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Shh-Bmp2 Signaling Module and the EvolutionaryOrigin and Diversification of Feathers

MATTHEW P. HARRIS,1n JOHN F. FALLON,1 AND RICHARD O. PRUM2

1Department of Anatomy, University of Wisconsin, Madison, Wisconsin 537062Department of Ecology and Evolutionary Biology, and Natural History Museum,University of Kansas, Lawrence, Kansas 66045

ABSTRACT To examine the role of development in the origin of evolutionary novelties, weinvestigated the developmental mechanisms involved in the formation of a complex morphologicalnoveltyFbranched feathers. We demonstrate that the anterior-posterior expression polarity of Sonichedgehog (Shh) and Bone morphogenetic protein 2 (Bmp2) in the primordia of feathers, avian scales,and alligator scales is conserved and phylogenetically primitive to archosaurian integumentaryappendages. In feather development, derived patterns of Shh-Bmp2 signaling are associated with thedevelopment of evolutionarily novel feather structures. Longitudinal Shh-Bmp2 expression domainsin the marginal plate epithelium between barb ridges provide a prepattern of the barbs and rachis.Thus, control of Shh-Bmp2 signaling is a fundamental component of the mechanism determiningfeather form (i.e., plumulaceous vs. pennaceous structure). We show that Shh signaling is necessaryfor the formation and proper differentiation of a barb ridge and that it is mediated by Bmp signaling.BMP signaling is necessary and sufficient to negatively regulate Shh expression within formingfeather germs and this epistatic relationship is conserved in scale morphogenesis. Ectopic SHH andBMP2 signaling leads to opposing effects on proliferation and differentiation within the feathergerm, suggesting that the integrative signaling between Shh and Bmp2 is a means to regulatecontrolled growth and differentiation of forming skin appendages. We conclude that Shh and Bmpsignaling is necessary for the formation of barb ridges in feathers and that Shh and Bmp2 signalingconstitutes a functionally conserved developmental signaling module in archosaur epidermalappendage development. We propose a model in which branched feather form evolved by repeated,evolutionary re-utilization of a Shh-Bmp2 signaling module in new developmental contexts. Featheranimation Quicktime movies can be viewed at http://fallon.anatomy.wisc.edu/feather.html. J. Exp.Zool. (Mol. Dev. Evol.) 294:160–176, 2002. r 2002 Wiley-Liss, Inc.

INTRODUCTION

Evolutionary novelties are derived features thatare qualitatively different from, and not homo-logous with, antecedent structures (Muller andWagner, ’91; Raff, ’96). The mechanistic basis ofthe origin of morphological novelties remains acentral question in evolutionary biology. Variationin developmental processes is thought to be animportant means of macroevolutionary change(Muller and Wagner, ’91; Raff, ’96; Wagner,2000). Furthermore, the innovative use of pre-existing developmental processes or molecularsignaling pathways has been hypothesized to bean important mechanism for the evolution ofnovel morphological structures (Raff, ’96; VonDassow and Munro, ’99). However, in only a fewinstances have mechanistic changes in geneexpression that are associated with the evolutionof a morphological novelty been documented(Keys et al., ’99; Wagner, 2001). The feather is a

branched integumentary appendage that is anexcellent example of a complex assemblage ofmorphological evolutionary novelties (Prum, ’99).The feather has been hypothesized to have evolvedfrom scutate scales of archosaurian ancestors(Lucas and Stettenheim, ’72). The fundamentalquestion is how novel structural diversity aroseduring the evolution of the integumentalappendages of the archosaurs (the reptilianclade including birds, crocodilians, dinosaurs,and pterosaurs).

Based on a morphological analysis of feathermorphogenesis, Prum (’99) recently hypothesized

Grant sponsor: National Institutes of Health; Grant number:32551; Grant sponsor: National Science Foundation; Grant number:DBI-0078376.

nCorrespondence to: Matthew Harris, 351 Bardeen Hall, 1300University Avenue, Madison, WI 53706. E-mail: [email protected]

Received 3 June 2002; Accepted 24 June 2002Published online in Wiley InterScience (www.interscience.wiley.

com). DOI: 10.1002/jez.10157

r 2002 WILEY-LISS, INC.

JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 294:160–176 (2002)

that feathers originated and diversified through aseries of developmental processes that are hier-archically and historically contingent. This devel-opmental model of the origin of feathers makestestable predictions about both the morphologiesof primitive feathers and the role of developmentalchange in the origin of complex feather structure.Recent discoveries of fossil feathers from nonaviantheropod dinosaurs have documented severalplesiomorphic feather morphologies, includingbranched filamentous structures and central fila-ments hypothesized to be a rachis (Chen et al., ’98;Ji et al., ’98; Xu et al., ’99; Ji et al., 2001; Xu et al.,2001; Norell et al., 2002). These findings demon-strate the phenotypic complexity of early integu-mental appendages during the early evolution offeather complexity (Prum, ’99; Brush, 2000;Chuong et al., 2000), and they are congruent withpredictions from the developmental model (Ji et al.,2001; Sues, 2001; Xu et al., 2001; Norell et al., 2002).Epidermal appendages are initiated by signaling

between an epithelium and subjacent mesench-yme leading to the formation of an epithelialplacode. Recent studies of epidermal appendagemorphogenesis have concentrated on the molecu-lar mechanisms associated with placode inductionand the feather bud as a morphological entity.Little to no attention has been paid to themolecular events that pattern and define themorphogenesis of the definitive feather structure.Here we present a comparative analysis of thedevelopment of feathers and scales in extantarchosaurian lineages to test the hypothesis thatchanges in developmental mechanisms were cri-tical to the evolution of feathers; specifically, todetermine if there are common developmental andmolecular mechanisms reutilized in the develop-ment of novel feather structures. This moleculardevelopmental analysis provides a new context tounderstand the evolutionary origin of feathersfrom keratinized scales, and the diversification ofcomplex feather morphologies, including the evo-lution of barbs, the rachis, and the planarpennaceous vane.We focus on the function and regulation of two

intercellular signaling moleculesF Sonic hedge-hog (Shh) and Bone Morphogenetic Protein 2(Bmp2)Fduring feather development. Thesegenes are known to be involved in the develop-ment of many organ systems, including vertebrateintegumentary appendages (Bitgood and McMa-hon, ’95). In many developing systems, Bmps,members of the TGFb gene superfamily, havebeen shown to act in concert with Shh, or its

invertebrate ortholog hedgehog (Basler andStruhl, ’94; Borod and Heberlein, ’98; Murtaughet al., ’99; Dahn and Fallon, 2000; Drossopoulouet al., 2000; Zhang et al., 2000; Zhang and Yang,2001) and the two genes are expressed concomi-tantly in the development of nonfeather integu-mentary appendages of amniotes (Bitgood andMcMahon, ’95; St-Jaques et al., ’98; Jung et al.,’99).

