7379 Shining Light on the Teleost Pituitary: Generating a Zebrafish Prop1 Reporter

Abstract Disclosure: J.J. Martínez Mayer: None. M. Balakrishnan: None. D. Stainier: None. M. Perez-Millan: None. Anterior pituitary gland development is conserved across vertebrates, with zebrafish possessing all hormone-producing cell populations found in mammals. The transcription factor PROP1 is the primary transcription factor defining the anterior pituitary hormone-producing lineages, and PROP1 mutations in mammals cause congenital hypopituitarism. Here, we aimed to establish a transgenic zebrafish line to study pituitary development by tracking prop1+ cells in vivo. We first decided to use Tol2 mediated transgenesis to express EGFP under a prop1 promoter region. To define which sequences drive prop1 expression, we looked for open chromatin marks (H3K4me1) and interspecies conservation using the UCSC Genome Browser Zv9/danRer7. We found a conserved element 6 kb upstream of the promoter as well as a conserved sequence in the first intron, with a putative GLI2 binding site. Because these two regions were separated by a highly repetitive sequence and extended over 10kb, three different constructs were tested separately: 1) 2kb upstream conserved sequence (construct 1); 2) upstream promoter region up to 6 kb upstream (construct 2 - which includes construct 1) and 3) a minimal promoter (500 bp) plus exon 1 and intron 1 (construct 3). After microinjection we observed transient EGFP expression in pituitary cells at 30 and 48 hpf in transgenic animals for constructs 1 and 3, respectively. Transgenic zebrafish for construct 2 displayed no EGFP expression. These results suggest that the repetitive region in the prop1 promoter may act as a silencer and that prop1 expression relies on several genetic elements that can not be easily captured in a single construct. These observations led us to opt for a knock-in approach, which would recapitulate prop1 expression more faithfully. Using CRISPR-Cas9 coupled with modified double stranded DNA donors, a P2A-GAL4-VP16 construct was inserted into exon 2 of prop1. After founder screening by PCR, positive fish were crossed with UAS:mCherry-CAAX to enable live imaging of early pituitary development. The fluorescent signal was detectable from 26 hpf and persisted until day five, which was the final time point assessed. We performed live-imaging of early anterior pituitary development in prop1 reporter zebrafish at high resolution. Interestingly, we found a previously undescribed population of prop1 expressing cells that resides outside the pituitary gland. This population is likely to represent a unique feature of teleost development and is currently under investigation. Presentation: 6/2/2024