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Lineage tracing analysis of cone photoreceptor associated cis-regulatory elements in the developing chicken retina.

ABSTRACT: During vertebrate retinal development, transient populations of retinal progenitor cells with restricted cell fate choices are formed. One of these progenitor populations expresses the Thrb gene and can be identified by activity of the ThrbCRM1 cis-regulatory element. Short-term assays have concluded that these cells preferentially generate cone photoreceptors and horizontal cells, however developmental timing has precluded an extensive cell type characterization of their progeny. Here we describe the development and validation of a recombinase-based lineage tracing system for the chicken embryo to further characterize the lineage of these cells. The ThrbCRM1 element was found to preferentially form photoreceptors and horizontal cells, as well as a small number of retinal ganglion cells. The photoreceptor cell progeny are exclusively cone photoreceptors and not rod photoreceptors, confirming that ThrbCRM1 progenitor cells are restricted from the rod fate. In addition, specific subtypes of horizontal cells and retinal ganglion cells were overrepresented, suggesting that ThrbCRM1 progenitor cells are not only restricted for cell type, but for cell subtype as well.

SUBMITTER: Schick E

PROVIDER: S-EPMC6597718 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Lineage tracing analysis of cone photoreceptor associated cis-regulatory elements in the developing chicken retina.

Schick Estie E McCaffery Sean D SD Keblish Erin E EE Thakurdin Cassandra C Emerson Mark M MM

Scientific reports 20190627 1

During vertebrate retinal development, transient populations of retinal progenitor cells with restricted cell fate choices are formed. One of these progenitor populations expresses the Thrb gene and can be identified by activity of the ThrbCRM1 cis-regulatory element. Short-term assays have concluded that these cells preferentially generate cone photoreceptors and horizontal cells, however developmental timing has precluded an extensive cell type characterization of their progeny. Here we descri ...[more]

PMID: 31249345

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Project description: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.

Project description:BackgroundCell specific gene expression is largely regulated by different combinations of transcription factors that bind cis-elements in the upstream promoter sequence. However, experimental detection of cis-elements is difficult, expensive, and time-consuming. This provides a motivation for developing bioinformatic methods to identify cis-elements that could prioritize future experimental studies. Here, we use motif discovery algorithms to predict transcription factor binding sites involved in regulating the differences between murine rod and cone photoreceptor populations.ResultsTo identify highly conserved motifs enriched in promoters that drive expression in either rod or cone photoreceptors, we assembled a set of murine rod-specific, cone-specific, and non-photoreceptor background promoter sequences. These sets were used as input to a newly devised motif discovery algorithm called Iterative Alignment/Modular Motif Selection (IAMMS). Using IAMMS, we predicted 34 motifs that may contribute to rod-specific (19 motifs) or cone-specific (15 motifs) expression patterns. Of these, 16 rod- and 12 cone-specific motifs were found in clusters near the transcription start site. New findings include the observation that cone promoters tend to contain TATA boxes, while rod promoters tend to be TATA-less (exempting Rho and Cnga1). Additionally, we identify putative sites for IL-6 effectors (in rods) and RXR family members (in cones) that can explain experimental data showing changes to cell-fate by activating these signaling pathways during rod/cone development. Two of the predicted motifs (NRE and ROP2) have been confirmed experimentally to be involved in cell-specific expression patterns. We provide a full database of predictions as additional data that may contain further valuable information. IAMMS predictions are compared with existing motif discovery algorithms, DME and BioProspector. We find that over 60% of IAMMS predictions are confirmed by at least one other motif discovery algorithm.ConclusionWe predict novel, putative cis-elements enriched in the promoter of rod-specific or cone-specific genes. These are candidate binding sites for transcription factors involved in maintaining functional differences between rod and cone photoreceptor populations.

Dataset Information

Lineage tracing analysis of cone photoreceptor associated cis-regulatory elements in the developing chicken retina.

Publications

Lineage tracing analysis of cone photoreceptor associated cis-regulatory elements in the developing chicken retina.

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