Volume 123, Issue 3, Pages 493-505 (November 2005) Divergence of Melanocortin Pathways in the Control of Food Intake and Energy Expenditure  Nina Balthasar, Louise T. Dalgaard, Charlotte E. Lee, Jia Yu, Hisayuki Funahashi, Todd Williams, Manuel Ferreira, Vinsee Tang, Robert A. McGovern, Christopher D. Kenny, Lauryn M. Christiansen, Elizabeth Edelstein, Brian Choi, Olivier Boss, Carl Aschkenasi, Chen-yu Zhang, Kathleen Mountjoy, Toshiro Kishi, Joel K. Elmquist, Bradford B. Lowell  Cell  Volume 123, Issue 3, Pages 493-505 (November 2005) DOI: 10.1016/j.cell.2005.08.035 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Generation of loxTB Mc4r Mice (A) A disrupted Mc4r null allele was generated by inserting a loxP-flanked transcriptional blocker (loxTB) between the transcription initiation (+1) and the ATG of the Mc4r coding sequence. Expression of Cre-recombinase removes the transcriptional blocker and allows Mc4r transcription. (B) In situ hybridization detecting Mc4r mRNA (darkfield photomicrograph of 35S-silvergrains) in mice homozygous for the disrupted Mc4r null allele (loxTB Mc4r, right panels) and their wild-type littermates (left panels) (PVH—paraventricular hypothalamus, NLOT—nucleus of the lateral olfactory tract, DMV—dorsal motor nucleus of the vagus). Scale bar = 1mm. (C) Body-weight curve of male loxTB Mc4r mice (wt, closed circles, n = 10 and loxTB Mc4r, open circles, n = 8; mean ± SEM). Cell 2005 123, 493-505DOI: (10.1016/j.cell.2005.08.035) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Reactivation of the loxTB Mc4r Allele (A) loxTB Mc4r mice were crossed with Zp3-Cre mice, causing germline deletion of the transcriptional blocker and reactivation of the Mc4r allele (female wt, closed circles, n = 9; loxTB Mc4r, open circles, n = 8; Zp3-Cre, loxTB Mc4r, open diamond, n = 10; mean ± SEM). (B) loxTB Mc4r mice were crossed with Nes-Cre mice, causing deletion of the transcriptional blocker in neurons and reexpression of neuronal MC4Rs (female wt, closed circles, n = 9; loxTB Mc4r, open circles, n = 7; Nes-Cre, loxTB Mc4r, open squares, n = 8; mean ± SEM). (C) Mc4r in situ hybridization in wild-type, loxTB Mc4r, and Nes-Cre, loxTB Mc4r mice, demonstrating reactivation of Mc4r mRNA in neurons. Scale bar = 0.5 mm. (D) Snout-anus length in 13-week-old female anaesthetized mice (female wt, n = 9; loxTB Mc4r, n = 7; Nes-Cre, loxTB Mc4r, n = 8; mean ± SEM; ***p < 0.001). Cell 2005 123, 493-505DOI: (10.1016/j.cell.2005.08.035) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Stereotaxic AAV-Cre Injections (A) Cre immunohistochemistry in loxTB Mc4r mice stereotaxically injected with AAV-Cre on one side of the PVH. (B) Mc4r mRNA in situ hybridization confirms Mc4r mRNA reexpression at the site of AAV-Cre injection. Scale bar = 0.5 mm. (C) As control groups, wt mice were injected with AAV-Cre and loxTB Mc4r mice with AAV-eGFP. loxTB Mc4r body weights 80 days after AAV-Cre injection were separated into PVH “miss” and “hit” by Mc4r mRNA presence or absence in the PVH (wt AAV-Cre “miss,” n = 7; wt AAV-Cre “hit,” n = 5; loxTB Mc4r AAV-eGFP “miss,” n = 6; loxTB Mc4r AAV-eGFP “hit,” n = 9; loxTB Mc4r AAV-Cre PVH “miss,” n = 8, loxTB Mc4r AAV-Cre PVH “hit,” n = 11; mean ± SEM; *p < 0.05). Cell 2005 123, 493-505DOI: (10.1016/j.cell.2005.08.035) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Sim1-Cre