Horse Hooves and Bird Feathers: Two ModelSystems for Studying the Structure andDevelopment of Highly Adapted IntegumentaryAccessory OrgansFThe Role of the Dermo-Epidermal Interface for the Micro-Architectureof Complex Epidermal Structures

HERMANN BRAGULLAn and RUTH M. HIRSCHBERGDepartment of Veterinary Anatomy, Freie Universitat Berlin,D-14195 Berlin, Germany

ABSTRACT Accessory organs of the integument are locally modified parts of the potentiallyfeather-bearing skin in birds (e.g., the rhamphotheca, claws, or scales), and of the potentially hairyskin in mammals (e.g., the rhinarium, nails, claws, or hooves). These special parts of the integumentare characterised by a modified structure of their epidermal, dermal and subcutaneous layers. Thedevelopmental processes of these various integumentary structures in birds and mammals showboth similarities and differences. For example, the development of the specialised epidermalstructures of both feathers and the hoof capsule is influenced by the local three-dimensionalconfiguration of the dermis. However, in feathers, in contrast to hooves, the arrangement of thecorneous cells is only partially a direct result of the particular arrangement and shape of the dermalsurface of the papillary body. Whereas the diameter of the feather papilla, as well as the number,length, and width of dermal ridges on the surface of the feather papilla influence the three-dimensional architecture of the feather rami, there is no apparent direct correlation between thedermo-epidermal interface and the development of the highly ordered architecture of the radii andhamuli in the feather vane. In order to elucidate this morphogenic problem and the problem oflocally different processes of keratinisation and cornification, the structure and development offeathers in birds are compared to those of the hoof capsule in horses. The equine hoof is the mostcomplex mammalian integumentary structure, which is determined directly by the dermal surface ofthe papillary body. Perspectives for further research on the development of modified integumentarystructures, such as the role of the dermal microangioarchitecture and the selective adhesion andvarious differentiation pathways of epidermal cells, are discussed. J. Exp. Zool. (Mol. Dev. Evol.)298B:140151, 2003. r 2003 Wiley-Liss, Inc.

INTRODUCTION

The integument is a complex organ whichmediates between an organism and its environ-ment. Depending on the environmental conditionsand the animals habits, the integument has tofulfil a multitude of functions (Starck, 82;Homberger, 2001). In birds and mammals, themost obvious roles of the skin are protection fromharmful environmental influences and thermore-gulation, which are achieved by various degrees ofcornification or pigmentation and through thedevelopment of specialised integumentary acces-sory organs (i.e., skin appendages), such as

feathers or hairs, that cover most parts of thebody. Specific localised skin modifications enableparticular functions. For example, the modifiedmicro-anatomy of the oral or nasal openings (e.g.,avian bills, mammalian lips, and rhinarium)facilitate specific feeding habits, and differentforms of digital end organs (e.g., claws, nails,hooves) enable specific locomotory modes, such as

nCorrespondence to: Dr. Hermann Bragulla, Department ofVeterinary Anatomy, Freie Universitat Berlin, Koserstrabe 20,D-14195 Berlin, Germany.

E-mail: bragull2@zedat.fu-berlin.dePublished online in Wiley InterScience (www.interscience.wiley.

com). DOI: 10.1002/jez.b.00031

r 2003 WILEY-LISS, INC.

JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 298B:140151 (2003)

plantigrade, digitigrade or unguligrade weight-bearing stances (Starck, 82; Homberger, 2001).The epidermis as outermost layer of the integu-

ment is particularly adapted to withstand variousmechanical forces and, hence, produces a mechani-cally resistant cornified superficial layer. Thecorneous layer can contain either soft or hardhorn, resulting from two different keratinisationproceduresFwith (soft) or without (hard) forma-tion of a granular layer. Integumentary structuresexposed to high local mechanical stress undergohard keratinisation (for example the nail plate).Whereas in mammals, only a-keratins are pro-duced in the keratinising layers of the epidermis,in reptiles and birds b-keratins prevail in hardhorn structures (Lucas and Stettenheim, 72;Alibardi, 2002; Sawyer et al., 2003).The functional diversity of the skin is, therefore,

complemented by a structural diversity dependingon its biomechanical needs at the specific localisa-tion on the surface of the body. Nevertheless, allintegumentary structures in birds and mammalsshare a common basic construction (Lucas andStettenheim, 72; Junqueira et al., 92). The skinconsists of three distinct tissue layers: (1) theepidermis, a stratified keratinised and oftencornifying epithelium; (2) the dermis, a predomi-nantly collagenous connective tissue layer withdense vascular and nervous networks; and (3) thesubcutis, a more loosely arranged connectivetissue layer that may contain adipose tissue.Generally, the skin covering most parts of thebody contains particular skin-appendages, such ashairs and associated glands in mammals, orfeathers in birds. Depending on the species andthe location on the body, one, two or all threelayers of the integument may exhibit structuralpeculiarities that are characterised as skin mod-ifications, such as beaks, scales, claws, or hooves(Budras et al., 2002).Formation of the various integumentary acces-

sory organs starts before the terminal differentia-tion and cornification of the embryonic epidermis,that is, when the intermediate layer beneath theperiderm has formed (reviewed by Byrne et al.,2003). The epithelial-mesenchymal interactionsinvolved in the patterning of distal integumentaryappendages, such as the digital end organs, appearto be similar to those observed in the patterning ofother vertebrate integumentary structures, suchas feathers, hairs, and scales (reviewed by Ham-rick, 2001). Early factors regulating skin appen-dage formation in birds and mammals belong tothe Fibroblast Growth Factor (FGF) and Bone

