article: 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.
  Recent advances in the high throughput production of targeted mutant mice using gene targeting in ES cells have made it possible to rapidly and effectively produce knockout mouse lines for use in phenotypic screening [1], [2], [3], [4]. Such approaches are now beginning to yield significant insights into mammalian gene function [1], [5]. Central to recent community efforts has been the Wellcome Trust Sanger Institute' s Mouse Genetics Project (Sanger-MGP), which is undertaking a high throughput reverse genetic screen using the knockout ES cells generated by the International Knockout Mouse Consortium, which has now merged with the International Phenotyping Mouse Consortium (www. mousephenotype. org). To maximise the value of this resource, it is important to develop organ specific secondary screens to complement the broad phenotyping efforts applied in existing production pipelines. To this end, we implemented a multi-parameter, multi-test skin screen to identify mouse models of skin dysfunction and to elucidate genetic pathways important for skin development and function. In this study, we detail the skin assessment pipelines internal to the Sanger-MGP as well as skin analyses performed externally, with the exception of the fluorescent tail whole mount analysis, published elsewhere [6]. A flowchart for the pipeline is summarised in Figure 1A and presented in more detail in Figure S1A. The skin is the largest of the intermediate sized organs in the human body [7] and it plays integral roles in immune surveillance, wound healing and protection from environmental challenge [8]. Skin related conditions account for significant health expenditure and top the list of reasons for general practice appointments in the U. S. [9]. A considerable number of skin conditions have genetic underpinnings [10]. Therefore, understanding the genetic basis of skin development and skin disorders has the potential to greatly impact both the diagnosis and treatment of skin related conditions. Moreover, a significant number of other systemic diseases or those principally affecting other organs also manifest themselves in the skin. Such conditions include diabetes mellitus, chronic kidney disease, celiac disease, and rheumatoid arthritis. As a highly regenerative organ in which developmental gene expression is often recapitulated during adulthood, the skin also provides a window into many of the programs central to differentiation and homeostasis. The skin and its associated “mini organ”, the hair follicle, are particularly attractive targets for such a screen because of their ready accessibility and dynamic nature [11]. Furthermore, the clinical pathology and physiology based mouse phenotyping “pipelines” are not designed to find subtle lesions affecting the skin [12]. For these reasons a skin specific phenotyping program was implemented on the genetically modified mice produced by the Sanger-MGP with a goal of defining new and previously unappreciated genetic determinants of skin disease. In this paper mouse lines carrying mutations in each of 562 genes were investigated, and phenotypes were identified as a result of mutations in 23 genes (Table S1). These skin studies have identified genes not previously associated with skin phenotypes and ascribe further functions to genes which were previously associated with skin disease. We also used these tests to assign 25 Mammalian Phenotype (MP) ontology terms [13] (http: //www. informatics. jax. org/searches/MP_form. shtml), 2 of which are novel (Table S1). The scale of the screen also provides the opportunity to assess the relative value of different phenotyping approaches, with a view to implementing these screens in other proposed and ongoing high throughput reverse genetic screens.
