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](Notch1tm2Pst). 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.

中文翻译：

---

129S2 / SvPasCrl基因组中的修饰子负责Notch1 [12f / 12f]小鼠的生存能力。

背景技术小鼠NOTCH1在胞外域的表皮生长因子样重复序列12（EGF12）中的Thr466上携带高度保守的O-岩藻糖聚糖。X射线晶体学显示该位点的O-岩藻糖可被DLL4和JAG1 Notch配体识别。我们以前显示，Notch1 Thr466Ala突变体在报告基因分析中显示出极少的配体诱导的NOTCH1信号传导，而Thr466Ser突变使O-岩藻糖转移并恢复了NOTCH1信号传导缺陷。随后，我们生成了具有Thr466Ala突变的突变小鼠，称为Notch1 [12f]（Notch1tm2Pst）。令人惊讶的是，混合背景上的纯合Notch1 [12f / 12f]突变体是可行的和可育的。结果我们现在报道，在与C57BL / 6 J小鼠回交11-15代后，几乎没有纯合的Notch1 [12f / 12f]胚胎诞生。定时交配表明，胚胎致死率在胚胎第（E）〜E11.5天发生，与缺少Notch1或Pofut1（将O-岩藻糖添加到Notch受体的O-岩藻糖基转移酶）的小鼠相比，小鼠的致死性有所延迟，后者死于〜E9.5。C57BL / 6 J Notch1 [12f / 12f]胚胎的表型比受规范的Notch通路成员丢失影响的突变体轻，但卵黄囊中的血管生成紊乱，延缓的体发生和发育是其特征。Notch靶基因的原位杂交Uncx4.1和Dll3或NOTCH1切割的蛋白质印迹分析未显示E9.5的显着差异。然而，头部cDNA的qRT-PCR显示E9.5 Notch1 [12f / 12f]胚胎中Dll3，Uncx4.1和Notch1的表达增加。从Notch1 [12f / 12f]胚胎头部进行cDNA测序和Southern分析表明，Notch1 [12f]基因座在回交后是完整的。因此，我们寻找通过将C57BL / 6 J Notch1 [12f / +]小鼠与129S2 / SvPasCrl小鼠杂交来修饰基因的证据。F1子代的杂交产生了可行的F2 Notch1 [12f / 12f]小鼠。结论我们得出的结论是129S2 / SvPasCrl基因组包含一个显性修饰基因，可拯救NOTCH1 [12f]的信号传导功能。当NOTCH1的EGF12中不存在O-岩藻糖聚糖时，修饰基因的鉴定有可能阐明促进Notch信号传导的新因子。结论我们得出的结论是129S2 / SvPasCrl基因组包含一个显性修饰基因，可拯救NOTCH1 [12f]的信号传导功能。当NOTCH1的EGF12中不存在O-岩藻糖聚糖时，修饰基因的鉴定有可能阐明促进Notch信号传导的新因子。结论我们得出的结论是129S2 / SvPasCrl基因组包含一个显性修饰基因，可拯救NOTCH1 [12f]的信号传导功能。当NOTCH1的EGF12中不存在O-岩藻糖聚糖时，修饰基因的鉴定有可能阐明促进Notch信号传导的新因子。