Anthony Wynshaw-Boris - US grants

The UC San Diego Superfund Basic Research Program will identify and characterize molecular events that contribute to the onset of environmental disease resulting from exposure to Superfund site contaminants. In the course of studies in Projects 1, 4, 5, 6 and 7, genes with altered patterns of gene expression after exposure to Superfund site chemicals will be identified. To understand the function of these novel genes in vivo, the Mouse Genetics and Phenotyping Core will produce mice with altered expression patterns of these genes, and assist investigators in the analysis of the phenotypes of these mutant mice. It is anticipated that the organization structure of this Core will foster collaborations among the five projects producing and characterizing mutant mice. The specific aims of the Mouse Genetics and Phenotyping Core are to: 1) aid in the design and production of knock-out and transgenic mice; 2) provide expertise in husbandry of these mutant mouse strains, including breeding the mice to be sure that the mutant allele is transmitting through the germline, and to homozygosity for knock-outs; 3) maintain mice as inbred strains, and provide other inbred strains to produce relevant hybrid backgrounds; 4) in close conjunction with the investigator, perform a phenotyping screen on each of the mutant strains that will include histopathology, anatomic pathology, blood and urinalysis, and physiological monitoring; 5) assist the investigator in a further analysis of phenotypes when detected.

DESCRIPTION (Applicant's abstract): To understand the complex regulatory network responsible for neuronal migration, it is necessary to identify the genes of that network. We hypothesize that regulatory networks of genes important for a biological process, such as neuronal migration, can be defined and examined by determining the relative expression of genes in the network when it is perturbed by mutation of a key element in the pathway. We have elected to examine regulator networks for neuronal migration in mouse mutants for Lis1. Lis1 is the mouse homolog of a human gene (LIS1) mutated in isolated lissencephaly sequence (ILS), a human neuronal migration defect. We have made several different Lis1 mutant mice that have varying levels of LIS1 activity, and have used these alleles to produce mice with graded reduction of Lisl activity. We have also produced mice with conditional inactivation of Lis1 (floxed Lis1 allele), as well as transgenic mice that produce Cre in defined regions of the brain during development. Matings between the Cre mice with mice containing the floxed Lisl allele will allow us to inactivate Lisl in defined temporal and spatial patterns in mice. RNA will be isolated from these mice containing graded reduction of Lis1 in the whole brain from birth or in distinct spatial and temporal patterns of the brain. These RNA samples will be used as probes against cDNA microarrays representing 8700 independent mouse genes. The patterns of expression of these genes will be compared throughout development in brain regions in vivo and in granule cell cluster cultures in vitro. Patterns of global gene expression will be compared with those in brains from other mouse mutants with neuronal migration defects, such as reeler and cdk5. This is will allow us to distinguish the importance of LIS1 to the neuronal migration regulatory network. The specific aims of this proposal are: 1) to collect RNA samples from developing and adult brains, subregions of the brains and migrating neurons in vitro from wildtype mice, Lisl mutants with varying dosage of LIS1, conditional mutants with regional specific deletion of the Lisl gene, and other murine neuronal migration mutants, 2) to determine relative levels of global gene expression in these various mutant samples compared with wildtype and other control samples using cDNA microarrays of the UniGene set of 8700 mouse cDNAs; 3) to cluster gene expression profiles based on similar patterns of expression, dose dependent changes in expression, and by mouse mutant by comparing results of these hybridization experiments during development and adulthood in brains and brain regions, untreated cerebellar cluster cultures and cultures treated with PAF from wildtype and different Lisl mutant mice.

