Anti-Flt4 monoclonal antibody, clone 6C7

The development of arteries, veins and lymphatics from pre-existing vessels are intimately linked processes controlled by a number of well-studied reiteratively acting signalling pathways. To delineate the mechanisms governing vessel formation in vivo, we performed a forward genetic screen in zebrafish and isolated the mutant expando. Molecular characterisation revealed a loss-of-function mutation in the highly conserved kinase insert region of flt4. Consistent with previous reports, flt4 mutants were deficient in lymphatic vascular development. Recent studies have demonstrated a role for Flt4 in blood vessels and showed that Dll4 limits angiogenic potential by limiting Flt4 function in developing blood vessels. We found that arterial angiogenesis proceeded normally, yet the dll4 loss-of-function arterial hyperbranching phenotype was rescued, in flt4 signalling mutants. Furthermore, we found that the Flt4 ligand Vegfc drives arterial hyperbranching in the absence of dll4. Upon knockdown of dll4, intersegmental arteries were sensitised to increased vegfc levels and the overexpression of dll4 inhibited Vegfc/Flt4-dependent angiogenesis events. Taken together, these data demonstrate that dll4 functions to suppress the ability of developing intersegmental arteries to respond to Vegfc-driven Flt4 signalling in zebrafish. We propose that this mechanism contributes to the differential response of developing arteries and veins to a constant source of Vegfc present in the embryo during angiogenesis.

Prenatal testosterone (T), but not dihydrotestosterone (DHT), excess disrupts ovarian cyclicity and increases follicular recruitment and persistence. We hypothesized that the disruption in the vascular endothelial growth factor (VEGF) system contributes to the enhancement of follicular recruitment and persistence in prenatal T-treated sheep. The impact of T/DHT treatments from Days 30 to 90 of gestation on VEGFA, VEGFB, and their receptor (VEGFR-1 [FLT1], VEGFR-2 [KDR], and VEGFR-3 [FLT4]) protein expression was examined by immunohistochemistry on Fetal Days 90 and 140, 22 wk, 10 mo (postpubertal), and 21 mo (adult) of age. Arterial morphometry was performed in Fetal Day 140 and postpubertal ovaries. VEGFA and VEGFB expression were found in granulosa cells at all stages of follicular development with increased expression in antral follicles. VEGFA was present in theca interna, while VEGFB was present in theca interna/externa and stromal cells. All three receptors were expressed in the granulosa, theca, and stromal cells during all stages of follicular development. VEGFR3 increased with follicular differentiation with the highest level seen in the granulosa cells of antral follicles. None of the members of the VEGF family or their receptor expression were altered by age or prenatal T/DHT treatments. At Fetal Day 140, area, wall thickness, and wall area of arteries from the ovarian hilum were larger in prenatal T-and DHT-treated females, suggestive of early androgenic programming of arterial differentiation. This may facilitate increased delivery of endocrine factors and thus indirectly contribute to the development of the multifollicular phenotype.