Maf is a family of oncogenes originally identified in an avian oncogenic retrovirus, AS42, encoding bZIP transcription factors. Recently, two maf-related clones, c-maf (maf-2) and mafB (maf-1), were isolated from genes of the rat liver complementary DNA library. It has been reported that genes of the maf family have essential roles in embryonic development and cellular differentiation. Both genes are expressed in a wide variety of tissues including spleen, kidney, lens, brain and liver; but so far, maf gene expression has never been investigated in skin development. The present study was performed to analyze the expression of c-maf-1 and mafB genes in rat skin in embryonic stages from 15 days onwards using in situ hybridization.

Fischer rats were used in the present study and were purchased from Nippon Clea (Shizuoka, Japan). The embryos were obtained from pregnant rats of embryonic days (ED) 15, 16 and 19 and were rinsed briefly with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and fixed in the same solution at 4°C overnight. The skin of rats on postnatal days (PD) 3 and 5 were also used to investigate maf gene expression. Fixed tissues were embedded in paraffin. Sections, 5 ?m thick, were cut. Digoxigenin (DIG)-labeled single-strand RNA probes were prepared using a DIG RNA labeling kit (Boehringer Mannheim, Germany) according to the manufacturer’s protocol. To prepare the rat maf probe, the full-length fragment of rat c-maf and mafB cDNA were subcloned into a pBluescript SK (?) plasmid (Stratagene, La Jolla, CA, USA). This plasmid was either linearized with EcoRI and transcribed with T7 RNA polymerase to prepare an anti-sense probe or linearized with HindIII and transcribed with T3 RNA polymerase to prepare the sense probe. Hybridization was carried out as described previously.

The expression of c-maf mRNA was first detected on ED 16 in the nuclei of cells in the basal layer of developing epidermis. On day 19, high expression was found in the nuclei of basal keratinocytes and developing hair germs. On PD 3, c-maf mRNA could not be demonstrated in the epidermis and hair follicles. Nonspecific staining was found at the edge of the corneal layer with both DIG-labeled anti-sense and sense RNA probes. On the other hand, high expression of c-maf mRNA was detected in the nuclei of cells in developing cerebral cortex on ED 19 which has been used as positive control. MafB has similar expression patterns as c-maf (data not shown). Table 1 summarizes expression patterns of both mafs.

Figure 1. Expression of c-maf mRNA in the skin of rat embryos and neonatal rats. On ED 19, high c-maf mRNA levels were detected in the nuclei of developing epidermal keratinocytes of the basal layer (open arrows) (A), and in the developing hair germs (B). On PD 3, c-maf mRNA could not be detected in basal epidermal keratinocytes (arrow heads) (C), and in hair follicles (open arrows) (D). A negative control section incubated with the DIG-labeled sense c-maf RNA probe did not contain any positive staining in the skin at ED 16 (E). High expression of c-maf mRNA was detected in the nuclei of cells in the developing cerebral cortex on ED 19 as positive control (F). Bars, 25 ?m.

As already mentioned, the maf oncogene (v-maf) was initially identified in an avian oncogenic retrovirus, AS42, which induces musculoaponeurotic fibrosarcoma in vivo and transforms chicken embryo fibroblasts in vitro. Several maf-related genes have been identified, and the maf gene family includes the large maf genes (c-maf, mafB and Nrl) and the small maf genes (mafK, mafG and mafF). Recently, it was found that both c-maf and mafB genes participate in transcriptional regulation during the development of the lens in rats. In the present study, high expression of both maf mRNAs was detected in the nuclei of basal keratinocytes and in the hair germs in developing rat epidermis on ED 19. Maf mRNA was not detected in the upper layer of differentiated epidermis on ED 19. Furthermore, maf mRNA expression was lost in postnatal rat skin. On the basis of data already available including the present results, it is concluded that c-maf and mafB may be involved in the embryonic development of the epidermis and hair follicles, but not in cellular differentiation of the skin.