DESCRIPTION (provided by applicant): Haploinsufficiency of human LIS1 (or PAFAHIB1) is responsible for the human neuronal migration defect lissencephaly. Recently, we found that LIS1 interacts with NUDEL, a homologue of the A. nidulans nuclear distribution mutant NudE. LIS1 and NUDEL together bind the cytoplasmic dynein heavy chain (CDHC) to regulate dynein motor function in non-neural cells. NUDEL is phosphorylated by Cdk5/p35, a complex essential for neuronal migration in mice, suggesting that the LIS1/NUDEL/CDHC complex is regulated by phosphorylation. Phosphorylated NUDEL binds to 14-3-3epsilon, a member of a large family of binding proteins that mediate subcellular localization or stability of phosphoproteins. These recent studies have provided a pathway through which LIS1 acts to regulate dynein motor function in non-neural cells. However, it is unknown if this pathway is critical for neuronal migration. It is tempting to speculate that the dysregulation of dynein motor function by the LIS1/NUDEL/CDHC complex is also responsible for the neuronal migration defects in mammals with reduced doses of LIS1, mice deficient for the Cdk5/p35 complex, and mice deficient for 14-3-3epsilon. We hypothesize that LIS1 forms a complex (LIS1/NUDEL/CDHC) that regulates dynein motor function during neuronal migration. The function of this complex is regulated by phosphorylation of NUDEL by Cdk5/p35 via binding and regulation of the cellular distribution and/or stability of NUDEL by 14-3-3epsilon. To address these hypotheses, we will utilize genetic and gene transfer approaches to modulate the levels of specific components of this pathway in mice or cells. The effects of these modifications on neuronal migration will be determined in vivo and in vitro using quantitative migration assays. To prove whether effects of these modifications on neuronal migration result from alterations in the LIS1/NUDEL/CDHC complex, we will employ cell biological and biochemical assays of cytoplasmic dynein function, including the cellular localization and biochemical interaction of members of this complex. The specific aims of this application are: 1) determine whether LIS1 participates in neuronal migration by regulation of dynein motor function, 2) determine whether NUDEL participates with LIS1 to regulate dynein motor functions via Cdk5/p35 phosphorylation during neuronal migration; and 3) to determine if 14-3-3epsilon regulates the cellular distribution and/or stability of Cdk5-phosphorylated NUDEL during neuronal migration to regulate the activity of LIS1 and CDHC.

DESCRIPTION (provided by applicant): This proposal describes a predoctoral genetics training program on the La Jolla Mesa including the University of California, San Diego (UCSD) School of Medicine, the UCSD Division of Biology, and The Salk Institute for Biological Studies. The goal of this program is to train graduate students for future careers as academic or industrial scientists investigating genetic phenomena, or using genetic methods to understand biological problems important for human health. Our training faculty have joined together to create a common vision of contemporary genetics training in which we first build a foundation of basic biological and biomedical science. This foundation will then support a tripod of integrated educational principles of genetics and genomics including: 1) rigorous education in the classical principles and intellectual methods of genetics; 2) research training in the newest methods of classical and molecular genetics including genomics; and 3) development of an appreciation of the problems, outlooks, and ethical issues associated with modem clinical and medical genetics. The development of this unique program is tied to continued expansion of our graduate programs based on substantial new faculty recruitment in genetics and genomics and as a companion to the integration of previously distinct training efforts at UCSD. Thus, we are requesting financial support for 20 trainees in the first year phasing into 26 trainees after four years to be educated in genetics laboratories at UCSD and the Salk Institute.

DESCRIPTION (provided by applicant): Neural tube defects (NTDs) are common birth defects in humans. Although it is known that genetic susceptibility factors are associated with NTDs in humans, they have yet to be identified. The mouse can be used as a model system to study genetic factors associated with NTDs and the mechanism of neural tube closure. In the course of studying mice with inactivation of each of the three mouse Disheveled (Dvl) genes, we discovered that Dvl2 -/- and Dvl1 -/- Dvl2 -/- display NTDs. Disheveled is an important gene in the evolutionarily conserved Wnt/wingless signal transduction pathway and the planar cell polarity (PCP) pathways. All eukaryotic Dvl proteins contain three highly conserved domains: DIX, PDZ and DEP. Studies in Drosophila and Xenopus indicate that while the DIX and N-terminal portion of the DEP domain are required for Wnt pathway signaling, the PDZ and C-terminal DEP domain are essential for the PCP/convergent extension pathway. Disruption of either the Wnt or PCP pathways may result in NTDs. We propose to determine the spatial and temporal requirements for Dvl proteins during neural tube closure, and whether the NTDs displayed by Dvl mutant mice are caused by Writ signaling defects and/or PCP pathway defects. We will use the following specific aims. 1) To further characterize Dvl-dependent pathways responsible for the NTDs in Dvl 1/2 mutants, we will examine Wnt and PCP/convergent extension pathway signaling in cells and in vivo during neural tube closure in wild-type and Dvl 1/2 double mutants. 2) We will test for genetic interactions between Loop tail/strabismus (Stbm) and fused/axin with Dvl1 and Dvl2 in neural tube closure, to genetically distinguish PCP and Wnt pathway effects, respectively. 3) To determine the sites in neural tube that require Dvl2 function for normal closure, we will produce mice with specific loss-of-function of Dvl2 or specific expression of Dvl2 in spatially and temporally restricted patterns. The effect of these mutations on neural tube closure in wild type and Dvl mutants will be assessed. 4) To determine the domains of the Dvl2 protein that are required for neural tube closure, we will produce an allele series of Dvl2 mutants in mice using a BAC transgenic strategy. Precise mutations in Dvl2 will be engineered using homologous recombination of BACs in bacteria, and the resultant mutant Dvl2 BACs will be used to produce transgenic mice. The effect of these mutations on neural tube closure in Dvl -/-Dvl2 -/- mutants will be assessed.