  A number of the screens identified new players in cutaneous biology or identified new phenotypes not previously associated with these genes. Some individual examples are discussed. A critical question for any screening approach is the relative value it brings in respect of identifying new phenotypes. While all parts of the skin screen identified mutant lines that were also identified in other tests, there were still sets of genes that were unique to each specific test (Figure 5A). For example, three out of the five genes identified in the hair follicle cycling screens were not identified using any other screening approach in our skin phenotyping pipeline (Figure 5A). This is also particularly true of the histopathology screen in which 11 of the 14 positive genotypes recorded no other hits in other skin pipeline – although the depth of screening by this approach was limited to 1–2 mice per line (Figure 5A). We also noted that each test identified MP terms that were not described by any other tests (Figure 5B). That is to say, the addition of extra screens for subtle phenotypes that were not reflected in gross changes to appearance were reliably able to identify both new genes and new phenotypes (Figure 5A, B, C; Table S1). Taken together this strongly argues for the inclusion of these more involved screens to flag skin related abnormalities, particularly in light of the relatively low hit rates achieved using simple dysmorphology. The screen in its current form may also underestimate the frequency of abnormal phenotypes due to the limitations imposed by sample size, albinism in strains, the stage of the hair cycle the necropsy samples, and a consequence of homozygous embryonic lethality. More detailed screens employing larger numbers of mice, together with skin-specific conditional sub-strains to overcome problems of complete knock-out lethality, will be required to accurately define a greater number and range of phenotypes. We detail one of the most comprehensive reverse genetic skin screens yet reported in mice. The gene targeting strategy, combined with a robust skin phenotyping protocol, has allowed immediate establishment of a preliminary connection between a genotype and a phenotype. Such approaches have only become feasible through the recent advent of high throughput, targeted mouse mutagenesis screens. Clearly, more detailed follow-up studies are necessary to elaborate on these initial findings and to investigate in greater detail the contribution of a given gene to the biology of the skin. One such follow-up study on Krt76 is provided in an accompanying manuscript in this issue of PLoS Genetics [16]. Nonetheless high throughput approaches of this type will be central to flagging genes for further study. An argument might be made that molecules important to something as visually evident as skin development and homeostasis would already have been identified through mutagenesis approaches, or historically as a result of sporadically occurring mutations. However, the identification of two new genes that cause defects in pigmentation (Mysm1 and Vangl1) and a further additional pigmentation phenotype in Myo5a mutants shows that even for the most obvious cutaneous feature, there remains considerable value in ongoing screening for new causative mutations. In particular, clinical and histological presentations vary between alleles based on the type of mutation (null, hypomorph) or inbred strain background (strain specific modifier genes), and so comparison between these “knockout ready” targeted mouse lines and existing spontaneous mutants present in various public repositories will be particularly informative in respect of gene function. Similarly the skin might be considered an “easy” target for phenotypic screens, and that important genes could simply be identified based on a casual observation of the mutant animal in question. Our results argue that this is not the case. Instead we find that there is significant value in including secondary, organ specific screens in large scale projects of this nature. In deciding which phenotypic screens to apply to a given mutant mouse population one has to consider the likelihood that a given test will identify a phenotype not also apparent in other primary phenotyping screens. The object of such an assessment is therefore to reduce the costs and processes associated in examining such large cohorts of mice, whilst maximising the value of the resource (i. e. in robust phenotypes flagged). Interestingly, based on our experience, each of the phenotypic screens utilised added significantly to the base level of hits in the screen (Figure 5). While gross skin abnormality assessments shared the greatest overlap with the other screens employed, there were considerable numbers of genes identified in the more subtle screens that did not result in an obvious, visually assessable, phenotype in the mouse. The hair follicle cycling screen is a case in point. To all intents and purposes the visual phenotype of those animals which display abnormal hair follicle cycling is normal. However, a simple estimation of cycling stage uncovered a further class of mutants whose involvement in this process was never previously appreciated. The same is true of the section based histopathology screen, which was able to identify subtle but potentially valuable phenotypes. In both cases, whether through the subtle temporal regulation of follicle cycling or via the cellular diversity assessed in the pathology screen, looking a little deeper, can be a particularly valuable exercise. Taken together, our screen has identified a diverse collection of genes which will form the basis of further studies aimed at elucidating gene function in more detail. They also highlight the value of including detailed skin phenotyping approaches in any future efforts of this nature.
