James B. Skeath - US grants

In both vertebrates and invertebrates a progressive series of events culminate in the creation of the intricate pattern of neurons and their connections in the mature central nervous system (CNS). These steps include the separation of neural from epidermal precursors; the proliferation of neural precursors; and the determination (restriction of developmental potential) of group of or individual neurons. Subsequent steps include axon guidance, synaptogenesis, cell death and synaptic plasticity. Our research focuses on the processes of neural determination in Drosophila. Through a classical saturation screen we identified 204 mutations in 46 genes that control neural determination of the subset of CNS neurons that express the homeobox containing gene even-skipped (eve). We are focusing on 22 mutations in six genes, representing five novel genes and one recently identified gene (sampodo), that induce ectopic Eve-positive neurons. These mutations more likely disrupt cell fate choices than those that remove eve positive neurons, which might just block cell division. The specific aims of this proposal are: (1) to perform a detailed genetic analysis of the CNS function of sanpodo, a gene that modulates Notch signaling to specify the fate of many CNS sibling neurons; (2) to characterize at the molecular, genetic and phenotypic level the five novel genes that control neural determination; (3) to identify additional genes that control neural determination via screening large collections of extant lethal P element strains; and, (4) to improve present lineage tracing techniques to facilitate following the entire lineage of CNS neural precursors in normal and mutant backgrounds. Recent studies have found a remarkable conservation of structure, expression and function between many Drosophila genes expressed within the nervous system and their vertebrate homologs. This studies as well as initial insights from genome projects support the idea that the fundamental architecture of many biological processes are conserved between flies and humans. Thus, the identification, isolation and characterization of genes that control neural determination in Drosophila should yield general insights into the underlying molecular mechanisms that control either cell proliferation or differentiation during nervous system development. Such information may provide useful clues into the various causes of neurological or neuromuscular dysfunctions.

DESCRIPTION (provided by applicant): The cellular complexity of the nervous system sets it apart from other tissues. Asymmetric cell divisions, in which a precursor cell divides to produce two sibling cells of different fates, are central to the generation of cell diversity in nervous systems. Most asymmetric divisions in Drosophila and vertebrate nervous systems depend on the opposing activities of the Notch signaling pathway and the cytoplasmic determinant Numb. During precursor divisions Numb segregates exclusively into one sibling cell where it blocks Notch signaling to prevent adoption of the Notch-dependent fate. The absence of Numb in the other sibling allows Notch signaling, and thus, adoption of the Notch-dependent fate. Present models suggest that Numb blocks Notch activity by promoting endocytosis of the Notch receptor. However, there are significant caveats to this model. For example, both sibling cells exhibit equivalent levels of Notch at the cell membrane and a truncated form of Numb deleted for all known endocytic motifs is functional during Notch-mediated asymmetric divisions. Work from our lab on Sanpodo, a novel transmembrane protein required for Notch signaling only during asymmetric divisions, suggests a different model. Sanpodo localizes to the cell membrane of the sibling cell whose fate depends on Notch activity, while in the other cell Numb blocks Sanpodo from localizing to the cell membrane. These observations led to the model that Sanpodo acts at the cell membrane to promote Notch signaling, and that Numb blocks Notch activity by keeping Sanpodo off of the cell membrane. However, the molecular mechanisms by which Sanpodo promotes Notch activity arid Numb regulates Sanpodo localization remain unknown. This proposal seeks to elucidate the molecular basis of Sanpodo function and regulation. Specifically, we propose to (i) identify the functional domains of Sanpodo via structure/function studies, (ii) identify and characterize factors that interact genetically or physically with sanpodo via complementary biochemical and genetic approaches and (iii) search computationally for vertebrate sanpodo genes. Defects in Notch activity are being implicated in a growing number of diseases, including multiple types of brain cancer. And while no homolog of Sanpodo has yet been identified in vertebrates, based on (i) the high degree of divergence between the amino acid sequences of Sanpodo in insects and (ii) the conservation of the Notch/numb molecular machinery as a fundamental mechanism regulating asymmetric divisions, we hypothesize the existence of a functional Sanpodo homolog with limited primary sequence homology. Thus, identifying vertebrate sanpodo genes and elucidating the molecular basis of Sanpodo function and regulation will provide key insight into the molecular control of asymmetric divisions. Such insight should help us understand the etiology of diseases in which this process is de-regulated and design new methods to treat these diseases.

