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

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

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

Background: Mouse 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](Notch1 tm2Pst). Surprisingly, homozygous Notch1[12f/12f] mutants on a mixed background were viable and fertile. Results: We 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. Conclusion: We 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.

Original languageEnglish (US)
Article number19
JournalBMC Developmental Biology
Volume19
Issue number1
DOIs
StatePublished - Oct 7 2019

Keywords

  • 129S2/SvPasCrl
  • Backcross
  • C57BL/6J
  • Embryogenesis
  • Modifier gene
  • Notch signaling
  • Notch1[12f]

ASJC Scopus subject areas

  • Developmental Biology

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    Varshney, S., Wei, H. X., Batista, F., Nauman, M., Sundaram, S., Siminovitch, K., Tanwar, A., & Stanley, P. (2019). A modifier in the 129S2/SvPasCrl genome is responsible for the viability of Notch1[12f/12f] mice. BMC Developmental Biology, 19(1), [19]. https://doi.org/10.1186/s12861-019-0199-3