KRT76 interacts with Claudin1.

<p>(A) HIS-tagged KRT76 tail domain and HIS-tag alone where produced in E.coli, purified and immobilised on nickel-resin. Resin was then incubated with mouse paw pad lysates and the specific pull-down of CLDN1 with the KRT76- tail domain and not HIS-tag was shown by anti-Claudin1 WB. (B) Soluble extracts were prepared from A549 cells and anti-CLDN1 or non-immune IgG antibody immunoprecipitated. IP and lysate/input samples were then blotted for ZO-1, CLDN1 and KRT76. (C) A549 cells co-express CLDN1 and KRT76 and these colocalise in cytoplasmic punctate structures -see arrowheads.</p

<i>Krt76</i> gene trap disruption causes gross epidermal defects.

<p>(A) Schematic showing <i>Krt76</i> gene trap (knock-out first) targeting construct. (B) Whole mount LacZ staining of <i>Krt76^tm1a/+</i> reporter mice, shows <i>Krt76</i> expression in the dorsal and ventral snout and palate, eyelid, and vagina. (C) Mice homozygous for <i>Krt76</i> gene trap disruption (<i>Krt76^tm1a/tm1a</i>) exhibit flaky skin following birth (see arrow-insert). Adult <i>Krt76^tm1a/tm1a</i> mice exhibit a scruffy coat and smaller body weight (n = 3 males, age 9 weeks, ***p&lt;0.004) (D, E), as well as tail scaling (F). <i>Krt76^tm1a/tm1a</i> mice exhibit paw pad hyperpigmentation (G), concurring with regions of LacZ reporter expression (H). LacZ expression within paw pads is detected in exocrine glands (H′) and suprabasal epidermal layers (I). (J, J′) Haemotoxylin and Eosin (H&amp;E) staining of paw pads from WT (J) and <i>Krt76^tm1a/tm1a</i> (J′) mice. Yellow arrowheads indicate abnormal dermal pigmentation. (K, L) Immunofluorescence analysis with indicated antibodies in wild type and <i>Krt76^tm1a/tm1a</i> mouse paw pad. Samples are counter stained with nuclear dye DAPI (4',6-diamidino-2-phenylindole). Coloured brackets indicate approximate distribution of FLG and KRT76 expression around the granular layer. (M) Western blot analysis of WT and <i>Krt76^tm1a/tm1a</i> dorsal skin and face skin extracts. (N) Immunofluorescence analysis with anti-KRT76 and anti-K14 antibodies in wild type mouse dorsal skin at E14.5, E18.5, P1 and adult time points and adult <i>Krt76^tm1a/tm1a</i> dorsal skin (N′). Asterisks indicate non-specific basal layer staining. (O) <i>Krt76</i> mRNA qRT-PCR analysis of p3 dorsal skin relative to <i>Gapdh</i>. Scale bars represent 50 µm.</p

<i>Krt76^mutant</i> mice show barrier function defects and KRT76 stabilises Claudin1 at tight junctions.

<p>(A) Transepidermal water loss assay on P3 dorsal skin from wild type and <i>Krt76^tm1a/tm1a</i> mice. (B) P3 paw pad skin was dermally injected with a biotin tracer and diffusion through the epidermis assessed, with Filaggrin (FLG) and DAPI co-staining for tissue orientation. Yellow arrowhead shows diffusion in suprabasal keratinocytes into cornified layer. (C) Biotin tracer was assessed alongside TJ component, Claudin1 (CLDN1). Tracer exclusion indicated by flanking yellow arrowheads. (D) Immunofluorescence analysis of CLDN1 and Ecadherin (ECAD) distribution in wild type and <i>Krt76^tm1a/tm1a</i> mouse dorsal skin. (E) Image quantification at the cellular surface shows an inward shift and a decrease in intensity of CLDN1 not observed with ECAD. (F) Further quantification by image analysis of CLDN1 co-localisation at the cell surface with ECAD or DAPI in the nucleus. (G, H) Immunofluorescence analysis of CLDN1 localization in dorsal skin of wild-type and <i>Krt76^tm1a/tm1a</i> mice in early phenotype and biopsy wounded adult dorsal skin of wild-type and <i>Krt76^tm1a/tm1a</i> mice. (I) Dorsal skin fractionation assay showing localisation of different proteins to different fraction; relative lcoalisation of CLDN1 are indicated in (I′). (J, K) Immunofluorescence analysis of CLDN1 localization in adult dorsal skin and paw pads of 4OHT-treated conditional <i>Krt76</i> knock-out mice and control sibling. Note paw pad phenotype from grooming transfer of tamoxifen. *p&lt;0.05, **p&lt;0.01. Error bars  =  S.E.M. Scale bars represent 50 µm.</p

Skin histopathology overview.

