Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Zebrafish retinal development

ABSTRACT: Retinal cells are specified in a zebrafish recessive mutant called young (yng) but they fail to terminally differentiate; i.e. extend neurites and make synaptic contacts. A point mutation in a brahma-related gene 1 (brg1) is responsible for this phenotype. In this microarray study, a three-factor factorial design was utilized to investigate the effects of 1) mutation, 2) change in time (36 vs. 52hpf), and 3) change in tissue (retina vs. whole embryos), and their interactions on gene expression. Significant probesets were inferred by using both specific contrasts of the fitted Analysis of Variance (ANOVA) models and a corresponding 2-fold expression cutoff. The probesets were grouped into three broad categories: 1) Brg1-regulated retinal differentiation genes (731 probsets), 2) Retinal specific genes but independent of Brg1 regulation (3038 probesets) and 3) Genes regulated by Brg1 but outside the retina (107 probesets). Four gene groups/pathways including neurite outgrowth regulators, Delta-Notch signalling molecules, Irx family members and specific cell cycle regulators were identified in the first group, and their relevance for retinal differentiation functionally validated. This study demonstrates that an approach such as ours can identify relevant genes and pathways involved in retinal development as well as the development of other tissues at the same time. Experiment Overall Design: The gene expression levels of three independent replicates of retina consisting of ten samples each at 36 and 52hpf retinas from WT (WR36 & WR52) and yng (YR36 & YR52) larvae, and stage-matched whole embryos: WT at 36hpf (WA36), WT at 52hpf (WA52), yng at 36hpf (YA36), and yng at 52hpf (YA52) were measured by Affymetrix Zebrafish Whole Genome Arrays. We analyzed the effects of these factors on gene expression levels: 1) mutation (M), 2) tissue (R), and 3) time (T). Each of these factors have two levels: mutant and WT (M); retinas and whole body (R); and 36 and 52 hpf (T). As a result, there are totally eight conditions for all the comparisons and the design is called a 2x2x2 factorial design. The aim of a factorial analysis is to delineate the effects of each factor and their combinations (interaction) on gene expression. The overall analysis strategy is to first fit a full Analysis of Variance (ANOVA) model with all possible main effects (T, M, R) and their interactions (T*M, M*R, R*M, T*M*R) to the gene expression data for each probeset. The estimates of the model are obtained through a maximum likelihood estimation method. Then a backward elimination strategy is used to remove insignificant main effects and interactions to get the most parsimonious models. Finally, a group of contrasts and their corresponding fold changes are used to infer whether that probeset is significantly associated with a particular biological process.

ORGANISM(S): Danio rerio

SUBMITTER: Yuk Fai Leung

PROVIDER: E-GEOD-8874 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: The purpose of this study was to develop a framework for analyzing RPE expression profiles from zebrafish eye mutants. Methods: The fish model we used was smarca4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4), a retinal dystrophic mutant that the retinal phenotype and expression profiles were previously characterized. Histological and Affymetrix GeneChip analyses were conducted to define the RPE defects and underlying differential expression respectively. Results: Histological analysis indicates that smarca4 RPE was formed but its differentiation was abnormal. In particular, ultra-structural analysis of smarca4 RPE by transmission electron microscopy showed a number of defects in melanogenesis, suggesting that the cytoskeletal dynamics was impaired. To compare the expression profile of normal wild-type (WT) and smarca4 RPE, their retinas and RPE-attached retinas were microdissected and the gene expression values of these tissues measured by Affymetrix GeneChip analysis. The RPE expression values were then estimated from these samples using an approach previously established by us. A factorial analysis was conducted using the expression values of RPE, retinal as well as the whole-embryo samples. Specific rules (contrasts) were built using the coefficients of the resulting fitted model to select for three groups of genes: 1) Smarca4-regulated RPE genes, 2) Smarca4-regulated retinal genes, and 3) Smarca4-regulated RPE genes that are not differentially expressed in the retina. The latter group consists of 39 genes that are highly related to cytoskeletal dynamics, melanogenesis, paracrine and intracellular signal transduction. Conclusions: Our analytical framework can potentially identify genes in zebrafish mutants that both retina and RPE are affected by the underlying mutation. The gene expression levels of three independent replicates of WT whole embryos (WA52), WT retinas (WR52), WT RPE-attached retinas (WRR52), smarca4/yng retinas (YR52), smarca4 RPE-attached retinas (YRR52) and smarca4 whole embryos (YA52) were measured by Affymetrix Zebrafish Whole Genome Arrays. All samples were collected at 52 hpf. The probe-level data of these sample groups were background adjusted, normalized and summarized by a robust multiarray average (RMA) algorithm. RPE expression values were estimated from the comparison between the retinal and RPE-attached retinal samples of the same genotype based on a method we previously developed (Leung et al., Invest Ophthalmol Vis Sci. 2007; 48:881). Then, a 3x2 factorial design was used to analyze the expression values of three kinds of tissue (T): whole embryo, retina and RPE in two kinds of mutation (M) background: WT and smarca4. Using the coefficients from the fitted models, contrasts were built to identify candidate genes that fulfilled specific criteria. The false discovery rate (FDR) q-value cutoff for a contrast was 0.001. Several contrasts were ultimately combined to allow for selection of genes that were regulated by Smarca4 in the retina and RPE. These include 1) Smarca4-regulated RPE genes, 2) Smarca4-regulated retinal genes, and 3) Smarca4-regulated RPE genes that are not differentially expressed in the retina.

Project description:The early retinal progenitor-expressed gene Sox11 regulates the timing of the differentiation of retinal cells. Sry-related HMG box (Sox) proteins play diverse and critical roles in a variety of morphogenetic processes during embryonic development. Sox11 and Sox4 are members of the SoxC subtype, and we found that Sox11 was strongly expressed in early retinal progenitor cells, and that when expression of Sox11 subsided around birth, Sox4 expression began. To analyze the role of Sox11 and Sox4 in retinal development, we perturbed their expression pattern by expressing them ectopically in retinal explant culture. Overexpression of Sox11 or Sox4 in retinal progenitors resulted in similar phenotypes, that is, increased cone cells and decreased Muller glia. Sox11-knockout retinas showed delayed onset and progress of differentiation of early-born retinal cells during the embryonic period. After birth, retinal differentiation took place relatively normally, probably because of the redundant activity of Sox4, which starts to differentiate around birth. Neither overexpression nor loss-of-function analysis gave any evidence that Sox11 and Sox4 directly regulate transcription of genes critical to early-born retinal cells. However, histone H3 acetylation status of the early neurogenic genes was lowered in knockout retinas, suggesting that Sox11 regulates the timing of differentiation in early-born retinas by creating an epigenetic state that helps to establish the competency to differentiate. We also found that the unique expression patterns of Sox11 and Sox4 may be achieved by the Notch signaling pathway and by epigenetic regulation. Taking our findings together, we propose that the precise regulation of Sox11 and Sox4 expression during retinogenesis by multiple mechanisms leads to the fine adjustment of retinal differentiation. To delineate the molecular mechanisms underlying the retinal action of Sox11, we performed microarray analysis of E18 retinas from wild-type and Sox11 knockout mice. Total RNA was obtained from each one retina of Sox11 knockout and wild-type littermate embryos at E18.

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Transcription profiling of Zebrafish retinal development

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