Paganos P et al. , A New Model Organism to Investigate Extraocular...

ECB-ART-50914

Cells null;1117:. doi: 10.3390/cells11172636.

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A New Model Organism to Investigate Extraocular Photoreception: Opsin and Retinal Gene Expression in the Sea Urchin Paracentrotus lividus.

Paganos P , Ullrich-Lüter E , Caccavale F , Zakrzewski A , Voronov D , Fournon-Berodia I , Cocurullo M , Lüter C , Arnone MI .

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Molecular research on the evolution of extraocular photoreception has drawn attention to photosensitive animals lacking proper eye organs. Outside of vertebrates, little is known about this type of sensory system in any other deuterostome. In this study, we investigate such an extraocular photoreceptor cell (PRC) system in developmental stages of the sea urchin Paracentrotus lividus. We provide a general overview of the cell type families present at the mature rudiment stage using single-cell transcriptomics, while emphasizing the PRCs complexity. We show that three neuronal and one muscle-like PRC type families express retinal genes prior to metamorphosis. Two of the three neuronal PRC type families express a rhabdomeric opsin as well as an echinoderm-specific opsin (echinopsin), and their genetic wiring includes sea urchin orthologs of key retinal genes such as hlf, pp2ab56e, barh, otx, ac/sc, brn3, six1/2, pax6, six3, neuroD, irxA, isl and ato. Using qPCR, in situ hybridization, and immunohistochemical analysis, we found that the expressed retinal gene composition becomes more complex from mature rudiment to juvenile stage. The majority of retinal genes are expressed dominantly in the animals' podia, and in addition to the genes already expressed in the mature rudiment, the juvenile podia express a ciliary opsin, another echinopsin, and two Go-opsins. The expression of a core of vertebrate retinal gene orthologs indicates that sea urchins have an evolutionarily conserved gene regulatory toolkit that controls photoreceptor specification and function, and that their podia are photosensory organs.

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	Figure 1. Cell type atlas of the P. lividus mature rudiment. (A) ScRNA-seq pipeline from larvae collection, rudiment dissection and dissociation to 10× capturing and computational analysis. (B) Overlay UMAP showing the overlap of the libraries originating from the two biological replicates. (C) Dotplot showing the average expression of genes used as markers to identify specific cell clusters. (D) UMAP showing mature rudiment cell types, colored by their assignment to the initial set of 20 distinct cell clusters. Average expression gradient for opsin genes is depicted in purple, and for the rest of the genes, in red.
	Figure 2. Photoreceptor cell type families and retinal molecular signature. (A) Heatmap and Venn diagram showing the differentially expressed marker genes between the two opsin2/opsin4 double positive clusters (PCRs 2 and PCRs 3). (B) Dotplot showing the averaged scaled expression of common marker genes of the two opsin2/opsin4 double positive clusters against the whole P. lividus mature rudiment atlas.
	Figure 3. Distinct molecular signature of the two opsin2/opsin4 double positive cell type families. (A) Sub-clustering analysis of the two opsin2/opsin4 double positive cell type families resulted in the generation of the three and two distinct sub-clusters, respectively. (B) Dotplot showing the average scaled expression of sea urchin orthologues of retinal genes distributed in the different sub-clusters. Average expression gradient for opsin genes is depicted in purple, while the rest of the genes expressed in PRCs 2 and PRCs 3 sub-clusters are shown in green and magenta, respectively.
	Figure 4. Immunolocalization (IHC) of sea urchin Opsin4 and expression of opsin4 mRNA (FISH) in mature rudiments and juveniles. (A) Immunohistochemical detection of Opsin4 at the 8-arm mature rudiment stage. (B) FISH using an antisense probe against Pl-opsin4 at the mature rudiment stage. (C) Immunohistochemical detection of Opsin4 protein at juvenile stage. (D–F) FISH using a specific antisense probe for Pl-opsin4 paired with IHC for Opsin4 protein at juvenile stage. (G,H) IHC detection of antigens indicative of the nervous system (1e11), musculature (MHC) and PRCs (Opsin4). (G,H) are a result of compilation of different stacks corresponding to the same individual. (I) Podium close-up showing the immunolocalization of Opsin4 and the pan-neuronal marker 1e11. (J–L) IHC detection of antigens indicative of the nervous system (1e11), musculature (MHC) and PRCs (Opsin4) focusing on a podium and the surrounding spines. DAPI was used to visualize nuclei (gray). Orientation: (A), the specimen is viewed from the top; (B), the specimen is viewed from the side; (D,G,H), juveniles are in oral view.
	Figure 5. Retinal transcription factors pax6, neuroD1 and six3 RNA: expression in whole mount mature rudiments and juveniles. (A–E) FISH using a specific antisense probe against Pl-pax6 at rudiment (A,B) and juvenile (C–E) stages. In B and E, FISH are also paired with IHC detection of Opsin4. (F) FISH detection of the neuroD transcripts at juvenile stage. (G–I) FISH using an antisense probe for six3 at rudiment (G) and juvenile (H,I) stages. In I, FISH is paired with IHC for Opsin4. DAPI was used to visualize nuclei (gray). Orientation: (A), the specimen is viewed from the top; (B,G) the specimens are viewed from the side; (C–F,H,I) juveniles are in oral view.
	Figure 6. Expression analysis of Opsins in P. lividus juveniles. (A) Relative gene expression of different opsins in juveniles as revealed by qRT-PCR. Sample size: 3 biological replicates; Statistical significance cut-off criteria: * p < 0.05; ** p <0.01. (B) Gene expression visualization of Opsin1, opsin2 and Opsin4 at juvenile stage. (B1–B3) double IHC detection of Opsin1 (magenta) and acetylated tubulin labeling ciliated structures (green). (B4) FISH using a specific antisense probe for Pl-opsin2 (cyan) paired with IHC for acetylated tubulin (green). (B5,B6) FISH for Pl-Opsin2 (cyan) transcripts paired with IHC detection of Opsin4 (red). DAPI was used to visualize nuclei (gray).
	Figure 7. Schemes with the opsin expression on them and the molecular signature of the 2 clusters of PRC identified at mature rudiment stage. Nervous system in magenta, Opsin4+ PRCs in green, opsin2+ PRCs in cyan, Opsin1+PRCs in yellow.

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Agca, Neurosensory and neuromuscular organization in tube feet of the sea urchin Strongylocentrotus purpuratus. 2011, Pubmed, Echinobase