Mapping the Multi‐Modal Distribution of Craniofacial Phenotypes in NOSIP Mutants

Nitric Oxide Synthase Interacting Protein (NOSIP) is an E3 mono‐ubiquitin ligase that regulates the activity of protein phosphatase 2A. Deletion of NOSIP leads to severe and variable malformations of the craniofacial complex. Our goal was to use NOSIP mutant mice to understand genetic and cellular mechanisms of phenotypic variation similar to human disease. The phenotype of an individual embryo is the result of the developmental trajectory that it previously traversed. We propose that this concept of canalization is essential for understanding the etiology of complex phenotypic outcomes. We tested this by creating a 3D morphospace of NOSIP heterozygous and homozygous mutants to determine if embryos occupy unique morphospace regions based on affected craniofacial structures. Embryos between embryonic day 10.5 (E10.5) to E17 were collected, fixed in PaxGene, and imaged (mCT). Anatomical features were reconstructed using Avizo. RNAscope was used to assess Nosip expression, and RNA was the extracted from the head of embryos and prepared for sequencing. While some heterozygous embryos appear completely unaffected, both homozygous null and heterozygous embryos presented with a mild to severe spectrum of phenotypic malformations, with no homozygous null embryos appearing unaffected. Embryos on the more severe side of the spectrum has a single telencephalic lobe with severe facial malformations, including loss of maxillary processes, micrognathia and obstructed airway. Embryos on the milder spectrum had normal telencephalic vesicle separation and had less severe facial phenotypes, including mild left‐right asymmetry and clefting. NOSIP expression between E10.5 and E12.5 appears to fairly ubiquitously, with high expression in the neuroepithelium, lower jaw mesenchyme, nasal tissue. Expression was markedly reduced in normal NOSIP heterozygotes. Mutant transcriptomes at E10.5 cluster based on genotype, but by E12.5, transcriptomes cluster based on phenotype severity, suggesting that transcriptional profiles cannot be solely predicted based on genotype alone. Pathways most affected include protein function, SHH and canonical WNT signaling. Six3 and Shh appear to be negatively affected in NOSIP mutants, and correlates with more severe brain defects. Overall, it appears that Nosip ‐KO creates a multi‐modal distribution of craniofacial phenotypes along the developmental timeline by affecting early brain development, suggesting that understanding canalization may provide insight into the complexity of genotype‐phenotype map. Future work will focus on identifying specific dysregulations in protein function of mutants and normalizing early signaling pathways to rescue more normal phenotypes.