Date of Graduation

5-2024

Document Type

Thesis

Degree Name

Bachelor of Arts

Degree Level

Undergraduate

Department

Biological Sciences

Advisor/Mentor

Nakanishi, Nagayasu

Committee Member/Reader

Kral, Timothy Alan

Committee Member/Second Reader

Alwood, Nancy

Committee Member/Third Reader

Brown, Mitch

Abstract

Due to their unique phylogenic position as sister to Bilateria, Cnidaria are often credited with the utility of allowing for reconstruction of ancestral biology based on characteristics shared with bilaterians and other animals. This factor makes investigation into the nervous systems of cnidarians critical in understanding early neural evolution. Wamides, a class of neuropeptides, have been shown to play a regulatory role in life cycle transitions across many different species. The cnidarian specific Wamide neuropeptide, GLWamide, has previously been identified to play an accelerator role in the metamorphic timing of a specific species of sea anemone, Nematostella vectensis. However, the mechanism by which GLWamide signaling regulates developmental timing remains unknown. Preliminary data has established the ETS gene as a particular gene of interest. This transcription factor-encoding gene was identified as a downstream target for GLWamide signaling. Thus, an ETS-like transcription factor serves as a candidate molecule that controls life cycle transition upon the activation of GLWamide signaling in this sea anemone species. Here, we used CRISPR/Cas 9-mediated targeted mutagenesis to create ETS knock-out mutant lines in order to evaluate phenotypic differences that were induced by this partial loss-of-function gene. We established two heterozygous mutant lines. The first line carries an allele of a targeted transgenic cassette via meganuclease (I-SceI)-assisted integration, while the other carries an allele containing ETS site-specific DNA modifications induced through CRISPR/Cas 9-mediated transgenesis. In this line, we found two distinct mutation patterns within our ETS F1 samples that could potentially alter the protein synthesis occurring in the N. vectensis genome. In addition, in-situ hybridization was used to evaluate the expression pattern of ETS transcription factor during N. vectensis development. Early life cycle stages (namely the planula stage) demonstrated little to no expression. However, expression increased throughout later life stages, with endodermal expression seen in the tentacle budding stage, and endodermal and region-specific expression seen in the primary polyp stage. Upon analysis of the expression pattern seen, evidence suggests this transcription factor could be heavily involved in the tentacle formation and development processes in N. vectensis. Based on these results, we hypothesize that this ETS-like transcription factor could control life cycle transitions in N. vectensis upon activation of GLWamide signaling, though further research is required to verify our findings. The establishment of the two mutant lines discussed here will set the foundation for the generation of homozygous null mutants, which will be found in the F2 generations. These mutants will be examined for phenotypic differences in developmental timing, the pattern of tentacular morphogenesis, and/or the pattern of postembryonic cell type development to better assess the ETS gene function in N. vectensis.

Keywords

cnidarian neuropeptides, life cycle transition, GLWamide signaling, ETS gene, transcription factor, gene expression

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