Cocurullo M et al. (2023), Single-Cell Transcriptomic Analysis Reveals the...

ECB-ART-51763

Cells 2023 Aug 23;1217:. doi: 10.3390/cells12172134.

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Single-Cell Transcriptomic Analysis Reveals the Molecular Profile of Go-Opsin Photoreceptor Cells in Sea Urchin Larvae.

Cocurullo M , Paganos P , Annunziata R , Voronov D , Arnone MI .

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The ability to perceive and respond to light stimuli is fundamental not only for spatial vision but also to many other light-mediated interactions with the environment. In animals, light perception is performed by specific cells known as photoreceptors and, at molecular level, by a group of GPCRs known as opsins. Sea urchin larvae possess a group of photoreceptor cells (PRCs) deploying a Go-Opsin (Opsin3.2) which have been shown to share transcription factors and morphology with PRCs of the ciliary type, raising new questions related to how this sea urchin larva PRC is specified and whether it shares a common ancestor with ciliary PRCs or it if evolved independently through convergent evolution. To answer these questions, we combined immunohistochemistry and fluorescent in situ hybridization to investigate how the Opsin3.2 PRCs develop in the sea urchin Strongylocentrotus purpuratus larva. Subsequently, we applied single-cell transcriptomics to investigate the molecular signature of the Sp-Opsin3.2-expressing cells and show that they deploy an ancient regulatory program responsible for photoreceptors specification. Finally, we also discuss the possible functions of the Opsin3.2-positive cells based on their molecular fingerprint, and we suggest that they are involved in a variety of signaling pathways, including those entailing the thyrotropin-releasing hormone.

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	Figure 1. Molecular characterization of the Opsin3.2 cells at different developmental stages. (A) FISH of Sp-Opsin3.2 stains 1 cell at 3 dpf. (B) FISH for Sp-Trh was combined with double TRH/Opsin3.2 immunolocalization at 3 dpf, highlighting the bipolar structure of these sensory/neurosecretory neurons, with the Opsin3.2 localized on a ciliated-like structure located on the external side of the larva. The TRH peptide, instead, is concentrated in the opposite side of the cell, and it appears to be transported along the projections of the cells (directed toward the larva apical organ). (B′) Details of the cell showed in (B) on the left. (C) FISH of Sp-Opsin3.2 at 4 dpf. (D,D′) FISH for Sp-Trh was combined with double TRH/Opsin3.2 immunolocalization at 4 dpf. All three cells have a bipolar organization. (E,F) Double FISH of Sp-Opsin3.2 and Sp-Trh at 5 dpf stains three–four cells. FISH of Sp-Opsin3.2 at 4 wpf (G,G′) and 5 wpf (H–H″) detect two clusters of cells located at the base of the oral arms. All images are full projections of merged confocal Z sections. Nuclei are shown in white. Abbreviations: ao, apical organ; m, mouth; oh, oral hood.
	Figure 2. Cell type atlas of the S. purpuratus larvae at 3 and 5 dpf pluteus stages. (A) Schematic representation of the scRNA-seq pipeline. (B) UMAP representing the cell clustering obtained through harmony of the 3 and 5 dpf pluteus single-cell datasets and their localization in a schematic representation of a four-armed S. purpuratus larva. (C) Dotplot showing the average expression of a subset of genes used as markers to annotate the different cell clusters.
	Figure 3. Identification of the Opsin3.2 and Opsin2 PRCs. (A) Dotplot showing the expression of Sp-Opsin3.2 and Sp-Opsin2 and of the neuropeptide Sp-Trh and of the immune cell marker Sp-Pks1. (B) UMAP illustrating that Opsin3.2 PRCs have been isolated from the Neuron (4) cluster, while Opsin2 have been selected from the immune cells cluster. (C) Dotplot showing the expression pattern of genes selected from [18].
	Figure 4. Molecular signature of S. purpuratus PRCs at pluteus stages. (A) Dotplot showing the average expression of genes putatively involved in the phototransuction cascade activated by the opsins stimulation. (B) Dotplot showing the average expression of genes involved in the regulation circadian rhythms. (C) Dotplot showing the average expression of genes selected to further describe the molecular identity of the Opsin3.2 and Opsin2 PRCs. (D) Dotplot showing the percentage of TFs common to the two PRCs. (E) Dotplot showing the average expression of known TFs involved photoreceptor specification.
	Figure 5. Opsin3.2 PRCs summary. Schematic reconstruction of the morphology, topology and molecular signature of the Opsin3.2 PRCs.

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