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J Anat. May 2006; 208(5): 621–642.
PMCID: PMC2100222

Cell structure of developing downfeathers in the zebrafinch with emphasis on barb ridge morphogenesis

Abstract

The present ultrastructural and immunocytochemical study on the embryonic feathers of the zebrafinch, an altricial passerine bird, describes cellular differentiation of developing downfeathers. Barb ridges are folds of the original epidermis of the embryonic feather germ in which the basal–apical polarity of epidermal cells is upset. The result is the loss of most germinal activity of basal cells of the barb ridges so that only the embryonic epidermal layers remain. The more external layer is the primary periderm, followed by 4–6 layers of inner-periderm cells that mature into feather sheath and barb vane ridge cells. The following layer, the subperiderm, produces a small type of beta-keratin typical of feathers. In barb ridges, the subperiderm layer is displaced to form barbule plates and barb cells. The formation of branching barbules occurs by the presence of barb vane ridge cells that function as spacers between barbule cells. The fourth layer is homologous to the germinal layer of the epidermis, but in barb ridges it rapidly loses the germinal capability and becomes the cyclindrical layer of marginal plates. The study indicates that a necrotic process determines the carving out of the final feather shape, although apoptosis may also play a role. In fact, after barb and barbule cells have formed a keratinized syncitium, retraction of the vascular bed determines anoxia with the resultant necrosis of all feather cells. Only those of the keratinized syncitium remain to form the feather while supportive cells disappear. The sheath covering the barb and barbule syncitium is lost by the formation of a sloughing layer following degeneration of external barb ridge vane cells and loss of the sheath. It is proposed that the evolution of the morphogenetic process of barb ridge formation was peculiar to tubular outgrowths of the integument of archosaurian reptiles that evolved into birds. Once established in the embryonic programmes of skin morphogenesis of ancient birds, variations in the process of barb ridge morphogenesis allowed the fusion of ridges into large or branched ridges that originated the rachis. This process produced pennaceous feathers, among which were those later used for flight. The present study stresses that the morphogenetic process of barb ridge formation determines the concomitant appearance of barbs and barbules. As a consequence, intermediate forms of evolving feathers with only barbs but not barbules are unlikely or are derived from alteration of the above basic morphogenetic mechanism.

Keywords: feather development, feather evolution, immunocytochemistry, ultrastructure, zebrafinch

Introduction

From archosaurian ancestors birds evolved in the early middle Mesozoic Jurassic (Chiappe, 1995; Padian & Chiappe, 1998). Feathers are the most typical skin appendages of birds and show many variations in shape and dimension (Lucas & Stettenheim, 1972; McGowan, 1989; Brush, 1993, 2000; Prum, 1999; Maderson & Alibardi, 2000; Prum & Brush, 2002; Bartels, 2003). Feathers represent the most complex epidermal structure known in vertebrates, due to the precise organization of the network of barb and barbules to form the vane (Lucas & Stettenheim, 1972). Barbules, barbs, calamus and rachis are completely keratinized and dead structures, created by a particular type of resilient keratin termed beta-keratin (Gregg & Rogers, 1986; Presland et al. 1989; Sawyer et al. 2000, 2003, 2004; Sawyer & Knapp, 2003).

Feathers derive from specific cell layers of the embryonic epidermis (Sawyer et al. 2004) localized in feather tracts, after the epidermis has received an inductive action from the mesenchyme (Lucas & Stettenheim, 1972; Sengel, 1975, 1986; Chuong & Widelitz, 1999; Yu et al. 2002, 2004; Chuong et al. 2003; Sawyer et al. 2003; Widelitz et al. 2003; Dhouailly et al. 2004). Recently, it has been proposed that feathers derive from specific cell populations of the embryonic layers of the general avian epidermis (Sawyer et al. 2003, 2004). This hypothesis is based on knowledge of the epidermal layers present in embryos of extant archosaurians (crocodilians and birds), and on the expression of specific keratins. The embryonic epidermis of scales of crocodilians, avian scales, and apteric and interfollicular epidermis comprises temporary layers, destined to be lost around the time of hatching (Sawyer et al. 1986, 2000; Alibardi & Thompson, 2001, 2002; Alibardi, 2002; Sawyer & Knapp, 2003). The fate of embryonic layers is different in developing feathers (Sawyer et al. 2004).

