text: To confirm our impression that Numb can be asymmetrically segregated between the two daughter cells of a neuroepithelial cell division, we dissociated P0 retinas and cultured the cells at low density in serum-free medium in the presence of basic FGF and EGF. In these conditions, some neuroepithelial cells continued to divide. After 3–4 hr, any cells that were in clusters of two or more were ringed and excluded from the analysis. After 24–28 hr, the cells were fixed and stained for Numb. We observed many cell doublets in which the two cells seemed either to be in the process of cytokinesis or to have recently completed cytokinesis. The proportion of cell doublets increased from 6% at 3–4 hr to 24% at 24–28 hr, suggesting that a significant proportion of single cells divided during the culture period.
  In many single cells, Numb staining was confined to one side of the cell (Fig. 5A,a–c), whereas in others it was diffusely distributed around the cell periphery (data not shown). In most cell doublets, Numb staining was found in both cells (Fig. 5A,d–f), which were probably produced by a symmetric division during the culture period; the staining was usually polarized and always at the same side of the two cells (Fig.5A, d--f). In some doublets, however, only one of the two cells stained for Numb, and the staining was confined to one pole of the cell, which was usually the pole opposite to the cell–cell contact zone (Fig. 5A, g–i), consistent with the cell being produced by an asymmetric division during the culture period. Thus, some dissociated retinal neuroepithelial cells can retain their polarityin vitro and apparently segregate Numb asymmetrically to their daughter cells when they divide.
  Asymmetric distribution of Numb and symmetric distribution of Notch-1 in dissociated retinal cell cultures.A, Differential-interference contrast images of dissociated retinal cells (a, d,g), immunofluorescence images of Numb staining (red) of the corresponding cells (b,e, h), and overlaid images of both (c, f, i). Some single cells show Numb confined to one pole (a–c). In most cell doublets, Numb is found at the same pole in both cells, consistent with the cells being produced by a symmetric division in culture (d–f). In some doublets, however, Numb is found only in one of the cells, with a distribution consistent with the two cells being produced by an asymmetric division in which only one daughter cell inherited Numb (g–i).B, Numb (a, c,e) and Notch (b, d,f) immunofluorescence staining in dissociated retinal cells. The DNA is stained in blue with Hoescht dye in a, c, and e. In a single cell in which Numb is segregated to one pole (a), Notch-1 is evenly distributed on the cell surface (b). In cell doublets (c–f), Notch-1 is always observed evenly distributed on the surface of both cells (d, f), irrespective of whether Numb is found on both cells (c) or only on one (e).
  Because Numb inhibits Notch signaling (Frise et al., 1996; Guo et al., 1996; Spana and Doe, 1996; Wakamatsu et al., 1999) and Notch has been reported to be asymmetrically segregated to the basal daughter cell of vertical divisions in the developing ferret cortex (Chenn and McConnell, 1995), we double-labeled dissociated retinal cells for Notch-1 and Numb. Irrespective of the distribution of Numb, Notch-1 was always diffusely distributed on the cell surface (Fig. 5B). Thus, Notch-1 is apparently always distributed to both daughter cells during cytokinesis, at least in cultured retinal cells.
  In examining propidium-iodide-stained sections of the developing retina, it is difficult to be certain of the orientation of a mitotic spindle. We have circumvented this problem by using confocal microscopy and three-dimensional coordinate geometry to determine spindle orientation. Our results indicate that some dividing neuroepithelial cells in the developing rat retina have their mitotic spindle oriented 45–90° away from the plane of the neuroepithelium and seem to distribute Numb preferentially to the apical daughter cell.
  Numb plays a crucial part in cell-fate determination duringDrosophila development, and there is some indirect evidence that it may also do so in vertebrates. Two mammalian homologs of Numb have been identified in mouse, both of which are expressed in the nervous system: Numblike is cytosolic in postmitotic neurons of the cortical plate (Zhong et al., 1997), whereas m-Numb is bound to the cytosolic face of the plasma membrane at the apical pole of mitotic cortical neuroepithelial cells (Zhong et al., 1996, 1997). Mice homozygous for a loss-of-function mutant allele ofm-numb exhibit severe defects in cranial neural tube closure and precocious neuron production in the forebrain, and they die at approximately E11.5, suggesting a role for Numb in maintaining a progenitor cell character during cortical neurogenesis (Zhong et al., 2000).
