The alien stem cells of Octopus vulgaris - From blood cell to neuron

Multicellular animals show diverse architectures of nervous systems, ranging from diffuse nerve nets in cniderians and sea-urchins to centralized nervous systems in annelids, arthropods and vertebrates. Cephalopods are often omitted from this list, but their brains are unusually big, centralized and connected when compared with other mollusks and invertebrates. How this is achieved and whether there is a common ground to building complex nervous systems, is currently unknown. Therefore, studying the mechanisms of neurogenesis across Bilateria can help to unravel how nervous systems have evolved. However, neurogenesis research in the last decades has largely focused on vertebrates (especially mice and fish) and arthropods (Drosophila). Indeed, data from species in the third major bilaterian clade, the Spiralia, are scarce and mostly originate from annelids. The lack of data from this clade, and especially from the molluscan phylum makes it impossible to reconstruct the ancestral state of the urbilaterian nervous system. Therefore, this thesis aims at inferring the cellular and molecular mechanisms that underlie neurogenesis in the cephalopod Octopus vulgaris. Most research on the central nervous system of cephalopods so far, has focused on morphological descriptions and behavioral paradigms. However, genomic and transcriptomic data published in the last 5 years, now allow studies on the molecular level. This thesis contributes to the establishment of Octopus vulgaris as upcoming model species, using traditional as well as state-of-the art techniques.

The first aim in this thesis was to establish a standardized system that allows off-shore development of octopus embryos. We also provided an updated atlas of O. vulgaris embryonic development and fused multiple staging scales of cephalopods, permitting unambiguous staging and easy comparison of developing octopuses, squids and cuttlefish. The second aim was to generate a molecular map of neurogenesis in the developing octopus embryo. By studying the expression of homologs involved in neurogenesis across bilaterians, we characterized the spatiotemporal patterning of the developing central nervous system in octopus. Specifically, we used in situ hybridization to map the expression of the HMG-box transcription factor soxB1, bHLH transcription factors ngn, ascl1 and neuroD, the RNA-binding protein elav and markers of maturing neurons zfh1, t3mo and slc6A1, during O. vulgaris organogenesis and maturation. This study showed that markers for neural progenitors (Ov-soxB1, Ov-ngn and Ov-ascl1) are expressed outside the developing cords, in the lateral lips that surround the eyes. Subsequent cell cycle studies (expression mapping of Ov-pcna and presence of phosphorylated histone H3) revealed absence of proliferating cells in the developing cords, but omnipresence in the lateral lips. These findings suggests that cells with a molecular fingerprint of neural progenitors are also dividing. Markers for differentiating neurons (Ov-soxB1, Ov-zfh1, Ov-neuroD, Ov-elav) on the other hand, were upregulated in the anterior and posterior transition zones, that connect the lateral lips to the central brain. Finally, expression of markers of post-mitotic neurons (Ov-elav, Ov-slc6A1, Ov-t3mo) was highest in the developing cords. Our preliminary lineage tracing study then showed that the neural progenitors in the lateral lips indeed give rise to cells in the central nervous system (at least the optic lobes), with progeny first passing through the posterior transition zone. This indicates extensive neural migration in the octopus embryo, unseen in other invertebrates. We also found that neuronal differentiation starts early in embryonic development, with Ov-elav and Ov-t3mo expressing cells present at Stage IX and Stage XI, respectively. In addition, the O. vulgaris brain is still immature at hatching, with high level Ov-elav expression in all lobes. Taken together, this thesis provides novel insights into the neurogenic process in Octopus vulgaris, which will be crucial for understanding octopus and cephalopod neurogenesis, and by extension evolution of bilaterian neurogenesis.