Avian and squamate scales exhibit morphological diversity
First, we aimed to investigate the diversity of both avian and squamate epithelial appendages. To do this, we used scanning electron microscopy (SEM) to examine morphological variations in the epithelial appendages of these evolutionarily distinct groups. Birds and squamates share a common ancestry within Diapsida, but their respective lineages diverged from each other approximately 255 million years ago [28]. Diverse feather types develop in tracts from the proximal–distal elongation of feather buds, covering most of the chicken embryo’s body (Fig. 1A). Scutate scales are large, overlapping, approximately rectangular structures found on the metatarsal shank and dorsal surface of the foot [20, 29]. Both feathers and scutate scales display anterior–posterior asymmetry (Fig. 1A, B) after developing from a radially symmetrical placode (Fig. 2A–P, Fig. 3A–H) [20]. Reticulate scales form on the ventral surface of the footpad and digits (Fig. 1C). Unlike feathers and scutate scales, they maintain radial symmetry in their adult form.
We next examined the morphology of squamate scales belonging to three lizard species, to discern the diversity of these structures. This included the veiled chameleon (Chamaeleo calyptratus) and the bearded dragon (Pogona vitticeps) which are members of Acrodonta, and the blue-headed anole (Anolis allisoni), which belongs to Pleurodonta [30]. Hatchling C. calyptratus possess bilateral overlapping scales on the dorsal surface of the feet (Fig. 1D). Scales on the ventral foot surface retain a similar shape to the dorsal scales, but do not overlap and appear thicker than those on the dorsal surface (Fig. 1D). These ventral foot scales are morphologically similar to chicken reticulate scales (Fig. 1C). Scales of hatchling A. allisoni are large, overlapping and approximately rectangular, with those on the ventral foot surface appearing comparable to chicken scutate scales, in terms of their general morphology (Fig. 1E). The scales of pre-hatchling (E46) P. vitticeps are similar to those of A. allisoni, as they are large, overlapping structures on both the dorsal and ventral foot surfaces (Fig. 1F).
Overall, there appears to be less morphological diversity between the scales present on ventral and dorsal foot surfaces of the lizard species examined here than observed in the chicken. Furthermore, we observed no clear boundary separating dorsal and ventral squamate scale types. Therefore, the scales on lizard dorsal and ventral foot surfaces may be modifications of a similar squamate scale morphology, whereas the chicken possesses morphologically distinct scale types: the scutate and reticulate scales [20].
Conserved gene signalling is observed throughout the development of reticulate scales and other avian appendages
Next, we aimed to compare and understand the developmental pathways and mechanisms underlying the early formation of different avian epithelial appendages, including reticulate scales. Most epithelial appendages have been shown to develop from the initial formation of an anatomical placode, which arises within an initiatory field such as a feather tract [1, 5, 8]. The anatomical placode is defined by an epithelial thickening with columnar cells exhibiting a reduced rate of proliferation, along with conserved molecular signalling in both the epithelium and underlying mesenchyme [5]. First, to investigate cellular proliferation rate, we examined immunoreactivity of proliferating cell nuclear antigen (PCNA) during the early development of avian epithelial appendages (Fig. 2).
As shown previously, avian feathers and scutate scales both develop from anatomical placodes which first arise within initiatory fields at embryonic day 7 (E7) and E10, respectively [6, 8, 31]. These placodes exhibit columnar cells of the basal epithelium with a characteristically reduced rate of proliferation compared to surrounding cells [5] (Fig. 2A, I, white arrowheads). Notably, PCNA immunoreactivity indicated that reticulate scales first develop from comparatively larger epithelial thickenings that emerge along the ventral side of the footpad and digits at E10.5. These placodes also possess columnar basal epithelial cells with a slightly reduced proliferation compared to surrounding cells (Fig. 2Q, white arrowhead, Additional file 1: Figure S1).
We next aimed to investigate whether conserved molecular signalling in the epithelium and mesenchyme underlies the development of chicken epithelial appendages. First, we examined expression of the transcriptional cofactor β-catenin (β-cat), one of the earliest known epithelial regulators of primordium-specific gene expression [32] (Figs. 2, 3). Whole-mount ISH revealed β-cat demarcates the development of feathers, scutate and reticulate scales, from initiation through to morphogenesis (Fig. 3) [32, 33]. Whilst feather development involves anterior to posterior and lateral addition of primordia (Fig. 3A–D), similar to zebrafish scale patterning [34], scutate scale patterning occurs through the spread of placodes proximally along the metatarsal shank and distally along the digits (Fig. 3E–H). Some scutate scale placodes may fuse to produce enlarged scale buds [26]. Notably, localised expression of β-cat marks restricted circular domains along the ventral footpad and digits (E10.5, Fig. 3I–K), which appear to subsequently subdivide into individual reticulate scales (E12, Fig. 3L).
