(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).