Gene Expression Profiling During Optic Fissure Closure

Abstract

Purpose:: Regulation of optic fissure closure during the 5^th week of human gestation is poorly understood. Failure of the fissure to close at this time can lead to uveal coloboma with varying loss of visual function. Our current study seeks to identify specific genes or pathways directing approximation and fusion of the optic fissure.

Methods:: Wild-type C57BL/6J mouse embryos were harvested at E10.5, 11.5, and 12.5. Presumptive RPE and neural retina was isolated specifically from the margins of the optic fissure using laser capture microdissection (LCM) with the proximal lens as an anatomic reference point. RNA was isolated from pooled tissue at each embryonic timepoint, amplified linearly, labeled according to manufacturer's protocol (Arcturus) and hybridized to Affymetrix MOE 430 2.0 chips. Expression data was extracted using Affymetrix GCOS 1.4. Statistical analysis was performed using JMP 6.0 with scripts from MSCL Analyst's Toolbox (CIT, NIH). Results obtained were verified with real-time PCR and whole-mount in situ hybridization.

Results:: Gene expression in the margins of the optic fissure is highly regulated in wild type mice over three embryonic days which represent the fissure in unfused (E10.5), closing (E11.5) and fused states (E12.5). A total of 297 genes were found to be differentially expressed as measured by at least a two-fold change over the three days (FDR<.15). Biological significance of this subset was evaluated using Ingenuity Pathways Analysis from which several possible mechanisms involved in fissure closure emerge: non-canonical Wnt signaling/planar cell polarity, regulation of cell adhesion (e.g., cadherins), integrity of the basement membrane and differential cell proliferation and apoptosis. In addition, many previously uncharacterized transcripts display differential expression during optic fissure closure. Real-time PCR and whole-mount in situ hybridization confirm the spatiotemporal expression of several transcripts meeting both statistical and biological indices.

Conclusions:: The highly regulated expression of these genes leads us to conclude that they may play a critical role in normal closure of the optic fissure. Disruption of these genes in animal models may lead to failure of the optic fissure to close, yielding a coloboma phenotype. Characterizing these mechanisms will illuminate our understanding of human uveal coloboma.

Keywords: development • gene microarray • genetics