Morphogenic Protein (BMP) families, and otherfactors, such as sonic hedgehog (ssh), Msx1, Msx2,Wnt or Eda, also seem to play important roles inthis process (Widelitz et al., 99; Millar et al., 99;reviewed by Hamrick, 2001; and Byrne et al.,2003). The final shape and internal structure ofthe integumentary accessory organs are largelydefined by the configuration of the dermo-epider-mal interface (i.e., the papillary body), whichdetermines the three-dimensional arrangementof the overlying epidermal cells (Bragulla, 96;Homberger, 2001; Bragulla and Hirschberg, 2002).Thus, this interface has a great influence on themechanical properties of corneous structures,such as surface smoothness or roughness, plia-bility, resilience, resistance to abrasion, or trans-missibility of mechanical stimuli (Nickel, 38;Homberger and Brush, 86; Pellmann et al., 93;Bonser and Witter, 96; Bragulla, 96; Budras andSchiel, 96; Bragulla, 98; Budras et al., 98; Patanand Budras, 2003). The effect of the three-dimensional configuration of the dermo-epidermalinterface on corneous structures is further en-hanced because the supply of the avascularepidermis is completely dependent on the dermalmicrovasculature for nutrients and for the dis-tribution of growth factors and cytokines perdiffusionem for systemic regulatory feedbackmechanisms (Imayama, 81; Hirschberg et al.,99; Hirschberg et al., 2001). The development ofthe dermo-epidermal interface, mutual influenceof the epidermis and dermis, and dermal micro-vasculature during the development of specialisedintegumentary structures are, therefore, of greatinterest, especially from a comparative point ofview.

Equine hooves (Figs. 1, 2), the most complex andspecialised form of digital end organs, and avianfeathers (Figs. 3, 4), the most complex andspecialised form of skin appendages, are amongthe best studied integumentary structures. Be-cause sound and healthy hooves are vital for aproper performance for both recreational andprofessional horse riding, the structure of thehorse hoof (reviewed by Pellmann, 95; Bragulla,96; and Henke, 97) is a main focus for bothclinical and basic research groups. The evolutionand origin of the feather has become a centralquestion in biology, and the original primaryfunctions of early feathers are being vigorouslydebated (Prum, 99; Brush, 2000; Bock, 2000;Homberger and de Silva, 2000; Tarsitano et al.,2000; Xu et al., 2001; Prum and Brush,2002; Sawyer et al., 2003). Knowledge of the

INTEGUMENTARY ACCESSORY ORGANS IN BIRDS AND MAMMALS 141

development of the feather, particularly of thegenes, growth factors, or signalling moleculesinvolved in this process, could provide new in-sights for an evolutionary model of this skin

appendage. While much is already known aboutfeather development, in particular about theformation of the feather shaft (rachis) and barbs(rami) (Lucas and Stettenheim, 72; Chuong and

H. BRAGULLA AND R.M. HIRSCHBERG142

Edelmann, 85 a, b), little is understood about theprocess of forming the highly complex epidermalstructures of the feather barbules (radii) andbarbicels (hamuli).Although at first sight there are great differ-

ences between hooves and feathersFstructuresthat are neither homologous, nor analogous, norconvergentFthere are also some striking simila-rities in the structure of these integumentaryorgans. On the one hand, the equine hoof consistspredominantly of tubular horn growing from agerminative layer that is shaped by a complex andregionally variable dermal papillary body, parti-cularly in those regions of the hoof that aredesigned to absorb compressive forces during theweight-bearing phase of locomotion. The feather,on the other hand, can also be considered as aspecial case of (modified) tubular horn (Prum, 99;Homberger, 2001). Both, feather and hoof, initi-ally develop from a plane dermo-epidermal anlageand display very complex interdigitating dermo-epidermal branching structures during their de-velopment (Lucas and Stettenheim, 72; Hamrick,2001; Bragulla, 2003).In this study, we focused, therefore, on the

development and structure of the dermo-epider-mal interface of the equine hoof and avian feather,as two highly specialised examples of mammalianand avian skin modifications or appendages,respectively, in order to elucidate the influence ofthe dermo-epidermal interface on the epidermaldifferentiation and the microarchitecture of hard

horn structures. Another aim was to gain greaterinsight into the development and evolution ofintegumentary accessory organs in general andto suggest further research directions usingthe equine hoof and avian feathers as modelsystems.

Structure and function of the equinehoof wall

In the course of the evolution of horses,numerous morphological characteristics have de-veloped in order to enhance locomotor speed, oneof which is the weight-reduction of the distal partof the limb by eliminating all but a single digit.The tip of the digit is protected by a light-weight,cone-shaped horn structure, i.e., the hoof capsule,thatamongst other functionsenables the specificweight-bearing stance of equines (i.e., the walkingon the edge of the nail plate). The hoof in a strictsense is a modified integumentary structureenabling the various functions of the end organ,and the horny hoof capsule is produced by thehighly modified epidermal layer, which is deter-mined by the very complex three-dimensionalsurface of the dermal layer covering the tip ofthe distal phalanx. Because different parts of thehoof serve different functions, the hoof is sub-divided into three main parts: the wall (paries),the sole, and the frog. The hoof wall (Fig. 1), inturn, is formed by three contributary regions: theStratum externum or periople, the Stratum med-ium or coronet, and the Stratum internum sive