We show that Shh-Bmp2 signaling in theformation of integumentary appendages of birdsconstitutes a molecular signaling module1, andthat there is conservation of expression of thismodule early in the development of integumentaryappendages of extant archosaurs (i.e., birds andcrocodiles). Furthermore, the development of thenovel tubular feather germ and feather branchedstructure is associated with the evolutionaryreutilization of this signaling module in noveldevelopmental contexts during the development ofa feather. We demonstrate that this signalingmodule is necessary for the morphogenesis of barbridges, and thus, is critical to the morphogenesis ofthe branched structure of the feather. The datasuggest that the repeated evolutionary re-utiliza-tion of this modular molecular signaling unitwithin the epidermis of early scale developmentfacilitated the origin of the novel epithelialstructures that characterize feather morphology.In this paper, we give an introduction to featherstructure and development. Then, we describepatterns of Shh-Bmp2 expression in archosaurianfeathers and scales, and document the associationbetween variation in Shh-Bmp2 expression andthe growth of feather branched structure. Finally,we present experimental data documenting themodularity of Shh-Bmp2 signaling and function ofthis module in feather morphogenesis.

METHODS

To investigate the molecular mechanisms un-derlying the development of feathers and scales,we analyzed the regulation of the expression ofShh and Bmp2 by whole mount in situ hybridiza-tion (WMISH). To document downstream effectsof these signaling molecules, we used WMISH toexamine gene expression of the Shh receptorPatched (Ptc) and Patched2 (Ptc2), and the Shh-activated transcription factor Gli1. We assessed

1We use the term module after Gilbert and Boker (2001): A signalingmodule represents a developmental unit of integrated signalingsystems that is shared between species and tissues, exhibitingconservation and descent with modification.

Shh/Bmp2 MODULE IN FEATHER EVOLUTION 161

the function of Shh and Bmp2 signaling in featherdevelopment by experimentally treating formingembryonic feather germs with ectopic SHH orBMP2 protein loaded on to beads, or by blockingthe endogenous signaling of Shh and Bmp2 byusing pharmacological agents and viral vectors tooverexpress molecular regulators of their intracel-lular signaling.Observations and experiments were conducted

on chick and duck natal down feathers, chick andduck scutate scales, and alligator scales to inves-tigate the comparative patterns in gene expressionand function among species, different appendages,and among morphologically distinct morphologiesof the same appendage (e.g., plumulaceous vs.pennaceous natal down).

Specimen collection and preparation

Avian embryos of the Babcock strain of WhiteLeghorn chickens (Gallus gallus; Madison WI),SPAFAS strains (Charles River Laboratories), andPekin and Khaki Campbell ducks (Anas platyr-hynchus; Metzger Farms) were incubated at 391Cuntil harvest. Alligator (Alligator mississippien-sis) embryos were obtained from the ChenierNational Refuge. Embryos were fixed overnightin 4% PFA and dehydrated in a methanol seriesand stored at �201C until use. SPAFAS embryoswere used solely for RCAS studies.

Whole mount in situ andimmunohistochemistry

Whole mount in situ labeling was performed asdescribed by (Nieto et al., ’96) with the addition of10% polyvinyl chloride to the color reaction andsubsequent clearing in methanol. Whole mountimmunohistochemistry followed the in situ proto-col omitting the hybridization and proteinasesteps. Overnight incubation of primary and sec-ondary antibodies was used. Signal was detectedusing alkaline phosphatase conjugated antibodiesand NBT/BCIP as a substrate. Probes used in thisstudy were chick Shh (P. Beachy); Bmp2 (E.Laufer); Ptc2 (C. Tabin); Ptc (C. Tabin); and Gli1(C. Tabin). Interspecific hybridizations were per-formed at 651C. Primary antibodies used in thesestudies were Shh 5E1 (gift of C. Chiang, 1:100),phosphorylated histone H-3 (ser10) (UpstateBiotechnology Institute, 1:100), and AMV-3C2(Iowa developmental hybridoma bank developedby D. Boettiger). For detection of viral antigen inE16 feathers, feathers were removed and washedin PBS/0.1% Tween, 0.1% Triton-X to aid in

antibody penetration into tissue. Anti-mouse-APand anti-rabbit-AP secondary antibodies wereused at 1:2,000 dilutions. Histological sections oftissue after immunohistochemical and in situanalysis were conducted using isopropanol as anantimedium for paraffin embedding.

Feather explants and chorio-allantoicmembrane grafts

Skin explants from stage HH36 (Hamburgerand Hamilton, ’51) spinal tract were selected forsize of the forming feather buds. Only tissue thathad formed medium sized buds were chosen forfurther analysis in order to avoid affecting earlyformation of polarity and growth of the featherbud. Explants were folded dermal side in andcultured at 371C in 2%FBS, 1% Pen-Strep,Dulbecco modified minimal essential medium(DMEM). After 24 hours the explants were treatedwith Noggin protein (5 mg/ml, gift from R. Har-land). Similar preparation of tissue was performedfor chorioallantoic membrane (CAM) grafts ofchick tissue. CAM Explants were treated withcyclopamine (10 mg/ml, gift from W. Gaffield), in1% DMSO/PBS, drop-wise on the surface of theexplant four times daily until harvest.

Skin explants for bead implant studies weremade from embryonic day 14 (E14) Pekin orKhaki Campbell duck rectrices (tail feathers), andE10 chick dorsal metatarsal skin and placed inPBS. Size selected heparin acrylic beads loadedwith recombinant proteins were placed in aforming feather or forming scale and grafted onto the CAM of chick hosts until harvest. The beadswere preincubated for 1 hour in recombinanthuman BMP2 protein (1mg/ml, gift from GeneticsInstitute), N-SHH protein (1mg/ml, gift fromP. Beachy), and Noggin protein (300mg/ml).

Viral mediated overexpression

RCAS virus constructs were grown and concen-trated as described (Morgan and Fekete, ’96).RCAS-caPKA was a kind gift of Drs. S. McKnight,Y. Brun, and B. Olwin; RCAS BMPR1b (K231R),described by (Zou and Nisewander, ’96), was a giftfrom Dr. J. Lough. All virus infections were intothe amnionic fluid of a stage HH31 SPAFAS chickto maximize expression only to the epidermallayers of feather follicles (Morgan et al., ’98).

