Distinct behaviors of neural stem and progenitor cells underlie cortical neurogenesis

Abstract

Neocortical precursor cells undergo symmetric and asymmetric divisions while producing large numbers of diverse cortical cell types. In Drosophila, cleavage plane orientation dictates the inheritance of fate-determinants and the symmetry of newborn daughter cells during neuroblast cell divisions. One model for predicting daughter cell fate in the mammalian neocortex is also based on cleavage plane orientation. Precursor cell divisions with a cleavage plane orientation that is perpendicular with respect to the ventricular surface (vertical) are predicted to be symmetric, while divisions with a cleavage plane orientation that is parallel to the surface (horizontal) are predicted to be asymmetric neurogenic divisions. However, analysis of cleavage plane orientation at the ventricle suggests that the number of predicted neurogenic divisions might be insufficient to produce large amounts of cortical neurons. To understand factors that correlate with the symmetry of cell divisions, we examined rat neocortical precursor cells in situ through real-time imaging, marker analysis, and electrophysiological recordings. We find that cleavage plane orientation is more closely associated with precursor cell type than with daughter cell fate, as commonly thought. Radial glia cells in the VZ primarily divide with a vertical orientation throughout cortical development and undergo symmetric or asymmetric self-renewing divisions depending on the stage of development. In contrast, most intermediate progenitor cells divide in the subventricular zone with a horizontal orientation and produce symmetric daughter cells. We propose a model for predicting daughter cell fate that considers precursor cell type, stage of development, and the planar segregation of fate determinants.

Fig. 1

Radial glial (RG) cells undergo symmetric self-renewing divisions during early stages of cortical development. A: Classification of cleavage plane analysis. B: Time-lapse imaging of a single RG cell in an organotypic slice culture prepared from an embryonic day (E)13 rat. The RG cell is shown entering metaphase at the surface of the lateral ventricle at t = 0. The dotted white line indicates the surface of the lateral ventricle. A vertically oriented cleavage furrow (red line) begins developing t = 30 m, and can be clearly visualized at t = 33 m and 36 m. Both daughter cells acquired the morphology of RG cells and remained in the ventricular zone (VZ). C: Horizontal cleavage plane divisions at the ventricular surface also produce daughter cells with symmetric morphologies and behaviors. A single bipolar RG cell at E13 is shown in G1-phase at t = 0. The RG cell body rose slightly in the VZ before descending to the ventricular surface (t = 5h:20m). The pial fiber became very thin and faint during mitosis but was detected post hoc (see Suppl. Movie 2). The RG cell divided with a horizontal cleavage plane (red line) at t = 5h:40m. After division the soma of the basal daughter cell (red arrowhead) moved away from the ventricle at a faster rate than its apical sibling. This behavior might be interpreted to signify an asymmetric fate for the daughter cells. Nonetheless, extended time-lapse imaging demonstrated that both daughter cells retained contact with the ventricle, resumed IKM, and divided at the ventricular surface (t = 40 h). We therefore classified this RG division as symmetric self-renewing. An unrelated cell with the morphology of a tangentially migrating cell was present in the marginal zone of the viewing field from 5–11 hours. Time elapsed is shown in either minutes (m), or hours and minutes (hh:mm) as indicated below each sequence. The entire time-lapse sequences can be viewed in Supplemental Movies 1, 2. A magenta-green version of this figure can be viewed online as Supplementary Figure 1.

Fig. 2

Intermediate progenitor (IP) cells divide with horizontal cleavage orientation, and produce symmetric daughter cells. Time-lapse sequence of a single eGFP-labeled IP cell at embryonic day (E)13 (A), and E17 (B), that divided horizontally away from the ventricle. The left panels in both A and B are transmitted light images showing the ventricular zone (VZ), the ventricular surface, and also the pial surface in A. The IP cell shown in panel B divided in the subventricular zone (SVZ). In both examples, the IP cells divided with horizontally oriented cleavage planes (red line), and daughter cells exhibited similar morphologies and behaviors that included the extension of processes toward the ventricle (A, t = 8h; B, t = 1h:30), before the extension of a new process oriented toward the cortical plate. Time elapsed is shown in either minutes (m), or hours and minutes (hh:mm) as indicated below each sequence. The entire time-lapse sequences can be viewed in Supplemental Movies 3, 7. A magenta-green version of this figure can be viewed online as Supplementary Figure 2. Scale bar in A applies to B.

