Development 110, 1021-1030 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

1021

Gradients of homeoproteins in developing feather buds

CHENG-MING CHUONG1 *, GUILLERMO OLIVER2 t, SHEREE A. TING1,

BEATRICE G. JEGALIAN2, HAI MING CHEN1 and EDDY M. DE ROBERTIS2

1 Department of Pathology, University of Southern California, School of Medicine HMR, 2011 Zonal Avenue, Los Angeles, CA 90033, USA2Department of Biological Chemistry, University of California Los Angeles, School of Medicine, Los Angeles, CA 90024, USA

* Author for correspondencet Present address: Facultad de Humanidades y Ciencias, Dept de Bioquimica, Tristan Narvaja 1674, Montevideo, Uruguay

Summary

Homeoproteins are functionally involved in patternformation. Recently, homeoproteins have been shown tobe distributed in a graded fashion in developing limbbuds. Here we examine the expression of homeoproteinsin chicken feather development by immunocytochemicallocalization. We find that XlHbox 1 antigen is present incell nuclei and is distributed in a gradient in themesoderm of developing feather buds, with strongestexpression in the anterior-proximal region. The gradi-ent is most obvious in feather buds from the mid-trunklevel. Feather buds from the scapular level express veryhigh levels of XlHbox 1 and feather buds from the caudalregion express no XlHbox 1, suggesting that a broad

gradient along the body axis is superimposed on asmaller gradient within each individual feather bud.Feather ectoderm also expresses XlHbox 1 antigen butwithout an obvious graded pattern. Another homeopro-tein, Hox 5.2, is also expressed in developing featherbuds in a graded way, and its distribution pattern ispartially complementary to that of XlHbox 1. Theseobservations suggest that homeoproteins may be in-volved in setting up the anteroposterior polarity of cellfields at different levels, first for the body axis, then forthe limb axis and finally for the feather axis.

Key words: homeoprotein, feather, pattern formation.

Introduction

Pattern formation is the process by which specificstructures of the correct shape, size, position andorientation are generated. It has long been suggestedthat morphogenetic 'fields' and 'gradients' contribute tothe establishment of patterns (Huxley and De Beer,1934; Weiss, 1939; Wolpert, 1969; Malacinski andBryant, 1984), but their molecular basis remainsunknown. In Drosophila, homeobox genes are involvedin pattern specification and are usually expressed indefined bands along the anteroposterior (A-P) axis(reviewed by Lewis, 1978; Gehring, 1987). Using lowstringency hybridization, homeobox genes have beenisolated from vertebrates (Carrasco et al. 1984) andshown to have position-specific expression patternsalong the body axis (reviewed by Holland and Hogan,1988; De Robertis et al. 1990). Recently, XlHbox 1protein was shown to be distributed in a gradient indeveloping limb buds with strongest expression in theanterior and proximal region of the forelimb bud(Oliver et al. 19886). Another homeobox gene product,called Hox 5.2, is also expressed as a gradient of nuclearprotein in developing limb buds, but with the oppositepolarity to that of XlHbox 1 (Oliver et al. 1989). Inaddition, there is orderly and sequential expression of

genes of the Hox-5 complex along the distal to proximalaxis of the limb, which correlates precisely with the 5' to3' order of these genes in the genome (Dolle et al.1989). Therefore, the same family of genes that controlsthe A-P body axis appears to also regulate limb axisformation.

Feather development is an excellent model to studythe inductive processes that lead to the generation ofcomplex structures because of their distinct patterns,accessibility to experimentation (reviewed by Lucusand Stettenheim, 1972; Sengel, 1976) and availability ofmutants (Goetinck and Abbott, 1963). Feather budsexpress clear anteroposterior polarity and, althoughmesenchymal cells within a feather bud appear to besimilar, there are molecular heterogeneities which areposition-specific. For example, N-CAM is concentratedin the anterior part of the bud mesoderm (Chuong andEdelman, 1985a) while fibronectin is enriched in theposterior part of the bud mesoderm (Mauger et al.1982). Because of the recent findings suggesting thathomeoproteins are involved in determining patterns invertebrates in several instances (Ruiz i Altaba andMelton, 1989; Kessel etal. 1990; Wright et al. 1989), weasked whether homeoproteins are also involved infeather pattern formation. First, are there homeopro-tein gradients in feather buds? If so, are these gradients

1022 C.-M. Chuong and others

reflecting the position the feather occupies along thebody axis or the orientation of the feather axis?

In this study, we examined the distribution ofhomeoprotein XlHbox 1 during feather developmentand found that there is indeed a graded distribution ofXlHbox 1 protein within individual developing featherbuds. We compared the distribution of XlHbox 1 withthat of Hox 5.2 antigen and found a partiallycomplementary pattern. The polarized distribution ofN-CAM in feather buds was compared to the XlHbox 1gradient, and a partially overlapping, but clearlydistinct, expression pattern was observed.

