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Fig. 7. Rbpsuh deletion reduces the generation of astrocytes and increases the generation of oligodendrocytes in the E19.5 mouse spinal cord without depleting progenitors that could form multilineage colonies in culture. (A-F) The differences observed at E14.5 persisted in the E19.5 spinal cord, including decreased numbers of Gfap⁺ astrocytes (A,B) and increased numbers of Mbp⁺ oligodendrocytes (C,D) in the Nestin-Cre⁺ Rbpsuh^fl/fl spinal cord as compared with littermate controls. We also observed more Sox10⁺ Pdgfr⁺ oligodendrocyte lineage cells in the Nestin-Cre⁺ Rbpsuh^fl/fl spinal cord (E,F, arrows). C,D represent montages of multiple non-overlapping images of the same section. (G) Quantification revealed a significant (^*, P<0.05) reduction in the number of BFABP⁺ astrocytes and significant increases in the numbers of Olig2⁺, Sox10⁺ and Mbp⁺ oligodendrocyte lineage cells in the Nestin-Cre⁺ Rbpsuh^fl/fl spinal cord (three to seven sections per mouse, three to five mice per genotype). Because glia are difficult to count in sections based on filamentous Gfap staining, we acutely dissociated cells from E19.5 spinal cord and plated them in culture at low density for 6 hours then stained for Gfap expression. (H) We observed a significantly lower percentage of Nestin-Cre⁺ Rbpsuh^fl/fl cells in culture that were Gfap⁺ (^*, P<0.05; 1000 cells counted per mouse, three mice per genotype). There was no significant difference in the number of cells undergoing cell death (activated caspase 3⁺) in control and Nestin-Cre⁺ Rbpsuh^fl/fl mice (data not shown). (I-P) Dissociated E19.5 thoracic spinal cord cells were cultured at clonal density then stained for neurons (TuJ1+), astrocytes (Gfap⁺) and oligodendrocytes (O4+). Representative multilineage colonies from control (I-L) and Nestin-Cre⁺ Rbpsuh^fl/fl (M-P) spinal cord cells show more Gfap⁺ cells and stronger Gfap staining in control colonies. (Q) The percentage of spinal cord cells that formed each type of colony. Nestin-Cre⁺ Rbpsuh^fl/fl spinal cord cells were significantly more likely than control cells to form multilineage colonies (N+A+O) and oligodendrocyte-containing colonies (O-containing), but significantly less likely to form astrocyte-only colonies (A). P<0.05. The increase in neuron-containing colonies reflected the increase in multilineage colonies, not an increase in neuronal-restricted progenitors. Scale bars: 50 µm in A-F.

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