Project Summary/Abstract: During development, cellular diversity is achieved via the dynamic interpretation of a fixed DNA template, a process that incorporates both straightforward genetic and obscure epigenetic concepts. Cell identity is in part governed by transcription factor-directed programs, but these interact with elaborate “codes” of post-translational modifications to chromatin that index regions of the genome for activity or repression. To date, the general relationships between local chromatin remodeling and gene regulation have been primarily modeled in cell lines and cannot yet account for the intricacy of the developing embryo. Moreover, although many epigenetic regulators are essential for viable embryogenesis, it remains unclear how these enzymes participate in specific developmental processes given the generic nature of their target substrates. A detailed accounting of how epigenetic regulation operates during gestation is critical to understand complex congenital or fetal disorders, which often have unclear penetrance, affect multiple cell types or tissue systems, and can be highly influenced by the maternal environment. Here, I propose a transformative new strategy for comprehensively phenotyping mutant embryos that incorporates detailed micromanipulation techniques, novel molecular systems, and single cell analysis to recover high resolution morphological, molecular and temporal information from many replicates simultaneously. My flexible approach permits rapid transition from hypothesis to validation and eliminates many cumbersome aspects of traditional transgenics, as well as the frequent limitation of examining only a limited number of lineages via a handful of pre-specified marker genes. I will apply this pipeline to dissect the specific regulatory impact of maternal dietary folate and of the Polycomb group repressors on early organogenesis, which represent “top-down” and “bottom-up” approaches to interpret developmental robustness, the deliberate and reproducible generation of a sophisticated organism under uncertain and fluctuating conditions. To do so, I will leverage my ability to recover data from mutant cohorts and optimize new analytical strategies to quantify phenotypic variation and incomplete penetrance. These factors are central concerns of reproductive medicine and have been extraordinarily challenging to reduce to clear molecular mechanisms or pathways, subsequently limiting innovation of novel interventions and diagnostics. In addition to addressing fundamental human health issues, these efforts will also substantially advance a suite of new tools for exogenously titrating specific regulatory components as well as for recording the historical relationship between single cells. If successful, this proposal will uncover previously opaque dimensions of embryonic regulation, including shared and cell-type specific consequences of epigenetic or environmental insults, as well as transform general methods for characterizing previously intractable congenital abnormalities.

Project Narrative: Epigenetic regulators have limited target specificity and yet are essential for mammalian embryogenesis, complicating efforts to discriminate context-specific functions that may cause phenotypic abnormalities. Here I propose a foundational new strategy to generate and comprehensively analyze complex mutant embryos across multiple scales, allowing me to directly connect breakdowns in chromatin regulation or the maternal environment to key molecular drivers of reproductive and fetal pathologies. In addition to substantially advancing these critical human health concerns, I will also optimize a series of new technologies specifically designed to recover previously opaque dimensions of development, including the emergence of phenotypic variation that is typical of many congenital disorders.