Shift of Localized Growth Zones Contributes to Skin Appendage Morphogenesis: Role of the Wnt/β-catenin Pathway
2003; Elsevier BV; Volume: 120; Issue: 1 Linguagem: Inglês
10.1046/j.1523-1747.2003.12008.x
ISSN1523-1747
AutoresRajas Chodankar, Chung‐Hsing Chang, Zhicao Yue, Ting‐Xin Jiang, Sanong Suksaweang, Laura W. Burrus, Cheng‐Ming Chuong, Randall B. Widelitz,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoSkin appendage formation represents a process of regulated new growth. Bromodeoxyuridine labeling of developing chicken skin demonstrated the presence of localized growth zones, which first promote appendage formation and then move within each appendage to produce specific shapes. Initially, cells proliferate all over the presumptive skin. During the placode stage they are organized to form periodic rings. At the short feather bud stage, the localized growth zones shifted to the posterior and then the distal bud. During the long bud stage, the localized growth zones descended through the flank region toward the feather collar (equivalent to the hair matrix). During feather branch formation, the localized growth zones were positioned periodically in the basilar layer to enhance branching of barb ridges. Wnts were expressed in a dynamic fashion during feather morphogenesis that coincided with the shifting localized growth zones positions. The expression pattern of Wnt 6 was examined and compared with other members of the Wnt pathway. Early in feather development Wnt 6 expression overlapped with the location of the localized growth zones. Its function was tested through misexpression studies. Ectopic Wnt 6 expression produced abnormal localized outgrowths from the skin appendages at either the base, the shaft, or the tip of the developing feathers. Later in feather filament morphogenesis, several Wnt markers were expressed in regions undergoing rearrangements and differentiation of barb ridge keratinocytes. These data suggest that skin appendages are built to specific shapes by adding new cells from well-positioned and controlled localized growth zones and that Wnt activity is involved in regulating such localized growth zone activity. Skin appendage formation represents a process of regulated new growth. Bromodeoxyuridine labeling of developing chicken skin demonstrated the presence of localized growth zones, which first promote appendage formation and then move within each appendage to produce specific shapes. Initially, cells proliferate all over the presumptive skin. During the placode stage they are organized to form periodic rings. At the short feather bud stage, the localized growth zones shifted to the posterior and then the distal bud. During the long bud stage, the localized growth zones descended through the flank region toward the feather collar (equivalent to the hair matrix). During feather branch formation, the localized growth zones were positioned periodically in the basilar layer to enhance branching of barb ridges. Wnts were expressed in a dynamic fashion during feather morphogenesis that coincided with the shifting localized growth zones positions. The expression pattern of Wnt 6 was examined and compared with other members of the Wnt pathway. Early in feather development Wnt 6 expression overlapped with the location of the localized growth zones. Its function was tested through misexpression studies. Ectopic Wnt 6 expression produced abnormal localized outgrowths from the skin appendages at either the base, the shaft, or the tip of the developing feathers. Later in feather filament morphogenesis, several Wnt markers were expressed in regions undergoing rearrangements and differentiation of barb ridge keratinocytes. These data suggest that skin appendages are built to specific shapes by adding new cells from well-positioned and controlled localized growth zones and that Wnt activity is involved in regulating such localized growth zone activity. Feather formation, a topologic transformation from a flat epithelium to a complex three-dimensional branched skin appendage, begins with reciprocal interactions between the epithelium and the underlying mesenchyme (Saunders, 1948Saunders Jr, J.W. The proximo-distal sequence of origin of the parts of the chick wing and the role of the ectoderm.J Exp Zool. 1948; 108 (reprinted 1998. J Exp Zool 282: 628–668): 363-404Crossref PubMed Scopus (695) Google Scholar;Dhouailly, 1984Dhouailly D. Pattern formation: a primer in developmental biology.in: Malincinski G.M. Bryant S.V. Specification of Feather and Scale Patterns. Macmillan Publications, New York1984: 581-601Google Scholar;Chuong, 1993Chuong C.M. The making of a feather: homeoproteins, retinoids and adhesion molecules.Bioessays. 