Noelle Dwyer - US grants

DESCRIPTION (provided by applicant): The mechanisms by which polarized neural stem cells divide to produce various progenitor, neuronal, and glial cell types at precise times during development remain among the most compelling mysteries in biology and medicine. How these cells split to produce two daughters with symmetric or asymmetric fates, while still maintaining polarity and epithelial structure, remains unclear despite much progress. Cytokinesis, the actual partitioning of cytoplasmic and membrane components, has rarely been directly studied in neural stem cells. The objective of this particular application is to directly study and perturb cytokinesis in mammalian neural stem cells, both in vivo and in vitro. The central hypothesis is that cytokinesis mechanisms differ between early proliferative and later neurogenic division phases, contributing actively to the generation of a cerebral cortex of the proper area and layer structure. We will test this hypothesis through three Specific Aims: 1) characterize and perturb spatial and temporal parameters of cytokinesis in cortical neural progenitors at different stages of development, 2) correlate midbody inheritance patterns with symmetric and asymmetric fates, and test the fate consequences of experimentally increasing midbody retention, and 3) elucidate phenotypes produced when cytokinesis is specifically disrupted during early or late stages of development. Our long-term goal is to elucidate how specific alterations in cell division mechanisms in different progenitor types during development can lead to variations in brain size and structure, malformations, or other disorders. The contributions of the proposed research are expected to be a foundation of innovative approaches and tools for studying cytokinesis in normal and abnormal brain development, and a more detailed understanding of how cytokinetic structures partition apical components in neural progenitors. These contributions will be significant because they may uncover novel mechanisms that contribute to differential fate determinant segregation for neural stem cell renewal or neurogenesis, and make predictions for human brain phenotypes that may arise from global or localized defects in cytokinesis.

Abstract: The mechanisms by which neural stem cells in the embryonic brain divide and generate daughter cells to build the cerebral cortex of the proper size and structure remain among the most compelling mysteries in biology and medicine. How these long, polarized cells split to produce two daughters with symmetric or asymmetric fates, while still maintaining the epithelial structure, remains unclear despite much progress. Proper mitosis (chromosome segregation) and cytokinesis (cytoplasm, organelle, and membrane segregation) of neural stem cells is essential for their proliferation and survival. Indeed, neural stem cells trigger apoptosis more easily than mature neurons or other dividing cell types. Yet how apoptosis is triggered and how it can be prevented are not understood. This proposal focuses on a specific genetic model of microcephaly in which neural stem cells appear to have difficulty with a late step of cell division (abscission), and trigger their own apoptosis. Surprisingly, the small brain size and survival of these mutant animals can be rescued by knockout of the tumor suppressor gene p53. The objective of this particular application is to elucidate how p53- dependent apoptosis is triggered in this model of microcephaly. The central hypothesis is that neural stem cells that cannot complete the last step of division of the daughters (abscission) trigger p53-dependent apoptosis. We will test this hypothesis through two Specific Aims: 1) determine the temporal and causal relationship between cytokinetic abscission defects and p53 activation in NSCs, and 2) elucidate this novel pathway of p53 activation. Our long-term goal is to elucidate how specific alterations in cell division mechanisms in neural stem cells during development can lead to variations in brain size and structure, malformations, or other disorders. The contributions of the proposed research are expected to be a novel pathway for p53 activation and apoptosis, and a more detailed understanding of how cell division defects lead to brain malformations. These contributions will be significant because they may uncover novel mechanisms that contribute to neural stem cell survival or death, and make predictions about possible treatments for specific types of microcephaly or other brain malformations resulting from excess apoptosis.

