Distinct Wnt members regulate the hierarchical morphogenesis of skin regions (spinal tract) and individual feathers - PubMed (original) (raw)

Distinct Wnt members regulate the hierarchical morphogenesis of skin regions (spinal tract) and individual feathers

Chung-Hsing Chang et al. Mech Dev. 2004 Feb.

Abstract

Skin morphogenesis occurs in successive stages. First, the skin forms distinct regions (macropatterning). Then skin appendages with particular shapes and sizes form within each region (micropatterning). Ectopic DKK expression inhibited dermis formation in feather tracts and individual buds, implying the importance of Wnts, and prompted the assessment of individual Wnt functions at different morphogenetic levels using the feather model. Wnt 1, 3a, 5a and 11 initially were expressed moderately throughout the feather tract then were up-regulated in restricted regions following two modes: Wnt 1 and 3a became restricted to the placodal epithelium, then to the elongated distal bud epidermis; Wnt 5a and 11 intensified in the inter-tract region and interprimordia epidermis or dermis, respectively, then appeared in the elongated distal bud dermis. Their role in feather tract formation was determined using RCAS mediated misexpression in ovo at E2/E3. Their function in periodic feather patterning was examined by misexpression in vitro using reconstituted E7 skin explant cultures. Wnt 1 reduced spinal tract size, but enhanced feather primordia size. Wnt 3a increased dermal thickness, expanded the spinal tract size, reduced interbud domain spacing, and produced non-tapering "giant buds". Wnt 11 and dominant negative Wnt 1 enhanced interbud spacing, and generated thinner buds. In cultured dermal fibroblasts, Wnt 1 and 3a stimulated cell proliferation and activated the canonical beta-catenin pathway. Wnt 11 inhibited proliferation but stimulated migration. Wnt 5a and 11 triggered the JNK pathway. Thus distinctive Wnts have positive and negative roles in forming the dermis, tracts, interbud spacing and the growth and shaping of individual buds.

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Figures

Fig. 1

Expression of Wnt 1, 3a, 5a and 11 during skin development. Wnt expression during chicken embryo feather formation. All views are of the dorsal tract unless otherwise noted. Wnt 1 demonstrated a restrictive to bud expression mode. Wnt 1 expressed in the dorsal midline as a continuous stripe (arrow) with lower expression elsewhere at stage 29 (E6) was then restricted to the bud epithelium surrounded by a clear halo at stage 33 (E7.5), and moved to the distal short buds and long buds at late stage 34 (E8.5). Wnt 3a generally followed a similar expression pattern, as a continuous stripe in the stage 29 (E6) dorsal midline epithelium (arrow), then disappearing from the interbud area and only staining the bud domain at stage 31 (E7). Densely stained morphogenetic zones besides the primary row appeared before the newly emerging rows. Wnt 3a was localized to the distal short buds at stage 34 (E8). Insets show cross sections. Wnt 11 demonstrated a restrictive to interbud expression mode. Wnt 11 initiated as a continuous stripe in the primary row and then disappeared from the bud domain leaving clear circles as seen in the stage 31 (E7) cervical and femoral tracts. As the feather rows propagated laterally (arrow indicates direction), Wnt 11 strongly stained the interbud area of newly formed feather rows, faded from the interbud area of older rows, and finally appeared within the mesenchyme of short buds. Stage 35 (E9) embryo side view, Wnt 11 stains the apteric zones between the femoral/spinal (yellow asterisk) and humeral/spinal tracts (green asterisk). Wnt 11 appeared as a transient ring surrounding the feather bud base, then stained throughout the mesenchyme before disappearing under the early bud placodes (stage 31 sections). It then concentrated in the interbud mesenchyme, was lost from the bud areas (stage 34 sections), and gradually appeared in the distal and posterior short and long bud mesenchyme at stage 35—late 35 (E9–9.5). Wnt 5a was expressed all over the epithelium first at stage 30 (E6.5), then transiently disappeared in the bud epithelium at stage 30 (E6.5) and reappeared in the bud again at stage 31 (E7). In the short bud, Wnt 5a was expressed in the anterior bud epithelium and posterior bud mesenchyme, and retained the early stage interbud epithelial staining at stage 34 (E8). As the buds matured, the epithelial staining decreased while the mesenchymal staining increased, shifting the expression pattern to the posterior bud domain at stage 35 (E9). Wnt 5a was concentrated as a ring around the base of developing asymmetric feather buds, and stained the distal mesenchyme of long buds (E9, stage 35). Size bar = 500 μm, Size bar for insets = 100 μm. H and H developmental staging is indicated. Schematic drawing summarizing the expression patterns of Wnt 1, 3a, 5a and 11.

Fig. 2

RCAS-DKK inhibited the formation of dermis and feather buds. RCAS-DKK transduction inhibited feather bud formation in vivo and in vitro. RCAS-DKK was injected into the body and amniotic cavity of stage 19–20 (E3) embryos. Feather formation was inhibited over much of the body compared to controls (A). Sections show that feather buds normally elongate with a thickened dermis, but the DKK transduced cultures had a thin dermis (B). RCAS-DKK was then applied to cultured skin explants prepared from stage 31 (E7) chicken embryos. After 4 days in culture, control skin explants formed elongated feather buds, but the feather primordia in DKK transduced cultures remained small and did not elongate (C). Size bars = 500 μm.