1993; 15: 513-521Crossref PubMed Scopus (96) Google Scholar;1998;Chuong et al., 2000aChuong C.M. Chodankar R. Widelitz R.B. Jiang T.X. Evo-Devo of feathers and scales: building complex epithelial appendages.Curr Opin Dev Genet. 2000; 10: 449-456Crossref PubMed Scopus (104) Google Scholar). Neither the epithelium nor the mesenchyme is individually potent to form appendages (Dhouailly, 1975Dhouailly D. Formation of cutaneous appendages in dermo-epidermal interactions between reptiles, birds and mammals.Roux Arch Dev Biol. 1975; 177: 323-340Crossref Scopus (46) Google Scholar;Sengel, 1976Sengel P. Morphogenesis of Skin. Cambridge University Press, Cambridge1976Google Scholar;Jiang et al., 1999Jiang T.X. Jung H.S. Widelitz R.B. Chuong C.M. Self organization is the initial event in periodic feather patterning. Roles of signaling molecules and adhesion molecules.Development. 1999; 126: 4997-5009PubMed Google Scholar). The initial signal specifying the location, size, and structural identity of an appendage arises from the mesenchyme and the responding epithelium forms the appropriate appendage and determines its orientation (Novel, 1973Novel G. Feather pattern stability and reorganization in cultured skin.J Embryol Exp Morphol. 1973; 30: 605-633PubMed Google Scholar;Chuong et al., 1996Chuong C.M. Widelitz R.B. Ting-Berreth S. Jiang T.X. Early events during skin appendage regeneration. Dependence of epithelial mesenchymal interaction and the order of molecular reappearance.J Invest Dermatol. 1996; 107: 639-646Crossref PubMed Scopus (113) Google Scholar). Early feather bud primordia through the short bud stage are radially symmetric. Around the late short bud stage the feathers are transformed to bilaterally symmetric structures with the molecular determinants distributed across the anterior–posterior (AP) axis. Shortly thereafter, the proximal–distal axis forms leading to an elongation phase of growth where new cells are added to the distal and flanking regions. Balanced interactions between the anterior and posterior compartments play a major part in the formation of the posterior–distal axis (Widelitz et al., 1999Widelitz R.B. Jiang T.X. Chen C.W.J. Stott N.S. Chuong C.M. Wnt 7a in feather morphogenesis. involvement of anterior-posterior asymmetry and proximal-distal elongation demonstrated in an in vitro reconstitution model.Development. 1999; 126: 2577-2587PubMed Google Scholar). In the late long bud stage, the epithelium invaginates into the dermis to form the feather follicle. The growth and structure of feathers during their initial formation and subsequent feather cycles is thought to be due to the presence of a localized growth zone (LoGZ) that contains cells with a higher mitotic potential than the rest of the bud (Chuong et al., 2000aChuong C.M. Chodankar R. Widelitz R.B. Jiang T.X. Evo-Devo of feathers and scales: building complex epithelial appendages.Curr Opin Dev Genet. 2000; 10: 449-456Crossref PubMed Scopus (104) Google Scholar; Widelitz and Chuong, in pressWidelitz RB, Chuong CM: Complex pattern formation: regulation of the size, number, spacing and symmetry during feather morphogenesis. Int J Dev Biol Rev, in pressGoogle Scholar). Scales are also chicken epidermal integument derivatives, but do not contain a LoGZ and remain flat (Chuong et al., 2000aChuong C.M. Chodankar R. Widelitz R.B. Jiang T.X. Evo-Devo of feathers and scales: building complex epithelial appendages.Curr Opin Dev Genet. 