Summary / Abstract Cleft lip with or without cleft palate (CL/P) is known to develop from a combination of genetic, epigenetic and environmental influences, yet most animal models are based only on genetic alteration. Here, we develop and extend an existing, but understudied, model of mouse cleft lip and palate with a known epigenetic contribution, A/WySn. By using novel methods to examine the relationships between DNA methylation, gene expression, proliferation and morphology in a single embryo we can directly relate these parameters. This allows us to study how genetically identical individuals enter differing developmental trajectories, including the presence or absence of a structural birth defect. We present two aims which focus on the development of the model and show how these aims can progress into aims for a future R01 application. These aims dissect the complex genetics of this model so that future grants can model how epigenetic changes lead to signaling changes and morphological changes which cause orofacial clefting. Aim 1 focuses on the understanding the molecular mechanism leading to CL/P in the model, while Aim 2 focuses on cellular level changes leading to CL/P development. Aim 1 has two subaims which focus identifying the gene or genes responsible for the epigenetic modification of the Wnt9bIAP from the Clf2 locus and understanding how it functions as a modifier of the cleft lip phenotype in the A-strain mouse lines. In the future we can perturb methylation to understand its role as a driver of CL/P and knock out a gene in the Clf2 domain to confirm their role as a modifier of Wnt9bIAP methylation. Aim 2 builds imaging tools and pipelines to model how changes in a cell biological property, such as proliferation and apoptosis, lead to a phenotypic change such as CL/P. In the future, we can use this method to test hypotheses around how altered growth may lead to birth defects using computational models. Together, these aims expand our knowledge of epigenome-phenotype maps by expanding our mechanistic understanding of how variation flows through development to generate phenotypic variation.