Effects of varying Notch1 signal strength on embryogenesis and vasculogenesis in compound mutant heterozygotes

Abstract

Background: Identifying developmental processes regulated by Notch1 can be addressed in part by characterizing mice with graded levels of Notch1 signaling strength. Here we examine development in embryos expressing various combinations of Notch1 mutant alleles. Mice homozygous for the hypomorphic Notch1(12f) allele, which removes the single O-fucose glycan in epidermal growth factor-like repeat 12 (EGF12) of the Notch1 ligand binding domain (lbd), exhibit reduced growth after weaning and defective T cell development. Mice homozygous for the inactive Notch1(lbd) allele express Notch1 missing an aproximately 20 kDa internal segment including the canonical Notch1 ligand binding domain, and die at embryonic day approximately E9.5. The embryonic and vascular phenotypes of compound heterozygous Notch1(12f/lbd) embryos were compared with Notch1+/12f, Notch1(12f/12f), and Notch1(lbd/lbd) embryos. Embryonic stem (ES) cells derived from these embryos were also examined in Notch signaling assays. While Notch1 signaling was stronger in Notch1(12f/lbd) compound heterozygotes compared to Notch1(lbd/lbd) embryos and ES cells, Notch1 signaling was even stronger in embryos carrying Notch1(12f) and a null Notch1 allele.

Results: Mouse embryos expressing the hypomorphic Notch1(12f) allele, in combination with the inactive Notch1(lbd) allele which lacks the Notch1 ligand binding domain, died at approximately E11.5-12.5. Notch1(12f/lbd) ES cells signaled less well than Notch1(12f/12f) ES cells but more strongly than Notch1lbd/lbd ES cells. However, vascular defects in Notch1(12f/lbd) yolk sac were severe and similar to Notch1(lbd/lbd) yolk sac. By contrast, vascular disorganization was milder in Notch1(12f/lbd) compared to Notch1(lbd/lbd) embryos. The expression of Notch1 target genes was low in Notch1(12f/lbd) yolk sac and embryo head, whereas Vegf and Vegfr2 transcripts were increased. The severity of the compound heterozygous Notch1(12f/lbd) yolk sac phenotype suggested that the allelic products may functionally interact. By contrast, compound heterozygotes with Notch112f in combination with a Notch1 null allele (Notch1(tm1Con)) were capable of surviving to birth.

Conclusions: Notch1 signaling in Notch1(12f/lbd) compound heterozygous embryos is more defective than in compound heterozygotes expressing a hypomorphic Notch1(12f) allele and a Notch1 null allele. The data suggest that the gene products Notch1(lbd) and Notch1(12f) interact to reduce the activity of Notch1(12f).

Figure 1

Generation of Notch1^12f/lbd embryos. (A) Diagram of the Notch1^12f and Notch1^lbd alleles. (B) Diagram of mouse Notch1 EGF repeats in Notch1^12f and Notch1^lbd extracellular domains. The EGF repeats with putative O-fucosylation sites are shaded in gray and the mutation in EGF12 is shown. (C) Genotyping by PCR from E9.5 yolk sac DNA of a litter from a Notch1^12f/12f × Notch1^+/lbd cross. Primers 5F and 6R detect the Notch1^12f and Notch1⁺ alleles, primers 5F and 9R detect the Notch1^lbd allele.

Figure 2

A graded reduction in Notch1 signaling in Notch1^12f/lbd ES cells. (A) Notch1 expression on the surface of ES cells (Notch1^+/+, Notch1^12f/12f, Notch1^12f/lbd and Notch1^lbd/lbd) was analyzed by flow cytometry using anti-Notch1 mAb 8G10 (solid line). Shaded profiles are secondary Ab only. (B) Delta1-Fc binding to ES cells. Control is secondary antibody alone. 5 mM EDTA inhibited ligand binding to control levels (gray). Data are mean ± SEM (n = 4), * p < 0.05 between Notch1^+/+ and all mutant lines. (C) Delta1-induced Notch signaling and (D) Jagged1-induced Notch1 signaling were determined by co-culturing ES cells with Delta1/L or Jagged1/L cells compared to control L cells after transfection of a Notch reporter construct. Bars represent fold-activation ± SEM (n = 4), * p < 0.05; ** p < 0.01, *** p < 0.001 based on the two-tailed Student's t test; (E) Whole cell lysates from ES cells were subjected to western analysis using the Val1744 antibody for activated Notch1 and the 8G10 antibody for full length Notch1. The histogram shows the relative expression of activated Notch1 after normalization to β-tubulin III (mean ± SEM from 4 experiments).