2000; 10: 449-456Crossref PubMed Scopus (104) Google Scholar,Chuong et al., 2000bChuong C.M. Hou L.H. Chen P.J. Wu P. Patel N. Chen Y. Dinosaur's feather and chicken's tooth? Tissue engineering of the integument.Eur J Dermatol Rev. 2000; 11: 286-292Google Scholar). To begin to understand these different phases of feather growth, we sought to characterize the LoGZ and to examine the molecular determinants that regulate its size and placement within developing feathers. The signaling of secreted, soluble factors through membrane bound receptors has been implicated in the morphogenesis of a number of vertebrate systems. The Wnt family falls into this class of molecules and signals through the frizzled receptors. In humans there are 19 Wnts and 10 frizzled receptors (Malbon et al., 2001Malbon C.C. Wang H. Moon R.T. Wnt signaling and heterotrimeric G-proteins: strange bedfellows or a classic romance?.Biochem Biophys Res Commun. 2001; 287: 589-593Crossref PubMed Scopus (81) Google Scholar). As they have not been purified as yet, it is unknown which frizzled receptors mediate the functions of each of the Wnts. The Wnts have been implicated in regulating growth control and development in a number of experimental systems. Here, we focus on skin appendages. In the hair, Wnt 3a and 10b are expressed in the hair matrix (St Jacques et al., 1998St Jacques B. Dassule H.R. Karavanova I. et al.Sonic hedgehog signaling is essential for hair development.Curr Biol. 1998; 8: 1058-1068Abstract Full Text Full Text PDF PubMed Google Scholar). Ectopic expression of Wnt 3a produces shortened hairs (Millar et al., 1999Millar S.E. Willert K. Salinas P.C. Roelink H. Nusse R. Sussman D.J. Barsh G.S. WNT signaling in the control of hair growth and structure.Dev Biol. 1999; 207: 133-149Crossref PubMed Scopus (223) Google Scholar). Wnt 4 may play a part in epidermal–mesenchymal interactions (Saitoh et al., 1998Saitoh A. Hansen L.A. Vogel J.C. Udey M.C. Characterization of Wnt gene expression in murine skin: possible involvement of Wnt-4 in cutaneous epithelial–mesenchymal interactions.Exp Cell Res. 1998; 243: 150-160Crossref PubMed Scopus (41) Google Scholar). Wnt 5a is regulated by sonic hedgehog during dermal condensation formation (Reddy et al., 2001Reddy S. Andl T. Bagasra A. Lu M.M. Epstein D.J. Morrisey E.E. Millar S.E. Characterization of Wnt gene expression in developing and postnatal hair follicles and identification of Wnt5a as a target of Sonic hedgehog in hair follicle morphogenesis.Mech Dev. 2001; 107: 69-82Crossref PubMed Scopus (344) Google Scholar). β-catenin expression was found to cause hair tumors (Gat et al., 1998Gat U. Das Gupta R. Degenstein L. Fuchs E. De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin.Cell. 1998; 95: 605-614Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar). Recently, blockage of Wnt activity via overexpression of Dickkopf 1 was shown to block hair initiation (Andl et al., 2002Andl T. Reddy S.T. Gaddapara T. Millar S. Wnt signals are required for the initiation of hair follicle development.Dev Cell. 2002; 2: 643-653Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar). In feathers, Wnt 7a was found to regulate the polarity of development (Widelitz et al., 1999Widelitz R.B. Jiang T.X. Chen C.W.J. Stott N.S. Chuong C.M. Wnt 7a in feather morphogenesis. involvement of anterior-posterior asymmetry and proximal-distal elongation demonstrated in an in vitro reconstitution model.Development. 1999; 126: 2577-2587PubMed Google Scholar), whereas ectopic β-catenin expression led to ectopic feather formation and the conversion of scales to feathers (Noramly et al., 1999Noramly S. Freeman A. Morgan B.A. Beta-catenin signaling can initiate feather bud development.Development. 1999; 126: 3509-3521PubMed Google Scholar;Widelitz et al., 2000Widelitz R.B. Jiang T.X. Lu J. Chuong C.M. Beta-catenin in epithelial morphogenesis: conversion of part of avian foot scales into feather buds with a mutated beta-catenin.Dev Biol. 2000; 219: 98-114Crossref PubMed Scopus (133) Google Scholar). To explore further the LoGZ and its regulation, we have extended our investigation to examine the expression domains of Wnt 6 during feather formation. We have also compared Wnt 6 with Wnt 5a, 8c, 11, and 14 and characterized the expression of some frizzled receptors and their soluble competitor sfrp 2. Furthermore, we have misexpressed Wnt 6 to examine its function in feather growth. Antibodies against bromodeoxyuridine (BrdU) were from Sigma (St Louis, MO), against proliferating cell nuclear antigen (PCNA) were from Chemicon (Temecula, CA), and against retroviral GAG were from Spafas Charles River Laboratories (North Franklin, CT). Each was used following the manufacturer's recommended protocols. This procedure was carried