DESCRIPTION (provided by applicant): Social interaction is a fundamental characteristic of most eukaryotic organisms, and it is modulated by both genetic and environmental factors. Social interaction has been productively studied in several animal model systems, leading to the identification of brain regions and neural systems important for this behavior, such as the amygdala and associated structures. However, the genetic contribution to social behavior is currently not well understood. The mouse has been used to define specific genes important for a variety of behaviors. We feel that the mouse can be used as an important model to study the genetics of social interaction for two reasons: it is easy to manipulate mice genetically; and mice display a wide repertoire of social behaviors. In the course of investigating the normal in vivo role of Dishevelled-1 (Dvl1), one of three murine Dvl genes, we discovered that Dvl1-deficient mice exhibit reduced social interaction (Lijam et al. 1997). We recently found that these mice have abnormalities in the amygdala, a brain region known to participate in social behaviors. Thus, Dvl1 mutant mice provide an entry point into pathways important for mammalian social behavior. Dvl1 is a component of the Wnt/Wg pathway that is essential for cell fate determination and proliferation in all multicellular eukaryotic organisms. In addition, Dishevelled from Drosophila positively regulates the planar cell polarity (PCP) pathway. In Drosophila and Xenopus, it is known that distinct domains of Dvl proteins are important for distinguishing between Wnt/Wg and PCP signaling, suggesting that similar mutational analysis can define Dvl functions in mammals. An important outstanding question regarding these mice is: what pathways are mediated through Dvl1 that regulate social behavior? We propose to use the Dvl1-deficient mice to determine the genetic pathways and genes that interact to modify social behavior, and to investigate in detail the effects of these genes on social behavior as well as brain structure and function. Our specific aims are: 1) determine the spatial/temporal expression pattern of the Dvl1 protein required for normal social behavior by using a Dvl1 conditional knockout mouse and relevant Cre lines; 2) determine the domains of the Dvl1 protein required for normal social behavior by producing an allele series of Dvl1 mutants in Dvl1 -/- mice using a novel BAC transgenic strategy; 3) search for suppressors of the Dvl1 -/- social interaction phenotype by performing ENU mutagenesis on Dvl1-/- mice (sensitized screen); and 4) determine regional brain structural and functional defects of mutants produced in Aims 1-3 compared with Dvl1-/- mutants as well as other Dvl mutants.

DESCRIPTION (provided by applicant): Recently our understanding of the molecular mechanisms governing the development of the brain has been facilitated by genetic approaches in human and mouse that have identified several genes and protein products required for neocortical development and neuronal migration. Many of these genes and protein products have been placed into three major functional pathways, based on genetic, biochemical and cell biological studies in mouse models: the RELN pathway, the Cdk5/p35 pathway and the LIS1 pathway. Recent studies from this program project have identified some important cross-talk between these three pathways. However, there are several critical gaps in our understanding. First, it is unknown if these pathways and interactions regulate exclusively neuronal migration or other processes involved in brain development such as neurogenesis and survival. Second, the manner and degree in which these various components and signaling pathways are interconnected are not known. Finally, the relationships of OCX to the three major pathways of neuronal migration are unknown. It is critical to determine the integration of the gene products and their signaling pathways during neuronal migration for a more comprehensive understanding of the molecular intricacies that govern neocortical development. Therefore, we propose to investigate the integration of these pathways by the following Specific Aims: Aim 1. Test the hypothesis that LIS1 has several important functions during brain development and in the adult by examining the dosage dependent effects of LIS1 during neurogenesis, neuronal migration, cell survival and adult neuronal function in vivo. Based on our published and preliminary data, we predict that LIS1 is critical for processes at all stages of brain development, and even in the postmitotic adult brain, although not in non-neuronal somatic tissues. Aim 2 Test the hypothesis that the phosphorylation of NUDEL by Cdk5/p35 and binding to 14-3-3epsilon are critical for neuronal development and migration in vivo by producing specific Cdk5 phosphorylation site mutants in mice by BAG transgenesis. Aim 3 Test the hypothesis that OCX is part of the LIS1 pathway. Based on our preliminary data, we predict that with LIS1, OCX plays a role in the regulation of dynein motor function, and that regulation of OCX activity via phosphorylation by Cdk5/p35 may be analogous to NUDEL regulation.

