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. 2004 Nov 9;101(45):15949-54.
doi: 10.1073/pnas.0407290101. Epub 2004 Nov 1.

Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development

Nicholas W Gale  1 , Melissa G Dominguez, Irene Noguera, Li Pan, Virginia Hughes, David M Valenzuela, Andrew J Murphy, Niels C Adams, Hsin Chieh Lin, Jocelyn Holash, Gavin Thurston, George D Yancopoulos
Affiliations

Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development

Nicholas W Gale et al. Proc Natl Acad Sci U S A. .

Abstract

Vascular development depends on the highly coordinated actions of a variety of angiogenic regulators, most of which apparently act downstream of vascular endothelial growth factor (VEGF). One potential such regulator is delta-like 4 ligand (Dll4), a recently identified partner for the Notch receptors. We generated mice in which the Dll4 gene was replaced with a reporter gene, and found that Dll4 expression is initially restricted to large arteries in the embryo, whereas in adult mice and tumor models, Dll4 is specifically expressed in smaller arteries and microvessels, with a striking break in expression just as capillaries merge into venules. Consistent with these arterial-specific expression patterns, heterozygous deletion of Dll4 resulted in prominent albeit variable defects in arterial development (reminiscent of those in Notch knockouts), including abnormal stenosis and atresia of the aorta, defective arterial branching from the aorta, and even arterial regression, with occasional extension of the defects to the venous circulation; also noted was gross enlargement of the pericardial sac and failure to remodel the yolk sac vasculature. These striking phenotypes resulting from heterozygous deletion of Dll4 indicate that vascular development may be as sensitive to subtle changes in Dll4 dosage as it is to subtle changes in VEGF dosage, because VEGF accounts for the only other example of haploid insufficiency, resulting in obvious vascular abnormalities. In summary, Dll4 appears to be a major trigger of Notch receptor activities previously implicated in arterial and vascular development, and it may represent a new opportunity for pro- and anti-angiogenic therapies.