out according to procedures described in Nieto et al., 1996Nieto M.A. Patel K. Wilkinson D.G. In Situ Hybridization Analysis of Chick Embryos in Whole Mount and Tissue Sections Methods Cell Biol. Vol. 51. Academic Press, San Diego1996: 291Google Scholar. Embryos or skins were dissected in RNase free phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde at 4°C overnight. Samples were dehydrated and rehydrated through a series of increasing and decreasing concentrations of methanol, respectively. They were bleached with hydrogen peroxide and digested with proteinase K and then fixed again in 0.2% gluteraldehyde in 4% paraformaldehyde. Samples were treated with prehybridization buffer at 65–70°C before hybridizing them in hybridization buffer containing 2 μg per ml digoxigenin-labeled riboprobes at 65–70°C overnight. Posthybridization washes were carried out the following day in 2×sodium citrate/chloride buffer containing 0.1% Chaps three times, 0.2×sodium citrate/chloride buffer containing 0.1% Chaps thrice and twice in phosphered buffered saline with 0.1% Tween-20. They were then blocked in 20% goat serum in phosphered buffered saline with 0.1% Tween-20 before incubation with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Indianapolis, IN) at 4°C overnight. Samples were washed with phosphered buffered saline with 0.1% Tween-20 containing 1 mM levamisole for 1 h each for at least five times. For the color reaction, the samples were first equilibrated in 100 mM Nacl, 100 mM Tris-Cl, pH 9.5, 50 mM MgC12, 0.1% Tween-20 (NTMT) solution containing 100 mM Tris–HCl, pH 9.5, 50 mM MgCl2, 0.1% Tween-20. Alkaline phosphate substrates 4-nitroblue tetrazollum chloride (NBT) and 5-bromo-4-chloro-3-indoyl phosphate (BCIP) were added 4.5 μl and 3.5 μl per ml of 100 mM Nacl, 100 mM Tris-Cl, pH 9.5, 50 mM MgCl2, 0.1% Tween-20 (NTMT) respectively. This procedure was carried out according to procedures described in Nieto et al., 1996Nieto M.A. Patel K. Wilkinson D.G. In Situ Hybridization Analysis of Chick Embryos in Whole Mount and Tissue Sections Methods Cell Biol. Vol. 51. Academic Press, San Diego1996: 291Google Scholar. Briefly, embryos were fixed in 4% paraformaldehyde, dehydrated through a series of ethanol, and embedded in paraffin wax. Sections were cut at 7–10 μm thickness and then mounted on positively charged coated slides. The sections were dewaxed in xylene, rehydrated through an ethanol series prior to the start of the experiment. The specimens were postfixed in 4% paraformaldehyde after digested with 10 μg proteinase K per ml for 5 min. The tissue sections were then hybridized overnight at 60°C in the hybridization buffer containing 1 ng per μl of digoxigenin-labeled RNA probes. Posthybridization washes were carried out using 100 mM Nacl, 100 mM Tris-Cl, pH 9.5, 50 mM MgCl2, 0.1% Tween-20 and 2×sodium citrate/chloride buffer. Digestion with RNase A (10 μg per ml) was followed by blocking of slides in blocking solution. The samples were then incubated with alkaline phosphatase conjugated sheep anti-digoxigenin antibody (Roche). The bound antibody was detected using an alkaline phosphatase substrate, BM Purple. Eggs were incubated in a humidified incubator at 37°C and collected in a Petri dish containing Dulbecco's modified Eagle's medium without serum according to Hamburger and Hamilton, 1951Hamburger V. Hamilton H.L. A series of normal stages in the development of the chick embryo.J Morphol. 1951; 88: 49-91Crossref PubMed Scopus (9734) Google Scholar. Dorsal skins were dissected from embryos, stage 28–34 with the help of watchmaker's forceps. In multiwell plates, the tissues were pulsed with 20 μM BrdU diluted in Dulbecco's modified Eagle's medium at 37°C in 5% carbon dioxide and 95% air for 30–90 min.This was followed by fixing the tissue in 100% methanol and bleaching with 10% hydrogen peroxide in 1: 4 dimethyl sulfoxide/100% methanol, rehydrating in PBS and denaturing them with 2 M HCl and neutralizing the acid by immersing the tissues in 0.1 M sodium borate (pH 8.5). The tissues were incubated with anti-BrdU antibody diluted 1: 100 in 0.1% bovine serum albumin in PBS. The secondary and tertiary antibodies used were biotinylated anti-mouse IgG diluted 1: 300 and streptavidin horseradish peroxidase diluted 1: 400 in 0.1% bovine serum albumin