Directed differentiation of embryonic stem (ES) cells to a particular cell type requires an understanding of the mechanisms underlying the development of that cell type. Genes critical for lineage specification in embryogenesis can be manipulated in ES cells to direct their differentiation. Several genes have been identified as key players in the development of midbrain dopaminergic neurons, including NURR1 (NR4A2), which is essential for the genesis and survival of midbrain dopaminergic neurons. Little is known, however, about the mechanisms by which this gene is regulated. We set out to identify transcription factors that regulate NURR1 expression so that their utility in directing the differentiation of ES cells and/or embryonal carcinoma cells (ECs) into dopaminergic neurons could be assessed.

We chose to study NURR1 regulation in zebrafish due to their rapid development and relative ease of transgenesis. We identified two zebrafish orthologs of NURR1, nr4a2a and nr4a2b . By comparing the expression patterns of these genes to markers known to be found in mammalian midbrain dopaminergic neurons (tyrosine hydroxylase, the dopamine transporter, and Pitx3), we determined that the dopaminergic neurons in the ventral diencephalon of the zebrafish correspond to the dopaminergic neurons found in the mammalian midbrain. We then sought to identify cis-acting regulatory sequences that mediate NURR1 transcription. We identified thirty putative NURR1 regulatory elements based on their conservation across mammalian species and tested these elements for function using in vitro luciferase assays and transgenic zebrafish. We identified four sequences that effectively drove reporter gene expression in nr4a2-expressing tissues in transgenic zebrafish embryos. Notably, the sequence that drove the strongest expression in the zebrafish ventral diencephalon was the NURR1 promoter.

To identify critical subregions within the NURR1 promoter, we generated a systematic series of truncated promoter constructs that we evaluated in luciferase assays. This was followed by transcription factor binding site identification and mutagenesis, electrophoretic mobility shift assay, and finally siRNAs to identify specific transcription factors. We demonstrate that CUX1 and ZIC3 directly repress NURR1 transcription, suggesting their potential value to the improvement of methods to differentiate ES cells into dopaminergic neurons for cell-based therapies.