Hugo J. Bellen - US grants

9413237 Eichele This award will support a training program for undergraduate and graduate students that focuses on the use of invertebrate model systems to inform investigation of vertebrate development, and vice versa. The relatively recent and surprising discovery that a series of genes that control development in drosophila have homologues with similar structure, function and organization in vertebrates has set the stage for this type of approach. The 18 faculty associated with the program have basic research programs that study the development of a variety of vertebrates and invertebrates, including drosophila, nematode, slime mold, mouse, chicken and ferret; the group includes a few individuals at nearby institutions whose participation is expected to broaden the impact of the program. The training will include existing, expanded and new course offerings, as well as lab rotations aimed at helping students become conversant with principles of vertebrate and invertebrate studies. An annual two-day retreat will help build the program. Students will be encouraged to undertake research that includes study of two organisms, one vertebrate and the other invertebrate. This project represents a modern approach to training in one of the most exciting areas of modern biology. ***

nerve /myelin protein; tissue /cell culture

DESCRIPTION (Adapted from applicant's description): The goal of this predoctoral training program in Developmental Biology is to produce scientists committed to careers in biological and biomedical research related to Developmental Biology, including basic developmental mechanisms, developmental pathways, morphogenesis, cancer, and human genetic disease that cause developmental defects. Courses in molecular biology, classical and molecular genetics, biochemistry, developmental biology, cell biology, and soon functional genomics will be part of this training. The trainees will have a thorough foundation in all these areas and laboratory research experience. In addition, trainees have opportunities to attend seminars and journal clubs in the Texas Medical Center. Funds are requested for eight predoctoral trainees. The Developmental Biology Training Program will be based in the Graduate School of Biomedical Sciences at Baylor College of Medicine.

The focus of this project is to study the role that endocytosis plays in regulating of developmental signaling pathways. We will be focusing on the highly conserved Notch and Wingless signaling pathways. These pathways play critical roles in regulating cell number and cell fate specification during development and adulthood. Given these important functions, it is not surprising that misregulation of these pathways has been implicated in numerous human diseases including cancer. We are interested in how these signaling pathways are so precisely regulated. Our preliminary data focuses on Hrs, an endocytic protein that mediates endosomal sorting and multivesicular body formation. We have found that altered Hrs function results in mislocalization of Notch and Wingless signaling members and results in aberrant Notch and Wingless signaling. These findings demonstrate the critical link between protein intracellular trafficking and signaling regulation. In this proposal, we plan to investigate the mechanism through which this endocytic regulation occurs. First, a detailed structure-function analysis of Hrs will be performed, including a study of the functional implications of Hrs localization and protein associations. These studies will provide insight not only into the regulation of Notch and Wingless signaling, but also the mechanism of multivesicular body formation. For the Notch pathway, we will then perform detailed analyses of protein intracellular localization by confocal and electron microscopy. Very little is currently known about the intracellular trafficking of Notch signaling proteins. Our studies will expand on what is current known and investigate the implications of protein localization on signaling. The Wingless protein is known to form morphogen gradients that induce different developmental fates depending on morphogen concentration. We will characterize the role that endocytosis plays in forming the morphogen gradient and regulating Wingless signaling by investigating the effects on Wingless transcription, secretion, spread, and degradation. These findings will elucidate not only the regulation of Wingless signaling, but also what has long been an issue of controversy - how the Wingless gradient forms. Taken together, this work will help us determine the intracellular trafficking of key signaling protein and the effect that these trafficking events play in regulating signaling. Since misregulation of the Notch and Wingless pathways is implicated in human disease, we anticipate that insights gained from this study will aid in better understanding the molecular pathogenesis of these diseases.

DESCRIPTION (provided by applicant): This project will provide genetic tools to the public research community by constructing a library of Drosophila strains (ultimately 14,000) in which each fruit fly gene has been tagged or disrupted with a single transposable element insertion. Approximately 40,000 new single P-element insertion strains and 13,000 new single piggyBac insertion strains will be constructed. The transposon insertion site in each strain will be identified by DNA sequencing, computational analysis and manual curation. A subset of about 4,000 lines will be selected, based on the criterion that the gene tagged by a transposon has not been disrupted in other strains in the collection. This subset of strains will be saved, balanced and transferred to the public stock center at Bloomington, Indiana, for distribution to the research community. During the proposed project period, we will increase the size of the current library to 10,000 strains, bringing 10,000/13,600 = 74 percent of Drosophila genes, regardless of phenotype, under experimental control. This number of new lines generated (4,000) is more than the number of all the Drosophila genes for which both molecular structure and mutant phenotype are currently known. While a wide range of biological and medical problems have already begun to be analyzed using Drosophila, far more is yet to come. These new tools will empower and advance productive research affecting most NIH Institutes and myriad human medical conditions. In strictly financial terms this proposal will benefit NIGMS as well as other NIH institutes, as equivalent strains will otherwise have to be inefficiently generated under individual lab R01s at much greater expense.