Materials and methods

AntibodiesRabbit antibodies to XlHbox 1 (or against its humanhomologue Hox 3.3.) and Hox 5.2 were made against fusionproteins and affinity purified as described in Oliver et al. 1988aand 1989. Rabbit antibodies against chicken N-CAM wereprepared as described in Chuong and Edelman, 1985a.

ImmunostainingImmunostaining was processed as described in Oliver et al.1988a. Briefly, chicken embryos were staged according toHamburger and Hamilton (1951) and skin from differentlocations was dissected. Specimens were fixed in Bouin'sfixative, embedded in paraffin and sectioned at 6^m. Afterde-paraffination, sections were incubated overnight withprimary antibodies, followed by alkaline phosphatase-conju-gated goat anti-rabbit antibodies (Promega). NBT and BCIP(Promega) were used as substrate, with a positive reactionresulting in purple color.

Although endogenous alkaline phosphatase activity existsin the dermal condensation and feather buds (Hamilton andKonig, 1956) and we did observe it in our frozen sections ofembryonic chicken skin, the paraffin embedding procedurewe used completely abolished the endogenous alkalinephosphatase activity. This is most obviously shown in Fig. 3F,in which no dermal reactivity is seen after long incubation(lh) with substrate.

Chicken embryosFertilized eggs were obtained from Red Wing Farm (LosAngeles) and kept in an egg incubator (Humidare). Weinitially used White Leghorn chicken eggs in these studies.Later, we also examined a different strain (Sex-linked) ofchicken embryos, and obtained the same results. Thus theexpression is not specific to a particular strain.

Results

Normal feather developmentThe process of feather formation in the chicken embryois briefly summarized in Fig. 1. For detailed descrip-tions, please refer to Lucus and Stettenheim (1972),Sengel (1976), Sawyer and Fallon (1983), and Mayersonand Fallon (1985). Feathers start as a flat sheet ofectoderm (Fig. 1A). The underlying mesoderm, calledthe feather field (according to the concepts of Weiss,1939), acquires inductive properties and interacts withthe ectoderm, initiating the differentiation of theepithelial feather placode (Fig. IB). The placodeepithelium undergoes rapid cell proliferation, while themesenchymal cells beneath it increase by cell recruit-ment, as well as by cell proliferation (Chevallier, 1977;Gallin et al. 1986). Thus the growing feather bud hasectodermal and mesodermal components (Fig. 1C).The whole bud then invaginates into the skin to formthe feather follicle (Fig. ID) with the mesodermal coreforming the dermal papilla and the feather pulp, andthe bud epithelium becoming the collar and the featherfilament. The collar is the induced epithelium wherenew epithelial cells are added to the growing feather(Fig. IE). Therefore, in the feather filament, the tip ismore mature than the base. The epithelium sheetinvaginates and fuses, forming the alternating marginalplates and barb plates (Fig. IF). The fused marginalplate cells later die, generating the space between barbplates. Most of the barb plate epithelium becomeskeratinized and forms the feather barbs themselves,generating the branched pattern of the mature feather(Fig. 1G).

A. Ectoderm G. Mature Feather F.

- rachis

-barb

pulp.

Feather Filament

marginalplate

B. Placode

C. Feather Bud

E

Pr

dermalpapilla

Fig. 1. Schematicrepresentation of featherdevelopment. Counter-clockwise. In the beginning,there is just a flat piece ofectoderm (A). After induction,the feather placode and dermalcondensation form (B). Bothectoderm and mesodermcomponents increase in sizeand protrude from the skin,forming the feather bud (C).Later, it invaginates into theskin to form the feather follicle(D). Cross sections at thedermal papilla level andfeather filament level areshown in E and F separately.The feather filament eventuallybecomes the mature feather(G).

Homeoprotein gradient in feather buds 1023

In adult feathers, the barbs (secondary branches)grow outward from the rachis (the primary branch,Fig. 1G), which is located in the anterior end of thefeather bud. This side of the bud forms an obtuse angleto the body surface, while the posterior end forms anacute angle (Fig. 1C). It is important to note thisbecause the anterior of the feather bud, the anterior ofthe body, and the anterior of the limb can point todifferent directions as development advances.

