Supplementary Figure 6: Representative markers for hypothalamic neuronal subtypes and their localization

(a) For each neuronal cluster, the most specific markers were calculated (gene names are on top). To identify the most unique markers for each neuronal cluster, we used power = 0 analysis to identify topmost-expressed unique genes. To force uniqueness, we excluded genes that appear in the list of top 5 markers in other clusters. Since we often observed clusters that are characterized by gene combinations rather than unique global markers, some of the top 5 markers showed low specificity. All genes were found to be statistically significant by the Wilcoxon rank-sum test (q < 0.05) with the exception of L3hypdh (p = 0.04), Prkd1 (p = 0.007) and Ing2 (p = 0.01). The color scale to the right presents values after log transform, which were centered and normalized to mean = 0 and s.d. = 1 for each gene. Saturated colors represent the upper and lower 1% (range 1-99%). (b-c₁) Novel neuropeptide identities in the hypothalamus. Hypocretin (Hcrt, b) and galanin (Gal, c)-containing neuronal clusters (#35 and #37, respectively) uniquely co-express mRNAs for pyroglutamylated RFamide peptide (Qrfp; b₁) and neuropeptide VF precursor (Npvf, c₁). Note that Hcrt⁺ cluster #36 lacks Qfrp expression. In situ hybridization identifies Qrfp⁺ or Nvpf⁺ neurons in the arcuate nucleus (Arc)-lateral hypothalamic area (LHA) and dorsomedial hypothalamus (DMH), respectively. Histochemical data are from the Allen Brain Atlas (www.brain-map.org). Scale bars = 150 μm. mRNA copy numbers were expressed as means ± s.e.m. (log₂(mRNA copies + 1); power = 1). *q < 0.05 (Wilcoxon rank-sum test corrected for multiple testing).