[unreadable] DESCRIPTION (provided by applicant): The aim of this proposal is to create novel reagents for the Drosophila research community to facilitate mapping and identification of genes on the X chromosome and to facilitate manipulation of large autosomal genes. Mapping of genes on the autosomes has been greatly facilitated by genome-wide projects to generate molecularly defined deletions. Unfortunately, deletion mapping is not easy to apply to the X chromosome, as males carry only one copy and so are hemizygous. The alternative to deletion mapping is duplication mapping, i.e. one can use duplications of the X chromosome that are translocated onto the Y chromosome or an autosome to map essential genes on the X chromosome. If these duplications are molecularly defined, as is the case for the deletions, then one can map a gene quickly and precisely. The simplest way to create such a set of defined duplications would be to generate a collection of overlapping large transgenic fragments that cover the entire X chromosome and are inserted onto the Y chromosome or an autosome. Here, we propose to create 350 fly stocks that carry molecularly defined ~100 kb transgenic fragments inserted on the Y chromosome and that cover the entire euchromatic portion of the X chromos- ome. To achieve this goal, we have developed a set of seminal new tools by introducing recombineering technology into a set of vectors (the Pfacman] vectors) that allow transformation via phiC31-mediated integration. We show that genomic constructs in the 75-133 kb range can be engineered through recomb- ineering mediated gap-repair in bacteria, that these DNA fragments can be easily manipulated, and that they can be inserted into the genome efficiently. Hence, we propose to construct a BAG library with a P[acman] vector, sequence and map the ends of the cloned inserts, and create transgenic fly stocks. These reagents will greatly facilitate mapping and identification of genes on the X chromosome, one of the key priorities in the current Drosophila white paper. We also propose to identify or engineer 220 P[acman] BACs to create transgenic strains that carry large autosomal or heterochromatic genes. Mutations in large genes cannot be rescued by P element transgensis, but can easily be manipulated by recombineering and inserted efficiently into the genome through phiC31-mediated integration. All the vectors, the BAG library, and the transgenic stocks will be made available to the research community as soon as they are generated and tested. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application is to seek support to continue the interdepartmental and interinstitutional graduate and postgraduate Program in Developmental Biology (DB Program) at Baylor College of Medicine. The DB Program was founded on a commitment to excellence in research, with strict guidelines for both students and faculty of the Program 1992. The DB Program has 36 faculty members from seven basic science and three clinical departments, including a balanced mix of full, associate, and assistant professors. It provides students with a wide spectrum of exciting research possibilities. During the 14 years of the DB Program, 74 students have entered the program, 36 students are currently enrolled, and 31 students have so far graduated. The program has succeeded in attracting excellent students and has gradually grown from classes of three students to classes of seven to eight students. Numerous students from the DB Program have graduated with an excellent to outstanding publication record, and the average number of publications per graduate student is above 4.5, with an average of more than 2.5 first author papers. These publications have an average impact factor of higher than 10 per publication. Since the current students are recruited from a better and larger applicant pool, it is anticipated that these data will improve even more in the future. For example the GPA of the incoming students has steadily grown from an average of 3.21 in 2000 to 3.66 in 2006. The program emphasizes a rigorous educational program with numerous courses in many different areas of science. Students must pass two qualifying exams, one of which testing general knowledge and one examining their mastery of the ongoing research in the laboratory they select, which is an R01 type application. The goal is to prepare students for a multidisciplinary research environment in biomedical sciences. The program has selected and monitored students carefully and has a very low attrition rate (less than 7%) when compared to most graduate programs. It also has been able to recruit talented underrepresented students in the past five years. During the past training period there were four predoctoral positions awarded each year. Given the expansion of the program and the number of excellent eligible students, seven predoctoral positions are being requested now. The goal is to support first and second year graduate students. For example, there are six eligible incoming students for 2006. Also requested is a modest postdoctoral training component to the program since there are more than 35 eligible candidates per year performing research in the laboratories of the training faculty members. A better integrate of these postdoctoral fellows in the DB Program would entice them to attend some classes offered by the program, invite them to the annual retreat, promote their interactions with other developmental biologists, allow them also to teach in some of the courses, and hence prepare them better for a career in developmental biology.

Confocal microscopy complements the work of most BCM-IDDRC investigators whose work entails analysis and description of protein distribution within cells or tissues. Among the specific strengths of this system are the capacity to use multiple markers concurrently and the ability to study the three-dimensional character of one or several related proteins with sub-cellular resolution. For members of the BCM-IDDRC, the three confocal systems have facilitated discovery and elucidation of genes involved in human disorders such as Rett and SCA1, functional analyses of genes including Mathi, Mecp2, Ataxin, Gfi-1, shar-pei, APP, VAP-33A, endophilin, crumbs, hrs, CSP, atonal, syntaxin, senseless, neurexin, ROP, discs lost, skittles, gutfeeling, synaptobrevin, synaptotagmin, apterous, Dpp and protein structure/function studies of potassium channel domains. These and similar studies have involved thirteen BCM-IDDRC laboratories on an ongoing basis and nine other BCM-IDDRC laboratories on an occasional basis. Cost Effectiveness: The original cost of the two Zeiss confocal microscope systems and related equipment was approximately $1.2 million. Operating costs per year including primarily service contracts, salary costs for user training and day to day maintenance and technical support of histological services are currrently approximately $128,000. Very few BCM-IDDRC laboratories could individually justify either the expense of purchasing and maintaining any one of the confocal systems or would fully utilize the full capacity of a single system. Quality Assurance: All of the microscopes in the Confocal core were purchase new, were under warranties during the first year and have since been continuously under service contracts with the manufacturers to assure all systems meet or exceed performance quality requirements. Preventative maintenance of the equipment includes regular cleaning of user inaccessible portions of the microscope systems and revision of microscope software as needed. Where feasible, we have in the past and will continue to improve the software and hardware associated with the microscopes to provide additional features, attain better equipment performance, and better meet new or changing user and regulatory requirements. The microscopes are observed on a daily basis and user serviceable portions of the systems are examined, aligned, cleaned and repaired on a regular basis by the core manager. Most of our users experience their initial use of Confocal microscopy using our equipment and training to obtain optimal images from the equipment is a significant component of quality. Each of our new users is provided with at least two hours of individual hands-on training with the core manager followed by required solo sessions and ongoing support as needed. F. UTILIZATION The confocal microscopy core will provide training, equipment and technical support to any BCM-IDDRC investigator who would benefit from use of the confocal microscope systems. We expect that BCM-IDDRC users will continue to benefit from the preferential access to the systems and because of the favorable fee structure in comparison with available alternatives

