The evolution of the vertebrate head in chordate organisms involved genetic mutations that permitted new gene interactions of the existing developmental toolkit. Compared to other phyla, the vertebrate head contains complex somatosensory receptors, as well as innovative mechanical and sensory structures. The emergence of these structures resulted from the evolution of the neural crest, a transient cell population exclusive to vertebrates, with qualities such as pluripotency, the capacity for a cell to give rise to other cell types, as well as the ability to delaminate and migrate. This ectodermal cell type can give rise to a multitude of adult structures including those that are putative mesoderm derivatives, such as craniofacial bone and cartilage. This project focused on the evolutionary aspect of the neural crest in chordate organisms addressed the question of how the neural crest emerged. The twist gene is essential in chordate development and encodes a basic transcription factor that plays an important role in the development of embryos. An experiment was conducted to determine what kind of mutations may have integrated twist in the neural crest gene network. This project focuses specifically on mutations within twist cis-regulatory elements (CREs), non-coding DNA sequences that regulate transcription of genes, that may have facilitated its involvement in neural crest cells. Our current understanding is that the CREs controlling twist expression in neural crest cells are different from 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 in 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 2 twist it is possible to functionally test these elements using standard transgenic techniques. The putative cis-regulatory element sequences of around 1000-2000 base-pairs in length were tested functionally within zebrafish using the gateway cloning method 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 along with transcriptase into single-cell embryos highlights twist CRE activity by driving fluorescent signals in a given region of the embryo revealing how the neural crest gene network was co-opted, the expression of new function derived from an old gene.