David R. Beier, MD, PhD - US grants

The long-term objectives of this proposal are to identify and to study a locus that is required for normal murine development in utero and for normal hepatic function after birth. This locus is identified by an insertional mutation due to a transgene integration; homozygous transgenic mice are runted at birth, feed poorly, and usually die during the first two days of life with a severely necrotic liver. The morphology of the hepatic injury appears similar to that seen in toxic liver injury or fulminant hepatitis. The affected locus is called ple, for perinatal lethality. The transgene has been mapped to a region of mouse chromosome 15 whose genetic organization has been conserved during evolution, suggesting the likelihood that there is a human homolog of the ple locus. The insertion site of the transgene has been cloned, which will facilitate the characterization of the gene(s) whose expression is affected by the transgene integration. The specific aims of the proposal are to use well-proven strategies for the identification of transcribed genes in cloned genomic DNA sequences to obtain a full-length cDNA to the locus (or loci) that are disrupted by the transgene. Since there is evidence (by cross-hybridization) that there are related murine loci and the suggestion (by conservation of synteny) that there is a human homolog, these genes will also be pursued. These will then be characterized by biochemical and expression analysis in order to gain understanding of their role in normal murine development and hepatic metabolism. Because the region of chromosome 15 to which the ple locus maps contains a number of mouse mutations, and because this region has been conserved in humans, it is furthermore a specific aim to generate a detailed physical and genetic map of this region using pulsed-field gel electrophoresis and interspecific recombination mapping analysis, respectively. Additionally, it should be feasible to use crosses of ple with recombinant inbred mouse strains to map loci which influence expression of the ple phenotype. Based on genetic and physical analysis, it is possible that the disrupted locus is a member of the type II cytokeratin gene family. While this class of intermediate filaments has been studied extensively biochemically, no specific mutations in a cytokeratin have been identified, and their role in cellular physiology is not well understood. If the ple insertional mutation proves to be a cytokeratin, it will serve as a valuable resource for the study of inter-mediate filament gene function. Finally, a number of abnormal phenotypes, some clearly due to spontaneous mutations that are unlinked to the transgene, have been identified in this transgenic line. This transgenic line may be genetically unstable and have an elevated mutation frequency. Since there is presently insufficient evidence to prove this hypothesis, it will be necessary to continue to characterize the morphology and heritable nature of the abnormal phenotypes.

The long-term objectives of this proposal are to genetically map with high resolution a recessive mouse mutation that predisposes to the development of polycystic kidney disease. This analysis can presently be expedited using PCR-based strategies, and may serve to identify candidate genes which, when mutated, result in this disease. Genetic mapping has directly facilitated the molecular characterization of a number of human disorders. Examples include cystic fibrosis and neurofibromatosis, for which the identification of flanking markers enabled investigators to identify the mutant gene by positional cloning. Similarly, mapping of the dominant familial cardiac hypertrophy trait to human chromosome 14 served to identify cardiac myosin as a candidate gene for a role in this disease. At present, little is known about the molecular basis of polycystic disease. One locus has been identified on human chromosome 16 which appears responsible for the development of dominantly inherited polycystic disease, and efforts to clone this gene are in progress. Linkage analysis for the localization of the genes which cause recessively-inherited polycystic disease is presently not feasible, due to the early lethality and relative rarity of this disorder. An alternative approach to the identification of genes that predispose to polycystic kidney diseases may potentially be accomplished by the study of mouse mutations. There are at least five mutations which cause polycystic kidneys: cpk (congenital polycystic kidney), pcy (progressive cystic kidney), spk (spongy kidney), 1330 (not named), and jck (juvenile cystic kidney). This last mutation is one that I have discovered and am presently characterizing. Recent developments in genetic analysis and molecular techniques make it technically feasible to map the jck trait with high resolution. This will be done using PCR-based analysis of its linkage with genetic markers in a cross between inbred laboratory strains (C57B1/6J and DBA/2J) and in an interspecific cross between C57B1/6J and M. castaneus mice. This mapping analysis may identify candidate genes that are responsible for the development of polycystic kidney disease. These candidate loci can then be tested for abnormalities using molecular techniques such as SSCP (single-strand conformer polymorphism) analysis to assess if these genes are mutant in the jck mice.