The path from the discovery of a gene involved in mental retardation (MR) to understanding the mechanism of its functions often follows a similar process requiring each of the specialties of the sub cores. Following the initial identification of the responsible gene, a mouse knock in and/or knock out model is generated. If human disease causing mutations are known, transgenesis of these mutations may be used to determine whether similar etiology may be inferred from the mouse model. In parallel with the other research activities, each of the gene expression sub cores are engaged in the sequential process of discovery. In situ hybridization is used to determine the normal distribution pattern of RNA from the gene, particularty in the mouse brain. The in situ core is able to provide comprehensive analysis of the normal, knock out or transgenic mouse brain RNA distribution pattern in a format that allows precise comparison with the distribution pattern of other known genes. Since the in situ core produces data and images that are directly comparable with the major mouse atlases currently being made available to researchers, the overall data resource is robust and rapidly expanding. Detailed analysis of the pathology of the knockout or transgenic mouse brain is supported by the neuorpathology core. Here brain and other tissue samples are examined by specialists using conventional histology and immunohistochemistry often leading to a detailed understanding of the specific abnormalities. The confocal microscopy core facilities are used to prepare physically and/or optically sectioned samples used to study protein distribution, to compare normal and abnormal patterns of distribution, to determine whether other proteins are affected by the gene and to study the three dimensional distribution of the gene product. Very often the linear pattern of discovery described is an extreme oversimplification and the actual process is iterative because the results of one core both inform and are informed by the results of another. This is one of the key benefits of engaging the several cores in long term collaborative arrangements. Among the long term studies relevant to IDD that have benefited from the above processes through the BCM-IDDRC gene expression core are Rett Syndrome (Zoghbi lab), forms of Fragile X (Nelson lab), and Angelmen syndrome (Beaudet lab), SCA1 (Zoghbi, Bellen, Botas labs). As an example, alteration of the MeCP2 gene as the primary genetic basis of Rett syndrome was discovered by the Zoghbi laboratory in 1999 following more than a decade of collaborative work on the disease spanning many disciplines including clinical observations, pathology, analysis of behavior and molecular biology (1) . Throughout this period the pathology core provided invaluable support through histology, identification and analysis. More recently, the confocal microscopy core and the in situ core have come to play a significant role in studying the relationship of MeCP2 to other proteins, the effects of X linkage in Rett and the mechanisms of Mecp2 function. Work during the current grant period has gradually been unraveling individual components of the wide range of abnormalities associated with Rett but has focused particularly upon aspects of the disease that may be susceptible to remediation. Unusual susceptibility to stress is a hallmark of Rett syndrome and an elevated stress response is also present in mice models with an truncated MeCP2 transgene (2) . In support of the Zoghbi lab' efforts to study the stress response of these mice the in situ core provided histology and statistical analysis demonstrating that the CrH gene is elevated in regions of the brain associated with corticosteroid release (2) . Continuing with this information, the Zoghbi laboratory found that regulatory elements of the CrH promoter region interact directly with MeCP2 but do not bind the MeCP2(308) truncated gene. Using a Cre-loxP technology, MeCP2 was then eliminated exclusively from Sim-1 expressing hypothalamic cells reproducing the abnormal stress response found in mice lacking MeCP2(3) . Results from the In situ core and confocal microscopy core were then used to determine that these conditional knock out mice have reduced expression of MeCP2 in the same stress response associated regions in which MeCP2 is under-expressed in Mecp2 (308) mice and that stress response related genes such as glucocorticoid-inducible kinasel and FK-506-binding protein are mis-regulated in the Mecp2 null brain. The potential for accurate clinical diagnosis is complicated in X-linked diseases such as Rett because X chromosome inactivation (XCI) may be skewed by genetic and epigenetic factors to mask phenotypic expectations. Work by the Zoghbi laboratory, motivated by prior clinical evidence of imbalanced XCI in some human Rett patients and high phenotypic variability in some mouse models of Rett, employed the confocal core facilities for detailed analysis of XCI at the cell and tissue levels in Mecp2 (308) mice (4) . Using an antibody that recognized wild type MeCP2 but not MeCP2(308) together with antibodies that recognize specific neurons, it could be shown that XCI is skewed throughout the brain in Mecp2(308)/X mice and that in Mecp2(308) mice the degree of skewing toward the wild type genetic background observed at the cellular and tissue level was inversely correlated with the penetrance of mutant phenotypes. The importance of precise regulation of Mecp2 expression was explored in studies comparing neuronal function in mouse neurons where MeCP2 is over-expressed or under-expressed (5) . In this study the confocal microscopy core equipment was used to correlate synapse density with synapse function and to confirm that MeCP2 is involved in early postnatal synaptogenesis and maintenance of glutamatergic neurons in vitro and in vivo. This work has important implications because it provides evidence for the early role of synaptogenesis in IDD generally and because it offers another avenue for therapeutic intervention.

