Fig. 4. Developmental change in the direction of Numb segregation at P/N divisions. (A) E10-E14 cortical cells were plated at clonal density fixed 24 hours after plating and stained for Numb and ß-tubulin III or Nestin. Progenitor cell/neuron (P/N) pairs were analyzed for Numb distribution. Numb segregates into the neuronal daughter less frequently at E10 compared to cells isolated from the active neurogenic period, E13-14. (B-D) An E10 cell pair stained for Nestin and ß-tubulin III. (B) Phase-contrast image. (C) Both daughter cells are Nestin⁺ (green), but only one is ß-tubulin III⁺ (red). Arrow indicates the cell with double staining (yellow). (D) Diagram illustrating how P/N divisions at E10 generate a Nestin⁺ß-tub^- cell and a Nestin⁺ß-tub⁺ cell. (E-G) An E13 cell pair stained for Nestin and ß-tubulin III. (E) Phase-contrast image. (F) One daughter cell is Nestin⁺ (green), the other one is ß-tubulin III⁺ (red). (G) Diagram illustrating how P/N divisions at E13 generate a Nestin⁺ß-tub^- cell and a Nestin^-ß-tub⁺ cell. (H-I) BrdU incorporation into E13 P/N pairs. A pair of sister cells (boxed) was stained for ß-tubulin III (red) and BrdU (green). Only the ß-tubulin III^– cell had incorporated BrdU (arrow), consistent with it being a proliferating progenitor cell, while the neuron was BrdU^-. (J-L) A pair of E10 cortical sister cells stained for Numb and Nestin. (J) Phase-contrast image. (K) Numb staining is asymmetric. (L) Both daughter cells are Nestin⁺. (M-O) A pair of E13 cortical sister cells stained for Numb and Nestin. (M) Phase-contrast image. (N) Numb staining is asymmetric. (O) The Nestin^- daughter is Numb⁺. Arrows point to the Nestin⁺ Numb^- daughter cell. Scale bars: 20 µm.

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