Given the rapid progress of murine genetic analysis, it is appropriate to consider the alternative directions that this research should go. It is reasonable at this point to propose that future mapping efforts focus on the identification and localization of polymorphisms within expressed sequences. The simplest argument for this is that the ultimate purpose of mapping analysis is to localize genes. As such, if sufficient polymorphism can be readily identifiable in cDNAs such that they are practical for linkage studies, they are a priori potentially more useful than anonymous DNA sequences. We have recently demonstrated that such polymorphism can be readily found in untranslated regions of expressed loci (such as introns or 3' untranslated sequence) using a PCR-based analysis of single-strand confirmation polymorphism (SSCP). In this technique, PCR primers are made which amplify fragments of between 100-300 bp. These fragments are denatured by incubation at high temperature and are then electrophoresed on a non-denaturing acrylamide gel, which permits the formation of internal secondary structure in the separated PCR single-strands. It has been shown that the formation of these secondary structures is very sensitive to the nucleotide sequence of the PCR fragment. This allows the discrimination between regions with very small differences in DNA sequence, and can often detect single base changes. In addition to using SSCP as a simple and rapid means to map cDNAs in RI strains, we have found that this is an efficient way of identifying polymorphism between species. We have begun a systematic analysis of this strategy in order to assess the generality of the technique and we are able to demonstrate that sequences obtained from either published databases or from randomly selected brain cDNAs can be readily used to obtain and map polymorphic loci in an interspecific cross. In our preliminary studies, we have generated PCR-typable markers for 36 loci, including 21 that have not been previously been mapped. Since this strategy permits the integration of sequence analysis, linkage analysis, and physical mapping (since the primer sequences represent STS's) using a simple, easily transferrable PCR-based technology, we submit that it is ideally suited to the development of an expressed sequence map of the mouse genome. We therefore propose to use SSCP analysis to characterize polymorphisms in and map at least 2000 expressed genes during the course of this work.

DESCRIPTION (Directly taken from the application) The long-term objectives of this proposal are: 1) to identify by positional cloning the product of the murine autosomal recessive juvenile cystic kidney (jck) mutation, 2) to localize and analyze candidate gene corresponding to modifier loci which affect severity of polycystic kidney disease (PKD) in this mouse model, and 3) to identify genes whose expression is altered in cystic kidneys. A novel method of chromosomal exclusion was used to map jck to chromosome 11 using an intercross between (C57BL/6 X DBA/2)F1 jck/+ mice; this mutation has been further localized to an interval of <1 cM and found to be very tightly linked with a microsatellite marker (no recombinants in over 1000 mice tested). Direct selection and CpG-island PCR are being used to analyze YAC clones to identify candidate transcripts for jck. In addition, the severity of PKD in the F2 progeny was significantly more variable than that found in the parental C57BL/6 strain, suggesting that a modifier locus introduced from DBA/2 affects expression of jck. Two regions one from DBA/2 on chromosome 10 and a second from C57BL/6 on chromosome 1 - are associated with inheritance of a more severe PKD phenotype. The finding of a highly significant association of inheritance of a C57BL/6-related locus with disease severity (with a LOD score of 16.8) was unexpected (since the disease phenotype in this background is not severe), and our results that it is the combination of loci from different genetic backgrounds that results in the more severe phenotype, presumably as a consequence of an interaction between the protein products. Since a positional cloning strategy is less appropriate for the characterization of loci that affect quantitative traits, congenic strains are being generated to more specifically localize the modifying genes. Additional crosses are also being analyzed to test the generality of the effects of these modifying loci. We have also used a modified differential display PCR technique that targets the entire gene (and not just 3' UTR) to test for genes whose expression is altered in jck mutant mice. Our first experiments using this approach have served to identify a novel putative transporter gene that appears to be required for normal bone development, since its expression is absent in the osteosclerosis (oc) mutant mouse, which has osteopetrosis and rickets as part of its phenotype. We propose to use this approach at very early time points in order to identify genes whose expression is immediately affected by the absence of the jck product. Understanding the molecular basis of this mutation should provide insight into a fundamental mechanism of cystogenesis that may be relevant to human disease. In addition, the importance of characterizing the modifying loci should not be underestimated, since these genes may provide useful targets for therapeutic intervention that could significantly ameliorate disease-related morbidity. Finally, the characterization of genes whose expression is altered in these mutant mice should serve to identify pathways of gene interactions.

