2019 |
Nowakowski, Tomasz |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Mapping Developmental Lineage Relationships in the Cerebral Cortex @ University of California, San Francisco
Summary/Abstract The cerebral cortex contains an astonishing diversity of neuronal cell types distributed across dozens of functional areas, which emerge during early development for an apparently uniform neuroepithelium, and the radial glia cells, which act as neural stem cells. Neurogenesis in the cortex follows highly orchestrated and carefully controlled programs that establish a highly reproducible patterns of the six cortical layers. It has long been hypothesized that developmental histories of the cells are instrumental for establishing normal patterns of neuronal connectivity necessary for establishing the primitives of cortical transformation of sensory information. Many genetic mutations underlying brain development abnormalities and neurodevelopmental psychiatric disorders have long been hypothesized to affect early stages of brain development, including neuronal differentiation and circuit formation. However, we currently lack scalable tools for interrogating the developmental processes, especially the lineage relationships, and relating them to the adult brain cell types atlas, which would serve as a framework for interrogating the impact of disease mutations or developmental perturbations on brain development in a highly scalable manner. Here, we propose to leverage two emerging technologies for developmental lineage tracing that are amenable with single cell RNA sequencing. We will deploy these methods in the context of developing mouse cerebral cortex to survey the developmental lineage relationships in distinct functional areas of the cortex for which reference adult cortical single cell data are readily accessible through the BRAIN Initiative Cell Census Network, to address the hypothesis that distinct neurogenesis patterns underlie the development of area- specific excitatory cortical neurons. Most scalable developmental lineage tracing studies do not preserve the spatial position information, which is traditionally critical for the understanding of cellular organization in the brain, including the cortex. To overcome this limitation, we will contextualize the developmental lineage information in situ by performing spatial transcriptomics analysis in primary tissue. We will focus this effort on mapping lineage relationships in the primary visual cortex. We expect that this approach will enable mapping of the tangential dispersion of clonally related neurons. This innovative research program will, if successful, contribute to the development of scalable technologies for mapping developmental lineage relationships in the cerebral cortex by comparing two scalable methods for developmental lineage tracing, and serve as a step towards integrating developmental lineage tracing technologies with spatial tissue mapping, which will facilitate integration of developmental lineage data into the mouse brain cell atlas.
|
0.915 |
2021 |
Kriegstein, Arnold (co-PI) [⬀] Nowakowski, Tomasz Sanders, Stephan [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Assessing Genomic, Regulatory and Transcriptional Variation At Single Nuclei Resolution in the Brains of Individuals With Autism Spectrum Disorder @ University of California, San Francisco
ABSTRACT Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder of unknown etiology and with limited effective therapeutic options that affects millions of individuals. Our research team has a longstanding commitment to understanding the cause of ASD and the molecular processes underlying brain development, function, and pathology. We will use this experience to apply the latest molecular techniques to samples from a new repository of brain tissue from individuals with ASD to create the largest and most detailed analysis of the molecular consequences of ASD. Genetic analyses of gene disrupting de novo mutations have identified over one hundred genes associated with ASD with three main functional groups: regulation of gene expression, neuronal communication, and cytoskeleton. Prior analyses of brain tissue from individuals with ASD have identified a group of downregulated neuronal communication genes, that overlap with ASD-associated genes, and a group of upregulated glial genes that do not overlap with ASD-associated genes or variants. It is unclear if these changes reflect altered cell composition or cell function and how they relate to genetic factors. We propose to analyze post-mortem brain samples from 40 individuals with ASD and 40 unaffected controls, sourced from the Autism BrainNet BioBank, to assess the molecular changes that occur. We will use whole-genome sequencing to identify gene disruptive variants in genes previously associated with ASD and to identify rare and common variants that may alter gene expression or splicing. In tissue samples the prefrontal cortex and striatum in from 40 cases and 40 controls, we will use recently developed single-nuclei methods to perform RNA-seq and ATAC-seq at single-cell resolution to identify ASD-related changes in gene regulation and expression in specific cell types and brain regions. For tissue samples from the prefrontal cortex of 20 cases and 20 controls we will also use cutting-edge single nuclei long-read RNA-seq (Iso-seq), along with bulk tissue RNA-seq, for an in-depth analysis of how gene isoforms differ between ASD cases and controls. Finally, we will assess how single-nuclei gene expression varies in brain organoids grown from pluripotent stem cells edited to contain mutations in three ASD-associated genes. Integrating these data, we will profile the molecular changes associated with ASD and assess how these changes vary by cell type, brain region, age, sex, seizure status, and genotype. We will use RNAscope in situ hybridization to validate the molecular and cell composition changes we observe and a lentivirus-based massively parallel reporter assay to test the function of regulatory regions or variants in proximity to genes with ASD-related differences in expression to validate these effects and assess causality. We hope that these insights will provide a basis for understanding the heterogeneity of ASD and the neurobiological features of this disorder and provide molecular signatures that could be developed into future biomarkers for ASD model systems.
|
0.915 |