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The homeobox gene Msx-2 is expressed specifically in sites of skin appendage formation. To explore its part in skin morphogenesis, we produced transgenic mice expressing Msx-2 under the control of the cytomegalovirus promoter. The skin of these transgenic mice was flaky, exhibiting desquamation and shorter hairs. Histologic analysis showed thickened epidermis with hyperproliferation, which was restricted to the basal layer. Hyperkeratosis was also evident. A wide zone of suprabasal cells were misaligned and coexpressed keratins 14 and 10. There was reduced expression of integrin β1 and DCC in the basal layer. Hair follicles were misaligned with a shrunken matrix region. The dermis showed increased cellularity and empty vacuoles. We suggest that Msx-2 is involved in the growth control of skin and skin appendages. [ABSTRACT FROM AUTHOR]

The hair cycle involves remodeling of cells and of cell groups into a complex follicular structure. During skin appendage development, adhesion molecules such as neural cell adhesion molecule (NCAM) and deleted in colon carcinoma (DC) participate in the formation of cell groups. NCAM has been found to be expressed in the mesenchyme during mouse hair follicle induction. DCC expression has been observed in the epithelial cells of the developing feather. We postulate that these two molecules may also define cell groups in the cycling hair follicle. Here we report their spatio-temporal expression patterns during the depilation-induced murine hair cycle. NCAM expression was also examined in positive and negative hair-inductive follicular papilla cell lines. Throughout the hair cycle, DCC expression was confined to the basal keratinocytes of the epidermis and the epithelial portion of the hair follicle. During mid-anagen, two types of deleted in colon carcinoma staining were observed. One was a cell surface pattern seen in the epithelial cells in the bulge region where the follicular stem cells reside. The other was a diffuse cytoplasmic staining pattern in the transient hair follicle epithelia located below the bulge region. Prominent NCAM staining was observed in the follicular papilla throughout the hair cycle and was accompanied by weak staining of the matrix epithelia. NCAM expression correlated with hair induction by a follicular papilla cell line. The results suggest that DCC and NCAM define the permanent cell groups of the hair follicle and that NCAM is important for hair induction.

Induction of skin appendages involves a cascade of molecular events. The fibroblast growth factor (FGF) family of peptide growth factors is involved in cell proliferation and morphogenesis. We explored the role of the FGFs during skin appendage induction using developing chicken feather buds as a model. FGF-1, FGF-2, or FGF-4 was added directly to the culture medium or was released from pre-soaked Affigel blue beads. Near the midline, FGFs led to fusion of developing feather buds, representing FGFs' ability to expand feather bud domains in developing skin. In lateral regions of the explant where feather placodes have not formed, FGF treatment produces a zone of condensation and a region with an increased number of feather buds. In ventral epidermis that is normally apteric (without feathers), FGFs can also induce new feather buds. Like normal feather buds, the newly induced buds express Shh. The expression of Grb, Ras, Raf, and Erk, intracellular signaling molecules known to be downstream to tyrosine kinase receptors such as the FGF receptor, was enriched in feather bud domains. Genistein, an inhibitor of tyrosine kinase, suppressed feather bud formation and the effect of FGF. These results indicate that there are varied responses to FGFs depending on epithelial competence. All the phenotypic responses, however, show that FGFs facilitate the formation of skin appendage domains.

Early molecular events during the development and regeneration of skin appendages were studied using cultured chicken skin explants with epithelial-mesenchymal recombination. The explant epithelium was separated from the mesenchyme, rotated 90 degrees or 180 degrees, recombined with the mesenchyme, and cultured. After this procedure, existing feather buds disappeared and new buds were regenerated. The location of the new buds is determined by the original dermal condensations, whereas the orientation is dictated by the original epithelium. The temporal expression of key morphogenetic molecules was examined 3, 6, and 20 h after recombination by whole-mount in situ hybridization and immunostaining. The results showed the following. (i) Placode formation and the expression of wingless-int (Wnt) 7a and Msx-1 in the placode epithelium are mesenchyme dependent. (ii) Hox C6 and neural cell adhesion molecule (NCAM) expression in the anterior mesenchyme is placode epithelium dependent. (iii) Bone morphogenetic protein (BMP)-2, BMP-4, and fibroblast growth factor (FGF)-4 expression in the original dermal condensations was unaffected by recombination. (iv) Old dermal condensations can induce new placodes with new Wnt 7a, sonic hedgehog (Shh), and Msx-1 and -2 expression. (v) The new placode epithelium can then induce new Hox C6 and NCAM microgradients in the feather bud mesenchyme. (vi) The order of appearance can be classified into four groups in the following order: BMP-2, BMP-4, and FGF-4 (peptide growth factors); Wnt 7a and Shh (Drosophila segment polarity gene homologs); Msx-1 and Msx-2 (Msx class homeobox genes); and then Hox C6 (Hox class homeobox genes) and NCAM (adhesion molecules). These results suggest an order for the molecular cascade during the inductive phase of skin appendage development.

The mechanism involved in the morphogenesis of skin appendages is a fundamental issue underlying the development and healing of skin. To identify molecules involved in the induction and growth of skin appendages, we studied the expression of two homeobox genes, Msx-1 and Msx-2, during embryonic chicken skin development. We found that i) both Msx-1 and Msx-2 are early markers of epithelial placodes for skin appendages; ii) both Msx-1 and Msx-2 are expressed in the growing feather bud epithelia but not in the interbud epithelia; iii) although mostly overlapping, there are differences between the expression of the two Msx genes, Msx-1 being expressed more toward the anterior whereas Msx-2 is expressed more toward the distal feather bud; iv) there is no body-position-specific expression pattern as was observed for members of the Hox A-D clusters; v) in the feather follicle, Msx-1 and 2 are expressed in the collar and barb ridge epithelia, both regions of continuous cell proliferation; vi) when feather-bud growth was inhibited by forskolin, an activator of adenylyl cyclase, the expression of both genes was reduced. These results showed that Msx genes are specifically expressed in epithelial domains destined to become skin appendages. Its function in skin-appendage morphogenesis may be twofold, first in making epithelial cells competent to become skin appendages and, second, in making epithelial cells maintain their potential for continuous growth.

We examined the roles of adhesion molecules and homeoproteins in the morphogenesis of skin appendages using feather as a model. The expression pattern of these molecules in different stages of feather development were very dynamic. For example, neural cell adhesion molecules are present first in the dermal condensations, then in distal bud epithelium, then in the dermal papilla, and finally in the marginal and axial plates. Tenascin is present first in the placode, then in the anterior bud epithelium and mesoderm, and then in the dermal papilla. The expression patterns suggest that the adhesion molecules are involved in forming the boundary of cell groups that interact to form skin appendages. Antibody perturbation of embryonic skin-explant cultures showed that liver cell adhesion molecules are involved in establishing the hexagonal pattern, neural cell adhesion molecules are involved in the formation of dermal condensations, tenascin appears to be involved in the growth of feather buds, and integrin is essential for epithelial-mesenchymal interactions. Using antibodies to XlHbox 1 (similar to Hox 3.3 or C6) and Hox 4.2 (or D4), we showed that there is a homeoprotein gradient within the feather buds, and that the expression pattern is position-specific. It is hypothesized that Hox codes, derived from the combined expression pattern of homeoproteins, determine the phenotypes and orientation of skin appendages. Experiments using retinoids in the media or retinoid-soaked beads to create a local retinoid gradient are consistent with this hypothesis. As demonstrated here, feather development provides an excellent opportunity to analyze the molecular cascade of skin-appendage morphogenesis.