Extended Data Fig. 1: Validation of Cre-mediated recombination in tanycytes.

a. Expression profiles of tanycytes specific Cre-expressing rAAVs in reporter mice across the brain. Expression of AAV-Dio2-iCRE-GFP was assessed in ROSA26|STOP|tdTomato^fl/fl reporter mice 3 weeks post i.c.v. injection in ARC, d3V, LV and AP. AAV-Dio2-Cre was injected i.c.v. in mice expressing Cre-dependent ZsGreen reporter from ROSA26 locus (Rosa26|STOP|ZsGreen^fl/fl) and assessed in ARC, SFO, LV and AP. Virus expression and fluorescence of the ZsGreen reporter was assessed 3 – 4 weeks post injection in 3 – 5 mice. b. AAV-Dio2-GFP representative images were acquired from IR^GFP-Tan mice 6 weeks post injection. AAV-Dio2-mKate2 expression in tanycyte nuclei was verified in IR^fl/fl mice 3 weeks post injection. Because AAV-Dio2-mKate2 exhibited a weak fluorescent signal, it was amplified with immunohistochemistry staining for red fluorescent protein (RFP). All viruses were expressed in the lateral and basal tanycytes of the 3^rd ventricle. n = 4/group. c. Representative images of smISH of ZsGreen mRNA expression in mediobasal hypothalamus of Rosa26|STOP | ZsGreen^fl/fl-mice 3 weeks post i.c.v. injection of AAV-Dio2-Cre virus in 5 mice. Expression of Zsgreen reporter gene was assessed in the anterior and posterior hypothalamus (c1 – medial dorsal, c2 – medial ventral, c3 – posterior dorsal, c4 – posterior ventral) by co-labelling for tanycyte- (Dio2) and astrocyte- (GFAP) specific genes. Dashed lines indicate the outline of the ARC. ARC – arcuate nucleus, d3V – dorsal third ventricle, SFO - subfornical organ, LV – lateral ventricle, AP – Area Postrema.