Project description:The rd1 mouse retina is a well-studied model of retinal degeneration where rod photoreceptors undergo cell death beginning at postnatal day P10 until P21. This period coincides with photoreceptor terminal differentiation in a normal retina. We have used the rd1 retina as a model to investigate early molecular defects in developing rod photoreceptors prior to the onset of degeneration. Using a microarray approach, we performed gene profiling comparing rd1 and wild type retinas at four time points starting at P2, prior to any obvious biochemical or morphological differences, and concluding at P8, prior to the initiation of cell death. We have identified genes that are differentially regulated in the rd1 retina at early time points, which may give insights into developmental defects that precede photoreceptor cell death. This is the first report of PRA1 expression in the retina. Our data support the hypothesis that PRA1 plays an important role in vesicular trafficking between the Golgi and cilia in differentiating and mature rod photoreceptors.
Project description:The rd1 mouse retina is a well-studied model of retinal degeneration where rod photoreceptors undergo cell death beginning at postnatal day P10 until P21. This period coincides with photoreceptor terminal differentiation in a normal retina. We have used the rd1 retina as a model to investigate early molecular defects in developing rod photoreceptors prior to the onset of degeneration. Using a microarray approach, we performed gene profiling comparing rd1 and wild type retinas at four time points starting at P2, prior to any obvious biochemical or morphological differences, and concluding at P8, prior to the initiation of cell death. We have identified genes that are differentially regulated in the rd1 retina at early time points, which may give insights into developmental defects that precede photoreceptor cell death. This is the first report of PRA1 expression in the retina. Our data support the hypothesis that PRA1 plays an important role in vesicular trafficking between the Golgi and cilia in differentiating and mature rod photoreceptors. Retinal samples were harvested from both rd1/le and wt animals at postnatal days 2, 4, 6, and 8 for microarray. Each sample included 8-14 retinas and experiments were performed in quadruplicate. Ten micrograms of total RNA was used for cDNA systhesis in target molecule production.
Project description:The rod photoreceptor-specific neural retina leucine zipper protein Nrl is essential for rod differentiation and plays a critical role in regulating gene expression. In the mouse retina, rods account for 97% of the photoreceptors; however, in the absence of Nrl (Nrl-/-), no rods are present and a concomitant increase in cones is observed. Using mouse GeneChips (Affymetrix), we have generated expression profiles of the wild-type and Nrl-/- retina at three time-points representing distinct stages of photoreceptor differentiation. Experiment Overall Design: Both the Nrl-/- mice and wild type controls were of a matched mixed genetic background (R1 and C57BL/6 strains).
Project description:Hmx1 is a transcription factor expressed in the developing eye and ear and in some other parts of the nervous system. Dumbo mice are carrying the Hmx1 p.Q64X loss-of-function mutation (Munroe et al., 2009. BMC Developmental Biology). Transcriptomic analyses of this mouse model allows to decipher biological pathways under the control of Hmx1. In our study, we used it to better understand the role of Hmx1 in the retina and to identify several of its target genes. Wild-type and dumbo mice were maintained in a 12-hours light/12-hours dark cycle with free access to food and water. Animals were sacrified at P15 for retina isolation and RNA extraction.
Project description:This study provides a comprehensive transcript analysis of the developing mouse retina with a focus on normalization, differentially expression, cell-type specific gene expression, transcription factor co-expression, alternative splicing, and novel transcript discovery. RNA-seq profiling was performed on four embryonic and eight postnatal time points during mouse retinal development and maturation. Alignment, transcript quantitation, normalization and differential expression, alternative splice usage, and novel transcript discovery was performed. Quantitative analysis revealed that 25,901 total transcripts encoding for 13,714 genes were expressed in the mouse retina between embryonic day 11 and postnatal day 28. Of these expressed transcripts, 12,075 were significantly differentially expressed (10,069 genes) at some point during development corresponding to ~73% of the expressed genes.