A homeoprotein gradient in the feather buds, whichvaries along the body axisTo examine homeoprotein expression in feather budson the trunk along the anteroposterior axis, weprepared mid-sagittal sections of chicken trunks fromstage 31 to stage 36 (embryonic days 7 to 10), whenfeather buds develop on the trunk. The sections werestained with anti-XlHbox 1 and an interesting patternwas observed (Figs 2, 3). At stage 31, XlHbox 1expression in the mesenchymal cells of the formingdermis was graded along the anterior-posterior (A-P)axis of the body, with a sharp anterior border of intensestaining and gradually decreasing amounts in moreposterior regions (Figs 2B, 3A-C). This is reminiscentof the graded mesodermal pattern of XlHbox 1 protein

A

D

A • 1 * PV

B

observed in zebrafish embryos (Molven et al. 1990). Atstage 34, the feather buds have formed. Feather budsfrom anterior parts of the trunk expressed high amountsof • XlHbox 1 antigen. This expression graduallydecreased toward the mid-trunk level and eventuallydisappeared in the buds from the caudal part of thebody (Figs 2C, 3D-F). The most interesting obser-vations came from mid-trunk region buds. Each ofthese expressed XlHbox 1 as a gradient, with higheramounts toward the anterior and proximal region of thebud (Fig. 3E). Mesodermic nuclei of feather buds fromthe scapular level expressed XlHbox 1 antigen sostrongly that it is difficult to distinguish whether itsdistribution is graded or not (Fig. 3D). The patternremained the same when we used excess amount ofantibody to XlHbox 1, suggesting that the pattern is notdue to insufficient amount of antibody. The unstainedregions in the central cores of more mature feather buds(Fig. 3D,G) are newly formed blood vessels. Featherbuds from the caudal regions expressed no XlHbox 1 inthe mesoderm (Fig. 3F).

Thisvariation in XlHbox 1 protein content in featherbuds along the A-P axis of the body is not due todifferences in the timing of A-P maturation of trunkfeathers, which is known to proceed in an orderly way

Ca

Fig. 2. Low power view ofXlHbox 1 homeoprotein gradientin developing feather buds alongthe body axis. (A) Schematicdrawing showing the orientationof the body (A, anterior; P,posterior; D, distal; V, ventral)and the corresponding positionsfrom where higher magnificationswere derived. Squares a (scapularlevel), b (mid-trunk level), c(caudal region), correspond topanels A,B,C of Fig. 3. Sagittalsections of stage 31, 34, 36chicken embryo are shown in B,C, D. Note that XlHbox 1expression is graded along theanterior-posterior axis of thebody with the highest levels in theanterior body (B). As featherbuds develop, the buds from thescapular region express XlHbox 1throughout the mesoderm.Several buds in a row from themid-trunk region express anXlHbox 1 gradient with higherlevels in the anterior andproximal region of the featherbuds (C,D, arrows). Tail featherbuds even at this late stage donot express XlHbox 1 (D).Triangular, regions of artifacts.Bar, lmm. X6.25.

1024 C. -M. Chuong and others

A B

H

Fig. 3. High-power view of XIHbox 1 homeoprotein gradient in developing feather buds along the body axis. Sagittalsections of stage 31 (A,B»C)> 34 (D,E,F) and 36 (G,H.I) embryos. They are from scapular level (A,D,G), mid-trunk level(B,E,H), and caudal level (C,F,I) which correspond to squares a, b and c of Fig. 1A. In the feather placode stage, XIHbox1 appears evenly distributed under the placode whether they are from scapular or mid-trunk region, although higheramounts are observed in those from the scapular region (A,B)- The mesenchyme in the caudal region is devoid of XIHbox1 (C). In later stages, while feather bud mesoderm from the scapular level expresses XIHbox 1 intensely (D,G. The core offeather bud is not stained because it corresponds to the region of newly formed blood vessels and will become feather pulplater.), the feather bud mesoderm from the mid-trunk level displays a gradient of XIHbox 1 maximal at the antero-proximal ends of feather buds (E,H, curved arrows). Tail feather bud mesoderm does not express XIHbox 1 (F) even atlater stages (I), showing that the differences observed are not due to differences in feather maturation. The ectodermexpresses XJHbox 1 antigen in all regions. Some uneven distribution in epidermis was observed, but the precise patternawaits further study. In both epidermis and mesenchyme, XIHbox 1 protein has a cell nucleus staining pattern. 100 fim.X50.

(Mayerson and Fallon, 1985). This is known becausevery similar results were obtained when embryos ofolder stages were examined (Figs 2D, 3G-I). At stage36, mesoderm nuclei from feather buds at the scapularlevel still expressed high amounts of XIHbox 1. Thecore region of the buds was negative because it wasoccupied by blood vessels (Fig. 3G). The gradedpattern of XIHbox 1 within feather buds can be seen inseveral consecutive buds in Fig. 3H. Caudal feather

buds, although well developed, were still entirelydevoid of XIHbox 1 mesodermic staining (Fig. 31) evenat these late stages.

