Cytochemical and molecular characteristics of the process of cornification during feather morphogenesis

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Abstract

Feathers are the most complex epidermal derivatives among vertebrates. The present review deals with the origin of feathers from archosaurian reptiles, the cellular and molecular aspects of feather morphogenesis, and focus on the synthesis of keratins and associated proteins. Feathers consist of different proteins among which exists a specialized group of small proteins called beta-keratins. Genes encoding these proteins in the chick genome are distributed in different chromosomes, and most genes encode for feather keratins. The latter are here recognized as proteins associated with the keratins of intermediate filaments, and functionally correspond to keratin-associated proteins of hairs, nails and horns in mammals. These small proteins possess unique properties, including resistance and scarce elasticity, and were inherited and modified in feathers from ancestral proteins present in the scales of archosaurian progenitors of birds. The proteins share a common structural motif, the core box, which was present in the proteins of the reptilian ancestors of birds. The core box allows the formation of filaments with a different molecular mechanism of polymerization from that of alpha-keratins. Feathers evolved after the establishment of a special morphogenetic mechanism gave rise to barb ridges. During development, the epidermal layers of feathers fold to produce barb ridges that produce the ramified structure of feathers. Among barb ridge cells, those of barb and barbules initially accumulate small amounts of alpha-keratins that are rapidly replaced by a small protein indicated as “feather keratin”. This 10 kDa protein becomes the predominant form of corneous material of feathers. The main characteristics of feather keratins, their gene organization and biosynthesis are similar to those of their reptilian ancestors. Feather keratins allow elongation of feather cells among supportive cells that later degenerate and leave the ramified microstructure of barbs. In downfeathers, barbs are initially independent and form plumulaceous feathers that rest inside a follicle. Stem cells remain in the follicle and are responsible for the regeneration of pennaceous feathers. New barb ridges are produced and they merge to produce a rachis and a flat vane. The modulation of the growth pattern of barb ridges and their fusion into a rachis give rise to a broad variety of feather types, including asymmetric feathers for flight. Feather morphogenesis suggests possible stages for feather evolution and diversification from hair-like outgrowths of the skin found in fossils of pro-avian archosaurians.

Introduction

Among extant vertebrates, special keratinaceous appendages, namely the feathers, characterize the class of a specialized subclass of archosaurian reptiles, the birds (Padian and Chiappe, 1998; Colbert et al., 2001). The evolution of these archosaurs was characterized by a summation of specialized adaptations that made their body light and well-adapted to be lifted in the air for an active flight. Among these anatomical adaptations are the extremely hollowed bones, the specialized skeleton for flight, take-off and landing, the loss of teeth, the tiny branching of air sac to make bones pneumatics, the highly efficient lungs, and the integument with feathers. Besides skeletrical features, the skin of birds reveals a reptilian origin, in particular the presence of scales in the tarsal–metatarsal region where different types have been described, namely scutate, scutellate, and reticulate (Lucas and Stettenheim, 1972; Spearman and Hardy, 1985; Sawyer et al., 1986).

Feathers represent the most peculiar derivatives of avian skin, probably as an evolutionary derivation from scales and keratins of reptilian ancestors (Fig. 1; see Landmann, 1986). During embryogenesis, feathers derive from a pimp-like evagination of the embryonic skin, the feather germ, which elongates to produce hair-like feather filaments (Matulionis, 1970; Chuong and Widelitz, 1999). This results in the most complex epidermal derivative ever found in vertebrates. Feathers consist of an ordinate collection of minute branching ramifications indicated as barbs, which remains separated in plumulaceous feathers or merged with a central rachis in pennaceous feathers (Figs. 1A (e–f) and 2). Each barb comprises a central axis, indicated as the ramus, and lateral branching projections indicated as barbules. No follicle is present during the initial stages of feather morphogenesis when the feather filament elongates and the first cell differentiation occurs from their apex to more ventral regions. The linear epidermis of feather filaments initiates to fold inward into barb ridges, starting from the more apical part of feather filaments and moving down. The ramification of feathers derives from the geometrical differentiation of barb and barbule cells within each barb ridge that produces barbs. The first feathers formed in the embryo are termed downfeathers, a type of plumulaceous feathers that lack an axial structure (the rachis) or possess a very short rachis. At a later stage of feather development, the basal part of a downfeather sinks into the skin together with the surrounding epidermis and forms a ring-like groove termed follicle. The latter progressively deepens into the dermis and becomes deeply embedded in the dermis by the end of the morphogenesis of downfeathers. Stem cells present in the basal part of the feather filaments remain inside the follicle, and from this structure a new feather is later regenerated to replace the initial downfeather. Regenerating feathers can assume very large dimensions, shapes and patterns of pigmentation (Bartels, 2003).

