Neurogenese en neurale patroonvorming in de octopus arm

Coleoidea is a particularly notable subclass of phylum Mollusca as its members, such as octopuses, cuttlefishes and squids, evolved the largest nervous system amongst invertebrates and possess a wide range of behaviors. The total number of neurons in an octopus nervous system is equivalent to that of a small mammal. One-third of the neurons in an adult octopus nervous system are in the brain, while the rest are distributed in the periphery, mainly in the arm crown. The arm crown is a novel evolutionary feature in coleoid cephalopods. How the nervous system develops in an octopus arm crown is still a mystery. To understand the embryonic nervous system development in an octopus arm, it is vital to decipher the regionalization of different tissues which form the arms during embryogenesis and study the intrinsic and extrinsic factors regulating neuronal and glial cell differentiation. The first aim was to develop a molecular tool to study gene expression patterns of selected marker genes in three dimensions (3D), as the complexity of an octopus arm cannot be documented in two dimensions. Therefore, we optimized a protocol for whole mount in situ hybridization chain reaction (WMHCR) in combination with immunohistochemistry and aqueous-based tissue clearing in the common octopus (Octopus vulgaris) embryos to study embryonic neurogenesis. Moreover, we developed an automated tool, Easy_HCR, for HCR probe design and processing. We assessed the precision of visualization of neural anatomy between the 3D rendered gene expression imaging based on WMHCR with 3D view obtained via manual segmentation based on DAPI nuclear staining. Furthermore, we analyzed the robustness and clearing performance of 4 different aqueous-based clearing protocols and their compatibility with WMHCR on octopus embryonic and paralarval tissues. The second aim was to unravel the embryonic nervous system development and neural patterning in embryonic octopus arms from a molecular perspective. We investigated the temporospatial expressions of different neurogenic, gliogenic, and myogenic marker genes. We demonstrated a strong resemblance between neurogenesis in octopus arms and peripheral neurogenesis observed in arthropods. Furthermore, we observed that, initially, the cell proliferation covers the entire arm bud; however, as the arm develops during organogenesis, proliferation is confined to the lateral neurogenic zones, and towards the end of the embryogenesis, it diminishes. We also identified two putative neuropil glial subtypes with different distribution. Additionally, we discovered the spatial patterning of the previously unidentified myogenic zones, which gradually place themselves in the octopus arm tips during the embryogenesis. Finally, we revealed that disturbing notch signalling increases neurogenesis and decreases glial differentiation, which is similar to roles of Notch in the cephalopod retina, and in fly and vertebrate neurogenesis.