Problems in the development of the neural crest (NC) can cause abnormalities in the skin and craniofacial structure such as cleft palette. The neural crest cells (NCs) are pluripotent and derive from the ectoderm to form cartilage, bone, connective tissue, sensory neurons, glia, and pigments cells amongst many other cell types and tissues.  NC differentiation is induced in the ectodermal germ layer during gastrulation.  A transcription factor is a gene that activates the expression of another gene in the gene regulatory network.  The activation of transcription factor activator protein 2 (Tfap2) causes the induction of the NC.  Tfap2 is essential in the control of cell induction, differentiation, survival and proliferation in various developmental contexts, including the neural crest. The lack of expression of Tfap2a and Tfap2c results in the loss of neural crest development. 

Our work in the lab is focused on identifying if any Zebrafish Regulatory Elements (ZRE) act as a possible enhancer. Zebrafish Regulatory Elements are downstream of our gene of interest, Tfap2. If the ZREs are possible enhancers, they regulate by binding with Tfap2a and Tfap2c. In order to identify the specific ZREs responsible for regulation, we are testing the ZRE genetic sequences through amplification using polymerase chain reaction (PCR).  We hope to perform Gibson Assembly transformation to insert the ZRE sequence into a plasmid containing green fluorescence as a measurement marker.  If we are successful and the specific ZREs is a neural crest enhancer, we will see fluorescence in the melanocytes.