PROJECT SUMMARY E15.5 fetuses of 6 homozygous mutants from each of the 85 sci-RNA-seq lines will be acquired from Project 1 to be imaged via high-resolution microCT following established diffusible iodine contrast enhanced scan protocols for fetal mice. Up to hundred fetuses of unaffected littermates will also be imaged to derive a synthetic population template of normative samples (Figure1) and will be used to assess the ‘normal’ phenotypic variation in mouse development at E15.5 (Aim 1). The template will be segmented to the finest anatomical detail possible and will contain more anatomical structure and detail than the existing KOMP2 fetal atlas. Each sci-RNA-seq line will be assesses for overall phenotypic variability (Figure 3) and then be subjected to region-specific statistical (Figure 4) analysis of tissue volume and shape differences (Aim 2). We will also train a deep-learning model to achieve automated segmentation of fetal mice and compare the results to the current-state-of-the-art, template-based analysis (Aim 3). Our goal is to leverage recent advances in the 3D imaging, computer vision and machine-learning to make the 3D morphological characterization of mouse mutants to be more accurate, quantitative, reproducible and accessible. Combining molecular phenotype and anatomical phenotypes will provide a comprehensive characterization of mice with mutations relevant to human structural birth defects.