Embryonic feathers of zebrafinch
The zebrafinch is an altricial bird, and long downfeathers develop only in few regions of the body (head, lower back, thigh, flank, etc.) as in other altricial species (Romanoff, 1960). The embryonic feathers are the only feathers that grow during embryonic life, while the other feather germs remain quiescent until hatching. The existence of altricial and precocial avian species shows that a mechanism that controls the timing of growth of feather germs is present in birds. In altricial species such as the zebrafinch only the few germs of embryonic feathers are stimulated to grow while feather germs in the other areas remain quiescent until after birth. The controlling mechanism of this specific temporal inhibition of growth of feathers in different regions, to the best of our knowledge, as yet unknown.
In precocial birds, most germs are instead activated during embryonic life, but a second generation of feathers (juveniles) form around 14 days of development in the wing and a few other areas, while no juvenile feather germs are activated in other areas (Lucas & Stettenheim, 1972). This also suggests the presence of a controlling mechanism of inhibition of follicle growth in precocial birds.
The general morphogenesis of zebrafinch embryonic feathers follows that described in other birds: above a mesenchymal condensation the embryonic epidermis forms a placode that evaginates into a dome-like feather germ and continues to grow into a feather filament (Fig. 13A–C; see Chuong & Widelitz, 1999). Barb ridges start to form near the tip of feather filaments and progressively extend down (Fig. 13C,C1,C2,C3). Feather filaments at 13–15 days post-deposition present a basal cellular zone, a variably extended intermediate and keratogenous zone, and an apical keratinized zone (Fig. 13C). The keratinized region quickly spreads down to most of the lower regions between 15 and 16 days post-deposition. Therefore, at hatching (around 18 days post-deposition) when the sheath is shed, barbs and barbules can distend to form the ramifications of embryonic feathers (downfeathers).
Figure 13. Schematic drawing showing a feather germ (A) elongating into a conic feather germ (B), and a feather filament (C). See text for details. The cell layers of horizontally orientated embryonic epidermis (A1) are maintained in the vertically orientated layers of the feather filament (B2), including the fusiform shape of subperiderm cells (B3) as seen in section (asterisk) and complete (triangle). Different levels of the feather filament show that the linear epidermis in its basal portion (B1, C1) become folded into barb ridges in more apical, older, levels (C2,C4). Details of forming (C3) and maturing (C5) barb ridges are shown. The same colour represent the same lineage of cells (despite the fact that cells may change shape and disposition).
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Development of the cellular structure of the downfeather: barb ridge formation
The primary periderm becomes circularly orientated on the external part of the feather sheath (Fig. 13C2–3). Cells of the sheath become corneous via the accumulation of bundles of alpha-keratin that merges with periderm granules to form a compact mass at 13–15 days post-deposition (Matulionis, 1970; Alibardi, 2002, 2005). These bundles of keratins have a circular orientation along the perimeter of the feather filament, and probably act as a cytoskeletal belt that resists the deformations of the innermost epidermal sheet where new cells are still produced (Lucas & Stettenheim, 1972; Sengel, 1975; Chodankar et al. 2003).
The subperiderm layer of embryonic epidermis at 14–16 days post-deposition contains some feather-beta keratin, confirming previous ultrastructural and immunocytochemical studies on the chick embryo (Sawyer et al. 1986, 2003, 2004), and zebrafinch embryo (Alibardi, 2002). The subperiderm layer is formed from the germinal layer of the embryonic epidermis, and the sequence of embryonic layers is preserved in the vertically orientated epidermis of the feather filament (Fig. 13A1,B2).