At the end of their differentiation, feathers consist of a regular network of dichotomically branched barbs and barbules that produce a plumulaceous or pennaceous vane (Lucas and Stettenheim, 1972; Brush, 1993; Prum and Brush, 2002; Bartels, 2003). Barb and barbule cells are corneocytes containing a resistant type of keratin, termed feather keratin (Gregg and Rogers, 1986; Brush, 1993; Sawyer et al., 2000, Sawyer et al., 2005a). These skin appendages are derived from inductive mesenchymal–epidermal interactions in the embryonic skin that produced feather germs (Chuong, 1993; Chuong and Widelitz, 1999; Chuong et al., 2003; Wu et al., 2004). Germs elongate into feather filaments in which some cells keratinize by the accumulation of numerous proteins indicated as feather keratins (Matulionis, 1970; Sawyer et al., 2003, Sawyer et al., 2005b). Feather keratins have been classically included within a more general class of small proteins also present in reptiles, and indicated as beta- or phi-keratins as they form filaments and bundles that recall those of alpha-keratins in epidermal cells (Gregg and Rogers, 1986; Brush, 1993; Sawyer et al., 2000). These proteins contain beta-pleated sheets in some regions of their molecule, give an X-ray pattern of β-type, and are capable of forming filaments (Baden and Maderson, 1970; Fraser and Parry, 1996). Recent studies, while confirming the presence of regions with a type of beta-pleated sheet, distinguish these small proteins from true keratins (Alibardi, 2006c; Alibardi and Toni, 2006a, Alibardi and Toni, 2006b, Alibardi and Toni, 2006c, Alibardi and Toni, 2006d, Alibardi and Toni, 2006e; Alibardi et al., 2007; Toni et al., 2007a, Toni et al., 2007b; Fig. 1).

The modern classification of all vertebrate corneous proteins comprises two categories. The first types are filamentous proteins of 40–70 kDa (alpha-keratins or intermediate-filament proteins, often called cytokeratins, see Fuchs et al., 1987; Steinert and Freedberg, 1991; Powell and Rogers, 1994; Coulombe and Omary, 2002). The second type are inter-keratin or keratin-associated proteins (KAPs) of 6–40 kDa (Resing and Dale, 1991; Powell and Rogers, 1994; Rogers et al., 2006). The latter are smaller proteins that form an amorphous matrix around alpha-keratins or may replace most of alpha-keratin to form a dense corneous material.

Different from the mammalian hard corneous material of hairs, nails, and horns, where keratin-associated proteins are mainly amorphous, in reptilian and avian keratinocytes of scales and feathers matrix proteins, indicated as beta- or phi-keratins, form fibrous polymers and filaments (Fraser et al., 1972; Brush, 1983). The latter, however, are very different from intermediate filament proteins in composition, chemical–physical properties, and in the mechanism of polymerization. According to the above classification, small proteins previously indicated as beta-keratins (fibrous proteins with some regions with a beta-pleated conformation) should be considered as a fibrous type of “keratin-associated or matrix proteins” (sauropsid KAPs, Alibardi, 2007b, Alibardi, 2007f; Toni et al., 2007a, Toni et al., 2007b). It is believed that, during evolution from stem reptiles, one evolutive lineage of amniotes leading to mammals (the theropsids) produced non-fibrous keratin-associated proteins, here indicated as mammalian KAPs (Powell and Rogers, 1994; Rogers et al., 2006). The other lineage of amniotes, the sauropsids, leading to modern reptiles and birds, instead produced fibrous keratin-associated proteins (the “beta-keratins”). In particular in the lineage of archosaurian reptiles from which birds were originated, a small type of fibrous protein, indicated as feather keratin, was produced and its characteristics permitted the formation of barbs and feathers.

