In vivo consequences of deleting EGF repeats 8-12 including the ligand binding domain of mouse Notch1.
BACKGROUND
Notch signaling is highly conserved in the metazoa and is critical for many cell fate decisions. Notch activation occurs following ligand binding to Notch extracellular domain. In vitro binding assays have identified epidermal growth factor (EGF) repeats 11 and 12 as the ligand binding domain of Drosophila Notch. Here we show that an internal deletion in mouse Notch1 of EGF repeats 8-12, including the putative ligand binding domain (lbd), is an inactivating mutation in vivo. We also show that maternal and zygotic Notch1(lbd/lbd) mutant embryos develop through gastrulation to mid-gestation.


RESULTS
Notch1(lbd/lbd) embryos died at mid-gestation with a phenotype indistinguishable from Notch1 null mutants. In embryonic stem (ES) cells, Notch1(lbd) 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 Notch1(lbd) and Notch1 null cells. However, the Notch1(lbd/lbd) allele was expressed similarly to wild type Notch1 in Notch1(lbd/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.


CONCLUSIONS
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 Notch1(lbd/lbd) mutant embryos die at approximately 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.
Ge
Changhui
C
Department of Cell Biology, Albert Einstein College of Medicine, New York, NY-10461, USA. gechanghui@gmail.com
Liu
Tongyi
T
Hou
Xinghua
X
Stanley
Pamela
P
D000818:Animals; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D008024:Ligands; D051379:Mice; D017354:Point Mutation; D011485:Protein Binding; D051881:Receptor, Notch1 / Q000235:genetics*; D020449:Repetitive Sequences, Amino Acid; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D015398:Signal Transduction
D004268:DNA-Binding Proteins; D008024:Ligands; C497762:Notch1 protein, mouse; D051881:Receptor, Notch1; D004815:Epidermal Growth Factor