DESCRIPTION (provided by applicant): We propose to map and deposit a collection of well defined chemically induced mutations in essential genes on the Drosophila melanogaster X chromosome for distribution by the Bloomington Drosophila Stock Center (BDSC) to the fly community. This resource will be very valuable as the mutations were induced on an isogenized y w FRT19A chromosome using low concentrations of ethyl methanesulfonate to minimize genetic load. The chromosomes were screened for the presence of lethal mutations and subsequently assayed in an ey-FLP and Ubx-FLP FRT screen. We screened for mutations that affect numerous biological processes, including eye, head, wing, thorax and bristle development, photoreceptor growth cone guidance, synapse formation, synaptic transmission, and neurodegeneration. We also identified mutations that cause a head overgrowth phenotype as well as tumorous unpatterned growth. We propose to map the molecular lesion in 380 different complementation groups, to rescue the phenotype with small X chromosome duplications, to deposit the stocks in the BDSC, and to provide all the data relevant to each mutant to FlyBase. This collection will cover 40-45% of all the essential genes on the X chromosome. This is a very valuable collection of mutations that will be much used by the members of the Drosophila community (see 22 letters of support). This collection of mutations will promote our basic understanding of many different biological processes, as well as help us better understand the pathogenesis of cancer, developmental, and neurological diseases. PUBLIC HEALTH RELEVANCE: Basic research using Drosophila melanogaster has led to discovery of numerous genes that affect many biological processes, including homeotic genes, genes controlling numerous signaling pathways, trp and potassium channels, and circadian rhythm genes. It has now become apparent that much of this research is very beneficial to our understanding of numerous diseases, including common and rare genetic disorders, neurological diseases, and cancer. This proposal aims at broadening the tool set in the fruit fly to perform better and more sophisticated experiments more efficiently. It will undoubtedly help us better understand numerous disease mechanisms.

PROJECT SUMMARY The Drosophila Gene Disruption Project (GDP), since its foundation in 2000, has produced a large, publicly available library of individual, sequence-mapped transposable element (TE) insertions that have become an essential resource for fly research. Generating and sequencing 180,000 TEs allowed the most useful ~16,000 (located in/near 13,000 genes) to be selected and deposited in the Bloomington Drosophila Stock Center, where they comprise 25%-30% of all stocks. >700,000 GDP cultures have been distributed to thousands of labs nationally and internationally, facilitating the analysis of thousands of genes. The features of the TEs newly developed by GDP greatly enhance their value as they allow characterization of gene expression, protein distribution, tissue specific knock down, isolation of interacting proteins, assessing function of homologues of other species and other sophisticated, state of the art manipulations. GDP?s MiMIC TEs contain ?C31 target sites that permit in vivo genetic swapping of the cassette. The flexibility to swap cassettes into existing MiMIC sites provides a genetic toolkit that is unrivaled in other species, greatly advancing the field of functional genomics and impacting our understanding of gene function across species. During the proposed budget period, GDP will provide tools that will be used to analyze gene function and constitute a new resource for medicine, aiding with the discovery and study of new human diseases and the underlying mechanisms. A critical prerequisite for modeling disease in Drosophila is the ability to express each of the 9,000 homologous human genes in the normal fly gene expression pattern. This can currently be achieved by using MiMIC and the SA-T2A-GAL4-polyA cassette (T2A-GAL4). When inserted in introns between two coding exons, this cassette is highly mutagenic and produces a GAL4 that can be used to drive the cDNA of a fly or human homolog, frequently rescuing the mutant phenotype and allowing disease modeling. The major focus of the GDP is to expand the number of genes with an inserted T2A-GAL4, especially those with recognized human homologs. To enable tagging of all genes, we developed new genetic strategies, one utilizing CRISPR (CRIMIC) to insert T2A-GAL4 in introns and two that replace the coding regions of genes with GAL4. Here we propose to tag 3,000 Drosophila genes using these strategies depending on the structure of the locus and the nature of the cassette that we wish to insert. The vast majority of genes will be tagged with GAL4 because it permits numerous applications including disease modeling. The resulting lines will be characterized genetically and molecularly and the expression pattern of the genes will be documented in third instar larval and adult brains. Progress will be delayed or denied if it is left to individual laboratories to generate the technically demanding strain construction, rather than having it carried out efficiently by GDP on a large scale. The generation and distribution of these reagents is highly appreciated by the Drosophila community and many letters of support are provided.

? DESCRIPTION (provided by applicant): This proposal describes the establishment of a model organism screening center (MOSC) for the Undiagnosed Diseases Network (UDN). The proposed MOSC will play a role in variant selection in close collaboration with the centers and sites of the UDN. The Leadership will obtain from the UDN a list of 300 candidate variants per year, and use independent genomic data available at Baylor College of Medicine (BCM) from rare disease cohorts to compare variants, human phenotypes and ultimately select 200 variants per year for experimentation. We will then assign variants (estimated 130 per year) conserved in Drosophila to the Drosophila Core at BCM. In the Drosophila Core, innovative technology will be used to tag proteins with GFP in the endogenous locus and examine expression pattern. These lines are to be generated by another large collaborative effort through the Drosophila Gene Disruption Project (GDP), saving cost for the MOSC. We will also use this technology to produce mutations that will be evaluated in a battery of phenotypic assays. This will guide experimental design and the generation of transgenics in Drosophila carrying human cDNAs. The 70 estimated variants per year assigned to the Zebrafish Core at the University of Oregon will be prioritized using existing expression and phenotypic data in fish and mice by analyzing the Human Phenotype Ontology annotations of the UDN patients and take advantage of the Zebrafish Core connection to the Zebrafish Informatics Network (ZFIN). The Zebrafish Core will then gathers existing mutations or generate new mutations with CRISPR/Cas9 and perform high throughput phenotypic analyses. The proposed MOSC also plans to share data with the UDN sites and centers every two months and to establish a website accessible to the entire UDN showing results and work in progress. No personal information, patient information or symptoms will be mentioned and we will only share it with investigators that are directly involved in the project. We also outline a plan for the Leadership to interact with the physicians for diagnostic o medically actionable data. Therefore the proposed center uses the most innovative technology in human genomics, Drosophila and zebrafish to provide diagnostic information for UDN patients.

