June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Single cell RNA sequencing reveals heterogeneity of Müller glia-derived progenitors and partial recapitulation of the developmental retinogenesis program in the light injured zebrafish retina
Author Affiliations & Notes
  • Laura Celotto
    Center for Regenerative Therapies Dresden, Zentrum fur Regenerative Therapien Dresden, Dresden, Sachsen, DE, academic/medres, Germany
  • Fabian Rost
    Center for Regenerative Therapies Dresden, Zentrum fur Regenerative Therapien Dresden, Dresden, Sachsen, DE, academic/medres, Germany
  • Stefan Hans
    Center for Regenerative Therapies Dresden, Zentrum fur Regenerative Therapien Dresden, Dresden, Sachsen, DE, academic/medres, Germany
  • Michael Brand
    Center for Regenerative Therapies Dresden, Zentrum fur Regenerative Therapien Dresden, Dresden, Sachsen, DE, academic/medres, Germany
  • Footnotes
    Commercial Relationships   Laura Celotto None; Fabian Rost None; Stefan Hans None; Michael Brand None
  • Footnotes
    Support  DFG BR1746/3 and BR1746/6 and ERC advanced grant (Zf-BrainReg)
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1366 – F0297. doi:
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    • Get Citation

      Laura Celotto, Fabian Rost, Stefan Hans, Michael Brand; Single cell RNA sequencing reveals heterogeneity of Müller glia-derived progenitors and partial recapitulation of the developmental retinogenesis program in the light injured zebrafish retina. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1366 – F0297.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : In zebrafish, retinal progenitors (RP) derive from Müller glia (MG) and restore the entire retinal architecture upon lesion. Detailed information on the underlying genes orchestrating this process is missing. Here, we profile the transcriptome of RPs and MGs and track their differentiation in the early regenerating zebrafish retina.

Methods : Adult zebrafish expressing mCherry in MG and GFP in all proliferating cells were used. Retinae of either untreated controls or light lesioned animals were dissociated at 44 hours post lesion (hpl), 4 and 6 days post lesion (dpl). Subsequently, mCherry as well as mCherry/GFP double-positive cells were isolated via Fluorescence Activated Cell Sorting and single cell RNA sequencing (scRNAseq) was performed using 10x Genomics. To validate the results of the scRNAseq, we applied immunohistochemistry and in situ hybridization.

Results : Bioinformatic analysis of 11690 cells identified 15 different cell clusters. Four clusters represent MG. Two clusters contain resting, gfap-positive MG, which are found at all four time points. The others contain reactive, proliferating and reactive, non-proliferating MG obtained at 44 hpl and 4 dpl, respectively. Heterogeneous RP clusters appear at 4 and 6 dpl. Multipotency markers, like pax6a, pax6b, rx1 and vsx2, are expressed in early RPs and resting or reactive MG. Both, early RPs and reactive MG upregulate proliferative, Notch-related and inflammatory markers, like pcna, her4.1 and hmgb2b. A second cluster of RPs is proliferative and expresses early neurogenic markers like atoh7 and pou2f2a. A third RP cluster upregulates markers of several neuronal lineages, like onecut1, onecut2 (horizontal cells), otx5 (photoreceptors) and neurod4 (amacrine cells). Retinal ganglion cells, horizontal cell RPs (pcna, lhx1a, prox1) and photoreceptor RPs (pcna, nr23, thrb) branch from neurogenic RPs already at 4 dpl. Red and blue cones derive from photoreceptor RPs as well as amacrine and bipolar cells from neurogenic RPs at 6 dpl. Rods were not present in the sampled progeny.

Conclusions : MG and early RPs have highly similar transcriptomes, whereas neurogenic and fate restricted RPs initiate markers of retinogenesis in the regenerating retina. Regenerated progeny arises following the developmental retinogenic order.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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