In embryogenesis, the transformation of a single-cell to a multicellular organism is the preeminent example of coordinated, fluctuating gene expression controlled by heavily regulated intercellular communication. This is directed by several signaling pathways such as the Notch Signaling Pathway. Notch is contact mediated and has been shown to be involved in differentiation, survival/apoptosis, proliferation, and the cell cycle. Primary target genes of Notch include the hairy and enhancer of split (HES) transcription factor family. Hes1 regulates the cell cycle, specifically in the timing of cell fate and differentiation. Studies point to intertwined roles between Hes1 and Notch in taste bud development. Understanding the relationship between Notch and taste bud marker genes, such as calretinin (CALB2), at critical stages in taste bud development may help reveal their function. Ambystoma mexicanum, or axolotl, is a great candidate for modeling taste bud patterning and differentiation. The goal of my honors thesis was to examine Notch in taste bud development through quantification and location of Hes1, Hes6, and CALB2. First, a CALB2 primer for RT-qPCR was successfully designed and employed to generate a gene expression profile during taste bud development in oropharyngeal endoderm (OPE) explants. These results confirm CALB2 expression is an excellent reporter gene for quantification studies during taste bud development. Second, quantification and location of Hes1, Hes6, and CALB2 mRNA was studied using in situ hybridization (ISH). Two approaches for (ISH) were explored: first through synthesizing a self-designed DIG riboprobe and second by utilizing ACD’s RNAscope Double Z probe technology and proprietary protocol. Results yielded from RNAscope show promise for future ISH attempts to study Hes1, Hes6, and CALB2 with this technology.
Pfennig, Sarah, "Understanding the Notch Signalling Pathway: Connecting Hes Gene Expression to Taste Bud Development in Ambystoma mexicanum" (2019). Biology Honors Papers. 28.
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