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. 2020 Apr 18;37:100985. doi: 10.1016/j.molmet.2020.100985

Figure 1.

Figure 1

Generation of a mouse line expressing Pomc restricted to GABAergic-POMC neurons. (A)Pomc− allele contains an insertion of a neomycin resistance cassette (neo), flanked by loxP sites (triangles), interrupting Pomc neuronal enhancer activity (blue circle: nPE1 enhancer; gap after neo: Deleted nPE2 enhancer). Gad2-CreER line drives Pomc reactivation restricted to GABAergic-POMC neurons after i. p. injection of tamoxifen (TAM) in arcPomc−/−:Gad2-CreER mice (rescued). The POMC-cellular specificity of Pomc reactivation is due to the endogenous Pomc regulatory elements in the knock-in arcPomc– allele. Gray circle: Pomc pituitary promoter. Arc: arcuate Pomc transcription. Black rectangle: Pomc exon 1. (B) Representative coronal brain sections of Gad2-CreER, Pomc−/−, and rescued arcPomc−/−:Gad2-CreER mice showing ACTH immunopositive neurons. The arrows indicate rescued POMC expression in GABAergic neurons. 3V, third ventricle; Arc: arcuate nucleus. Magnification bars: 200 μm. (C) Percentage of POMC neurons in arcPomc−/−:Gad2-CreER-rescued mice (GABAergic-POMC neurons), relative to POMC neurons in Gad2-CreER control mice, at different coronal levels of the hypothalamus. The rostrocaudal analysis shows no significant differences (OWA). Error bars: ± SEM; n = 3. (D) Representative coronal brain section of an arcPomc−/−:Gad2-CreER-rescued mice subjected to fluorescent hybridization for Gad1 (GABAergic neurons, green) and immunohistochemistry for ACTH (POMC neurons, red). The white rectangle depicts the area of magnified images shown below. Magnification bars: 200 μm (upper picture) and 50 μm (bottom pictures). The white arrowheads point to GABAergic-POMC neurons. (E) Representative coronal brain section of Ai14:Gad2-CreER mice showing recombination in GABAergic cells (red, “Gad2CreER-Tom”) and ACTH immunopositive neurons (green, “POMC”). The white rectangle depicts the area of the magnified image shown on the right. The white arrows point to GABAergic-POMC neurons. Magnification bars: 200 μm (left picture) and 50 μm (right picture).