdicaria) embryos were provided by the Hancock Wildlife Foundation (Surrey, Canada). Chicken and duck eggs were incubated vertically at 37.8°C with no rotation. Emu and ostrich eggs were incubated horizontally at 36.4°C and rotated 90° periodically until day of extraction. Embryonic developmental stages were scored according to Hamburger and Hamilton for chicken [75] and Hamburger Hamilton equivalent for emu [76].

Skin explant culture and manipulation of intercellular signalling

Dorsal skin from the base of the skull to tail, comprising the dorsal tract and parts of both left and right femoral tracts, were dissected from embryos of the required developmental stage in PBS and affixed onto nitrocellulose filters (Millipore). For basic culture, explants were submerged in 2 ml of standard medium (DMEM supplemented with 2% FBS and 1% penicillin-streptomycin) or supplemented medium at 37°C, 5% CO₂.

Recombinant human FGF9, BMP2, BMP4, BMP7, BMP2/7, and BMP4/7 proteins were from R&D Systems. Recombinant human FGF20 protein was obtained from two commercial suppliers, but both had no significant effects on skin development or on expression of the FGF signalling target gene ETV5 in our hands. Fc-chEDA1, Fc-chEDA2, Ecto-D2, Aprily2, and mAb-EDAR3 were as described [52,54]. Small molecules used were SU5402 (Sigma-Aldrich), LDN193189 (Stemgent), CHIR99021 (Axon Biochem), Latrunculin A (Sigma-Aldrich), and Methotrexate hydrate (Sigma-Aldrich).

Compressed versus taut skin culture

Skin explants at E6.5 were placed into TRI reagent (Sigma-Aldrich) immediately (as time 0 hour) or cultured for 2 or 4 hours either mounted on a nitrocellulose filter (basic culture method) or free-floating in standard culture medium. Cultured explants were subsequently placed into TRI reagent and processed for qRT-PCR.

Explant compression culture

Rectangular segments (1 mm wide) were excised from triangular nitrocellulose filters (Millipore), leaving a thin strip of filter to connect the two parts. Dissected E6.5 TPZ dorsal skin explants were mounted to these prepared filters with the dorsal midline directly parallel to the excised section of the filter. Skin was placed across the excised gap and cultured for 2 hours under standard conditions to allow for the adherence of the explant to the filter. A cut was then made perpendicular to the excised section of the filter on the “free-floating” side of the culture (explant is untouched by the cut), separating the two parts of the filter. The “free floating” side of the culture was then manually compressed by narrowing the filter-excised gap. Compressed explants were subsequently cultured over a total period of 48 hours under standard culture conditions.

Localised delivery of proteins

Affi-Gel Blue Gel beads (Bio-Rad) were washed twice in PBS for 10 minutes and incubated in either recombinant proteins or an equal concentration of BSA diluted in PBS overnight at 4°C. Beads were transferred to nitrocellulose filters, and dissected dorsal skin explants were mounted, dermis side down onto the beads, to the filter. Explants were then cultured using the standard culture method. For Latrunculin A cotreatments, skin explants were cultured for 2 hours prior to the application of protein-loaded beads.

Time-lapse imaging and PIV analysis

For time-lapse imaging, E6.5 explants prepared from CAG-GFP chicken embryos were mounted onto nitrocellulose filters and cultured skin side down in a 6-well dish containing 2 ml standard or supplemented medium at 37°C, 5% CO₂. Real-time imaging was performed using the Zeiss Live Cell Observer/Deconvolution system, imaged every 10 minutes for the duration of the experiment, and analysed using ZenBlue2012 (Zeiss) software.

For the PIV analyses [77,78], microscope image frames of skin showing fluorescent (red and green) cells were superposed in one image to achieve higher density. PIV was conducted using MatPIV, implemented in Matlab, with global, local, and signal-to-noise filter. Interrogation areas (32 × 32 pixel size) with 50% overlap were chosen. PIV yields displacement (velocity) fields for every pair of consecutive frames, which were used to derive velocity and angle kymographs as well as streamlines.