<p>(A) The dorsal skin of 44 wild type mice and 514 mutant mice were assessed by an expert dermatologist. 3 wild type mice and 30 targeted alleles (in 30 mutant strains) showed abnormalities in one or more of the phenotypic categories listed (35 abnormalities total). The phenotypes of abnormal hair shaft morphology and skin inflammation were observed in both wild type and mutants and taken to be a background phenotype, therefore were not considered significant in the overall phenotypic analysis. Wild type images for comparison are shown in (B) and (F). Phenotypes unique to mutant animals included abnormal hypodermis dermis morphology (C,D), abnormal pigmentation (E) and abnormally prominent arrector pilli muscle (G). More specifically, <i>Aldh18a1^tm1a/+</i> mice presented with mild, multifocal mixed inflammatory cell infiltration with mild fibrosis and distortion of adipocytes in the hypodermal fat layer potentially indicating abnormalities in white fat cells (C). <i>Prmt3^tm1a/tm1a</i> mutant mice exhibited granulomatous steatitis (red arrowheads) (D). <i>Arpc1b^tm1a/tm1a</i> mutant mice had mild to moderate multifocal areas of dermal fibrosis with pigment laden macrophages (yellow arrows) suggesting hair follicle rupture (E). <i>Rad18^tm1a/tm1a</i> mutant mice had normal skin but unusually prominent arrector pili muscles (black arrows) (G). Scale bars are 50 µm.</p

High throughput screening provides insights into molecular mechanisms of exogen in hair follicle cycling.

<p><i>Nsun2</i> expression was reported by <i>lacZ</i> staining in <i>Nsun2^tm1a/+</i> mice in the hair follicles of murine dorsal skin (A,B), the outer ear pinna (C), and tail (D). <i>Nsun2</i> mice demonstrated evidence of premature separation of the club hair from the surrounding follicle, leaving an empty area or breakage (E see arrowhead, Fi-v) in contrast to a normal follicle (G). <i>Lrig1</i> expression was reported by <i>lacZ</i> staining in <i>Lrig1^tm1a/+</i> mice in the developing epidermis and dermis of E14.5 skin (H), the developing hair follicle and upper dermis of E18.5 skin (I), the upper dermis and junctional zone above sebaceous glands in adult skin (J), the hair follicles of adult murine dorsal skin (K), the outer ear pinna (L) and inguinal fat pads (M). <i>Lrig1</i> mice also demonstrate premature separation of the club hair (N). <i>Farp2</i> expression was reported by weak <i>lacZ</i> staining in footpads and dermis of <i>Farp2^tm1a/+</i> mice (O,P see arrowhead). <i>Farp2</i> mice also demonstrated premature separation of the club hair (Q). Scale bars are 50 µm.</p

Multi parameter, multi test, organ specific screens add to the number of genes identified in skin biology.

<p>Each of the 3 different tests identified genes that were represented in other screens (shared) as well as genes that were unique to the test (A). Histopathology screen identified the highest number of unique genes flagged with 79% (n = 11/14) of genes not represented in any other skin screen. Different screens highlighted 25 unique MP terms, with only 1 MP term represented in multiple tests (B). Twenty three unique genes were identified across the 3 different skin tests with 4 represented in multiple tests (C).</p

Gene-FileHeader-Genotype-Phenotype Reference List

Master reference file to link Gene to Files and Noted Phenotypes

Examples of pigmentation phenotypes and expression patterns in genes with novel roles in skin biology.

<p><i>Mysm1^tm1a/tm1a</i> mice exhibited a range of pigmentation defects, including belly spots, and foot pad hyper-pigmentation (A, B, C). <i>LacZ</i> reporter expression of <i>Mysm1</i> is detected in the paw pad (D), dorsal skin and tail (E,F) whole mounts. Tail whole mount labelled with Keratin 14 (KRT14, red) and Keratin 15 (KRT15, green) indicate defects in hair follicle organization and associated structures in <i>Mysm1</i> knockouts (H) compared to heterozygotes (G). I) Analysis of footpad pigmentation in male <i>Vangl1^tm1a/tm1a</i> mice. J,K) <i>LacZ</i> reporter expression of <i>Vangl1</i> in the ear skin (pinna) and tail whole mounts.</p

Overview of pipeline, hair follicle cycling baseline and phenotypes.

<p>A) Flow-chart illulstrating pipeline of skin phenotyping. B–E) Examples of obvious coat phenotypes in <i>Myo5a, Lrig1</i> and <i>Nsun2</i> strains relative to wild-type mice. F) 10 male and 10 female pigmented wild type mice were shaved and skin colour assessed from 35–53 days of age. Results are shown in a grid where cell color represents skin colour. Black indicates anagen, shades of grey (and dark pink) indicate catagen, pale pink indicates telogen for males, pink indicates telogen for females, and crosses indicate days mice were not assessed. Mutant mice and matched wild type controls were next shaved in weekly cohorts and dorsal skin assessed for hair cycle phase using the skin color method above at an age of 41–43 days. Pigmented mice were assessed as normal (grey skin/in catagen), non synchronous (mixed patches of hair cycle phases), and anagen (black skin). Albino mice could not be assessed and were excluded from the analysis. G–K) Shows <i>Nf1^tm1a</i>; <i>Nsun2^tm1a</i>; <i>Myo5a^tm1e</i>; <i>Trpc4ap^tm1a</i>; <i>Nom1^tm1a</i> demonstrated signs of abnormally in constrast to their wild type controls. L) Table summarises wild type findings for baseline reference.</p

HEpart2E-N

Package contains HE images of genes from E-