M.P. HARRIS ET AL.162

RESULTS

Feather structure and development

Feathers are branched integumentary appen-dages. A typical feather is characterized by acentral rachis, a series of barbs connected to therachis, and numerous barbules connected to thebarbs (Fig. 1M). Feather and scutate scale devel-opment (reviewed in Lucas and Stettenheim, ’72;Sawyer, ’72; Prum, ’99) begin with a thickenedepidermal placode. In feathers, distal outgrowth ofthe placode leads to the formation of a nascentfeather bud, which elongates into a tubularstructure called the feather germ. Subsequently,feather barbs develop by longitudinal compart-mentalization of the feather germ epithelium intobarb ridges, that become the barbs, or primary

branches, of the feather (Fig. 1M). In plumulac-eous feathers (e.g., down feathers), barb ridgesgrow longitudinally within the feather germ andhave a rudimentary rachis. In pennaceous feath-ers (i.e., feathers with a prominent rachis and aplanar vane), the barb ridges grow helicallyaround the tubular feather germ and fuse at thedorsal surface of the feather germ to form therachis. Animations of both plumulaceous andpennaceous feather growth and developmentmay be viewed at http://fallon.anatomy.wisc.edu/feather.html. The evolutionary innovations in-volved in the development of a feather from anarchosaurian scale have been proposed to behierarchically dependent (Prum, ’99). Specifically,a tubular outgrowth necessarily precedes the sub-division of the tubular epidermis into barb ridges,and the formation of barb ridges necessarily

Fig. 1. Whole mount in situ hybridization (WMISH) ofShh and Bmp2 in developing feathers and scales of thechick. Panels (A–B) and (E–F) are oriented with rostral atthe top, and (C–D) and (G–H) are oriented with distal axis atthe top. Expression of Shh and Bmp2 is polarized withinindividual scale (A–E) and feather (B–F) placodes of an E11chick shank and E10 pteryla, respectively. Shh is expressed ina posterior domain and Bmp2 is expressed in an anteriordomain within each placode. As feather buds grow distally,Shh (C) and Bmp2 (G) expression is localized to the distaltip of the growing feather germs. Subsequently, Shh (D) andBmp2 (H) expression in feather germs becomes refined intolongitudinal stripes along the proximal-distal axis of thefeather. Histological sections through elongated feathershybridized to Shh (I) and Bmp2 (J) show that Shh isexpressed throughout the folded marginal plate epithelium

(arrowheads) of the forming barb ridge (outlined with dottedline), but that Bmp2 expression is restricted to the peripheralfolds. Histological sections through elongated feather germshybridized to the Shh regulated genes Ptc (K) and Ptc2 (L)show that genes regulated by Shh are expressed only in theperipheral sections of the marginal plate. (M), Schematic ofthe structure of a developing feather germ showing formingbarb ridges (br) and expression domains of Shh and Bmp2within the marginal plate epithelium (mp) of a single barbridge. The polarity of expression of Shh and Bmp2 expressionin the central to peripheral marginal plate epithelium isillustrated in a gradient model (bar). The barb ridges becomethe formed barbs (b) and barbules (bbl) of the feather. Therachis (r) forms through fusion of the barb ridges as they growhelically around the circumference of the feather(see Fig. 4F–J).


precedes the formation of the rachis by the fusionof barb ridges. Further innovation of barbuleswould lead to the formation of the closed, planar,pennaceous feather.

Shh and Bmp2 expression polarityin archosaur placodes

To investigate the molecular mechanisms in-volved in the morphogenesis and evolution ofbranched feather structure, we examined thedevelopment of integumentary appendages ofextant archosaurian lineages represented by thechick (Gallus gallus), duck (Anas platyrhynchus),and alligator (Alligator mississippiensis). Througha comparative analysis of the molecular mechan-isms of integumentary appendage developmentwithin the chick, other species of birds, orphylogenetically related archosaurs, we can beginto ask how change in developmental mechanismshave facilitated the formation of morphologicalcomplexity during feather evolution.We centered our analysis on two patterning

genes, Shh and Bmp2, that we found to be closelylinked in epithelial appendage morphogenesis.Previous studies outlined the expression domainsof Shh and Bmp2 in early feather development(Nohno et al., ’95; Ting-Berreth and Choung, ’96;Noramly and Morgan, ’98). We extended theseanalyses by demonstrating that the timing andpolarity of expression of these two genes areconserved in development of placodes of feathersand scales in the chick, and that this initialpattern of expression is conserved in the placodesof the integumentary appendages of chick, duck,and alligator.Initially, in the chick, Bmp2 is expressed

throughout the forming placode of feathers of thechick and its expression subsequently becomespolarized as Shh is expressed in the placode (datanot shown) (Noramly and Morgan, ’98). Shh hasbeen shown to be expressed in a posterior domainwithin the thickened epithelial placode of avianscutate and scutellate scales (found on the tarsiand dorsal surface of the feet), and in the posteriordomain of feather placodes (Figs. 1 and 2) (Nohnoet al., ’95; Ting-Berreth and Choung, ’96). At thesame stages of appendage development, Bmp2 isexpressed along the anterior border of the epithe-lial placode of both scales and feathers (Figs. 1 and2) (Morgan et al., ’98). Thus, the expression of Shhand Bmp2 in scale and feather placodes of thechick exhibits conservation of timing and anterior-

posterior polarity of Shh-Bmp2 expression withinthe epithelial placode (Fig. 1A,B,E, and F).

In order to see if these mechanisms areconserved among archosaurs, we analyzed theexpression of Shh and Bmp2 in the embryonicintegumentary appendages of the duck and alli-gator. Analysis of Shh and Bmp2 expression informing duck scale and feather placodes showedsimilar expression polarity and timing as seen inchick embryos (Fig. 2). This suggests a conserva-tion of the spatial and temporal regulation of Shhand Bmp2 expression in early feather and scaledevelopment among birds. Alligators are membersof the extant sister group of birds, and they havekeratinized integumentary scales that are homo-logous with those of birds. Alligator scales exhibita distinct scale ridge and morphological polaritysimilar to avian scutate scales (Ferguson, ’85;Alibardi and Thompson, 2000). Inter-specifichybridization of chick antisense RNA probes forShh and Bmp2 showed specific hybridization tothe scale placodes on the ventral surface of the tailof stage 21 alligator embryos. The expression ofShh and Bmp2 exhibited the same anterior-posterior polarized pattern seen in both duckand chick (Fig. 2A,F).

Collectively, these data suggest that expressionof Shh and Bmp2 was regulated in a similarfashion in the scales of the common ancestor ofbirds and crocodilians, and that this plesiomorphicexpression pattern has been maintained in theinitial placode stages of feather bud morphogen-esis (Fig. 2K).

Novel expression of Shh and Bmp2 duringfeather development

As feather development proceeds, the featherplacode grows into a conical bud, and patternedsubdivision of the conical epithelium of the feathergerm forms the barb ridges that become thefeather barbs and barbules. These morphogeneticprocesses and the structures formed are evolutio-narily derived and unique to feathers (Prum, ’99).The developmental transition from placode to theoutward growth of the feather bud is correlatedwith co-expression of Shh and Bmp2 in the distalfeather bud epithelium as shown by sectioning(data not shown; Fig. 1C,G); co-expression is alsoseen in the early feather placode, and precedesformation of polarized expression of Shh andBmp2 (Morgan et al., ’98). Subsequently, theexpression of Shh and Bmp2 becomes subdividedinto longitudinal, or proximal-distal, stripes that


are parallel to the primary growth axis of thefeather bud (Fig. 1D,H). These longitudinaldomains of Shh and Bmp2 expression mark theboundaries of the forming barb ridges as thetubular epithelium of the feather germ foldsinward to compartmentalize portions of theepidermis into distinct barb ridges (Fig. 1M).The expression of Shh and Bmp2 is identical inthe embryonic natal down of chicken and ducks(data not shown), suggesting that the regulation ofexpression in this novel context is common tonatal feathers.Shh is expressed in the folded basal layer of the

feather epidermis between barb ridges, which iscalled the marginal plate (Nohno et al., ’95; Ting-Berreth and Choung, ’96). Shh expression firstappears peripherally and spreads into two stripesalong the juxtaposed basal layers of neighboringbarb ridges (arrowheads, Fig. 1I,M; and data notshown). The expression of Bmp2, however, is

restricted to the peripheral folds of the marginalplate (Fig. 1J,M). The expression of the Shhreceptors Ptc, Ptc2 (Pearse et al., 2001) (Fig.1K,L) and the down stream transcriptionfactor, Gli-1 (data not shown), which are tran-scriptionally regulated by Shh signaling, alsoexhibit a peripheral bias in their expression inthe feather germ as the formation of the barbridges proceeds.

