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Description
There are two main objectives of this dissertation, which were explored in the marine invertebrate chordate developmental model Ciona intestinalis. First, to identify new genes involved in epidermal sensory neurons (ESN) of the ascidian peripheral nervous system (PNS) and to infer their function. To do this, we performed a comparative RNA-sequencing (RNA-SEQ) experiment on wild type embryos, and embryos that overexpressed an important regulatory gene, Pou4, that is involved in the ESN regulatory network. Ectopic expression of Pou4 in the epidermis converts epidermal cells into ESNs and comparing these embryos to wild type embryos will allow us to discover Pou4 gene targets, as they will be differently expressed. We identified over 2000 differentially expressed genes which are important components of the mechanoreceptor sensory hair cells found in the vertebrate inner ear supporting my hypothesis that ESN are evolutionarily similar to vertebrate mechanoreceptors. Second, we aimed to establish the fundamental molecular tool of RNA interference (RNAi) in ascidians. This was achieved by generating artificial microRNA (miRNA) hairpins to target genes of interest and, therefore, disrupt their function. We were successful in effectively silencing three different genes which resulted in expected phenotypes, one of which was Pou4. Artificial hairpins targeting Pou4 caused a decrease in the Pou4 mRNA and cilia of ESN, of which Pou4 is an important factor in their development. Performing RNAi in C. intestinalis through the use of artificial miRNA hairpins will serve as an instrumental technique to add to the toolkit available in this developmental model. This dissertation work is significant for the ascidian model in that it aims to determine the function of ESN, suggesting that they are a model for chordate mechanorecptors. Furthermore, this work establishes an important tool for silencing genes of interest in ascidians. This work will further propel C. intestinalis as an ideal model organism for studying developmental biology.
