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 reverse transcriptase polymerase chain reaction 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.

Figure 1

Patterning of the limbal vasculature and expression of pro- and anti-angiogenic factors by the anterior eye during avian corneal development. A–C: Expression of H2B-eYFP by angioblasts and vasculature in whole mount Tg(tie1:H2B:eYFP) quail eyes at E3, E5, and E7. Remodeling of the temporal ciliary artery forms a vascular plexus (arrows) while the nasal ciliary artery regresses (asterisk). D–F: Cross sections through E3, E5, and E7 Tg(tie1:H2B:eYFP) quail eyes showing localization of angioblasts and vasculature in the periocular region. Sections were counterstained with DAPI. G: Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of tissues isolated from E3, E5, and E7 chick eyes for pro- and anti-angiogenic factors. Abbreviations: C, cornea; vr, vascular ring; tca, temporal ciliary artery; nca, nasal ciliary artery; ir, iridial ring artery; L, lens; OC, optic cup; pm, periocular mesenchyme; ep, corneal epithelium; en, corneal endothelium; st, corneal stroma. Scale bars in (A–C) = 500µm; (D–F) = 50µm.

Figure 2

Expression of VEGFA and its receptors in the anterior eye during vasculogenesis and corneal development. A–L: Section in situ hybridization was used to determine the expression of VEGFA (A–C), VEGFR1 (D–F), VEGFR2 (G–I), and sFlt1 (J–L) in the anterior eye at E3, E5, and E7. VEGFR1 and VEGFR2 are strongly expressed by angioblasts at E3 (arrows, D`, G`) and in the vascular ring at E5 (E`, H`). Brown tissue at E5 and E7 indicates natural coloration of the retinal pigmented epithelium (asterisk). Abbreviations: L, lens; OC, optic cup; pm, periocular mesenchyme; pi, presumptive iris; ep, corneal epithelium; en, corneal endothelium; st, corneal stroma; vr, vascular ring; ir, iridial ring artery. Scale bar in (A–L) = 100µm; (D`, E`, G`, H`) = 20 µm.

Figure 3

Expression of FGF1, FGF2, and their receptors in the anterior eye during vasculogenesis and corneal development. A–L: Section in situ hybridization was used to determine the expression of FGF (A–C), FGF2 (D–F), FGFR1 (G–I), and FGFR2 (J–L) in the anterior eye at E3, E5, and E7. Abbreviations: L, lens; OC, optic cup; pm, periocular mesenchyme; pi, presumptive iris; ep, corneal epithelium; en, corneal endothelium; st, corneal stroma; vr, vascular ring; ir, iridial ring artery. Scale bar in (A–L) = 100µm; (G`, H`, J`, K`) = 20 µm.

Figure 4

Expression of PDGFB and PDFGR-β in the anterior eye during vasculogenesis and corneal development. A–F: Section in situ hybridization was used to determine the expression of PDGFB (A–C), and PDGFR-β (D–F) in the anterior eye at E3, E5, and E7. Abbreviations: L, lens; OC, optic cup; pm, periocular mesenchyme; pi, presumptive iris; ep, corneal epithelium; en, corneal endothelium; st, corneal stroma; vr, vascular ring; ir, iridial ring artery. Scale bar in (A–F) = 100µm; (D`, E`) = 20 µm.

Figure 5

Expression of Sema3E, Sema3G and their respective receptors PlexinD1 and Npn2 in the anterior eye during vasculogenesis and corneal development. A–L: Section in situ hybridization was used to determine the expression of Sema3E (A–C), PlexinD1 (D–F), Sema3G (G–I), and Npn2 (J–L) in the anterior eye at E3, E5, and E7. Sema3E is expressed in the mesenchyme of the iridocorneal angle at E7 (arrowheads). PlexinD1 and Sema3G are strongly expressed by angioblasts at E3 (arrows, D`, G`) and in the vascular ring at E5 (E`, H`). PlexinD1 is also expressed in the mesenchyme of the presumptive iris at E5 and E7 (long arrows). Abbreviations: L, lens; OC, optic cup; pm, periocular mesenchyme; pi, presumptive iris; ep, corneal epithelium; en, corneal endothelium; st, corneal stroma; vr, vascular ring; ir, iridial ring artery; ca, ciliary artery. Scale bar in (A–L) = 100µm; (D`, E`, G`, H`, J`, K`) = 20 µm.

Figure 6

Expression of Netrin1, Netrin4, Neogenin, and Unc5 in the anterior eye during vasculogenesis and corneal development. A–F, H–K: Section in situ hybridization was used to determine the expression of Unc5B (A–C), Neogenin (D–F), Netrin1 (H–I), and Netrin4 (J–K) in the anterior eye at E3, E5, and E7. Unc5B is strongly expressed by angioblasts at E3 (arrows). Neogenin is expressed in the iridocorneal angle (long arrows). G: Schematic representation of the expression patterns of Netrin1 and Netrin4 in the eye. Netrin1 is expressed at the optic cup along the choroid fissure (asterisk). Cross-section in (I) is perpendicular to the choroid fissure, as depicted in (G, dotted line). J`, K`: Netrin4 is expressed in the optic stalk (arrowheads), as depicted in G. Abbreviations: L, lens; OC, optic cup; pm, periocular mesenchyme; pi, presumptive iris; ep, corneal epithelium; en, corneal endothelium; st, corneal stroma; vr, vascular ring; ir, iridial ring artery. Scale bar in (A–F, H, J, J`, K, K`) = 100 µm; (A`, B`, D`, E`) = 20 µm; (I) = 500 µm.

Figure 7

Schematic diagram summarizing gene expression and the putative role of pro- and anti-angiogenic factors during vasculogenesis of the anterior eye. Pro- and anti-angiogenic factors are expressed in the lens, optic cup, and periocular mesenchyme (blue and red, respectively). These expression patterns suggest that corresponding proteins are secreted into the presumptive cornea and periocular region (blue and red arrows, respectively). Angioblasts, blood vessels (green), and periocular neural crest cells (gray) express receptors for the pro- and anti-angiogenic factors. Absence of angioblast migration and vasculogenesis in the developing cornea suggest a strong response to anti-angiogenic factors probably secreted by the lens and optic cup. In contrast, there is a strong response to angiogenic factors in the periocular region, which favor angioblast migration and proliferation. Brown coloration in the optic cup represents the retinal pigment epithelium. Abbreviations: pc, presumptive cornea; PM, periocular mesenchyme; OC, optic cup; A/BV, angioblasts and blood vessels; NC, neural crest cells.