Abstract
BackgroundIdentifying 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 Notch112f 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 Notch1lbd allele express Notch1 missing an ~20 kDa internal segment including the canonical Notch1 ligand binding domain, and die at embryonic day ~E9.5. The embryonic and vascular phenotypes of compound heterozygous Notch112f/lbd embryos were compared with Notch1+/12f, Notch112f/12f, and Notch1lbd/lbd embryos. Embryonic stem (ES) cells derived from these embryos were also examined in Notch signaling assays. While Notch1 signaling was stronger in Notch112f/lbd compound heterozygotes compared to Notch1lbd/lbd embryos and ES cells, Notch1 signaling was even stronger in embryos carrying Notch112f and a null Notch1 allele.ResultsMouse embryos expressing the hypomorphic Notch112f allele, in combination with the inactive Notch1lbd allele which lacks the Notch1 ligand binding domain, died at ~E11.5-12.5. Notch112f/lbd ES cells signaled less well than Notch112f/12f ES cells but more strongly than Notch1lbd/lbd ES cells. However, vascular defects in Notch112f/lbd yolk sac were severe and similar to Notch1lbd/lbd yolk sac. By contrast, vascular disorganization was milder in Notch112f/lbd compared to Notch1lbd/lbd embryos. The expression of Notch1 target genes was low in Notch112f/lbd yolk sac and embryo head, whereas Vegf and Vegfr2 transcripts were increased. The severity of the compound heterozygous Notch112f/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 (Notch1tm1Con) were capable of surviving to birth.ConclusionsNotch1 signaling in Notch112f/lbd compound heterozygous embryos is more defective than in compound heterozygotes expressing a hypomorphic Notch112f allele and a Notch1 null allele. The data suggest that the gene products Notch1lbd and Notch112f interact to reduce the activity of Notch112f.
Highlights
Identifying developmental processes regulated by Notch1 can be addressed in part by characterizing mice with graded levels of Notch1 signaling strength
Previous studies showed that Notch112f/lbd compound heterozygotes die by ~E12.5 [12]
To examine Notch ligand binding and the strength of Notch signaling in more detail, Embryonic stem (ES) cells were derived from Notch112f/lbd compound heterozygous blastocysts and compared to ES cells derived from Notch112f/ 12f and Notch1lbd/lbd homozygous blastocysts and wild type ES cells (Fig. 2)
Summary
Identifying developmental processes regulated by Notch can be addressed in part by characterizing mice with graded levels of Notch signaling strength. Mice homozygous for the hypomorphic Notch112f allele, which removes the single O-fucose glycan in epidermal growth factor-like repeat 12 (EGF12) of the Notch ligand binding domain (lbd), exhibit reduced growth after weaning and defective T cell development. Compound heterozygotes carrying Notch112f and the inactive Notch1lbd allele, which lacks the ligand binding domain and generates an inactive ~280 kDa Notch receptor at the cell surface, are not born [12]. Heterozygous Notch1+/lbd and Notch1+/tm1Con mice are viable and fertile whereas Notch112f/lbd compound heterozygotes die between E11.5 and E12.5, significantly later than either Notch1lbd/lbd [12] or Notch null embryos [13,14] that do not express Notch1 [15,16,17]
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