Little is known about the signal transduction pathways controlling skin architecture and hair development, although these processes are likely to be very important for the understanding of mechanisms that are disturbed in human skin diseases such as psoriasis. We propose to investigate these questions using a mouse model called waved 3 (wa3). This spontaneous recessive mutation is characterized by wavy fur and open eyes (unfused eyelids) at birth. This phenotype is similar to that previously described for the mouse mutants waved 1 (wa1) and waved 2 (wa2), which respectively represent molecular defects in the growth factor TGF-alpha and in its receptor, EGFR. We have determined that wa3 maps to mouse chromosome 7, and is not allelic with wa1 or wa2. We propose to use a positional cloning approach in order to determine the molecular basis of the wa3 mutation, which will facilitate the analysis of its role in normal skin development as well as in skin diseases. We also propose to use genetic crosses of the wa3 mutant with transgenic mice that overexpress TGF-alpha in order to directly test the possibility that the wa3 product is required for normal expression of this growth factor.

DESCRIPTION: (adapted from abstract) ENU has been well documented as an extremely efficient agent for the introduction of mutations in the mouse genome. This mutagenesis approach, coupled with the rapid development of mapping strategies and genomic reagents, are facilitating the mapping and cloning of mutations in the mouse and provide a powerful means for the development of a new repertoire of mouse mutations. In this proposal the applicant plans to use ENU mutagenesis to generate novel recessive mutations which effect the structure of the mouse brain, heart and kidney. Since mice carrying these mutations will frequently not be viable after birth, the investigator is proposing to screen for mutations in late embryonic development [day 18 of gestation] from a cross predicted to produce animals homozygous for the mutant allele. By using different defined inbred strains in the crosses, a mapping step is built into the screen which facilitates the localization of mutations as they are phenotypically identified. The combination of mutagenesis, anatomical analysis, and genetic mapping represents a novel and powerful means to study mammalian development and organogenesis. In addition, limiting the analysis to mutants that survive to late gestation maximizes the likelihood that the mutations which are found will be relevant to models of human congenital defects. In parallel to these analyses the applicant proposes to use a sequence-based assay of mutation frequencies as a means to monitor and optimize mutagenesis protocols.

Advances in technology have resulted in a dramatic change in the nature of genetic investigation. Innovation in bioinformatics has made a wide variety of databases, which include nucleotide and protein sequence data, genetic map data, genetic markers, and many other resources, accessible to investigators. The refinement of high-throughput sequencing protocols has also had its effect, with the result that the relative ease of obtaining sequence data has promoted a sequence-based characterization of the genome of a nature such that information is readily transferable from laboratory to laboratory. The infrastructure that is necessary to utilize this technology-intensive investigation requires a considerable investment in computers, instrumentation, and technical support. To address this change in the nature of genetic analysis in the context of an academic medical center, we have initiated a genome analysis effort to support investigations at the Brigham and Women's Hospital. This effort is intended to provide necessary infrastructure and genetic expertise to expedite the execution of the significant number of NIH- supported research efforts that are presently in progress, and to inform the development of future studies. This center will also serve as a nucleus for introduction of additional technologies in genetic analysis as they are inevitably developed, and will provide a focus for the education of academic physicians, both at the trainee and more senior investigator levels, in the appropriate technologies of contemporary and future genetic analysis. This shared instrument grant (SIG) application requests and ABI377 automated sequencing instrument. Given the crucial role of high through- put DNA sequence analysis in gene discovery, this instrument represents the cornerstone of our planned genetics program. To implement this effort, the Brigham and Women's Hospital has committed sufficient space and resource to equip a state-of-the-art molecular biology laboratory, as well as support for both faculty-level and technical support personnel.

computer assisted sequence analysis; informatics; genetic mapping; biomedical facility;

kidney; histogenesis; genotype; polycystic kidney; biomedical facility; animal genetic material tag; genetic mapping;