PROJECT SUMMARY This proposal describes the establishment of a model organism screening center (MOSC) for the Undiagnosed Diseases Network (UDN). The proposed MOSC will play a role in variant selection in close collaboration with the centers and sites of the UDN. The Leadership will obtain from the UDN a list of 300 candidate variants per year, and use independent genomic data available at Baylor College of Medicine (BCM) from rare disease cohorts to compare variants, human phenotypes and ultimately select 200 variants per year for experimentation. We will then assign variants (estimated 130 per year) conserved in Drosophila to the Drosophila Core at BCM. In the Drosophila Core, innovative technology will be used to tag proteins with GFP in the endogenous locus and examine expression pattern. These lines are to be generated by another large collaborative effort through the Drosophila Gene Disruption Project (GDP), saving cost for the MOSC. We will also use this technology to produce mutations that will be evaluated in a battery of phenotypic assays. This will guide experimental design and the generation of transgenics in Drosophila carrying human cDNAs. The 70 estimated variants per year assigned to the Zebrafish Core at the University of Oregon will be prioritized using existing expression and phenotypic data in fish and mice by analyzing the Human Phenotype Ontology annotations of the UDN patients and take advantage of the Zebrafish Core connection to the Zebrafish Informatics Network (ZFIN). The Zebrafish Core will then gather existing mutations or generate new mutations with CRISPR/Cas9 and perform high throughput phenotypic analyses. The proposed MOSC also plans to share data with the UDN sites and centers every two months and to establish a website accessible to the entire UDN showing results and work in progress. No personal information, patient information or symptoms will be mentioned and we will only share it with investigators that are directly involved in the project. We also outline a plan for the Leadership to interact with the physicians for diagnostic or medically actionable data. Therefore the proposed center uses the most innovative technology in human genomics, Drosophila and zebrafish to provide diagnostic information for UDN patients.

PROJECT SUMMARY The proposed Phase II continuation of the Model Organism Screening Center (MOSC) for the Undiagnosed Diseases Network (UDN) builds on extensive tools, reagents, and pipelines that we established in Phase I. The leadership team represents international expertise in Drosophila, zebrafish, and human medical genetics. In Phase I, the MOSC developed an interface in the UDN Gateway that supports submission of cases, genes, and variants that the clinical sites propose for model organism studies. The MOSC also developed MARRVEL (www.MARRVEL.org), an online tool that integrates human and model organism data and helps to prioritize variants. The MOSC further established collaborations with independently funded collections of rare disease cohorts at Baylor College of Medicine (BCM) that we use to identify matches to UDN phenotypes and genetic variants. The MOSC holds regular conference calls with clinical sites and provides tools that help them to select variants. The MOSC will continue calls with the sites to review informatic analyses and then assign variants (estimated at 45 per year) for in-depth model organism studies of variants and genes. In the Drosophila Core at BCM, approximately 30 genes and variants per year are studied with innovative technology in flies. For 15 additional genes/variants that affect vertebrate-specific genes or biology, the Zebrafish Core at the University of Oregon will induce new mutations in zebrafish with CRISPR/Cas9 followed by high throughput phenotypic analyses. The MOSC will also develop tools and reagents for future variant studies in Drosophila and zebrafish for an additional 100 genes per year. We will share these additional resources with the broader research community through an innovative ModelMatcher service and will also prioritize genes for generation of mouse knock-outs in collaboration with the Knockout Mouse Phenotyping Project (KOMP). Thus, the proposed center will provide informatics selection, human genetics expertise, broad versatile model organism resources, and in-depth studies of UDN cases to aid in diagnosis. The MOSC employs the most innovative technologies in human genomics and Drosophila and zebrafish genetics, based on extensive previous experience modeling human disease.

PROJECT SUMMARY The overall aim of this proposal is to develop a toolkit designed to facilitate the functional annotation of human genes using Drosophila genetic studies. Numerous evolutionarily conserved genes can be studied in flies using a simple strategy. First, one inserts a T2A-GAL4-polyA artificial exon in the gene of interest. This can be done by converting the insertion sites of MiMIC transposable elements with T2A-GAL4-polyA or by direct integration of this cassette using CRISPR. These insertions typically create strong loss of function mutations. Moreover, the GAL4 transactivator is expressed in the same tissue and at the same time as the gene of interest. This can then be used to test if the fly or the homologous human cDNA is able to rescue the phenotype associated with the loss of the fly gene. If the human cDNA rescues, one has established that the two genes are orthologous. One can then determine the effect of human variants of interest (point mutations and polymorphisms) for functionality in flies, an approach that has already been shown to be extremely valuable for studies of human disease. To perform these experiments systematically we need to produce a library of human cDNAs that can be expressed in flies. We plan to create a resource for expressing ~8,000 epitope tagged human cDNAs of genes that are conserved between human and Drosophila. These cDNAs under the control of the UAS-GAL4 system will be inserted into a specific locus using the ?C31 integrase. We will make this library available to researchers via the Drosophila Genomics Resource Center. In addition, we will produce transgenic flies from a subset of these (1,500), using the following criteria: genes associated with known human diseases, genes with fly homologs that can be easily manipulated with available tools, and genes prioritized by other researchers (Drosophila biologists and human geneticists). These experiments will allow Drosophila and human researchers to test the functional replacement of genes for which some information is already available. The corresponding stocks will be deposited in the Bloomington Drosophila Stock Center. Finally, we will use the T2A-GAL4-polyA strategy to create strong loss of function mutations for about 500 Drosophila genes for which a MiMIC insertion is available. We will assess the phenotypes of a subset of these and test if the human cDNA is able to rescue the observed phenotypes in about 20 cases. This will establish how well the strategy works and provide valuable data for the community. Our goal is to provide molecular, genetic and transgenic resources for fly and human geneticists to accelerate the identification of human gene functions.

