Kobielak and Fuchs. 10.1073/pnas.0510422103. |
Supporting Figure 5
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 5
Fig. 5. Additional characterization of differentiation and proliferation defects associated with loss of a-catenin. (A and B) E18.5 WT and cKO skin grafts at days indicated after grafting. (C) Lesional area of a rare cKO genetically homozygous mouse that survived because it is mosaic for ablation of the floxed a-catenin allele. (A-C) Sections were either stained for hematoxylin and eosin or subjected to immunofluorescence microscopy with Abs against K1, a suprabasal epidermal differentiation marker; b4 integrin, a hemidesmosomal marker; Lef1, a hair matrix marker; trichohyalin (AE15), a marker of the hair follicle inner root sheath; and hair-specific keratins (AE13), a marker of the cortex and hair shaft, to exclude a hair follicle origin of the epithelial invaginations. In B note that K6 is expressed in cKO epidermis. It is normally restricted to the outer root sheath of the hair follicle, but it is in the epidermis of hyperproliferative skin disorders. Arrows denote typifying features of the WT state, aberrant in the cKO skin. Asterisks denote keratinized pearls, typical of hyperproliferative, papilloma-like epidermal undulations. (C) In areas of reduced K14-GFP staining, Ecadherin staining was also reduced. These areas tended to be concentrated at the invasive front of older grafts and in this case, a-catenin null patches of mosaic mice. Color coding is according to secondary Abs. Abbreviations are as in Fig. 1.
Supporting Figure 6
Fig. 6. Semithin sections of skin grafts. Shown are 1-mm Epon sections stained with toluidine blue of 40d grafts from E18.5 WT (A and A’) and cKO (B and B’) mouse skins. (A) WT graft displays a normal morphology with the presence of numerous hair follicles (hf). (A’) All layers of epidermis are present. (A’ Inset) The same magnification as B’. (B) No hair follicles are detected in the cKO graft but masses of epidermal cells invaginate into the dermis. Boxed area in is magnified in B’. Boxed areas in A’ and B’ are the regions magnified in the ultrastructural images shown in Fig. 1. The different epidermal layers can be recognized in these invaginations (B’). epi, epidermis; bc, basal cells; sp, spinous layer; gr, granular layer; sc, stratum corneum.
Supporting Figure 7
Fig. 7. Testing for signs of Wnt activity in a-catenin cKO skin. Nuclear b-catenin can be detected by immunohistochemistry of paraffin-fixed tissue. (A) E18.5 WT skin sections display nuclear b-catenin only in the developing hair placodes, where it is known to participate in hair follicle morphogenesis. By contrast, E18.5 cKO epidermis shows only cell border staining with b-catenin Abs (boxed area is magnified in Inset). This finding is consistent with the lack of hair placode formation in the absence of a-catenin (1). b-Catenin/Lef1/Tcf transcriptional activity can be detected by a Wnt reporter gene, encoding b-galactosidase under the control of a minimal promoter and an enhancer (referred to as TOP) composed of multimerized Lef1/Tcf binding sites. When b-galactosidase is expressed, its activity can be detected by X-Gal, a dye that turns blue when cleaved (see ref. 2). Reporter gene activity is strong in the precortical cells, which give rise to the hair shaft in WT skin. By contrast, no activity was detected in the a-catenin null skin, consistent with the lack of hair follicle morphogenesis in this tissue, and with the absence of nuclear b-catenin. (B) When nuclear b-catenin and Lef1 are present, cells will activate the TOP enhancer, which can be sensitively assayed for by using a TOP-FLASH reporter gene, encoding firefly luciferase. The level of TOP-FLASH activity was low in WT and KO keratinocytes, even when supplemented with a K14-Lef1 transgene. When supplemented with a truncated version of b-catenin, known to be constitutively stable in keratinocytes (3), activity was elevated markedly. Interestingly, the KO cells were significantly more sensitive to the addition of exogenous stabilized b-catenin than the WT cells, possibly reflecting the lack of competition between Lef1/Tcf proteins and E-cadherin/a-catenin for b-catenin. Based on these data, it is even more likely that we would have detected this activity in vivo had it been present. (C) Recently, it was shown that stabilized b-catenin can directly associate with and repress the activity of p50 NF-kB homodimers, which control a novel set of genes including the tumor suppressor gene KAI1 (4). KAI1 mRNAs did not score on our microarrays as appreciably decreased in the a-catenin null state. Real-time PCR of mRNA isolated from either GFP+, FACS-sorted cells or cultured keratinocytes from cKO epidermis did not exhibit a major repression of KAI1 over WT epidermal cells. epi, epidermis; der, dermis.
1. Vasioukhin, V., Bauer, C., Degenstein, L., Wise, B. & Fuchs, E. (2001) Cell 104, 605-617.
2. DasGupta, R. & Fuchs, E. (1999) Development (Cambridge, U.K.) 126, 4557-4568 .
3. Gat, U., DasGupta, R., Degenstein, L. & Fuchs, E. (1998) Cell 95, 605-614.
4. Kim, J. H., Kim, B., Cai, L., Choi, H. J., Ohgi, K. A., Tran, C., Chen, C., Chung, C. H., Huber, O., Rose, D. W., et al. (2005) Nature 434, 921-926.
Supporting Figure 8
Fig. 8. Correlation between up-regulated genes and immunodetection of their encoded proteins in E18.5 skin. Immunohistochemistry was used for detection of nuclear Snail protein; immunofluorescence was used for the rest. Asterisks denote artifactual staining of outer epidermal layers caused by the secondary Ab used. Arrow denotes K6 staining in outer root sheath of hair follicles, which is normal. Suprabasal K6 staining in epidermis is abnormal, characteristic of hyperproliferative conditions. Arrowhead denotes staining of the vascular endothelial marker VCAM(+) staining in some cells within the cKO epidermis. TenC, tenascin C; other abbreviations are as in Fig. 1.
Supporting Figure 9
Fig. 9. Ultrastructural evidence of an inflammatory response in a-catenin cKO skin. E18.5 WT and cKO mouse skins were grafted onto Nude mice, and after 40 days, tissues were processed for electron microscopy. Shown are representative images. Dashed lines denote the dermo-epidermal border. WT (A and C) and cKO (B and D) grafts. In WT skin, most of the dermal cells in proximity with the epidermis (epi) are fibroblasts (f). In cKO graft, inflammatory cells are found in the dermis and also infiltrating the epidermis macrophages (mac), mast cells, granulocytes (gran) of the eosinophil (eo), and neutrophil (n) types, lymphocytes (lym). Mi, mitosis.