The basal–apical polarity of cells in the epidermis, where cells migrate from the basal to the corneous layer, become a central–lateral polarity in the vertically orientated epithelium of the feather filament. In the latter, keratinocytes migrate laterally from the basal layer toward the circularly orientated periderm. The shape of cells, in particular that of the subperiderm (elongate beta-keratin cells), initially remains similar in the vertically orientated epithelium of the feather filament (Fig. 13B2,B3). However, the stratification of epidermal layers is altered by the formation of barb ridges. The latter represent infolds of the epidermal sheet inside the narrow cylinder of the feather filament that terminates in a dome-like tip. In this condition it is likely that the epidermal sheet takes a conformation that reduces the ‘tension’ between the epidermal cylinder and its domed surface at the tip of the feather filament. The resistance of the sheath, due to the circularly orientated tonofilaments, allows the epidermal sheet to fold inside in order to accommodate for the increased surface area of the epithelial sheath resulting from cell division (Sengel, 1975; Chodankar et al. 2003). The effect of mechanical forces (Fristom, 1988) acting on the epithelial sheath of feathers has not been explored, although it has been stressed that cell division is mainly concentrated in the inner part of the ridge (Yu et al. 2004). Whether the mesenchyme inside the feather filament has a role in fold formation remains to be experimentally demonstrated. The initial barb ridges are located in the dorsal (proximal or rostral) position of the feather filament, and rapidly extend to the whole circular epidermis.
The outer periderm remains monolayered while the secondary epidermis is distorted by the folding process and gives origin to 2–4 layers of sheath cells and to barb ridge vane cells (Fig. 13C3,C5). The latter penetrate into the axial plate among barbule cells. Cells of the subperiderm are displaced by the folding movement but tend to remain contiguous and form the alar or barbule plate and the axial plate, the barb area (Fig. 13C3,C5). The basal layer forms the flat cylindrical cells of the marginal plates, which rapidly lose germinal activity in mature barb ridges. Therefore, few cells are produced to replace those already present in differentiating barb ridges.
Morphogenesis of barb ridges alters the fate of embryonic epidermis in feather filaments
The morphogenetic process that determines the formation of barb ridges displaces barb vane ridge cells and subperiderm cells. The latter, which are able to synthesize feather beta-keratin, differentiate into barb and barbule cells (Sawyer et al. 2003, 2004). At 14–15 days post-deposition, in elongated feather filaments of the zebrafinch embryo, subperiderm cells are transforming into keratinized feather cells. In particular, those localized in the basal portion of the axial plate and those in the alar plates elongate by the accumulation of feather keratin that forms long filaments along the direction of cell elongation (Kemp et al. 1974; Gregg & Rogers, 1986; Alibardi, 2002, 2005; Figs 13C5 and 14A,A3).
Within each barb ridge, barb and barbule cells fuse into a syncitium forming the ramus (made of barb cells) from which branched barbules (made of barbule cells) are joined together as a result of disappearance of their cell membranes (Fig. 14A5,A7). This process seems to initiate with the formation of adherens or even tight junctions among piled cells. The frequently occurring particle-free vesicles between barbule cells resemble those observed during myoblast fusion to fom myotubes (Kalderon & Gilula, 1979). The process of barb and barbule fusion into syncitia deserves further cellular and molecular studies.
Figure 14. Schematic drawing showing cell differentiation within forming barb ridges (long arrows indicate directions of apical basal formation) of elongated feather filament (A). In A3 the cell structure of the subperiderm layer within the outline of the barb ridge is highlighted. In A5 the fusion of barbule cells to form barbules separated by barb vane ridge cells is nearly complete. The hollowed ramus comprises fused barb cells. In A7 the syncitum of barb and barbule cells is completed, and supportive cells are degenerating, including those of the sloughing layer beneath the sheath. See text for details.
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In the process of formation of branched barbules, barb vane ridge cells function as spacers among barbules. Barb vane ridge cells enwrap barbule cells, often very finely, as far as the ramus (Fig. 14A5). The three-dimensional process of cell differentiation within a barb ridge therefore derives from the displacement into axial and alar plates of cells of the subperiderm (Fig. 13C3,C5; Alibardi, 2005). As a consequence of the morphogenetic process of barb ridge formation, barbs and barbules are produced at the same time. The displacement of barbule cells into two symmetric rows, the barbule plates, allows symmetric branching from the ramus (Figs 14A3,A5,A7 and 15A2). The aggregation of barb cells in the innermost part of the axial plate allows the formation of the rod-like structure of the ramus (Figs 13C5 and 14A3,A5). The presence of barb cortical cells allows the branching of barbule cells and their continuity with the ramus. The symmetric branching of barbules from the ramus gives the biplanarity of each barb ramus with its barbules (Figs 14A5,A7 and 15A1,A2). However, it is the presence of supportive cells (barb vane ridge and cylindrical cells) among keratinizing cells that later permits carving out of the syncitium of the barb, barbules (and calamus) that represents the feather.