It is important to realize that, besides the specific proteins, feathers originated only after the evolution of a special morphogenetic process. The latter transformed some folds of the embryonic or adult epidermis of feather germs into barb ridges (Fig. 2). Barb ridges are formed within feather filaments and within barb ridges special chains of cells are assembled to form barbules and barbs. Despite previous morphological (Lucas and Stettenheim, 1972) and ultrastructural studies (Matulionis, 1970; Kemp et al., 1974; Bowers and Brumbaugh, 1978; Alibardi, 2002), details on the formation of barb and barbule cells have only been recently presented (Alibardi, 2005a, Alibardi, 2005c, Alibardi, 2006a, Alibardi, 2006c; Alibardi and Sawyer, 2006; Alibardi, 2007b, Alibardi, 2007f). In particular, the latter studies have stressed the role of supportive cells within barb ridges. Supportive cells do not accumulate keratins but mainly lipids and their following degeneration allows the emergence of barb ramification. Detailed knowledge of all different cell types present in developing feathers is necessary to understand the origin of the three-dimensional organization of barb and barbule cells. This in turns allows one to fully appreciate how variations in the three-dimensional disposition of feather cells can change the branching pattern of barbs, thereby producing different types of barbs and consequently feathers.

The present review summarizes the information on the fine structure of developing and regenerating feathers in relation to their molecular composition and localization of alpha- and beta-keratins. The complex inductive and signaling molecules involved in the morphogenesis and regeneration of feathers will not be dealt in the present review (see Harris et al., 2002; Chuong et al., 2003; Widelitz et al., 2003). The review stresses the similarity of avian keratins with those of other reptiles, indicated as beta-keratins, that represent a multi-gene family of small proteins originated in stem reptiles from which modern reptiles and birds evolved. The study confirms morphological data, indicating that feather keratins were derived from epidermal keratins of pro-avian reptiles (Maderson, 1972; Maderson and Alibardi, 2000). During feather evolution a small beta-keratin, suited better than previous proteins, was selected and allowed the elongation of feather cells to form a branching pattern at the origin of barbs.

Among archosaurian reptiles, which were flourishing in the Mesozoic, today only crocodilians and birds remain (Colbert et al., 2001; Pough et al., 2001). Members of these two groups have some basic characteristics (initial diapsid skull, four heart chambers, initial procoelous vertebrae, double blood circulation, hollowed bones, scales containing beta-keratin, telolecytic development, mesobronch-based lungs, egg, and parental care, etc.). However, they also differ in many other characteristics such as habits, shape, movements, heterothermy versus homeothermy, feathers, flight, air sacs, and air lung circulation, etc. In fact, while birds fly, are light, bipedal, and covered by feathers, crocodilians appear strongly scaled, heavy and streamlined dwellers.

The skin in the two groups is apparently very different, being heavily scaled in crocodilians versus the delicate, smooth, and feathered skin of birds. Only the tarsal–metatarsal region is scaled in birds, and this characteristic is considered a reminiscence of their reptilian ancestors. In fact, the available morphological and biochemical data indicate that avian scales are the closest to those of reptiles, and of crocodilians in particular (Brush and Wyld, 1980; Alibardi and Toni, 2007b). Avian and crocodilian scales not only have similar shape but also their keratins and gene organization is very similar (Brush, 1993; Sawyer et al., 2000; Alibardi and Toni, 2007b; Dalla Valle et al., 2007c). Some hypotheses and experimental studies indicate that scales of extant birds derived from the inhibition of feather morphogenesis in the tarsal–metatarsal region of legs (see summaries in Lucas and Stettenheim, 1972; Prin and Dhouailly, 2004). The presence of beta-keratins and morphogens such as Sonic Hedge Hog (Shh), Wingless-integrated (Wnt) and Bone Morphogenetic Protein 4 (BMP4), in reptilian and avian scales (Chuong, 1993; Harris et al., 2002, Harris et al., 2005; Chuong et al., 2001) has further suggested that scales and feathers of birds derived from pro-avian archosaurian reptiles (Maderson, 1972; Brush, 1993; Maderson and Alibardi, 2000).

