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Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts

¹ Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, NY 12208, USA
² Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA
³ Howard Hughes Medical Institute, Departments of Physiology and Biochemistry, University of California San Francisco, San Francisco, CA 94143, USA

View larger version (100K): [in a new window]   Fig. 1. Numb expression in the E10.5 mouse cortex in vivo and in acutely isolated cells. (A) Numb staining is seen throughout the E10.5 mouse cerebral cortex. Some ventricular zone cells show strong staining at their apical border. LV, lateral ventricle. (B-H) E10.5 cortical cells were isolated, fixed acutely, stained with anti-Numb antibody and double labeled with an antibody to Nestin or ß-tubulin III. (B) Quantification of Numb+ cells in the progenitor cell population (Nestin+) and immature neuronal population (ß-tubulin III+). (C,F) Phase-contrast images of the fields stained for Numb (D and G, in red), Nestin (E, in green), or ß-tubulin III (H, in green). Arrows indicate cells double labeled for Numb and Nestin (C-E) and Numb and ß-tubulin III (F-H). Scale bars: 100 µm.

View larger version (72K): [in a new window]   Fig. 2. Symmetric and asymmetric Numb distribution is observed during cortical cell divisions. (A) Illustration of the pair assay. The left panel shows a phase image of a field of single cortical cells 2 hours after plating into Terasaki microwells. The cells were monitored under the microscope and cells that divided were identified: arrows point to pairs of sister cells after 1 day in vitro (DIV). (B-I) Numb staining in cortical progenitor cells. E10-E12 cortical cells were dissociated, plated into Terasaki plates, fixed at different time-points, and stained for Numb. (B-E) Phase-contrast images of the fields shown in F-I. (B,F) A cortical progenitor cell with Numb distributed in a crescent on one side of the cell cortex. (C,G) A pair of sister cells fixed within 10 minutes after mitosis that was in the process of separating. Numb is seen in only one of the daughter cells, demonstrating that it can be asymmetrically segregated during mitosis. Arrow indicates the Numb-negative cell. (D-I) Pairs of sister cells identified 24 hours after plating. Numb may be asymmetrically (H) or symmetrically (I) distributed into the two daughter cells. Scale bars: (A) 100 µm; (B-H) 20 µm.

View larger version (59K): [in a new window]   Fig. 3. Asymmetric Numb distribution is correlated with asymmetric cell divisions generating a cortical progenitor cell and a neuron. (A-C) An E10 cell pair consisting of a neuron and a progenitor cell that has asymmetric Numb expression. (A) Phase-contrast image; (B) ß-tubulin III staining is present in one daughter; (C) Numb is present only in the neuronal daughter (arrow). In other pairs, Numb was found in the progenitor and not the neuronal daughter (not shown). (D-F) An E10 cell pair consisting of two neurons that have symmetric Numb expression. (D) Phase-contrast image; (E) ß-tubulin III staining is present in both daughter cells; (F) symmetric Numb staining. Scale bars: 20 µm.

View larger version (37K): [in a new window]   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.

View larger version (7K): [in a new window]   Fig. 5. Lineage trees constructed from time-lapse recordings reveal that Numb is expressed at multiple stages in cortical stem cells and neuroblasts. E10-E12 cortical cells were followed with time-lapse video microscopy for up to 7 days then fixed and stained for Numb and ß-tubulin III. The lineages were constructed from the recorded images, and Numb+ cells within the trees were identified (indicated by star). (A) A neuroblast clone. Numb was expressed in neuronal progeny generated at various stages within the lineage trees. (B) A stem cell clone. Numb was seen in neurons and also in the dividing ß-tubulin III– glial progenitor cells found at the end of the lineage. In terminal neuroblast divisions that generated two post-mitotic neurons, Numb could be distributed asymmetrically or symmetrically. Examples are seen in A and B respectively, although both types of distribution occurred in neuroblast and stem cell lineages. N, ß tubulin III+ neuron; -, non-neuronal cell; X, cell died; ?, cell migrated out of field.

View larger version (45K): [in a new window]   Fig. 6. Numb expression in pairs of sister neurons is related to their morphology. (A) E14 cortical pairs were identified after 24 hours in culture, fixed and stained for Numb (not shown) and ß-tubulin III (green). Examples of neuron pairs with asymmetric Numb are shown in the upper panel, and with symmetric Numb in the lower panel. Note that upper panel pairs usually have different morphologies, while those in the lower panel have similar morphologies. (B) E10.5 cortical cells were cultured in serum-free medium with 10 ng/ml bFGF. In some experiments, C6 membrane homogenate was added to enhance neurite outgrowth and neuron survival (Temple and Davis, 1994). Pairs of sister cells were identified after 1 day in vitro, cultured for a further 3 days, and then stained for Numb (red) and ß-tubulin III (green). Arrows indicate Numb- neurons. (a-d) Neuron pairs with asymmetric Numb distribution usually had different morphologies. (e-h) Neuron pairs with symmetric Numb distribution usually had similar morphologies. (C) Differences between sister neurons in total process length, average primary process length, number of primary processes and branch points (mean±standard deviation) were plotted for Numb asymmetric pairs and Numb symmetric pairs. *P<0.05, **P<0.001, Student’s t-test. Scale bars: (A) 20 µm; (B) a: 100 µm; b-h: 20 µm.

View larger version (14K): [in a new window]   Fig. 7. Neurons with Numb have longer neurites than those without Numb. E10.5 cortical cells were cultured at low density for 7 days, then fixed and stained for Numb and ß-tubulin III. The total neurite length of Numb+ neurons was compared with that of Numb– neurons. (A) The percentage of neurons within each neurite length category is shown. Generally, Numb+ neurons had longer processes than Numb– neurons. (B) The mean neurite length per neuron was calculated; there was a significant difference, approximately three fold, for Numb+ versus Numb– neurons. *P<0.001. Bars represent standard errors.

View larger version (17K): [in a new window]   Fig. 8. Model of Numb function during cortical development. Numb asymmetry generates differences between sister cells at different stages of development. At E10, asymmetric Numb distribution generates different Nestin+ progenitor cells. At E13, besides asymmetric progenitor divisions (not shown) Numb generates asymmetric P/N and N/N divisions. Numb may function by inhibiting Notch activity in some or all of these divisions. For example, during E13 P/N divisions Numb may inhibit Notch activity in one daughter of a radial glial cell to make it differentiate into a neuron. At N/N divisions, Numb may inhibit Notch in one daughter to generate a different neuron type.