Abstract
Regeneration is the renewal, regrowth, or restoration of a body part, tissue, or whole body. The ability to fully restore the function or structure of a tissue is restricted to a small number of taxa. However, most can facilitate tissue repair through adult stem cells. There is generally a relationship between an animal’s ability to regenerate and its morphological complexity, where the more complex the body plan, the less likely it will have the ability to regenerate. One exception is Botrylloides diegensis, an invertebrate that is the closest related chordate to the vertebrate clade. This species can undergo whole-body regeneration (WBR). This marine species can regenerate from a single fragment of 0.1 mm of a blood vessel that contains only a few hundred cells to form a fully functioning zooid within 8 days. For regeneration to occur, an injury must result in only vascular tissue with no adult structures remaining; if any adult structures remain, the colony will undergo wound healing. Very little is understood about the cellular and molecular biology of whole-body regeneration. Not much is known about the triggers of WBR except the presence or absence of adult zooids. The progenitor cells that drive WBR have been hypothesised to arise in two possible ways, either; (1) there is a stem cell niche within the vascular lining that becomes active and migrates to the vascular lumen when regeneration is induced, or (2) a small circulatory population of pluri- or multipotent cells exist.
Early gene expression was investigated through RNA-Sequencing of 1, 3, 5, and 10 h post-injury regenerating and wound healing fragments. Differential expression analysis showed a range of developmental signaling pathways involved in the early stages of regeneration compared to wound healing, including calcium signaling, Wnt and non-canonical Wnt signaling. Further investigation showed that inhibition of calcium signaling resulted in decreased formation of regeneration niches from which the adult will form. In contrast, calcium activation resulted in multiple regeneration niches developing or forming while an adult is already present in the colony. Investigation of the non-canonical Wnt signaling pathway through RT-qPCR, in situ hybridisation and inhibition, revealed the expression of associated genes in the developing buds during asexual reproduction. This expression, combined with the inhibition of JNK, which showed disorganisation of the developing buds during asexual reproduction, may indicate a role of the non-canonical pathway during asexual reproduction. The temporal and spatial expression of pluripotency genes was investigated using phylogenetics, RT-qPCR, and in situ hybridisation to expand current stem cell knowledge.
This thesis is the first to investigate the early timepoints of regeneration and compare them to wound healing to begin to understand the triggers and pathways essential for regeneration. This research has contributed to the establishment of a model organism for regeneration. Regeneration research has many exciting implications about evolution, gain and loss of regenerative potential, and transferable insights into human medicine that may prove hugely beneficial.