Fig 2: Neuroanatomical distribution of CRABP1 across the mouse brain. a Enrichment analysis of genes preferentially expressed in the “arcuate nucleus of hypothalamus” compared to all other brain regions in mouse. Crabp1 expression is enriched 107-fold in the ARC. b Rostrocaudal analysis of CRABP1 expression across the mouse brain using immunohistochemistry. Brain areas where CRABP1+ signal was detected are identified. Images are representative of n = 2 male mice. c Representative fluorescent in situ hybridization images of Crabp1 expression across the ARC. d Number of Crabp1-expressing cells in the ARC according to bregma. Each dot refers to the number of Crabp1-expressing cells within the ARC of one coronal brain section. Each bregma level includes a minimum of 8 brain sections coming from at least n = 6 male mice. Data are presented as mean ± SEM. For all images, scale bars correspond to 500 µm.

Fig 3: Crabp1 ARC neurons express receptors for metabolic hormones. a Representative fluorescent in situ hybridization image of Lepr-b and Crabp1 expression within the ARC. b Higher magnification of the framed region highlighted in (a). Cells co-expressing Crabp1 and Lepr-b are indicated by white arrows. Cells denoted by blue arrows (i and ii) are presented in higher magnification below. c 10.8 ± 1.7% of Crabp1ARC neurons express Lepr-b. Each dot refers to quantification of one coronal brain section. For Crabp1/Lepr-b, 12 brain sections from n = 3 male mice were quantified. d Representative fluorescent in situ hybridization image of Glp1r and Crabp1 expression within the ARC. e Higher magnification of the framed region highlighted in (d). Cells co-expressing Crabp1 and Glp1r are indicated by white arrows. Cells denoted by blue arrows (iii and iv) are presented in higher magnification below. f 35.1 ± 2.5% of Crabp1ARC neurons express Glp1r. Each dot refers to quantification of one coronal brain section. For Crabp1/Glp1r, 12 brain sections from n = 3 male mice were quantified. Data are presented as mean ± SEM. Scale bars correspond to 100 µm for all images except for higher magnification images of single cells (i, ii, iii, and iv) where scale bars correspond to 10 µm.

Fig 4: Crabp1-expressing neurons overlap with yet uncharacterized neuronal clusters of the ARC. a Representative fluorescent in situ hybridization image of Htr3b and Crabp1 expression within the ARC. b Higher magnification of the framed region highlighted in (a). Cells co-expressing Crabp1 and Htr3b are indicated by white arrows. c Representative fluorescent in situ hybridization image of Tbx19 and Crabp1 expression within the ARC. d Higher magnification of the framed region highlighted in (c). Cells co-expressing Crabp1 and Tbx19 are indicated by white arrows. e Representative fluorescent in situ hybridization image of Tmem215 and Crabp1 expression within the ARC. f Higher magnification of the framed region highlighted in (e). Cells co-expressing Crabp1 and Tmem215 are indicated by white arrows. For all images, scale bars correspond to 100 µm. Images are representative of n = 3 male mice for each experiment.

Fig 5: Crabp1 ARC neurons are GABAergic and distinct from prototypical POMC and AgRP neurons. a Representative fluorescent in situ hybridization image of Slc32a1 (Vgat) and Crabp1 expression within the ARC. b Higher magnification of the framed region highlighted in (a). c 98.9 ± 0.3% of Crabp1ARC neurons express Slc32a1. Each dot refers to quantification of one brain section. For Crabp1/Slc32a1, 11 brain sections from n = 3 male mice were quantified. d Representative fluorescent in situ hybridization image of Pomc and Crabp1 expression within the ARC. e Higher magnification of the framed region highlighted in (d). f 2.6 ± 0.4% of Crabp1ARC neurons express Pomc. Each dot refers to quantification of one coronal brain section. For Crabp1/Pomc, 12 brain sections from n = 3 male mice were quantified. g Representative fluorescent in situ hybridization image of Agrp and Crabp1 expression within the ARC. h Higher magnification of the framed region highlighted in (g). i 3.8 ± 0.6% of Crabp1ARC neurons express Agrp. Each dot refers to quantification of one coronal brain section. For Crabp1/Agrp, 11 brain sections from n = 3 male mice were quantified. Data are presented as mean ± SEM. For all images, scale bars correspond to 100 µm.