We have discovered a novel murine mutation which causes open eyes at birth and wavy hair. This phenotype is nearly identical to the waved 1 (wa1) and waved 2 (wa2) mutations. The gene affected in wa1 mice is transforming growth factor alpha (Tgfa), a widely expressed growth factor. Epidermal growth factor receptor (Egfr), the receptor for Tgfa, is mutated in wa2/wa2 mice. We mapped our waved mutation in an intercross and have localized it to proximal mouse Chr. 7. This shows that this mutation is not allelic with either wa1 or wa2, which map to Chr. 6 and 11 respectively, and we have designated it as waved 3 (wa3). The Tgfa/Egfr ligand/receptor pair has been investigated extensively; however, little is known about other genes that participate in the signaling process. Both of these genes are known to be over-expressed in many tumor cells, as well as in the skin disease psoriasis. While wa3 has not been proven to participate in the Tgfa signaling pathway, the phenotype is strikingly similar to wa1 and wa2. Furthermore, a number of studies of the biochemistry and genetics of Tgfa suggest that there are additional genes that function in the Tgfa signaling pathway. We suggest that the wa3 mutation is in such a gene, and propose experiments to test this hypothesis. Even in the case that wa3 is not in the Tgfa signaling pathway, its function is clearly important for normal development. We have been able to make considerable progress with respect to positional cloning of wa3. The wa3 non-recombinant interval has conserved homology with human chromosome 19q13.2, and the human genomic DNA corresponding to this interval has been nearly completely sequenced. Additionally, we have generated a complete mouse BAC contig across the wa3 interval. Analysis of the sequence in the region has already suggested several genes that can be characterized as candidates for the wa3 mutation. The combination of a positional cloning approach with a detailed characterization of the wa3 phenotype should provide insight into understanding genes that control normal skin development.

DESCRIPTION (provided by applicant): With the renewed interest in the use of ENU mutagenesis has come the recognition that this technique creates an unexpected (if not undesirable) problem; namely, how to efficiently maintain a large number of mutants and prioritize these for further investigation. The determination of the genetic map position of a mutant, even at low resolution, can effectively serve these ends. Map position can be very important for assessing whether a mutant represents a re-mutation of a known gene or a mutation at a novel locus. Map location can also suggest candidate loci for examination by sequence or expression analysis. Additionally, the definition of a recombinant interval carrying a mutation allows one to identify heterozygous carriers by genotype analysis, rather than the more tedious, expensive, and time-consuming strategy of progeny testing. In this project, the investigators propose to generate and import mice carrying mutations affecting development, and map these loci using a haplotype analysis approach that requires small numbers of affected animals. Mice that appear to be re-mutations at known loci (based on phenotype and map position) will then be excluded, and mice with presumptive novel mutations will be further analyzed at various embryonic stages by histological analysis and using in situ or immunohistochemical analysis. All of these results will be posted and mice will be available to any interested investigator. Finally, for a small number of mice, the investigators will attempt to identify the causal locus by positional cloning, with an aim towards developing the most efficient protocols for this.

DESCRIPTION (provided by applicant): While considerable progress has been made in the characterization of the genes that cause PKD, the mechanism of the pathobiology in this disorder is still unknown. In particular, the potential utility of gene-based therapy for this disorder is not at all clear. In this regard, it is useful to consider alternative approaches to inhibit the progression of cystic degeneration in PKD. It is well known that the same underlying genetic defect in the PKD gene can have different degrees of severity, presumably as a function of the genetic background of the affected individual. This has led to the recognition of potential importance of modifier loci, which can influence the expression of the PKD defect. The localization of such modifying loci has been most successful in animal models of recessive PKD. While these are clearly different than human ADPKD, there exists the possibility that these modifying loci may have their effect on cystic disease progression irrespective of the underlying molecular defect. This possibility, combined with the powerful genomic tools that have been developed for positional cloning of genes in model organisms, provides strong support for a proposal to localize and clone modifiers of PKD progression in model organisms. We have extensive experience both in the analysis of modifying loci of PKD in mice, as well as the utilization of genomic technology for gene discovery, and we propose to apply this expertise to the characterization of a locus that appears to influence disease progression in several mouse mutant systems. Specifically, we plan to use congenic strains to identify a recombinant interval carrying the modifying loci. We will also use haplotype analysis, DNA sequencing and bioinformatic analysis to identify genes across the region, which can be considered as candidates for the modifying locus. In addition, we suggest that for the comprehensive analysis of genetic factors modulating PKD it is necessary to have a better model of the dominantly inherited PKD-predisposition characteristic of the human disease. It is therefore an aim of this proposal to develop mutant mice that develop cysts as a consequence of loss of heterozygosity, using either a spontaneous or inducible system.

