Journal: eLife
Article Title: Directed differentiation of functional corticospinal-like neurons from endogenous SOX6+/NG2+ cortical progenitors
doi: 10.7554/eLife.100340
Figure Lengend Snippet: ( A ) Confocal micrograph of mouse neocortex at postnatal day 7 (P7) showing expression of SOX6 by a subset of BrdU+ proliferative cells. See also . ( B ) In situ hybridization of Neurog2 in Sox6 wild-type (wt) (left) and knockout (KO) (right) cortex at P6. ( B’ ) Insets showing the boxed areas in B. Loss of Sox6 results in widespread ectopic expression of Neurog2 ( B’ ). See also . ( C ) Immunofluorescence showing expression of SOX6 by DsRed+ NG2+ progenitors in cortex at P5. ( D ) Immunostaining of NG2 proteoglycan in NG2-DsRed cortex at P5 shows expression of DsRed by NG2+ progenitors. Inset: a cell with a strong DsRed signal in the cell body and NG2 proteoglycan around the main cell body and in cellular processes. See also . ( E ) Representative FACS plot of neocortical cells from NG2-DsRed transgenic cortex showing distinct DsRed-Bright, -Dim, and -negative populations. ( F ) qPCR analysis of Sox6 , Olig2 , and Cspg4 from acutely sorted DsRed-Negative, -Dim, and -Bright populations, as well as cultured DsRed-Bright cells (5DIV), demonstrates that SOX6+/NG2+ progenitors are enriched in DsRed-Bright population and maintain key gene expression in vitro . See also . Data are presented as mean ± SD, n=4, biological replicates, Actb normalized data relative to DsRed-negative population. ∗∗∗∗ p <0.0001, p ≥0.05, no statistically significant difference (n.s.); ANOVA Tukey’s post hoc test. ( G ) Volcano plot comparing fold difference in average expression of progenitor genes between acutely sorted DsRed-Bright and -Dim populations (RNA-seq, n=5, biological replicates). See also . ( H ) Representative brightfield image of cultured SOX6+/NG2+ (DsRed-Bright) progenitors at 5 DIV showing preserved progenitor multipolar morphology. See also . ( I, J ) Cultured progenitors continue expressing the key progenitor-specific molecules NG2, SOX10 ( I ), OLIG2, and SOX6 ( J ) at 7 DIV. ( K ) Quantification of TUJ1+ and GFAP+ cells at 3-, 5-, and 7 DIV shows essentially no contaminant cells in culture. Data are presented as mean ± SD, n=2, biological replicates. See also . ( L ) Pearson correlation analysis of progenitor genes shows high similarity between acutely sorted and cultured SOX6+/NG2+ (DsRed-Bright) progenitors ( R =0.84, p <2.2e-16). Data points represent log2 fold differences in gene expression relative to acutely sorted DsRed-Dim population. See also . ( M ) Heatmap of the top five marker genes for seven major cell types in brain shows that SOX6+/NG2+ progenitors are enriched in DsRed-Bright populations and that progenitor cultures are free of potential contaminants. Counts are variance-stabilizing transformed (vst) normalized data in log2 scale. ( N ) Volcano plot comparing fold differences in average expression of the top 500 genes for major cell types between cultured SOX6+/NG2+ (DsRed-Bright) progenitors and acutely sorted cells. n=5/6, biological replicates. See also . Scale bars ( A, C, H ) 50 μm; ( D, I, J ) 100 μm. cc: corpus callosum, ctx: cortex.
Article Snippet: Brightfield images were acquired using a Nikon ECLIPSE Ts2R-FL inverted microscope.
Techniques: Expressing, In Situ Hybridization, Knock-Out, Immunofluorescence, Immunostaining, Transgenic Assay, Cell Culture, Gene Expression, In Vitro, RNA Sequencing, Marker, Transformation Assay
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