Abstract
When challenged by adverse conditions such as thermal stress, many invertebrates can enter a period of prolonged dormancy, followed by revival once conditions improve. Among these branches, colonial ascidians can switch between a dormant state and an active state in response to environmental challenges for survival. Botrylloides diegensis, a colonial tunicate of the Tunicata subphylum, the closest invertebrate clade to the vertebrates, is capable of whole-body regeneration from a small fragment of vasculature within 8 days after amputation. Changes in seawater temperature back to the organism’s desired temperature range have established roles in inducing revival from dormancy; however, not much is known about the changes driving this process. This project sought to uncover the molecular and cellular mechanisms underlying revival from dormancy in B. diegensis.
Using in situ hybridisation on dormant tissues, expression of multiple thermosensitive channels, Trp, was identified in different cell types, including endothelial cells, phagocytes, cytotoxic cells, transport cells, mast cell-like cells, and undifferentiated cells. In addition, Trp expression was observed in different tissue structures such as the tunic, ampullae, and vascular lining.
A small chemical inhibitor, 2-aminoethoxydiphenyl borate, was used at 3 µM, 15 µM, and 30 µM concentrations to inhibit TRP channel activity and colony growth in different tissue states. Chemical inhibition modulated colony growth in a concentration-dependent manner, with 3 µM allowing colony growth, 15 µM suppressing colony growth, and 30 µM showing complete inhibition of growth.
Calcimycin, a calcium ionophore, was used to treat dormant colonies to observe morphological changes in response to calcium, as previous studies found that calcium signalling and ion binding genes were differentially expressed during the early stages of regeneration and wound healing. Continuous and transient exposure to calcimycin resulted in no observable morphological changes in dormant tissues of B. diegensis, as the fragments remained compact throughout the observation period.
Five candidate genes known to change transcripts in response to calcium (e.g., Wnta5, JNK, CamKII, Atf2, and Calr) were investigated using reverse transcription quantitative polymerase chain reaction, and existing single-cell RNA sequencing datasets, and bulk RNA- seq datasets. RT-qPCR analyses revealed that relative gene expression of Wnt5a was higher in the active state compared to the dormant state. Furthermore, Atf2 and Calr showed different results, with Atf2 showing increased expression at 1 h, while Calr revealed increased expression at the 24 h timepoint. Expression of JNK and CamKII expression remained the same in both timepoints. Existing scRNA-seq data identified the spatial expression of candidate genes in single-cell resolution, showing their presence across conserved cell clusters during whole-body regeneration and in adult tissues. Bulk RNA-seq revealed differential gene expression between two tissue states, hibernating and active zooids. Normalised counts of Wnt5a and Calr increased in active-zooids compared to hibernation, while Atf2 showed the opposite trend. In contrast, JNK and CamKII exhibited similar expression levels across both tissue states.
Altogether, this thesis provides a foundation for understanding the potential molecular and cellular processes underlying revival from dormancy in B. diegensis, contributing to the broader field of dormancy-revival research and offering insights into how environmental conditions influence gene expression.