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. 2001 May 8;98(10):5643-8.
doi: 10.1073/pnas.091584598.

Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium

H Uyttendaele  1 , J Ho, J Rossant, J Kitajewski
Affiliations

Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium

H Uyttendaele et al. Proc Natl Acad Sci U S A. .

Abstract

Notch proteins function as receptors for membrane-bound ligands (Jagged and Delta-like) to regulate cell-fate determination. We have investigated the role of Notch signaling in embryonic endothelium of the mouse by expressing an activated form of the Notch4 protein in vasculature under the regulation of the Flk1 (VEGFR) locus. Expression of activated Notch4 results in a growth and developmental delay and embryonic lethality at about 10 days postcoitum. The extent of the developing vasculature in mutant embryos was restricted, fewer small vessels were seen, and vascular networks were disorganized. The brain periphery of mutant embryos contained large dilated vessels with evidence of compromised vessel-wall integrity and large areas of necrosis; yolk-sac vasculature was abnormal. Expression of an activated form of Notch4 in embryonic vasculature leads to abnormal vessel structure and patterning, implicating the Notch pathway in phases of vascular development associated with vessel patterning and remodeling.

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Figures

Figure 1
Figure 1
Presentation of ES-cell generation and analysis of ES-cell clones. (A) Schematic representation of ES-targeting strategy. (B) Targeting construct. The targeting vector is designed to replace the exon of Flk1 that encodes the initiating methionine with a cassette containing int-3HA and the neomycin (neo) gene cassette. (C) Genotype analysis of ES clones by Southern blotting. The targeted locus places int-3HA under the gene-regulatory region of Flk1 and introduces a NotI (N) site at the junction of the int-3HA and neo genes. Genomic DNA was digested with NotI, and hybridization was done with an Flk1-specific probe. (D) Expression analysis for int-3HA transcripts in embryoid bodies (EBs) by RT-PCR. Analysis was conducted with control RNA from kidney, from parental line 1D5, and from a homologous recombinant line, 1A12.
Figure 2
Figure 2
Embryo whole-mounts composite. Control Tie1-lacZ (A and C) and Flk1/int-3 (B and D) embryos at 9.5 dpc were processed for whole-mount β-galactosidase staining to visualize the endothelial cells. At 9.5 dpc, vasculature in mutant embryos appears to be more restricted and disorganized. Yolk-sac vasculature is shown for Tie1-lacZ (E) and Flk1/int-3 (F). Analysis of vessels in the umbilical cord (arrow) is shown for Tie1-lacZ (G) and Flk1/int-3 (H).
Figure 3
Figure 3
Embryo sections composite. β-Galactosidase staining of histological sections of Tie1-lacZ (A, C, and E) and Flk1/int-3 embryos (B, D, and F) at 9 dpc. A and B show cross sections through the brain. C and D show cross sections through the spinal cord, cardinal vessels, and heart. E and F show cross sections through yolk sacs. In Flk1/int-3 embryos, enlarged and collapsed vessels are observed, large areas of necrosis are seen, and fewer β-galactosidase-staining cells are visible (B and D). (Bars = 1 mm.)
Figure 4
Figure 4
Staining of embryo whole mounts and sections. Analysis of genes expressed in vasculature in Tie1-lacZ embryos (A, C, E, and G) and Flk1/int-3 embryos (B, D, F, and H). Whole-mount embryos were stained with probes for Tie2 (A and B), with antibodies for α-SMA (C and D), and with probes for Jagged1 (G and H). Histological sections were stained with antibodies for α-SMA (E and F).
Figure 5
Figure 5
EBs composite. (A) EBs were generated from either Tie1-lacZ or Flk1/int-3 (40× or 100× magnification, respectively). The endothelial cells in EBs were visualized by β-galactosidase staining. (B) RT-PCR analyses for Tie1, Notch3, and Notch4 were carried out by using RNA derived from kidney (k), control EBs (1D5), and Flk1/int-3 EBs (1A12). (C) Northern blot analysis was used to detect Flk1, Jagged1, and β-actin transcripts in control EBs (1D5) or Flk1/int-3 EBs (1A12).
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Comment in

  • Notch signaling during vascular development.
    Gridley T. Gridley T. Proc Natl Acad Sci U S A. 2001 May 8;98(10):5377-8. doi: 10.1073/pnas.101138098. Proc Natl Acad Sci U S A. 2001. PMID: 11344278 Free PMC article. No abstract available.

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