The epithelial layer overlying each feather bud waspositive for XIHbox 1 over the entire trunk region(Figs 2, 3). In some cases ectodermal expressionseemed to be stronger in the posterior portion of thebud epidermis, but the pattern was not clear- cut andmust await further studies. In both epidermis and

Homeoprotein gradient in feather buds 1025

B

Fig. 4. Generation of the XlHbox 1gradient in feather buds from a stage 33embryo. Both panels were from the sameembryo. Sagittal sections. (A) XlHbox 1is expressed in the dermal condensationbefore the placode morphology inectoderm is obvious (left feather germ inA). XlHbox 1 appears to behomogeneously distributed in the wholefeather field at this stage, but thedistribution gradually becomes polarizedtowards the anterior end of the feathergerm. At the feather placode stageshown in the right feather germ in A,XlHbox 1 is more enriched in theanterior half of the feather field (straightarrow) than the posterior half. (B) Asthe feather buds form, XlHbox 1becomes localized to the anterior (ant,small arrow) and proximal end of eachfeather bud (curved arrows). XlHbox 1 isalso present on the ectoderm. Staining isexclusively nuclear in both ectoderm andmesoderm. The affinity-purified rabbitantibody was make against XlHbox 1peptides derived from a cDNA clone ofhuman origin (Oliver et al. 1988a). Bar,100^m. X125.

mesenchyme, the subcellular staining pattern ofXlHbox 1 was nuclear. This is perhaps best seen inFig. 4A for epidermis and Fig. 4B for mesenchyme.The nuclear localization raises interesting questionsconcerning the nature of the intercellular communi-cation signals that establish a gradient of nuclearprotein along an apparently homogeneous expanse ofmesodermic cells.

We also examined the expression of XlHbox 1 infeather buds of the wing and observed an anterior andproximal gradient from the middle region of the wing.Feather buds from proximal regions of the wingexpressed a large amount of XlHbox 1 in themesoderm, while those from more distal regionscontained much less XlHbox 1 protein (data notshown). Thus, the expression pattern of XlHbox 1 inthe distal to proximal axis of the wing is analogous tothe pattern of feather buds along the A-P axis of thebody.

Generation of the XlHbox 1 gradient in feather budsIn this section we are solely concerned with develop-ment of feather buds from the dorsal feather area(Mayerson and Fallon, 1985) at the mid-trunk level,

which normally display a clear XlHbox 1 gradient. Thefirst indication of feather formation is the appearance ofXlHbox 1 antigen in discontinuous dermal conden-sations (Fig. 4A left) in the feather field. As far as wecan tell, these mesodermal changes precede anymorphological changes in the ectoderm. The XlHbox 1antigen is uniformly distributed throughout the featherfield, even as the ectodermal placode begins to form.When the placode is more developed, XlHbox 1 beginsto show higher expression in the anterior half of thefeather field (Fig. 4A, right). When a distinct featherbud forms, the graded expression in the anterior andproximal mesoderm becomes obvious (Fig. 4B, curvedarrows). Thus, apparently, the gradient is generated bydecreasing the amount of antigen in the posteriormesoderm.

Distribution of Hox 5.2 antigen in feather budsTo explore whether other homeoproteins also express asimilar graded distribution, we examined the expressionof Hox 5.2 (Oliver et al. 1989). Similarly to the situationin the limb bud (Oliver et al. 1989), Hox 5.2 is negativein the ectoderm, but an interesting pattern is observedin the mesoderm (Fig. 5). In developing feather bud

1026 C. -M. Chuong and others

*"<:

¥ 4

"**f-f^ - ; ^ :

*-&&$$&

Fig. 5. Distribution of Hox 5.2antigen in feather buds from astage 33 chicken embryo. In A,the feather mesoderm expressHox 5.2 homogeneously in theentire feather field. In B, thegraded distribution of Hox 5.2 isshown. In an adjacent section(C), the same two feather buds(pointed by curved and straightarrows) express a complementarydistribution of XlHbox 1 protein.The ectoderm is negative of Hox5.2. Note the nuclear stainingpattern of Hox 5.2. A, bar,100 fjm; X125. B,C, bar,x94.

mesoderm, Hox 5.2 antigen is expressed very stronglythroughout the feather field (Fig. 5A). Thus, allmesodermal cells in the feather field appear to expressboth homeodomain antigens at the early placode stage.As development proceeds, Hox 5.2 decreases inanterior and proximal regions of some feather buds(Fig. 5B, arrows), adopting a distribution that isopposite to that of XlHbox 1 (Fig. 5C). While thiscomplementarity is clear in the feather buds shownhere, in other buds it is only partial. This is due, at leastin part, to the fact that Hox 5.2 is expressed throughouttrunk as well as tail feathers, while the XlHbox 1gradient is present only in feather buds of anterior andmid-trunk regions. Further studies are required toelucidate the complex patterns of Hox 5.2 in differentfeather areas. However, we can conclude that at leastone other homeoprotein is expressed in the feather budin a graded pattern, which in some buds is opposed tothat of XlHbox 1.