PROJECT SUMMARY Genetic screens in mice and the naturally occurring genetic variation in humans have provided a valuable resource to identify genes implicated in hair cell function and deafness. Two examples is the identification of genes involved in Usher syndrome, the most common form of deaf-blindness, and the role of Myh9 in both syndromic and non-syndromic deafness. We have used the power of Drosophila genetics to identify new genes involved in hearing and deafness. The auditory organs of Drosophila and vertebrates have a number of molecular and functional similarities despite being widely separated in evolutionary time. We identified mutations in Ubr3, an E3 ubiquitin ligase, that cause a physical detachment of the sensory components of Johnston's organ from the fly antenna. Strikingly, this phenotype is identical to that seen in mutations in Drosophila Myosin VIIa. Since Myosin VIIa mutations in humans cause Usher Syndrome type IB, it is possible that Ubr3 may regulate Myosin VIIa function in invertebrates and vertebrates. Our data suggest that Ubr3 genetically interacts with Myosin VIIa and Ubr3 and Myosin VIIa physically and genetically interact with Drosophila homologues of two other Usher syndrome proteins, PCDH15 and Sans. However, we have found that Ubr3 does not modify Myosin VIIa, but instead mono-ubiquitinates non-muscle Myosin II. This modification increase an interaction between the two myosins, and fine-tuning the level of this interaction appears critical for Myosin VIIa function. In the present proposal, we will expand on the use of Drosophila as a model system to understand deafness by characterizing the roles of Ubr3, Myosin II and Myosin VIIa in hearing in Drosophila (Aim 1). We will then test the function of Ubr3 in the development and function of mouse hair cells, and will test whether the interaction of Myosin II and Myosin VIIa is conserved in mice (Aim 2). Finally, we will carry out a genetic screen of 3000 Drosophila genes that may be involved in human disease using newly developed protein knockdown technology to identify genes that play a role in hearing in Drosophila (Aim 3).

Gene annotations in model organisms such as Drosophila are important contributors to our understanding of the functions of human genes, including human disease-associated genes. Paralogs, which share a common ancestor, present a challenging case for identification of gene function in model organisms as in some cases, loss of function of one paralog is masked by compensatory function of the other(s), such that only when both are disrupted will informative phenotypes be observed. In other cases, redundancy is partial, such that knockout of one paralog has a subset of the phenotypes observed when more than one member of the group is disrupted simultaneously. Recent advances in CRISPR technology by our group and others makes it now possible to systematically knockout paralog pairs in Drosophila, including in a stage- and tissue-specific manner. We will use our existing infrastructure for bioinformatics-based identification of orthologs and paralogs, efficient large-scale production of fly stocks, and fly stock and data sharing to develop a resource useful for double-knockdown of paralogs. Our initial characterization of the genes with regards to signal transduction and neurodegeneration, as well as in-depth analyses by the community, will uncover function for paralogous genes, helping to close the ?phenotype gap? (lack of associated loss-of-function phenotypes) that currently exists for nearly half of all genes in this important genetic model system. The result will be a fly stock resource for further study by Drosophila experts, a bioinformatics pipeline and methods that can be applied to other model systems, and a data resource that will inform annotation of fly and human genes

Project Summary This application is being submitted in response to NOT-RM-19-009 as a supplement to the parent award U54NS093793. The Common Fund supports a number of resources that can significantly enhance gene and variant prioritization for study in the Model Organisms Screening Center of the Undiagnosed Diseases Network and beyond. To facilitate the use of these resources, we propose to create a tool that can be easily accessed by clinical geneticists and model organism scientists alike. MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration) was created two years ago because important data that is necessary for rare variant analysis for personalized medicine is spread throughout the internet in tens of different locations. To improve efficiency and streamline access to these data sources, we created a web-tool that allows users to query tens of data sources at once, including GTEx, and links to IMPC, the display portal for KOMP2. In this proposal, our goal is to develop version 2 of MARRVEL to promote the use of Common Fund resources in the rare disease research community for manual and automated data analysis. This goal will be accomplished by developing MARRVEL 2.0 by integrating KOMP2 (IMPC) and PHAROS data and using the aggregated dataset to develop a machine-assisted gene and variant prioritization for diagnosis and animal model generation. Our goals align with those of the NIH Common Fund to increase the utility of resources for broader use in the biomedical community.

PROJECT SUMMARY The aim of this proposal is to continue to develop a toolkit designed to facilitate the functional annotation of human genes and disease associated variants through genetic studies in Drosophila melanogaster. We initiated this project three years ago through support of an R24 funded by ORIP. The Drosophila genome contains ~8,500 genes that are evolutionarily conserved in vertebrates including human. To model human diseases, we typically start by creating a severe loss-of-function mutation of a fly gene that is likely to be an ortholog of the human gene that is known or suspected to be pathogenic. We insert a SA-T2A-GAL4-polyA artificial exon into an early intron common to all transcripts of the gene of interest (GOI) using CRISPR mediated homologous recombination. This typically creates a strong loss-of-function allele that expresses the GAL4 transactivator in the same spatial and temporal pattern as the mutated gene. Hence, a UAS-nuclear or membrane GFP permits us to determine the cell types in which the gene is expressed through co-staining with known cell identity markers or based on cellular morphology. Importantly, GAL4 often allows us to rescue the phenotypes associated with the loss-of-function allele by driving a UAS-fly or human cDNA. If the human cDNA rescues we can test human variants of interest for functionality in flies, an approach that has already greatly helped in the identification of many new human diseases in the past few years. These experiments also allow detailed functional analyses to better understand the pathogenic mechanisms and to test FDA approved or experimental drugs. We have also produced a library of just over 2,000 T2A-GAL4 stocks and ~3,000 UAS- human cDNAs lines to perform these experiments systematically. We assembled a library of 33,000 full length human cDNAs from different sources, generated and sequenced ~4,000 plasmids containing the UAS-human cDNA for transformation in the fly. Nearly 3,000 of these constructs have been inserted in the fly genome in defined loci using the ?C31 integrase, and transgenic stocks have been established. The UAS constructs are available from the Drosophila Genomics Resource Center (DGRC) and the stocks are available from the Bloomington and Kyoto stock centers. Here we propose to expand the UAS-human cDNA collection and clone the remaining 4,000 human cDNAs of the 8,500 conserved genes and establish an additional 3,000 transgenic stocks for distribution. We also propose to generate 1,000 SA-T2A-GAL4-polyA insertions in homologous fly genes using a new method that we developed to accelerate the testing of the UAS-human cDNAs by the research community and promote the systematic study of human disease associated genes. Our goal is to provide molecular, genetic and transgenic resources to the fly research community and human geneticists to accelerate the discovery of human diseases and help unravel human gene function.