The most external layer of the embryonic epidermis is the primary periderm, followed by the secondary periderm, the subperiderm and the germinal layer. How are these embryonic layers modified in the epidermis of developing feathers to produce the final downy (incoherent) or pennaceous (coherent) network of barb–barbules that constitute the feather? A detailed cytological study during avian ontogenesis can trace the epidermal layers at the origin of barb and barbule cells in the embryonic epidermis: this is the main topic of the present study. Although the histology and ultrastructure of the developing feather is known (Hoosker, 1936; Watterson, 1942; Goff, 1949; Matulionis, 1970; Lucas & Stettenheim, 1972; Kemp et al. 1974; Maderson & Alibardi, 2000; Chuong et al. 2003; Sawyer & Knapp, 2003), the ultrastructure of differentiating barb and barbule cells has only been recently studied (Alibardi, 2005).

In the present study the zebrafinch, a small passerine bird, has been selected as a model to study the cellular basis for feather formation. This altricial species (hatched mostly naked) possesses few downy feathers with a small number of barbs than in feathers of the chick (Alibardi, 2002). A detailed ultrastructural analysis on cells of the embryonic layers of downfeathers has been made.

The study shows the progressive transformation of cells within the barb ridge into the keratinized cells of the downfeather. Detailed knowledge of the cellular layers and of their three-dimensional aspect add to our understanding of the origin of barbs and barbules. The study is centred on the morphogenetic mechanism at the base of downfeather formation: the barb ridge. After a detailed analysis of the barb ridge into its cellular and three-dimensional structure, we proceed towards an understanding of downy or pennaceous feathers. The formation of the barb ridge is utilized to formulate speculations about how this process of feather morphogenesis evolved during the Mesozoic to generate feathers.

Materials and methods

Embryos of the zebrafinch (Taeniatopygia guttata castanotis) were collected as previously reported (Alibardi, 2002). Briefly, from fertile eggs embryos were collected at progressive days post-deposition at seasonal temperature in May–July. Fertile eggs were collected at 11–12 days (n = 3), 13–14 days (n = 3), 14–15 days (n = 2) and 16–17 days (n = 3).

Regions of the embryos including the embryonic feathers (head, neck, wing, low back) were studied. Pieces were fixed at 4 °C for 5–8 h in 2.5% glutaraldehyde, 4% paraformaldehyde in 0.12 m phosphate buffer at pH 7.2–7.4. The tissues were rinsed in the buffer for 30 min, post-fixed in 1% OsO4 for 90 min, rinsed in distilled H2O for 10–15 min, post-fixed in 4% uranyl acetate for 60–90 min, dehydrated and embedded in Durcupan resin.

The tissue containing feathers, or isolated feathers, were sectioned at 1–3 µm thickness in cross-, oblique or longitudinal sections with an ultramicrotome, and semithin sections were stained with 0.5% toluidine blue for light microscopic study. In particular, cross-sections of the apical regions (about one-fifth of the whole feather filament), intermediate regions (about four-fifths of the feather filament) and from the basal portion of the feather filament (in the follicle until about the level of exit of the feather filament from the follicle) were collected for study. Thin sections (60–90 nm thick) were collected on copper grids, stained with uranyl acetate–lead citrate, and observed in a Philips CM-100 transmission electron microscope operating at 60–80 kV.

Other embryos of 13 days (n = 2) and 15 days (n = 2) post-deposition were fixed in 4% paraformaldehyde as above, dehydrated in ethanol and embedded in Bioacryl resin under UV polymerization at 0 °C (Scala et al. 1992). From the latter tissues, 2–4-µm-thick sections were used for immunocytochemistry using a feather beta-keratin antibody (anti-FβK, see Sawyer et al. 2000). Tissues were fixed for 3–4 h in 4% paraformaldehyde in 0.1 m phosphate buffer at pH 7.5, dehydrated in ethanol and embedded in Bioacryl resin (Scala et al. 1992). Using a microtome and glass knives, 2–4-µm-thick sections were collected for immunocytochemical detection using a rabbit polyclonal antibody against feather keratin (anti-FβK; Sawyer et al. 2000). The primary antibody (1 : 200 dilution in buffer with 2% BSA and 5% NGS) was applied overnight on the sections at 4 °C (in controls the antibody was omitted). After rinsing, a secondary, fluorescein-conjugated anti-rabbit IgG (1 : 40 dilution) was applied for 1 h at room temperature, and sections were studied under an epifluorescence microscope equipped with a fluorescein filter.

For ultrastructural immunocytochemistry, thin sections were collected over nickel grids, and pre-incubated for 10 min in 0.05 m Tris buffer at pH 7.6 containing 2% bovine serum albumin and 5% normal goat serum. The sections were then incubated overnight at 0–4 °C with the feather-keratin antibody (diluted 1 : 200 with the buffer), rinsed in the buffer and incubated with the secondary antibody (10 nm gold-conjugated IgG anti-rabbit, diluted 1 : 40) for 1 h, and rinsed in buffer and distilled water. Sections were briefly stained in 2% uranyl acetate, dried and observed under the electron microscope (CM-100 Philips).