Airway hyperresponsiveness (AHR) is a component of asthma, and the complex inheritance of both asthma and AHR have made it difficult to find the genetic etiology of these important problems. Analysis in a less heterogeneous genetic system than the human population could be useful for identifying causal loci. We and others have attempted this using quantitative trait locus (QTL) analysis in the mouse, which provides an excellent model for naTve AHR, but the results of these studies have been inconsistent. As part of this Program, we have used a very different approach to address this. We created phenotypically selected recombinant congenic mice to identify loci associated with increased naTve AHR. The seventh generation hyperresponsive mice retained A/J loci on chromosomes 2, 6, and 10. Surprisingly, analysis of unselected N8 progeny demonstrated that the naive AHR phenotype was not significantly associated with any of the loci individually, but was highly significantly associated with an interaction of loci on chromosomes 2 and 6. These findings were confirmed in an independent analysis of consomic mice. Also, as part of the Program, we generated A/J mice that are genetically depleted of mast cells. These mice do not show naive AHR, demonstrating that this trait is mediated by mast cells. The identification of genomic regions containing loci causally associated with AHR, the demonstration that this trait requires their interaction, and the observation that mast cells are required for expression of the disease phenotype has important implications for the dissection of the genetic etiology of asthma in humans. Of interest is that the protease ADAM33, which has been associated with the human disease by genetic analysis, is within the retained A/J region. An advantage of model systems is that they can facilitate functional analysis, and we propose to examine the role of ADAM33 using haplotype analysis and transgenesis. We also propose to further examine the role of mast cells in mediating the naive AHR observed in A/J mice by reciprocal bone marrow transplant and adoptive transfer. Lastly, we will continue to narrow the genetic interval in which the causal loci reside, with the aim of ultimately identifying the genes using a positional cloning strategy.

DESCRIPTION (provided by applicant): The importance of the mouse for biomedical research cannot be overstated. Genetic investigations of mouse mutations were initiated at the turn of the century, shortly after the rediscovery of Mendel's laws. More recently, the development of technologies to manipulate the mouse germ line by transgenesis or homologous recombination has made the mouse the definitive system for the study of mammalian gene function. With the rapid progress in the characterization of the mouse genome, and the application of efficient methods of mutagenesis such as ENU treatment or gene trapping, potential for further progress is unparalleled. This is manifested in the stated aim of several consortia of investigators to generate large numbers of mutant mice. We propose an annual workshop to facilitate the interaction of researchers who have invested their research efforts in these various technologies. The aim will be to discuss progress, problems, new technologies, and logistical issues related to the distribution and utilization of these resources. This meeting will be the successor to a series of workshops that have emphasized several of the component technologies, such as ENU mutagenesis and gene-trap mutagenesis. Importantly, this forum will aim to include a wide variety of technologies, so that their potential advantages and disadvantages can be considered and discussed. This workshop will also aim to carry on the tradition of international representation that has been characteristic of the previous meetings. Ultimately, this workshop will serve the goal of improving public health by facilitating the generation and utilization of model systems for biomedical investigation.

DESCRIPTION (provided by applicant): We have undertaken a project to generate models of human congenital defects by screening ENU-mutagenized mice for recessive mutations affecting late embryonic development. The screen incorporated a genetic mapping component, with an aim to facilitate the positional cloning and functional characterization of the mutant genes. The strategy has worked well, and we have generated many mutant lines with phenotypes similar to human malformation syndromes and birth defects. The spectrum of abnormalities found to date is remarkably varied; for example, we have generated models of spondylocostal dysostosis, Robin sequence, congenital diaphragmatic defect, non-syndromic cleft palate, polycystic kidney disease, epidermal bullosa, non-bullosa congenital icthyosiform erythroderma, and structural heart disease. We have mapped a number of these, and identified the mutated locus in 9 lines. To accomplish this, we have taken advantage of efficent technologies for genetic mapping and positional cloning. The functions for many of the genes we have identified are not well understood, and we have initated a variety of biochemical and developmental studies to explore them. In addition to characterizing the biology of the defects in the mutant mice, we have in several cases established that the genes we identified play a role in the causation of human disease. Thus, all of the premises that were the basis of our original proposal have been experimentally validated. In this continuation proposal we hope to refine the specificity and sensitivity of the screen and optimize several aspects of the analysis, while maintaining the fundamental approach that has thus far proven so productive.