in PBS, respectively. The tissues were washed with PBS before each antibody was applied. Peroxidase was detected using the 3-amino-9-ethylcarbazole substrate. Replication competent avian sarcoma virus (RCAS) directing the constitutive expression of Wnt 6 was prepared following the method of Morgan and Fekete, 1996Morgan B.A. Fekete D.M. Manipulating gene expression with replication-competent retroviruses.Methods Cell Biol. 1996; 51: 185-218Crossref PubMed Google Scholar. Briefly, chicken embryo fibroblasts were transfected by lipofection with lipofectamine (Invitrogen, Carlsbad, CA). Cells were maintained in the logarithmic phase of growth in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Once the culture reached approximately 70% confluence, the media was replenished and the medium containing virus was collected at 24 h. Viral titers were determined by staining for the viral GAG product and by assessing the expression of the exogenous gene. The virus was introduced to the amniotic fluid of E2–E3 chicken embryos to allow transduction of the skin. Transduction at this time produced more obvious phenotypes in the growth of the feather follicles. Short-term BrdU labeling was used to demonstrate the localization of growth zones in developing feathers. The LoGZ is defined as high BrdU labeling in a localized area of developing organ primordia. Placodes form in the absence of cell proliferation (Wessels, 1965Wessels N.K. Morphology and proliferation during early feather development.Dev Biol. 1965; 12: 131-153Crossref PubMed Scopus (107) Google Scholar). At the placode stage, the feather primordia have radial symmetry. At the early short bud stage most of the proliferating cells localized to the posterior epithelial compartment (Figure 1a,a′), which led to the development of AP asymmetry. At the molecular level, these two compartments are strikingly different and activate different sets of genes. After establishing the AP orientation, the proximal–distal axis was established. At the late short bud stage, the LoGZ was present in the distal epithelial zone (Figure 1b,b′, arrow). During the early long bud phase, proliferation continued to be located in the distal epithelium (Figure 1c,c′, arrows). The proliferation zone gradually shifted down the feather from the distal zone of the bud during the long bud stage to the flank region (Figure 1d′). At this transition stage, nonproliferative areas surrounded the LoGZ, proximally and distally (Figure 1d, arrows indicate the LoGZ). Later, in the follicle stage, a layer of cells invaginated into the dermis and initiated the formation of the feather follicle. At this stage growth occurred at the feather base (Figure 1e, see arrows; Figure 1e′ was cutaique angle to show just the base of the follicle). In a later staged follicle, proliferation remained at the proximal end of the feather, called the collar (equivalent to the hair matrix; Figure 1). Above this proliferative collar is the ramogenic zone, which is the region where the feather filament epithelial cylinder starts to form barb ridges and rachidial ridges. Whereas the majority of proliferation takes place at the base of the feather filament some proliferation was observed further up the feather filament. A cross-section of the feather filament in the early barb ridge (indicated as the plane in Figure 1f) shows that proliferation also was localized to the germinative basal epithelial layer of the forming barbs, or the barb ridge growth zone (Figure 1g). A higher magnification view shows that these basal layer cells were invaginating to produce the feather barbs (Figure 1h). BrdU incorporation showed more staining in the epidermis. At different developmental stages through development, proliferation in both the epithelium and mesenchyme shift positions. For example, BrdU labeled the posterior feather mesenchyme clearly, whereas the short buds grew to become the long buds (Chen et al., 1997Chen C.W. Jung H.S. Jiang T.X. Chuong C.M. Asymmetric expression of Notch/Delta/Serrate is associated with the anterior-posterior axis of feather buds.Dev Biol. 1997; 188: 181-187Crossref PubMed Scopus (57) Google Scholar). Here we also used PCNA staining to show more clearly proliferation within the mesenchyme (Figure 1i–k). This is particularly clear in early and late follicle