During embryogenesis, scales derived from the interaction between a localized inductive mesenchyme and a large surface of the epidermis. It has been hypothesized that these areas of dermal–epidermal interactions (ADEIs) were relatively large in pre-avian archosaurians (Alibardi, 2004, Alibardi, 2005c; Fig. 1A (a)). Reptilian scales were either of the scute-like type as in extant crocodilians or appeared as tuberculate or pebble-like scales as those described in fossilized archosaurians, including dinosaurs (Martin and Czerkas, 2000). During the evolution of the avian skin (which became lighter), the rigid corneous layer of archosaurian scales became progressively restricted to smaller and smaller ADEIs while most of the skin became non-specialized, softer, and gave rise to inter-follicular and apteric skin. The flat scale in archosaurian ancestors became progressively narrower, tuberculate, or hair-like, and the epidermis was nourished from a vascularized mesenchyme. The latter also exchanged regulative or inductive signals with the epidermis, and this activity determined the formation of specialized layers of cells in both scales and feathers (Alibardi, 2004). The mesenchyme of expanded ADEIs, in scales, maintained the links with the epidermis and, as the ADEI reduced, the mesenchyme became restricted to dermal condensations and later formed the dermal papilla (Fig. 1A (b, c)). During this hypothetical evolution, dermal cells of the unspecialized dermis came to occupy larger and larger areas of the skin indicated as apterii. The small-scaled area that remained, further elongated into a hair-like structure filled with mesenchyme, a condition that resembles that of developing feather filaments (Fig. 1A (d)). The small, coniform appendages eventually became hollow and could be homologous to present-day feather filaments. Finally, when folds appeared in the epidermis of these hypothesized filaments, the formation of true feathers began (see further detail in the last section on feather evolution) (Fig. 1A (e, f)).

The above hypothetical stages are supported by morphological and biochemical data. Reptilian and avian scales, like feathers, are mainly composed of beta-keratins, and scales can produce feathers under some natural or experimental circumstances (Chuong and Widelitz, 1999; Sawyer et al., 2005a, Sawyer et al., 2005b). Also, early feather germs under treatment with retinoic acid are turned into scales (Chuong, 1993). It is still believed that feathers might be derived from archosaurian scales, probably by a drastic alteration of the morphogenetic pattern of dermo-epidermal interactions (Lucas and Stettenheim, 1972; Maderson, 1972; Regal, 1975; Maderson and Alibardi, 2000). Recent studies on the embryonic epidermis of alligator and birds have produced further indication that feathers derive from a generalized archosaurian epidermis (Alibardi, 2003; Sawyer et al., 2003, Sawyer et al., 2005b; Alibardi, 2006b; Alibardi et al., 2006b).

During development, different cell populations with a specific fate are produced in sequence in the epidermis (Sawyer et al., 2000, Sawyer et al., 2003; Sawyer and Knapp, 2003). These studies have suggested that specific cell populations were selected from the generalized archosaurian epidermis to produce specific epidermal structures such as scales, beaks, claws, and feathers. In particular, some cell populations containing a specific type of beta-keratin (feather keratin) were selected among others to produce barb and barbule cells of feathers.

As in reptilian embryonic epidermis, also in the embryonic epidermis of birds, an outer and an inner periderm layer are initially formed (Fig. 2). The number of layers of the inner periderm varies according to different regions of the body. Over most of the body, especially in inter-follicular, apteric, and scale skin only one layer of inner periderm cells is present (Sawyer and Borg, 1979; Sawyer et al., 1986). In the claw 2–4 layers of inner periderm cells are instead present while they increase to 6–8 in the beak (Kingsbury et al., 1953; Alibardi, 2002). Beneath the inner periderm one subperiderm layer is present in scales, and probably in claw and beak epidermis, which contains both alpha-keratin and feather-keratin immunoreactivity (Fig. 3, Fig. 4). In a recent hypothesis (Sawyer et al., 2003, Sawyer et al., 2005b), it is suggested that downfeathers derive from the expansion of cells of the subperiderm. In downfeathers, the origin of barb ridges determines a re-organization of cells of the subperiderm, which are displaced into barbule plates and a central ramus that give rise to barbs (Fig. 2). These studies have found that the subperiderm layer in all archosaurian epidermis contains feather or a feather-like keratin that disappears at hatching except in feathers. Therefore this protein or its epitope recognized by the FBK antibody may be an ancient component of the epidermis of archosaurian reptiles.