Wnt gene /protein; child with disability

DESCRIPTION (provided by applicant): We propose a postdoctoral Program to provide research training in medical genetics. The primary objective of the program is the training of physicians in research in medical genetics, to enable them to become independent investigators, and academic and scientific leaders in human genetic disease. A special and unique strength of the Program is the integration of clinical and research activities in medical genetics at UCSF, resulting in the interactions of research trainees with trainees and faculty in clinical genetics. Research trainees are thereby provided with the broadest possible background upon which to formulate and develop future research problems. The major component (z 80%) of the Training Program is the conduct of a clearly defined and substantive research project under the supervision of a Program Faculty investigator. The Training Faculty includes investigators from both clinical and basic science departments in multiple schools at UCSF. The research activities of the Training faculty are truly diverse, and include basic, clinical, clinical laboratory, and epidemiologic research spanning virtually all of contemporary human genetics and genomics. Faculty members of this Training Program have an excellent record in pre- and postdoctoral training in genetics, impressive laboratory facilities, and extensive funding for their research. This faculty can therefore provide the space, laboratory resources, and programmatic environments for the successful research training of postdoctoral trainees under this Program. Additional components of the Training Program include attendance at scheduled research and medical genetics conferences;graduate level courses in research methodology, and in basic genetics and advanced human genetics;and targeted instruction in clinical and clinical laboratory genetics. The vast majority of applicants and entrants are physicians (MD or MD-PhD) interested in research careers in medical genetics. The Program has also been able to accommodate scientists (PhD) interested in research, teaching, and clinical laboratory careers in medical genetics. The Program is overseen at UCSF by a Program Director, and Executive Committee and an Advisory Group, who regularly evaluate and monitor the progress of trainees. Finally, the Program will immeasurably benefit from the recent broad expansion and enhancement of the academic presence of human genetics at UCSF, especially the establishment of the Institute for Human Genetics.

DESCRIPTION (provided by applicant): The well-conserved canonical and non-canonical Wnt pathways are important for all aspects of mammalian development, including the development of the central nervous system. An outstanding question remains: how are the various Wnt pathways that regulate development integrated in vivo? Dvls are outstanding candidates to address this question, since these conserved proteins are required in all eukaryotes for both canonical and non-canonical Wnt pathways. We have uncovered partially unique but predominantly redundant functions among the three Dvl genes. Single mutants display some unique defects in social behavior and conotruncal heart development, while double Dvl mutants display severe neural tube defects (craniorachischisis) and severe cochlear defects. In further support of redundancy, Dvl1/2/3 triple mutants are unable to undergo gastrulation and do not form mesoderm. We plan to dissect the in vivo pathways that Dvls regulate normal development and are disrupted in the Dvl mutants to produce these phenotypes. We produced in vivo conditional alleles in mice for each of the Dvl genes as well as in vivo alleles that can distinguish either canonical Wnt of non-canonical Wnt/PCP pathway function. We used these alleles to provide definitive evidence that the craniorachischisis phenotype displayed by Dvl1;Dvl2 double mutants resulted from disruption of convergent extension movements via the Wnt/PCP pathway. We will use these tools to provide a comprehensive analysis of the role of the canonical Wnt and non-canonical Wnt/PCP pathways during neuronal development from the first development of neural folds during gastrulation and neurulation throughout neurogenesis and neuronal migration. Based on our published and preliminary data, we predict that Dvls and the pathways they regulate are critical at all stages of brain development. We will use the following specific aims: 1) Determine the role of canonical Wnt and non-canonical Wnt/PCP pathways during gastrulation in vivo;2) Characterize the Dvl dependent pathways responsible for neural tube closure during neurulation;3) Determine the role of Dvls and the canonical Wnt and non-canonical Wnt/PCP pathways during forebrain/midbrain-hindbrain development using double Dvl mutants;and 4) Determine the role of Dvls and the canonical Wnt and non-canonical Wnt/PCP pathways during forebrain/midbrain-hindbrain development using triple Dvl mutants. PUBLIC HEALTH RELEVANCE: Understanding cellular mechanisms and pathways that mediate neuronal development by the Wnt pathways using Dvl mutant mice will likely provide important insights into human neural tube defects and the development of the central nervous system. The use of sophisticated mouse mutants that inactivate each of the Dvls or express conditional or mutant alleles that express fluorescently tagged proteins will allow for the detailed study of these mechanisms and pathways in ways that are impossible in the human.