Effects of varying Notch1 signal strength on embryogenesis and vasculogenesis in compound mutant heterozygotes.

BACKGROUND: Identifying developmental processes regulated by Notch1 can be addressed in part by characterizing mice with graded levels of Notch1 signaling strength. Here we examine development in embryos expressing various combinations of Notch1 mutant alleles. Mice homozygous for the hypomorphic Notch1(12f) allele, which removes the single O-fucose glycan in epidermal growth factor-like repeat 12 (EGF12) of the Notch1 ligand binding domain (lbd), exhibit reduced growth after weaning and defective T cell development. Mice homozygous for the inactive Notch1(lbd) allele express Notch1 missing an aproximately 20 kDa internal segment including the canonical Notch1 ligand binding domain, and die at embryonic day approximately E9.5. The embryonic and vascular phenotypes of compound heterozygous Notch1(12f/lbd) embryos were compared with Notch1+/12f, Notch1(12f/12f), and Notch1(lbd/lbd) embryos. Embryonic stem (ES) cells derived from these embryos were also examined in Notch signaling assays. While Notch1 signaling was stronger in Notch1(12f/lbd) compound heterozygotes compared to Notch1(lbd/lbd) embryos and ES cells, Notch1 signaling was even stronger in embryos carrying Notch1(12f) and a null Notch1 allele. RESULTS: Mouse embryos expressing the hypomorphic Notch1(12f) allele, in combination with the inactive Notch1(lbd) allele which lacks the Notch1 ligand binding domain, died at approximately E11.5-12.5. Notch1(12f/lbd) ES cells signaled less well than Notch1(12f/12f) ES cells but more strongly than Notch1lbd/lbd ES cells. However, vascular defects in Notch1(12f/lbd) yolk sac were severe and similar to Notch1(lbd/lbd) yolk sac. By contrast, vascular disorganization was milder in Notch1(12f/lbd) compared to Notch1(lbd/lbd) embryos. The expression of Notch1 target genes was low in Notch1(12f/lbd) yolk sac and embryo head, whereas Vegf and Vegfr2 transcripts were increased. The severity of the compound heterozygous Notch1(12f/lbd) yolk sac phenotype suggested that the allelic products may functionally interact. By contrast, compound heterozygotes with Notch112f in combination with a Notch1 null allele (Notch1(tm1Con)) were capable of surviving to birth. CONCLUSIONS: Notch1 signaling in Notch1(12f/lbd) compound heterozygous embryos is more defective than in compound heterozygotes expressing a hypomorphic Notch1(12f) allele and a Notch1 null allele. The data suggest that the gene products Notch1(lbd) and Notch1(12f) interact to reduce the activity of Notch1(12f).