Fig. 1. Schematic drawing of the structure and function ofthe equine hoof. The surface of the distal phalanx (7) iscovered by a regionally modified skin. The hoof wall iscomprised of three different components: the periople, thecoronet, and the wall proper. Each of these regions displays aspecific configuration of the dermo-epidermal interface, thusenabling the formation of either pressure-resistant tubularhorn over dermal papillae (in the periople, coronet, and wall-sole junction), or tensile-resistant lamellar horn over dermallamellae (in the wall proper). a. Schematic drawing of thedermo-epidermal interface and horn configuration in thecoronet and proximal wall proper. The coronary dermo-epidermal interface displays long and slender dermal papillae(4), while the overlying epidermal cells produce matching horntubules (2), each consisting of a medulla (M) and a cortex (C).In the wall proper, the superficial dermal layer displaysprimary and secondary lamellae (5) over which lamellar horn(3) is produced in the epidermis. b. Schematic drawing of thesuspensory apparatus of the hoof wall. The body weight-induced pressure force rests on the distal phalanx. From theparietal surface of the distal phalanx, this pressure istransformed into a tensile force via a system of collagenousdense fibres traversing the reticular layer (6) of the dermisand inserting on the surface of the dermal primary and

secondary lamellae of the wall proper (5). At the lamellarydermo-epidermal interface, the tensile force is then trans-mitted to the adjacent secondary and primary epidermalcornifying lamellae (3) and on to the proximo-distally orientedcoronary horn tubules (2), where it is re-transformed into apressure force resting on the weight-bearing margin of thehoof wall. c. Schematic drawing of the weight-bearing marginof the hoof (white-line-area). At the wall-sole junction,terminal papillae (8) arise from the distal ridge of the primarydermal lamellae (5). The overlying epidermis producesmatching terminal horn tubules (10). d. Schematic drawingof the white-line area at the wall-sole junction. The hornylamellae (3) and terminal horn tubules (10) of the wall properfill the space between inner coronary (2) and solear (11) horntubules. The fine structure of the coronary horn tubules withcortex (C) and medulla (M) together with intertubular horn (I)is indicated. 1=perioplic horn, 2=coronary horn, 3= lamel-lar horn of the wall proper, 4=dermal coronary papillae,5=dermal lamellae of the wall proper, 6= reticular layer ofthe dermis, 7= surface of the distal phalanx, 8= terminalpapillae of the wall proper, 9= solear dermal papillae,10= terminal horn tubules of the white-line, 11= solear horn,C=cortex of horn tubule, M=medulla of horn tubule,I= intertubular horn.

INTEGUMENTARY ACCESSORY ORGANS IN BIRDS AND MAMMALS 143

lamellatum or wall proper. Each of the hoofregions, or segments, are characterised by specificmodifications of one, two or all three integumen-tary layers. Thus, the equine hoof is structurally

and functionally the most complex form of digitalend organs.

The body weight of the horse rests on the distalrim of the hoof wall, i.e., the weight-bearingmargin of the hoof capsule, which corresponds tothe edge of the nail plate in other mammals. Thisis achieved by a unique modification of the threeregions of the hoof wall, which form the so-called

Fig. 2. The dermo-epidermal interface and its influence oncornifying structures. a. Schematic drawing of the distalequine limb indicating the localisation (boxed area) of thecoronary structural elements shown in Figures 2 b - e. b.Schematic drawing: papillary body with finger-like dermalpapillae containing superficial dermal blood vessels. Con-centric colored rings indicate the diffusion, or concentration,gradient of nutrients supplying the keratinising epidermalcells. Arrows indicate the direction of tubular horn produc-tion. Horn tubules with cortex (C) and medulla (M) areembedded in intertubular (I) horn. c. Surface of coronarydermal papillae with tertiary longitudinal dermal ridges(arrows). The epidermis and hoof capsule has been maceratedand removed. Scanning electron microscopy. d. Dermal papillacovered by adjacent epidermal cells. The basal epidermal cellsinvaginate the papillary surface (arrows). A=arteriole,V=venule. Light microscopy, transverse section as indicatedin Fig. 2c. e. Horn tubule with marrow (M) and cortex (C)embedded in intertubular horn (I). Light microscopy, trans-verse section.

Fig. 3. The development of a contour feather and its parts.a. Basal third of a pennaceous feather follicle of the chicken(Gallus gallus forma domestica). 1= feather papilla, 2=barbridge. The square indicates the sector shown in figure 2b. Thelines indicate the levels of the cross sections shown in Figures2ce. Light microscopy, longitudinal section. b. ongitudinalsection of a barb ridge. 2=basal layer (pulp epithelium),3= ramogen cells (barb), 4= radiogen cells (barbules), 5=ha-mulogen cells (barbicels), 8= feather sheath cells. Lightmicroscopy, longitudinal section. ce. 1= feather papilla,2=basal layer (pulp epithelium), 3=barb cells, 4=barbulecells, 5=barbicel cells, 6=marginal cells (marginal plate),7=axial cells (axial plate), 8= feather sheath cells, 2 5= intermediate cells (intermediate plate). Note the thindermal ridges separating the developing epidermal structures,and the capillaries at the base of the dermal ridges. Lightmicroscopy, cross sections.

H. BRAGULLA AND R.M. HIRSCHBERG144

Fig. 4. The fine structure of the contour feather. a.Contour feather of the chicken (Gallus gallus forma domes-tica): 1= feather shaft (rachis), 2=barbs (rami), 3=barbules(radii), 4=barbicels (hamuli). Scanning electron micrograph.b. Magnification of boxed area in Fig. 4a: barbicels (4)

originating at the distal tip of a barbule. Scanning electronmicrograph. c. Microstructure of a barb (2) with polyhedralmedullary and flat, sickle-shaped cortical cells. Cross fracture,scanning electron micrograph.

INTEGUMENTARY ACCESSORY ORGANS IN BIRDS AND MAMMALS 145

suspensory apparatus of the distal phalanx. Thisapparatus transfers the body weight from thesurface of the distal phalanx to the wall of the hoofcapsule, thereby suspending a horse in its hooves(Fig. 1b). Depending on the respective biomecha-nical requirements, the local configuration of thehoof horn is adapted to the pressure and tensileloads exerted on the tissue and, thus, forms eithertubular (pressure-resistant) or lamellar (tensile-resistant) horn over dermal papillary or lamellarformations, respectively (Fig. 1a). Papillary struc-tures are formed in the periople, coronet, sole andfrog regions of the hoof, whereas the wall properdisplays predominantly a lamellar surface and so-called cap and terminal papillae at the junctionsbetween the coronet and wall and between thewall and the sole (Fig. 1 ad). The morphology ofthe hoof is reviewed by Pollitt (94), Pellmann(95), Bragulla (96), Henke (97), Douglas andThomason (2000), Thomason et al. (2001) andBragulla (2003).The coronary region, or coronet, of the hoof as