Therefore, competence to respond to Shhsignaling within the marginal plate epithelium islocalized to the peripheral epidermis (Fig. 1M). Asimilar restriction in the competence to respond toShh signaling is seen in the placode stage offeather development and may have a role inestablishing axial polarity within the epithelium(Morgan et al., ’98).

Barb ridges have an intrinsic polarity; theramus, the core filament, is formed centrally andbarbules differentiate towards the periphery of the

Fig. 2. Comparative phylogenetic analysis of Shh andBmp2 expression in placodes of integumentary appendages ofarchosaurs. (A, F), Expression of Shh and Bmp2, respectively,in the ventral tail epidermis of the embryonic alligator(Alligator mississippiensis). Gene expression is shown byWMISH using interspecific hybridization with chick probesfor Shh and Bmp2. Picture insets in A and F are highermagnification images of the alligator tail shown. Arrows pointto polarized expression of Shh or Bmp2 in forming scalerudiments. The expression of Shh (B–E) and Bmp2 (G–J) in:E15 Pekin duck (B, G) and E10 chick scutate scales (D, I); and

from E15 duck (C, H), and E10 chick (E, J) feather pteryla.Dotted lines in (C) and (E) outline the forming feather placodecontaining Shh expression. (K), Phylogeny of the archosaur-ian orders (a) Crocodylia (alligator), (b) Anseriformes (duck),and (c) Galliformes (chick) showing the conservation ofanterior-posterior expression polarity within the epidermalplacodes of their integumentary appendages. The transcrip-tional regulation of both Shh and Bmp2 from the ancestralarchosaurian scale placode has been conserved in the derivedfeather placodes of birds. The n indicates the origin of feathersin an ancestor of the avian clade.


barb ridge. Polarized Shh and Bmp2 expression inthe marginal plate, which is the folded portion ofthe basal epithelium, may function in formationand maintenance of central-peripheral polaritywithin the barb ridges themselves.

Shh-Bmp2 prepattern and the developmentof branched feather morphology

Natal down of the chick exhibits stereotypicalvariations in barb branching (Watterson, ’42), butthe developmental basis for these morphologicalvariations are not known. We found that thelongitudinal domains of Shh and Bmp2 expressionalong the feather germ show patterns that presagethese variations in down morphology.

The longitudinal Shh expression domains de-monstrate four distinct variations from simplelinear propagation of the stripes at the base of thefeather germ: bifurcation, termination, de novoinitiation, and fusion (Fig. 3A–E). Because theselinear domains of Shh expression mark themarginal plate epithelia that border neighboringbarb ridges, the specific variations in Shh expres-sion represent four distinct mechanisms of barbridge morphogenesis: Shh expression domainbifurcation yields new barb ridge formation (Fig.3A,F); expression domain termination leads tobarb ridge fusion (two barbs connected to a singlebasal barb) (Fig. 3B,G); de novo domain initiationof Shh expression domain yields division of a barbridge into two (two barbs connected by a single

Fig. 3. Variation in patterns of Shh expression correlatewith the phenotypic variation in natal down of chick. (A–E),Feather germs of comparable stage from the femoral tract of astage HH35 White leghorn chick embryo illustrate fourvariations in the propagation of longitudinal Shh prepatternin feather germs within a single tract of an embryonic chick.(A) Bifurcation of Shh expression domain; (B) Termination ofShh expression domain; (C) Fusion of Shh expressiondomains; (D) de novo initiation of new Shh expressiondomain; and (E) Simultaneous cessation and initiation events.These longitudinal Shh expression domains indicate thefolded marginal plate epithelium that differentiates neighbor-

ing barb ridges. (F–J), Each class of Shh expression patternobserved corresponds to an observed phenotype in day-oldchick natal down. (F) New barb ridge formation resulting inaddition of a barb; (G) Barb fusion in which two barbs areconnected proximally to form a single branched barb; (H)Barb loss in which a free barb is unconnected to the rest of thefeather; (I) Barb division in which a single barb splitsproximally into two basal bars; and (J) Simultaneous barbfusion and barb division. Evidence of barb loss was found inensheathed down feathers of newly hatched chicks. In G, I,and J, the barb shown has been removed from the downfeather to illustrate the phenotype of interest.


distal tip) (Fig. 3D,I); and fusion of Shh expressiondomains leads to barb ridge loss (a free barbunconnected to the rest of the feather) (Fig. 3E,J).We found four classes of feather phenotypes innatal down from the femoral tract of newlyhatched chicks that correlate precisely with theobserved pattern variation of Shh expression inthe feather germs of the embryonic femoral tract(Fig. 3F– J). The longitudinal domains of Bmp2expression in the feather germ show identicalpattern variations to those seen with Shh (datanot shown). The correlation of the observed Shhand Bmp2 expression pattern and the branchingphenotypes of chick natal down suggests thatregulation of Shh and Bmp2 expression andfunction is a critical component for the formationof the distinct and varied branching phenotypes offeathers. Experiments testing the necessity of Shhand Bmp2 signaling in barb ridge formationsupport this hypothesis (see below).In the duck, most of the first embryonic feathers

to grow are pennaceous (i.e., they have a promi-nent rachis and planar vane), whereas embryonicfeathers in a chick are plumulaceous [i.e., theyhave a minimal rachis and no planar vane (Lucasand Stettenheim, ’72)]. The natal down feathersfrom chick and duck, which have these majorstructural differences in branching morphology,also exhibit the specific, predicted variations inShh expression. Serial histological sectionsthrough an E20 duck rectrix (tail feather), whichhas a prominent rachis, document that the rachisforms by the fusion of barb ridges, beginning atthe periphery and proceeding towards the interior,as they grow helically towards the dorsal surface(Fig. 4F– J). The longitudinal Shh expressiondomains along the dorsal surface of the feathergerm of E16 duck rectrices show a gradualdecrease and cessation of the Shh expressionstripes which precede the initial barb ridge fusionevent that forms the rachis ridge and thesubsequent fusions of adjacent barb ridges to therachis ridge (Fig. 4K). In serial histologicalsections, these fusion events are correlated witha decrease in Shh expression that proceeds in aperipheral to central manner (Fig. 4L–N) as inbarb ridge formation (Fig. 4B–E). These resultssuggest that the longitudinal Shh expressiondomains constitute a molecular prepattern forthe development of the barb ridge branchingpattern in feathers.Helical growth of barb ridges, the formation of a

rachis, and the pennaceous vane are all correlatedwith the formation of a ventral locus of new barb

ridge formation (Lucas and Stettenheim, ’72). Aspredicted by variations in longitudinal Shh ex-pression domains in plumulaceous chick down, theventral locus of new barb formation is identifiableearly in the pennaceous duck rectrix by therepeated, localized bifurcation of Shh expressiondomains on the ventral surface of the featherfollicle (Fig. 4P). An analysis of cell division inembryonic duck down shows a localized focus ofcell division (as detected by staining for the mitoticepitope, phosphorylated histone H3 (p-H3) (Hend-zel et al., ’97) on the ventral surface of feathergerms at the site of new barb formation (Fig. 4S).

