Sustained Expression of Snail Results in Epidermal Hyperproliferation and Differentiation Defects in Tg Mouse Skin
The striking spike of Snail expression coincident with hair bud formation and enhanced proliferation prompted us to examine the consequences of ectopically expressing Snail elsewhere in mouse skin epidermis. To distinguish Tg from endogenous Snail, we used the HA-epitope, shown previously not to alter Snail's transcriptional activity [12]. Of 20 K14-Snail[HA] Tg animals generated, three expressed the transgene and all exhibited analogous phenotypes. Mice that integrated the transgene at the single-cell stage died at or shortly after birth. The three surviving full-Tg founder mice harbored transgene integrations that gave stable transmission of mosaic Snail gene expression through the germline. Progressively poor health necessitated our sacrificing most offspring from these lines within a year of birth.
As Snail Tg animals grew, they became distinguished by their small size, short tails, and flaky skin (Figure 2A). Histological analyses of 3-d old (P3) mice revealed mosaic patches marked by epidermal thickening (Figure 2B). The mosaic morphology was reflected at the level of Tg Snail protein, with only the hyperthickened regions expressing nuclear HA-tagged Snail (Figure 2C). These hyperthickened areas were marked by excessive proliferation, as revealed by antibodies against the proliferating nuclear antigen Ki67 (Figure 2D and 2E). Activated, pMAPK-positive cells were also prevalent in these areas (Figure 2F and 2G), as were cells expressing keratin 6, a keratin induced in the suprabasal layers of hyperproliferative skin (Figure 2H and 2I).
Figure 2  Misexpression of Snail in Mouse Skin Epidermis Results in Hyperproliferation
Three different surviving Tg founder mice harbored a K14-Snail transgene that was integrated into a locus that resulted in inheritable, mosaic expression of the transgene in skin epidermis. All displayed similar abnormalities, as did their offspring.
(A) P16 WT and K14-Snail Tg mice. Insets denote magnified tail segments, which displayed a mosaic, flaky appearance in Tg mice. Size differences appeared with age, and are likely due to K14-promoter activity in the tongue and oral epithelium, resulting in progressive defects and reduced food intake. Hence, skin sections from young (P3) mice were analyzed (B–I).
(B) Hematoxylin- and eosin-stained Tg skin section. Double arrows demarcate the border of mosaic histology, with seemingly normal epidermis (epi) and a mature hair follicle (hf) at left and hyperthickened epidermis at right.
(C) Immunofluorescence of Tg skin section labeled with antibodies as color-coded on frame. Double arrows demarcate the border of mosaic anti-Snail (green), revealing Snail expression coincident with regions of hyperthickened epidermis (at left) and absent in regions of normal epidermis (at right).
(D–I) Sections of P3 WT or Tg skin (affected region) subjected to either immunofluorescence (D, E, H, and I) or immunohistochemistry (F and G) with antibodies as indicated on the panel. Anti-keratin 5 indicates K5, normally restricted to the basal layer of the epidermis; anti-keratin 6 detects keratin 6, expressed in postnatal epidermis under conditions such as wounding, in which hyperproliferation occurs. All other antibodies are as in the legend to Figure 2. Comparison of D and E provide representative examples that illustrate that pMAPK is found in only a subset of all proliferating (Ki67-positive) cells. Note also the presence of Ki67- (E) and pMAPK-positive (G) cells in some suprabasal areas; Ki67-positive cells colabeled with anti-Snail (E). Expression of the Snail transgene did not block terminal differentiation in the hyperproliferative epidermis, but it distorted it markedly (Figure 3A–3H). Typical of most hyperproliferating conditions, Snail expression led to a large expansion in layers with spinous and granular morphology. Additionally, however, was a marked and variable expansion of keratin 5 (K5), normally restricted to the innermost basal layer (see Figure 3). Although the failure of Snail-null mice to develop past gastrulation [21] precluded our ability to study the loss of Snail function in skin development, a good correlation emerged between the expression of Snail protein and the extension of K5, Ki67, and pMAPK suprabasally (compare data in Figures 2 and 3).
Figure 3  Alterations in the Differentiation Program and Basement Membrane Organization in Snail-Expressing Tg Epidermis
(A–H) Immunofluorescence of skin sections from P3 WT and Tg mice. Shown are affected areas of Tg skin; in areas where Snail protein was not expressed, stainings were normal. Sections were labeled with antibodies as indicated and color-coded on each frame. Antibodies are against markers of normal epidermal differentiation, and include K5 (a basally expressed keratin), K1 (a suprabasal keratin, expressed in spinous layer cells), involucrin (Inv; a suprabasally expressed cornified envelope protein found in upper spinous and granular layer cells), loricrin (Lor; a cornified envelope protein expressed in the granular layer), and filaggrin (Fil; a protein that bundles keratin filaments in the granular layer and stratum corneum). Note abnormal extension of anti-K5 suprabasally, often present in anti-K1 positive suprabasal Tg cells.
(I–N) Immunohistochemistry (I and J) or immunofluorescence (K–N) of sections of P30 Wt (I, K, and M) and Tg (J, L, and N) (affected areas) skins using the antibodies indicated. Note that with age, affected areas of the Tg epidermis became increasingly undulating, often exhibiting papilloma-like invaginations (J). Insets in I and J are magnified views of the boxed areas, illustrating the absence (Wt) or presence (Tg) of nuclear anti-cyclin D staining. With age, affected areas of the Tg epidermis also displayed perturbations within the basement membrane, as judged by antibody labeling against either basement membrane (K and L) or hemidesmosomal (M and N) components. Double arrows in L demarcate mosaic zones, revealing that perturbations were restricted to hyperthickened, i.e., Snail-positive zones (to left of double arrows). Other abbreviations are as noted in the legend to Figure 2. The changes in hyperproliferation and differentiation were not initially accompanied by gross signs of epithelial invaginations. With age, however, epidermal folds and undulations developed in areas where Snail was expressed, and proliferative markers persisted in these regions (Figure 3I and 3J; anti-cyclin D staining). The undulations were accompanied by partial dissolution of the underlying basement membrane (Figure 3K and 3L). Aberrant staining was also observed with antibodies against components of the hemidesmosomes, which provide strong adhesion of basal epidermal cells to the underlying basal lamina (Figure 3M and 3N). Interestingly, similar types of alterations occur in the basement membrane in the hair bud of embryonic and newborn mice when Snail is normally expressed. The fact that the basement membrane separating the epidermis from the dermis is altered only in the adult Tg animals suggests the involvement of intermediary factors not as readily available in the epidermis as they are in the follicle.