Expressed sequence tags and chromosomal localization of cDNA clones from a subtracted retinal pigment epithelium library

Expressed sequence tags (ESTs) provide useful molecular landmarks for physical mapping and identify the position of an expressed region in the genome. The use of subtracted cDNA libraries enriched for tissue-specific genes as a source of ESTs should reduce the repetitive isolation of constitutively expressed sequences. We report here the sequence tags from the 3'-end region of 58 new directionally cloned cDNAs from a substracted human retinal pigment epithelium (RPE) cell line library. Eight of the cDNAs have been assigned to human chromosomes using PCR-based EST assays. Chromosomal mapping of subtracted RPE cDNA clones may also help in identifying candidate genes for inherited eye diseases.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29973/1/0000336.pd

Oligodeoxyribonucleotide probe accessibility on a three-dimensional DNA microarray surface and the effect of hybridization time on the accuracy of expression ratios

BACKGROUND: DNA microarrays are now routinely used to monitor the transcript levels of thousands of genes simultaneously. However, the array fabrication method, hybridization conditions, and oligodeoxyribonucleotide probe length can impact the performance of a DNA microarray platform. RESULTS: We demonstrate solution-phase hybridization behavior of probe:target interactions by showing a strong correlation between the effect of mismatches in probes attached to a three dimensional matrix of a microarray and solution-based, thermodynamic duplex melting studies. The effects of mismatches in the probes attached to the microarray also demonstrate that most, if not all, of the oligodeoxyribonucleotide is available for hybridization. Kinetic parameters were also investigated. As anticipated, hybridization signals increased in a transcript concentration-dependent manner, and mismatch specificity increased with hybridization time. Unexpectedly, hybridization time increased the accuracy of fold changes by relieving the compression observed in expression ratios, and this effect may be more dramatic for larger fold changes. CONCLUSIONS: Taken together, these studies demonstrate that a three-dimensional surface may enable use of shorter oligodeoxyribonucleotide probes and that hybridization time may be critical in improving the accuracy of microarray data

Retinal Organoids derived from hiPSCs of an AIPL1-LCA Patient Maintain Cytoarchitecture despite Reduced levels of Mutant AIPL1

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor-specific chaperone that stabilizes the effector enzyme of phototransduction, cGMP phosphodiesterase 6 (PDE6). Mutations in the AIPL1 gene cause a severe inherited retinal dystrophy, Leber congenital amaurosis type 4 (LCA4), that manifests as the loss of vision during the first year of life. In this study, we generated three-dimensional (3D) retinal organoids (ROs) from human induced pluripotent stem cells (hiPSCs) derived from an LCA4 patient carrying a Cys89Arg mutation in AIPL1. This study aimed to (i) explore whether the patient hiPSC-derived ROs recapitulate LCA4 disease phenotype, and (ii) generate a clinically relevant resource to investigate the molecular mechanism of disease and safely test novel therapies for LCA4 in vitro. We demonstrate reduced levels of the mutant AIPL1 and PDE6 proteins in patient organoids, corroborating the findings in animal models; however, patient-derived organoids maintained retinal cell cytoarchitecture despite significantly reduced levels of AIPL1.This work was supported by Institute of Health Carlos III (ISCIII)/ ERDF (European Research Development Fund), Spain, ((PI16/00409 (DL); DL, AAC, and SE are members of PRB3 supported by a grant (PT17/0019/0024) of the PE I + D + i 2013–2016, funded by ISCIII and ERDF. The work was also supported by ISCIII-ERDF (PI16/00425), CIBERER 06/07/0036, IIS-FJD Biobank PT13/0010/0012, RAREGENOMICS funded by Regional Government of Madrid, (CAM, B2017/BMD3721) and ERDF, the University Chair UAM-IIS-FJD of Genomic Medicine, the Spanish National Organization of the Blind (ONCE), the Spanish Fighting Blindness Foundation (FUNDALUCE), and the Ramon Areces Foundation. MC is supported by the Miguel Servet Program (CPII17_00006) from ISCIII. DL is supported by Miguel Servet I Program (CP18/00033). VR is supported by National Institute of Health (R01 EY028035, R01 EY025536). Transcriptome profiling and analyses were supported by the Intramural Research Program of the National Eye Institute (ZIAEY000450, ZIAEY000474) and utilized the high-performance computational capabilities of the Biowulf Linux cluster at NIH (http://biowulf.nih.gov)

NRL-Regulated Transcriptome Dynamics of Developing Rod Photoreceptors

SummaryGene regulatory networks (GRNs) guiding differentiation of cell types and cell assemblies in the nervous system are poorly understood because of inherent complexities and interdependence of signaling pathways. Here, we report transcriptome dynamics of differentiating rod photoreceptors in the mammalian retina. Given that the transcription factor NRL determines rod cell fate, we performed expression profiling of developing NRL-positive (rods) and NRL-negative (S-cone-like) mouse photoreceptors. We identified a large-scale, sharp transition in the transcriptome landscape between postnatal days 6 and 10 concordant with rod morphogenesis. Rod-specific temporal DNA methylation corroborated gene expression patterns. De novo assembly and alternative splicing analyses revealed previously unannotated rod-enriched transcripts and the role of NRL in transcript maturation. Furthermore, we defined the relationship of NRL with other transcriptional regulators and downstream cognate effectors. Our studies provide the framework for comprehensive system-level analysis of the GRN underlying the development of a single sensory neuron, the rod photoreceptor

Transcriptional Regulation of Rod Photoreceptor Homeostasis Revealed by In Vivo NRL Targetome Analysis

A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP–Seq) data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP–Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP–Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis

A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants.

This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.3448Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 × 10(-8)) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10(-10)). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.We thank all participants of all the studies included for enabling this research by their participation in these studies. Computer resources for this project have been provided by the high-performance computing centers of the University of Michigan and the University of Regensburg. Group-specific acknowledgments can be found in the Supplementary Note. The Center for Inherited Diseases Research (CIDR) Program contract number is HHSN268201200008I. This and the main consortium work were predominantly funded by 1X01HG006934-01 to G.R.A. and R01 EY022310 to J.L.H

Retinal Degeneration Triggers the Activation of YAP/TEAD in Reactive Müller Cells.

International audienceDuring retinal degeneration, Müller glia cells respond to photoreceptor loss by undergoing reactive gliosis, with both detrimental and beneficial effects. Increasing our knowledge of the complex molecular response of Müller cells to retinal degeneration is thus essential for the development of new therapeutic strategies. The purpose of this work was to identify new factors involved in Müller cell response to photoreceptor cell death.Whole transcriptome sequencing was performed from wild-type and degenerating rd10 mouse retinas at P30. The changes in mRNA abundance for several differentially expressed genes were assessed by quantitative RT-PCR (RT-qPCR). Protein expression level and retinal cellular localization were determined by western blot and immunohistochemistry, respectively.Pathway-level analysis from whole transcriptomic data revealed the Hippo/YAP pathway as one of the main signaling pathways altered in response to photoreceptor degeneration in rd10 retinas. We found that downstream effectors of this pathway, YAP and TEAD1, are specifically expressed in Müller cells and that their expression, at both the mRNA and protein levels, is increased in rd10 reactive Müller glia after the onset of photoreceptor degeneration. The expression of Ctgf and Cyr61, two target genes of the transcriptional YAP/TEAD complex, is also upregulated following photoreceptor loss.This work reveals for the first time that YAP and TEAD1, key downstream effectors of the Hippo pathway, are specifically expressed in Müller cells. We also uncovered a deregulation of the expression and activity of Hippo/YAP pathway components in reactive Müller cells under pathologic conditions