stages (Figure 1j,k), but less so in the long feather buds (Figure 1i). We initially surveyed the expression of patterns of several Wnt pathway members at the short feather buds stage, the long feather bud stage, and in the feather follicle stage. The localization of Wnt 6 was examined in growing feathers using whole-mount and section in situ hybridization and its distribution was compared with Wnt 5a, 8c, 11, 14, Fz 1, and Fz 7. At E8–E9, Wnt 6 exhibited partially overlapping patterns with Wnt 5a, and cFz-1 in the LoGZ. Wnt 6 expression initiated along the caudal midline and concentrated in the forming feather buds while diminishing in the interbud domains (Figure 2a). Expression then bifurcated at the spinal tract (Figure 2b). Later, as feathers developed bilaterally from the midline, Wnt 6 was newly expressed in the forming feather buds and continued to be expressed in the growing buds near the midline (Figure 2c). Wnt 6 was strongly expressed in the posterior compartment of the developing bud (Figure 2d). With further progression and growth, Wnt 6 expression shifted to the distal compartment epithelium (Figure 2e,f). The dermal components and the interbud areas were negative. As the Wnts are secreted molecules, however, they may influence the adjacent mesenchyme. Wholemount section in situ hybridization shows Wnt 6 staining toward the tip of short buds (*), in the flank of early long feather buds (#) and toward the base of feather follicles (+) (Figure 2g). Wnt 5a also was expressed in the posterior compartment during the early short bud stage (Figure 2h). As the bud lengthened and the posterior domain extended along the AP axis, its expression moved to the distal zone at the late short bud stage (Figure 2i). A ring of cells expressing Wnt 5a appeared at the base of the feathers at this stage. Wnt 8c and Wnt 14 were all over the short bud epithelium (data not shown). Wnt 11 was initially expressed in the interbud area and was absent from the feather buds. At the late short bud stage its expression was seen in the distal mesenchyme and also in the interbud epithelium (Figure 2j,k). Consistent with the presence of the LoGZ, Fz-1 and Fz-7 expression were seen in the posterior epithelium at the early short bud stage (Figure 2l,m) and later in the distal epithelium (Figure 2l, inset). The long bud stage is characterized by the acquisition of the proximal–distal axis and manifested by height > base. BrdU incorporation and PCNA staining at this stage were seen to progressively shift down the feather filament to the lower section of the bud (Figure 1c,d,i,j). Whereas several Wnts were expressed in the vicinity of the LoGZ at this time, Wnt 6 was expressed in regions undergoing differentiation. It was expressed in stripes along the proximal–distal axis of the bud marking the barb (Figure 3a). Wnt 14 and sfrp 2 were expressed within the BrdU and PCNA defined zone (Figure 3b,c, between the arrows). They were both initially expressed all over the bud epithelium at the short bud stage (not shown) and later were restricted to the proliferation zone in the long bud. At the late long bud stage, invagination of the follicle has begun. Wnt 14 was strongly expressed in the epithelium (Figure 3e). Its expression was marked in the developing germinative layer of the early follicle (see arrows). Fz-1 was expressed more extensively all over the epithelium at this stage (Figure 3f). It was also seen in the ramogenic zone, highlighting the invaginated basal layer of proliferating cells and the central blood vessel. Wnt 11 was expressed in the distal feather mesenchyme and the interbud epithelium at this stage (Figure 3d). In the adult chicken, each feather is rooted in the dermal region with a follicular structure, which is fixed to the dermis by a dense network of connective tissues and muscles. At the base of the follicle, the dermal papilla interacts with the papillary ectoderm and generates new epithelial cells (Figure 4a). Above the dermal papilla is the collar epithelium that is equivalent to the hair matrix and contains the transit amplifying cells. The follicle epithelium is stratified into the basal layer, intermediate layer, and follicle sheath (Figure 4b). PCNA and short-term BrdU labeling localized to the basal layer of the follicle epithelium containing