Detailed ultrastructural and immunocytochemical analysis using the A1 antibody against general alpha-keratins of the chick, and the specific FBK antibody, which recognizes a feather-specific antigen of 24 amino acids (AVGSTTSAAVGSILSEEGVPINSG), have confirmed this hypothesis on alligator and avian epidermis (Alibardi, 2006b; Alibardi, 2006a; Fig. 3). In crocodilians the sequence of embryonic layers of the epidermis resembles that of avian embryonic epidermis, in particular the presence of a subperiderm layer that contains feather or feather-like keratin.

The A1 antibody, against a chick alpha-keratin isolated from scales (O’Guin and Sawyer, 1982; Knapp et al., 1993), shows some labeling associated with periderm granules in the outer and inner periderm, but not with the diffuse alpha-keratin filaments present in these cells (Fig. 3A). The labeling becomes intense over the numerous beta-keratin packets formed in cells of the subperiderm layer (Fig. 3B). A similar pattern of immunolabeling is seen using the beta-1 (rabbit polyclonal serum against an avian beta-keratin, data not shown) or the feather-keratin antibody (Fig. 4A, B). Immunopositive keratin filaments are randomly packed within the subperiderm, forming a tangled mass of corneous material.

The distribution of feather keratin is observed in both the outer and inner periderm layers, suggesting that the synthesis of this type of keratin is precocious in archosaurian epidermis (Sawyer and Knapp, 2003). This immunolabeling indicates that some little, feather-like, beta-keratin accumulates among a loose keratin network (made by 3–5 nm-thick filaments) in the outer periderm. In the inner periderm, the labeling is mainly associated with sparse keratin filaments or with coarse filaments. These organelles resemble periderm granules of avian epidermis (Matulionis, 1970; Sawyer and Borg, 1979; Alibardi, 2002), and it is possible they are homologous or have similar roles in the epidermis of other archosaurians. It is possible that coarse filaments surround thinner beta-keratin filaments or even produce the initial aggregation of beta-keratin molecules into filaments. Other proteins associated with keratin are probably present in these organelles, which disappear in mature cells, before the embryonic layers are sloughed at hatching. Recent ultrastructural autoradiographic studies have however indicated that periderm granules do not contain histidine-rich molecules (Alibardi, 2006b, Alibardi, 2006d). These granules appear in sheath and supporting cells of feathers that are destined to degenerate, but their specific role remains unsolved.

Since birds are phylogenetically related to archosaurian reptiles, their corneous proteins probably still conserve some amino acid sequences also present in reptilian proteins. The composition of corneous structures (scales, claws, ramphotheca, etc.) in reptilian epidermis was unknown until recently. Some information on the percentage of amino acids and molecular weight were available for some reptilian species (Wyld and Brush, 1979, Wyld and Brush, 1983; Thorpe and Giddings, 1981; Sawyer et al., 2000, Sawyer et al., 2005a; Alibardi and Toni, 2005a, Alibardi and Toni, 2005b; Toni and Alibardi, 2007a, Toni and Alibardi, 2007b, Toni and Alibardi, 2007c; Toni et al., 2007a, Toni et al., 2007b). No alpha-keratins were sequenced, and only a small keratin from a lizard claw was known in its primary amino acid structure (Inglis et al., 1987). However, it was unclear whether this protein was representative of reptilian scales or merely reflected the composition of a specialized, claw keratin.

