Plane of cell cleavage and numb distribution during cell division relative to cell differentiation in the developing retina

Abstract

Progenitor cells in the early developing nervous system can divide symmetrically, giving rise to two daughter cells that divide again, or asymmetrically, giving rise to one cell that differentiates and one that divides again. It has been suggested that the orientation of the cell cleavage plane during mitosis determines the type of division. A marker of early cell differentiation, the RA4 antigen, was used to identify regions of the developing chick retina with and without differentiating cells, and the orientation of the cleavage plane was characterized for mitotic figures in each region. No difference was found in the frequency of any orientation between the regions with or without differentiating cells. Furthermore, in the region of the retina with differentiating cells, the RA4 antigen was present in mitotic figures with every possible orientation. Thus, the orientation of the cleavage plane appears to be unrelated to whether or not a division produces a cell that differentiates. It has also been suggested that the intracellular protein Numb mediates neurogenesis via asymmetric localization during cell division. Numb localization was compared with expression of markers of early cell differentiation, the RA4 antigen and Delta. Differentiating and nondifferentiating cells were found both with and without Numb expression. Cells with a cleavage plane parallel to the retinal surface were polarized, such that Numb and/or the RA4 antigen, when present, were only in the daughter cell farthest from the ventricle. These findings indicate a need to reconsider current hypotheses regarding the key features underlying symmetric and asymmetric divisions in the developing nervous system.

Fig. 1.

Identification of areas of the retina with and without differentiating cells based on immunostaining with the RA4 antibody. The RA4 antibody recognizes ganglion cells as they begin to differentiate. A, A differential interference contrast micrograph of a section of a stage 23 (E3.5) embryonic chick eye. Box, The region shown in the fluorescence micrographs at higher magnification in B andC. B, DAPI staining shows the nuclei of all cells. PE, Pigment epithelium; R, neural retina; L, lens. C, RA4 immunostaining shows differentiating ganglion cells. White lines indicate the division between the central area of the retina (to the left and below theline), where cell division is likely to produce daughter cells that differentiate, and the peripheral area (to theright and above the line), where cell division only produces cells that continue to divide. Scale bars, 50 μm.

Fig. 2.

Orientation of the plane of cell cleavage during cell division in the developing retina. These fluorescence micrographs show mitotic figures (arrows) in DAPI-stained sections of retina. The plane of cell cleavage ranges from parallel (A) to perpendicular (B) relative to the ventricular surface of the retina. These two mitotic figures are in the RA4⁻ area of the retina, where cells are not differentiating. This indicates that cells generated by division with a parallel cleavage plane do not necessarily differentiate. The cell-free, subretinal space separates the neural retina from the cells of the pigment epithelium, which is along thebottom of the micrographs. Scale bar, 10 μm.

Fig. 3.

Orientation of cleavage planes relative to differentiation. Bars indicate the percentage of mitotic figures with a plane of division perpendicular (white), parallel (black), or at an intermediate angle (gray) to the ventricular surface of the retina. Cells were considered to have a perpendicular or parallel cleavage plane when their axis was within 30° of the major axis; cells outside this range were considered to have an intermediate cleavage plane. There was no significant difference in the percentage of cells with parallel or perpendicular cleavage planes between the RA4⁻ and RA4⁺ areas of the retina at any of the three ages examined. In addition, there was no significant change between the different ages examined in the ratio of mitotic cells with perpendicular or parallel cleavage planes. These results suggest that the plane of cleavage does not correlate with divisions that generate differentiating cells. Error bars indicate SE.

Fig. 4.

Range of orientations of the cleavage plane of mitotic figures. The percentage of mitotic figures with each possible angle of cleavage in 10° increments is indicated for dividing cells in areas of the retina with (RA4⁺) and without (RA4⁻) differentiating cells. This analysis was performed on horizontal sections through the center of stage 27 retina. A total of 250 cells was analyzed in each area of the retina. This suggests that cells with a parallel plane of division are the extreme of a continuum weighted toward a perpendicular orientation and that they do not represent a separate group of cells.

Fig. 5.

RA4 and Numb distribution in dividing cells in the area of the retina with cell differentiation. Pairs of micrographs of the same mitotic cell show immunoreactivity for RA4 (green) on the left side and for chicken Numb (red) on the right side. DAPI staining (blue) shows all nuclei on both sides. Arrows indicate the plane of division.A, A mitotic figure with a perpendicular cleavage plane and symmetric distribution of RA4 and Numb. B, A mitotic figure with a perpendicular cleavage plane, asymmetric distribution of RA4, and no expression of Numb. C, A mitotic figure with a parallel cleavage plane, asymmetric distribution of Numb, and no expression of RA4. Figure 6 summarizes all combinations observed. Through-focus confirmed the expression pattern of Numb and RA4 in each cell. Scale bar, 10 μm.

Fig. 6.

The distribution of Numb and RA4 relative to different cleavage planes during cell division. Each pair of circles represents a mitotic figure with the orientation of the cell cleavage plane indicated. The subretinal ventricle would be just below the cells. Cells with a cleavage plane within 30° of perpendicular or parallel to the surface of the retina were grouped; cells outside this range were not classified.Gray circles indicate the presence of RA4 immunoreactivity, and black crescents indicate the presence of Numb immunoreactivity. Numbers indicate the relative frequency of each combination for 500 mitotic bodies in the RA4⁺ area of 18 retinas from stage 23 embryos.

Fig. 7.

Numb distribution in dividing cells in the area of the retina without cell differentiation. Micrographs of fluorescence show immunoreactivity for chicken Numb (red) and DAPI-stained nuclei (blue). Cells were not differentiating in this area of the retina, as indicated by a lack of RA4 staining. Numb staining was most abundant near the vitreal surface of the retina (toward the top). In mitotic bodies, Numb was distributed to both daughter cells (arrow inA), to one daughter cell (arrow inB), or to neither daughter cell (arrowhead in B). Because no cells were differentiating in this area, Numb clearly was not expressed exclusively by differentiating cells. The cell-free, subretinal space separates the neural retina from the cells of the pigment epithelium along the bottom of the micrographs. Scale bar, 10 μm.

Fig. 8.

Delta and Numb expression by cells in areas of the retina with cell differentiation. Micrographs of fluorescence show immunoreactivity for chicken Delta-1 (red), chicken Numb (green), and DAPI-stained nuclei (blue). Differentiating cells expressed Delta, but only a portion of these also expressed Numb. The row of cells along thebottom of the micrograph are pigment epithelium (PE). Scale bar, 10 μm.