Ali Murat Elagoz

Research on cephalopod early ontogeny has significantly advanced in recent decades, including embryo organogenesis and neurogenesis and early behavioural adaptations, particularly in commercially important and coastal species. Within this context, here we compiled current knowledge on the collection, handling and care under experimental conditions and monitoring in the field of cephalopod eggs and egg masses. It covers field observations and egg collection methods, as well as laboratory incubation protocols for eggs maintained with and without maternal care. It is emphasized how abiotic and biotic factors, including temperature, salinity, oxygen and maternal condition, shape embryonic development and hatchling survival and viability. Monitoring methods for cephalopod egg masses in the wild and the effects of natural threats such as storms and predators are reviewed. Anthropogenic impacts such as pollution, fisheries and climate change are also discussed. Technological advances have enabled finer analysis of neural development as the embryo grows, while ethical considerations regarding embryonic sentience capacity and welfare conditions are becoming central to current experimental designs. Based on current knowledge, it is recommended to apply ethical considerations and the 3Rs principle (Replacement, Reduction, Refinement) for cephalopod embryos following the onset of organogenesis. Thus, a list of potential indicators of health and welfare that could be used for assessing and monitoring cephalopod embryos and hatchlings is proposed. Overall, this synthesis aims to serve as a guide for advancing egg collection and laboratory incubation methodologies, as well as adopting ethical handling protocols to improve cephalopod embryo care and quality.

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.

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.

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 whole-mount 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 an 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.

Octopuses are mollusks that have evolved intricate neural systems comparable with vertebrates in terms of cell number, complexity and size. The cell types within the octopus brain that control their amazingly rich behavioral repertoire are still unknown. Here we profile cell diversity of the paralarval Octopus vulgaris brain to build a comprehensive cell type atlas that comprises mostly neural cells, as well as multiple glial subtypes, endothelial cells and fibroblasts. Moreover, we spatially map cell types within the octopus brain, including vertical and optic lobe cell types. Investigation of cell type conservation reveals a shared gene signature between glial cells of mice, fly and octopus. Genes related to learning and memory are enriched in vertical lobe cells, which show molecular similarities with Kenyon cells in Drosophila . Taken together, our data sheds light on cell type diversity and evolution of the complex octopus brain. Highlights & Key findings: Characterization of different cell types present in the early paralarval brain Cross-species comparisons reveal a conserved glial gene expression signature Vertical lobe amacrine cells in octopus have molecular similarities to fly Kenyon cells Homeobox genes are defining transcription factors for cell type identity Recently expanded gene families may underlie cellular diversification

Cephalopods have evolved nervous systems that parallel the complexity of mammalian brains in terms of neuronal numbers and richness in behavioral output. How the cephalopod brain develops has only been described at the morphological level, and it remains unclear where the progenitor cells are located and what molecular factors drive neurogenesis. Using histological techniques, we located dividing cells, neural progenitors and postmitotic neurons in Octopus vulgaris embryos. Our results indicate that an important pool of progenitors, expressing the conserved bHLH transcription factors achaete-scute or neurogenin, is located outside the central brain cords in the lateral lips adjacent to the eyes, suggesting that newly formed neurons migrate into the cords. Lineage-tracing experiments then showed that progenitors, depending on their location in the lateral lips, generate neurons for the different lobes, similar to the squid Doryteuthis pealeii. The finding that octopus newborn neurons migrate over long distances is reminiscent of vertebrate neurogenesis and suggests it might be a fundamental strategy for large brain development.

Cephalopods have evolved nervous systems that parallel the complexity of mammalian brains in terms of neuronal numbers and richness in behavioral output. How the cephalopod brain develops has only been described at the morphological level, and it remains unclear where the progenitor cells are located and what molecular factors drive neurogenesis. Using histological techniques, we located dividing cells, neural progenitors and postmitotic neurons in Octopus vulgaris embryos. Our results indicate that progenitors are located outside the central brain cords in the lateral lips adjacent to the eyes, suggesting that newly formed neurons migrate into the cords. Lineage tracing experiments then showed that progenitors, depending on their location in the lateral lips, generate neurons for the different lobes. The finding that octopus newborn neurons migrate over long distances is reminiscent of vertebrate neurogenesis and suggests it might be a fundamental strategy for large brain development.

Diabetic nephropathy (DN) is the primary cause of end-stage renal disease, worldwide, and oxidative stress has been recognized as a key factor in the pathogenesis and progression of DN. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has the most important contribution to reactive oxygen species generation during the development of DN. Bioactive compound use has emerged as a potential approach to reduce chronic renal failure. Therefore, a red orange and lemon extract (RLE) rich in anthocyanins was chosen in our study, to reduce the toxic renal effects during the development of DN in Zucker diabetic fatty rat (ZDF). RLE effects were examined daily for 24 weeks, through gavage, in ZDF rats treated with RLE (90 mg/kg). At the end of the experiment, ZDF rats treated with RLE showed a reduction of the diabetes-associated up-regulation of both NOX4 and the p47-phox and p22-phox subunits, and restored the BAX/BCL-2 ratio respect to ZDF rats. Furthermore, RLE was able to reduce the oxidative DNA damage measured in urine samples in ZDF rats. This study showed that RLE could prevent the renal damage induced by DN through its capacity to inhibit NOX4 and apoptosis mechanisms.

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.