Interestingly, SHH protein immunoreactivity isalso localized to the ventral region of new barbridge formation, suggesting an additional functionfor Shh signaling in initiation of helical growth,which is a signaling event independent of expres-sion in barb ridges (Fig. 4T). The dorsal-ventralpolarized organization of embryonic duck featherscontrasts with chick natal down in which barbaddition is random around the circumference ofthe feather germ after the origin of the firstcomplement of barb ridges (data not shown;Watterson, ’42). Chick down also has localizedfoci of cell division and SHH protein expression,but notably without a ventral bias (Fig. 4U–V).

In summary, longitudinal expression of Shh andBmp2 in the developing feather germ prepatternsbarb ridge morphogenesis, exhibiting variationsthat mark a variety of branched barb mor-phologies in plumulaceous down. Coordinatedutilization of two of these prepattern mechanisms,i.e., dorsal serial Shh-Bmp2 stripe cessationcoordinated with rachis formation and barbridge fusion, and repeated Shh-Bmp2 stripebifurcation ventrally resulting in new barbridge formation, produces helical growth of barbridges, the rachis, serial barb fusion, the ventralnew barb locus, indeterminate barb number, andthe planar vane that characterize pennaceousfeathers. Thus, control of Shh-Bmp2 expressionduring feather morphogenesis is a fundamentalcomponent of the mechanisms determining feath-er form (e.g., plumulaceous and pennaceousstructure).

Shh and Bmp2 signaling integrationand function

The expression patterns of Shh and Bmp2shown above suggest that the functions of thesetwo genes are related during feather and scalemorphogenesis. Previous studies in the develop-


ment of integumentary appendages of amniotesindicate that Shh maintains a consistent role incell proliferation and patterning, whereas epider-mal expression of Bmps are often associated withdifferentiation (Bitgood and McMahon, ’95; St-Jaques et al., ’98; Chiang et al., ’99; Jung et al.,’99; Park and Morasso, 2002). We suggest thatShh and Bmp2 signaling functions as a conserved,integrated signaling system, or module, in themorphogenesis of epidermal appendages. To testthis hypothesis, we analyzed the conservation of

function of Shh and Bmp2 during scale andfeather morphogenesis.

We tested the effect of Bmp2 on Shh function byplacing beads loaded with recombinant humanBMP2 (rBMP2) protein into follicles of duck E14rectrices and assayed for Shh expression afterthey were cultured on the chorio-allantoic mem-brane (CAM) of chick hosts. Treatment of featherbud explants with ectopic rBMP2 protein causedlocalized, reduced Shh expression within thefeather after 24 hours (Fig. 5A,B). Conversely,

Fig. 4. Shh signaling and the formation of featherbranched structure. (A), Close up of new barb ridge additionas indicated by division of longitudinal expression domain ofShh. (B–E), 10mm serial histological sections through a singlefeather hybridized for Shh showing the formation of a newbarb ridge from distal (developmentally oldest) (B) toproximal (developmentally youngest) (E) region. Formationof a new barb ridge progresses from peripheral to central. (F–J) Serial 10mm histological sections of an E20 duck rectrix (tailfeather) showing the formation of the rachis by fusion ofneighboring barb ridges to the presumptive rachis ridge onthe dorsal side of the feather germ (at the left). r, presumptiverachis; *, fusing barb ridge. Barb ridge formation shows aperipheral to central bias in morphogenesis as well. (K), Shhexpression in E14 duck rectrices showing prepattern of rachisformation by barb fusion to the rachis through cessation ofShh expression (arrowheads). (L–N), Serial 10mm histologicalsections of a E14 duck rectrix showing the pattern of Shhexpression during the initial formation of the rachis by fusion

of neighboring barb ridges from distal (L) to proximal (N).Cessation of Shh expression progresses from the periphery,inward (arrow heads). Formation of the rachis is accompaniedby helical growth of barb ridges. (O –P), Shh prepattern in apennaceous E16 duck feather germ demonstrates the ven-trally localized area of new barb ridge addition, indicated by aseries of bifurcations of Shh domains (P, arrows), juxtaposedto the dorsal barb ridge fusion, indicated by a series of Shhdomain cessations (O, arrowheads). (Q–R), This pattern ofhelical barb ridge growth, ventral new barb ridge addition,and dorsal barb ridge fusion is not seen in E13 chick remigeswhich have a plumulaceous structure and lack a prominentrachis (Q–R, dorsal and ventral views respectively). Inpennaceous duck feather germs, the foci of p-H3 (S) andSHH (T) are primarily seen on the ventral-proximal orienta-tion (92%, n¼36). In chick down (U–V), SHH protein andareas of cell division localize to discrete foci of E13 chicks inno particular orientation around the follicle.


treatment of feather bud explants in vitro with theBMP antagonist, Noggin, caused increased Shhexpression only within forming feather germs andnot in inter-follicular tissue (Fig. 5E,F). Thus,BMP2 is sufficient and necessary to negativelyregulate Shh activity within the forming feathergerm. Similar to our results in the feather germ,there is evidence that Noggin overexpression inthe epidermis of the early placode of the feathercan lead to the expansion of Shh expressiondomains leading to trilobed feathers (Noramlyand Morgan, ’98). Given the conservation ofexpression of Shh and Bmp2 between featherand scale placodes, we tested the effect of rBMP2protein on Shh expression in forming chick scalesto see if regulation of Shh may be similar to thatseen in the feather follicle and feather placode.Treatment of chick scutate scale CAM explantswith rBMP2 protein loaded beads suppressed Shhexpression in forming scale placodes demonstrat-ing conservation of the signaling integrationbetween Shh and Bmp2 in both integumentarystructures (Fig. 5C,D).

Several lines of evidence indicate that Shh playsan important role in regulating epidermal prolif-eration and patterning. Increased Shh signalingleads to epidermal overgrowth, or cancer (Dah-mane et al., ’97; Fan et al., ’97; Oro et al., ’97; Xieet al., ’98), and altered specification of epidermalcell fates (Rowitch et al., ’99). Additionally, Shh,and its Drosophila ortholog hedgehog are neces-sary for pattern formation and differentiationwithin epithelia of forming ectodermal appendages(Basler and Struhl, ’94; St-Jaques et al., ’98;Chiang et al., ’99). Given the conservation ofintegrated Shh and Bmp2 signaling documentedabove, we examined the role of Shh and Bmp2 inregulating the cell cycle and differentiation ofepidermis during feather morphogenesis.

