32 research outputs found
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
Mouse models in cutaneous biology
The recent advent of large scale reverse genetics and phenotyping projects has signalled a new era in the application of mouse genetics to understand gene function. International efforts to elucidate gene function are exemplified in The Sanger Mouse Genetics Project. This project has created a genetically mutant mouse resource of unprecedented size for the phenotype-based investigation of gene function in vivo. The integration of organ- specific screening pipelines is critical for the continued existence and maximization of reverse screening efforts to ascribe gene function. Using this approach, the analysis of the genetic complement to skin biology is examined. Phenotype data gathered as a part of this screen initiated the in depth investigation of the contribution of two genes, Krt76 and Mad2, to skin barrier function and skin stem cells, respectively. This is the first and largest tissue-specific reverse genetic screen ever to be carried out, and accordingly, has identified novel genes and novel roles for known genes required for normal skin function. At the genome level, a multi-test, multi-parameter reverse genetic screen was applied to over 500 different knockout mouse strains with the aim of identifying defects in development or homeostasis of the skin. The dynamic, indispensable, and accessible nature of the skin make it a valuable model system in which to study genetic contributions to its function and model cell biology. This screen identified more than 20 different affected lines, many of which carry mutations in genes that have not previously been implicated in the biology of the organ. This work highlights the promise of high throughput reverse genetic screens to give critical insights into the genetics of skin biology. The regenerative properties of the skin are regulated by multiple stem cell populations that reside there, making the skin an excellent organ in which to model stem cell biology. Mad2 is a known component of the spindle assembly checkpoint (SAC) whose loss in mitotically active cells leads to aneuploidy. Because aneuploidy and disruptions in spindle assembly are incompatible with early mouse development, Mad2 was conditionally inactivated in the skin. This allowed for the in vivo investigation of Mad2, and showed that defects in the checkpoint are tolerated in the dividing cells of the interfollicular epidermis, but not in the stem cell population in the hair follicle. This study is an elegant example combining mouse genetics and skin biology to understand the differential response to aneuploidy within a single tissue. The healing and protective properties of the skin were examined using germline inactivation of Keratin76 (Krt76). It was shown that Krt76, a poorly characterized intermediate filament protein, is absolutely required to maintain the integrity of the skin and is essential for postnatal survival. The progressive development of cutaneous wounds occurs on the background of a primary defect in the maintenance of the skin barrier. Here, a novel mechanism by which intermediate filaments interact with tight junction components to maintain barrier function in the skin is described
Mouse models in cutaneous biology
The recent advent of large scale reverse genetics and phenotyping projects has signalled a new era in the application of mouse genetics to understand gene function. International efforts to elucidate gene function are exemplified in The Sanger Mouse Genetics Project. This project has created a genetically mutant mouse resource of unprecedented size for the phenotype-based investigation of gene function in vivo. The integration of organ- specific screening pipelines is critical for the continued existence and maximization of reverse screening efforts to ascribe gene function. Using this approach, the analysis of the genetic complement to skin biology is examined. Phenotype data gathered as a part of this screen initiated the in depth investigation of the contribution of two genes, Krt76 and Mad2, to skin barrier function and skin stem cells, respectively. This is the first and largest tissue-specific reverse genetic screen ever to be carried out, and accordingly, has identified novel genes and novel roles for known genes required for normal skin function.
At the genome level, a multi-test, multi-parameter reverse genetic screen was applied to over 500 different knockout mouse strains with the aim of identifying defects in development or homeostasis of the skin. The dynamic, indispensable, and accessible nature of the skin make it a valuable model system in which to study genetic contributions to its function and model cell biology. This screen identified more than 20 different affected lines, many of which carry mutations in genes that have not previously been implicated in the biology of the organ. This work highlights the promise of high throughput reverse genetic screens to give critical insights into the genetics of skin biology.
The regenerative properties of the skin are regulated by multiple stem cell populations that reside there, making the skin an excellent organ in which to model stem cell biology. Mad2 is a known component of the spindle assembly checkpoint (SAC) whose loss in mitotically active cells leads to aneuploidy. Because aneuploidy and disruptions in spindle assembly are incompatible with early mouse development, Mad2 was conditionally inactivated in the skin. This allowed for the in vivo investigation of Mad2, and showed that defects in the checkpoint are tolerated in the dividing cells of the interfollicular epidermis, but not in the stem cell population in the hair follicle. This study is an elegant example combining mouse genetics and skin biology to understand the differential response to aneuploidy within a single tissue.
