Cellular Expression of Smarca4 (Brg1)-regulated Genes in Zebrafish Retinas

Abstract

BackgroundIn a recent genomic study, Leung et al. used a factorial microarray analysis to identify Smarca4 (Brg1)-regulated genes in micro-dissected zebrafish retinas. Two hundred and fifty nine genes were grouped in three-way ANOVA models which carried the most specific retinal change. To validate the microarray results and to elucidate cellular expression patterns of the significant genes for further characterization, 32 known genes were randomly selected from this group. In situ hybridization of these genes was performed on the same types of samples (wild-type (WT) and smarca4a50/a50 (yng) mutant) at the same stages (36 and 52 hours post-fertilization (hpf)) as in the microarray study.ResultsThirty out of 32 riboprobes showed a positive in situ staining signal. Twenty seven out of these 30 genes were originally further classified as Smarca4-regulated retinal genes, while the remaining three as retinal-specific expression independent of Smarca4 regulation. It was found that 90.32% of the significant microarray comparisons that were used to identify Smarca4-regulated retinal genes had a corresponding qualitative expression change in the in situ hybridization comparisons. This is highly concordant with the theoretical true discovery rate of 95%. Hierarchical clustering was used to investigate the similarity of the cellular expression patterns of 25 out of the 27 Smarca4-regulated retinal genes that had a sufficiently high expression signal for an unambiguous identification of retinal expression domains. Three broad groups of expression pattern were identified; including 1) photoreceptor layer/outer nuclear layer specific expression at 52 hpf, 2) ganglion cell layer (GCL) and/or inner nuclear layer (INL) specific expression at both 36 & 52 hpf, and 3) GCL and/or INL specific expression at 52 hpf only. Some of these genes have recently been demonstrated to play key roles in retinal cell-type specification, differentiation and lamination. For the remaining three retinal-specific genes that are independent of Smarca4 regulation, they all had a subtle expression difference between WT and smarca4a50/a50 retinas as detected by in situ hybridization. This subtle expression difference was also detected by the original microarray analysis. However, the difference was lower than the fold change cut-off used in that study and hence these genes were not inferred as Smarca4-regulated retinal genes.ConclusionsThis study has successfully investigated the expression pattern of 32 genes identified from the original factorial microarray analysis. The results have demonstrated that the true discovery rate for identifying Smarca4-regulated retinal genes is 90.3%. Hence, the significant genes from the microarray study are good candidates for cell-type specific markers and will aid further investigation of retinal differentiation.

The Role of egr1 in Early Zebrafish Retinogenesis

“…In situ hybridization was conducted as described [13]. The riboprobes that were used in this study are as follows: early growth response 1 (egr1); cone-rod homeobox ( crx ); neurogenic differentiation ( neurod ); nuclear receptor subfamily 2 group E member 3 ( nr2e3 ); pancreas specific transcription factor 1a ( ptf1a ); opsin 1 (cone pigments) , short-wave-sensitive 1 (opn1sw1) ; opsin 1 (cone pigments) , short-wave-sensitive 2 ( opn1sw2 ); opsin 1 (cone pigments), long-wave-sensitive 1 ( opn1lw1 ) and rhodopsin ( rho ).…”

“…For example, our group characterized the zebrafish mutant of smarca4, which encodes the ATPase of SWI/SNF chromatin remodeling complex [11], by gene expression analysis. A number of genes were found to be differentially expressed in the mutant dystrophic retinas [12], [13]. One of these downstream targets, irx7 , was subsequently demonstrated to be essential for retinal differentiation [14]; therefore, further characterization of these smarca4 -regulated genes will provide new insights into the mechanistic details of retinal development.…”

p35 promotes the differentiation of amacrine cell subtype in the zebrafish retina under the regulation of egr1

“…In situ hybridization was conducted as described (Hensley et al, ). The riboprobes used in this study are as follows: p35 (this study) and egr1 (Zhang et al, ).…”

“…To identify candidate genes regulated by smarca4 in the retinas, Zhang and colleagues microdissected the WT and smarca4 retinas and determined their differential gene expression by microarrays analysis (Zhang and Leung, ; Leung et al, ). The expression pattern and function of a number of the identified Smarca4 ‐ regulated genes were then subsequently validated by in situ hybridization (Hensley et al, ) and gene perturbation experiments (Leung et al, ; Zhang et al, ; Zhang et al, ) respectively. For example, we recently determined two transcription factors (TF) ‐ irx7 and egr1 were essential for differentiation of INL cells (Zhang et al, ) and Parv+ and GABA+ ACs (Zhang et al, ), respectively.…”

Slc7a14 Is Indispensable in Zebrafish Retinas

“…Embryos for in situ hybridization (ISH) of slc7a14 were collected at 5 dpf as previously described (Hensley et al, 2011;Zhang et al, 2013). Slc7a14-specific DNA fragments 584 bp in length were amplified from the zebrafish cDNA library and cloned into the pGEM-T Easy vector (Promega, United States).…”