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The cells that give rise to melanoma, melanocytes, originate from a pluripotent population of embryonic cells called the neural crest (NC). During embryogenesis, neural crest cells undergo an epithelial to mesenchymal transition (EMT) allowing them to leave the neuroepithelium, invade underlying tissues and migrate to their terminal locations. The microphthalmia-associated transcription factor (MITF) has been identified as the master regulator of melanocyte fate and as being actively repressed in other neural crest cell lineages. Previous studies suggest that varying levels of MITF drive cell invasion and the progression of melanoma. Understanding the ways in which MITF expression is gulated during development may further elucidate the mechanisms that promote oncogenesis. In zebrafish, melanocytes share a bipotent precursor with iridescent iridophores, another NC-derived pigment cell. Aristaless-like transcription factors (ALX), alx4a and alx4b, have recently been implicated in promoting iridophore fate, possibly by repressing mitfa expression. Our lab used CRISPR/Cas9 to knockout alx4a in zebrafish and found that alx4a mutants did not develop iridophores. Based on these findings, the goal of my research project was to confirm that the resulting mutant phenotype is in fact due to the loss of alx4a and further investigate the role of alx4a in promoting iridophore fate. To confirm that the loss of iridophores was due to the loss of alx4a, I performed rescue experiments using a sox10 promoter to drive alx4a cDNA in all neural crest cells. To determine if alx4a is sufficient to drive iridophore fate, I performed overexpression experiments using an hsp70l promoter to drive alx4a cDNA throughout the embryo at 25 hpf. Whole mount in situ hybridization (WISH) was used to examine and locate where alx4a is expressed at different developmental stages. Counts of mitfa-egfp cells were lastly obtained to assess the expression of melanophore precursors in alx4a mutants. Understanding the underlying mechanisms that promote iridophore specification and repress melanocyte fate, may potentially highlight significant future therapeutic treatments for neural-crest derived diseases like malignant melanoma.
Cepeda, Yenelsy, "Investigating the role of alx4a in zebrafish iridophore development" (2022). Honors Projects. 210.
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