Individual cell tracking

Dorsal skin explants were prepared from transgenic Chameleon E6 chicken embryos and placed onto a nitrocellulose filter. TAT-CRE (62.5 units; Millipore) diluted in phenol red–free DMEM (Sigma-Aldrich) to a final volume of 100 μl was delivered under the dermis. Explants were cultured in a 6-well plate containing 500 μl of phenol red–free medium supplemented with 2% FBS and 1% penicillin-streptomycin for 2 hours at 37°C, 5% CO₂. Explants were submerged in a total of 2 ml of supplemented phenol red–free medium and cultured for 20 hours. Recombination was confirmed via fluorescence microscopy prior to time-lapse confocal imaging using the Zeiss LSM710 confocal microscope. Images were taken every 15 minutes over a period of 24 hours using the red and green channels. Data were processed using ZenBlue2012 software (Zeiss) followed by cell-tracking analysis (Brownian motion) using IMARIS software.

Automatic cell tracking was performed using Fiji. The cell diameters were estimated for each slice and each signal (green, red) separately. For preprocessing, the images were converted to 8-bit. Salt-and-pepper noise was removed with a median filter (3-pixel kernel). Cell motion analysis was performed for each slice and each signal separately. For each analysis, an image sequence with a spot and track overlay (fast backward tracks and uniform colouring) was saved. Exported files from TrackMate include track and spot statistics. The statistics for the two processed channels of each slice were combined. For time points with missing spots, the positions were interpolated using the mean of the preceding and following time point.

To measure individual cell behaviour specifically in loose dermis or primordium rows before and during condensate formation, manual cell tracking was performed using the Manual Tracking tool in the Fiji distribution version of ImageJ. Maximum-intensity Z-projection of time-lapse images of only the uppermost dermal layer from TAT-Cre-induced Chameleon skin explants were cropped and separated into two regions, loose dermis and primordium row–forming (225-μm-wide blocks). The first row lateral to the primary midline row was designated the row-forming region, whereas loose dermis was the skin region that never experienced condensate formation throughout the imaging period (determined from the Nuclear Blue signal at the end of the time lapse). Three independent skin samples were analysed. Tracking periods were 10 hours before and during primordium formation. Only cells that were tracked for the full duration of each period were included in the analysis.

Epidermal-dermal recombination

Interspecies epidermal-dermal recombinations were performed on HH29-equivalent embryos. Dissected dorsal skins were incubated in 2X magnesium and calcium-free saline containing 0.25% EDTA for 20 minutes at 37°C followed by separation of the epidermal and dermal layers in PBS. The dermis from one species was placed onto a nitrocellulose filter, and the epidermis from the other species was stretched across the dermis and pinned to the filter. Recombinants were cultured in a 6-well plate with 500 μl of standard medium for 2 hours at 37°C, 5% CO₂ and then submerged in a total of 2.5 ml of standard medium and cultured for a maximum of 6 days. Medium was replaced every 48 hours.

In ovo embryo injection

Eggs incubated to E5.5 were windowed and intravenously injected with 200 μg of Aprily2 or Ecto-D2 antibody in PBS. For mAb-EDAR3 treatment, 50 μg of the antibody or an equivalent volume of PBS was intravenously injected into windowed E5.5 eggs. PBS (200 μl) containing 1% penicillin/streptomycin was pipetted over the embryo prior to injection. Glass microcapillaries were loaded with antibody and injected directly into either the left or right vitelline vein using the Femto-Jet4i microinjector. Eggs were sealed and reincubated until the desired stage followed by fixation of embryos in 4% paraformaldehyde (PFA) for processing. For analysis of older embryos (≥E11.5), a second round of injection was performed at E7.5.

Tissue processing and paraffin sectioning

Skin explant samples or embryos fixed in 4% PFA were washed in PBS and then dehydrated through an ethanol series, followed by incubation in 1:1 (v/v) ethanol:chloroform. Samples were incubated in chloroform overnight prior to embedding in paraffin blocks. Transverse sections (8 μm) were prepared from both embryo and skin explant samples.

Tissue processing and cryosectioning

Hetero-specific epidermal-dermal recombinant and TAT-Cre-induced Chameleon explants were fixed in 4% PFA, embedded in 15% sucrose/7.5% gelatin in 0.12 M sodium phosphate buffer, and cryosectioned at 20 μm. Gelatin was removed by washing in prewarmed (37°C) PBS. Recombinant sections were stained with DAPI mounted in ProLong Gold antifade reagent (Life Technologies) and imaged with an inverted confocal microscope (Zeiss LSM 710) using ZenBlue2012 software (Zeiss).

