Abstract
Different organisms have varying regenerative capacities, some species are only able to regenerate several cell types, while others can regrow organs or multiple tissues. This is predicted to be a result of the inverse correlation between tissue complexity and regenerative ability. Therefore, more complex organisms, such as humans are weaker regenerators especially compared to species like Botrylloides diegensis. B. diegensis is a marine colonial chordate/ascidian that can complete whole-body regeneration (WBR) in ~2 weeks from a piece of vascular tissue. Ascidians are a part of the tunicate clade, the closest invertebrate relative to vertebrates. The renewed adult body (zooid) fully restores tissues/organs like the neural complex, stomach, intestine, heart, germline, and branchial tissues in a short time. In this thesis, the mechanisms of B. diegensis WBR were explored using single-cell highthroughput sequencing and ATAC-seq combined with the histological investigation of WBR of Botrylloides species. I first conducted DNA barcoding analysis on the colonies collected from seven sampling points of Aotearoa which confirmed our model organism as B. diegensis (previously named, B. leachii), along with the identification of two novel records, B. anceps and B. jacksonianum. Through monitoring and histological analysis, the WBR contents of these three species were exposed comparatively. Twin-body regeneration of B. anceps was elucidated histologically along with the single zooid regeneration of B. jacksonianum, both demonstrated significant similarities in regeneration bud structure and cellular phenotypes of the regeneration vesicle forming cells. Cell transcriptome profiles of individual cells were calculated from specific regeneration points (stages 1, 2 and 4). Marker genes were predicted based on their expression levels and specificity for certain clusters. SoxC was hypothesised to be a stem cell marker, whereas Alkbh8 was predicted to be a germline marker. Transitionary cell states were observed during early regeneration which were reduced in the later WBR process. Trajectories were consistent with these hypotheses. Zooid tissue markers were identified by profiling the intact whole colony during stage A of blastogenesis. Genes like Ctrb1 and Kng1 were particularly expressed in the stomach, and Cnfn and Tubb were upregulated in the branchial tissues. Alkbh8+ cells were found in the ovary and oocyte, in situ, consistent with the WBR data. Additionally, chromatin accessibility profiling was performed using ATAC-seq for stages 0-2 and following histone deacetylation inhibition (HDACi) during early WBR. Part of the motivation for the ATAC-seq study was due to the dynamic changes observed in histone variants and the previous work showed HDACi interrupts WBR. ATAC-seq confirmed changes to chromatin accessibility and supported that HDAC activity was associated/required for regeneration, as the differentially accessible regions were associated with genes with known roles in WBR in Botrylloides and other animals. Peak signals were linked to diverse mechanisms such as Ddx58 signalling which resulted to be affected significantly because of HDACi. FoxG-motif enriched for the regions with decreased accessibility. This might be due to FoxG binding to those regions and working with HDACs. Gata3 was predicted to be a TF that might play a key role in WBR. Overall, this thesis provides the first single-cell and accessibility atlas of B. diegensis during WBR combined with an evolutionary perspective on Botrylloides species and their WBR.