In vivo consequences of deleting EGF repeats 8–12 including the ligand binding domain of mouse Notch1

Notch1lbd/lbd embryos died at mid-gestation with a phenotype indistinguishable from Notch1 null mutants. In embryonic stem (ES) cells, Notch1lbd was expressed on the cell surface at levels equivalent to wild type Notch1, but Delta1 binding was reduced to the same level as in Notch1 null cells. In an ES cell co-culture assay, Notch signaling induced by Jagged1 or Delta1 was reduced to a similar level in Notch1lbdand Notch1 null cells. However, the Notch1lbd/lbd allele was expressed similarly to wild type Notch1 in Notch1lbd/lbd ES cells and embryos at E8.75, indicating that Notch1 signaling is not essential for the Notch1 gene to be expressed. In addition, maternal and zygotic Notch1 mutant blastocysts developed through gastrulation.Mouse Notch1 lacking the ligand binding domain is expressed at the cell surface but does not signal in response to the canonical Notch ligands Delta1 and Jagged1. Homozygous Notch1lbd/lbd mutant embryos die at ~E10 similar to Notch1 null embryos. While Notch1 is expressed in oocytes and blastocysts, Notch1 signaling via canonical ligands is dispensable during oogenesis, blastogenesis, implantation and gastrulation.Notch1 is a heterodimeric, type I transmembrane receptor that is required for cell fate decisions throughout the metazoa [1,2]. The Notch1 extracellular domain contains 36 tandem epidermal growth factor-like (EGF) repeats, and three Lin/Notch repeats. Of the 36 EGF repeats in Drosophila Notch, deletion of only EGF repeats 11 and 12 prohibits the binding of the Notch ligands Delta and Serrate in in vitro binding assays [3,4]. Notch signaling in mammals is also initiated by binding to canonical Notch ligands (Delta and Jagged) on adjacent cells. Ligand binding activates Notch signaling through two proteolytic cleavage events, first in the extracellular domain by the ADAM10 metalloprotease [5], and subsequently in the transmembrane domain by a presenilin complex with γ-secretase activity [6,7]. The released Notch intracellular doma