Neuronal COUP-TFII Regulation Figure 4. COUP-TFII protein expression in adult mouse ventromedial hypothalamus. (A) Identification of the hypothalamic structures by Kluver Barrera staining. Black square: Arcuate nucleus, red square: Ventromedial hypothalamic nucleus. (B–F) Immunostaining of brain sections from adult mice using mouse monoclonal antibodies directed against COUP-TFII (PP-H7147-00, dilution 1:50). The secondary antibody is coupled with the horse radish peroxydase and revealed with diaminobenzidine leading to a brown precipitate. The sections shown are from the same experiment with the same exposure times. Magnification 620. (C) Secondary antibody incubation control for COUP-TFII antibody specificity (D) Enlargement from the VMH region, magnification 640. (E) Enlargement of the arcuate nucleus region (ARH), magnification 640. (F) Enlargement from the LHA region, magnification 640. These pictures are representative of 6 independent experiments. (G–J) Immunofluorescence staining of adult mice hypothalamic cryostat sections, magnification 620. (G) DAPI staining of the ARH-VMH region (blue fluorescence) (H) Identification of COUP-TFII positive neurons in the VMH-ARH region of adult mice using the COUP-TFII monoclonal antibody (green fluorescence) (I) Identification of COUP-TFI positive neurons in the VMH-ARH region of the hypothalamus using a COUP-TFI monoclonal antibody (red fluorescence) (J) Identification of COUP-TFI positive neurons in the cortex region of the same cryostat section than (I) as a positive control for COUP-TFI antibody. Scale bars = 100 mm. doi:10.1371/journal.pone.0013464.g004 COUP-TFII negative neurons with 65% of the SF1 positive cells expressing COUP-TFII (figure 5E). On the other hand, the COUP-TFII neurons were all SF1 expressing neurons (figure 5E, yellow fluorescence). Even so it was known that COUP-TFII mRNA and SF1 mRNA were present in the VMH, this study allowed the first identification of a subpopulation among the VMH neurons expressing both the COUP-TFII and SF1 proteins. The SF1 positive neurons have been characterized previously as part of the circuitry controlling energy homeostasis and reproductive behavior and are known to express the melanocortin receptor 4 (MC4-R) [22,23,24,25]. We confirmed the presence of this receptor in the COUP-TFII positive neurons by co-localization experiments and found that COUP-TFII neurons (figure 5F, red fluorescence) are indeed MC4-R positive neurons (green fluorescence) (figure 5G), PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13464