Previous studies using ectopic expression of Shhin chick epidermis indicate that an increase in thelevels of Shh signaling lead to an overgrowth ofthe feather germ (Ting-Berreth and Choung, ’96;Morgan et al., ’98). Similarly, we report that duckfeathers treated with heparin beads loaded withN-SHH protein showed a distinct hypertrophy ofthe epidermis surrounding the bead (Fig. 6B).Analysis of explants 48 hours after bead placementshowed an increase of cells staining for the mitoticepitope p-H3, indicating that the phenotype is due,in part, to increased cell division (Fig. 5H).Feather follicles treated with beads loaded withNoggin protein showed an increase in p-H3labeling supporting the conclusion that BMP

Fig. 5. Shh and Bmp2 signal integration and effect on cellproliferation. (A–B), WMISH detection of Shh transcripts24 hours after bead implantation (circles) show a specificreduction of Shh expression by (B) rBMP2 (85%; n¼7)in embryonic feathers when compared to (A) PBS controlbeads (0%; n¼6); circles indicate location of bead. In asimilar experiment in embryonic scale rudiments, rBMP2treatment leads to a reduction of (D) Shh transcripts (80%;n¼5) when compared to (C) control (16%; n¼6). (E–F),WMISH detection of Shh transcripts in chick featherexplants treated with (E) PBS (0%; n¼6) or (F) Noggin(75%; n¼8) indicate that BMP signaling is necessary forregulation of the level of Shh transcripts in feather germs.(G–I), Increased Shh signaling in the feather by treatmentwith (H) N-SHH (100%, n¼4) or (I) Noggin (100%, n¼5)loaded on beads (arrows) leads to an increase in cell division asmeasured by detection of p-H3 when compared to (G)control beads (0%, n¼4). A cicle outlines the location of thebead in (G).


signaling abrogates Shh activity (Fig. 5I). Incontrast, beads loaded with rBMP2 protein in-duced a localized increase in epidermal differen-tiation of the feather follicle (Fig. 6C). This alteredhistology was demonstrated by a thickened stra-tum corneum around the bead. These data suggestthat there is a negative feedback control mechan-ism between Shh and Bmp2 signaling whichfunctions in the homeostasis of epidermal growthand differentiation during appendage develop-ment. Bmp2 regulates Shh expression in develop-ing feather germs and, thus, cell division. Inaddition, BMP2 treatment leads to an increase indifferentiation, which may be a consequence ofsuppressing cell proliferation, but Bmp2 may alsoaffect immediate downstream genes associatedwith differentiation [e.g., Dlx3 (Park and Morasso,2002)]. A recent report (Kulessa et al., 2000)showed similar effects of Noggin misexpression inhair follicles; they demonstrate a release ofinhibition on the expression of Shh as well asincreased proliferation in hair follicles. Along withour data, these observations raise the possibilitythat the regulatory mechanism seen in feathersand scales may be common to the whole class ofvertebrate epidermal appendages.Our data indicate that Shh and Bmp2 signaling

are integrated to control proliferation and differ-entiation within forming feathers and that nega-tive regulation of Shh expression, and hencefunction, by BMP signaling is conserved in theinitial development of scale and feather placodes.To test the role of these integrated signalingsystems on barb ridge morphogenesis, we usedantagonists of Shh signaling on feather explantsgrown in culture or grafted to the CAM of thechick. The alkaloid cyclopamine is a potentinhibitor of Shh signaling (Incardona et al., ’98).Cyclopamine treatment of feather CAM explantsaltered feather morphogenesis showing an inhibi-tion of epidermal invagination (a morphologicalindicator of barb ridge formation) in histologicalsections (Fig. 6D–G). A comparable result wasachieved by treating explants with the Shhblocking antibody 5E1 showing similar inhibitionof epidermal invagination (data not shown). Thesedata demonstrate that Shh signaling is necessaryfor barb ridge formation.To further investigate the effects of Shh and

Bmp2 on barb ridge morphogenesis, we used viralconstructs to locally increase the expression ofnegative mediators of both Shh and Bmp2 signal-ing within forming feathers. Protein kinase A(PKA) is a cAMP dependent kinase that has been

found to be a general inhibitor of Shh signaling inmany developing systems, including feathers(Hammerschmidt et al., ’96; Noveen et al., ’96).We found that areas of forced overexpression of aconstitutively active variant of PKA (RCAS-CaP-KA) in the epidermis of embryonic chick feathersled to aberrant feather growth demonstrated bysharp bends in the growing feather germ thatlocalized to foci of viral expression (Fig. 6I). Thesedeformed feathers showed a consistent alterationof cell differentiation within the epidermis thatlocalized to areas of viral infection. In histologicalsection, regional differentiation towards a ‘‘bar-bule-like’’ fate was seen in areas of viral infectionin a non cell-autonomous manner (see eosinophiliccells, white arrowheads, Fig. 6L). Parallel experi-ments using virus containing a dominant negativeBmpr1B variant that abrogates BMP signaling(RCAS-dnBMPR1B; Zou and Nisewander, ’96)resulted in feathers with bends in the follicle thatwere associated with an overgrowth of the folli-cular epidermis, similar to the overgrowths seenaround N-SHH loaded beads (Fig. 6J). Analysis ofhistological sections revealed that, in areas of viralinfection, a noncell autonomous effect on differ-entiation is evidenced by the presence of none-osinophilic ‘‘centralized’’ cells in the peripheralepithelium and/or heightened growth of thesheath ectoderm. In the dnBMPR1B infectedfeather, nuclei are seen throughout the epidermissuggesting an inhibition of cell differentiation inthe cornefied cell layers (Fig. 6M). These datademonstrate that Shh and Bmp signaling isnecessary for feather barb morphogenesis andsuggest that integrated Bmp2 and Shh signalingcontrols polarity and differentiation within theforming barb ridges (Fig. 6N). Interestingly,Haake et al. (’84) describe a peripheral to internalwave of differentiation in the forming feather withbeta keratin expression in peripheral domainssimilar to expression of Shh responsive genes thatwe report in this study.

We conclude that Shh and Bmp2 are anintegrated signaling system that mediates cellgrowth and differentiation of the feather. Earlypolarity in the expression of these signalingsystems may permit differential signaling withinepithelia. Bmp2 is expressed prior to Shh and maylimit the expression of Shh to the posteriordomain of forming placodes and to the centraldomains of barb ridge epidermis. Our resultssuggest that the effect of Bmp2 on Shh expression,and hence Shh function, permits controlledmorphogenesis of epidermal appendages.


