6 research outputs found
Identification and characterization of early photoreceptor cis-regulatory elements and their relation to Onecut1
Background: Cone and rod photoreceptors are two of the primary cell types affected in human retinal disease. Potential strategies to combat these diseases are the use of gene therapy to rescue compromised photoreceptors or to generate new functional photoreceptors to replace those lost in the diseased retina. Cis-regulatory elements specific to cones, rods, or both types of photoreceptors are critical components of successful implementation of these two strategies. The purpose of this study was to identify and characterize the cell type specificity and activity of cis-regulatory elements active in developing photoreceptors.
Methods: Cis-regulatory elements were introduced into the developing chicken and mouse retina by electroporation. Characterization of reporter activity in relation with cell type markers was determined using confocal microscopy. In addition, two high-throughput flow cytometry assay were developed to assess whether these elements were downstream of Onecut1 in the photoreceptor specification network.
Results: The majority of cis-regulatory elements were active in both cone and rod photoreceptors and were largely uninfluenced by a Onecut1 dominant-negative construct. Elements associated with the Thrb, Nr2e3, and Rhodopsin genes showed highly enriched activity in cones or rods, and were affected by interference in Onecut1 signaling. Rhodopsin promoter activity was the most highly influenced by Onecut1 activity and its induction could be modulated by the Maf family transcription factor L-Maf. Nr2e3 elements were observed to have activity in cone photoreceptors and Nr2e3 protein was expressed in developing cone photoreceptors, suggesting a role for this predominant rod gene in cone photoreceptor development.
Conclusions: The analysis presented here provides an experimental framework to determine the specificity and strength of photoreceptor elements within specific genetic networks during development. The Onecut1 transcription factor is one such factor that influences the gene regulatory networks specific to cones and rods, but not those that are common to both
OTX2 represses sister cell fate choices in the developing retina to promote photoreceptor specification
During vertebrate retinal development, subsets of progenitor cells generate progeny in a non-stochastic manner, suggesting that these decisions are tightly regulated. However, the gene-regulatory network components that are functionally important in these progenitor cells are largely unknown. Here we identify a functional role for the OTX2 transcription factor in this process. CRISPR/Cas9 gene editing was used to produce somatic mutations of OTX2 in the chick retina and identified similar phenotypes to those observed in human patients. Single cell RNA sequencing was used to determine the functional consequences OTX2 gene editing on the population of cells derived from OTX2-expressing retinal progenitor cells. This confirmed that OTX2 is required for the generation of photoreceptors, but also for repression of specific retinal fates and alternative gene regulatory networks. These include specific subtypes of retinal ganglion and horizontal cells, suggesting that in this context, OTX2 functions to repress sister cell fate choices
Molecular Analysis of Cone Photoreceptor Genesis from a Specific Retinal Progenitor Population
There are two types of photosensitive cells of the retina that contribute to image formation: Cone photoreceptors that mediate color discrimination and rods that provide photosensitivity in low-light conditions. Given the importance of cones in high acuity and color vision, deficiencies in this cell type that result from ailments such as retinitis pigmentosa and macular degeneration can lead to a debilitating loss of vision. Currently, one of the most pressing goals in the field of retinal development is the elucidation of the gene regulatory networks (GRN) involved in inducing an undifferentiated cell into becoming a functional cone photoreceptor.
Recently, an enhancer element that drives transcription of the THRB gene in the developing retina, THRBCRM1, was found to be active in a specific lineage of progenitor cells that are restricted to produce only cones and horizontal cells5. Further exploration revealed that the transcription factors OC1 and OTX2 are required simultaneously for activation of THRBCRM1 and both are present in cells positive for THRB mRNA. This work largely focuses on a closer exploration of the transcriptional changes that underlie the emergence of this class of restricted progenitor cells from multipotent progenitors and the production of cone photoreceptors.
First, collecting and sequencing the mRNA of the THRBCRM1 restricted progenitors allows for a snapshot of the gene expression levels in this cell population. Thorough analysis of this data, I determined some of the changes necessary for the transition of these progenitors to a restricted state, namely the downregulations of a class of proteins known to be involved in the multipotent state.
Second, using an artificial version of OC1 designed to exclusively repress its targets, a new sequencing dataset was produced and, in combination with our THRBCRM1 data, candidate genes downstream of OC1 were screened for importance in the establishment of cone fate. A novel cone-related gene involved in the regulation of rod fate in early retinal development was identified.
Lastly, the initial dataset was combed for enriched cone genes that might serve as a novel marker for early cones. With the use of our genetic toolset in chicken retinas and different strains of transgenic mice, the expression pattern and putative marker specificity of a novel cone gene is described. Additionally, a BAC-GFP transgenic line is shown to be a faithful reporter of early cones. This tool was used for the collection of cone precursors at an early developmental time point (E14.5) for single-cell RNA sequencing, providing a dataset enriched in early cone precursors