Identification of genes important for cutaneous function revealed by a large scale reverse genetic screen in the mouse.

The skin is a highly regenerative organ which plays critical roles in protecting the body and sensing its environment. Consequently, morbidity and mortality associated with skin defects represent a significant health issue. To identify genes important in skin development and homeostasis, we have applied a high throughput, multi-parameter phenotype screen to the conditional targeted mutant mice generated by the Wellcome Trust Sanger Institute's Mouse Genetics Project (Sanger-MGP). A total of 562 different mouse lines were subjected to a variety of tests assessing cutaneous expression, macroscopic clinical disease, histological change, hair follicle cycling, and aberrant marker expression. Cutaneous lesions were associated with mutations in 23 different genes. Many of these were not previously associated with skin disease in the organ (Mysm1, Vangl1, Trpc4ap, Nom1, Sparc, Farp2, and Prkab1), while others were ascribed new cutaneous functions on the basis of the screening approach (Krt76, Lrig1, Myo5a, Nsun2, and Nf1). The integration of these skin specific screening protocols into the Sanger-MGP primary phenotyping pipelines marks the largest reported reverse genetic screen undertaken in any organ and defines approaches to maximise the productivity of future projects of this nature, while flagging genes for further characterisation

Spindle checkpoint deficiency is tolerated by murine epidermal cells but not hair follicle stem cells

The spindle assembly checkpoint (SAC) ensures correct chromosome segregation during mitosis by preventing aneuploidy, an event that is detrimental to the fitness and survival of normal cells but oncogenic in tumor cells. Deletion of SAC genes is incompatible with early mouse development, and RNAi-mediated depletion of SAC components in cultured cells results in rapid death. Here we describe the use of a conditional KO of mouse Mad2, an essential component of the SAC signaling cascade, as a means to selectively induce chromosome instability and aneuploidy in the epidermis of the skin. We observe that SAC inactivation is tolerated by interfollicular epidermal cells but results in depletion of hair follicle bulge stem cells. Eventually, a histologically normal epidermis develops within ∼1 mo after birth, albeit without any hair. Mad2-deficient cells in this epidermis exhibited abnormal transcription of metabolic genes, consistent with aneuploid cell state. Hair follicle bulge stem cells were completely absent, despite the continued presence of rudimentary hair follicles. These data demonstrate that different cell lineages within a single tissue respond differently to chromosome instability: some proliferating cell lineages can survive, but stem cells are highly sensitive

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

Novel skin phenotypes revealed by a genome-wide mouse reverse genetic screen

reverse genetic scree

INPP5E regulates phosphoinositide-dependent cilia transition zone function

Human ciliopathies, including Joubert syndrome (JBTS), arise from cilia dysfunction. The inositol polyphosphate 5-phosphatase INPP5E localizes to cilia and is mutated in JBTS. Murine Inpp5e ablation is embryonically lethal and recapitulates JBTS, including neural tube defects and polydactyly; however, the underlying defects in cilia signaling and the function of INPP5E at cilia are still emerging. We report Inpp5e(-/-) embryos exhibit aberrant Hedgehog dependent patterning with reduced Hedgehog signaling. Using mouse genetics, we show increasing Hedgehog signaling via Smoothened M2 expression rescues some Inpp5e(-/-) ciliopathy phenotypes and "normalizes" Hedgehog signaling. INPP5E's phosphoinositide substrates PI(4,5)P-2 and PI(3,4,5)P-3 accumulated at the transition zone (TZ) in Hedgehog-stimulated Inpp5e(-/-) cells, which was associated with reduced recruitment of TZ scaffolding proteins and reduced Smoothened levels at cilia. Expression of wild-type, but not 5-phosphatase-dead, INPP5E restored TZ molecular organization and Smoothened accumulation at cilia. Therefore, we identify INPP5E as an essential point of convergence between Hedgehog and phosphoinositide signaling at cilia that maintains TZ function and Hedgehog-dependent embryonic development

HEpart3O-S

Package contains HE images of genes from O-

Data from: Identification of genes important for cutaneous function revealed by a large scale reverse genetic screen in the mouse

The skin is a highly regenerative organ which plays critical roles in protecting the body and sensing its environment. Consequently, morbidity and mortality associated with skin defects represent a significant health issue. To identify genes important in skin development and homeostasis, we have applied a high throughput, multi-parameter phenotype screen to the conditional targeted mutant mice generated by the Wellcome Trust Sanger Institute's Mouse Genetics Project (Sanger-MGP). A total of 562 different mouse lines were subjected to a variety of tests assessing cutaneous expression, macroscopic clinical disease, histological change, hair follicle cycling, and aberrant marker expression. Cutaneous lesions were associated with mutations in 23 different genes. Many of these were not previously associated with skin disease in the organ (Mysm1, Vangl1, Trpc4ap, Nom1, Sparc, Farp2, and Prkab1), while others were ascribed new cutaneous functions on the basis of the screening approach (Krt76, Lrig1, Myo5a, Nsun2, and Nf1). The integration of these skin specific screening protocols into the Sanger-MGP primary phenotyping pipelines marks the largest reported reverse genetic screen undertaken in any organ and defines approaches to maximise the productivity of future projects of this nature, while flagging genes for further characterisation