Comparative distribution of XlHbox 1 and N-CAMIn our previous study, N-CAM was found to beexpressed in a polarized way toward the anterior part of

the feather bud (Chuong, 1985a). This posed aninteresting question as to whether cells expressingXlHbox 1 in the anterior part of the bud coincide withthose expressing N-CAM. We compared adjacentsections stained with anti-XlHbox 1 and anti-N-CAM(Fig. 6)'. N-CAM is expressed in the anterior portion ofthe bud mesoderm (Fig. 6A). In buds from the mid-trunk region, XlHbox 1 was expressed in some of thecells in the anterior bud mesoderm that expressedN-CAM, but the border of expression of XlHbox 1extended more posteriorly than that of N-CAM(Fig. 6D, curved arrow). The staining seen in theposterior mesoderm of the buds here corresponds to themost anterior region of the next feather bud(Fig. 6A,D, inserts). In other sections, when the twobuds were further separated, the enhanced expressionswere seen to be located solely in the anterior buds (e.g.Fig. 4B). N-CAM was present on all the feather budsregardless of their position in the body. Feather budsfrom the caudal region still express N-CAM in theanterior part of the bud but were totally negative ofXlHbox 1. This also served as a control, showing thatthe feather buds from the anterior part of the trunk arenot entirely different from buds of the posterior part of

Homeoprotein gradient in feather buds 1027

Fig. 6. Comparative distribution of XlHbox 1and N-CAM during feather development.(A,B,C) N-CAM; (D,E,F) XlHbox 1. (A,D)Stage 34, sagittal sections; (B,E) stage 41,sagittal sections of feather follicles;(C,F) stage 41, cross sections of featherfilaments. In the feather bud stage (A,D), N-CAM is present in the anterior (ant, straightarrow) feather mesoderm and part of theectodermal placode as described previously(Chuong and Edelman, 1985a). The adjacentsection shows that the distribution of XlHbox1 overlaps partially with that of N-CAM butthe two do not correlate exactly. Thepresence of XlHbox 1 extends to moreposteriorly than that of N-CAM (curvedarrow). N-CAM staining starts moreanteriorly than that of XlHbox 1. Inserts arethe lower power view which shows that thestaining in posterior buds is due to theextension of the anterior end of the nextbuds. In others when two buds are widerapart, the enriched XlHbox 1 is seen in theanterior end only (Fig. 4B). In the featherfollicle stage (B,E), N-CAM is concentratedin the dermal papilla (dp) and weaklyexpressed in the collar (cl). XlHbox 1 isenriched in the collar region but is alsoexpressed in more distal ectoderm. Insert ofE is the high magnification to show nucleusstaining pattern of XlHbox 1 in collar region(square). In the feather filament (C), N-CAMis present in the marginal plate (mp) andabsent in the barb plate (bp). An adjacentsection (F) shows that XlHbox 1 has acomplementary pattern: it is present in thebarb plate and absent in the marginal plate.A,D, bar, 100 fm; xl25. B,C,E,F, andinserts in A and D, bar, 100 fan; x50. Insertsin E, x250.

the body, at least as far as the expression of a differentmolecule, N-CAM, is concerned.

As the feather bud continued to grow in height andthe base of the feather bud begins to invaginate,forming the feather follicle, XlHbox 1 disappears frommesodermal cells but continues to be strongly expressedin nuclei of the collar region which is the ectodermalcomponent of the proximal part of the feather wherenew epithelial cells are produced (Fig. 6E). The nuclearlocalization of XlHbox 1 in feather filament epidermis

can be seen at higher magnifications (Fig. 6E insert).XlHbox 1 is negative in the mesenchymal components(dermal papilla and feather pulp) of the mature feather(Fig. 6E and F).

Interestingly, in the feather follicle stage, N-CAMand XlHbox 1 are expressed in a complementary way:N-CAM is concentrated in the dermal papilla (Chuongand Edelman, 19856), the inducing mesenchymalcomponent, while XlHbox 1 is absent from the dermalpapilla but present in the collar epithelium (compare

1028 C. -M. Chuong and others

Fig. 6B and E). In the proximal feather filament,N-CAM is present in the marginal plate cells (which aredestined to die, forming the space between featherbarbs) showing a dramatic spoke wheel pattern intransverse sections of the feather filament (Chuong andEdelman, 19856, Fig. 6C). In contrast, XlHbox 1 isabsent from the marginal plate cells but is present in thebarb plate cells (Fig. 6F). Thus there is a complemen-tary distribution pattern of N-CAM and XlHbox 1 inepithelia at later stages of feather development.

Discussion

In this study we provide evidence that the XlHbox 1homeodomain protein is expressed as an anterior (andproximal) gradient during development of chickenfeather buds of the mid-trunk region. This nuclearprotein gradient is similar to that previously reportedfor XlHbox 1 in a very different structure: the forelimbbud mesoderm of Xenopus, mice and chicks (Oliver etal. 19886, 1989). An anterior and proximal gradient isalso present in the developing zebrafish pectoral fin bud(Molven et al. 1990). The conservation of homeoboxproteins throughout vertebrate evolution enables oneto use polyclonal antibodies to study homologousproteins in a variety of species, as discussed elsewhere(Oliver et al. 1989; Molven et al. 1990).