Administrative Supplement Request for U54NS093793 based on PA-18-591 https://grants.nih.gov/grants/guide/pa-files/PA-18-591.html Title: A global matchmaking platform for collaborative research on rare and undiagnosed diseases Project Summary: Rare disease patients often experience painstaking diagnostic and therapeutic odysseys. State-of-the-art genome sequencing technologies may provide answers for ~30-40% of these cases, but many are often left with a handful of candidate genetic variants that require experimental follow-up studies to establish causality. In this proposal, we will build a centralized website and database called ModelMatcher (https://www.modelmatcher.net/) that can be used by clinicians and other stakeholders of undiagnosed disease research (e.g. patients, family members, patient organizations, funding agencies, pharma) to identify basic scientists who are interested in collaborations to facilitate diagnostic, translational and therapeutic research to support the mission of the Undiagnosed Diseases Network and beyond. Basic scientists can register their information regarding their contact, experimental systems used in their labs, areas of expertise, and genes/pathways of interest in ModelMatcher, and clinical users can mine this database to identify experts who can perform functional studies on disease candidate variants identified in patients? genomic DNA. Such registries can also be useful for other stakeholders of rare disease research to identify a group of scientists who are willing to work collaboratively on translational and therapeutic research for a specific rare disease that does not have efficient treatments. Besides sparking studies of specific genes and diseases, this first-of-its- kind global registry of scientists with diverse expertise linked to medical genetics databases will create an unprecedented opportunity to build strong collaborative networks and initiatives far beyond what we envision.

Title: Integration of MARRVEL and ModelMatcher to facilitate undiagnosed disease research Project Summary: Rare disease patients often experience painstaking diagnostic and therapeutic odysseys. State-of-the-art genome sequencing technologies may provide answers for ~30% of these cases, but many are often left with a handful of candidate genetic variants that require experimental follow-up studies to establish causality. In addition to performing functional studies of candidate variants identified by the Clinical Sites and the Sequencing Core of the Undiagnosed Diseases Network (UDN), the Model Organisms Screening Centers (MOSCs) has been developing bioinformatic tools to support the overall mission of the UDN. For the past four years, we have been developing a bioinformatic tool MARRVEL, to gather and display important data that is necessary for rare variant analysis based on variety of databases that are scattered around the web for personalized medicine. In addition, the MOSC just built and launched a centralized registry of collaborative scientists called ModelMatcher that can be used by clinicians and other stakeholders of undiagnosed disease research (e.g. patients, family members, patient organizations, funding agencies, pharma) to identify basic scientists who are interested in collaboration to facilitate diagnostic, translational and therapeutic research. Although both MARRVEL and ModelMatcher are valuable resources, the two have been built on distinct platforms due to technical reasons and there is currently no cross-talk between these services. In this project, we will modify and upgrade MARRVEL and ModelMatcher by extensively linking the two websites to increase utility, value, and user-experience by updating the online portals and through development of APIs (Application Programming Interfaces). Upon completion, MARRVEL users will be able to instantaneously identify scientists who are actively working on a specific gene in model organisms, and ModelMatcher users will be able to gather comprehensive information about their gene of interest from diverse human databases and in various model organisms when they search the registry. The integration of these two one-of-its-kind websites that have been developed through the support of the UDN will not only have a large impact on studies of rare and undiagnosed diseases, but will stimulate information exchange and collaborations on genes involved in common diseases as well as other genetic disorders including cancer. Finally, newly developed APIs will allow other database to computationally access information stored in the ModelMatcher and MARRVEL, further facilitating collaborations internationally and throughout multiple scientific and clinical disciplines.