cells of unlimited replicative potential (Figure 1f) and to the pulp and dermal papilla (Figure 1k). Liver cell adhesion molecule was expressed throughout the follicle epithelium (Figure 4c). Neural cell adhesion molecule was expressed in the mesenchyme, including the dermal papilla and dermal sheath (Figure 4d). Cytokeratin 1 (Presland et al., 1989Presland R.B. Whitbread L.A. Rogers G.E. Avian keratin genes: II. Chromosomal arrangement and close linkage of three gene families.J Mol Biol. 1989; 209: 561-576Crossref PubMed Scopus (42) Google Scholar), a feather keratin differentiation marker, was expressed throughout the intermediate layer of cells (Figure 4e). The Wnt 6 expression profile was generated to see how its distribution correlated with the different feather domains. The distribution then was compared with that of β-catenin and other Wnt family members. Wnt 6 (Figure 4h) expression was weak in the basal layer and strong in the intermediate layer of the follicle and overlapped with the distribution of Wnt 5a (Figure 4g), Wnt 14 (not shown), Fz 1 (Figure 4j), and Sfrp 2 (Figure 4k). β-catenin was expressed exclusively in the proliferative basal epithelial layer (Figure 4f). β-catenin was shown to be in the feather germs (Noramly et al., 1999Noramly S. Freeman A. Morgan B.A. Beta-catenin signaling can initiate feather bud development.Development. 1999; 126: 3509-3521PubMed Google Scholar;Widelitz et al., 2000Widelitz R.B. Jiang T.X. Lu J. Chuong C.M. Beta-catenin in epithelial morphogenesis: conversion of part of avian foot scales into feather buds with a mutated beta-catenin.Dev Biol. 2000; 219: 98-114Crossref PubMed Scopus (133) Google Scholar). Previous work focused more on the protein subcellular localization and considered that β-catenin mRNA should be ubiquitous. This was shown not to be the case. We were one of the first to show the remarkable β-catenin mRNA expression from a homogeneous expression pattern to a restricted circular pattern in the developing feather germ with a negative halo surrounding it (Jiang et al., 1999Jiang T.X. Jung H.S. Widelitz R.B. Chuong C.M. Self organization is the initial event in periodic feather patterning. Roles of signaling molecules and adhesion molecules.Development. 1999; 126: 4997-5009PubMed Google Scholar;Widelitz et al., 2000Widelitz R.B. Jiang T.X. Lu J. Chuong C.M. Beta-catenin in epithelial morphogenesis: conversion of part of avian foot scales into feather buds with a mutated beta-catenin.Dev Biol. 2000; 219: 98-114Crossref PubMed Scopus (133) Google Scholar). Subsequently, a similar mRNA expression pattern was found in developing hair germs (Huelsken et al., 2001Huelsken J. Vogel R. Erdmann G. Cotsarelis G. Birchmeier W. Beta-catenin controls hair follicle morphogenesis and stem cell differentiation in the skin.Cell. 2001; 105: 533-545Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar). Wnt 8c was strongly expressed throughout the follicle epithelium, including the follicle sheath, intermediate, and basal layers (Figure 4i). Branching morphogenesis occurs above the collar region in the ramogenic zone. The epithelial sheet invaginates periodically to segregate regions that will either apoptose or keratinize (Chuong et al., 2000aChuong C.M. Chodankar R. Widelitz R.B. Jiang T.X. Evo-Devo of feathers and scales: building complex epithelial appendages.Curr Opin Dev Genet. 2000; 10: 449-456Crossref PubMed Scopus (104) Google Scholar) (Figure 1f and Figure 4l). Proliferating cells were localized to the basal layer of the forming barbs ridges (Figure 1g,h). The intermediate layer contains cells that undergo differentiation to form the intricate branch pattern of the feather. Cytokeratin 1 expression was weakly expressed in the barbule plate cells of the differentiating ramogenic zone (Figure 4m), but feather keratin A was strongly expressed in the barbule plate cells (Figure 4n). Wnt 6 was also expressed in the barbule plate of the barb ridge (Figure 4o) and may contribute to differentiation at this time, although it may also be involved in maintaining the few proliferating cells retained in this layer. The expression pattern of Wnt 6 and several other Wnt members closely coincided with the pattern of the LoGZ. We tested the possible involvement
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