DESCRIPTION (provided by applicant): Mouse mutants that perturb cortical patterning have provided considerable insight into the development of the mammalian brain. We have previously undertaken a project to generate models of human congenital defects by screening ENU-mutagenized mice for recessive mutations affecting late embryonic development. The screen incorporated a genetic mapping component, with the intent to facilitate the positional cloning and functional characterization of the mutant genes. The strategy has worked well, and we have generated many mice with phenotypes similar to human malformation syndromes and birth defects. In this proposal we aim to target cortical development, with the goal of identifying and cloning additional mutants that will be useful for understanding how the mammalian brain is patterned. Specifically, we plan to screen for mice that have abnormalities of brain morpholology and histology. We will also screen for mutations that perturb patterning of reporter genes that mark specific anatomical structures, A third strategy is to screen for mutations that have a genetic interaction with known cortical patterning genes such as Lis1. We have developed efficient strategies for genetic mapping and positional cloning, and we are currently working on rapid methods of mutation validation using RNAi. Finally, for a subset of mutations that have relatively specific effects on cortical development, we plan to pursue in-depth analysis, including a collaborative effort with investigators at the Allen Institute for Brain Science utilizing high-throughput technologies for in situ expression analysis. Depending upon the nature of the mutated gene, we will pursue functional studies as appropriate. PUBLIC HEALTH RELEVANCE. We propose to treat mice with the chemical ethyl-nitrosourea (ENU), which causes mutations in DNA. We will examine the progeny of treated mice to assess whether they have disorders of brain development. We are particularly interested in those that have relatively subtle effects;that is, which appear to specifically affect the formation of the brain, but not other organs. Once we have generated these mutants, we will use state-of-the-art methods of genome analysis to identify the mutated gene. With these in hand, we can begin to evaluate what developmental pathways are affected in these abnormal mice. Understanding the formation of the brain will likely help us to understand the roots of the many brain diseases affecting one in five Americans today (www.ninds.nih.gov). These diseases include developmental disorders (including autism), degenerative diseases of adult life, metabolic diseases, and brain tumors. Defects in neuronal migration within the forebrain lead to mental retardation, epilepsy and severe learning disabilities. The forebrain is also primarily affected in many other diseases such as Parkinson's and Huntington's diseases and schizophrenia. Significant efforts, both public and private, are currently underway to begin to identify more of the genes expressed in the brain at various stages as well as their expression patterns. This information, combined with the mutational analysis we plan, will help uncover the basic steps required for normal brain development.

DESCRIPTION (provided by applicant): Genetic analysis in mouse models is a means to investigate how modifying loci cause variation in phenotypic expression. We have shown that polycystic kidney disease (PKD) progression in the juvenile cystic kidney (jck) mutation can be influenced by different strain backgrounds. We have localized one of these modifier loci to proximal chromosome 4, in a region previously found to modify disease progression in two different mouse PKD mutations. The evidence that the same locus can influence disease progression in three different murine PKD mutations is of considerable significance, since this suggests this gene might influence PKD severity irrespective of its cause. As such, this locus represents a potential target for therapeutic intervention in human PKD. We propose to continue our ongoing high-resolution localization of this PKD modifier in congenic strains. We also propose to test PKD modifier candidate loci by analysis of genetically targeted mutant mice. Lastly, we propose a novel strategy of analysis using outbred mice that will potentially increase the speed and resolution of genetic localization of modifying loci. PUBLIC HEALTH RELEVANCE: It is well known that the same underlying genetic defect in the PKD gene can have different degrees of severity, presumably as a function of the genetic background of the affected individual. This has led to the recognition of potential importance of modifier loci, which can influence the expression of the PKD defect. Positional cloning of such loci may suggest alternative avenues of therapeutic intervention.