The intensity of XlHbox 1 staining in the individualfeather buds varies with their position along theanteroposterior axis of the body. While feather buds inthe mid-trunk region show the aforementioned gradi-ent, those in the caudal region have no mesodermalstaining, and feathers in the anterior trunk haveXlHbox 1 antigen over most of the mesodermal region(Figs 2, 3). Thus, the amount of expression of thisantigen in feather buds reflects its distribution along thebody axis. Since feather formation follows a temporalsequence with anterior feather buds forming beforemore posterior ones in the dorsal feather area(Mayerson and Fallon, 1985), it was important todistinguish whether our observations were due toposition-specific expression of XlHbox 1 or to differentmaturation stages of feather formation. Time coursestudies indicated that the mesoderm of feather budsfrom the posterior body do not express XlHbox 1 at anypoint of their differentiation (Fig. 3C,F,I). Thus thegradient of expression of XlHbox 1 protein in featherbuds reflects a position-dependent property, not amaturation gradient that changes with time.

In the wing feathers, differences in expressionintensity are also observed, with mesodermal ex-pression being maximal in the proximal region andminimal at the distal tip. This is in agreement with thenormal distribution of XlHbox 1 in the mesoderm alongthe proximodistal axis of the forelimb (Oliver et al.1989). While the feather mesenchyme in the dorsal areaof the trunk is thought to derive from somitic mesoderm(demonstrated by transplantation experiments usingsomites labelled with [ H]thymidine or transplantedfrom Japanese quail embryos, Mauger, 1972), the

feather mesenchyme in the wing derives entirely fromthe somatopleure (Chevallier etal. 1977). Despite thesedifferent embryological origins, the XlHbox 1 gradientis present in feather buds in the trunk and in the wing.

It can be expected that other homeoproteins will alsobe expressed in feather buds. We have shown that asecond homeobox antigen, Hox 5.2, is intenselyexpressed in feather buds, with a distribution clearlydifferent from that of XlHbox 1. The distribution ofHox 5.2 antigen in some feather buds is complementaryto that of XlHbox 1 (Fig. 5B,C). This is reminiscent ofthe complementary distribution of these two proteinsduring development of the forelimb bud (Oliver et al.1989), in which XlHbox 1 protein is in the anteriorproximal bud while Hox 5.2 adopts an oppositegradient with a maximum in the posterior and distallimb bud. The recent observation that several genes ofthe Hox 5 complex are coordinately expressed in thelimb bud (Dolle et al. 1989) suggests that coordinatedexpression of other homeoproteins may also occur indeveloping feather buds. Presumably, in tail featherbuds, which are totally negative for XlHbox 1, differenthomeoproteins may adopt the role performed byXlHbox 1 in the mid-trunk feather buds.

Our previous work had shown that the cell adhesionmolecule N-CAM had a polarized expression in thefeather buds, localized in particular to the anterior andproximal region (Chuong and Edelman, 1985a,b). Thesimilarities of this distribution to that of XlHbox 1 inlimb buds provided the initial reason for examining theexpression of homeobox proteins during feather devel-opment. Careful examination of the pattern of N-CAMand XlHbox 1 expression (Fig. 6) has shown that, whilethe patterns partially overlap at the feather bud stage,they are distinct from each other. Furthermore,N-CAM has the same polarized distribution in featherbuds from all regions of the body, while XlHbox 1forms a gradient only in the mid-trunk region.

In the epithelial-mesenchymal interaction of featherformation, the pattern is determined by the mesenchy-mal components (Sengel, 1976). It would be interestingto explore what positional signals determine the extentof XlHbox 1 expression, how the homeoproteingradient on the feather buds is set, and how this relatesto the polarized expression of N-CAM. One candidateregulator, retinoic acid, is known to influence homeo-protein expression in teratoma cells (Mavilio et al.1988). In some chicken strains, but not in all, retinoicacid can alter the morphology of scales, which becomefeather-scales (Dhouailly, 1984). This leads to aninteresting question as to the causal relationship amongretinoic acid, expression of homeoproteins, expressionof N-CAM and the determination of feather patterns.The accessibility of feather development (Gallin et al.1986; Goetinck and Carlone, 1988) to experimentalmanipulation makes it an ideal model for further workon pattern formation.

