Expression of pro‐ and anti‐angiogenic factors during the formation of the periocular vasculature and development of the avian cornea

Abstract

Background During embryonic development, endothelial precursor cells (angioblasts) migrate relatively long distances to form the primary vascular plexus. The migratory behavior of angioblasts and localization of the primitive blood vessels is tightly regulated by pro-angiogenic and anti-angiogenic factors encountered in the embryonic environment. Despite the importance of corneal avascularity to proper vision, it is not known when avascularity is established in the developing cornea and how pro- and anti-angiogenic factors regulate this process. Results and Discussion Using Tg(tie1:H2B:eYFP) transgenic quail embryos to visualize fluorescently labeled angioblasts, we show that the presumptive cornea remains avascular despite the invasion of cells from the periocular region where migratory angioblasts reside and form the primary vasculature. Semiquantitative RT-PCR analysis and spatiotemporal examination of gene expression revealed that pro- and anti-angiogenic factors were expressed in patterns indicating their potential roles in angioblast guidance. Conclusions Our findings show for the first time that chick corneal avascularity is established and maintained during development as the periocular vasculature forms. We also identify potential candidate pro- and anti-angiogenic factors that may play crucial roles during vascular patterning in the anterior eye.

PlexinD1 is required for proper patterning of the periocular vascular network and for the establishment of corneal avascularity during avian ocular development

“…Fixed eye balls were dehydrated through ethanol series, embedded in paraffin, and sectioned at 12 μm. Riboprobes for PlexinD1 , Sema3C , and Sema3E were generated as previously described (Kwiatkowski et al, 2013). RNA in situ hybridization was performed in sectioned tissue as described (Etchevers et al, 2001).…”

“…This process occurs when neural crest cells migrate between the lens and overlying ectoderm to form the corneal endothelium and stroma (Creuzet et al, 2005; Hay, 1980; Lwigale et al, 2005), while migratory angioblasts undergo vasculogenesis in the periocular region. The angioblasts and nascent blood vessels do not enter the cornea, but instead form a dense vascular network in the periocular region (Hiruma, 1996; Kwiatkowski et al, 2013), which distributes oxygen and nutrients to the tissues in the anterior eye including the cornea.…”

“…Besides Sema3A, other antiangiogenic factors are expressed in the anterior eye including Sema3E , Sema3F (Chilton and Guthrie, 2003), sFlt1 , Netrin1 , Netrin4 (Kwiatkowski et al, 2013), and CXCL14 (Ojeda et al, 2013), however their putative function during the formation of the periocular vascular network and in establishing corneal avascularity alongside Sema3A have not been demonstrated. Pertinent to this study is the vivid expression of Sema3E in the optic cup and iridocorneal angle, and the concomitant expression of PlexinD1 by angioblasts and the forming periocular vascular network (Kwiatkowski et al, 2013). Interestingly, PlexinD1 expression coincides with Nrp1 during the formation of the periocular vasculature, but it is not clear whether PlexinD1 functions independently or in conjunction with Nrp1 to regulate vascular patterning during ocular development.…”

Corneal Development

“…3A and B). 92 Proangiogenic factors including vascular endothelial growth factor (Vegf ), fibroblast growth factor (Fgf ), and platelet-derived growth factor (Pdgf ) are expressed in the anterior eye, and the periocular angioblasts express their respective receptors (VegfR1/2, Nrp1/2, FgfR1/2, and PdgfB/RB). This suggests potential roles of the proangiogenic factors in recruiting angioblasts into the anterior eye and initiating the vasculogenesis and angiogenesis processes required for the formation of the limbal vascular plexus.…”

“…However, antiangiogenic factors including the Semaphorins (Sema3A, Sema3E, Sema3G) and Netrins (Netrin1, Netrin4, Neogenin) are also expressed in the anterior eye, whereas the angioblasts express their respective receptors (Nrp1, PlexinD1, Nrp2, and Unc5B). 92 With the exception of Sema3A, the role of antiangiogenic factors during ocular development is yet to be determined. Our recent study indicates that the presence of Sema3A in the lens plays a role in preventing Vegf-induced angioblast invasion and subsequent vascularization of the developing cornea.…”