Fig. 1

Phenotypic characterization of Nurr1⁺/NeuN⁺ dopaminergic neurons. a (top) Coronal midbrain section including substantia nigra (SN). b Representative FACS plot showing subtypes of sorted nuclei including double positive Nurr1⁺/NeuN⁺ (fraction P8). c Midbrain section immunohistochemically stained with anti-Nurr1 antibody, showing Nurr1 immuno-reactive nuclei associated with melanin-positive somata (black arrows) in the SN. d Nurr1 western blot comparing SN versus non-specific midbrain control region. e Dopaminergic marker genes (adopted from single-cell RNA-seq study [42]) were quantified for expression in the six cell type-specific nuclei fractions collected by FACS from ventral midbrain and anterior cingulate cortex, as indicated. Note significantly higher expression of dopaminergic marker genes (y-axis) in midbrain Nurr1⁺/NeuN⁺ nuclei as compared to other nuclei populations (Wilcoxon test, p < 5 × 10⁻² to 10⁻⁵). Note the subtle increase in expression of dopaminergic marker genes in midbrain Nurr1⁻/NeuN⁺ nuclei compared to the remaining four cell types, suggesting that this fraction of nuclei represents a more heterogenous admixture of cell types including subset of dopaminergic intermingled with non-dopaminergic neuron nuclei. f Representative genome browser screenshots of nucRNA-seq coverage for dopaminergic marker genes, (top) TYROSINE HYDROXYLASE (TH) and (bottom) ENGRAILED1 (EN1) in four midbrain nuclei fractions based on Nurr1 and NeuN immunotagging as indicated and two cortical nuclei fractions based on NeuN immunotagging as indicated: midbrain: (red track) Nurr1⁺/NeuN⁺, (pink track) Nurr1⁻/NeuN⁺; (orange track) Nurr1⁻/NeuN⁺, (blue track) Nurr1⁻/NeuN⁻; anterior cingulate cortex (ACC): green track NeuN⁺ and brown track NeuN⁻