Abstract: Airway hyperresponsiveness (AHR) is a key physiological component of asthma. While AHR is inducible in experimental animal models by sensitization to antigen and can be potentiated by antigen challenge in sensitized humans, it is also evident in non-sensitized (na¿ve) individuals. The genetic basis of this complex trait has remained elusive. Analysis of mice that demonstrate heritable na¿ve airway hyperresponsiveness provides a means to identify its underlying molecular basis. We have previously used genetic mapping to identify quantitative trait loci (QTLs) that confer AHR. We have also used genetic and pharmacological analysis to demonstrate that this trait is mediated by mast cells. In this proposal, we plan to continue our investigation of the heritable loci that predispose to AHR using state-of-the-art genetic strategies, and to assess the role of mast cells in conferring the strain-specific AHR trait.

DESCRIPTION (provided by applicant): The power of the zebrafish system stems from its utility as a developmental biology model combined with the ease of its genetic manipulation and experimentation. Our understanding of key genetic mechanisms of vertebrate development has been propelled by the phenotypic characterization, genetic mapping and positional cloning of induced and spontaneous mutations in zebrafish. However, the potential of this system has not been fully realized, as inefficient microsatellite-based mapping remains the primary method in the field. We propose to apply technological and computational advances of present day genomics to genetic mapping in the zebrafish system. Specifically, we propose to develop a method for rapid and accurate mapping of recessive zebrafish mutants using Next Generation Sequencing (NGS) of pooled samples. We also propose to investigate parameters of screen design and sample analysis to optimize the use of this protocol. Finally, we aim to develop methods for identification of the causal mutation among the variants discovered within the mapping interval. Application of NGS technology, complemented by specifically developed computational techniques, will provide an efficient, accurate and inexpensive method for genetic mapping in zebrafish. This approach will enable the simultaneous identification of informative genetic markers, mapping of the mutation position, and potential identification of the causal sequence change in a single experiment. The data obtained in these genomic analyses and the methods developed will be made available to the zebrafish community. Importantly, these approaches will also be widely applicable to genetic analysis of other model systems.

ABSTRACT There is abundant evidence from the analysis of human populations and mouse models that the severity of Polycystic Kidney Disease (PKD) can be modified by interacting genetic loci. The identification of these loci should provide insight into our understanding of the basic pathobiology of cystogenesis and disease progression. Importantly, they can potentially reveal novel pathways of therapeutic intervention. We have extensive experience in the characterization of a mouse model of cystic kidney disease, and specifically the investigation of strain-specific modifiers of its severity. However, the yield of proven causal genes in mouse studies of this type has been low. In contrast, we have been very successful using a different approach for novel disease gene discovery, namely mutagenesis with the chemical ethyl-nitrosourea (ENU). We have recently modified this method so that we can do our screen entirely on an inbred background, using Whole Genome Sequencing methodology for positional cloning. The recent characterization of the PKD1RC mutant mouse as having slowly progressive PKD, which is sensitive to strain-specific modifiers, compels our proposal that we use ENU mutagenesis for the generation and discovery of modifiers of PKD1-induced cystic kidney disease. To complement this phenotype-driven approach, we will also pursue an analysis of candidate loci that may modify PKD severity. We have data to suggest that Sonic Hedgehog (SHH) signaling plays a role in cystogenesis, and we will test whether the deletion of genes in this pathway affects disease severity in the PKD1RC mouse model.

