Differential expression of anti-angiogenic factors and guidance genes in the developing macula

[摘要] Purpose: The primate retina contains aspecialized, cone-rich macula, which mediates high acuity and colorvision. The spatial resolution provided by the neural retina at themacula is optimized by stereotyped retinal blood vessel and ganglioncell axon patterning, which radiate away from the macula and reduceshadowing of macular photoreceptors. However, the genes that mediatethese specializations, and the reasons for the vulnerability of themacula to degenerative disease, remain obscure. The aim of this studywas to identify novel genes that may influence retinal vascularpatterning and definition of the foveal avascular area. Methods: We used RNA from human fetalretinas at 19–20 weeks of gestation (WG; n=4) to measure differentialgene expression in the macula, a region nasal to disc (nasal) and inthe surrounding retina (surround) by hybridization to 12 GeneChip®microarrays (HG-U133 Plus 2.0). The raw data was subjected to qualitycontrol assessment and preprocessing, using GC-RMA. We then used ANOVAanalysis (Partek®Genomic Suite™ 6.3) and clustering (DAVID website) to identifythe most highly represented genes clustered according to “biologicalprocess.” The neural retina is fully differentiated at the macula at19–20 WG, while neuronal progenitor cells are present throughout therest of the retina. We therefore excluded genes associated with thecell cycle, and markers of differentiated neurons, from furtheranalyses. Significantly regulated genes (p<0.01) were thenidentified in a second round of clustering according tomolecular/reaction (KEGG) pathway. Genes of interest were verified byquantitative PCR (QRT–PCR), and 2 genes were localized by in situhybridization. Results: We generated two lists ofdifferentially regulated genes: “macula versus surround” and “maculaversus nasal.” KEGG pathway clustering of the filtered gene listsidentified 25 axon guidance-related genes that are differentiallyregulated in the macula. Furthermore, we found significant upregulationof three anti-angiogenic factors in the macula: pigment epitheliumderived factor (PEDF), natriuretic peptide precurusor B (NPPB),and collagen type IVα2. Differential expression of several members ofthe ephrin and semaphorin axon guidance gene families, PEDF,and NPPB was verified by QRT–PCR. Localization of PEDFand Eph-A6 mRNAs in sections of macaque retina shows expressionof both genes concentrates in the ganglion cell layer (GCL) at thedeveloping fovea, consistent with an involvement in definition of thefoveal avascular area. Conclusions: Because the axons ofmacular ganglion cells exit the retina from around 8 WG, we suggestthat the axon guidance genes highly expressed at the macula at 19–20 WGare also involved in vascular patterning, along with PEDF and NPPB.Localization of both PEDF and Eph-A6 mRNAs to the GCLof the developing fovea supports this idea. It is possible thatspecialization of the macular vessels, including definition of thefoveal avascular area, is mediated by processes that piggyback on axonguidance mechanisms in effect earlier in development. These findingsmay be useful to understand the vulnerability of the macula todegeneration and to develop new therapeutic strategies to inhibitneovascularization.

[学科分类] 生物化学/生物物理