Varshney Shweta, Wei Hua-Xing, Batista Frank, Nauman Mohd, Sundaram Subha, Siminovitch Katherine, Tanwar Ankit, Stanley Pamela
Department of Cell Biology, Albert Einstein College of Medicine, New York, NY, 10461, USA.
Present address: ETHOS Health Communications, Yardley, PA, 19067, USA.
BMC Dev Biol. 2019 Oct 7;19(1):19. doi: 10.1186/s12861-019-0199-3.
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 Notch112f. Surprisingly, homozygous Notch1[12f/12f] mutants on a mixed background were viable and fertile.
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.
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.
小鼠NOTCH1在细胞外结构域的表皮生长因子样重复序列12（EGF12）中的苏氨酸466位点携带一个高度保守的O-岩藻糖聚糖。X射线晶体学研究表明，该位点的O-岩藻糖可被DLL4和JAG1这两种Notch配体识别。我们之前的研究表明，在报告基因检测中，Notch1苏氨酸466位点突变为丙氨酸（Thr466Ala）的突变体几乎没有配体诱导的NOTCH1信号传导，而苏氨酸466位点突变为丝氨酸（Thr466Ser）的突变则能使O-岩藻糖转移并恢复NOTCH1信号传导缺陷。随后，我们构建了携带Thr466Ala突变的突变小鼠，命名为Notch1[12f]（Notch1）。令人惊讶的是，在混合背景下的纯合Notch1[12f/12f]突变体是可存活且可育的。
我们现在报告，在回交至C57BL/6 J小鼠11至15代后，很少有纯合Notch1[12f/12f]胚胎出生。定时交配显示，胚胎致死发生在胚胎期（E）约E11.5，与缺乏Notch1或Pofut1（向Notch受体添加O-岩藻糖的O-岩藻糖基转移酶）的小鼠相比有所延迟，后者在约E9.5死亡。C57BL/6 J Notch1[12f/12f]胚胎的表型比受经典Notch信号通路成员缺失影响的突变体要轻，但卵黄囊中血管生成紊乱、体节发生延迟和发育异常是其特征。在E9.5时，对Notch靶基因Uncx4.1和Dll3进行原位杂交或对NOTCH1切割进行蛋白质印迹分析均未发现显著差异。然而，对E9.5 Notch1[12f/12f]胚胎头部cDNA进行qRT-PCR分析显示，Dll3、Uncx4.1和Notch1的表达增加。对Notch1[12f/12f]胚胎头部cDNA进行测序和Southern分析表明，回交后Notch1[12f]基因座是完整的。因此，我们通过将C57BL/6 J Notch1 [12f/+]小鼠与129S2/SvPasCrl小鼠杂交来寻找修饰基因的证据。F1代后代的杂交产生了可存活的F2 Notch1[12f/12f]小鼠。
我们得出结论，129S2/SvPasCrl基因组包含一个显性修饰基因，可挽救NOTCH1[12f]在信号传导中的功能。鉴定该修饰基因有可能揭示当NOTCH1的EGF12中不存在O-岩藻糖聚糖时促进Notch信号传导的新因子。
