In-situ hybridization to detect Corin transcripts (blue) in FVB mice. Corin is first detected when the dermal condensate segregates from surrounding dermis during the first (A, E15.5) and second (B, E16.5) waves of follicle formation. Follicles derived from the first, second and third waves of follicle formation are indicated. Throughout the growth phase of the hair cycle, Corin transcripts are expressed in the DP and not detected elsewhere in the skin. Samples from E17.5 (C) P0 (D,E), P3 (F) and P11 (G) are shown. The field enclosed by the red square in D is shown in higher magnification in panel E to reveal that Corin is expressed throughout the DP and not in the surrounding hair matrix.

(A) Schematic representation of corin mRNA and protein. The mRNA of Corin (shown in red) comprises 22 exons encoding a type-II transmembrane serine protease. The single-pass transmembrane domain (TM) of Corin resides in close proximity to the N-terminus, and the large extracellular portion includes two frizzled-like cysteine-rich motifs (designated Frizzled 1 and 2), eight LDL receptor repeats (LDLR 1-8), a macrophage scavenger receptor-like domain (SRCR) and a catalytic domain of trypsin-like serine protease at the C-terminus. The active site residues of the catalytic triad (H, D and S) are shown. The regions corresponding to the RNA probe and the fragment of Corin protein used to immunize rabbits are indicated. (B–F) Immonuhistochemical detection of Corin (green) in DP at E17.5 (B), P0 (C), P3 (D), P6 (E) and P11 (F). Red stain highlights nuclei. Occasional staining was observed outside the DP (white arrowheads) but this was also detected in controls in which only the secondary antibody was used (data not shown and Fig. S2). In E and F, higher magnification of a bulb region is shown (upper right) to reveal that the majority of the protein is localized at the periphery of DP cells.

(A) Gene targeting approach used for corin ablation. A YFP-Neo cassette replaced 64bp downstream of the ATG in exon 1(red box). Primers (arrowheads, see table S1) and expected PCR fragments are indicated. Primer WT51 binds in the deleted-64bp segment and the primers SC3 and SC5 bind outside the targeting construct. The NcoI fragments used for Southern analysis are indicated. The FRT flanked pgkNeo^R cassette was oriented in parallel to Corin transcription. (B,C,D) Genomic analysis of wild-type, heterozygous and homozygous Corin mice. (B) Southern analysis of NcoI-digested genomic DNA. The 5'-probe reveals 18.6kb and 10kb bands from wild-type and mutant alleles respectively. (C) PCR with primers SCNEO5 and SC3 generates a 6075bp band from the targeted allele (upper panel) while WT51 and SC3 generate a 5266bp fragment from wild type. (D) PCR with WT51, WT31, KILY5 and SEQFP1 detects both wild-type (312bp) and mutant (478bp) alleles for routine genotyping. (E,F) Immunostaining of frozen P3 skin-sections from wild-type and mutant mice with anti-Corin antibodies. Corin-green; Nuclei-red.

Mice lacking Corin (−/−) exhibit lighter coat-color than corresponding wild type (+/+) on an Agouti background. (A,B) Mice during the first (A) and second (B) hair cycles are shown. (C) Two litters at P16 are shown each containing one a/a (black), one tyrC/tyrC (white), and two A/A with Corin genotypes indicated. (D) Pairs of mice homozygous and heterozygous for a functional agouti allele and wild-type or mutant for Corin are shown with higher magnification views at right. On both Agouti genotypes lack of Corin leads to a lighter coat color and in the absence of Corin, A^W/A^W are lighter than A^W/a.

(A) The longest yellow band found in zigzag hairs lacking Corin (Mut) and the shortest subapical yellow band found in wild-type zigzag hairs (WT) are shown. (B) The approach used to quantify the differences between wild-type and mutant zigzag hairs. The ratio (R) between the length of the yellow band (Y) and the length of the apical segment (Z) was scored and assigned to three categories: R≤0.5 (C and D), 0.5<R≤0.75 (E and F), 0.75<R≤1 (G and H). (C–H) The apical segment of representative hairs in these categories is shown. The tip of each hair is shown in higher magnification in the upper left corner. Both the length of the yellow band and the length of the apical segment vary in both wild-type and mutant mice (compare D and F). (I) The distribution of zigzag hairs in wild-type and mutant mice among the above categories is shown including the proportion of black versus yellow tips in each category (black vs. yellow shading respectively). All wild-type zigzag hairs end with a black tip (C,E), while 70% of mutant zigzag hairs exhibit a yellow tip (D,F,H). The hair population of R≤0.5 with black tips that predominates in wild type is almost completely absent in mice lacking Corin. The category of 0.75<R≤1 is essentially unique to mice that lack Corin. The p-values for R≤0.5, 0.5<R≤0.75 and 0.75<R≤1 are <0.0001, 0.0017 and 0.0002 respectively.

(A) Representative examples of three categories of awl hairs used in this analysis. In contrast to zigzag hairs, awl hairs may lack a subapical yellow band. (B) The ratio (R) between the length of the yellow band (Y) and the length of the whole hair (A) was calculated and assigned to the categories: R=0, R≤0.25, 0.25<R≤0.5. (C) The distribution of awl hairs among these categories in wild-type (black) and mutant mice (pink). Although a small proportion of 0.25<R≤0.5 is present in wild-type mice, most of these fall close to R=0.25 (data not shown). The p-values for R=0, R≤0.25 and 0.25<R≤0.5 are 0.0002, 0.01 and <0.0001 respectively.

(A–F) The relative RNA levels determined by Real-time PCR (y-axis) of Corin (A), agouti (B), Mc1r (C), Pomc1 (D), Atrn (E) and Mgrn1 (F) are shown from birth to P9 (x-axis) for Corin +/+ (black) and Corin −/− (pink). The RNA levels of all genes tested were normalized to the same units, but different scales employed in A,E,F vs. B,C vs. D. Thus the level of Atrn mRNA is about 5 fold higher than the RNA levels of agouti at its peak (B and E), while Pomc1 levels are similar to those of agouti at its baseline levels from P7 on (B and D). (G) In-situ hybridization of agouti in P5 skin. Stars indicate hair follicle bulbs with agouti expression in the DP and eumelanin deposition. Note that in the absence of Corin, eumelanin deposition has not been detected in the bulb region surrounding agouti-expressing DPs. In the right upper corner, higher magnification of the region enclosed by the red square is shown.