Abstract: The mechanisms by which neural stem cells build the brain from a simple epithelial tube is a compelling mystery of biology. Mouse genetics has been one of the most powerful tools to discover the genes and processes involved in building a healthy brain, or in neurodevelopmental disorders that affect brain structure or function. The library of targeted mutations created by the International Mouse Phenotyping Consortium (IMPC) provides a highly valuable resource for understanding those genes and processes. Many mutations that cause neurodevelopmental phenotypes are lethal, either due to the brain defect or to pleiotropic functions of the gene in other organs essential for viability. Thus, the bank of lethal mutants at IMPC provides an enriched set of candidate genes required for brain development. However, changes in brain structure or wiring are difficult to detect without the right tools and expertise. We propose to apply our expertise to phenotype selected knockout lines that are likely to have neurodevelopment defects, based on gene expression, function, and/or known mutation in a human developmental disorder. This is in response to an FOA to characterize developmental defects in lethal IMPC mutants. We will combine non-hypothesis driven gross phenotyping with hypothesis-driven analysis of a few selected brain development mutants. To add value and efficiency to this screen, subsets of mutants will also be tested by co-investigators for inner ear development phenotypes, and placenta, gastrulation, or neural tube defects. For the first tier of phenotyping, lines will be tested for age of lethality and phenotyped for gross abnormalities of brain, inner ear, spinal cord, and body development. Tier 2 phenotyping will incorporate histopathology of brain, inner ear, and placenta, since many structural defects in these organs cannot be ascertained without sectioning and staining. We will test for proliferation, layering, and axon tract defects. Tier 3 phenotyping will focus on a small number of mutants with both abnormal brain phenotypes and gene functions in cytokinesis, to address our hypothesis: that different defects in cytokinesis of cortical neural stem cells underlie a variety of brain malformations. We will make use of methods we have established for quantitative analysis of cytokinetic furrowing and abscission defects in developing mouse cortex. In all stages of phenotyping, heterozygotes and homozygotes will be compared to controls quantitatively and with statistical rigor. Data will be shared with IMPC for the benefit of the community. Our team of three investigators has a combined >50 years of expertise working on cellular bases of organ development in the mouse model, and forward and reverse genetics. Through this project , we will discover new mouse models for developmental disorders of the brain, inner ear, and placenta, and will provide important new insights into the mechanisms of neural stem cell divisions and cellular defects underlying brain malformations.

Abstract: In order for the brain to develop with proper size and structure, neural stem cells (NSCs) at early ages must make proliferative divisions and maintain their stemness to expand the stem cell pool, but then switch to undergo neurogenic divisions at the correct time to create neurons. However, the mechanism of this fate choice from remaining an NSC early on, to later choosing to exit the cell cycle and become a neuron, is still poorly understood. The last step of cell division is abscission, which severs the daughter from the mother cell. Abscission occurs during the time when the fate decision is made, and at the apical membrane, where many fate signals are located. We developed methods and tools to quantitatively analyze abscission in cortical NSCs, in vivo and in vitro. We found that abscission is not simply necessary to cut cells apart and keep them alive. Rather, we made the surprising discovery that both abscission duration and remnants of abscission (midbody remnants) are developmentally regulated, changing as development proceeds. Furthermore, we found that a small-brained mouse mutant with altered abscission duration has a reduced proportion of proliferative NSC divisions. These data led to our central hypothesis that changes in abscission duration and midbody remnant persistence can shift NSC daughter cells fate choices as development proceeds. We will test this hypothesis through the use of innovative genetic and cell biological approaches, on single NSC divisions and whole tissue analyses. We will utilize two mouse mutants that perturb abscission specifically, affecting duration and midbody remnants differentially. We will carry out three Specific Aims: 1) test whether abscission duration is correlated with daughter cell fate outcomes in vitro, 2) dissect the primary and secondary effects of dysregulated abscission on cortical NSC daughter cell fates, morphologies and lineage progression in vivo, and 3) investigate a candidate signaling mechanism at the apical membrane that could link abscission regulation to stem cell maintenance. The contributions of the proposed research will be to increase understanding of the fundamental question of how stem cells in developing tissues maintain high proliferative capacity early and then reduce it later in favor of differentiated daughter cell types. It will also elucidate how regulation of NSC divisions affect daughter cell fates, structures, and subsequent divisions. These contributions will be significant because they will reveal novel mechanisms and gene pathways that regulate how brain size and structure are controlled, and will elucidate how specific alterations in NSC division mechanisms during development can lead to brain malformations, or other neurodevelopment phenotypes.