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I.B. Lobov, R.A. Renard, N. Papadopoulos, G. Thurston, N.W. Gale, G.D. Yancopoulos, S.J. Wiegand; Deletion of a Single Dll4 Allele Alters Retinal Vascular Development in Mice . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3877.
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Genetic deletion of Dll4, a Notch ligand prominently expressed in the arterial endothelium, causes severe abnormalities in vascular development and remodeling. Deletion of even a single allele results in almost complete embryonic lethality in the C57BL/6:129 hybrid mixed background and less than 50% of embryonic lethality in ICR background. Therefore, to evaluate the role of Dll4 in retinal vascular development, we analyzed retinas of ICR mice heterozygous for Dll4 deletion / lacZ substitution.
VelociGene technology was used to replace the entire Dll4 coding region with the ß–galactosidase reporter gene in C57BL/6:129 hybrid mouse embryonic stem cells [Gale N et al., PNAS 101:15949, 2004]. Chimeric males were bred to ICR females. Mice backcrossed for 3 generations to ICR (87.5% ICR) were used for this study. Dll4lacZ/+ mice and wild type littermates were humanely euthanized between postnatal days 5 and 10. After enucleation, retinas were dissected, flat mounted and stained for ß–gal or X–gal to reveal sites of Dll4 expression and/or with FITC–labeled Griffonia simplicifolia lectin I to visualize the vasculature.
In the developing superficial retinal vasculature, Dll4 was most prominently expressed in the endothelium of differentiated arteries and in specialized "tip cells" located at the leading edge of the growing vascular net. Dll4 was also widely expressed in capillaries throughout the superficial plexus, as well as in the developing deep retinal bed, particularly at the tips and distal stalks of sprouting vessels. Relative to wild–type controls, the retinas of Dll4lacZ/+ mouse pups exhibited several characteristic vascular abnormalities, including delayed arterial differentiation, delayed outgrowth of the superficial plexus, increased numbers of filopodia and vessel sprouts at the leading edge of the vasculature, and increased capillary density in the newly formed primitive capillary network.
Dll4 negatively regulates vascular sprouting, and promotes the timely development and differentiation of the retinal vasculature.
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