Mapping the dynamics of mouse neurogenesis in autism models using high-resolution genomic barcoding technologies

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PROJECT SUMMARY/ABSTRACT The complexity of mammalian embryogenesis makes it challenging to determine the effect of genetic perturbations on development. The long-term goal is to better understand how genetic and environmental factors alter mammalian devel- opment to affect adult phenotypes or cause diseases. Toward achieving this long-term goal, the overall objective of this application is to develop a platform for high-throughput retrospective lineage reconstruction to quantitatively map devel- opmental alterations in a mouse model of autism. This platform will be based on developmental barcoding where random mutations accumulate in synthetic loci during embryogenesis. Each mutation is inherited by the descendants of the cell in which it occurs; each descendant can add new mutations to the ones it inherited. This process marks each cell with a set of mutations—a barcode—that can be used to resolve its lineage. The central hypothesis is that high-resolution lineage barcodes that are sequenced spatially in single cells can be used to retrospectively map proliferation and differentiation dynamics of neural progenitors to identify the differences between wildtype and mutant mouse models. The rationale for this research is that many genetic risk factors that are associated with birth defects remain mechanistically inexplicable based on cellular and molecular analyses of terminally differentiated tissues; this platform would enable retrospective mapping of these genetic effects after development to determine which progenitors they affect, when they affect those progenitors, and how they affect the behavior of those progenitors during development. The central hypothesis will be tested by pursuing three specific aims: 1) Establish high-resolution lineage recording in combinatorial and cumulative bar- codes embedded in each cell’s genome. This Aim will combine mutagenesis from double-strand breaks, which predomi- nantly lead to indels, with orthogonally induced point mutations to establish ultrahigh resolution lineage recording throughout mouse gestation. 2) Establish in situ single-cell barcode and identity readout directly from mouse tissues. This Aim will engineer barcoding loci to facilitate their amplification and sequencing in tissue sections together with molecular markers of cell state. Combining cell state and lineage barcodes will reveal proliferation and differentiation dynamics of their progenitors. 3) Determine the effects of Chd8 haploinsufficiency on the development of mouse brain using retro- spective lineage reconstruction. Chd8 haploinsufficiency causes autism but how it alters neurogenesis remains unclear. This aim will quantify the effects of Chd8 haploinsufficiency on proliferation and differentiation parameters of brain pro- genitors during mouse neurogenesis. The research proposed here is innovative because it establishes new strategies for high-resolution genomic barcoding of lineages and high-throughput spatial sequencing of these barcodes in tissue sec- tions. It further uses new theoretical concepts to convert terminal cells’ lineage barcodes and molecular identity infor- mation to quantitative insights about their progenitors. Additionally, it carries out in utero analysis of how Chd8 haploin- sufficiency alters progenitor fields that create the brain. This research is significant because it will enable determining how genetic perturbations alter mammalian embryogenesis to cause developmental anomalies such as autism.