Results

Gross morphology of downfeathers

A schematic drawing illustrating the development of embryonic feathers in zebrafinch is presented in Fig. 1. The drawing shows the regions of the feathers studied, as are further illustrated in the following photomicrographs (Figs 212).

Fig. 1
Schematic drawing illustrating feather development in zebrafinch, from the germ (A) to elongating germ (B, 8–10 days post-deposition), early feather filament (C, 10–13 days post-deposition), differentiating feather filament (D, 13–15 ...
Fig. 2
Gross aspect of late embryo with embryonic feathers (A–C) and histology of early stages of feather development (see Fig. 1A,B). A, embryo at 14–15 days post-deposition, showing the locations (arrows) of embryonic feathers. Scale bar, 3 ...
Fig. 12
Ultrastructural detail of mature cells within feather filaments (Fig. 1E). A, mature barbule branching (arrows) from a barb 15–16 days post-deposition. Scale bar, 2.5 µm. B, detail of the branching point between a barbule and its barb ...

The aspect of the embryo at 14–15 days post-deposition (2–3 days before hatching) is shown in Fig. 2(A). Feather germs were present over most of pterylae areas at this stage, but they mainly remained at the germ stage. A few embryonic feather filaments, 0.5–2.5 mm in length and with a diameter at the base of 70–100 µm, were seen. Feathers were localized in the forehead near the eye, in the occipital and neck areas, in the upper part of the wing and surrounding trunk area, in the proximal trunk area near the thigh of the hind limb, and in the central mid to lower back along the vertebral column (Fig. 2B,C). Embryonic feathers maintained their diameter but lengthened further (some were longer than 3 mm) before hatching, which is around 18–19 days post-deposition in this species.

Light microscopy: early stages of barb ridge morphogenesis

The development of embryonic feathers appeared at 8–10 days post-deposition with the formation of a dome-like germ comprising a thick epithelium covering a dermal condensation (Figs 1A,B and and2D).2D). The round germ elongated into a tilted germ made of stratified epidermis at 10–13 days post-deposition (Figs 1B,C and and2E).2E). Numerous blood vessels were present among the mesenchymal condensation within the germ that elongated to form filaments (Figs 2G and and3A).3A). The epidermis of feather filaments was stratified and numerous mitoses were seen in both basal and suprabasal layers. In embryos of 13–14 days, a follicle was present (Fig. 1D). The lowermost part of the epidermal wall, in contact with the dermal papilla, comprised a single epidermal layer and a flat periderm (Fig. 3B). The epidermis became stratified, moving in upper levels of the follicle and in feather filament.

Fig. 3
Histology of regions of early feather filament (see Fig. 1C). A, longitudinal section of feather filament near its base at 13 days post-deposition. In the thick epidermis numerous dividing cells (arrows) are present. Scale bar, 20 µm. B, detail ...

Between periderm and basal layer, 5–8 layers of cells were present in apical areas of the elongated germ or feather filament in longitudinal section. The more external 2–3 layers beneath the periderm were made of flat cells that represent the differentiating sheath. Both cross- and oblique sections of the medio-apical part of the feather filament showed barb ridges of various extension (Figs 1D3–D4, ,2G2G and 3C,D) that disappeared near the basal part of the filament where the epidermis was linear. The epidermis became thinner (1–2 suprabasal layers) by the tip of the feather filament where barb ridges were absent (Fig. 3C).

Longitudinal sections of feather filaments of 12–13 days (Fig. 1D) showed piled columns of cells, more cubic in the presumptive barbs (rami) and thinner and more fusiform than those of the branching barbules (Figs 3E and 4A,B). In cross-section, most of the epidermis of feather filaments was folded in 6–8 (thinner feathers) to 9–10 (larger feathers) barb ridges (Figs 1D and and3D).3D). Epidermal cells were more regularly stratified in completely formed barb ridges than in the initial ridges present at lower levels of the filament (Fig. 1D2–D3). Each barb ridge was delimited from the mesenchyme by a layer of flat or cubic cylindrical cells of the marginal plates, and had two symmetric barbule plates (Fig. 4A). The latter comprised larger cells (presumptive barb cells) in the centre, and two lateral rows of flat cells (presumptive barbule cells). The axial plate was formed by smaller cells than barb cells in more external areas connected with barb vane ridge and sheath cells.