major contributor to the hoof wall absorbs most ofthe pressure forces generated in the weight-bearing process and, thus, consists of very hardhorn comprised of slender horn tubules with aspecific medullary-cortical ratio produced over apronounced three-dimensional dermal papillarybody (Fig. 2c and d). It resembles the most highlydeveloped and complex papillary-tubular systemin the hoof, as the coronary dermo-epidermalinterface develops all kinds of surface modifica-tions: well-developed papillae, branching struc-tures (secondary and accessory papillae), andsmall ridges, i.e., lamellary structures (Bragulla,2003). The coronet of the hoof will therefore serveas an excellent comparative model for studying thedermal-epidermal interaction in the formation ofintegumentary accessory organs.Because knowledge of the development of a

distinct organ is a prerequisite to understandingits structure and function, the study of thedevelopment of the region-specific dermo-epider-mal interface will provide new information on thebasic developmental mechanisms and functionalprinciples of papillary body formation in general(Bragulla, 2003). The typical conformation of thepapillary body of the digital end organ is alreadyformed perinatally, therefore, studying the var-ious stages of its embryonic development, forexample in the coronary region, seems the mostpromising approach to this question.In the first semester of gestation, the dermo-

epidermal interface of all regions of the digital end

organ anlage is plane. In the second trimester ofgestation, the formerly plane dermo-epidermalcontact surface of the coronary region is trans-formed into low proximo-distally oriented dermalridges. These ridges get increasingly higher and,in later stages, are subdivided by the formation ofsmall shark-finlike projections at the crests of thedermal ridges. The development of this complexdermo-epidermal interface is initiated by anincreased mitotic activity of the basal epidermalcells that progressively invaginate the dermo-epidermal surface. The stimulus for this processseems to be arising from the growing diffusiondistance of the proliferating epidermal layers fromthe underlying dermal capillaries (Fig. 2a), caus-ing a latent nutritional deficiency of the prolifer-ating and differentiating epidermal cells (Bragulla,96). As the epidermal cells are completely depen-dent on the nutritional supply by the dermal bloodvessels, the superficial dermal capillary networksact as a guide-line for the basal epidermal cellsinverting the dermo-epidermal interface. This isdemonstrated by increased mitotic activity ofbasal epidermal cells in the vicinity of theperipheral dermal capillaries (Fig. 2d). The nextdevelopmental stage of the papillary body involvesthe formation of dermal papillae. Through furtherepidermo-dermal interactions, the shark-finlikeprojections of the dermal ridges become dome-shaped, then cone-shaped until they finally re-semble finger-like, i.e., papillary projections.Concurrently with the appearance of the firstdermal papillae, the surrounding epidermal cellsshow an increased rate of keratinisation. Appar-ently, increase of the dermo-epidermal interfacenot only supports the necessary supply of nutri-ents for the proliferating epidermal cells, but alsoenables these cells to stimulate keratin-productionand, thus, enter their prospective differentiationpathway. When the developing dermal papillaehave reached a certain diameter (approx. 100 mmthick and 250 mm long), they begin to influence themicrostructure of the fetal hoof horn, resulting inthe first faint horn tubules. With the furtherdevelopment of the dermal papillae in the thirdtrimester of gestation, the diameter of the horntubules and the proportions of the tubular cortexand medulla changes (Bragulla, 96). In laterdevelopmental stages (6th to 9th month of gesta-tion), low longitudinal tertiary ridges appear onthe surface of the papillae that further increasethe dermo-epidermal interface (Fig. 2c). Thesetertiary ridges develop in the same way as theother dermo-epidermal interface modifications,

H. BRAGULLA AND R.M. HIRSCHBERG146

i.e., by increased mitotic activity of the basalepidermal cells invaginating the dermal surface(Fig. 2d). But due to their small size, theirappearance does not result in correspondingepidermal horny structures (Bragulla andHirschberg, 2002).In conclusion, the complex and segment-specific

architecture of the coronary tubular horn ismainly directly determined by the shape of thepapillary body, i.e., the dermo-epidermal interface.A certain size and diameter of the dermal surfacemodifications (i.e., ridges or papillae) apparentlyare needed in order to influence the structure ofthe adjacent hoof horn. The primary function ofthe increased dermal-epidermal interface seems tobe the enlargement of the diffusion surface area inorder to promote the nutritional supply and is,therefore, closely related to the superficial dermalmicrovasculature.

Structure and function of the avianfeather

Feathers serve a multitude of functions. Apartfrom their function as a protective and thermo-regulatory body covering, they play a major role inbehaviour and communication, and, last but notleast, in forming a contiguous coat of feathers thatenables aerodynamic streamlining and flight(Homberger, 2002; Prum and Brush, 2002). Thephylogenetic appearance of feather-like struc-tures, and whether the penna, i.e., the contour-feather, which represents the most complex formof feathers, is derived from the less complexpluma, i.e., down, is still debated (Starck, 82;Chuong and Edelman, 85a,b; Prum, 99; Brush,2000; Xu et al., 2001).As in all integumentary accessory structures,

the feather coat is derived from all three, thoughmodified, layers of the skin. Although the epider-mal modifications, i.e., horny structures, of thefeather are the most eye-catching, the dermal andsubcutaneus contributions to this integumentaryaccessory organ have to be considered to under-stand the functions of the feathered skin(Homberger, 2002). Whereas dermis and epider-mis form the feather follicle, which generates formand structure of the feather itself, the dermis, andmore particularly the subcutis, form a verycomplex modified dermo-subcutaneuos apparatusfor anchoring and controlling individual feathersor feather groups, respectively (Homberger and deSilva, 2000).