The first event in feather development we detected isthe appearance of a group or 'field' of mesodermic cellsthat stains uniformly with homeobox antibodies. Thisprecedes the appearance of an ectodermal placode. The

Homeoprotein gradient in feather buds 1029

homeodomain proteins (XlHbox 1 and Hox 5.2) onlybecome graded after a feather bud has clearly formed(Figs 4 and 5). Based on the behavior of homeoboxantigens during limb development, it has been pro-posed that homeobox genes may provide at least part ofthe molecular mechanism by which morphogeneticfields are established in embryos (Oliver et al. 1989). Itis known (Harrison, 1918) that the mesodermal layer ofthe vertebrate embryo is divided at the neurula stageinto 'morphogenetic fields' that give rise, after trans-plantation to a host embryo, to organs such as gills,forelimbs, hindlimbs and tails. It has also beenproposed that each field would consist of a gradient oforganogenic potential, or gradient-field (reviewed byHuxley and De Beer, 1934). XlHbox 1 is expressed as aband in the lateral plate mesoderm in amphibianembryos when the forelimb field is established (DeRobertis et al. 1990). In the zebrafish, it then clearlyforms a circular field of XlHbox 1 expression, whichlater acquires an anteroposterior gradient of protein,and finally becomes the pectoral fin bud (Molven et al.1990). We show here that in later development, a newset of smaller fields appears during chicken featherformation and that each feather field is resolved into amesodermal gradient of nuclear protein at the featherbud stage.

Thus, as has been discussed most clearly by PaulWeiss, in vertebrate development the embryo issubdivided into increasingly smaller fields of differentdevelopmental potential (Weiss, 1939). In the case ofXlHboxl, the A-P body axis is first divided into a wideband of homeobox protein expression, which, in somecases, can be seen to gradually decrease towards theposterior end (Oliver et al. 1988ft; Molven et al. 1990).Second, an anteroposterior gradient of XlHbox 1expression is established during formation of theforelimb bud (Oliver et al. 1989). Third, a gradient ofXlHbox 1 antigen forms along the anterior-posterioraxis of the feather bud. The problem of patternformation seems to be resolved into increasinglysmaller fields, and homeodomain proteins may beutilized again and again in setting up the anteropos-terior polarity of cell fields undergoing pattern forma-tion.

This work is supported by NIH HD 24301 (C.M.C.), NIHHD 21502-05 (E.D.R.), NIH BRSG, American CancerSociety Institutional Grant, and Council for Tobacco Re-search (C.M.C.). C.M.C. is a recipient of American CancerSociety Junior Faculty Research Award. B.G.J. is supportedby NRSA Medical Scientist Training Program grant GM 8042-08.

References

CARRASCO, A. E., MCGINNIS, W., GEHRING, W. J. AND D EROBERTIS, E. M. (1984). Cloning of an X. laevis gene expressedduring early embryogenesis coding for a peptide regionhomologous to Drosophila homeotic genes. Celt 37, 409-414.

CHEVALLIER, A., KIENY, M., MAUGER, A. AND SENGEL, P. (1977).Developmental fate of the somitic mesoderm in the chickembryo. In Vertebrate Limb and Somite Morphogenesis (ed. D.

A. Ede, J. R. Hinchliffe, and M. Balls), pp. 421-432.Cambridge University Press, New York.

CHUONG, C.-M. AND EDELMAN, G. M. (1985a). Expression of celladhesion molecules in embryonic induction. I. Morphogenesis ofnestling feathers. J. Cell Biol. 101, 1009-1026.

CHUONG, C. M. AND EDELMAN, G. M. (1985ft). Expression of celladhesion molecules in embryonic induction. II. Morphogenesisof adult feathers. J. Cell Biol. 101, 1027-1043.

D E ROBERTIS, E. M., OLIVER, G. AND WRIGHT, C. V. E. (1990).

Homeobox genes and the vertebrate body plan. Sclent. Am.263, 46-52.

DHOUAILLY, D. (1984). Specification of feather and scale patterns.In Pattern Formation, a Primer in Developmental Biology (ed.G. M. Malacinski and S. V. Bryant), pp. 581-601. Macmillan,Publishing Co., New York.

DOLLE, P., IZPISUA-BELMONTE, J. C , FALKENSTEIN, H., RENUCCI,

A. AND DUBOULE, D. (1989). Coordinate expression of themurine Hox 5 complex homeobox-containing genes during limbpattern formation. Nature 342, 767-772.

GALLIN, W. J., CHUONG, C.-M., FINKEL, L. H. AND EDELMAN, G.

M. (1986). Antibodies to L-CAM perturb inductive interactionsand alter feather pattern and structure. Proc. natn. Acad. Sci.U.S.A. 83, 8235-8239.

GEHRING. W. J. (1987). Homeoboxes in the study of development.Science 236, 1245-1252.

GOETINCK, P. F. AND ABBOTT, U. K. (1963). Tissue interaction inthe scaleless mutant and the use of scaleless as an ectodermalmarker in studies of normal limb differentiation. / . exp. Zool.154, 7-19.