Figure 15. Schematic drawing showing how from non-merging barb ridges of the embryonic (downy) feather (A–A1) and the pennaceous feather could have evolved (large double arrow, B–B1). The morphogenetic process resulting from barb ridge fusion with the rachis before reaching the collar appears inherent to the primordial morphogenetic process in embryonic feathers. In A3 it is shown how the condensation of barb and barbule cells into a single mass, with the disappearance of axial and barbule plates, could have produced barbs with no barbules (non-ramified barbs, compare A4 with A2), and unbranched down feathers (A5). The latter may correspond to simple filamentous structures found in avian and theropod fossils. In B, the downgrowth of lateral barb ridges (indicated by arrows directed toward the rachis) is shown. The rachis has an intercalar growth, apically directed as indicated by the arrow. By this process in the ramogenic zone the next lateral barb ridges merge with the rachis after the previous barbs have moved upward. In this way the pennaceous feather in B1 is formed inside the sheath. See text for details.
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Specific mutations affecting the process of gene expression (BMP, SHH, Wnt, Beta-catenin, etc., see Harris et al. 2002; Chuong et al. 2003; Widelitz et al. 2003) during barb ridge morphogenesis and cell differentiation will allow us to understand and even simulate the development (and evolution) of feathers.
Terminal differentiation of the original embryonic epidermis of feathers
Terminal differentiation of keratinizing and supportive cells within barb ridges progress from apical to proximal regions of the feather filament, until all cells eventually die. Within barb ridges no cell replacement of supportive or keratinizing cells occurs (contrary to the process occuring in scales, beak, claws, and interfollicular and apteric epidermis): the downfeather is therefore an embryonic structure.
The process of terminal differentiation of barb cells involves a lipid degeneration followed by vacuolization. The integrity of the ramus is a result of the formation of the cortex around the vacuolated medulla. Cortical cells also form the connections with barbules and calamus and form the branched keratinized syncitium forming the feather.
Terminal differentiation in barb ridge vane cells among barbules and cylindrical cells delimiting the barb ridge eventually determines their death. Because they contain lipids mixed with little keratin, these cells do not keratinize and are reabsorbed (Lucas & Stettenheim, 1972; Bragulla & Hirschberg, 2003). Although previous studies have indicated that supporting cells are selectively eliminated by a process of apoptosis, the cytological characteristics of their degeneration observed in the present study are more similar to those seen in the process of necrosis (mitochondrial and cell vacuolization, cytoplasm dissolution, nuclear breakage, etc.; see also Matulionis, 1970; Alibardi, 2002, 2005). Cell necrosis may derive from the rapid shortage of blood supply as the central feather artery retracts during progressive morphogenesis (Goff, 1949; Lucas & Stettenheim, 1972). The involution of the vascular bed of the feather determines the death of all feather cells, both keratinized (barb and barbule cells) and non-keratinized (barb vane cells and cylindrical cells). Only the syncitium of keratinized beta-cells forming barbs and barbules remains after the loss of barb vane ridge and cylindrical cells and of the sheath (Figs 14A7 and 15A2).
Sheath cells accumulate bundles of alpha-keratin and are shed around hatching (Matulionis, 1970; Sawyer et al. 2003). With their degeneration beneath the sheath, a true sloughing layer is formed and allows the separation of the sheath from the innermost barbules (Fig. 14A7). The degeneration of cylindrical cells of marginal plates allows the separation of rami for the formation of downfeather (Fig. 14A1). Therefore, it is the combined degeneration of barb vane ridge and cylindrical cells that allows the feather to develop after sloughing of the sheath.
Barb ridges and the evolution of feathers
The symmetry of the organization of barb ridges in birds (schematically shown in Fig. 14A5) is essential for understanding the development and evolution of feathers. A key feature in the morphogenesis of feathers is the formation of barb ridges, which is here considered as an evolutionary landmark for feather evolution.
So far as the fossil record is concerned it seems that among reptiles only archosaurians were able to produce thin vertical tubes from the epidermis (Prum & Brush, 2002; Chuong et al. 2003; Kundrat, 2004; Wu et al. 2004). These tubes might have evolved from the narrowing and elongation of ancient tuberculate scales (scales with radial symmetry).