? DESCRIPTION (provided by applicant): This application requests the renewal of the Cellular and Molecular Biology Umbrella Training Program (T32 GM007067) in the Division of Biological and Biomedical Sciences at Washington University. The objective of the grant is to provide rigorous, interdisciplinary training in cell and molecular biology to a diverse cohort of students y providing support for 25 funded positions in years two and three of graduate school among students in four PhD programs - the Developmental, Regenerative and Stem Cell Biology Program, the Molecular Cell Biology Program, the Molecular Genetics and Genomics Program, and the Microbiology and Microbial Pathogenesis Program. Our program holds long-standing commitments to interdisciplinary training, cutting-edge research, and career development. Its organizational structure is designed to maintain effective communication and cooperation among the faculty and steering committees of the four PhD programs and to foster student and faculty interactions that span programmatic and departmental boundaries. Its educational mission is to ground students in the basic concepts and methodologies of cell and molecular biology and to train them to think critically and to write and speak effectively. We seek to evolve our program to keep pace with the ever-changing nature of basic research by helping our students pursue fundamental questions in cell and molecular biology. New initiatives aimed at accomplishing our mission include: 1) the introduction of forums that provide critical training in scientific presentation, 2) the implementation of a Bioinformatics Bootcamp, 3) the seamless integration of the CMB T32 program with a new IMSD R25 training program, 4) the creation of an Annual CMB Program Mini-Symposium, 5) the start of an evening Career Panel Discussion co- sponsored by the CMB and IMSD programs, 6) the genesis of two novel student-run career development organizations that provide short-term experiences in the biotechnology business and science policy, and 7) an ongoing process focused on streamlining graduate training in order to increase student productivity and decrease time to degree. Through these initiatives, we seek to enable our students to pursue careers at the vanguard of scientific research, education, and outreach by helping them establish a broad-based scientific foundation of knowledge and network of colleagues as they initiate their scientific career. In this effort, our guiding philosophy is to extend all successful program elements to as many students as soon as possible in order to maximize the training of all our students and thus the future impact of our students on society.

How the correct cell-types develop at the right time and place in an organism remains a central and enduring question in developmental biology. In no other tissue are the processes that generate and pattern cell-types taken to such extremes as in the developing CNS. Our research uses the Drosophila CNS as a model system to explore the genetic and molecular basis of cell-type diversity. We focus on the developmental mechanisms that act during post-embryonic neurogenesis to generate the neurons of the adult CNS. Post-embyronic neurogenesis commences during the first larval stage when neuroblasts reactivate division in response to the presence of dietary amino acids. Most post-embryonic neurons, however, are born during a rapid, prolonged burst of neuroblast proliferation that starts in the early third larval stage and that foreshadows, and continues past, the onset of metamorphosis. Between the time when neuroblast's reactivate proliferation and the onset of metamorphosis, larvae undergo a massive increase in size, and emerging studies suggest complex regulatory networks couple post-embryonic neurogenesis to physiology. However, both the systemic and intrinsic mechanisms that control post-embryonic neurogenesis are poorly understood. At present, studies on post-embryonic neurogenesis are limited by the inability to identify most adult neuroblast lineages based solely on gene expression and the paucity of genes known to regulate this process. We have identified ten transcription factors, the expression of which label most adult neuroblast lineages, providing the molecular tools to build the descriptive basis for detailed studies on post-embryonic neurogenesis. The overlapping expression profiles of these genes suggest they act in combination to specify the identity of individual neuronal hemilineages. And, via the wedding of forward genetic screens to whole genome sequencing methods, we have begun to identify the genes that control distinct steps of post-embryonic neurogenesis, including a putative GPCR that may couple the action of juvenile hormone to neuroblast division. Our proposed studies seek to transform these findings into an ever-clearer picture of the complex and coordinated regulatory networks that control post-embryonic neurogenesis. Our specific aims are to: ¿ Generate a gene expression map of all adult thoracic neuroblast lineages ¿ Test if a combinatorial code of transcription factors specifies the identity of neuronal hemilineages ¿ Identify genes that control post-embryonic neurogenesis via genetic screens and whole genome sequencing ¿ Characterize the mechanisms through which a GPCR governs post-embryonic neurogenesis