The healing and protective properties of the skin were examined using germline inactivation of Keratin76 (Krt76). It was shown that Krt76, a poorly characterized intermediate filament protein, is absolutely required to maintain the integrity of the skin and is essential for postnatal survival. The progressive development of cutaneous wounds occurs on the background of a primary defect in the maintenance of the skin barrier. Here, a novel mechanism by which intermediate filaments interact with tight junction components to maintain barrier function in the skin is described
TALEN-Mediated Gene Editing of the Thrombospondin-1 Locus in Axolotl
Loss-of-function genetics provides strong evidence for a gene's function in a wild-type context. In many model systems, this approach has been invaluable for discovering the function of genes in diverse biological processes. Axolotls are urodele amphibians (salamanders) with astonishing regenerative abilities, capable of regenerating entire limbs, portions of the tail (including spinal cord), heart, and brain into adulthood. With their relatively short generation time among salamanders, they offer an outstanding opportunity to interrogate natural mechanisms for appendage and organ regeneration provided that the tools are developed to address these long-standing questions. Here we demonstrate targeted modification of the thrombospondin-1 (tsp-1) locus using transcription-activator-like effector nucleases (TALENs) and identify a role of tsp-1 in recruitment of myeloid cells during limb regeneration. We find that while tsp-1-edited mosaic animals still regenerate limbs, they exhibit a reduced subepidermal collagen layer in limbs and an increased number of myeloid cells within blastemas. This work presents a protocol for generating and genotyping mosaic axolotls with TALEN-mediated gene edits.This article is from Regeneration 2 (2015): 37, doi:10.1002/reg2.29. Posted with permission.</p
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TALENâmediated gene editing of the thrombospondinâ1 locus in axolotl
Abstract Lossâofâfunction genetics provides strong evidence for a gene's function in a wildâtype context. In many model systems, this approach has been invaluable for discovering the function of genes in diverse biological processes. Axolotls are urodele amphibians (salamanders) with astonishing regenerative abilities, capable of regenerating entire limbs, portions of the tail (including spinal cord), heart, and brain into adulthood. With their relatively short generation time among salamanders, they offer an outstanding opportunity to interrogate natural mechanisms for appendage and organ regeneration provided that the tools are developed to address these longâstanding questions. Here we demonstrate targeted modification of the thrombospondinâ1 (tspâ1) locus using transcriptionâactivatorâlike effector nucleases (TALENs) and identify a role of tspâ1 in recruitment of myeloid cells during limb regeneration. We find that while tspâ1âedited mosaic animals still regenerate limbs, they exhibit a reduced subepidermal collagen layer in limbs and an increased number of myeloid cells within blastemas. This work presents a protocol for generating and genotyping mosaic axolotls with TALENâmediated gene edits
TALENâmediated gene editing of the thrombospondinâ1 locus in axolotl
Abstract Lossâofâfunction genetics provides strong evidence for a gene's function in a wildâtype context. In many model systems, this approach has been invaluable for discovering the function of genes in diverse biological processes. Axolotls are urodele amphibians (salamanders) with astonishing regenerative abilities, capable of regenerating entire limbs, portions of the tail (including spinal cord), heart, and brain into adulthood. With their relatively short generation time among salamanders, they offer an outstanding opportunity to interrogate natural mechanisms for appendage and organ regeneration provided that the tools are developed to address these longâstanding questions. Here we demonstrate targeted modification of the thrombospondinâ1 (tspâ1) locus using transcriptionâactivatorâlike effector nucleases (TALENs) and identify a role of tspâ1 in recruitment of myeloid cells during limb regeneration. We find that while tspâ1âedited mosaic animals still regenerate limbs, they exhibit a reduced subepidermal collagen layer in limbs and an increased number of myeloid cells within blastemas. This work presents a protocol for generating and genotyping mosaic axolotls with TALENâmediated gene edits
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 âź1 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<sup>tm1a/+</sup></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<sup>tm1a/tm1a</sup></i>) exhibit flaky skin following birth (see arrow-insert). Adult <i>Krt76<sup>tm1a/tm1a</sup></i> mice exhibit a scruffy coat and smaller body weight (nâ=â3 males, age 9 weeks, ***p<0.004) (D, E), as well as tail scaling (F). <i>Krt76<sup>tm1a/tm1a</sup></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 (Hâ˛) and suprabasal epidermal layers (I). (J, Jâ˛) Haemotoxylin and Eosin (H&E) staining of paw pads from WT (J) and <i>Krt76<sup>tm1a/tm1a</sup></i> (Jâ˛) mice. Yellow arrowheads indicate abnormal dermal pigmentation. (K, L) Immunofluorescence analysis with indicated antibodies in wild type and <i>Krt76<sup>tm1a/tm1a</sup></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<sup>tm1a/tm1a</sup></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<sup>tm1a/tm1a</sup></i> dorsal skin (Nâ˛). 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 Âľm.</p
<i>Krt76<sup> mutant</sup></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<sup>tm1a/tm1a</sup></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<sup>tm1a/tm1a</sup></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<sup>tm1a/tm1a</sup></i> mice in early phenotype and biopsy wounded adult dorsal skin of wild-type and <i>Krt76<sup>tm1a/tm1a</sup></i> mice. (I) Dorsal skin fractionation assay showing localisation of different proteins to different fraction; relative lcoalisation of CLDN1 are indicated in (Iâ˛). (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<0.05, **p<0.01. Error bars â=â S.E.M. Scale bars represent 50 Âľm.</p