Extended Data Fig. 8: In situ validation of synaptic signaling gene expression changes.

(a) mGreenLantern-tagged synaptophysin (Syp-GL) puncta (arrows) indicate putative presynapses in Opn5+ neurons in AvPe at P10. (b) Co-localization of synaptophysin and VIP protein puncta (arrows) in Gal+ neurons in MPN at P65 (left) and P10 (right). (c) MPN Gal+ neurons projecting to AvPe at P10 show dense synaptophysin puncta. (d) VIP puncta (arrows) at P10 (left) and P65 (right). (e) RNA in situ hybridization shows Lepr expression in Brs3+ and other AvPe neurons at P10. (f) Expression of Ghsr in Kiss1+ neurons at P65 (lower) but not P10 (upper). (g) Expression of Glp1r in Foxp2+ neurons of the PVN at P0 (upper) and P65 (lower). (h) Expression of Drd1 in Meis2+ HDB neurons at P65 (lower) but not P0 (upper). (i) Expression of Drd1 in Lhx1+ SCN neurons at P0 (upper) and P65 (lower). (j) Expression of Hrh3 in HDB neurons at P65 (right) but less so at P10 (left). (k) Expression of Ar is weak at P0, intermediate at P10, and widespread at P65. (l) At P10 and P65, Pnoc is expressed in MPN and Oprl1 is expressed in AvPe, consistent with potential Pnoc-Oprl1 signaling from MPN to AvPe as predicted by NeuronChat. (m) At P65 but not P10, Kiss1 is expressed in Pe, while Kiss1r is detectable at low levels in AvPe at both ages, consistent with P65 but not P10 Kiss1-Kiss1r signaling from MPN/Pe to AvPe as predicted by NeuronChat. (n) Calca expression is weak at P10 but nearly absent at P65 in MPN, while Calcr is expressed in AvPe at both ages, consistent with P10 but not P65 Calca-Calcr signaling from MPN to AvPe as predicted by NeuronChat.