Immunodetection

Sections were dewaxed in xylene, rehydrated into water through an ethanol series, and processed for antigen retrieval in the Bio-retriever2000 in either EDTA buffer or citrate buffer. Sections were washed in TBST, incubated in blocking buffer containing 5% goat serum in TBST for 2 hours at room temperature, and then incubated overnight at 4°C in TWIST1/2 antibody (GenTex) at 1/200 or phospho-Histone H3 (Ser10) antibody (Cell Signaling Technology) at 1/200 in blocking buffer. Sections were washed in TBST followed by incubation in biotinylated anti-rabbit secondary antibody (1/200 in blocking buffer) for 1.5 hours at room temperature. Sections were then washed in TBST and incubated at room temperature for 1 hour in HRP-conjugated streptavidin 1/200, followed by another TBST wash. Sections were stained in DAB solution until colour appeared and were washed in water for 20 minutes. DAB-stained sections were lightly counterstained with haematoxylin prior to mounting in hydromount. Images were collected by automated slide scanning (NanoZoomer, Hamamatsu).

For immunofluorescence, after removal of the HRP-conjugated streptavidin, Tryamide signal amplification was performed using TSA kit #26 Alexa fluor 647 (Thermo Fisher) for 10 minutes at room temperature as per manufactures guidelines. Sections were then counterstained with DAPI, mounted in ProLong Gold antifade reagent (Life Technologies), and imaged with an inverted confocal microscope (Zeiss LSM 710) using ZenBlue2012 software (Zeiss).

RNA in situ hybridisation

Cultured skin explants or embryos were fixed overnight in 4% PFA at 4°C, dehydrated through a methanol series, bleached in 5% hydrogen peroxide (H₂O₂), and then rehydrated into PBS. Samples were treated with 20 μg/ml proteinase K, postfixed in 4% PFA containing 0.2% glutaraldehyde, and hybridised with a digoxigenin (DIG)-labelled riboprobe for single in situ hybridisation or in combination with a 2, 4-dinitrophenyl (DNP)-labelled riboprobe for double in situ hybridisation. Unbound probe was removed via washing prior to DIG-labelled probe detection using 1/1,000 alkaline phosphatase (AP)-conjugated anti-DIG antibody (Roche) followed by signal detection using 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT).

For double in situ hybridisation, after the first colour reaction, samples were postfixed in 4% PFA for 30 minutes at room temperature. DNP-labelled riboprobe was detected using 1/1,000 AP-conjugated anti-DNP antibody (Vector) and then coloured using BCIP/2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT).

Analysis of sectioned tissue samples

For DAPI-stained samples, 0.71-μm optical slices were taken using an inverted confocal microscope (Zeiss LSM 710) with ZenBlue2012 software (Zeiss). Samples that had undergone in situ hybridisation were imaged with a stereo microscope (Olympus SZX10) using cell^B (Olympus, Tokyo, Japan) software. Images were analysed using Image-Pro Plus software (Media Cybernetics).

For determination of dermal cell density threshold permissive to feather patterning, FGF20 expression was detected in control, Fc-chEDA1-treated, and Ecto-D2-treated skin explants by in situ hybridisation. An incision was made on each explant at the margin of the FGF20-expressing regions of the dorsal tract using a razor blade. Samples were processed for paraffin sectioning, and sections were dewaxed in xylene followed by rehydration through an ethanol series into water. Sections were stained with DAPI and mounted in ProLong Gold antifade reagent (Life Technologies). Dermal cells were quantified from the skin regions immediately adjacent to the cut site in the FGF20-negative region (site of next presumptive primordium row). All dermal cells within the first 100 μm from the cut were counted from every sample and averaged.

For dermal cell density quantification, all sections analysed were taken from the posterior dorsal region of the embryo, between the femoral tracts. Sections were dewaxed and rehydrated followed by DAPI staining and mounted in ProLong Gold antifade reagent. Dorsal tracts were partitioned into 100 × 41-μm blocks for sections from whole embryos or 100-μm sections for skin explant sections across the entire presumptive tract, below the epidermis and beginning from the dorsal midline. Dermal cell numbers were quantified for each block across the entire dorsal tract for individual samples and then averaged across each treatment group/embryonic stage.

