Understanding the Establishment of TWIST Neural Crest Gene Regulatory Systems and Their Evolutionary Significance in Chordates

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Presenter(s)

Austin Katzer

Abstract or Description

This project will focus on evolutionary aspects of the neural crest in chordate organisms and try to help solve the question of how the neural crest emerged. The main focus of the experiment is to determine what kind of mutations integrated twist in the neural crest gene network. This project focuses specifically on mutations within twist cis-regulatory elements (CREs) that gave way to its involvement in neural crest cells. The working hypothesis is that the CREs controlling twist expression in neural crest cells are indeed different than those regulating twist expression in other regions within the developing organism. However, whether they are completely new or emerged from existing enhancer sequences is unknown. A new CRE would consist of a novel sequence that is not present invertebrate chordates and only acts in neural crest cells. Alternatively, an existing CRE could have been modified to permit twist expression in a novel tissue other than the ancestral mesodermal pattern. By identifying putative CREs using conserved teleost sequences outside of the coding region for twist it is possible to functionally test these elements using standard transgenic techniques. These sequences of 1000-1500 base pairs were tested functionally within zebrafish using gateway cloning to introduce the fragments into the reporter construct pGreene, which has a cFos basal promoter and eGFP flanked by Tol2 recombination arms. Injecting these constructs into single-cell embryos highlights twist CRE activity by driving fluorescent signal in a given region of the embryo revealing how the neural crest gene network was co-opted.