Fig. 6. Conserved Shh and Bmp2 signaling modulecontrols proliferation and differentiation during feathermorphogenesis. (A–C), Hematoxylin and eosin stained histo-logical sections of Khaki Campbell duck rectrix explantstreated with beads containing (A) PBS (0%; n¼3), (B) N-SHH(75%; n¼4), or (C) rBMP2 (100%; n¼2) and grafted to thechorio-allantoic membrane (CAM) of chick hosts. The beadsare marked with an asterisk; bar equals 10 mm in all threehistological sections. Arrows in (C) point to areas of increaseddifferentiation of the stratum corneum. (D–G), Chick feathergerm explants to the CAM treated with (F–G) cyclopamineshow inhibition of feather morphogenesis in comparison to(D–E) diluent control. Analysis of histological sections shows aspecific inhibition of barb ridge invagination by (G) cylopa-mine (52%, n¼21; X2, Po0.05) when compared to (E) diluentgroup (9%; n¼21). Arrows point to forming barb ridges in afeather of control explant. (H–M), Phenotypes of chick nataldown expressing (H, K) RCAN control, (I, L) RCAS-caPKA,and (J, M) RCA-BMPR1B viruses. Foci of viral expressionwere detected by looking at viral GAG protein expression(stained purple). (I), RCAS-caPKA infection showed a reduc-tion of the filament circumference and bending of the feathergerm. (J), RCAS-dnBMPR1B infection foci exhibited proximalepidermal overgrowth as seen in (B) N-SHH bead studies. (K),Hematoxylin and eosin stained transverse histological sec-tions of RCAN control samples showed no specific change inphenotype that correlated with epidermal viral infectionshown by blue-purple NBT-BCIP precipitate (0%, n¼4) (rm,

barb ramus; bbl, barbules; bar equals 10 mm). (L, M),transverse histological sections of areas of bends associatedwith viral infection shows a specific alteration in ramus andbarbule morphology in both (L) RCAScaPKA (75%; n¼4) and(M) RCAS dnBMPR1B (100%, n¼4) infected feathers. (N),Model of the effect of altering Shh signaling on barb ridgepolarity. RCAN control (vector) infected barb ridges exhibitnormal polarity containing a central ramus (ovals, rm) andbarbules (red circles, bbl) positioned more peripherally. Thebars represent the levels of Shh signaling along the central toperipheral axis. In RCAN treated feathers, the normal leveland polarity of Shh signaling within the forming barb ridge ismodeled as demonstrated in WMISH analyses (Fig 1). Thedata from histological analyses support a model in which alocalized reduction of Shh signaling by CaPKA expressionleads to a ‘peripheralization’ of the forming barb ridge asevidenced by the central presence of eosiniphilic (tissuestaining red), ‘‘barbule-like’’ cells in the epidermis (redcircles). This effect of CaPKA expression on Shh signalingin the forming barb ridge is modeled as a loss of polaritywithin the marginal plate epithelium (bar schematic). Con-versely, a localized activation of Shh signaling in the barbridge (modeled in bar schematic) by abrogation of BMPsignaling (dnBMPR1B) leads to a ‘centralization’ in theforming barb ridge with an increase in noneosinophilic cells(clear circles and ovals) in the epidermis and a lack of properepithelial differentiation.


DISCUSSION

Previous studies have investigated the molecu-lar mechanisms of early feather and scale devel-opment at the level of the placode and feather bud.Here we show that Shh and Bmp2 expression andthe negative regulation of Shh by Bmp2 isconserved among feather and scale placode pri-mordia. We demonstrate that the general tempor-al and spatial regulation of Shh and Bmp2expression is also conserved in the formation ofalligator scales. Thus, the initial formation of theplacode and the molecular determinants of placodeformation are likely to be pleisomorphic withinarchosaurs. The extension of our study to encom-pass an analysis of the later and derived mechan-isms of feather formation has shown that thecomplexity of feather branched morphology stemsfrom the iterative expression and function of thepleisomorphic integrated signaling between Shhand Bmp2 in novel developmental contexts. Wepropose that the reuse of this signaling module tocontrol cell division and differentiation of epithelialed to the morphogenesis of novel integumentarystructures.Our observations suggest that changes in cell

division and cell differentiation under control ofan integrated Shh and Bmp2 signaling modulelead to discrete changes in feather morphology.Shh and Bmp2 are expressed in a prepattern thatis a prelude to the development of the barb ridgesin a feather germ, and their signaling is necessaryfor barb morphogenesis. It is a critical observationthat these expression domains exhibit variationswithin a feather germ that correlate specificallywith the variation in branching patterns seen inplumulaceous chick natal down and pennaceousembryonic duck feathers. In addition, we providethe first evidence of the mechanism of helicalgrowth and the differentiation of branched formbetween plumulaceous and pennaceous feathers.We show that differential spatial control of celldivision in feather morphogenesis, which is asso-ciated with the formation of plumulaceous andpennaceous feather morphologies, correspondswith localized regulation of SHH protein expres-sion. Variations in the rate of new barb ridgeformation, barb ridge diameter, and the angle ofhelical growth are hypothesized to be among thedeterminants of the shape of the pennaceousfeather vane (Prum and Williamson, 2001). Wepropose that regulation of the Shh-Bmp2 signalingmodule and thus, proliferative capacity, is in-volved in, and essential for, determining the

tremendous diversity of pennaceous feather sizesand shapes found in extant birds.

The polarized expression of Shh and Bmp2 inplacode epithelia is conserved across archosaurintegumentary appendages and this polarity isobserved in the marginal plate epithelia of formingfeathers as well. Although several signalingsystems are involved in specification of placodeasymmetry upstream of Shh and Bmp2, b-cateninmediated signaling may be a critical mediator ofthis early expression, as overexpression of tran-scriptionally activated b-catenin is sufficient toinduce expression of Shh and Bmp2 in featherplacode epidermis and to initiate the establish-ment of polarity in their expression (Noramlyet al., ’99). It is notable that the control of Shh andBmp2 expression in the marginal plate epitheliumof forming barbs is unlikely to be controlled by thismechanism, since b-catenin is expressed in com-plementary domains to Shh and Bmp2 within thebarb ridge (Widelitz et al., 2000). Thus, variationin upstream regulation of the expression of theShh -Bmp2 module is likely to be critical for theevolution of morphological diversity in branchedfeather form.

Interestingly, as we show in feathers, Shh isthought to act as a prepattern for tooth cuspformation and its expression is differentiallyregulated in teeth of varied morphologies (Jernvallet al., 2000). Shh and Bmp2 are both expressedwithin the dental epithelium during the formationof a tooth anlagen and within patterning of toothcusps. Shh signaling is sufficient for epithelialinvagination and is necessary for tooth initiationand patterning (Hardcastle et al., ’98; Dassuleet al., 2000; Cobourne et al., 2001). Shh is thoughtto regulate proliferation within the forming toothand Bmp2 is associated with regulating differen-tiation factors [e.g., MSX and p21 (Dassule andMcMahon, ’98; Jernvall et al., ’98)]. It is possiblethat the antagonistic relationship of Shh andBmp2 signaling is conserved in the formation ofmammalian teeth, however, existing data are notsufficient to make a clear assessment of integra-tive Shh and Bmp2 signaling within the formingtooth anlagen.

