Abstract
Purpose: :
To investigate the cellular expression patterns by whole-mount in situ hybridization of Brg1-regulated retinal differentiation genes recently identified in our factorial microarray analysis of wild type (WT) and young (yng)/brg1 embryos.
Methods: :
Sixteen genes (olfm2, robo2, foxn4, elovl4, irx4a, rho, calb2l, fzd8b, gnat1, B-H2, cdh11, bhlbh5, lmo4l, tfap2a, pbx1a and id2a) whose changes in expression levels were found to be highly significant in our microarray analysis were randomly selected from 228 genes. All genes were identified by the analysis as Brg1-regulated except foxn4 and bhlbh5, which were specifically expressed in the retina but not regulated by Brg1. Primer pairs that flanked about 500 bps of the candidate genes and overlapped maximally with the corresponding target regions in the Affymetrix probesets were designed by Primer3 (http://frodo.wi.mit.edu/). These primers were used to amplify DNA fragments from a 2-day old zebrafish cDNA library and cloned into pGEM-T easy vector (Promega) for probe synthesis. Semi-automated whole-mount in situ hybridisations were performed on WT and yng embryos collected at 36 and 52 hours postfertilization (hpf).
Results: :
For the 14 Brg1-regulated genes, in situ hybridization showed corresponding qualitative expression changes in the yng retinas as compared to WT retinas in 12/14 cases at 36hpf and 14/14 cases at 52hpf. For the 2 non-Brg1-regulated genes expressed in the retinas, in situ hybridization showed corresponding qualitative expression changes in WT retinas as compared to the whole WT embryos in 2/2 cases at 36hpf and 1/2 cases at 52hpf. The cellular expression patterns of these genes at 52hpf were: ganglion cell layer (3 genes), amacrine cell layer (5 genes), ganglion & amacrine cell layer (2 genes), ventral patch photoreceptors (3 genes), marginal zone (1 gene), and not expressed (2 genes).
Conclusions: :
This study has qualitatively validated the change in expression of significant genes identified by our factorial microarray analysis in WT and yng embryos, and has localized spatial expression patterns to specific retinal layers. These results will assist further characterization of these candidate genes and their roles in retinal differentiation.
Keywords: gene microarray • in situ hybridization • retinal development