According to the different functions of thefeather coat in different bird species, it maycontain different types of feathers (pennae andplumae) in varying density and distribution(Lucas and Stettenheim, 72). All feather typesare comprised of a dermo-epidermal intracuta-neous and a cornified extracutaneous part. Thecomplex three-dimensional architecture of thecornified cells of the feather can be detected notonly in the penna but also in the less complexpluma. The advanced complex form of the penna isachieved by a progressing subdivision and inter-digitation of epidermal structures with the dermis,which is caused by a further development of thedermo-epidermal three-dimensional surface, i.e.,the papillary body in the feather papilla. The shaftis the central support of the feather, continued bythe vane with its barbs, barbules and barbicels.The barbicels stabilise the vane of the penna byinterdigitation with the barbules of the neighbor-ing barb. To save weight, the barbs are hollowtubular structures (Fig. 4), composed of polyhedralcells in the center (i.e., medulla) and flat cells inthe periphery (i.e., cortex).

The development of the very complex featherstructures starts with a distinct aggregation ofdermal cells beneath the undifferentiated epider-mal layer of the otherwise flat fetal prospectivefeather-bearing skin (Chuong and Edelmann, 85a, b). Further proliferation of the dermal cellsleads to the formation of a feather germ, aprotrusion of epidermal and underlying dermalcells. This feather germ then becomes a finger-likeprojection. Starting at the tip of the feather germ,i.e., in disto-proximal direction, the epidermal cellscovering the dermal core of the feather proliferateand differentiate, forming a multi-layered thickepithelium. In the next developmental stage, thefeather germ is then invaginated into the dermis,thus forming the feather follicle (Fig. 3a). At thesame time, longitudinal ridgesFreminiscent ofthe tertiary longitudinal ridges on the coronarypapillae of the equine hoofFoccur at the dermo-epidermal interface, again starting at the tip of thefeather germ. These epidermal (barb) ridges areformed by increased mitotic activity of the basalepidermal cells, which invaginate the dermal coreof the feather germ. This process is comparable tothat described in the development of the papillarybody in the equine hoof.

In the anlage of a penna, the epidermal ridges,which are separated by very thin dermal ridges,are getting higher and form marginal andintermediate cell plates. Differentiation of the

INTEGUMENTARY ACCESSORY ORGANS IN BIRDS AND MAMMALS 147

epidermal cells within the ramogen plates leads tothe formation of barbs, barbules and barbicels(Fig. 3be). The epidermal cells of a barb arerecognisable, with polyhedral cells forming themedulla, and flat and sickle-shaped horn cellsforming the cortex of the barb. These featherformations strongly resemble the cortex andmedulla of the coronary horn tubules in the hoof.While the epidermal cells of the future barbs,barbules and barbicels predominantly producekeratin filaments and associated proteins, and inthe final step cornify, the epidermal cells of theaxial and marginal plates show pycnotic nucleibesides an enhanced lipogenesis, and finallydisintegrate, thus separating barbs and barbulesfrom each other. In longitudinal sections of thefeather germ, the well-organized spiral course ofdifferentiating epidermal cell strands is revealed,whereas the papillary body of the feather remainsnearly smooth at the level of epidermal celldifferentiation.In conclusion, the dermal surface in the feather

follicle generates only very thin dermal ridges,separating the shaft and the barbs of the featherand influencing the point of origin and the angle ofeach barb. It is particularly noteworthy that thepapillary body of the feather follicle, comprisingthe small dermal lamellae, does not containcapillaries in the peripheral parts of the featherfollicle. And it apparently does not directlyinfluence separation, arrangement, and shape ofthe developing barbules and barbicels.

DISCUSSION AND RESEARCHPERSPECTIVES

In the development of the equine hoof, thestructure of a highly specialised dermo-epidermalinterface can be documented, with interactions ofboth upper layers of the skin (i.e., the dermis andepidermis) and a significant influence of the three-dimensional configuration of the very pronounceddermal papillary body on the adjacent epidermalstructures. In contrast, the dermal surface in thefeather follicle generates only thin dermal ridgesseparating the very complex epidermal structuresof barbs, barbules and barbicels in the developingfeather. The spiral course of these dermal ridgeson the feather papilla determines origin and angleof the barbs branching off from the feather shaft,but further differentiation of the epidermal struc-tures comprise degeneration or proliferation andspecial adherence of specific epidermal cell clus-ters, respectively, which seem to be independent of

the shape of the adjacent dermal surface. Ob-viously, structure and development of the highlyspecialised feather epidermis is achieved onlyinitially by a direct dermo-epidermal interaction.Later developmental stages are obviously influ-enced by other factors, most probably by differentcell adhesions at the basal/apical cell membranerelative to the lateral surface of the epidermal cellsthat generate the barbules and barbicels, as isindicated for the cells of the epidermal featherplacode by Chuong and Edelman (85a). Appar-ently, differentiation of the epidermal cells form-ing the prospective barbules and barbicels isalready determined in the basal epidermal layersin the depth of the feather follicle. A comparison ofthe location of the basal epidermal cells producingthe ramogen (presumptive barbs), radiogen (pre-sumptive barbules) and hamulogen (presumptivebarbicels) cells with the location of the axial andmarginal plate cells with respect to the position ofthe peripheral capillaries of the dermal featherpapilla suggests a primary influence of the diffu-sion gradient on the differentiation of the adjacentepidermal cells: while ramogen, radiogen andhamulogen cells are positioned close to thecapillary at the base of the dermal ridge with acorrespondingly high diffusion rate of nutrientsand growths factors, the axial and marginal platecells are comparatively remote from these vesselsand experience a correspondingly low diffusionrate of substances. Further research into thiscorrelation seems to be a promising new approachfor understanding the problem of feather develop-ment. Examinations of the occurrence and locali-sation of specific intercellular junctions andcomponents of the extracellular matrix, such asspecific adhesion molecules of these cells, appearlikewise to be promising in order to determine howthe cornifying cells of the presumptive branchingstructures of the feather (i.e., rami, radii, andhamuli) are separated, and how the differentcomponents of the feather vane unfold.

