GSE193622
Cell type diversity in the octopus brain
Octopuses are mollusks that evolved intricate neural systems which are comparable with vertebrates in terms of cell number, complexity and size. Exactly how an octopus increases its neural cell number so dramatically and whether an increase in cell type diversity enables higher cognitive function and complex behavior is still unknown. To profile the cell diversity of the developing octopus brain we applied 10x Genomics' single-cell/nuclei RNA sequencing technology. At hatching, the Octopus vulgaris brain possesses the main lobes and connections of an adult brain, but which cell types are present remains elusive. We were able to identify 42 robust cell types comprising mostly neural cells, as well as multiple glial subtypes and other non-neuronal populations such as endothelial cells and fibroblasts. In situ expression analysis of marker genes allowed spatial mapping of clusters, including vertical lobe cells and several optic lobe cell types. Investigation of cell type conservation indicated similar gene expression signatures between glial cells of mice, fly and octopus. Genes related to memory and learning were found enriched in vertical lobe cells, that showed molecular similarities with Kenyon cells in Drosophila but not to any mouse cell type. Lastly, we also analyzed the expression of newly expanded gene families (protocadherins, C2H2 zinc-finger transcription factors and G-protein coupled receptors) and found that these are enriched in specific cell types. Taken together, our data gives insight into cell type evolution and the composition of the complex octopus brain. Overall design: We performed single cell and single nuclei RNA sequencing on a pool of 30 paralarval brains of Octopus vulgaris. Library preparations for the sc/snRNA-seq experiments were performed using 10X Genomics Chromium Single Cell 3' Kit, v3 chemistry (10X Genomics, Pleasanton, CA, USA). We aimed for a targeted cell recovery of 6000-10 000 cells/nuclei. Post cell count and QC, the samples were immediately loaded onto the Chromium Controller. Single cell or single nuclei RNAseq libraries were prepared using manufacturers recommendations (Single cell 3' reagent kits v3.1 user guide; CG000204 Rev D), and at the different check points the library quality was assessed using Qubit (ThermoFisher) and Bioanalyzer (Agilent). With a targeted sequencing coverage of 25-50 K reads per cell, single cell libraries were sequenced on Illumina's NovaSeq 6000 platform  using paired-end sequencing workflow and with recommended 10X; v3.1 read parameters (28-8-0-91 cycles). A total of 202 402 758 reads were obtained for the nuclei and 247 457 191 reads for the cells.