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Transcriptional profiling of murine retinas undergoing semi-synchronous cone photoreceptor differentiation.

ABSTRACT: Uncovering the gene regulatory networks that control cone photoreceptor formation has been hindered because cones only make up a few percent of the retina and form asynchronously during development. To overcome these limitations, we used a ?-secretase inhibitor, DAPT, to disrupt Notch signaling and force proliferating retinal progenitor cells to rapidly adopt neuronal identity. We treated mouse retinal explants at the peak of cone genesis with DAPT and examined tissues at several time-points by histology and bulk RNA-sequencing. We found that this treatment caused supernumerary cone formation in an overwhelmingly synchronized fashion. This analysis revealed several categorical patterns of gene expression changes over time relative to DMSO treated control explants. These were placed in the temporal context of the activation of Otx2, a transcription factor that is expressed at the onset of photoreceptor development and that is required for both rod and cone formation. One group of interest had genes, such as Mybl1, Ascl1, Neurog2, and Olig2, that became upregulated by DAPT treatment before Otx2. Two other groups showed upregulated gene expression shortly after Otx2, either transiently or permanently. This included genes such as Mybl1, Meis2, and Podxl. Our data provide a developmental timeline of the gene expression events that underlie the initial steps of cone genesis and maturation. Applying this strategy to human retinal organoid cultures was also sufficient to induce a massive increase in cone genesis. Taken together, our results provide a temporal framework that can be used to elucidate the gene regulatory logic controlling cone photoreceptor development.

SUBMITTER: Kaufman ML

PROVIDER: S-EPMC7722777 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Transcriptional profiling of murine retinas undergoing semi-synchronous cone photoreceptor differentiation.

Kaufman Michael L ML Park Ko Uoon KU Goodson Noah B NB Chew Shereen S Bersie Stephanie S Jones Kenneth L KL Lamba Deepak A DA Brzezinski Joseph A JA

Developmental biology 20190601 2

Uncovering the gene regulatory networks that control cone photoreceptor formation has been hindered because cones only make up a few percent of the retina and form asynchronously during development. To overcome these limitations, we used a γ-secretase inhibitor, DAPT, to disrupt Notch signaling and force proliferating retinal progenitor cells to rapidly adopt neuronal identity. We treated mouse retinal explants at the peak of cone genesis with DAPT and examined tissues at several time-points by ...[more]

PMID: 31163126

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Project description:Transcription factors have been implicated in the specification and differentiation of all cells in the mammalian retina with several transcription factors controlling the development of multiple neuronal subclasses. Horizontal cells and cone photoreceptors share a particularly intimate functional relationship but have been previously thought to develop through separate and distinct intrinsic molecular processes. We demonstrate that the zinc finger transcription factor Sall3 regulates development of both S-cone photoreceptors and horizontal cells. Our data shows that loss of function of Sall3 down-regulates the horizontal cell specific transcription factor Lhx1 and subsequently causes ectopic formation of horizontal-like wide-field amacrine cells partially phenocopying Lhx1-/- mice. Additionally, horizontal cells which laminate appropriately in Sall3 knockout mice show abnormal horizontal cell gene expression and failures in dendritic arborization. Over-expression of Sall3 also partially re-specifies cells to a horizontal-like wide-field amacrine fate. Intriguingly, Sall3 loss of function experiments also generated a massive reduction in the number of S-cone photoreceptors with remaining S-cones showing deficiencies in S-cone gene expression and morphology. Conversely, over-expression of Sall3 resulted in the ectopic expression of the S-cone specific genes S-opsin (Sop) and cone arrestin (Arr3) in electroporated cells. Our studies reveal that Sall3 regulates aspects of horizontal cell development in two ways: first, by maintaining Lhx1 expression, and second, by directly regulating expression of horizontal cell specific genes. We also show that Sall3 is an essential regulator of S-cone development in the mammalian retina and a potent activator of Sop and Arr3. Sall3 wild-type and knockout retinas used for explant cultures were extracted by microdissection at P0 in sterile PBS. Four cuts were made using micro-dissection scissors and the retinas were transferred to Nucleopore Track-Etch membrane filters (Whatman) and the tissue flattened out evenly. Filters were then floated on DMEM/F12, 10% FBS, 1X penicillin/streptomycin in 12 well plates and incubated at 37oC, 5% CO2 for 7 days. Three retinas (Sall3 knockout or wild-type) were pooled and RNA extracted using the Qiagen RNeasy kit. This was performed in triplicate for both wild-type and Sall3 knockouts retinas. A total of three replicates of Sall3 knockout and Sall3 wild-type RNA were analysed (six total samples).

Project description:Functional vision restoration is within reach via stem cell therapy, but one of the largest obstacles is the derivation of colour-sensitive cone photoreceptors that are required for high-acuity daytime vision. To enhance progress made using nocturnal murine models, we instead utilize cone-rich zebrafish and herein investigate relationships between gdf6a and tbx2b in cone photoreceptor development. Growth/differentiation factor 6a (gdf6a), a bone morphogenetic protein family ligand, is an emerging factor in photoreceptor degenerative diseases. The T-box transcription factor tbx2b is required to specify UV cone photoreceptor fate instead of rod photoreceptor fate. Interactions between these factors in cone development would be unanticipated, considering the discrete phenotypes in their respective mutants. However, gdf6a positively modulates the abundance of tbx2b transcript during early eye morphogenesis, and we extended this conclusion to later stages of retinal development comprising the times when photoreceptors differentiate. Despite this, gdf6a-/- larvae possess a normal relative number of UV cones and instead present with a low abundance of blue cone photoreceptors, approximately half that of siblings (p<0.001), supporting a differential role for gdf6a amongst the spectral subtypes of cone photoreceptors. Further, gdf6a-/- larvae from breeding of compound heterozygous gdf6a+/-;tbx2b+/- mutants exhibit the recessive lots-of-rods phenotype (which also shows a paucity of UV cones) at significantly elevated rates (44% or 48% for each of two tbx2b alleles, χ2 p≤0.007 for each compared to expected Mendelian 25%). Thus the gdf6a-/- background sensitizes fish such that the recessive lots-of-rods phenotype can appear in heterozygous tbx2b+/- fish. Overall, this work establishes a novel link between tbx2b and gdf6a in determining photoreceptor fates, defining the nexus of an intricate pathway influencing the abundance of cone spectral subtypes and specifying rod vs. cone photoreceptors. Understanding this interaction is a necessary step in the refinement of stem cell-based restoration of daytime vision in humans.

Dataset Information

Transcriptional profiling of murine retinas undergoing semi-synchronous cone photoreceptor differentiation.

Publications

Transcriptional profiling of murine retinas undergoing semi-synchronous cone photoreceptor differentiation.

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