Neuronal COUP-TFII Regulation Figure 5. COUP-TFII protein co-localization studies with SF1 in the VMH. (A–G) Immunofluorescence staining of adult mice hypothalamic cryostat sections. (A–E) The sections shown are from the same experiment with the same exposure time (A) Identification of SF1 positive cells in the VMH by immunofluorescence using a SF1 polyclonal antibody (red fluorescence) (B) Identification of the COUP-TFII positive neurons in the VMH of adult mice using a COUP-TFII monoclonal antibody (green fluorescence) (C) Secondary antibody alone control for SF1 antibody specificity (D) Secondary antibody alone for COUP-TFII antibody specificity (E) Superimposed SF1 and COUP-TFII immunostaining. Magnification 640. (F–G) The sections shown are from the same experiment and the same exposure time. (F) COUP-TFII immunostaining (red fluorescence) (G) Superimposed MC4- R and COUP-TFII immunofluorescences. Magnification 6100. These pictures are representative of 4 independent experiments. Scale bars = 10 mm. doi:10.1371/journal.pone.0013464.g005 confirming the presence of MC4-R on SF1 neurons. To summarize, COUP-TFII is expressed in the VMH in the SF1 positive neurons, part of the melanocortin pathway. COUP-TFII gene expression is modulated by the melanocortin pathway in the GT1-7 hypothalamic cell line In the central nervous system, the fed state is characterized by the production of a melanocyte-stimulating hormone (aMSH), the hypothalamic POMC product which is synthesized by the neurons of the ARH in response to insulin [26]. The aMSH receptors, MC4-R and MC3-R, are present on numerous neurons from the different hypothalamic nuclei, including the lateral hypothalamus (LH) the paraventricular hypothalamus (PVN) and the VMH where the COUP-TFII neurons are located (figure 5) [27,28]. COUP-TFII being expressed on neurons known to express the MC4-R, we then checked whether an activation of this receptor could contribute to COUP-TFII expression. PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13464