Protein O-fucosyltransferase 1 is an essential component of Notch signaling pathways

Abstract

Notch receptor signaling regulates cell growth and differentiation, and core components of Notch signaling pathways are conserved from Drosophila to humans. Fringe glycosyltransferases are crucial modulators of Notch signaling that act on epidermal growth factor (EGF)-like repeats in the Notch receptor extracellular domain. The substrate of Fringe is EGF-O-fucose and the transfer of fucose to Notch by protein O-fucosyltransferase 1 is necessary for Fringe to function. O-fucose also occurs on Cripto and on Notch ligands. Here we show that mouse embryos lacking protein O-fucosyltransferase 1 die at midgestation with severe defects in somitogenesis, vasculogenesis, cardiogenesis, and neurogenesis. The phenotype is similar to that of embryos lacking downstream effectors of all Notch signaling pathways such as presenilins or RBP-J kappa, and is different from Cripto, Notch receptor, Notch ligand, or Fringe null phenotypes. Protein O-fucosyltransferase 1 is therefore an essential core member of Notch signaling pathways in mammals.

Figure 1

Expression of the mouse Pofut1 gene. (A and B) Northern analysis of adult mouse tissues (A) and embryos at different stages (B). (C) Whole-mount in situ hybridization of E11.5 wild-type embryos hybridized to antisense or sense Pofut1 coding region probe.

Figure 2

Targeting of the Pofut1 gene. (A) The endogenous Pofut1 gene contains 7 coding exons (black). A 560-bp region of genomic DNA containing exon 2 with a neomycin and thymidine kinase cassette between loxP sites (red) flanked by genomic DNA in the pFlox vector was used to obtain the targeted allele. Transfection with the Cre recombinase expression plasmid pMC1 gave ES cells with exon 2 deleted. B, BamHI; Xh, XhoI; Xb; XbaI. (B) Southern analysis after digestion with BamHI and hybridization with the genomic DNA probe P1 gave an 8.45-kb endogenous Pofut1 gene fragment with DNA from WW6 ES cells, and an 8.0-kb mutant band with DNA from a heterozygous clone with exon 2 deleted. (C) PCR genotyping with primers 644 and 645 of a litter from a cross between Pofut1^+/− heterozygotes; bands are endogenous allele (700 bp) and deleted allele (300 bp). (D) Whole-mount in situ hybridization with a Pofut1 gene antisense probe of E9.5 control (+/+) and Pofut1 null (−/−) embryos.

Figure 3

Morphological defects in Pofut1^−/− embryos. (A) Control wild-type or heterozygote embryo (left) and Pofut1^−/− null (right) embryo at E10 with distended pericardial sac (arrow) and unlooped heart (asterisk). (B) E8.5 control (left) and Pofut1^−/− (right) embryos. Bracket indicates the posterior region with fused somites; arrow points to a defective somite. (C) Sagittal sections (7 μm) of somites in E9.5 control (Upper) and mutant (Lower) embryos. (D) Vascular defect in yolk sac of E9.5 Pofut1-null embryo (right). (D) PECAM-1 antibody staining of E9.5 control (left) and mutant (right) embryos. PECAM-1 is missing from large brain and intersomitic vessels in the mutant (arrows). (E) Kinked neural tube of E8.25 Pofut1^−/− embryo (arrow; dorsal view).

Figure 4

Expression of Notch signaling pathway genes in Pofut1^−/− embryos. Control wild-type or heterozygote embryo (left) and Pofut1^−/− embryo (right) in all panels were probed together. (A and B) E8.75 lateral view (A) and E8.75 dorsal view (B) with a Dll1 probe. Dll1 was expressed in somites (arrowhead in B) and PSM (arrows) of control embryos and down-regulated in Pofut1^−/− embryos. Dll1 was up-regulated in neural tube (bracket in A), fore-, and hind-brain (asterisks) of Pofut1^−/− embryos. (C) Dll3 expression was down-regulated in PSM (arrow) but activated in the brain of E8.75 Pofut1^−/− embryos (arrowhead). (D) Expression of Jagged1 (Jag1) in the posterior region of the forming somite (arrowhead) and in otic vesicle (arrow) and brain of E8.75 control embryos was down-regulated in Pofut1^−/− embryos. (E) The Notch target Hes5 is expressed in the primitive streak (arrowhead) and forming somite (arrow) of E8.5 control (dorsal view). Hes5 expression was reduced and altered in E8.5 Pofut1^−/− embryo. (F) Myogenin in anterior somites of control embryos at E8.75 (arrow) was also present in the condensed somites of Pofut1^−/− embryos (arrow). (G) Myogenin was poorly and diffusely expressed in E9.5 Pofut1^−/− embryos (arrow). (H) Uncx4.1 was expressed in the caudal compartment of formed somites in control but was missing from the somitic region of E8.75 Pofut1^−/− embryos. Uncx4.1 was up-regulated in neural tube (arrow) and midbrain (asterisk) of E8.75 Pofut1^−/− embryos. (I) At E8.25 Notch1 expression in the prospective somite of Pofut1^−/− embryos (arrow) was higher and more diffuse than control (arrow). (J) At E8.75 Notch1 in the prospective somite (arrow) was similar but more diffuse in Pofut1^−/− embryos (arrow). (K) At E9.0, Notch1 expression was greater in control compared with Pofut1^−/− PSM (arrow). Notch1 was up-regulated in midbrain at all stages (asterisk in I, J, and K). (L) Lfng was expressed in PSM (arrow), the prospective somite (arrowhead), otic vesicle, and brain of E8.75 control embryos. In Pofut1^−/− embryos Lfng expression was missing from somitic areas but markedly up-regulated in neural tube (asterisk).