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{"project":"2_test","denotations":[{"id":"15630473-14644200-84810253","span":{"begin":309,"end":311},"obj":"14644200"},{"id":"15630473-12646922-84810254","span":{"begin":652,"end":653},"obj":"12646922"},{"id":"15630473-12646922-84810255","span":{"begin":749,"end":750},"obj":"12646922"},{"id":"15630473-7958926-84810256","span":{"begin":751,"end":753},"obj":"7958926"},{"id":"15630473-10655587-84810257","span":{"begin":6170,"end":6172},"obj":"10655587"},{"id":"15630473-10655586-84810258","span":{"begin":6173,"end":6175},"obj":"10655586"},{"id":"15630473-10498690-84810259","span":{"begin":7118,"end":7120},"obj":"10498690"},{"id":"15630473-12646922-84810260","span":{"begin":7230,"end":7231},"obj":"12646922"},{"id":"T89400","span":{"begin":309,"end":311},"obj":"14644200"},{"id":"T54398","span":{"begin":652,"end":653},"obj":"12646922"},{"id":"T55026","span":{"begin":749,"end":750},"obj":"12646922"},{"id":"T82528","span":{"begin":751,"end":753},"obj":"7958926"},{"id":"T99055","span":{"begin":6170,"end":6172},"obj":"10655587"},{"id":"T23817","span":{"begin":6173,"end":6175},"obj":"10655586"},{"id":"T93454","span":{"begin":7118,"end":7120},"obj":"10498690"},{"id":"T72430","span":{"begin":7230,"end":7231},"obj":"12646922"}],"text":"Snail mRNA and Protein Are Expressed Transiently at the Hair Bud Stage of Follicle Morphogenesis\nAlthough Snail family members are most frequently associated with EMTs, they also participate in many malignant processes involving a down-regulation but not a quantitative abrogation of intercellular junctions [18]. The range of developmental processes in which Snail family members have been implicated thus includes the type of epithelial remodeling that is observed in hair follicle bud formation. Given our prior observation that exogenously added Snail can participate with LEF-1/β-catenin in down-regulating E-cadherin expression in keratinocytes [4], coupled with the established requirement for LEF-1/β-catenin in hair follicle morphogenesis [4,19], we turned to addressing whether Snail/Slug family members might also participate in the process.\nPCR analyses identified transient Snail mRNA expression during a period of skin embryogenesis when waves of hair follicles are forming (unpublished data).To pinpoint specifically where Snail mRNA is expressed in the developing skin, we conducted in situ hybridization using a cRNA probe unique to the Snail 3′ untranslated region (UTR). Embryonic day 17.5 (E17.5) was chosen, since the multiple waves of follicle morphogenesis occurring at this time enabled us to evaluate Snail expression at different stages of the process. As shown in Figure 1A, specific hybridization was detected within the epithelium of nascent hair buds. By contrast, as follicles progressed further through their development (e.g., germ and peg stages), they exhibited no signs of hybridization (Figure 1A). The transient nature of Snail mRNA expression during follicle development was most apparent in hybridized skin sections containing follicles from two different waves of morphogenesis (as shown in Figure 1). Hybridizing hair buds from a later wave appeared juxtaposed with nonhybridizing follicles from an earlier wave.\nFigure 1 Snail Is Expressed Exclusively in the Hair Bud during Morphogenesis\nEmbryos were either frozen in OCT embedding compound (A, F, and H) or embedded in paraffin (C, D, E, and G), and then sectioned (8 μm).\n(A) In situ hybridizations with Snail sense or antisense cRNA probes. Black dotted lines demarcate the basement membrane that separates the epidermis (epi) from dermis (der). Arrows point to Snail RNA expression, restricted to the hair bud stage of follicle morphogenesis. It was not seen in later hair germ or peg stages.\n(B) Expression of Snail protein coincides with hair development. Protein extracts were prepared from keratinocytes transfected with empty expression vector (K14), containing the K14 promoter or with the vector driving HA-tagged Snail (K14-Snail); or from whole skin from E13.5 to P5 animals, including newborn (nb). Equal amounts of proteins were then resolved by SDS-PAGE through 12% gels and subjected to Western blotting using either an affinity-purified Snail polyclonal antiserum, which we generated, or anti-tubulin (loading control).