Fig. 4
Histology of regions of differentiating cells within barb ridges (Fig. 1D). A, detail of cross-sectioned barb ridge at 13 days post-deposition in mid level of feather filament. Scale bar, 10 µm. B, longitudinal section of mid part of feather filament ...

Light microscopy: late stages of cell differentiation within barb ridges

Both longitudinal, oblique and cross-sections of embryonic feathers at 13–15 days post-deposition showed the whole sequence of barb and barbule differentiation. Initially, barb medullary cells became paler, vacuolated and piled up to form the ramus (Figs 1D–E and 4B,C). Externally to barb medullary cells, barb cortical cells were fusiform with parallel orientation with respect to barb medullary cells (Fig. 4B,C). Barb cortical cells were in continuity with the external, fusiform barbule cells, and formed sincytial chains of cells (Fig. 4B,C). Barbule cells appeared more elongated than barb cortical cells (over 30 µm), and their chains branched from barbs.

The narrow spaces among the chains of barbule cells were occupied by pale barb vane cells, also present beneath the sheath (Fig. 4B–D). In more apical regions of feather filaments at 14 days post-deposition, barbule cells elongated to reach over 30 µm in length, and their points of fusion within the chain appeared as enlarged nodes (Figs 1D,E and 4C–E). In mid-apical regions of the feather filament, branching of barbules from centrally located barb cortical cells was clearly visible (Figs 1D and and5A5A).

Fig. 5
Histology of regions of differentiating cells within feather filaments (Fig. 1D). A, detail of branching points (arrows) of barbules from the axial barb in apical regions of feather filament at 14–15 days post-deposition. Scale bar, 10 µm. ...

Along the axial barb (the ramus), barbule chains overlapped extensively so that a barbule from a certain level climbed up over more than the next three or four more apical apical barbules. Barb vane ridge cells among chains of barbule cells were degenerating at 14–15 days post-deposition so that numerous spaces among barbules were mostly empty or contained cell debris (Figs 1D,E and and5B).5B). This was confirmed in cross-section (Fig. 5C–E).

Residual barb vane ridge cells and cylindrical cells formed septa that divided residual barb ridges from each other at 14–15 days post-deposition (Fig. 5C–E). At maturation, barb medullary cells were surrounded by cortical cells to form an axial ramus. From 6–7 to 9–10 rami surrounded by a cornified sheath were seen at this stage from the tip of feather filament down near the epidermal level of emergence of the filament (Fig. 1D). Blood vessels in the pulp were empty or absent in these regions.

At 16–17 days post-deposition, cells within each chain of barbule cells were cornified and cell boundaries were indistinct, including basal barbules fused with the ramus (Figs 1E and 6A–C). In cross-section each ramus was surrounded by large sections of basal barbule cells (those attached to the barb) and progressively thinner sections belonged to barbules originated at lower levels (apical barbules, Fig. 6D). The sheath was often broken, indicating fragility of the sheath under the effect of sectioning. The pulp showed mostly degenerated cells, and absence of blood vessels.

Fig. 6
Histology of regions of maturing feather filaments (Fig. 1E). A, detail of proximal (basal) barbule cells (arrows) branching from barb in feather at 15 days post-deposition. Scale bar, 10 µm. B, barb medulla (asterisk) and keratinized barbules ...

In the intrafollicular calamus at 16 days post-deposition, it appeared that the calamus wall was largely keratinized, the pulp did not contain blood vessels and many pycnotic cells were present. The thickness of the calamus wall was uneven, and cornified blocks formed the base of barbs (Figs 1E1 and and6E6E).

Light microscopic immunocytochemistry

The immunocytochemical study using the FBK-antibody showed that most cells of barb ridges at 13–15 days post-deposition were immunofluorescent (Fig. 7A,B). Spaces occupied by barb vane ridge cells and marginal plate cells among barb/barbule cells and the sheath were immunonegative (Fig. 7C–G). Longitudinal sections of barbule cells showed that keratin filaments were mainly packed along the main axis of cells (Fig. 7C,G). Barb cells showed most immunolabeling in the external cytoplasm surrounding the vacuolated, empty cytoplasm (Fig. 7G). The sheath was immunonegative.

Fig. 7
Immunofluorescence localization of fβk-reactive cells in embryonic feathers at 14–15 days post-deposition. A, cross-section of mid level of feather filament with labelled barb cells (arrowhead) and barbule cells (arrow). Scale bar, 10 ...

Electron microscopy and immunogold of early stages of barb ridge differentiation

Within a barb ridge at 12 days post-deposition, a thin elongation of barb vane ridge cells surrounded both single or groups of barbule cells (Figs 1C3 and 8A,B). Cylindrical cells of marginal plates contacted barb vane ridge cells in the fold beneath the sheath. Cells within the sheath, barb vane ridge cells and marginal plates appeared joined by desmosomes, but in barbule plates tight or adherens junctions were also frequently seen to join barbule cells together.