PROJECT SUMMARY Currently, ~5.7 million Americans live with Alzheimer?s disease (AD), representing a significant burden on society and our healthcare system. Despite long-standing knowledge that AD involves the aberrant accumulation of A?42-plaques and neurofibrillary tangles (NFT; composed of hyperphosphorylated Tau) a successful treatment for AD has yet to be defined. Successful therapies will likely involve the identification of AD-risk patients and early intervention prior to disease onset. Accumulating data support that early events that may contribute to AD-onset include the abnormal upregulation of reactive oxygen species (ROS) and dysregulation of lipid metabolism. These features were seemingly disconnected until we recently discovered that elevating ROS within neurons induces the formation of peroxidated lipids, which are transferred from the neurons to surrounding glia. Within glia, these lipids form LD and are, presumably, resolved. Inhibiting this process drives ROS-induced neurotoxicity. During chronic neuronal ROS, glia become overrun with LD and die, leaving the neurons vulnerable. This pathway is conserved in flies and mice, with supportive human data. Currently, we are uncovering the potential role of glial LD formation in AD. Preliminary data in Drosophila demonstrate that genes defined as risk factors for AD by GWAS converge onto the glial LD formation pathway. ABCA transporters, ABCA1 and ABCA7, are required in stressed neurons for glial LD formation, likely for the export of peroxidated lipids. Further, four genes ? LRP1, PICALM, CD2AP, and AP2A2 ? are required in glia, likely for the uptake of peroxidated lipids. We also found that the disease gene, Tau, is required within glia for LD formation. Last, preliminary and published data support that disrupting glial LD formation may drive extracellular A?42 accumulation, NFT formation, and disease. Overall, we hypothesize that glial LD formation is an early event that attenuates elevated ROS in healthy brains and this process becomes prematurely defective in AD. Building upon our current data, we will define potential contributions of glial LD formation defects on ROS-induced neurodegeneration, insoluble A?42 buildup, and NFT formation. Aim 1 will investigate AD-risk genes and AD-associated variants for involvement in glial LD formation during elevated ROS in neurons using novel humanized fly models. Aim 2 will focus on Tau as a potential mediator of glial LD formation in novel and established fly models, defining differing impacts of human Tau isoforms and mutations, and assessing if defects in glial LD formation can contribute to the phosphorylation/ aggregation of Tau. Last, Aim 3 will explore defined mechanisms from flies in mammalian systems. Initially, an established rat neuron:glia co-culture model that can measure lipid transfer from stressed neurons to glia and their accumulation into glial LD will be used to test if AD-risk genes and Tau can mediate this process. Further, pathological studies will be performed on post-mortem AD tissue to determine correlations between LD presence and disease features (e.g. A?42-plaques, NFT, disease severity, and presence of AD-risk variants).

PROJECT SUMMARY Here we propose to advance the goal of NHGRI to implement genomic medicine and focus on individuals who have not been able to afford DNA testing. The research takes place in the Department of Molecular and Human Genetics at Baylor College of Medicine (BCM) and Texas Childrens Hospital (TCH). Our team of clinicians, geneticists, computer scientists, genomicists and model organism researchers has had a five-year term of success with the Undiagnosed Diseases Network (UDN) Model Organisms Screening Center (MOSC). This has included successfully identifying a number of new disease genes such as EBF3, IRF2BPL, NACC1, TBX2, TOMM70, CDK19, ACOX1, WDR37, and ATP5F1D. We propose to recruit 100 individuals from an underserved population in Houston, Texas with suspected rare disease and without the means to pay for DNA sequencing through insurance. We will provide whole-exome sequencing which will generate a CAP/CLIA report that we anticipate could diagnose 35-40 individuals per year. The remaining individuals will then be converted to a family-based trio exome design. All the sequencing costs of this project will be covered by philanthrophic donation to our hospital and are not budgeted to the grant. We will make every effort to diagnose the remaining 60 individuals per year through machine learning and informatics using the MARRVEL platform, Drosophila functional studies of candidate genes and through ongoing 6 month, 12 month and 2 year follow-up with the patients where we will use matchmaking efforts such as GeneMatcher and Matchmaker exchange as well as our own genomic databases from the UDN and other studies to come to a genetic diagnosis. All subjects will receive genetic counseling from a trained team and will provide us with valuable medical, psychological and social data to guide how genomic implementation in an underserved population is perceived, impacts care and impacts the family. This work will not only produce novel insights into rare disease, diagnosis for undiagnosed families and an expanded role for genomics, it will guide us in the future to provide genomics and functional research to serve all individuals regardless of their ability to pay.

SUMMARY Alzheimer?s Disease (AD) is projected to affect 13 million people in the US by 2050 and remains neither curable nor preventable. Following remarkable recent progress, the genomic architecture of AD and related dementias (ADRD) is coming into focus. Similar to other common and genetically complex disorders, AD is characterized by substantial locus heterogeneity and polygenic susceptibility: risk or protective alleles are being identified in many distinct genes, and in most individuals, a subset of common and rare variants likely interact to trigger neurodegeneration. The critical next steps include confirmation of the responsible genes, understanding the functional impact of disease-associated variants, elaboration of the relevant cell types and pathways, and determining how polygenic interactions mediate disease risk. We propose an integrated computational and tiered experimental validation strategy to accelerate AD functional genomics, building on advances from the AD Sequencing Project (ADSP) and leveraging powerful technologies available in the fruit fly, Drosophila melanogaster. First (AIM 1), leveraging infrastructure developed for the Clinical Genome Resource and ENCODE projects, we will integrate ADSP results with other human data, including brain transcriptome and epigenome profiles, prioritizing genes and variants for experimental follow-up. Next (AIM 2), using high-throughput Drosophila screening, we will systematically manipulate 2,000 conserved, candidate AD genes in vivo to pinpoint causal modulators of age-dependent neurodegeneration, including interactions with Tau, Aß, and other pathologic triggers. Third (AIM 3), for a subset of 200 prioritized gene candidates, we will generate customized Drosophila strains and characterize cell-type expression and loss-of-function phenotypes. Lastly (AIM 4), for 50 high-priority targets, we will experimentally probe mechanisms in-depth, including testing of cell-type specific requirements (neurons vs. glia) and examining gene-gene interactions that define relevant pathways. We will broadly share all project data and resources with the research community (AIM 5). Our integrative, tiered, cross-species strategy promises rapid functional annotation of ADSP targets using powerful, in vivo assays in the aging nervous system of Drosophila, and is ideally suited for reciprocal cross-validation in complementary mammalian preclinical models. On a scale and timeframe not currently possible in other model systems, our innovative experimental strategy will transcend barriers to translation of human genetic discoveries and catalyze breakthroughs in our understanding AD pathobiology.