Project description:Transcriptional profiling of mouse retina comparing naïve mouse and experimental autoimmune uveoretinitis (EAU)-developing mouse. EAU was induced by immunization of retinal autoantigens in adjuvants. Retinas were harvested 14 days after immunization.
Project description:MicroRNA expression in the mouse eye.MicroRNAs (miRNAs) are key regulators of biological processes. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution. In this report, we present the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridization (ISH) procedures. We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina . This analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. The combination of the two approaches offers a valuable resource to decipher the contributions of specific miRNAs and miRNA clusters to the development of distinct ocular structures. microRNA profiling of ocular tissues from mouse. In particular we analysed the cornea, lens, Retina Pigment Epithelium (RPE) and retina and compared them against RNA extracted from the entire eye. The purpose of this experiment was to understand which microRNAs are present nd/or show differential expression in the various structures of the eye (cornea, lens, RPE, retina). The samples numbered 1 & 2 (i.e. CORNEA1, CORNEA2 etc ) are biological replicates, prepared from tissues dissecyed from different groups of wild-type animals. RNA extracted from the entire eye (EYE) served as the unique reference sample. For each tissue to be analysed we performed the following hybridizations: - 2 slides for lens (LENS1, LENS2) vs entire eye (EYE) - 2 slides for RPE (RPE1, RPE2) vs entire eye (EYE) - 2 slides for retina (RETINA1, RETINA2) vs entire eye (EYE) - 2 slides for cornea (CORNEA1, CORNEA2) vs entire eye (EYE) - 1 slide for entire eye (EYE) vs entire eye (EYE)
Project description:mRNA transcript levels in embryonic day 15.5 (E15.5), E17.5 and postnatal day 1 (P1) mouse Crb1KOCrb2ΔRPC against mouse Crb1KO neuroretina (Run1), and in E15.5 mouse Crb1KOCrb2ΔRPC against wild type neuroretina (Run2) were analyzed. A comparison between Crb1KOCrb2ΔRPC and Crb1KO retina, at E15.5, or E17.5, or P1 on 100% C57BL/6JOlaHsd, yielded only subtle persistent changes at the transcriptional level over time (Figure 1 G-I, respectively), despite significant differences in morphology. As inter-nal positive control at E15.5, E17.5 and P1 we used ATP-binding cassette, sub-family D (ALD), member 4 (Abcd4), which is substantially upregulated in mouse retina expressing the Cre recombinase fused to the Chx10 promoter, with the Abcd4 gene immediately adja-cent to the Chx10Cre locus in the mouse genome. To further examine potential transcriptional changes at E15.5, we compared Crb1KOCrb2ΔRPC mice in 100% C57BL/6JOlaHsd genetic background against wild type C57BL/6JRccHsd mice. In this setting, we observed 40 differentially expressed genes (DEGs) compared to the previously 0 DEGs (E15.5; adj. p-val < 0.01 & log2FC > 1.5). The Crb1KOCrb2ΔRPC mice on C57BL/6JOlaHsd genetic background, due to mutations in the synuclein alpha (Snca), multimerin-1 (Mmrn1), and Crb1 gene, do not express Snca, Mmrn1, or Crb1 gene transcripts, and express high levels of Abcd4 due to the adjacent Chx10Cre transgene on chromosome 6. We made use of all 4 genes as negative and positive controls in the gene expression profiling, since the mice on C57BL/6JRccHsd genetic background have no mutations in Snca, Mmrn1 or Crb1 and express low levels of Abcd4. Analysis of the E15.5 data revealed upregulated expression of the WD repeat and FYVE domain-containing protein 1 (Wdfy1) gene also known as FYVE domain-containing pro-tein localized to endosomes (Fens-1), which encodes a phosphatidylinositol 3-phosphate binding protein that contains a FYVE zinc finger domain and multiple WD-40 repeat do-mains.