Fig. 3

The pial fiber of mitotic radial glial (RG) cells can be identified by multiple varicosities. A: A single RG cell (red arrowhead) is shown dividing at the surface of the ventricle at t = 0. The ventricular surface is indicated by the white dotted line, the pial surface is indicated at the top of the panel. The white arrow indicates an intermediate progenitor (IP) daughter cell apposed to its parental pial fiber. The pial fiber becomes very thin during metaphase, but is identifiable by conspicuous varicosities (white arrowheads) along the entire length of the fiber. B: After division the pial fiber has become thicker, and most varicosities are no longer visible. The new RG daughter cell (red arrow) is located behind its parent RG cell in this projection image. Time is shown in hours and minutes (hh:mm) below each image. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate. A magenta-green version of this figure can be viewed online as Supplementary Figure 3.

Fig. 4

Radial glial (RG) cells divide vertically at the ventricular surface to produce asymmetric daughter cells in embryonic day (E)16–E19 rat neocortex. A: Time-lapse imaging recorded the proliferative and migratory behaviors of one RG cell and its progeny over 3 days. The RG cell divided with a vertically oriented cleavage plane (red line) at the ventricular surface (t = 2 h: 51 m). One daughter (cell 2a, red arrowhead) was a self-renewed RG cell that resumed IKM and divided a second time at the ventricle with a vertical cleavage plane orientation (t = 50h:53m). The second daughter (cell 2b, white arrowhead) was an intermediate progenitor cell that migrated away from the ventricle along the parental RG fiber and divided with a horizontal cleavage plane orientation (red line) at the bottom limit of the intermediate zone (IZ, t = 53h). In some cases only one cell in the clone was imaged to limit exposure to laser light (asterisk). The white dotted line represents the ventricular surface. Time elapsed is shown in hours and minutes (hh:mm) below the sequence. The entire time-lapse sequence can be viewed in Supplemental Movie 4. B: Whole-cell patch-clamp recordings performed after time-lapse imaging demonstrated that cell 2b generated two daughter cells that both expressed the inward voltage-gated currents (downward deflection of traces below red line) that are characteristic of sodium currents in immature neurons. C: In contrast, cell 2a produced two daughter cells that lacked inward voltage-gated currents, exhibiting only the outward voltage-gated currents that are characteristic of potassium currents in astroglial cells. D: Lineage tree depicting the progeny of the single RG cell shown in panel A. VZ, ventricular zone; IZ, intermediate zone. A magenta-green version of this figure can be viewed online as Supplementary Figure 4.

Fig. 5

Nissl analysis demonstrates that in vivo the majority of ventricular zone (VZ) surface divisions occur with a vertical cleavage plane orientation (A–C), and that the majority of abventricular divisions in the subventricular zone occur with a horizontal orientation (D–F). A: Nissl-stained tissue prepared from the neocortex of an embryonic day (E)17 rat. B: Higher power image showing a dividing precursor cell at the surface of the VZ. C: The percentage of ventricular surface divisions with vertical cleavage plane (black bars), oblique (gray bars), or horizontal (white bars) orientation did not change during cortical development. D: Nissl-stained tissue prepared from an E15 rat showing two examples of abventricular progenitor cells undergoing division away from the ventricle (arrow). E: Higher-power magnification of the boxed region in (D). F: The percentage of abventricular mitoses with vertical cleavage plane (black bars), oblique (gray bars), or horizontal (white bars) orientation changed across the period of cortical neurogenesis. Significantly more abventricular cells divided with a horizontal orientation at E15 and E17 (asterisk, P < 0.01), compared to either horizontal or oblique divisions. But the orientation of the abventricular mitoses was random at the end of neurogenesis at E20. Error bars depict the standard error of the mean. IZ, intermediate zone; CP, cortical plate.

Fig. 6

The embryonic subventricular zone (SVZ) is a neurogenic compartment. A: Nissl-stained tissue costained with doublecortin (DC) antibodies shows the pattern of DC-immunoreactivity in a coronal section of E17 rat cortex. The cortical plate (CP) is densely labeled while the ventricular zone (VZ) is lightly stained. B: Histogram showing the proportion of mitotic abventricular cells that express DC during embryonic cortical development. At E12 93% of SVZ mitoses are DC+. The proportion of DC+ mitoses decreased slightly to 83% at E20. By P7 the proportion of DC+ mitoses has fallen to less than 40%. Error bars depict the standard error of the mean. C–F: DC antibodies label abventricular mitoses. DC+ abventricular cells are shown in prophase, early anaphase, late anaphase, and telophase. G: Not all abventricular mitotic cells expressed DC. A telophase abventricular mitosis that does not express DC is shown. Brown DC-immunoreactive product can be seen in neighboring cells. H: Preadsorption of the doublecortin (DC) antibody with DC protein eliminated immunostaining. IZ, intermediate zone; SVZ, subventricular zone.