GOETINCK, P. F. AND CARLONE, D. L. (1988). Altered proteoglycansynthesis disrupts feather pattern formation in chick embryonicskin. Devi Biol. 127, 179-186.

HAMBURGER, V. AND HAMILTON, H. (1951). A series of normalstages in the development of the chick embryo. J. Morph. 88,49-92.

HAMILTON, H. L. AND KONIG, A. L. (1956). Effects of aphosphatase inhibitor on the structure of the developing downfeather. Am. J. Anat. 99, 53-80.

HARRISON, R. G. (1918). Experiments on the development of theforelimb of Amblystoma, a self-differentiating, equipotentialsystem. J. exp. Zool. 25, 413-461.

HOLLAND, W. H. P. AND HOGAN, B. (1988). Expression ofhomeobox genes during mouse development: a review. Genesand Dev. 2, 773-782.

HUXLEY, J. S. AND D E BEER, G. R. (1934). The Elements ofExperimental Embryology. Cambridge: Cambridge UniversityPress.

KESSEL, M., BALLING, R. AND GRUSS, P. (1990). Variations of

cervical vertebrae after expression of a Hox 1.1 transgene inmice. Ce//61, 301-308.

LEWIS, E. B. (1978). A gene complex controlling segmentation isDrosophila. Nature. 276, 565-570.

LUCAS, A. M. AND STETTENHEIM, P. R. (1972). Avian anatomy.Integument Part I and Part II. In Agriculture Handbook, 362.Agricultural Research Service, U. S. Department of Agriculture,Washington, D C. 1-750.

MALACINSKI, G. M. AND BRYANT, S. V. (eds) (1984). PatternFormation. Macmillan, New York.

MAUGER, A. (1972). The role of somitic mesoderm in thedevelopment of dorsal plumage in chick embryos. I. Origin,regulation capacity and determination. J. Embryo!, exp.Morphol. 28, 313-341.

MAUGER, A., DEMARCHEZ, M., HERBAGE, D., GRIMAUD, J. A.,

DRUGUET, M., HARTMANN, D. AND SENGEL, P. (1982).Immunofluorescent localization of collagen types I and III, andof fibronectin during feather morphogenesis in the chickenembryo. Devi Biol. 94, 93-105.

MAVILIO, F., SIMEONE, A., BONCINELLI, E. AND ANDREWS, P. W.

(1988). Activation of four homeobox gene clusters in humanembryonal carcinoma cells induced to differentiate by retinoidacid. Differentiation 38, 73-79.

MAYERSON. AND FALLON, J. F. (1985). The spatial pattern andtemporal sequence in which feather germs arise in the WhiteLeghorn chick embryo. Devi Biol. 109, 259-267.

1030 C.-M. Chuong and others

MOLVEN, A., WRIGHT, C. V. E., BREMILLER, R., DE ROBERTIS, E.M. AND KIMMEL, C. B. (1990). Expression of a homeobox geneproduct in normal and mutant zebrafish embryos: Evolution ofthe tetrapod body plan. Development 109, 279-288.

OLIVER, G., SIDELL, N., FISKE, W., HEINZMANN, C , MOHANDAS,T., SPARKES, R. S. AND DE ROBERTIS, E. M. (1989).Complementary homeo protein gradients in developing limb ,buds. Genes and Dev. 3, 641-650.

OLIVER, G., WRIGHT, C , HARDWICKE, J. AND DE ROBERTIS, E. M.(1988a). Differential antero-posterior expression of two proteinsencoded by a homeobox gene in Xenopus and mouse embryos.EMBO J. 7, 3199-3209.

OLIVER, G., WRIGHT, C , HARDWICKE, J. AND DE ROBERTIS, E. M.(1988b). A gradient of homeo domain protein in developingforelimbs of Xenopus and mouse embryos. Cell 55, 1017-1024.

Ruiz I ALTABA, A. AND MELTON, D. A. (1989). Involvement of

the Xenopus homeobox gene Xhox3 in pattern formation alongthe antero-posterior axis. Cell 57, 317-326.

SAWYER, R. H. AND FALLOW, J. F. (eds) (1983). Epithelial-Mesenchymal Interactions in Development. Praeger Publishers,New York. 147-161.

SENGEL, P. (1976). Morphogenesis of Skin. Cambridge UniversityPress, New York. 1-277.

WEISS, P. (1939). Principles of Development. Holt, New York.WOLPERT, L. (1969). Positional information and the spatial pattern

of cellular differentiation. J. theor. Biol. 25, 1-47.WRIGHT, C. V. E., CHO, K. W. Y., HARDWICKE, J., COLLINS, R.

H. AND DE ROBERTIS, E. M. (1989). Interference with functionof a homeobox gene in Xenopus embryos producesmalformations of the anterior spinal cord. Cell 59, 81-93.

{Accepted 14 September 1990)