Proopiomelanocortin gene (Pomc) expression at the vertebrate extremes: cis/trans comparative studies in mouse and zebrafish

DOMENE, SABINA; VIVIANA BUMASCHNY; FLAVIO JUNQUEIRA; MARCELO RUBINSTEIN

Rosario

Congreso; 2nd Meeting and Course of the Latin American Zebrafish Network (LAZEN); 2012

Latin American Zebrafish Network (LAZEN)

Proopiomelanocortin gene (POMC) expression at the vertebrate extremes: cis/trans comparative studies in mouse and zebrafish   Sabina Domene1, Viviana Bumaschny1, Flavio Junqueira de Souza1,2 and Marcelo Rubinstein1,2   1INGEBI-CONICET and 2 Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.   Pomc is mainly expressed in the pituitary gland and ventromedial hypothalamus of all jawed vertebrates. Transgenic studies in mice and zebrafish showed that pituitary Pomc expression is controlled by a proximal promoter that is unable to drive reporter gene expression in brain POMC neurons. In the mouse, neuronal Pomc expression is conveyed by two distal upstream enhancers that we named nPE1 and nPE2 which are highly conserved only in mammals. In zebrafish, the neuronal enhancers controlling Pomca expression are still unknown due to lack of sequence conservation. Since enhancer elements may evolve in the different lineages through the accumulation of small sequence changes or larger sequence rearrangements, we set out a comparative study to test the ability of mouse Pomc enhancers and zebrafish Pomca flanking regions to drive the expression of the fluorescent marker EGFP in transgenic zebrafish. To determine if there is functional conservation of the mammalian enhancers in fish, we studied their capacity to drive EGFP expression to the hypothalamus in transgenic zebrafish embryos. We performed transient transgenesis experiments using the tol2 transposon system, by injecting ptol2-nPE1-nPE2-zpomca-egfp, ptol2nPE1-ΔnPE2-zpomca-egfp, and ptol2ΔnPE1-nPE2-zpomca-egfp in transient transgenesis and GFP expression was determined at 5 days post fertilization. Interestingly, all constructs drove GFP expression to the pituitary and hypothalamus in zebrafish embryos, comparable to what is observed in mammals. The identity of POMC neurons was determined by double immunohistochemistry using antibodies against ACTH and EGFP. These results were confirmed in several nPE1-nPE2-zpomca-egfp F1 transgenic lines expressing GFP in the hypothalamus. These results show that functional information is conserved among vertebrates despite absence of sequence similarity.