Different proportions of specific monomers of beta-keratins were believed to produce scales with different rigidity, or concentrate in claw and ramphoteca (beak) (Brush and Wyld, 1980; Wyld and Brush, 1979, 1983). Beta-keratins have small molecular weight, mainly in the 14–20 kDa range, but components at higher molecular weight were also found (30–44 kDa), although in minor amount. Different forms (monomers) are present in diverse derivatives such as dorsal and ventral scales, shell scutes, claws, and ramphotheca in turtles, lizards, snakes, and crocodilians. The variability of beta-keratin monomers was also supported by other biochemical studies (Thorpe and Giddings, 1981; Gillespie et al., 1982; Marshall and Gillespie, 1982; Inglis et al., 1987). According to Wyld and Brush (1983), beta-keratins of reptiles were believed to be phylogenetically distant from beta-keratins of birds, and in particular unrelated to feather keratins (Brush, 1993). The latter study in particular pointed out that feather keratins are a completely new innovation of avian skin, related to the origin of feathers in birds.

Other data instead indicated that reptilian beta-keratins are relatively few in each species and in different skin appendages (Sawyer et al., 1986, Sawyer et al., 2000, Sawyer et al., 2005a). These beta-keratins, especially in archosaurians, were distinguishable into few main types: scale, beak, claw, feather, or feather-like keratins (Gregg and Rogers, 1986; Sawyer et al., 2000; Sawyer and Knapp, 2003). These proteins probably vary from species to species in their amino acidic sequences, and those in archosaurian skin (crocodilians) were strictly correlated with those of birds. Scale, claw, and beak keratins showed a relatively higher molecular weight, 14–18 kDa, while feather or feather-like keratins possessed a smaller molecular weight, 10–12 kDa (Gregg and Rogers, 1986).

In corneous structures of reptiles and birds it is thought that beta-keratins alone can originate both the fibrous component and matrix component of their hard corneous material. Therefore it was believed that a different mechanism of forming hard skin derivatives was present in reptiles and birds with respect to that of mammals (Gillespie, 1991; Brush, 1993). The recent proteomic and genomic studies of the reptilian beta-keratins have clarified this issue and suggested a new interpretation that unifies the mechanism of cornification in all amniotes (Alibardi and Toni, 2006a; Alibardi et al., 2007; Toni et al., 2007a, Toni et al., 2007b).

The determination of the amino acid sequence for beta-keratins from lizards, snake, turtle, and crocodile, together with that of a lizard claw (Inglis et al., 1987), has indicated that some regions of these proteins present high homology with avian keratins (Dalla Valle et al., 2005, Dalla Valle et al., 2007a, Dalla Valle et al., 2007b, Dalla Valle L et al., 2007c, and unpublished). Using different computer programs on the deduced amino acid sequences of these proteins, their predicted secondary structure can be shown graphically (Fig. 1). The predicted secondary structure of some representative beta-keratins in lizard and snake (lepidosaurian reptiles), and turtle has evidenced the presence of a central region (core box) where two close strand regions (beta-sheets) are present. The turtle proteins resemble those of crocodiles more than those of lizards and snakes. In turn, crocodilian proteins are more similar to avian scale, claw, and feather keratins than proteins from lepidosaurian and chelonian reptiles. This recent, molecular biology information (Dalla Valle et al., 2007c) supports the close relationships between crocodilians and birds, as will be discussed later (see Section 3.2).

The structure of genes so far sequenced coding for beta-keratins in sauropsids (reptiles and birds) shows two types of nucleotide organization, one without introns and the other with one intron in the 5′ non-coding region (Fig. 1). The first type, so far evidenced only in some lizards, presents a long, uninterrupted 5′ non-coding region, a coding region, and a 3′ variably long non-coding region. The messengers derived from this type of genes have a typical polyadenylation signal, and a downstream polyadenilated sequence. The second type, found in snake, crocodile, and turtle, differs in the presence of two 5′ non-coding regions between which an intron is present, followed by a coding region, and a variably long 3′ non-coding region. In the messenger of the latter, a typical polyadenylation signal is present. As will be later presented, these basic features (multiple copies of linearly arranged short genes) have been also evolved in the specialized lineage of archosaurian reptiles that gave rise to birds.