Similar principles of constructions are detect-able when the structures of the coronary tubularhorn of the equine hoof are compared with thehorny structures of a penna,. The formation of afeather barb with its hollow tubular structurestrongly resembles the formation of tubular hornin the papillary regions of the mammalian digitalend organs. In the equine hoof, the largestcoronary horn tubules actually become hollowstructures, because the inner medullary horn cellsof these tubules disintegrate and fade away(Budras et al., 98). Premature disintegration of

H. BRAGULLA AND R.M. HIRSCHBERG148

the inner horn cells is caused by only moderatekeratinisation, which might be due to the fact thatthese suprapapillary medullar cells are rapidlypushed away from the tip of the underlyingdermal papillae in the course of their differentia-tion and are, thus, separated from their supplysources (Fig. 2b). Disintegration of the innerfeather shaft and the inner barb cells might resultfrom the same driving forces.A spiral course of small longitudinal dermal

ridges comparable to those influencing the barbformation in the feather, are described in thebulbar papillae of the bovine hoof. Contrary to thecondition in the equine hoof, the solear and bulbar(pad) region in the bovine hoof participate in theweight-bearing process, acting as a highly derivedshock-absorbing system (Raber, 2000; Hirschberget al., 2002). The bulbar papillae display long-itudinal tertiary ridges following the spiral orconvoluted array of the bulbar papillae, which arethought to act as a shock absorbing devicecomparable to that of a spiral spring (Mulling,93, Hirschberg et al., 2001). These longitudinalridges contain capillaries only at the base of thepapilla, whereas the further course of these spiralridges towards the tip of the papilla is avascularlike in the spiral dermal ridges of the feather,possibly due to their small dimension (Hirschberget al., 2001).Even in the equine hoof, not all dermal surface

modifications result in an equivalent epidermalstructure. Dermal microstructures such as lowtertiary lamellae or tertiary longitudinal ridges onthe surface of dermal papillae apparently do notinfluence the structure of the horn produced in theoverlying epidermis (Bragulla, 96; Bragulla andHirschberg, 2002). Formation of a papillary bodyseems to start with an increased mitotic activity ofthe basal epidermal layers, so that a complexinteraction of the dermis and epidermis seems tobe responsible for the structure of integumentaryaccessory organs (Bragulla, 96, 98; Bragulla et al.,2001).Therefore, other factors than just the shape of

the dermo-epidermal interface are likely to play arole in the formation of epidermal cornifiedstructures. Recent studies (reviewed by Freedberget al., 2001) suggest two alternative pathwaysopen to epidermal keratinocytes: differentiationand activation. In unmodified mammalian skin,differentiation leads to succinct keratinisation andcornification of the epidermal cells as describedin healthy and fully developed skin. So-calledkeratinocyte activation described in injured or

pathologically altered skin is triggered by amultitude of growth factors and cytokines. Acti-vated keratinocytes express different keratins,change their cytoskeleton and surface receptors,and, more importantly in the present context, arehyperproliferative and migratory (Freedberg et al.,2001). In order to complement the common work-ing hypothesis on the regulatory processes ofdeveloping integumentary structures, the kerati-nocyte activation cycle might therefore be a newfocus for future research projects. Many hoofdiseases are characterised by a pathological sub-division of the papillary body (Marks, 84; Hirsch-berg, 2000), probably initiated by the same drivingforces as in the fetal development, i.e., lack ofoxygen and nutrients in the misaligned or over-loaded hoof regions. Differing keratin expressionin the germinative and keratinised epidermallayers of the modified skin of healthy and diseaseddigital end organs has been described (Wattle,2000; Hendry et al., 2001), likewise suggestingthat keratinocyte activation is involved in physio-logical as well as pathological adaptation of thedigital end organ, particularly in the developmentand maturation of the dermo-epidermal interface.Keratinocyte activation is also engaged in dermalangiogenesis (Detmar, 2000), and sprouting andintussusceptional, i.e., nonsprouting, remodellingangiogenesis have been reported both in thematuringand pathologically altered bovine pododerma(Hirschberg et al., 2003), indicating a tightregulatory vaso-hormonal association betweenthe dermal microvasculature and the formationof the dermo-epidermal interface.

In both avian and mammalian skin, lipid-producing cells are described besides the prevail-ing keratinocytes, i.e., sebokeratinocytes, in aviannon-feather-bearing skin (Menon and Menon,2000), or sebocytes in the sebaceous glands ofmammalian hairy skin, respectively (Junqueiraet al., 92; Budras et al., 2002). Moderate produc-tion of structural lipids in avian embryonic b-keratin-producing feathered skin is reported,apparently reflecting a primitive lipogenic activityof bird sebokeratinocytes (Alibardi, 2002). In orderto solve the problem how the cornified componentsof the developing feather branch and finallyseparate, the results of the present study suggesttwo different differentiation modes open to epi-dermal cells, i.e., keratinisation or lipidisation(lipogenesis). Keratinisation may result either incornification and, thus, production of a hard hornystructure, or in decay and disintegration of these

INTEGUMENTARY ACCESSORY ORGANS IN BIRDS AND MAMMALS 149

cells due to only moderate keratinisation. Thelatter then induces the formation of hollowstructures, as exemplified by the coronary horntubules of the equine hoof or the hollow barbs ofthe avian feather. In the feather, the epidermalcells of the intermediate and marginal platesapparently adopt another way of differentiation,i.e., lipidisation, by producing more lipids thankeratins. These lipids are released when the cellsfinally disintegrate, thus separating the adjacentfully keratinised and eventually cornifying struc-tures. The function of this production of structurallipids (in analogy to the production of structuralproteins in the process of keratinisation) in thefeather is comparable to that of avian seboker-atinocytes, and both pathways of differentiationmay be regarded as different forms of programmedcell death (i.e., apoptosis). Whereas cornifyingcellsFi.e., keratinocytes in a narrower context,regardless whether undergoing soft or hardcornification, or a- or b-cornificationFmaintainpart of their cellular structure and remainassociated with one another, the lipidisingcellsFthe sebokeratinocytes of the avian skinand the sebaceous cells of the mammalian skin-Floose both cellular contacts and cellular integ-rity. Adapting this concept of different ways ofkeratinocyte differentiation might prove veryfruitful for further research into the developmentof the avian feather, particularly as Wnt, a factorinvolved in early skin appendage formation, hasbeen suggested as being responsible for thetransdifferentiation of different epidermal celltypes to sebocyte phenotypes (reviewed by Byrneet al., 2003).