  On the basis of its apical distribution in mouse cortical neuroepithelial cells, Zhong et al. (1996) suggested that m-Numb would be distributed into either one or both daughter cells during cell division, depending on the orientation of the cell division. Our findings show that this is the case in the neonatal rat retina. We observed that Numb is asymmetrically distributed in both mitotic and interphase cells, suggesting that it is not degraded or redistributed in the cell after mitosis but remains apical at all stages of the cell cycle. Consequently, in mitotic cells with a horizontally oriented spindle, Numb seems to be present in about equal amounts in both of the nascent daughter cells, whereas in cells with a vertically oriented spindle it is preferentially segregated to the nascent apical daughter cell, with very little, if any, in the nascent basal cell. Even in low-density dissociated retinal cell cultures, Numb is asymmetrically localized in some cells and seems to be preferentially distributed to one of the two daughter cells when some retinal neuroepithelial cells divide. These results suggest that polarity can be maintained in dissociated cells in culture and support the conclusion that some retinal neuroepithelial cells in vivo divide asymmetrically and distribute Numb to only one daughter cell. BecauseDrosophila Numb is necessary for the daughter cells of asymmetric divisions in the fly to adopt distinct fates (Rhyu et al., 1994; Spana et al., 1995) and m-Numb can rescue the Numb-deficient fly mutant (Zhong et al., 1996), it seems likely that the asymmetric segregation of Numb plays a part in cell-fate choice during mammalian retinal development.
  Using time-lapse microscopy of dividing cells in slices of developing ferret cerebral cortex, Chenn and McConnell (1995) reported that the orientation of cell division could influence the fates of daughter cells. Horizontal divisions produced behaviorally and morphologically identical daughters that resembled precursor cells. In contrast, vertical divisions produced basal daughters that behaved like young migratory neuroblasts and apical daughters that remained within the proliferative zone. Putting these findings together with the results of Zhong et al. (1996) on the localization of m-Numb in the proliferative zone of the developing mouse cerebral cortex, it seems likely that the apical daughter cell that inherits m-Numb in vertical divisions would remain a neuroepithelial cell. In view of the well known role of Notch in lateral inhibition (for review, seeLewis, 1998), this conclusion may seem surprising, because cells that inherit Numb would be expected to have reduced Notch signaling (Frise et al., 1996; Guo et al., 1996; Spana and Doe, 1996). It is now clear, however, that Notch signaling can have diverse roles in development and can sometimes promote differentiation instead of inhibiting it (for review, see Wang and Barres, 2000).
  Our results show that the proportion of dividing retinal neuroepithelial cells with vertical spindles varies with developmental age. Few are observed at E18 (and at E14 or E16; data not shown), whereas many are observed at birth, and intermediate proportions are observed at P4. This timing might reflect the normal time course of retinal cell production. Different cell types are born within specific, but overlapping, time windows in the developing mammalian retina. In rodents, for example, retinal ganglion cells and cones are produced mainly prenatally, whereas Müller and bipolar cells develop postnatally (Young, 1985). Rod photoreceptors, which make up >70% of the cells in the adult rodent retina, are born over a prolonged period, spanning embryonic and postnatal development, but most are born around birth (Young, 1985). Thus, the high proportion of vertical divisions at birth could reflect the production of rods, Müller cells, or bipolar neurons (or a combination of these) around this time.
  Recently, Notch signaling has been found to promote the development of glial cells, including Schwann cells in the peripheral nervous system (Morrison et al., 2000) and radial glial cells (Gaiano et al., 2000) and Müller cells (Furukawa et al., 2000) in the CNS. In the retina, overexpression of Notch-1 in retinal precursor cells using a replication-incompetent retroviral vector resulted in bigger clones that contained an abnormally large number of cells expressing a Müller cell marker (Furukawa et al., 2000). Because Numb inhibits Notch signaling and Notch seems to be inherited symmetrically by both daughter cells of all retinal neuroepithelial cell divisions, at least in culture (Fig. 5B), the apical daughter cells of vertical divisions in the retina might be inhibited from developing into Müller cells as a consequence of inheriting Numb. A basal cell, which receives Notch but not Numb, by contrast, might be encouraged to differentiate into a Müller cell as a result of unopposed Notch signaling (Fig. 6). Lineage analysesin vivo, after retroviral infection of the P0 rat retina, have found that clones never contain more than one Müller cell (Turner and Cepko, 1987), consistent with a model in which Müller cells develop exclusively from Numb-deficient basal daughter cells produced by asymmetric cell divisions. Even at birth, however, the great majority of divisions are horizontal, suggesting that symmetric divisions produce most cells born postnatally, where Notch signaling is at least partly counteracted by Numb.
  A model for how asymmetric cell division in the retina may influence cell-fate. Some retinal neuroepithelial cells reorient their mitotic spindles and divide vertically. Because Numb is concentrated at the apical pole of the cells, it is likely to be inherited preferentially by the apical daughter cell of such divisions, where it inhibits Notch signaling. The basal daughter cell inherits little Numb, allowing unopposed Notch signaling and differentiation into a Müller cell. The apical cell might divide again or develop into another cell type.