The central region (core box) is also present in beta-keratins of all archosaurian reptiles so far sequenced (see later). The predictions of the secondary structure of these proteins show some of the structural homologies of reptilian beta-keratins with beta-keratin of birds, especially in a central region, the core box (Dalla Valle et al., 2007b, Dalla Valle L et al., 2007c; Alibardi and Toni, 2007a). The latter, which contains at least two beta-strands (beta-pleated sheets) in secondary conformation, is considered the site of polymerization of beta-keratin monomers into the long polymer that forms the filament of beta-keratin with the diameter of 3–4 nm (Brush, 1983; Gregg and Rogers, 1986; Fraser and Parry, 1996). Also the nucleotide structure presents in some of the beta-keratins cDNAs so far sequenced from reptilian epidermis show a general linear organization that resembles the avian nucleotide structure for their scale, claw, and feather genes (Dalla Valle et al., 2007a, Dalla Valle et al., 2007b, Dalla Valle L et al., 2007c).

From the above description and the recent data on the gene organization of crocodilian beta-keratins it is likely that these proteins derived from a genomic organization of this family of genes present in archosaurians that pre-dated the origin of birds. The latter inherited this basic organization and evolved their own specific sequences for the formation of scale and feathers keratins (see later description).

The above molecular data further indicate that reptilian and avian scales have a common evolutionary history. Recent molecular observations (Prin and Dhouailly, 2004) indicate that avian scales (and their keratins) develop when the basic feather morphogenetic program is altered. This has been interpreted as feathers represent the primitive appendages for birds and that scales derive from the inhibition of the basic feather program. However, the recent molecular data (Dalla Valle et al., 2007c) have shown that crocodilian beta-keratins and their genes have a very close structure and organization to avian scale keratin, and these studies re-confirm that scales are more primitive than feathers as scaled reptiles preceded feathered reptiles.

Section snippets

Development and regeneration of feathers

The histology of feather morphogenesis, as described by the first microscopists, is known with different degrees of details (Davies, 1889; Hoosker, 1936; Lillie, 1942; Watterson, 1942; Koning and Hemilton, 1954; Rawles, 1960; Espinasse, 1964; Lucas and Stettenheim, 1972; Bragulla and Hirschberg, 2003). More recent accounts on feathers developments have not found further microscopic details to simplify the understanding of feather morphogenesis, which remains very complex (Chuong and Widelitz,

Cytochemistry and biochemistry of feather corneification

The studies on the biochemical and cytochemical variations in composition in developing feathers indicated that both soft keratinization (based on alpha-keratin of intermediate filaments) and hard keratinization (based on small beta-keratin or sKAPs) occur during development and regeneration. Early studies showed that sulfhydryl groups, indicating newly synthesized unpolymerized keratin molecules, become abundant in feather filaments between the 13 and 14 day of embryonic development in the

Genes, proteins and feather evolution

The biochemical study on proteins isolated from different feathers and species of birds has shown that a different composition is present among calamus, rachis and vane (Fraser et al., 1972; Brush, 1993; King and Murphy, 1987). A detailed molecular biology analysis on beta-keratin genes in birds is not meant in this review, but feather keratin genes are presently under detailed studies analyzing nucleotide sequences from the database of the chick genome (Dalla Valle, personal communication).

Conclusive remarks and future directions

The present account has shown the relation of the more specialized and complex skin derivative, the feather, with epidermal layers of the generalized archosaurian epidermis. The study wants to draw attention to the fact that cornification of feathers follows the general scheme valid for amniotes: fibrous proteins (alpha-keratins) rapidly associate and are replaced by fibrous KAPs indicated as beta-keratins. Future studies on feather biology will try to know the regulation of gene expression for

Acknowledgments

The study was supported by a University of Bologna Grant (60%), by a FIRB Grant to Prof. V. Tomasi (University of Bologna, Italy, 2004) and by self-support (LA). Dr. L. Dalla Valle and her team at the University of Padua have collaborated on the molecular study on reptilian and avian keratins. Drs. RH Sawyer and LW Knapp (University of South Carolina, Columbia, USA) kindly provided the beta-1, FBK the and A1 antibodies. Nicodemo Mele (University of Bologna) made some drawings using the Adobe

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