Numerous archosaurian fossils have shown the presence of filiform structures. However, some filaments could have simply represented keratin filaments with no core mesenchyme, as in the bristles of the turkey beard (Lucas & Stettenheim, 1972; Sawyer et al. 2003). It is unknown whether the basic requirement for feather formation, namely the presence of barb ridges, was present in the epidermis of these ancient archosaurians. The mechanism of barb ridge formation (Figs 13 and 14) was probably an evolutionary innovation, and gave rise to the three-dimensional cellular organization for making barbs and barbules in feathers, typical avian and theropod characteristics (Prum & Brush, 2002; Wu et al. 2004). Those archosaurians that evolved barb ridges inside their tubular skin appendages, together with the other characteristics for flight (hollow bones, flying muscles, lungs, aerial sacs, etc.), were on the line that led to modern birds (Padian & Chiappe, 1998).
The passage from a plumulaceous to a pennaceous feather during evolution was a potential consequence derived from the evolution of the morphogenetic mechanism of barb ridge formation (Fig. 15A,A1,B,B1). The rapid formation of barb ridges in embryonic feathers generally impedes their fusion before they reach the collar region: therefore, no branching is formed and barbs remain separate (Fig. 15A,A1). Clearly, an alteration in the timing of barb ridge morphogenesis, or the prevalence of one barb ridge over the others, can determine the fusion of barb ridges before they reach the collar region (Fig. 15B). The point of fusion of barb ridges creates a larger barb ridge, the rachis (Hoosker, 1936), and eventually the formation of a pennaceous feather (Fig. 15B,B1). The inability of barb ridges of embryonic feathers to reach a potential rachis impedes the formation of a pennaceous vane. The inherent potential for fusion of barb ridges is shown in some embryonic feathers where barb ridges fuse before reaching the collar. In these cases some branching with a very short rachis is produced (Lucas & Stettenheim, 1972; Sengel, 1975; Harris et al. 2002).
As a consequence of the morphogenetic process of barb ridge formation, barbs and barbule are contemporaneously produced within a barb ridge (Figs 13 and 14). This condition suggests that intermediate forms of evolving feathers with only barbs and no barbules (Prum, 1999; Prum & Brush, 2002; Chuong et al. 2003; Wu et al. 2004) should be more carefully considered. In the fossil record of feathers and protofeathers, barbules are not described (Prum & Brush, 2002; Chuong et al. 2003; Kundrat, 2004; Wu et al. 2004). This is probably due to: (1) lack of preserved microscopical details; (2) the axial epidermal structures described are neither protofeathers nor feather remnants, but elongated keratinized structures similar to the bristle filaments of the wild turkey (Lucas & Stettenheim, 1972; Sawyer & Knapp, 2003). In this case, no barb ridges were probably present within these (fossilized) filaments, such that there was (3) alteration of the displacement of subperiderm cells within the barb ridge (Fig. 15A3–A5).
The latter process, a different three-dimensional displacement of (subperiderm) cells within barb ridges, could have formed barbs with no barbules (as in few feather types of extant birds; see Lucas & Stettenheim, 1972). A process of fusion of barbule plates with the central axial plate (Fig. 15A3) might be derived from the absence or modified cell–cell interactions between barb vane ridge and barbule cells. Probably, the perturbation of BMP, SHH, Nogging, etc., signals may produce different feather phenotypes (Harris et al. 2002; Chuong et al. 2003; Widelitz et al. 2003). The process of fusion of cells of the subperiderm layer into a single mass may originate a single ramus with no branching barbules (compare Fig. 15A3–A5, the ‘altrered’ phenotype, with Figs 14A3 and 15 A1,A2, the ‘wild’ phenotype). The central fusion of (subperiderm) cells may be the primitive morphogenetic process of barb ridge formation as it produced the typical, unbranched feathers of primitive theropod feathers (compare Fig. 15A4 with stage II of Prum, 1999). Using the latter model of cell displacement within barb ridges, fossilized unbranched feathers of theropods can be explained (Prum & Brush, 2002; compare Fig. 15A4 with the figures in table 1 of Wu et al. 2004).