Project I - Transcriptomic Analysis of Structural Birth Defects in Mouse Developmental Mutants

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PROJECT SUMMARY In this project we plan to apply a powerful and scaleable technique of combinatorial indexing to characterize gene expression at the single-cell level in mice carrying mutations that are either known to or likely to result in structural birth defects. We propose to assess whether single-cell expression can be utilized as a phenotype; specifically, we aim to organize these data sets to assess whether there are signatures of single-cell gene expression that facilitate grouping mutant lines based on presumptive pathways of developmental signaling perturbation. This analysis will be complemented by the anatomical analysis that is proposed in Project 2. Given the novelty of this method, we will initially analyze 10 lines that are mutated for genes in the Shh signaling pathway, in order to correlate single-cell transcriptomic data with well-studied developmental phenotypes. To maximize the opportunity for new gene discovery, we will also examine novel genes that have not been previously annotated with respect to human structural birth defects. Specifically, using an analysis of human exome sequencing data, we have identified a large cohort of genes that are likely haploinsufficient; i.e., they are not compatible with survival when heterozygous null. We have furthermore developed a heterozygote selection (shet) statistic that correlates remarkably well with human disease severity. We aim to characterize 75 lines from the top quintile shet set that have limited functional annotation; these genes will be chosen either a) based on evidence from single-cell expression during embryogenesis (Cao et al. 2019) that they are novel cell- type-specific index genes or b) are known lethal genes (in mice) that have a high frequency of protein interactions. As part of this effort we will develop bioinformatic tools to facilitate comparisons across different datasets. These can identify mutant lines with common abnormalities of developmental signaling, as well as potentially serving as a means to understand the mechanistic basis for human congenital abnormalities.