ED Figure 6. Molecular complexity and function of the Onecut3 regulon.

(a) Interlinked Onecut2 and Onecut3 regulons in hypothalamic neurons. Genes that had been biologically validated (see below) are shown in black. (b) Onecut2 and Onecut3 co-expression along the rostrocaudal axis of the hypothalamus. (c) Co-localization of Onecut3 and its target genes (from panel a). (d-d₂) Overexpression of Onecut3 and ATP-binding cassette D2 (Abcd2, to control promoter activity) in Neuro2A cells. Representative images by multiple fluorescence labelling-differential interference microscopy. (e) Quantification of Hoechst⁺ and phosphor-histone H3 (pHH3)⁺ Neuro2A cells revealed significantly reduced proliferation upon Onecut3 overexpression. No significant cell death was observed by either overexpressed plasmid or the transfection reagent alone. (e) qPCR analysis of genes regulated by Onecut3: CXCC-type zinc finger protein 5 (Cxxc5), transmembrane protease serine 9 (Tmprss9) and tyrosine hydroxylase (Th). All data were normalized to samples transfected with Abcd2, which were taken as technical controls. Scale bars = 50 μm (d,d₁) 20 μm (b,f), 10 μm (g).