Employed keratin immunostaining and BrdU incorporation assays (Fig. 3). In control skin

Employed BTZ043 Keratin immunostaining and BrdU incorporation assays (Fig. 3). In control skin, Keratin K14 expression is detected in the basal epithelial cells while keratin K1 reactivity was observed in all suprabasal cell layers (Fig. 3A). The mutant epidermis showed K14 labeling in more suprabasal layers (Fig 3A and 3B). BrdU-labeled cells were detected sporadically in the stratum basale in control epidermis, but more than twice as many BrdU-labeled cells were found in the mutant epidermis (Fig. 3B). We also assayed the epidermis for expression of Keratin K6, a marker of aberrant epidermal 25033180 differentiation. K6-labeled cells were strongly detected in the suprabasal layers of the mutant epidermis, but not in the control epidermis (Fig. 3C). These findings indicate that all layers of the skin are affected in the pigskin mutant.X-gal Staining of Whole Embryos and SkinTo assess the pattern of hair follicle induction, we used a BMP4lacZ reporter line [24] and we assayed for ?galactosidase activity by X-gal staining as described previously [25]. Briefly, males that were compound heterozygous for the Fatp4 mutation and for BMP4-lacZ, were mated to females heterozygous for the Fatp4 mutation. Embryos were genotyped by PCR using one pair of primers to amplify the wild type allele (Ex8 (S), 59-CCACTGAATG CAACTGTAGCC-39 and Ex9(WT,AS), 59TCCATTCCCTCCTGGGCAGACCT-39 and a different antisense primer (Ex9, pigskin AS, 59-TCCATTCCCTCCTGGGCAGACCA-39 to assay for the mutant allele. Amplification bands were 360 bp. Mouse embryos or peeled skin were harvested from timed pregnancies and fixed in 2 paraformaldehyde plus 0.2 glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) at 4uC for 1 hour. Embryos or skin were rinsed three times (30 minute each) in washing solution containing 0.1 M phosphate buffer (pH 7.3), 2 mM MgCl2, 0.01 sodium RE-640 deoxycholate, and 0.02 NP-40. Embryos were then stained at 4uC for 12 hours in X-gal staining solution (washing solution plus 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 1 mg/mL X-gal). Stained embryos or skin were rinsed in phosphate-buffered saline (PBS; pH 7.4) and stored in 70 ethanol. After staining, embryos were photographed using a 35 mm Nikon digital camera and images were processed with Adobe Photoshop. All of blue hair follicles in the lateral body (1 mm x 1 mm area) of E14.5 embryos were counted (at least three embryos in each genotype). A 1326631 strongstained blue dot with an unstained core and a distinctive ring shape from the skin of E16.5 embryos was counted as primary hair follicles (PHFs) while other smaller stained blue dots were counted as secondary hair follicles (SHFs). Statistical significance (p values) was computed by using Student’s t test. A p value of less than 0.05 was considered statistically significant. Image J software was used to count hair follicles [26].SNP Mapping of the Pigskin MutationThe pigskin mutation arose on an FVB background. In order to map the mutation, we mated pigskin carrier males to C57BL/6J partners. The F1 offsprings were used for test matings to identify mice that carried the pigskin mutation. Carriers were mated to each other, and the F2 offspring were again mated to identify carriers of the pigskin mutation. F2 carriers and their mutant offspring were used for SNP analysis [19]. We analyzed genomic DNA from four carrier parents, and nine affected newborns (Fig. 4) as well as the parental FVB and C57 lines. SNP mapping identified a candidate region of the genome c.Employed keratin immunostaining and BrdU incorporation assays (Fig. 3). In control skin, Keratin K14 expression is detected in the basal epithelial cells while keratin K1 reactivity was observed in all suprabasal cell layers (Fig. 3A). The mutant epidermis showed K14 labeling in more suprabasal layers (Fig 3A and 3B). BrdU-labeled cells were detected sporadically in the stratum basale in control epidermis, but more than twice as many BrdU-labeled cells were found in the mutant epidermis (Fig. 3B). We also assayed the epidermis for expression of Keratin K6, a marker of aberrant epidermal 25033180 differentiation. K6-labeled cells were strongly detected in the suprabasal layers of the mutant epidermis, but not in the control epidermis (Fig. 3C). These findings indicate that all layers of the skin are affected in the pigskin mutant.X-gal Staining of Whole Embryos and SkinTo assess the pattern of hair follicle induction, we used a BMP4lacZ reporter line [24] and we assayed for ?galactosidase activity by X-gal staining as described previously [25]. Briefly, males that were compound heterozygous for the Fatp4 mutation and for BMP4-lacZ, were mated to females heterozygous for the Fatp4 mutation. Embryos were genotyped by PCR using one pair of primers to amplify the wild type allele (Ex8 (S), 59-CCACTGAATG CAACTGTAGCC-39 and Ex9(WT,AS), 59TCCATTCCCTCCTGGGCAGACCT-39 and a different antisense primer (Ex9, pigskin AS, 59-TCCATTCCCTCCTGGGCAGACCA-39 to assay for the mutant allele. Amplification bands were 360 bp. Mouse embryos or peeled skin were harvested from timed pregnancies and fixed in 2 paraformaldehyde plus 0.2 glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) at 4uC for 1 hour. Embryos or skin were rinsed three times (30 minute each) in washing solution containing 0.1 M phosphate buffer (pH 7.3), 2 mM MgCl2, 0.01 sodium deoxycholate, and 0.02 NP-40. Embryos were then stained at 4uC for 12 hours in X-gal staining solution (washing solution plus 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 1 mg/mL X-gal). Stained embryos or skin were rinsed in phosphate-buffered saline (PBS; pH 7.4) and stored in 70 ethanol. After staining, embryos were photographed using a 35 mm Nikon digital camera and images were processed with Adobe Photoshop. All of blue hair follicles in the lateral body (1 mm x 1 mm area) of E14.5 embryos were counted (at least three embryos in each genotype). A 1326631 strongstained blue dot with an unstained core and a distinctive ring shape from the skin of E16.5 embryos was counted as primary hair follicles (PHFs) while other smaller stained blue dots were counted as secondary hair follicles (SHFs). Statistical significance (p values) was computed by using Student’s t test. A p value of less than 0.05 was considered statistically significant. Image J software was used to count hair follicles [26].SNP Mapping of the Pigskin MutationThe pigskin mutation arose on an FVB background. In order to map the mutation, we mated pigskin carrier males to C57BL/6J partners. The F1 offsprings were used for test matings to identify mice that carried the pigskin mutation. Carriers were mated to each other, and the F2 offspring were again mated to identify carriers of the pigskin mutation. F2 carriers and their mutant offspring were used for SNP analysis [19]. We analyzed genomic DNA from four carrier parents, and nine affected newborns (Fig. 4) as well as the parental FVB and C57 lines. SNP mapping identified a candidate region of the genome c.