Pattern analyses

Measurements of the width of the patterning region were performed on cultured skin explants or whole-mount embryos that were subjected to in situ hybridisation for FGF20 detection. The pattern-generating region of the dorsal tract on the explants was defined by FGF20 expression. Three measurements of the width of the FGF20 pattern were made for each skin and averaged, with a minimum of three skins per treatment.

For pattern fidelity analysis, explant or whole-mount samples processed for CTNNB1 detection through in situ hybridisation were imaged. Primordia from the dorsal tracts were outlined and given a binary mask, for which a 1 indicated the location of the primordium and a 0 indicated the interprimordium region (see Fig 2C). The centroid of each primordium was then extracted, and a Voronoi diagram was constructed from the point cloud data. The Voronoi diagram partitions the plane into regions based on the distance to the primordium centroids. Specifically, a primordium’s defined region consists of all spatial points closer to its centroid than to any other. These regions are called Voronoi cells. The Voronoi diagram has the property that the boundary cells are anomalous, with infinite (or exceedingly large) area. In order to remove such artefacts, we removed the most external primordium centroids from consideration within the data until the area of all Voronoi cells was less than three standard deviations from the mean. In practice, this eliminated only the anomalous cells. Once the appropriate cells had been chosen, statistics were autonomously extracted regarding the number of nearest neighbours of each primordium (Fig 2D, Fig 7B, S17B Fig). Specifically, if the primordia were perfectly aligned on a (nonsquare) grid, each placode would have six neighbours (and four neighbours in the singular case of a square grid). Thus, the spread and range away from six neighbours illustrates the variability in the pattern fidelity. Namely, a higher percentage of primordia with six neighbours implies a more regular placode pattern.

Visualisation of tinamou feather arrangement

E14 tinamou and chicken embryos were fixed in 4% PFA. Embryos were washed in PBS, and all feather filaments were cut off manually using fine scissors. Embryos were submerged in haematoxylin for 30 seconds, followed by PBS washes, and imaged with a stereo microscope (Olympus SZX10) using cell^B (Olympus, Tokyo, Japan).

qRT-PCR

Total RNA was isolated using TRI reagent and reverse transcribed using random hexamers and Superscript III Reverse Transcriptase (Invitrogen) in a final volume of 20 μl. cDNA was diluted 10-fold, 3 μl of which was used as template for each qRT-PCR. qRT-PCRs were performed using SYBR Green Universal Master Mix (Roche) in a final volume of 20 μl, and each was performed in triplicate, with a minimum of three biological repeats. A dilution standard curve was used to determine the relative expression levels.

Primer Sequences:

AXIN2 F: 5′-GCCGACTTCAAAAGCAAAAT-3′

AXIN2 R: 5′-GTAGGGGTTAACGGGATCGT-3′

BMP2 F: 5′-CCAACACCGTGTGCAGCTTCCA-3′

BMP2 R: 5′-GAAACGTCGTGCTGTTTTCCCAC-3′

BMP4 F: 5′-TGAGGAGCTTCCACCATGA-3′

BMP4 R: 5′-TGCTGAGGTTGAAGACGAAG-3′

BMP7 F: 5′-CGAACGCTTCGATAATGAAA-3′

BMP7 R: 5′-TTCTGGAGTCGAGCAGGAAC-3′

EDA F: 5′-TGGTCTCGCATCACTATGAAC-3′

EDA R: 5′-AATACTCCGAGTGCATTGCAG-3′

EDAR F: 5′-TTCTTTCGAGCCACTGTCCT-3′

EDAR R: 5′-CGAGGTCTGTTTTCCAGCAT-3′

ETV5 F: 5′-AGGCCTGGCTAGCTGAAGCTCA-3′

ETV5 R: 5′-ATCTTGGCTGGAGGTGGGGCAT-3′

FGF20 F: 5′-GCCAAGACCACAGCCTCTT-3′

FGF20 R: 5′-TTCCAAGGTAAAGGCCACTG-3′

GAPDH F: 5′-GACAACTTTGGCATTGTGGA-3′

GAPDH R: 5′-GGCTGTGATGGCATGGAC-3′