  All mouse studies were undertaken by Wellcome Trust Sanger Institute Mouse Genetics Project as part of the International Mouse Phenotyping Consortium and licensed by the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986. Animal models maintained under the auspices of ethics applications to Monash University were subject to the conditions of the Australian Bureau of Animal Welfare. Mice were generated from ES cells in the KOMP/EUCOMM pipeline and principally compromised conditional ready alleles in which a lacZ reporter cassette was integrated upstream of a floxed essential exon [5] (Figure S2). With a few exceptions (n = 24/532) the mice were generated from C57BL/6N ES cells and maintained on this genetic background. A proportion of the lines examined (n = 201), generally those that entered the pipeline in the initial stages of the project, were maintained on a mixed C57BL/6N; C57BL/6Brd-Tyrc-Brd background. For mice in the histopathology screen details of the strain background, age of sampling, diet, sex, time of tissue harvest, allele name and heterozygosity/homozygosity are detailed in Table S1. Animals were maintained in a specific pathogen free environment with ad libitum access to food (typically Mouse Breeders Diet (LabDiets 5021-3, IPS, Richmond, USA) ) and water. Specific details of housing and husbandry for individual lines are available from mouseinterest@sanger. ac. uk. For some lines (indicated in Table S1) mice at 4 weeks of age were transferred to a high fat (21. 4% fat by crude content; 42% calories provided by fat) dietary challenge (Special Diet Services Western RD 829100, SDS, Witham, UK). All mice generated by the Sanger-MGP underwent a broad primary phenotype screen (“dysmorphology screen”; see Table S1,), which included the visual assessment of skin and hair as described here. Tail skin (n = 2) was collected at necropsy for whole mount immunohistochemistry (IHC) analysis using antibodies against keratins (KRT) 15 and 14 (as per [52] [6]). The activity of the integrated beta-galactosidase (lacZ) cassette was assessed using heterozygous cohorts of mice (n = 2 male, 2 female) generated specifically for expression analysis. The pattern of β-gal reporter gene activity was determined by lacZ labelling of whole mount tissue preparations from heterozygous knockout. Mice were perfused with fresh cold 4% paraformaldehyde (PFA). Tissues were fixed for a further 30 min in 4% PFA, rinsed in phosphate buffered saline (PBS) and labelled with 0. 1% X-gal for 48 hours at 4°C. Samples were post-fixed overnight in 4% PFA at 4°C, cleared with 50% glycerol, transferred to 70% glycerol and imaged. Dorsal skin at the thoraco-lumbar region/interscapular region was collected at the time of necropsy, fixed in neutral buffered 10% formalin solution, process routinely, embedded in paraffin, sectioned at 6 µm, stained with hematoxylin and eosin (H&E). Samples from 1–2 female animals were reviewed by an experienced, board certified veterinary anatomic pathologist (JPS). Animals intended for histopathology were reserved for this purpose and not subjected to other phenotyping tests. All slides were scanned and images archived using Zeiss Mirax Slide Scanner and associated software. In addition, representative photomicrographs were taken of all H&E stained slides. Images are available immediately through the Dryad online database (doi: 10. 5061/dryad. mv34v) and progressively through the International Mouse Phenotyping Consortium website (www. mousephenotype. org) and on the Mouse Genome Informatics webpage (http: //www. informatics. jax. org/genes. shtml) (also see Table S1).
summary: Recent developments in high throughput applications to manipulate and inactivate specific genes in mouse embryonic stem cells (ES cells) have allowed for the initiation of large scale reverse genetic screens in the mouse. The immediate connection of a phenotype to a mutated (null) gene represents a paradigm shift in our ability to explore gene function. This study utilized such a screening approach to investigate the genetic contribution to skin development and homeostasis. Not only does this approach provide insight into the genetics of skin biology, it is also instrumental in generating novel models with which to study the genetic underpinnings of skin disease. Initial screening of 562 mutated genes in mice uncovered previously unrecognized genes involved in the biology of this organ and identified novel functions for previously studied genes associated with epidermal phenotypes. Taken together, these results highlight high throughput screening approaches that are valuable in reverse genetic screening and provide a pool of mouse mutants, available to the scientific community, that will serve as the basis for further detailed investigations into skin function and skin disease.
keywords: skin
  animal models
  model organisms
  integumentary system
  anatomy
  genetics
  biology and life sciences
  mouse models
  genomics
  research and analysis methods
title: Identification of Genes Important for Cutaneous Function Revealed by a Large Scale Reverse Genetic Screen in the Mouse