Fig. 3

Dual functions of Wnt 1 in regulating the size of feather tracts and feather primordia. RCAS-Wnt 1 transduction in early embryos decreased both the tract and feather bud size. RCAS-Wnt 1 was injected to the body and amniotic cavity of stage 14 embryos. At stage 36 (E10), Shh staining revealed that the sacral and caudal parts of the spinal tract were blocked or prematurely truncated, allowing the femoral tracts on both sides to invade into this affected zone (A). The zone between the normal and affected spinal tract showed a depressed integument with inhibited feather development. A section through this region showed underdeveloped and uncondensed mesenchyme, loss of the deep dermis layer and thinning of the superficial dermis, compared with controls (B). Immunostaining showed loss of collagen II formation in the mesenchyme, compared with controls (B). Mesenchymal NCAM expression levels were decreased, compared with controls (B). The presence of retrovirus was confirmed in RCAS control and Wnt 1 transduced embryos by staining for p27 (B). In vitro reconstituted explants transduced with RCAS-Wnt 1 had increased bud size, compared to RCAS-LacZ infected controls (C), while incubation in conditioned medium from a dominant negative Wnt 1 secreting cell line produced thinner and sparser feather buds (C). Transduction was confirmed by staining for retroviral p27. Size bars; wholemount = 500 μm, sections and explants = 200 μm.

Fig. 4

Wnt 3a promotes the formation of feather tracts, expands feather bud size and reduces interbud spacing. RCAS-Wnt 3a transduction induced feather tract expansion, feather primordia fusion or enlargement. Stage 18–22 chicken embryos transduced with RCAS-Wnt 3a virus (right side) in ovo were in situ hybridized to Shh to visualize the feather buds at stage 36–40. The transduced spinal tract was thickened and widened with increased bud size and decreased interbud space (left side) compared to controls (A). An enlarged view of the caudal tract shows that feather primordia were enlarged or fused with neighboring buds at the expense of interbud space. H and E staining showed that RCAS-Wnt 3a infected feather buds had a plateau-like shape with a thickened dermis and increased cellularity (B). RCAS antibody (P27) staining identified the retrovirus in both the epithelium and mesenchyme (B, brown). Diffuse staining of PCNA (B, brown), indicates a wide presence of cell proliferation. RCAS-Wnt 3a transduced reconstituted skin explants had enlarged and fused buds compared to controls (C). Size bars = 500 μm, Size bars for sections = 150 μm.

Fig. 5

Wnt 11 expands interbud spacing and decreases the feather number and filament diameter. RCAS-Wnt 11 was injected into the body and amniotic cavity of stage 19-20 (E3) embryos. Thinner and sparser feather filaments or focal loss of feathers were detected by stage 40 (E14) in the infected samples compared to controls (A). Control RCAS-alkaline phosphatase transduced embryos stained in wholemount for alkaline phosphatase. RCAS-Wnt 11 infected in vitro reconstituted explants prepared from stage 31 (E7) dorsal skin showed decreased bud size and number, and increased interbud spacing, compared to RCAS-LacZ infected controls (B) after 5 days in culture. Size bars = 500 μm.

Fig. 6

Relationship among molecular pathways. Reconstituted skin explant cultures. (A) At 5 days, endogenous Wnt 11 is expressed in the interbud epithelium (arrow) and distal bud mesenchyme (open arrow). Ectopic Wnt 3a suppresses the expression of Wnt 11 in the interbud region and in the distal bud mesenchyme. In contrast, Wnt 1 induces Wnt 11 in the bud epithelium and mesenchyme but suppresses Wnt 11 in the interbud epithelium. Wnt 3a expands the SHH expressing distal bud growth zone compared to controls (B). Size bar = 100 μm.

Fig. 7

Effect of Wnts on cell proliferation, signaling and migration. (A) Wnt 1, 3a and 5a increased while Wnt 11 inhibited cell proliferation compared to untransduced and RCAS controls. (B) Wnt 1 and Wnt 3a enhanced while Wnt 5a and Wnt 11 suppressed TCF4 promoter activity (OT). No activity was detected with a mutated TCF4 promoter (OF). (C) PKC activity was measured 2 and 4 days after chicken embryo fibroblasts were transduced with retrovirus directing Wnt expression. Only Wnt 5a induced increased PKC activity at these times. The data are presented as a PKC ratio comparing experimental groups to controls at each time point. (D) Wnt 5a and Wnt 11 induced increased JNK activity that was suppressed by SP600125. Three independent experiments were carried out with similar trends. One example is shown. (E) Effect of SP600125, the JNK inhibitor, on skin explant morphogenesis. Control and JNK inhibitor treated skin explants are shown 4 days after treatment. H and E stained cross sections (insets) show that JNK inhibition causes a broadening at the tip of the feather buds. (F) 32 h after scraping mitotically arrested cells from a center stripe, RCAS-Wnt 11 transduced cells reentered the available space faster than control or experimental cell types, demonstrating an increased migration rate. (G) Summary of Wnt effects on skin morphogenesis, molecular pathways and cell behavior. +, activity was detected; −, activity was not detected; ↑, activity was increased; ↓, activity was decreased; ↔, no change in activity. Size bar: E = 50 μm, F = 100 μm.

Fig. 8

Schematic summary of the positive and negative roles of Wnts during different hierarchical levels of skin morphogenesis. Four morphogenetic levels between five structural entities (dermomyotome, dermis, feather tracts, individual feather primordia, characteristic feather shape) are shown. Yellow spindles and circles represent dermal cells. Blue represents the epidermis. Two different tracts with different density and shape of skin appendages are shown. At each morphogenetic level, Wnts play either a positive (red) or negative role (green). Data for the dermatome to dermis migration is based on Olivera-Martinez et al., 2001, .

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Distinct Wnt members regulate the hierarchical morphogenesis of skin regions (spinal tract) and individual feathers - PubMed (original) (raw)