Transgenic Mice (A) Mice expressing Cre-recombinase under Sim1 promoter control were generated by engineering a Sim1 bacterial artificial chromosome using recombineering techniques. (B) Left panel: In situ hybridization for Sim1 mRNA (darkfield photomicrograph of 35S silvergrains) in wild-type mice. Right panel: eGFP immunohistochemistry in Sim1-Cre, Rosa-eGFP mice. Cre activity in Sim1-Cre transgenic mice was shown in all areas known to express Sim1. Note that staining in the DMV is axon terminal, not cellular, staining (MeA—medial amygdala). Scale bar = 0.5 mm. Cell 2005 123, 493-505DOI: (10.1016/j.cell.2005.08.035) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Sim1-Cre, loxTB Mc4r Mice (A) Mc4r mRNA in situ hybridization in wild-type (left panels) and Sim1-Cre, loxTB Mc4r mice (right panels). Sim1-Cre, loxTB Mc4r mice reexpress Mc4r mRNA in the PVH, NLOT, and cells of the medial amygdala, with no hindbrain Mc4r expression (PAG—periaqueductal gray; MPOA—medial preoptic area). Scale bar = 1 mm. (B and C) Body-weight curve of male and female wild-type, loxTB Mc4r and Sim1-Cre, loxTB Mc4r mice (male/female wt, closed circles, n = 12/10; loxTB Mc4r, open circles, n = 11/12; Sim1-Cre, loxTB Mc4r, open squares, n = 10/9; mean ± SEM). Cell 2005 123, 493-505DOI: (10.1016/j.cell.2005.08.035) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Body Composition and Energy Homeostasis in Sim1-Cre, loxTB Mc4r Mice (A) Snout-anus length in 12-week-old male wild-type (n = 10), loxTB Mc4r (n = 12), and Sim1-Cre, loxTB Mc4r mice (n = 9). (B) Fat and lean mass in male 14-week-old wild-type (n = 12), loxTB Mc4r (n = 12), and Sim1-Cre, loxTB Mc4r mice (n = 10) were analyzed by DEXA measurement. (C) Food intake was measured in 11-week-old male and female wild-type (male/female n = 12/5), loxTB Mc4r (n = 12/9), and Sim1-Cre, loxTB Mc4r (n = 9/7) for a week and is expressed here as 24 hr food intake. (D) Oxygen consumption was measured and averaged over a 10 hr dark or light period (wt, n = 5; loxTB Mc4r, n = 5; Sim1-Cre, loxTB Mc4r, n = 5; *p < 0.5; **p < 0.01; ***p < 0.001 compared to wild-type). All data are mean ± SEM. Cell 2005 123, 493-505DOI: (10.1016/j.cell.2005.08.035) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Food Intake and Energy Expenditure Responses to MTII (A) Mice were injected with saline or 80 μg MTII ip 30 min before lights out, and their 3.5 hr food intake was recorded. Data are represented as % saline intake per mouse. (n = 6, mean ± SEM; *p < 0.5 compared to saline). (B) Mice were injected ip with saline on day 1 and 400 μg MTII on day 2. Food was removed at the time of injection and oxygen consumption was measured and averaged for the ensuing 3 hr. Data are represented as % saline per mouse. (n = 9–11; mean ± SEM; *p < 0.5 compared to saline) (C) Functional divergence in melanocortin pathways. Our data show that α-MSH and AgRP neurons projecting to PVH and/or amygdala MC4R neurons regulate melanocortin-mediated food intake and body length. On the other hand, separate and different α-MSH and AgRP neuronal projections to other MC4R-containing areas must be important for melanocortin-mediated regulation of energy expenditure. Cell 2005 123, 493-505DOI: (10.1016/j.cell.2005.08.035) Copyright © 2005 Elsevier Inc. Terms and Conditions