A phenotype‐driven ENU mutagenesis screen identifies novel alleles with functional roles in early mouse craniofacial development

Abstract

Summary Proper craniofacial development begins during gastrulation and requires the coordinated integration of each germ layer tissue (ectoderm, mesoderm, and endoderm) and its derivatives in concert with the precise regulation of cell proliferation, migration, and differentiation. Neural crest cells, which are derived from ectoderm, are a migratory progenitor cell population that generates most of the cartilage, bone, and connective tissue of the head and face. Neural crest cell development is regulated by a combination of intrinsic cell autonomous signals acquired during their formation, balanced with extrinsic signals from tissues with which the neural crest cells interact during their migration and differentiation. Although craniofacial anomalies are typically attributed to defects in neural crest cell development, the cause may be intrinsic or extrinsic. Therefore, we performed a phenotype-driven ENU mutagenesis screen in mice with the aim of identifying novel alleles in an unbiased manner, that are critically required for early craniofacial development. Here we describe 10 new mutant lines, which exhibit phenotypes affecting frontonasal and pharyngeal arch patterning, neural and vascular development as well as sensory organ morphogenesis. Interestingly, our data imply that neural crest cells and endothelial cells may employ similar developmental programs and be interdependent during early embryogenesis, which collectively is critical for normal craniofacial morphogenesis. Furthermore our novel mutants that model human conditions such as exencephaly, craniorachischisis, DiGeorge, and Velocardiofacial sydnromes could be very useful in furthering our understanding of the complexities of specific human diseases.

Disrupting hedgehog and WNT signaling interactions promotes cleft lip pathogenesis

“…Genome sequencing revealed a T to A nucleotide change in intron 15 of Ptch1, which created a new splice acceptor site, resulting in a premature stop codon in exon 16 and generation of a truncated protein. Ptch1 wiggable embryos die in utero at around E12.0 as a result of various defects, including open neural tube and hypertelorism of the face (19). These phenotypes are consistent with a gain of function in HH signaling.…”

“…Consistent with this role, disruption of Ptch1 in mice results in an elevation of SHH signaling (22). Through an N-ethyl-N-nitrosourea mutagenesis screen in mice (19), we generated a novel allele of Ptch1. Genome sequencing revealed a T to A nucleotide change in intron 15 of Ptch1, which created a new splice acceptor site, resulting in a premature stop codon in exon 16 and generation of a truncated protein.…”

“…Ptch1 wiggable mice were generated through N-ethyl-N-nitrosourea mutagenesis in our lab (19). Hhat creface , TOPgal, and Ptch1-LacZ mice were maintained as described previously (18,22,23).…”

Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development

“…In addition to reverse genetic approaches, forward genetic screens utilizing N-ethyl-N-nitrosourea (ENU) mutagenesis in mice have identified new alleles that affect craniofacial development (Herron et al, 2002;Sandell et al, 2011). The ENU-induced recessive cleft secondary palate (csp1) allele was recently shown to be a loss-of-function mutation in the Prdm16 gene (Bjork et al, 2010b).…”

FOXF1 Transcription Factor Is Required for Formation of Embryonic Vasculature by Regulating VEGF Signaling in Endothelial Cells

“…Deletion of FOXF1 was confirmed by breeding FOXF1-deficient mice with LoxP-stop-LoxP-β-gal (R26R) and LoxP-tdTomato-LoxP-GFP ( mT/mG ) reporter mice (both from Jackson Lab.). Flk1-null mutant mice were previously described 23 . Animal studies were approved by the Animal Care and Use Committee of Cincinnati Children’s Hospital Research Foundation.…”