A modifier in the 129S2/SvPasCrl genome is responsible for the viability of Notch1[12f/12f] mice

Shweta Varshney,Subha Sundaram,Pamela Stanley,Frank Batista,Ankit Tanwar,Mohd Nauman,Hua-Xing Wei,Katherine Siminovitch

doi:10.1186/s12861-019-0199-3

Abstract

BackgroundMouse NOTCH1 carries a highly conserved O-fucose glycan at Thr466 in epidermal growth factor-like repeat 12 (EGF12) of the extracellular domain. O-Fucose at this site has been shown by X-ray crystallography to be recognized by both DLL4 and JAG1 Notch ligands. We previously showed that a Notch1 Thr466Ala mutant exhibits very little ligand-induced NOTCH1 signaling in a reporter assay, whereas a Thr466Ser mutation enables the transfer of O-fucose and reverts the NOTCH1 signaling defect. We subsequently generated a mutant mouse with the Thr466Ala mutation termed Notch1[12f](Notch1tm2Pst). Surprisingly, homozygous Notch1[12f/12f] mutants on a mixed background were viable and fertile.ResultsWe now report that after backcrossing to C57BL/6 J mice for 11–15 generations, few homozygous Notch1[12f/12f] embryos were born. Timed mating showed that embryonic lethality occurred by embryonic day (E) ~E11.5, somewhat delayed compared to mice lacking Notch1 or Pofut1 (the O-fucosyltransferase that adds O-fucose to Notch receptors), which die at ~E9.5. The phenotype of C57BL/6 J Notch1[12f/12f] embryos was milder than mutants affected by loss of a canonical Notch pathway member, but disorganized vasculogenesis in the yolk sac, delayed somitogenesis and development were characteristic. In situ hybridization of Notch target genes Uncx4.1 and Dll3 or western blot analysis of NOTCH1 cleavage did not reveal significant differences at E9.5. However, qRT-PCR of head cDNA showed increased expression of Dll3, Uncx4.1 and Notch1 in E9.5 Notch1[12f/12f] embryos. Sequencing of cDNA from Notch1[12f/12f] embryo heads and Southern analysis showed that the Notch1[12f] locus was intact following backcrossing. We therefore looked for evidence of modifying gene(s) by crossing C57BL/6 J Notch1 [12f/+] mice to 129S2/SvPasCrl mice. Intercrosses of the F1 progeny gave viable F2 Notch1[12f/12f] mice.ConclusionWe conclude that the 129S2/SvPasCrl genome contains a dominant modifying gene that rescues the functions of NOTCH1[12f] in signaling. Identification of the modifying gene has the potential to illuminate novel factor(s) that promote Notch signaling when an O-fucose glycan is absent from EGF12 of NOTCH1.

Highlights

Mouse NOTCH1 carries a highly conserved O-fucose glycan at Thr466 in epidermal growth factor-like repeat 12 (EGF12) of the extracellular domain
Conversion of Thr to Ala in the EGF12 O-fucose consensus site of Drosophila Notch precludes the addition of O-fucose to EGF12, and results in embryos that go through neurogenesis but fail to exhibit Notch signaling at the dorsal/ventral boundary of the wing disc [11]
In this paper we show that Notch1[12f] becomes a strong loss-of-function allele after backcrossing at least 11 generations onto the C57BL/6 J background, and we reveal the existence of a modifier gene in either the 129129S2/SvPasCrl or C57BL/ 6J genome

Summary

Introduction

Mouse NOTCH1 carries a highly conserved O-fucose glycan at Thr466 in epidermal growth factor-like repeat 12 (EGF12) of the extracellular domain. EGF12 of both Drosophila and mammalian Notch carry O-fucose [7, 8] and Xray crystallography of co-crystals between fragments of NOTCH1 and DLL4 [9] as well as NOTCH1 and JAG1 [10] revealed that O-fucose on EGF12 plays a key role in binding interactions between NOTCH1 and both ligands. In vitro binding assays revealed that there is a marked loss in the ability of Fringe (which adds a N-acetylglucosamine to Ofucose in EGF repeats) to inhibit the binding of Serrate to Notch lacking O-fucose in EGF12 [11]. A recombinant mammalian NOTCH1 fragment containing EGF12 lacking Ofucose does bind to Notch ligands in vitro in the presence of calcium [12]

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Results

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Conclusion