Transcriptome Dynamics Underlying Rod Photoreceptor Development The morphogenesis and functional differentiation of photoreceptors in the mouse retina span over period of three weeks. Dynamic yet precise changes in gene expression patterns are associated with each developmental stage. We generated transcriptome profiles of purified developing rods by taking advantage of a genetically engineered mouse line expressing GFP in rod cells. For comparison we also produced expression profiles of Nrl-/-:Nrl-GFP photoreceptors that exhibit S-cone characteristics due to the loss of Nrl. We have integrated the temporal transcriptome datasets to NRL ChIP-seq data. Our studies demonstrated a major shift in rod transcriptome from postnatal day, P6 to P10, consistent with the time of morphological differentiation. We have also identified novel NRL-regulated transcripts generated by alternate splicing or promoter usage. In addition, we discovered a large number of novel photoreceptor transcripts that are regulated by NRL. Based on the results we have identified NRL-centered gene regulatory networks and novel secondary regulators of rod cell development. Dynamic changes of chromatin state during development and aging The architecture of the various regions of the chromatin play an important role in gene expression. To study the open and closed regions of the rod photoreceptor chromatin, we performed ATAC- Seq experiments using flow sorted cells. The results indicated that chromatin structure tends to be more open in younger stages (3 months) and stays somewhat open until 12 months. At around 1yr we noticed a transition to a more closed state, which was unexpected. Currently, we are investigating the mechanism/s of this change in chromatin state and its possible triggers during aging. Epigenetic Regulation of Rod Development and aging To uncover the role of the epigenome during aging of rod photoreceptors, we generated genome wide profiles of DNA methylation and open chromatin changes in young and old rod photoreceptors. We found that the DNA methylation pattern of rods is dynamic during aging with a preferential loss of methylation in neuronal and metabolic genes and in chromosomal regions associated with pathways of longevity and cell homeostasis. Motif enrichment analysis suggest that the binding of the master genome architectural protein CTCF is altered in differentially methylated regions, possibly linking epigenomic changes to gene expression alterations with age. Noncoding Transcriptome of Developing Rods By mining RNA-seq data, together with de-novo assembly, we have identified several known and novel long noncoding RNAs (lncRNAs), which show dynamic expression during photoreceptor development. Our in situ hybridization studies suggest that several of the lncRNAS are predominantly localized to the nucleus, suggesting that this is their primary area of function. Using mass spectrometric analysis of retinal small proteome, we identified 13 lncRNAs that encoded small peptides. One of these, Platr6, was selected for further analysis based on its abundance, expression pattern, and evolutionary conservation. Transfection experiments in HEK 293 cells of a Flag-tagged Platr6 ORF showed it is epressed. Custom antibody developed in rabbit and chicken validated the presence of a peptide in human/swine/bovine retina, as well as HEK 293 and H9 cells. Chip-seq and Luciferase assay show that the gene is regulated by NRL and CRX transcription factors. qRT-RNA of mouse tissues show that the gene is expressed predominantly in the retina. CRISPR-Cas9 was used to generate two KO models: 1. targeting the ATG of the ORF to eliminate the potential to generate a peptide but preserve any lncRNA function (if one exists). 2. Deletion of a long non coding exon deleting half of the lncRNA but maintaining the ORF. IP experiments and KO models backcrossing are currently on their way to provide preliminary data as to the function of this peptide. Evolution of phototransduction genes and the fovea in Vertebrates Previously, we had shown molecular evidence for the evolution of rod photoreceptors from ancestral cones in mammals. We are now researching this concept further to study the evolution of phototransduction genes in different vertebrate lineages. We are using birds as a study system for for understanding the fovea, because birds display a diversity of foveal types. A goal of this project is the investigate the potential of birds as a study system for human macular degeneration. Rats and mice lack a fovea, and therefore the diversity of fovea displayed in avian species makes them a potentially very powerful model system for studying these diseases. Determinants of Photoreceptor Pre-Synapse Morphology Integration of photoreceptors in retinal architecture is essential for visual function. To develop a viable photoreceptor replacement therapy for retinal diseases, we must understand how photoreceptors make specific synaptic connections with inner retinal neurons. Though rods and cones originate from common precursors and perform similar functions, rods have small spherule like presynaptic structure (generally with a single ribbon) connecting to interneurons close to the ONL whereas cones have much larger pedicle with multiple ribbons and telodendrites and connect closer to interneurons. We took advantage of the pedicle-like morphology of the terminals in Nrl-/- retina to identify over 600 potential candidate genes that may be associated with determination of presynaptic structures. We have performed a genetic loss of function screen in rods for >10% of the genes to evaluate their impact on rod pre-synapse morphology. Of these, 18 genes affected the position of terminal within the OPL or the size of the spherule. Further studies are in progress to decipher how structural perturbations affect rod functionality. Cone-cells interaction Cellular interactions are critical for tissue development, function and homeostasis. Cone photoreceptors which are primary mediators of human vision, interact with different subtypes of bipolar and horizontal cells to transmit visual signals. Moreover, cones are tightly packed within the ONL and electrically coupled to rods via gap junctions. Importantly, secondary death of cone photoreceptors in retinitis pigmentosa is, in part, correlated to the loss of Rod Derived Cone Viability factor (Rdcvf), which is secreted by rod photoreceptors and protects cone cells. To investigate the molecular components involved in cone intercellular interactions, we took advantage of our transcriptome data and identified 823 genes that are enriched after P6 in Nrl-/- S-cones-like compared to rod photoreceptors and encode membrane or secreted proteins. To examine their relevance to cone function, we initiated a loss of function screen for 19 selected genes in the newborn Nrl-/- mouse retina, with a goal to identify genes associated with cone-cell communication. In vivo knockdown of one of the genes, the receptor tyrosine kinase Ephrin type-A receptoreceptor 10 (Epha10) altered cone morphology and cell body localization within the retina. Further studies are in progress to decipher the role of Epha10 in retinal development using a knockout mouse model (generated by Dr. Lijin Dong). Bioengineering Platforms for Development of Mature Neural Retina in vitro Our long-term goal is reconstruction of stem cell-derived retina in vitro for disease modeling and development of therapies. Our approach involves the use of NASA-developed rotating wall bioreactors to enhance nutrient and oxygen supply, and biomimetic scaffolds to provide physical support and mimic retinal microenvironment. We have observed faster differentiation and better maintenance of all cell types in mouse retinal organoids
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