Fig. 7

Tbr2+ intermediate progenitor (IP) cells are located in the ventricular zone (VZ) before the subventricular zone (SVZ) forms. A: The nuclear transcription factor Tbr2 (red) is detected in a few cells at embryonic day (E)12. Syto-11 (green) labels the chromatin of all cells. Interphase IP cells with dispersed chromatin that express Tbr2 appear yellow. B: At E15 the number of Tbr2+ cells increases dramatically throughout the VZ. Many Tbr2+ cells are close to the ventricle and some Tbr2+ mitoses can be seen at the VZ surface. C: By E17 the SVZ has fully formed and the majority of Tbr2+ cells are concentrated in the SVZ. Mitotic Tbr2+ cells are no longer present at or near the ventricle. D: The VZ has shrunk considerably by the end of cortical neurogenesis at E20. Tbr2+ cells are detected in the SVZ, but not in the VZ. E–H: The position of Tbr2+ cells in the proliferative zones of embryonic neocortex is plotted in these graphs. The distance of Tbr2+ cells was measured in a series of 10-μm bins from the VZ surface to the pia. The Y-axis depicts distance from the ventricle, the X-axis depicts the proportion of Tbr2+ cells that were present in each 10 μm bin. Error bars represent the standard error of the mean. The proportion of Tbr2+ cells undergoing division at the ventricle or near the surface of the ventricle is much higher at E15 (F), in comparison to the proportion earlier at E12 (E), or later at E17 (G). I: Examples of Tbr2+ cells are shown in the embryonic SVZ. J: A Tbr2-positive cell in the SVZ in telophase. Tbr2-immunoreactivity (red) is present between the sister chromatids. K–M: Examples of Tbr2+ cells undergoing division at the ventricle in the E15 rat. K: A Tbr2+ cell in prophase entering mitosis in near the VZ surface. L: A Tbr2+ cell in prophase at the surface of the VZ. The white arrowhead points to an adjacent Tbr2–negative cells undergoing division at the VZ surface. M, A Tbr2+ cell is at the margin of the ventricle undergoing division with an oblique orientation. A magenta-green version of this figure can be viewed online as Supplementary Figure 5. Scale bar in B refers to A–D; scale bar in M refers to I–L.

Fig. 8

Radial glial (RG) cells do not express Tbr2. A–C: GFP-labeled RG cells (green) in the embryonic ventricular zone (VZ) do not express Tbr2 (red). RG cells in interphase (A), G-2 phase (B), and prophase (C). Only RG daughter cells that are intermediate progenitor (IP) cells express Tbr2. The Tbr2 negative daughter cells (white arrowheads) may represent daughter neurons that are produced by RG divisions. D: A GFP-labeled IP cell in the subventricular zone migrating along its parental RG fiber (white arrows). E: The same cell shown in panel D expresses Tbr2 (red, F). G: Merged image showing colocalization. A magenta-green version of this figure can be viewed online as Supplementary Figure 6.

Fig. 9

The location, cleavage plane orientation, and mode of neural stem and progenitor cell divisions in the dorsal telencephalon during cortical development. Radial glial (RG) cells divide vertically at the surface of the ventricular zone (VZ) throughout cortical development. Before neurogenesis begins most RG divisions are symmetric self-renewing (red), expanding the founder population of VZ progenitor cells. At the onset of neurogenesis RG cells undergo asymmetric self-renewing divisions (dark blue) that produce either neurons or intermediate progenitor (IP) cells. Daughter neurons produced directly by RG cells may form the lower cortical layers. RG cells produce IP cells throughout the remainder of cortical neurogenesis. Daughter IP cells (light blue) divide close the ventricle during early stages of neurogenesis, but their location shifts away from the ventricle and the IP cells divide in the subventricular zone (SVZ) once that structure has formed. Most IP cells divide horizontally and produce symmetric pairs of neurons that form the upper cortical layers. After producing neurons RG cells translocate away from the ventricle and produce glial progeny (green). IZ, intermediate zone; MZ, marginal zone.