Pituitary Corticotroph Ontogeny and Regulation in Transgenic Zebrafish

Abstract

We characterized zebrafish proopiomelanocortin (POMC) gene promoter, and sequence analysis revealed that the promoter contains regulatory elements conserved among vertebrate species. To monitor the ontogeny of the pituitary POMC lineage in living vertebrates, we generated transgenic zebrafish expressing green fluorescent protein (GFP) driven by the POMC promoter. Zebrafish POMC-GFP is first expressed asymmetrically as two bilateral groups of cells most anterior to the neural ridge midline at 18-20 h post fertilization (hpf). POMC-GFP-positive cells then fuse into a single-cell mass within the pituitary anlage after 24 hpf and subsequently organize as distinct anterior and posterior domains between 48 and 64 hpf. Immunohistochemical studies with ACTH and alphaMSH antisera showed that POMC-GFP was mainly targeted to both anterior and posterior pituitary corticotrophs, whereas posterior pituitary region melanotrophs did not express GFP. To determine in vivo zebrafish corticotroph responses, dexamethasone (10(-5) m) was added to live embryos, which selectively suppressed POMC-GFP expression in the anterior group of corticotrophs, suggesting a distinct domain that is responsive to glucocorticoid feedback. Transgenic zebrafish with specific POMC-GFP expression in pituitary corticotrophs offers a powerful genetic system for in vivo study of vertebrate corticotroph lineage development.

How to make a teleost adenohypophysis: Molecular pathways of pituitary development in zebrafish

“…The pan-pituitary specification markers lim3 and pitx3 show reduced, but not completely abolished expression, while pit1 expression is completely absent (Pogoda et al, 2006b). This failure of pit1 activation is most likely the main reason for the later absence of prl, gh, and tsh- expression (see paragraph 9), whereas the absence of pomc expression might point to a direct role of Ascl1a as a transcriptional activator of pomc, consistent with the presence of E-boxes, consensus binding sites for bHLH proteins, in the promoter of the zebrafish pomc gene (Liu et al, 2003).…”

“…By trans-activating reporter assays it was shown that mouse Pitx1 can drive Lhx3 (the mouse homolog of zebrafish lim3) as well as prl and pomc expression (Lamonerie et al, 1996, Tremblay et al, 1998. Since Pitx binding sites have also been found in the zebrafish pomc promoter, and since lactotropes and corticotropes are the first differentiating AH cells of the zebrafish, one can speculate about a similar regulation of these genes by Pitx3 in teleosts (Liu et al, 2003).…”

“…As the primary site of endocrine action the AH harbors at least six hormone secreting cell types: the PD contains corticotropes producing adrenocorticotropin (ACTH), lacotropes producing Prolactin (PRL), thyrotropes producing thyrotropin (TSH), somatotropes producing Growth Hormone (GH), and gonadotropes that secrete the gonadotropins (LH, FSH). The PI consists of corticotropes and melanotropes, which generate -melanocyte stimulating hormone (-MSH) (Liu et al, 2003). As a specific endocrine feature, teleosts share an additional adenohypophyseal cell type: the Somatolactin producing cells, which can be found in the PD of the embryonic and in the PD as well as PI of the adult AH (Zhu et al, 2004, Lopez et al, 2006.…”

Enhancer turnover and conserved regulatory function in vertebrate evolution

“…Mammals, birds and other tetrapods have only one Pomc copy, but zebrafish and other teleosts, including fugu and Tetraodon nigroviridis, have two Pomc paralogues-pomca and pomcb-that date from the ancient whole-genome duplication in the teleost lineage around 320 Ma [51]. In zebrafish, pomca is expressed in the anterior and intermediate lobes of the pituitary as well as in the ventral hypothalamus, just like the tetrapod gene [52,53]. The expression pattern of zebrafish pomcb is unknown, but the Tetraodon homologue is expressed in the preoptic area of the brain and pituitary [51].…”

“…In the mammalian pituitary, there is a large body of evidence that Pomc expression is regulated by several TFBSs present in the proximal promoter [54][55][56], as well as by a mammalian-conserved distal enhancer [57]. In the zebrafish, similar to mammals, it has been found that a construct carrying the proximal rstb.royalsocietypublishing.org Phil Trans R Soc B 368: 20130027 promoter and first intron of the pomca gene can drive green fluorescent protein (GFP) expression to the anterior and intermediate lobes of the pituitary, but not to ventral hypothalamic neurons [52]. A similar construct carrying 500 bp of the Xenopus laevis Pomc gene promoter directs GFP expression to cells of the intermediate lobe, but not the hypothalamus, of transgenic frog embryos [58], suggesting that the function of the proximal Pomc promoter in driving pituitary expression is ancestral in vertebrates.…”