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Figures

Fig. 1.
Fig. 1.
Generation and preliminary analysis of Dll4 gene-targeted mice. (A) Schematic representation of Dll4 gene targeting strategy achieved by using VelociGene (19) technology. (Top) A 8.1-kb Dll4 genomic region spanning exons encoding entire coding region (red boxes) along with flanking homology boxes (yellow boxes) used for bacterial homologous recombination approach to incorporate LacZ/Neo reporter cassette shown (Middle), yielding modified Dll4 genomic region (Bottom), in which the Dll4 coding region has been precisely replaced by the LacZ/Neo reporter. Location of short LONA probes also depicted (red circles). (B) Representative screening data from LONA screen (19) for correctly targeted ES cells; clones scoring positive in 16 of 16 independent LONA assays are indicated (yellow arrowheads). (C) Representative genotyping data from F1 animals by using the LONA assay (19) showing clear quantitative distinction between WT and Dll4Lz/+ Het mice. (D) Quantitative RT-PCR results of levels of Dll4, Hes1, and Hey1 transcripts in E9.5 yolk sac RNA from WT and Het embryos. (E) Percentage of embryos and live births that are WT versus Het for the modified Dll4Lz allele, with the heterozygous offspring further subdivided between those that appear viable (blue bars) versus nonviable (red bars) as discussed in the text; all offspring are generated from breedings between male chimeras that are complete transmitters of ES-derived sperm, to either C57BL/6 females (Left and Center) or ICR females (Right). Note that whereas ≈50% of embryos (Left, yellow bar) are heterozygous for the Dll4Lz allele, as would be expected, the vast majority of these appear nonviable, defining haploid insufficiency embryonic lethality.
Fig. 2.
Fig. 2.
Characterization of Dll4 reporter expression patterns and vascular defects in Dll4Lz/+ embryos. (A and B) Freshly dissected E10.5 embryos representative of normal WT and nonviable Dll4Lz/+ heterozygous embryos (nvHet), with black arrowheads in B, indicating dramatic pericardial edema; H, heart. (C and D) Whole-mount β-gal staining reflecting Dll4 reporter gene expression at E10.5 in a viable Dll4Lz/+ heterozygous embryo (vHet) compared with an nvHet. In C, prominent reporter expression is notable in all of the major arterial structures [i.e., internal carotid artery (ICA), dorsal aorta (DA), aortic arch (AA), umbilical artery (UA), and intersomitic branches, unlabeled] as well as in the neural tube (NT), and the level of cross section depicted in E is also indicated, whereas in D arterial branches are less prominent or missing, and red arrowheads indicate the location of the dorsal aorta in nonconstricted region (F arrowhead) as well as constricted/atretic region (G arrowhead). (E) β-gal-stained cross section at the level depicted in C from a vHet embryo, showing that the Dll4 reporter is expressed exclusively in arterial endothelium (red arrowheads, indicating paired dorsal aortas) and neural tube but not in venous endothelium. (F) PECAM-1-stained cross section at the level depicted by the F arrowhead, in D, revealing relatively normal paired dorsal aortas (red arrowheads) and cardinal veins (blue arrowheads); perineural vessels around the neural tube are also seen. (G) PECAM-1-stained cross section at the level depicted by the G arrowhead, in D, revealing obvious defects in the dorsal aortas (red arrowheads) on the left, such that whereas arterial endothelial cells are in approximately correct position, they are not properly organized into a vessel structure, and no lumen can be seen; cardinal veins (blue arrowheads) appear relatively normal. (H and I) Whole-mount PECAM-1-stained WT versus nvHet embryos, comparing vasculature; in H, red arrowheads depict internal carotid artery, blue arrows in head region depict cephalic venous plexus, and blue arrowheads along back depict cardinal vein, all of which are missing or abnormal in nvHet in I, except for the cephalic venous plexus.
Fig. 3.
Fig. 3.
Abnormal yolk sac and placental vasculature in Dll4Lz/+ nvHet embryos compared with WT embryos. (A and B) Whole-mount views of freshly dissected embryos with intact yolk sacs showing lack of conducting arteries (red arrowheads in A) and orange peel-like appearance of yolk sacs in nvHet embryos. (C and D) Whole-mount flat views of PECAM-1-stained yolk sacs showing lack of conducting arteries (red arrowheads in C) as well as an almost sheet-like and fused primitive vascular network in nvHet embryos. (E and F) Hemotoxylin/eosin-stained paraffin cross sections of yolk sacs showing gross enlargement of the space between endoderm and mesoderm layers in nvHet embryos (black arrowheads), in part due to an almost complete lack of attachment sites (black arrows) between these layers, resulting in huge lacunae-like vessels in nvHet embryos as opposed to the normally sized and organized vessel cross sections seen in WT embryos. (G and H) High-power views of the periphery of the whole-mounted PECAM-1-stained yolk sacs from C and D. (I and J) Degenerative arterial changes in placentas derived from nonviable Dll4Lz/+ embryos (as compared with those derived from vHet Dll4Lz/+ embryos. (I and J Insets) Low-power whole-mount views of β-gal-stained placentas showing dramatic Dll4-reporter expression in umbilical arteries and their branches extending out to placental periphery, with corresponding high-power views highlighting apparent degenerative arterial changes in center of nvHet placentas that leave only atretic vascular remnants (red arrowheads).
Fig. 4.
Fig. 4.
Dll4 reporter gene expression reveals prominent expression in smaller arteries and capillaries but not venous vessels in normal adult tissues, with apparent induction during tumor angiogenesis. (A) Whole-mount views of Dll4 reporter expression in aorta and its intercostal artery branches (red arrowheads) of a viable Dll4Lz/+ adult animal. (B) Side-by-side views (from retinal whole mounts) of a small artery (red arrowheads) next to a small vein (blue arrowheads). (C) Prominent Dll4 reporter gene expression in most tissues (urinary bladder wall is shown) seems to extend from small arteries into their downstream microvascular networks. (D) Dll4 expression in an individual vascular circuit (visualized within wholemounts of the adult retina): small arteries, red arrowheads; capillaries, black arrowheads; and post capillary venules, blue arrowheads. (E) Lower-power views of s.c. tumor sections stained with anti-PECAM-1, demonstrating robust staining in all large and small vessels, both in tumor and overlying skin (arrowheads). (F) In contrast, Dll4 reporter analysis reveals stronger expression in tumor vessels as compared with the vessels in the adjacent skin. (G and H) High-power views of boxed areas in E and F highlighting small tumor vessels (black arrowheads) versus large tumor veins (blue arrowheads).
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