ACKNOWLEDGMENTS

The authors are grateful to Dr. Dominique G.Homberger, Department of Biological Sciences,Louisiana State University, for valuable sugges-tions and many fruitful discussions during thepreparation of this manuscript. We particularlythank Prof. Dr. Dominique G. Homberger andProf. Dr. Cheng-Ming Chuong, Department ofPathology, University of Southern California, LosAngeles, for the invitation to contribute an articleto the special edition of this journal. We also owemany thanks to Gisela Jahrmarker and Prof. Dr.Klaus-Dieter Budras, Department of VeterinaryAnatomy, Freie Universitat Berlin, for supportand time spent on designing Figure 1. Theexcellent support of our technical assistants,

Karin Briest-Forch and Ilona Kuster-Krehahn, isalso much appreciated.

LITERATURE CITED

Alibardi L. 2002. Keratinization and lipogenesis in epidermalderivatives of the zebrafinch, Taeniopygia guttata castanotis(Aves, Passeriformes, Ploceidae) during embryonic develop-ment. J Morphol 251:294308.

Bock WJ. 2000. Explanatory history of the origin of feathers.Amer Zool 40:478485

Bonser RHC, Witter MS. 1996. Comparative mechanics of bill,claw and feather keratin in the common starling, Sturnusvulgaris. J Avian Biol 27:175177.

Bragulla H. 1996. Zur fetalen Entwicklung des Pferdehufes.Habilitation. Faculty of Veterinary Medicine, University ofBerlin.

Bragulla H. 1998. Zur pranatalen Entwicklung der Hufkapsel.Wien Tierarztl Mschr 85:233244.

Bragulla H. 2003. Fetal development of the segment-specificpapillary body in the equine hoof. J Morphol (in press).

Bragulla H, Hirschberg RM. 2002. The fetal development ofthe dermal papillary body in the hoof and its influence onepidermal horn structure. Ann Anat Suppl 184:65.

Bragulla H, Ernsberger S, Budras K-D. 2001. On thedevelopment of the papillary body in the feline claw. AnatHistol Embryol 30:17.

Brush AH. 2000. Evolving a protofeather and featherdiversity. Amer Zool 40:631639.

Budras K-D, Schiel, C. 1996. A comparison of horn quality ofthe white line in the domestic horse (Equus caballus) andthe Przewalski horse (Equus przewalskii). Pferdeheilkunde4:641645.

Budras K-D, Schiel C, Mulling C. 1998. Horn tubules of thewhite line: an insufficient barrier against ascending bacter-ial invasion. Equine Vet Edu 10:8185.

Budras K-D, McCarthy PH, Fricke W, Richter R. 2002.Anatomy of the dog. Schlutersche Verlagsbuchhandlung,Hannover/Germany.

Byrne C, Hardman M, Nield K. 2003. Covering the limb formation of the integument. J Anat 202:113124.

Chuong C-M, Edelmann GM. 1985a. Expression ofcell-adhesion molecules in embryonic induction. I. Morpho-genesis of nestling feathers. J Cell Biol 101:10091026.

Chuong C-M, Edelmann GM. 1985b. Expression ofcell-adhesion molecules in embryonic induction. II.Morphogenesis of adult feathers. J Cell Biol 101:10271043.

Detmar M. 2000. The role of VEGF and thrombospondin inskin angiogenesis. J Dermatol Sci Suppl 1:S8784.

Douglas JE, Thomason JJ. 2000. Shape, orientation andspacing of the primary epidermal laminae in the hooves ofneonatal and adult horses (Equus caballus). Cells TissuesOrgans 166:304318.

Freedberg IM, Tomic-Canic M, Komine M, Blumenberg M.2001. Keratins and the keratinocyte activation cycle. JInvest Dermatol 116:633640.

Hamrick MW. 2001. Development and evolution of themammalian limb: adaptive diversification of nails, hooves,and claws. Evol Develop 3:355363.

Hendry KA, MacCallum AJ, Knight CH, Wilde CJ. 2001.Synthesis and distribution of cytokeratins in healthy andulcerated bovine claw epidermis. J Dairy Res 68:525537.

Henke F. 1997. Hufbeintrager und Hufmechanismus imSeiten-, Trachten- und Eckstrebenteil des Pferdehufes.

H. BRAGULLA AND R.M. HIRSCHBERG150

Doctoral dissertation. Faculty of Veterinary Medicine,University of Berlin.

Hirschberg RM. 2000. Microvasculature and microcirculationof the healthy and diseased bovine claw. Proc XIth Int SympDisorders Ruminant Digit & 3rd Int Conf Bovine Lameness.Parma, Italy, Sept. 2000. 9799.

Hirschberg RM, Mulling CKW, Bragulla H. 1999. Microvascu-lature of the bovine claw demonstrated by improvedmicro-corrosion-casting technique. Microsc Res Tech 45:184197.

Hirschberg RM, Mulling CKW, Budras K-D. 2001. Pododermalangioarchitecture of the bovine claw in relation to form andfunction of the papillary body: a scanning electron micro-scopic study. Microsc Res Tech 54:375385.