Fig. 8
Ultrastructural detail of differentiating cells within feather filaments (Fig. 1D) and scale (C). A, ultrastructural detail of cross-sectioned barb ridge at 14 days post-deposition. The arrows indicate pigment deposition. Arrowhedas indicate elongation ...

Beneath the inner periderm of the interfollicular or scale epidermis of embryos at 13–14 days post-deposition, the subperiderm layer contained small masses of FBK-immunolabelled beta-keratin (Figs 1D1 and and8C).8C). This immunoreactivity was shared with that of barb and barbule cells within feather filaments.

At 13–14 days post-deposition, beneath the stratified sheath barb and barbule cells contained long filaments of feather keratin among densely packed free ribosomes (Figs 1D and 9A,B). Sheath cells showed short filaments of alpha-keratin aggregates in some cytoplasmic areas, especially along the plasma membrane (Fig. 9B). Beneath the sheath, barb vane ridge cells were paler than barbule cells, and their elongations were also observed to extend among barbule cells located in more central areas of the feather filament. The cytoplasm of the narrow barb vane ridge cells contained a smaller number of ribosomes than barbule cells, sparse keratin filaments and occasional periderm granules (Fig. 9C–E). Numerous groups of smooth vesicles 0.05–0.2 µm in diameter were seen in the cytoplasm and cytoplasmic elongation of barb vane ridge cells (Fig. 9D,E). Elongation of barb vane ridge cells was also seen to insert between barbule cells, as was indicated by the occasional presence of periderm granules, and more frequently by the presence of smooth vesicles.

Fig. 9
Ultrastructural detail of differentiating cells within feather filaments (Fig. 1D). A, longitudinal section of apical part of feather filament at 14 days post-deposition showing outer periderm (arrowhead), sheath and pale barb vane ridge cells. Scale ...

In longitudinal section, barbule cells formed piled tubes of cells united by adherens or true tight junctions (Fig. 10A–C). Keratin bundles initially formed thin and long filaments orientated along the main axis of barbules.

Fig. 10
Ultrastructural detail of differentiating cells within feather filaments (Fig. 1D). A, more internal area of feather filament at 14 days post-deposition with parallel piled barbule cells joined by cell junctions (arrows). Arrowheads on thin keratin filaments. ...

At 15–16 days post-deposition, long keratin bundles with axial orientation enlarged to occupy progressively the cytoplasm of barb and barbules cells (Fig. 11A). Lipid droplets or extracted vacuoles were seen among keratin bundles. Keratin filaments were intensely immunoreactive using the feather keratin antibody, whereas the remaining cytoplasm and probably lipid vesicles among keratin bundles were immunonegative (Fig. 11B,C).

Fig. 11
Ultrastructural detail of differentiating cells within feather filaments (Fig. 1D). A, longitudinal section of barb cortical cells accumulating large and long keratin bundles at 15–16 days post-deposition. Scale bar, 1 µm. B, cross-section ...

Electron microscopy of late stages of barb and barbule differentiation

Degenerating barb vane ridge cells were seen among barbule cells at 14–15 days post-deposition, producing the separation between barbules (Fig. 12A–C). Barb vane ridge and cylindrical cells became vacuolated and their nucleus showed disappearance of nucleoplasm and clumping of heterochromatin (Fig. 12C). The cell membrane was discontinuous.

Barbule cells fused into compact tubes, as seen via light microscopy, and at 15–16 days post-deposition mature barbule cells were in continuity with the central and completely cornified barb (Fig. 12A). Few ribosomes remained within the darker cytoplasm localized among the majority of electron-pale feather keratin material (Fig. 12B–D). The remnants of the original plasma membrane among barbule cells after their fusion into a syncitium were seen at high magnification near the nodes, but membranes completely disappeared inside the keratin mass (Fig. 12B–D). A 90–150-nm layer of electron-dense material, different from the dense cytoplasm localized among the inner mass of keratin, was deposited along the cell perimeter: a cell membrane was not clearly seen in both barb and barbule cells.

Cell or nuclear remnants of supportive cells (barb vane ridge and cylindrical cells) were sometimes still present among barbules around 16 days post-deposition.

Discussion

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).

Fig. 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 ...

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).

Fig. 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 ...

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.

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.

Fig. 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 ...

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 and14)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 and14).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).

Acknowledgments

The study was partially supported by a UNIBO 60% grant and by self-support. Mr P. Giani and Dr M. Toni created Figs 1214.

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