\n(C–E) Immunohistochemistry shows expression of Snail protein in the nuclei of cells within the hair and skin. (C) E13.5 skin with a single layered epidermis (epi) shows no Snail expression. (D) The first morphological sign that cells have adopted a hair follicle fate is a cluster of cells called a placode in E16.5 skin. Snail is not expressed at this stage of development. (E) Snail is expressed in the hair bud of E17.5 skin but not in later stages of development such as the germ or peg.\n(F) Immunofluorescence with anti-Ki67 (green) identifies the proliferating cells of the skin, restricted to the basal layer of the epidermis and developing hair follicles. Anti-β4 int labeling reveals the presence of the hemidesmosomal integrin β4, restricted to the base of cells adhering to the underlying basement membrane. The white dotted line marks the outermost surface of the skin.\n(G) Immunohistochemistry with pMAPK marks a subset of proliferating cells within the epidermis and hair bud. Anti-pMAPK labeling was consistently robust within the hair bud.\n(H) Immunofluorescence with anti-laminin 5 (lam5), which demarcates the basement membrane, and anti-E-cadherin (E-cad), a component of AJs. At the leading edge of the growing bud, cell-cell borders show markedly diminished anti-E-cadherin labeling (arrowheads). To determine whether this transient nature of Snail mRNA expression is reflected at the protein level, we generated an antibody against the N-terminal sequence that resides upstream of the more conserved zinc finger domains. As judged by Western blot analysis, the antibody did not detect endogenous proteins from cultured keratinocytes, but it did yield a band of the expected size from keratinocytes transiently expressing a hemagglutinin (HA)-tagged Snail protein (Figure 1B). The antibody also recognized a band corresponding to the size of endogenous Snail (approximately 28 kDa) in lysates from embryonic mouse skin, the temporal appearance of which corresponded to the waves of hair follicle morphogenesis from E15.5 to newborn when over 90% of the hair on the mouse is formed (Figure 1B). Consistent with the Western blot data, immunohistochemical analysis did not detect Snail in single-layered E13.5 epidermis (Figure 1C) nor in the placode, which is the earliest morphological sign of the commitment of multipotent cells of the embryonic ectoderm to a hair cell fate (Figure 1D). Consistent with the in situ hybridization results, anti-Snail antibody labeled only hair buds and not follicles at more mature stages of development (Figure 1E). Taken together, the anti-Snail antibody appeared to be specific for its target protein. It did not detect other Snail family members known to be expressed in keratinocytes and/or skin (unpublished data). Furthermore, the immunohistochemical data paralleled our Snail in situ hybridization data revealing transient Snail expression at the hair bud stage (Figure 1A).\nAs judged by immunohistochemistry, Snail protein was localized to the nuclei of the hair bud cells (Figure 1E). This feature was consistent with Snail's known function as a transcriptional repressor [12,13]. Additionally, anti-Snail labeling was detected in only three of the four major waves of follicle morphogenesis. Snail was not found in the buds of guard hairs that are the earliest of all hairs to form (at E13.5), and which constitute less than 5% of the mouse coat (unpublished data).\nAs judged by immunofluorescence with antibodies against the proliferating nuclear antigen Ki67, the timing of Snail expression coincided with the stage at which the developing follicle enhanced its proliferation and down-growth (Figure 1F). Immunohistochemistry with antibodies against the active (phosphorylated) form of MAPK (pMAPK) marked a subset of the proliferating (Ki67-positive) cells, and pMAPK-positive cells were enriched in the hair bud (Figure 1G). The timing of Snail induction and Ki67 and pMAPK enrichment in the hair bud appeared to follow closely the induction of LEF-1/β-catenin activity, known to initiate in the hair placode stage [20]. However, like placodes, hair buds exhibited down-regulation in E-cadherin expression (Figure 1H; see also [4])."}

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