PROJECT SUMMARY The Administrative Core will coordinate the administrative, fiscal, and organization aspects of our PPG program. Personnel in this Core will facilitate scientific communication among the clinical and laboratory sites of the project, and will provide support services to each of the projects. Investigators in the Department of Pediatrics have faculty appointments at UW and scientific appointments at SCRI and SCH. Collaborative scientific projects involving investigators at SCRI, SCH, and UW are frequent, and the administrative staff at the have excellent working relationships, such that tasks such as inter-institutional financial and budgetary management are routine. As the overall Program Director, Dr. David Beier is responsible for scientific overview of the project, for overall budgetary and administrative matters, and for insuring the investigators adhere to the highest ethical standards in conducting their research. All of the senior investigators, Dr. Katrina Dipple, Dr. Jay Shendure, and Dr. Murat Maga, have experience with interdisciplinary multi-investigator, multi-site projects and are well-suited for the collaborative effort that we are proposing. In addition to logistic and financial management of the PPG, the Administrative Core will facilitate inter-project communication and meetings. To facilitate dissemination of results to the community, the Core will also assist in the organization of a local annual meeting that we anticipate will be of interest to the large number of investigators interested in technological development and its application to understanding mammalian development and human disease. Finally, the Core will facilitate the participation in the PPG of underrepresented minority students via the SCRI-sponsored URM Summer Internship Program.

PROJECT SUMMARY In this project we plan to apply a powerful and scaleable technique of combinatorial indexing to characterize gene expression at the single-cell level in mice carrying mutations that are either known to or likely to result in structural birth defects. We propose to assess whether single-cell expression can be utilized as a phenotype; specifically, we aim to organize these data sets to assess whether there are signatures of single-cell gene expression that facilitate grouping mutant lines based on presumptive pathways of developmental signaling perturbation. This analysis will be complemented by the anatomical analysis that is proposed in Project 2. Given the novelty of this method, we will initially analyze 10 lines that are mutated for genes in the Shh signaling pathway, in order to correlate single-cell transcriptomic data with well-studied developmental phenotypes. To maximize the opportunity for new gene discovery, we will also examine novel genes that have not been previously annotated with respect to human structural birth defects. Specifically, using an analysis of human exome sequencing data, we have identified a large cohort of genes that are likely haploinsufficient; i.e., they are not compatible with survival when heterozygous null. We have furthermore developed a heterozygote selection (shet) statistic that correlates remarkably well with human disease severity. We aim to characterize 75 lines from the top quintile shet set that have limited functional annotation; these genes will be chosen either a) based on evidence from single-cell expression during embryogenesis (Cao et al. 2019) that they are novel cell- type-specific index genes or b) are known lethal genes (in mice) that have a high frequency of protein interactions. As part of this effort we will develop bioinformatic tools to facilitate comparisons across different datasets. These can identify mutant lines with common abnormalities of developmental signaling, as well as potentially serving as a means to understand the mechanistic basis for human congenital abnormalities.

PROJECT SUMMARY The unifying theme of this proposal is the aim to use state-of-the-art technologies to investigate the basic biology of mammalian organ development and human structural birth defects. Our approach is wide-ranging, and aims to demonstrate how utilization of powerful technologies can inform many disorders. Importantly, this proposal marries a number of strengths of investigators at Seattle Children?s Research Institute and the University of Washington Department of Genome Sciences; specifically, expertise in the diagnosis and understanding of human congenital malformation syndromes and mammalian developmental biology, and the application of powerful new techniques for biological investigation. In Project 1, we propose to use single-cell RNA sequencing (sci-RNA-seq) technology to characterize mid- gestation embryos of mice carrying mutations relevant to human structural birth defects. Essentially, we are proposing to utilize sci-RNA-seq as a phenotype, with which one can annotate changes in expression and cell- type representation during abnormal organogenesis. Ideally, these profiles will be comparable to each other, and can potentially provide insight into fundamental biological pathways that are perturbed when developmentally important genes are lost. In Project 2, we will leverage recent advances in 3D imaging, computer vision and machine-learning to make the morphological characterization of mouse mutants more accurate, quantitative, reproducible and accessible. Progeny from the same lines studied in Project 1 will be harvested at E15.5 and imaged using microCT scanning. We will then employ several different data analysis techniques to identify differences in the tissue volume and shapes in the mutant mice compared to synthetic image constructed from a pool of ?normative? samples. The goal of Project 3 is to use novel technologies in prospective cohorts of children with structural birth defects to identify genetic variation not ascertained by current methods. These ?hidden? variants include structural rearrangements, as well as DNA mutations that arise post-zygotically and are not present in blood-derived DNA. We will use long-read based DNA and RNA sequencing methods, or deep short-read based DNA sequencing of multiple, non-blood derived tissues, on patients with structural birth defects whose clinical workup has been non-diagnostic.