Based on our results, we hypothesize thatfeathers originated and diversified from a primi-tive archosaurian scale through the repeated co-option, or reutilization, of a plesiomorphic Shh-Bmp2 signaling module in new developmentalcontexts (Fig. 7). The elongate, tubular feathergerm evolved by the derived, distal co-expressionof Shh and Bmp2 that mediates the elongation of


the placode. Subsequently, the evolution of a novelpattern of longitudinal stripes of polarized Shh-Bmp2 expression resulted in the differentiation ofthe conical epithelium of the feather germ intobarb ridges, yielding the primary branched struc-ture of the feather. The differential expression ofthis signaling module in separate longitudinaldomains creates several inherently different pat-terns of barb ridge morphogenesis (Fig. 3A–E).Two of these, new barb ridge formation and barbridge fusion (Fig. 3A–B), produced potentiallyadvantageous morphologies that subsequentlycombined to create the pennaceous feather witha planar vane composed of a rachis and barbs.Our combination of comparative and experi-

mental investigations strongly support these evo-lutionary conclusions about integumentaryappendage evolution. The experimental data docu-ment that the Shh-Bmp2 signaling module isnecessary for appropriate feather germ, barb

ridge, and rachis morphogenesis. Thus, the evolu-tion of the novel expression patterns of thismodule was a necessary and critical componentof the evolution of a tubular feather germ andfeather branched structure. An evolutionary mod-el of the origin of feathers must incorporate theorigin of novel regulatory mechanisms of theexpression of the Shh-Bmp2module and its effectson controlled cell proliferation and differentiationwithin the epithelia of the forming integumentaryappendage.

Our data and model are consistent with thehypothesis that feathers evolved from scutatescales. The anterior-posterior polarized patternof Shh-Bmp2 expression that is shared by allarchosaur integumentary appendage rudimentssupports their homology (Fig. 2K). However, weemphasize that the feather and archosaurian scaleare homologous only at the placode stage. Essen-tially all feather structures formed after the

Fig. 7. Sequential redeployment of Shh-Bmp2 moduleduring the evolution of a feather. Congruence betweenpatterns of expression of the Shh-Bmp2 module in feathersand archosaurian scales (top), and the developmental theoryof the origin and early evolution of feathers (bottom) (Prum,’99). The Stages I-IIIa of the developmental theory of featherevolution are proposed to have evolved by novel regulation ofShh-Bmp2 module expression. Stage I– the first, elongatetubular feather evolved from a primitive archosaurian scaleby the derived distal Shh-Bmp2 co-expression (Event 1). StageII- the first, branched plumulaceous feather evolved by theorigin of derived longitudinal Shh-Bmp2 expression domains

(Event 2) that created differentiated filaments from thetubular epithelium of the feather germ. The central-periph-eral (c-ph) polarity of Shh-Bmp2 expression in the marginalplate epithelium between the barb ridges is shown in theinset. Stage IIIa evolved by the controlled dorsal (d) cessationand ventral (v) division of the longitudinal Shh-Bmp2expression domains (Event 3) producing helical growth ofbarb ridges, indeterminate barb number, a rachis, serialfusion of barbs to the rachis, and a planar vane. Subsequentevents in the development and evolution of feathers, e.g.,origins and differentiation of barbules, will require furtherinvestigation.


placode stage are evolutionary novelties that haveno homologs within archosaurian or reptilianscales. Nevertheless, while there is divergence instructure after the placode stage, the develop-mental module of integrated Shh and Bmp2signaling used in placode specification of thearchosaurian scale has been re-utilized in theformation of derived feather structures. Thus, thissignaling modularity provides an inherent homol-ogy of developmental process among archosaurianintegumentary appendages (Gilbert et al., ’96).The developmental model of the origin of

feathers hypothesizes that feathers originatedand diversified through a series of evolutionarynovelties in developmental mechanisms (Prum,’99). This theory predicts that the most primitivefeathers were undifferentiated tubular cylinders(Stage I), which were followed by a downy, basaltuft of barbs (Stage II), and subsequently, by apennaceous vane with a rachis and barbs (StageIIIa) (Fig. 7). The predicted morphologies andhierarchical sequence of the stages of the devel-opmental model are entirely congruent with themolecular developmental results presented here.Stages I, II, and IIIa are each associated with anevolutionarily novel pattern of expression of theShh-Bmp2 signaling module during development(Fig. 7). Furthermore, the contingency of long-itudinal Shh-Bmp2 expression patterns upon theprior formation of an elongate feather predictsthat an undifferentiated tubular feather wasevolutionarily primitive to a basally branched, ordowny, structure. Likewise, helical growth, rachisformation, and indeterminate barb number aredevelopmentally contingent upon the prior estab-lishment of these longitudinal Shh-Bmp2 expres-sion patterns. Therefore, our data support thepredictions of the developmental model (Prum,’99) that a less organized, tufted, downy feathermorphology preceded the origin of the pennaceousfeather with a rachis and a planar vane (Fig. 7).Recent paleontological discoveries have docu-

mented that feathers evolved in coelurosauriantheropod dinosaurs before the origin of birds(Chen et al., ’98; Ji et al., ’98; Xu et al., ’99; Jiet al., 2001; Xu et al., 2001; Norell et al., 2002).The morphologies of these primitive, nonavianfeathers support predictions of the developmentaltheory of feather evolution (Prum, ’99; Ji et al.,2001; Sues, 2001), and their phylogenetic distribu-tions provide an emerging picture of the earlyhistory of feather evolution (Sereno, ’99; Padian,2001; Prum and Brush, 2002). Our data suggestthat the evolutionarily novel patterns of Shh-

Bmp2 signaling hypothesized here were derived inthe late Jurassic within specific lineages of basaland higher coelurosaurian theropod dinosaursthat were ancestral to birds.

The evidence of the repeated evolutionaryutilization of integrated Shh and Bmp2 signaling,reflects an inherent potential of this developmen-tal signaling module to permit controlled morpho-genesis within epidermal appendages ofarchosaurs. Thus, the re-utilization of this signal-ing module would facilitate the evolution of thephenotypic diversity of epidermal structures inamniotes. Our data support the conclusion thatqualitatively distinct morphological evolutionarynovelties can originate from the expression ofplesiomorphic molecular signaling systems in anovel context. Further, we present a mechanisticscenario for how the redeployment of an inte-grated signaling module can foster the evolution ofnovel morphological structures.

ACKNOWLEDGEMENTS

We wish to thank Ruth Elsey for her assistanceand expertise in collecting the alligator embryos,and R. Sawyer and T. Glenn for their help infacilitating the alligator work. We thank S & REgg Farm (Whitewater, WI) for providing theBabcock White Leghorn Flock. Feather anima-tions were created by David MacDougal at theUniversity of Wisconsin Department of MedicalIllustration. MPH would like to thank the supportby D. Maneval and A. Harris, and honor thememory of H. Pierce. The authors would like tothank the Fallon Lab and G. Wagner, K. Siegfried,D. MacGrogan, E. Haag, J. True, M. Christianson,and S. Blair for helpful comments on the manu-script. The research was supported by the Cremerfellowship from the UW Medical School to MPH.We especially want to thank the Florsheim familyfor their financial support of this –research.

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