Washington University is widely recognized for excellence in undergraduate and graduate education in the biomedical and behavioral sciences. As a consistent performer in diversity outcomes and one of the nation's leading research institutions, Washington University is particularly well suited to be part of the solution to under representation of minority students in these sciences. Our proposed MARC U-STAR program is a comprehensive, four-year program that capitalizes on the large number of our high-potential minority students and on our existing infrastructure that has been so successful at recruiting and retaining minority students in the biomedically-related sciences. Our recruitment and retention strategy builds upon existing infrastructure to attract underrepresented minorities to the sciences, build an early scholarly community for the students, empower them early in their choice to pursue science degrees, train them in biomedical research, and retain them in the biomedical and behavioral sciences pipeline to and through graduate school at Washington University or elsewhere. This program will be part of a critical component of a greater university-wide initiative to increase the percentage of Washington University graduates enrolling in and completing Ph.D. programs. Our rationale is that while we do not serve as many minority students as other universities with majority-minority populations, all of our minority students (830 students in 2006) have very high previous achievements and have high potential to complete Ph.D.'s in a biomedical or behavioral science. Further, around 60% of our minority students (130 per entering class) cite biomedically-related science as an academic and career interest upon entrance to the University, yet only a tiny fraction pursue biomedically-relevant Ph.D.'s. We propose to support 6 students per entering class in the training program; this is more than feasible, given our minority population, our high student interest in the biomedically-related sciences, and high student success rate in these fields. With even a modest, 75% success rate at MARC-scholar Ph.D. pursuits, we will realize an approximately four-fold increase in the number of Ph.D. pursuits of our minority students in the biomedically-relevant sciences.

Abstract: Diversity at the molecular level has created the diversity of all life forms ever to exist on this planet. Recent studies by Scott E. Page (Univ. Michigan) suggest increased diversity of thought, perspective and background among individuals working as part of a team enhances performance. The pursuit of knowledge and scientific excellence then demands the inclusion of students from all backgrounds. This application requests continued support for a successful `Initiative for Maximizing Student Development' (IMSD) program within the Division of Biological and Biomedical Sciences (DBBS) at Washington University. The mission of our program is to increase the matriculation, training, retention, graduation, and career outcomes of outstanding PhD students from groups historically underrepresented in the sciences in order to increase the diversity and power of the STEM workforce in the US, particularly at the professoriate level. Over the past grant cycle, our IMSD program has developed 15 training elements that integrate seamlessly with graduate student training and research, bolstering the academic, professional, and career training of all entering under- represented (UR) PhD students and in many cases all DBBS PhD students. In the next grant cycle, we propose to add five new activities to our program in order to further drive student success and reduce the achievement gap between UR and non-UR students in our PhD programs. These training elements span our students' graduate careers and focus on ensuring they surpass defined academic milestones (e.g., first-year courses, qualifying exams, and thesis proposals), are exposed to varied career options via career panels and job- shadowing opportunities, and develop strong vertically-integrated student support networks. Our IMSD Program is only in its fourth-year of existence, but already welcomes essentially all entering UR DBBS PhD students into it, even though program funds can support only a subset of these students, ensuring that our program is the focal point of academic and career development support for UR students in DBBS. Our IMSD Program supports students for up to their first two years and preferentially selects students for support who have demonstrated a talent for and determination in the pursuit of research-based science, while overcoming significant hardships. Despite its young age, our program already has helped to increase early stage UR student retention and academic success and to narrow the achievement gap between UR and non-UR students. Thus, our IMSD program harbors great potential to increase the diversity of scientists within the US workforce, and in so doing continuing and enhancing the tradition of scientific excellence in the US.

  Abstract: The union of form and function is a core tenet of biology. In animals, tissues often adopt their characteristic form early in development and maintain it through periods of tremendous growth, a process that often demands that cells remain largely fixed in place. Tissue shape and cell migration is dictated in large part by the basement membrane, a special type of extracellular matrix that surrounds tissues, bestowing on them structural support and resiliency. Yet, how tissues adopt and retain their form and how cells remain anchored in place during growth and development remain key questions in biology. The Drosophila larval CNS is an ideal system in which to explore the genetic and molecular mechanisms that govern tissue structure and anchor cells in place. The CNS is fully enwrapped by a thick basement membrane that provides structural support to the CNS via its physical properties and interactions with underlying surface glia; the CNS grows rapidly during larval stages, maintaining its form despite tremendous growth, and during this time, neural lineages remain largely fixed in place. The CNS basement membrane must then maintain its structure and that of the CNS while continually remodeling itself and its interactions with glia to allow for its growth and that of the CNS. The genetic and molecular principles that bestow upon the CNS basement membrane, and likely most basement membranes, this power remain cloudy ? their elucidation represents the focus of this proposal. We identify AdamTS-A, an extra-cellular metalloprotease, as a key organizer of CNS structure. Reduction in AdamTS-A function disrupts CNS structure and induces a mass exodus of neurons and cortex glia out of the CNS. Our studies indicate that AdamTS-A acts in surface glia, the outermost cell layer of the CNS that directly underlies the CNS basement membrane, to maintain CNS structure and to anchor the underlying neural cells in place by opposing the actions of collagen IV and integrin, which promote tissue stiffness. Increased tissue stiffness has been shown to promote cell migration. Thus, we hypothesize that reduction of AdamTS-A function in surface glia increases the stiffness of the overlying CNS basement membrane, which in a cell non- autonomous manner then triggers hundreds of CNS neurons and cortex glia to tunnel through the nerves that project from the CNS toward peripheral tissues fully enwrapped the entire time by the membranes of surface glia. In this grant, we leverage the strengths of the fly system to clarify the underlying genetic and molecular mechanisms through which AdamTS-A maintains tissue structure and keeps cells rooted in place in the CNS. Our specific aims are ? (i) to complete a systematic phenotypic characterization of AdamTS-A in the CNS, (ii) to identify the substrates and interacting proteins of AdamTS-A in the CNS, and (iii) to uncover the genes and pathways activated in the migrating cells in response to reduced AdamTS-A function in surface glia.

