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RNA-seq analysis of the developing chicken retina

ABSTRACT: The chicken embryo (Gallus gallus) is a classic model system for studying developmental biology. During chick development, embryonic day 8 (E8) retina are packed with multipotent neuronal precursors on the cusp of differentiation into all retinal cell types. By E18 the retina is nearly fully mature with all major retinal cell types differentiated and expressing cell type-specific genes. Whole retina were harvested from embryonic day 8 (E8), E16, and E18 developing chicken embryos. Whole cornea were also collected from E18 embryos as a reference tissue. Duplicates were obtained for each time point and total RNA was extracted from samples using a Qiagen AllPrep Mini Kit. To characterize differential gene expression in these tissues, Illumina stranded TrueSeq RNA-Seq libraries were prepared from total RNA and 125 PE sequencing reads were obtained. These data will be used to characterize retina-specific patters of gene expression as well as global changes in gene expression between critical developmental time points in the chicken retina.

ORGANISM(S): Gallus gallus

PROVIDER: GSE65938 | GEO | 2016/08/18

SECONDARY ACCESSION(S): PRJNA275440

REPOSITORIES: GEO

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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.

Project description:Purpose: The morphology of the RPE shows minimal change as the neural retina and choriocapillaris differentiate. Nonetheless, initial studies of barrier-related proteins suggest extensive remodeling of the RPE in response to this changing environment. A genomic approach was used to investigate the extent of this remodeling. Methods: RPE was isolated from E7, E10, E14 and E18 chick embryos and total RNA extracted for probing the entire genome on Affymetrix microarray chips. Statistical parameters using ANOVA were adjusted to yield a theoretical false discovery rate of 5%. STEM software was used to cluster genes into statistically related patterns of expression. Gene ontology clustering, using Affymetrix software was used for functional clustering. The proteinlounge.com database was used as a source of known biological pathways. Results: Of the 37,694 probe sets on the microarray, 17,199 were not absent. Of the 20,495 expressed probes, the expression of 8,889 was developmentally regulated. 4814 of these could be clustered into 12 patterns of expression that were statistically significant. The developmental patterns of expression for 22 tight and adherens junction proteins have been reported. Only two showed small variations from the patterns revealed by the microarray. Further, as would be predicted, expression of genes that promote progression through the cell cycle decreased and genes that inhibit progression increased with age. The data indicate extensive remodeling of the extracellular matrix, cell surface receptors, cell-cell junctions, transcellular ion transport and signal transduction pathways throughout development. Notably, the appearance of claudin 20, ZO-3 and cadherins 13 and 20 very late in development suggest barrier properties continue to change after functional junctions are formed. Conclusions: The data reveal a far more dynamic view of the RPE and its interactions with its environment than would be expected from morphological examination. The remodeling of junctional complexes, extracellular matrix interactions and transcellular transport capabilities indicates a continuous remodeling of the blood-retinal barrier. These data provide a standard whereby culture models of RPE function and regulation may be judged. SUBMITTER_CITATION: Rizzolo LJ, Chen X, Weitzman M, Sun R and Zhang H (2007) Analysis of the RPE transcriptome reveals dynamic changes during the development of the outer blood-retinal barrier. Mol. Vis. 13: in press. Experiment Overall Design: Sheets of RPE were isolated from chicken embryos on Embryonic day 7 (E7), E10, E14 and E18 and stored in RNAlatter (Qiagen, Valencia, CA), as described <Rahner C, Fukuhara M, Peng S, Kojima S, Rizzolo LJ. The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development. J Cell Sci. 2004;117:3307-18>. To isolate total RNA, the RNeasy Protect kit (Qiagen) was used according to the manufacturer's protocols. For each age, 3-4 independent preparations were used for analysis on Affymetrix microarrays of the chicken genome (Santa Clara, CA). For each preparation, sheets of RPE were pooled from 20-30 eyes. The quality of the total RNA was assessed by the Keck Center, Yale University using formamide gels and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA)

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RNA-seq analysis of the developing chicken retina

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