Figure 3

Embryogenesis in Notch1^12f/lbd embryos. (A-D) Vascularization of yolk sac in Notch1^+/12f, Notch1^12f/12f, Notch1^12f/lbd and Notchl^lbd/lbd embryos at E9.5. Large vitelline blood vessels were present in Notch1^+/12f and Notch1^12f/12f yolk sacs, but absent in the Notch1^12f/lbd and Notch1^lbd/lbd mutants. (E-H) Morphology of embryos at E9.5. Notch1^12f/12f are similiar to Notch1^+/12f, Notch1^12f/lbd are markedly underdeveloped, and Notch1^lbd/lbd are severely underdeveloped. (I-L) Notch1^12f/lbd embryos from E10.5-E12.5. White arrows show hemorrhaging in E10.5 and E11.5 embryos; most E12.5 embryos were resorbing. The number of embryos examined at each stage is given in Table 1.

Figure 4

Defects in vascular remodeling in Notch1^12f/lbd E9.5 embryos. All whole mount embryos were stained with Ab to Pecam1. (A-D) Morphogenesis of the main trunk of the anterior cardinal vein (arrow) in Notch1^12f/lbd and Notch1^lbd/lbd mutant embryos is defective compared to Notch1^12f/12f and control Notch1^+/12f embryos. (E-H) Vascular remodeling in brain in Notch1^+/12f and Notch1^12f/12f is similar but is defective in Notch1^12f/lbd and severely defective in Notch1^lbd/lbd embryos. (I-L) Vascular remodeling in heart is defective in Notch1^12f/lbd and more severely affected in Notch1^lbd/lbd embryos. (M-P) Intersomitic vessels (arrows) were well-organized in Notch1^+/12f and Notch1^12f/12f embryos but were mildly disorganized in Notch1^12f/lbd and essentially absent from Notch1^lbd/lbd embryos. The number of embryos examined was 3 - 4 of each genotype.

Figure 5

Real-time PCR of vasculogenic and Notch target genes in Notch1^12f/lbd yolk sac and embryo. Total RNA extracted from E10.5 yolk sac or embryonic head was reverse-transcibed and subjected to real-time PCR. Numbers of transcripts were normalized to β-actin, and the average relative expression of Notch1^+/12f samples was set to 1. (A-F) Relative expression of Pecam1, Vegf, Vegfr2, Hes5, Hey1, and Hey2 as marked. Bars represent SEM (n = 6). The two-tailed Student's t test was used in control versus mutant yolk sac and embryo head comparisons; a one-tailed Student's t test was used in mutant yolk sac versus mutant embryo head comparisons; * p < 0.05; ** p < 0.01

Figure 6

Notch1^12f/tm1Con embryos survive longer than Notch1^12f/lbd embryos. (A) PCR genotype of an E9.5 litter showed the 280 bp PCR product from Notch1^tm1Con allele and the 238 bp product from the Notch1^12f allele. (B) Yolk sac vascularization of E10.5 Notch1^12f/tm1Con and Notch1^+/12f embryos. (C) Notch1^12f/tm1Con embryos at E10.5 exhibit heamorrhaging around the heart (arrows). (D) Notch1^12f//tm1Con and control embryos at E15.5. One Notch1^12f/tm1Con embryo was defective but the other had no obvious defects. (E) Notch1^12f/tm1Con and control embryos at E17.5. One Notch1^12f/tm1Con embryo was defective but the other had no obvious defects. (F) Photo of a litter on postnatal day 1 (P1) which included one pup identified as Notch1^12f/tm1Con by PCR genotyping below. The pup was indistingishable but died within a few days. (G) PCR genotype of the P1 litter in panel F.

Figure 7

An allelic series of Notch1 mutants. Based on data reported herein and from the literature, the relative signaling strength of Notch1 mutant alleles in various combinations with wild type or other Notch1 mutant alleles is represented as discussed in the Discussion. The consequences with respect to time of death of embryos with severe Notch1 signaling defects, or more subtle defects in T cell, CNS or cardiac development are noted.