Hirschberg RM, Westerfeld I, Budras K-D. 2002. Thespecialised dermo-subcutaneous system of the bovine claw the suspensory and weight-bearing apparatus. Proc.XXIVth Congress EAVA, Brno, Czech Rebublik, July 2125th, 61.

Hirschberg RM, Budras K-D, Plendl J. 2003. PododermaleAngiogenese neue Ansatze zur Therapie von Klauenerk-rankungen. Kongressband, 25. DVG-Tagung, 03.-04. April2003, Berlin. DVG-Verlag, Gieben (in press).

Homberger DG. 2001. The case of the cockatoo bill, horse hoof,rhinoceros horn, whale baleen, and turkey beard: Theintegument as a model system to explore the concepts ofhomology and non-homology. Vertebrate functional mor-phology: Horizon of research in the 21st century. Dutta HM,Datta Munshi JS, eds. Science Publishers, Enfield, NewHampshire. 315341.

Homberger DG. 2002. The aerodynamically streamlined bodyshape of birds: Implications for the evolution of birds,feathers, and avian flight. Proceedings of the 5th Sympo-sium of the Society of Avian Paleontology and Evolution,Beijing, 1-4 June 2000. Eds. Z Zhou and F Zhang, SciencePress, Beijing. 227252.

Homberger DG, Brush AH. 1986. Functional-morphologicaland biochemical correlations of the keratinised structuresin the African Grey Parrot, Psittacus erithacus (Aves).Zoomorph 106:103114.

Homberger DG, de Silva KN. 2000. Functional microanatomyof the feather-bearing integument: Implications for evolu-tion of birds and avian flight. Amer Zool 40:553574.

Imayama S. 1981. Scanning and transmission electron micro-scope study on the terminal blood vessels of the rat skin.J Invest Dermatol 76:151157.

Junqueira LC, Carneiro J, Kelley RO. 1992. Basic histology.Appleton & Lange, Norwalk, Connecticut.

Lucas AM, Stettenheim PR. 1972. Avian anatomy: Integu-ment. Agriculture Handbook 362. U.S. Dept. of Agriculture,Washington, D.C.

Marks G. 1984. Makroskopische, licht- und elektronenop-tische Untersuchung zur Morphologie des Hyponychiumsbei der Hufrehe des Pferdes. Doctoral dissertation. Facultyof Veterinary Medicine, University of Berlin.

Menon GK, Menon J. 2000. Avian epidermal lipids: functionalconsiderations and relationship to feathering. Amer Zool40:540552.

Millar SE, Willert K, Salinas PC, Roelink H, Nusse R,Sussman DJ, Barsh GS. 1999. WNT Signaling in the controlof hair growth and structure. Development Biol 207:133149.

Mulling CKW 1993. Struktur, Verhornung und Hornqualitatin Ballen, Sohle und weisser Linie der Rinderklaue.Doctoral dissertation. Faculty of Veterinary Medicine,University of Berlin.

Nickel R. 1938. Uber den Bau der Hufrohrchen und seineBedeutung fur den Mechanismus des Pferdehufes. MorphJ 82:112160.

Patan B, Budras K-D. 2003. Segmentspezifitaten am Pferde-huf - Teil II: Zusammenhang zwischen Hornstruktur undmechanisch-physikalischen Horneigenschaften in denverschiedenen Hufsegmenten. Pferdeheilkunde 19:177184.

Pellmann R. 1995. Struktur und Funktion des Hufbeintragersbeim Pferd. Doctoral dissertation. Faculty of VeterinaryMedicine, University of Berlin.

Pellmann R, Reese S, Bragulla H. 1993. Wechselwirkungenzwischen Hornstruktur und Hornqualitat am Pferdehuf alsGrundlage fur das Verstandnis von Verhornungsstorungen.Mh Vet Med 48:623630.

Pollitt CC. 1994. The basement membrane at the equine hoofdermal-epidermal junction. Equine Vet J 26:399407.

Prum R. 1999. Development and evolutionary origin offeathers. J Exp. Zool 285:291306.

Prum RO, Brush AH. 2002. The evolutionary origin anddiversification of feathers. Quart Rev Biol 77: 261295.

Raber ME. 2000. Das Ballenpolster beim Rind. Ein Beitrag zurfunktionellen Anatomie der Klaue. Doctoral dissertation.Faculty of Veterinary Medicine, University of Zurich.

Sawyer RH, Salvatore BA, Potylicke TT, Frenche JO, GlennTC, Knapp LW. 2003. Origin of feathers: Feather betakeratins are expressed in discrete epidermal cell populationsof embryonic scutate scales. J Exp Zool Part B Mol Dev Evol295:1224.

Starck D. 1982. Integument und Anhangsorgane. Vergle-ichende Anatomie der Wirbeltiere auf evolutionsbiolo-gischer Grundlage, Band 3, Springer-Verlag, Berlin. P131257.

Tarsitano SF, Horne F, Plummer C, Millerchip K, Russel AP.2000. On the evolution of feathers from an aerodynamic andconstructional view point. Amer Zool 40: 676686.

Thomasson JJ, Douglas JE, Sears W. 2001. Morphology of thelaminar junction in relation to the shape of the hoof capsuleand distal phalanx in adult horses (Equus caballus). CellsTissues Organs 168:295311.

Wattle O. 2000. Cytokeratins of the stratum medium andstratum internum of the equine hoof wall in acute laminitis.Acta Vet Scand 41:363379.

Widelitz RB, Jiang TX, Chen CWJ, Stott NS, Jung HS,Chuong CM. 1999. Wnt-7a in feather morphogenesis:involvement of anterior-posterior asymmetry and proxi-mal-distal elongation demonstrated with an in vitro recon-stitution model. Development 126:25772587.

Xu X, Zhou ZH, Prum RO. 2001. Branched integumentalstructures in Sinornithosaurus and the origin of feathers.Nature 410:200204.

INTEGUMENTARY ACCESSORY ORGANS IN BIRDS AND MAMMALS 151