The U.S. has fallen behind several countries in STEM competiveness, where K-12 students are less proficient in science and math and earn fewer bachelor degrees in STEM than countries such as China and Korea. The maintenance of the U.S. economy relies heavily on innovation and technology. As well as our health systems rely on the discoveries of scientists and physicians. Thus STEM and health are critical to our nations welfare. Improving the education and STEM experiences for all students, but namely underrepresented minorities (URM) is also critical to our economy and healthcare as our country is projected to become a majority minority nation. We are proposing a program that will prepare low-income, URM high school students for careers in STEM and health. The Washington University Science Partnership Program will be housed at the McDonnell Genome Institute at Washington University in St. Louis and will partner with the Jennings School District, a largely African-American school district in St. Louis County. In this project we have proposed two aims to address the critical need for improved STEM education for URM. In Aim 1, we proposed to develop skills in these students in genomics and bioinformatics through authentic STEM experiences, both in class and through research internships. Science and health are approaching the era of personalized medicine due in part to the decoding of the human genome and the discoveries made as a result. However skilled persons in genomics and bioinformatics are in short supply compared to the demand and are especially lacking in URM groups. So this aim will seek to fulfill a direct need in science and prepare students for future, relatively lucrative jobs. In Aim 2 we will conduct workshops that focus on college and job readiness in STEM that will walk students through the process of applying to college as well as introduce them to the wide variety of career options in STEM. In total our proposed program is poised to increase the number of URM, namely African- Americans who enter college in STEM fields and who are retained in STEM and obtain careers in these fields. Our program directly addresses national priorities in STEM education: providing authentic research experiences to K-12 students, engaging underrepresented students in STEM and enhancing the STEM workforce.

Abstract: This proposal seeks the renewal of the Opportunities in Genomics Research (OGR) Program. Since 2007, the OGR Program has been run out of The McDonnell Genome Institute at Washington University in Saint Louis and funded through the NHGRI's Diversity Action Plan. The mission of the OGR Program is to increase the representation of students from underrepresented groups in genome science or genome science-related PhD and MD/PhD programs. We seek to accomplish our mission through the effective execution and evolution of summer undergraduate and academic year post-baccalaureate research programs for students from racial and ethnic backgrounds underrepresented in the sciences, students from disadvantaged backgrounds, and students with disabilities. Both OGR programs provide trainees with cutting-edge research experiences in genome science or related fields and 11 other educational activities that seek to train them to think critically and to write and speak effectively about their research. Workshops and classes are tailored to train students in the core concepts of genome science, bioinformatics, and scientific presentation. Professional development activities focus on helping students prepare for and excel in graduate school interviews and graduate school itself. Over its three funding cycles, student outcomes for the OGR program reveal great momentum. PhD matriculation of students in our summer program rose from 29% in the first cycle to 50% or above the last two cycles. More impressively, PhD matriculation of students in our post-baccalaureate program rose from 44% in the first cycle to 75% in the second cycle and 90% in the current cycle. We expect the implementation of new and modified activities to solidify and extend these gains. Specifically, new activities will promote integration among OGR trainees and trainees in the NHGRI T32 PhD Genomic Sciences program, enhance training in bioinformatics, provide trainees with greater exposure to the diversity of research careers available to PhD scientists, and initiate community outreach events to under-served area high schools and their students. Based on the past success and current plans of our program, we seek support for five more years of funding to continue our mission of enhancing diversity in PhD programs nation-wide. We propose to expand our post- baccalaureate program to five trainees per year, and with university support, to maintain the size of our summer undergraduate program at eight students per summer. We believe our programs will continue to help realize the great scientific and intellectual potential inherent within the diverse population of the United States, much of which currently lies latent due to the underrepresentation of many sectors of our nation's population within scientific research in general and genome science in particular.