Gene Nr2e1 as a possible stem CELL/PROGENITOR marker in the zebrafish (Danio rerio) pituitary

Abstract

This project aims to test the hypothesis that Prop1 regulates Nr2e1 expression that could be responsible for pituitary proliferation and differentiation. Nr2e1 is an orphan nuclear hormone receptor that is essential for neural stem cell maintenance and neogenesis, but none is known in pituitary. Ames dwarf mice harboring spontaneous mutation in Prop1 gene have congenital hypopitutiarism with deficiency in, LH/FSH PRL, TSH and GH associated to pituitary dysplasia/hypoplasia. Nr2e1 expression is increased in the pituitary of Ames mice. The available Nr2e1 knockout mouse, fierce, is expensive to import and maintain that make is unfit in our studies. Instead, I choose zebrafish to test the hypothesis due to low costs of genetic manipulations and ease maintenance. The FAPESP supported masters scholarship project to be carried out in Brazil, initially proposed to use morpholino to knockdown prop1 gene in order to evaluate Nr2e1 expression and then knockdown Nr2e1 to evaluate its targets. The morpholino approach is prone to non-specific effects and is only effective for a maximum of 7 days due to growth-related dilution. A better alternative to evaluate adult phenotype is CRISPR/Cas assay. This new genome-editing tool allows a specific disruption and single base replacement in the gene of interest, resulting in permanent, heritable mutations. Although the technique has been widely used in others countries, such expertise has not been available in Brazil. I therefore propose to generate mutations in Prop1 and Nr2e1 using CRISPR/Cas in a US lab with such expertise to study their function during periods with pituitary metabolic demand in animals with congenital hypopituitarism.Keywords: Zebrafish, CRISPR/Cas, congenital hypopituitarism, Prop1, Nr2e1. (AU)