Cyclical neurogenesis and neurodegeneration in the colonial tunicate Botryllus schlosseri

In this work, we studied the nervous and sensory system of the colonial ascidian Botryllus schlosseri during the development of embryos, buds, and in adult individuals belonging to young and old colonies. B. schlosseri is a colonial ascidian in which individuals (blastozooids) are organized in star-shaped systems. In a colony, sexual and asexual reproductions are coordinated and cyclical. Moreover, three generations of zooids coexist: adults (filter-feeding individuals), their buds (primary buds), and the budlets (secondary buds) produced by buds. Cyclically, during a phase called takeover, adult zooids are reabsorbed by the colony and are replaced in physiological activities by their buds that become adults. Cuncurrently, budlets mature into buds and produce a new generation of budlets. We first reviewed historical studies concerning B. schlosseri with the intent of characterizing the colony life cycle and bud development. We then compared nervous system formation in two different developmental pathways, embryogenesis and blastogenesis. Since B. schlosseri has internal fertilization and development, we developed a method for culturing embryos in vitro. Using a combination of in vivo, confocal, histological observations, and 3D reconstructions based on serial sections, we described the embryonic development and drafted a timetable. Next, we sequenced transcriptomes of embryos and buds at several stages and illuminated the strict temporal relationship between morphogenetic events and the expression pattern of genes associated with the nervous system during the formation of the larval brain, its degeneration at metamorphosis, the adult brain formation in embryo and bud, and its degeneration at takeover. We also studied the nervous system in adult individuals belonging to colonies of different ages. We observed that the number of brain cells changes throughout the adult zooid life following a specific trend. Transmission electron microscopy and TUNEL assays on adult brains showed that apoptosis is involved in neurodegeneration and the number of immunocytes contacting or infiltrating the brain increase in number during the adult life. Changes in brain cell number parallel changes in sensory cell number. We developed two novel behavioural experiments for B. schlosseri, which showed that zooid ability to respond to mechanical stimuli parallels changes in the number of brain and sensory cells. Then we compared adult individuals belonging to young and old colonies and found that aging influences both nervous system morphology and behaviour. We analysed differentially expressed genes in brains of individuals belonging to young and old colonies and found that the old colonies exhibit a gene pattern associated with several human neurodegenerative diseases, such as the Alzheimer’s disease. Finally, we studied, the coronal organ, a mechanoreceptor located on the tentacles, in the oral siphon. We analysed its cytodifferentiation during asexual reproduction and we documented the mechanosensorial impairment caused by gentamicin at morphological and behavioural level. In mammals gentamicin can destroy both hair cells and their innervating neurons. The pre-treatment with fenofibrate, a gentamicin protector, followed by a treatment with gentamicin, resulted in no significant effect on animal behaviour. These results support the hypothesis of homology between vertebrate hair cells and tunicate coronal sensory cells. In conclusion, the research presented here shows that B. schlosseri can be considered a useful model species for analysing the development of the central nervous system and sensory system, as well as its degeneration as caused by drugs, metamorphosis, takeover and aging. Additionally, the species’ different developmental pathways allow for interesting evolutionary comparisons, at both the morphological and molecular level, that can help improve scientific understanding of the origin of the animal phenotype