Factorial microarray analysis of zebrafish retinal development

Abstract

In a zebrafish recessive mutant young (yng), retinal cells are specified to distinct cell classes, but they fail to morphologically differentiate. A null mutation in a brahma-related gene 1 (brg1) is responsible for this phenotype. To identify retina-specific Brg1-regulated genes that control cellular differentiation, we conducted a factorial microarray analysis. Gene expression profiles were compared from wild-type and yng retinas and stage-matched whole embryos at 36 and 52 hours postfertilization (hpf). From our analysis, three categories of genes were identified: (i) Brg1-regulated retinal differentiation genes (731 probesets), (ii) retina-specific genes independent of Brg1 regulation (3,038 probesets), and (iii) Brg1-regulated genes outside the retina (107 probesets). Biological significance was confirmed by further analysis of components of the Cdk5 signaling pathway and Irx transcription factor family, representing genes identified in category 1. This study highlights the utility of factorial microarray analysis to efficiently identify relevant regulatory pathways influenced by both specific gene products and normal developmental events.

Fig. 1.

Factorial microarray experimental design and specific contrasts for inferring Brg1-regulated retinal terminal differentiation genes. See Factorial Analysis for details.

Fig. 2.

Heat maps of four kinds of pathways or gene families that are controlled by the Brg1-regulated retinal differentiation program. The log2 YR36/WR36 and YR52/WR52 expression ratios of four kinds of pathways, or gene families, are shown in a heat map format. The timing of significance is shown by the colored circles on the left hand side of the heat maps (see Factorial Analysis (Step 2) for details on significance inference). Orange circle: probesets inferred as significant at 36 hpf only; blue circle: probesets inferred as significant at 52 hpf only; orange and blue circle: probesets inferred as significant at both 36 and 52 hpf; black circle: probesets that are not significantly changed; open circle: no probeset on the microarray, expression values obtained by RT-PCR. (A) Processes directly related to terminal differentiation: neurite outgrowth and cytoskeletol regulation (Left), adhesion molecules (Top Right), synaptogenesis (Middle Right), and phototransduction (Bottom Right). (B) Signal transduction pathways: Delta-Notch and Fgf. (C) Transcription factor families: irx, tfap2, and id2. (D) Specific cell cycle regulators. Several genes (plasticin, evlb, syt11, gnat1, her4, notch1a, notch2, and fgfr2) have multiple probesets on the arrays, and the respective results are shown in the heat maps.

Fig. 3.

Characterization of Cdk5, p35, and p39's functions in zebrafish retinal development. Retinal histology of antisense morpholino knockdown experiments at 52 hpf. Top (left to right): Uninjected control, yng mutant, cdk5-SMO (0.56 ng). Bottom (left to right): p35-MO (8 ng), p39-MO (6 ng), p35-MO and p39-MO (5.1 ng each). The black arrows indicate the location of nuclear layers GCL, INL, and ONL, while the red arrow indicates the optic nerve (ON).

Fig. 4.

Irx7-MO knockdown experiment. Immunohistochemistry of uninjected control (Left) and irx7-MO (10 ng)-injected eyes (Right) at 52 hpf. The phalloidin F-actin stain (red) preferentially bound to the INL and ONL (white arrows) in the WT retina, but was severely reduced or absent, respectively, in irx7 morphants. The differentiation of RGC, as shown by the zn5 antibody (green), was compromised but not completely eliminated in the irx7 morphants. The DAPI nuclei staining of the control and irx7 morphants also shows the same adverse effects on the plexiform layers (red arrows) and GCL (green arrows).