Embryonic development of a centralised brain in coleoid cephalopods.

The last common ancestor of cephalopods and vertebrates lived about 580 million years ago, yet coleoid cephalopods, comprising squid, cuttlefish and octopus, have evolved an extraordinary behavioural repertoire that includes learned behaviour and tool utilization. These animals also developed innovative advanced defence mechanisms such as camouflage and ink release. They have evolved unique life cycles and possess the largest invertebrate nervous systems. Thus, studying coleoid cephalopods provides a unique opportunity to gain insights into the evolution and development of large centralised nervous systems. As non-model species, molecular and genetic tools are still limited. However, significant insights have already been gained to deconvolve embryonic brain development. Even though coleoid cephalopods possess a typical molluscan circumesophageal bauplan for their central nervous system, aspects of its development are reminiscent of processes observed in vertebrates as well, such as long-distance neuronal migration. This review provides an overview of embryonic coleoid cephalopod research focusing on the cellular and molecular aspects of neurogenesis, migration and patterning. Additionally, we summarize recent work on neural cell type diversity in embryonic and hatchling cephalopod brains. We conclude by highlighting gaps in our knowledge and routes for future research.

Cell type diversity in a developing octopus brain.

Octopuses are mollusks that have evolved intricate neural systems comparable with vertebrates in terms of cell number, complexity and size. The brain cell types that control their sophisticated behavioral repertoire are still unknown. Here, we profile the cell diversity of the paralarval Octopus vulgaris brain to build a cell type atlas that comprises mostly neural cells, but also multiple glial subtypes, endothelial cells and fibroblasts. We spatially map cell types to the vertical, subesophageal and optic lobes. Investigation of cell type conservation reveals a shared gene signature between glial cells of mouse, fly and octopus. Genes related to learning and memory are enriched in vertical lobe cells, which show molecular similarities with Kenyon cells in Drosophila. We construct a cell type taxonomy revealing transcriptionally related cell types, which tend to appear in the same brain region. Together, our data sheds light on cell type diversity and evolution in the octopus brain.

Optimization of Whole Mount RNA Multiplexed in situ Hybridization Chain Reaction With Immunohistochemistry, Clearing and Imaging to Visualize Octopus Embryonic Neurogenesis.

Gene expression analysis has been instrumental to understand the function of key factors during embryonic development of many species. Marker analysis is also used as a tool to investigate organ functioning and disease progression. As these processes happen in three dimensions, the development of technologies that enable detection of gene expression in the whole organ or embryo is essential. Here, we describe an optimized protocol of whole mount multiplexed RNA in situ hybridization chain reaction version 3.0 (HCR v3.0) in combination with immunohistochemistry (IHC), followed by fructose-glycerol clearing and light sheet fluorescence microscopy (LSFM) imaging on Octopus vulgaris embryos. We developed a code to automate probe design which can be applied for designing HCR v3.0 type probe pairs for fluorescent in situ mRNA visualization. As proof of concept, neuronal (Ov-elav) and glial (Ov-apolpp) markers were used for multiplexed HCR v3.0. Neural progenitor (Ov-ascl1) and precursor (Ov-neuroD) markers were combined with immunostaining for phosphorylated-histone H3, a marker for mitosis. After comparing several tissue clearing methods, fructose-glycerol clearing was found optimal in preserving the fluorescent signal of HCR v3.0. The expression that was observed in whole mount octopus embryos matched with the previous expression data gathered from paraffin-embedded transverse sections. Three-dimensional reconstruction revealed additional spatial organization that had not been discovered using two-dimensional methods.

De novo transcriptome of the diatom Cylindrotheca closterium identifies genes involved in the metabolism of anti-inflammatory compounds.

Diatoms are the most diverse and abundant group of phytoplankton species and represent a huge reservoir of marine natural products with possible application for human health. Several diatoms are known to have anticancer, anti-inflammatory, antioxidant and anti-microbial properties, but the compounds responsible of these activities are often still unknown. The diatom Cylindrotheca closterium showed anti-inflammatory properties inhibiting TNFα release in human monocytic leukemia cells. In this study, we present the full transcriptome of C. closterium, and used an -omic approach to identify transcripts coding enzymes that can be involved in the synthesis/degradation of anti-inflammatory compounds. This approach allowed to identify phosphatidylinositol-3-phosphatase, phosphatidylinositol 3-kinase catalytic subunit type 3, phosphatidylinositol N-acetylglucosaminyltransferase subunit A, monogalactosyldiacylglycerol synthase and violaxanthin de-epoxidase, which are known to be involved in anti-inflammatory compound metabolism. When C. closterium was cultured in silica-starvation conditions, selected as stress condition to potentially trigger the synthesis of bioactive metabolites, anti-inflammatory activity was lost and expression levels of the analyzed transcripts were reduced. These data suggested that the control culturing condition was the most active. This study used for the first time a transcriptomic-guided approach to identify enzymes involved in anti-inflammatory compound metabolism, directing future discoveries of marine natural products in microalgae.