Article
Extended Data Fig. 8 | Cell-autonomous mechanisms for the regional
specificity in neuronal conversion. a, TH+ neurons generated from cortical
astrocytes with normal and conditioned media from cultured midbrain
astrocytes. Scale bar, 100 μm. b, Quantification of cells in a. Three biological
repeats with at least 100 cells counted in each. Statistical significance was
determined by ANOVA; mean ± s.e.m. c–f, RT–qPCR analysis of DA
neuron-specific transcription factors in cortical and midbrain astrocytes
before and after lentiviral shPTB-induced neuronal conversion. c, The
indicated transcription factors were quantified by real-time PCR and
normalized against β-actin mRNA. d, To ensure that the isolated astrocytes
were free of contaminated neurons, RT–qPCR was also performed with the 3
indicated pan-neuron markers with isolated neurons as control. e, In response
to PTB knockdown, astrocyte-specific genes S100b and Gfap were repressed,
whereas pan-neuronal transcription factors Myt1l and Ascl1 were activated in
astrocytes derived from both cortex and midbrain. Dashed lines indicate levels
before shPTB treatment, which was set to 1 for comparison with levels after
shPTB treatment. f, Under the same conditions, the 4 DA-neuron-specific
transcription factors were more robustly induced in response to PTB depletion
in midbrain astrocytes compared to cortical astrocytes. Statistical significance
was determined by ANOVA with post hoc Tukey test (d) or two-sided Student’s
t-test (c, e, f), based on 3 biological repeats; mean ± s.e.m. P-values are
indicated. Results suggest higher basal levels and more robust induction of DA
neuron-specific transcription factors in midbrain astrocytes compared to
cortical astrocytes, providing evidence for the differences in cell-intrinsic
gene expression programs in giving rise to distinct subtypes of neurons.
g–i, Schematic of amperometric recording of monoamine release, showing
the placement of a carbon fibre electrode on a midbrain astrocyte-derived
neuron (g). Scale bar, 30 μm. h, Spike-like events were captured by holding the
electrode at +750 mV after K+ (25 mM) stimulation. i, A high-resolution view of
dopamine release events in h. Results demonstrate a key functional property of
midbrain astrocyte-derived DA neurons. Experiments were independently
repeated twice with similar results.