Sectioning of whole-mount ISH samples revealed that expression of β-cat was specific to the epithelium of developing feathers, scutate and reticulate scales, during both the primary epithelial thickening and morphogenesis stages (Fig. 2B, F, J, N, R, V). Additionally, we examined expression of a conserved regulator of epithelial appendage development, sonic hedgehog (Shh) [8, 35,36,37]. Shh expression was observed in the epithelium of developing appendages at both the placode and morphogenesis stages of development for feathers and scutate scales (Fig. 2C, G, K, O) [8]. Expression of Shh was not localised to the primary epithelial thickening stage of reticulate scales at E10.5, although we observed weak expression in the epithelium and underlying mesenchyme (Fig. 2S). During morphogenesis, expression of Shh was strong and specific to individual elevations of the epithelium (Fig. 2W). Finally, we charted the expression of bone morphogenetic protein 4 (Bmp4), a mesenchymal marker of placode development [5, 8]. Bmp4 expression was limited to the mesenchyme during the primary epithelial thickening stage of feathers, scutate and reticulate scales (Fig. 2D, L, T), before also shifting to the epithelium during morphogenesis (Fig. 2H, P, X). We also observed localised expression of additional conserved markers including bone morphogenetic protein 2 (Bmp2) and sprouty 2 (Spry2) during reticulate scale development (Additional file 1: Figure S2). Together, these results demonstrate that conserved molecular signalling in both the epithelium and underlying mesenchyme regulates the early development of chick epithelial appendages, including reticulate scales.
Overall, these results support previous research suggesting that feathers and scutate scales develop from an anatomical placode [8, 36, 37]. This character is typified by columnar epithelial cells exhibiting a reduced rate of proliferation and conserved molecular signalling in both the epithelium and mesenchyme [5, 6, 32]. Additionally, we provide new developmental evidence that reticulate scales may develop following a similar system, initiating at E10.5.
A derived patterning mechanism underlies chicken reticulate scale development
Previously, it has been suggested that reticulate scales do not develop from an anatomical placode but instead appear as symmetrical elevations at E12, although this event may be preceded by a placode spanning the entire foot or toe pad [6]. Here, we have provided evidence that circular domains of conserved localised gene expression arise upon the ventral surface of the footpad and digits before subsequent development of reticulate scales.
The epithelial thickenings that subsequently give rise to reticulate scales emerge along the digits at E10.5 (Figs. 2Q–T, 3I–L). These circular domains are larger than the initial placodes that give rise to feathers and scutate scales, and appear to subdivide into smaller, secondary units, which radiate outwards sequentially from a central unit (Fig. 4A–D). They subsequently undergo morphogenesis to become radially symmetrical reticulate scales (Fig. 1C). Such periodic patterning bears striking similarity to a RD system, similar to that which underlies avian feather patterning [8]. Feather patterning involves a bifurcating dorsolateral initiator row of placodes triggering the emergence of parallel, adjacent rows [8]. During reticulate scale patterning, we observed enlarged placode-shaped domains, which appear to subdivide into radially arranged smaller secondary units, as opposed to the emergence of placodes in parallel, adjacent rows in feather development [8] (Fig. 3I–L). Reticulate scale patterning may follow a derived RD mechanism, adapted from the system that underpins feather or scutate scale development.
Diverse vertebrate epithelial appendages are thought to be patterned through RD, in which interactions between diffusing activatory and inhibitory morphogens result in autonomous pattern formation [8, 13, 14]. Therefore, we examined whether RD simulation can explain the propagation of reticulate scales from a single, circular initiatory domain (Fig. 4E–H). We initialised a RD simulation with a central spot representing the primary epithelial thickening (Fig. 4E). Numerical exploration revealed a range of model parameter values for which waves of activatory and inhibitory signals radiated from the primary placode (Fig. 4E–H, see “Methods” for further details). From this simulation, we observed the enlarged primary domain subdividing into smaller secondary units, added sequentially from a central unit in a radial arrangement (Fig. 4E–H). This is comparable to expression patterns of β-cat observed from E10.5 to E12 (Fig. 4A–D). These results demonstrate that RD can theoretically explain the derived patterning mechanism underpinning the development of reticulate scales.
Squamates also possess distinct epithelial appendages on the ventral surfaces of their feet. This observation, in combination with the presence of reticulate scales in birds, led to the suggestion that the ancestral archosaur would have also possessed distinct reticulate scales [25]. To test this hypothesis, we examined scale development on the ventral footpad of a reptilian squamate, the bearded dragon (P. vitticeps) (Fig. 5A–J). Reptilian body scales are known to develop from anatomical placodes [5] (Fig. 5G–J). ISH of P. vitticeps samples revealed that scales of the ventral footpad and digits also develop from individual placodes that begin to emerge synchronously at E35, and express both Shh and β-cat (Fig. 5A–F). Therefore, the footpad scales of P. vitticeps are developmentally distinct from avian reticulate scales in terms of their patterning, as reticulate scales arise from restricted, circular domains which subdivide into individual units (Figs. 2, 3, 4). This provides evidence that reticulate scales are derived epithelial appendages that are not present in squamates, at least in the bearded dragon, rendering the condition in the ancestral archosaur ambiguous.