Analysing the process of whole body regeneration in Botrylloides leachi
Regenerative capacity differs greatly across organisms, with many species only being able to regenerate a limited number of cell types. Unfortunately, from an evolutionary perspective, regenerative power correlates inversely with tissue complexity, thus leaving more complex body systems such as humans with a very limited renewal response. An exception to this is the ascidian Botrylloides leachi, the closest related chordate to the vertebrate clade that can undergo whole body regeneration (WBR). This filter feeding marine animal, more commonly known as a sea squirt, can regenerate an entire new adult within 10-14 days from a miniscule piece of vascular tissue. Although its body plan appears rather simplistic, this invertebrate has tissue complexity as well as organs close to that of vertebrates, including a cerebral ganglion, heart, muscles and nerves. While other tunicates are also used for regeneration studies (e.g. Ciona intestinalis, Botryllus schlosseri), B. leachi is the only ascidian to retain its WBR potential throughout its lifecycle. Despite these unique properties, very little is understood about the cellular and molecular biology behind B. leachi’s regeneration process. This thesis analysed the process of WBR in B. leachi through next generation sequencing. RNA was collected and sequenced at key stages during the regeneration process. The transcriptome of B. leachi tissue undergoing WBR was analysed using differential gene expression, gene ontology and pathway analyses. We observed up-regulation in the expression of genes involved in wound healing. Known developmental pathways including WNT, TGF-β and Notch also had differentially expressed genes during the earliest stages of WBR. Later in WBR, the expression patterns in several pathways required for protein synthesis, biogenesis and the organisation of cellular components were up-regulated. Novel regeneration specific genes were also identified and functional siRNA experiments were carried out to determine if Bl-Wnt and Bl-RIG-IR-like were key to the regeneration process. Knockdown of both genes showed phenotypic changes to the normal regeneration process, however these putative results need to be further validated. Lastly, chromatin modifiers showed dynamic expression patterns throughout the regeneration process and were investigated further. Chromatin modifiers regulate chromosome conformation and thus can alter gene expression without change to the DNA sequence. As regeneration requires major changes to gene expression, this could be driven by histone modification and DNA methylation. An inhibitor experiment was carried out that demonstrated that histone deacetylase I/II activity was essential for B. leachi WBR. This thesis represents the first global overview of the genes and pathways whose expression is regulated during WBR in B. leachi by de novo transcriptomics.
Advisor: Wilson, Megan; Gemmell, Neil
Degree Name: Doctor of Philosophy
Degree Discipline: Genetics
Publisher: University of Otago
Keywords: Botrylloides; Leachi; Regeneration
Research Type: Thesis