Live imaging at the onset of cortical neurogenesis reveals differential appearance of the neuronal phenotype in apical versus basal progenitor progeny

Abstract

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin-driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors.

Figure 1. Intrinsic GFP fluorescence in the central nervous system of Tubb3-mGFP mouse embryos.

(A–F) Whole-mount images of unfixed Tubb3-mGFP embryos at E9.5 (A), E10.5 (B), E11.5 (C) and E12.5 (D–F; E, dorsal view; F, ventral view). Background has been darkened electronically. Scale bars, 500 µm. (G–K) Confocal scanning photomicrographs (1-µm single optical sections) of 12 µm-thick cryosections through the E11.5 dorsal telencephalon (G, J), midbrain (H), hindbrain (I) and E12.5 retina (K) of Tubb3-mGFP mice. Green, intrinsic Tubb3-mGFP fluorescence; blue, Hoechst staining of nuclei. Note the thicker preplate (PP) and neuronal layers (NL) in the hindbrain than midbrain and telencephalon; VZ, ventricular zone. Ventricular (apical) surface is down (dashed lines). The arrow indicates a Tubb3-mGFP expressing cell (i.e., a neuron) whose soma is close to the ventricular surface; arrowheads indicate neuronal processes. Scale bars: G–I, 20 µm; J, K 10 µm.

Figure 2. Tubb3-mGFP is specifically expressed in neurons and targeted to the Golgi complex and plasma membrane.

(A–F and H–K) Confocal scanning photomicrographs (1-µm single optical sections; H, confocal stack) of 12–16 µm-thick cryosections through the E11.5 dorsal telencephalon of Tubb3-mGFP mice, showing intrinsic Tubb3-mGFP fluorescence (A, C green, D, F green, H, I, K green) and nestin (B, C red), Tuj1 (E, F red), or giantin (J, K red) immunofluorescence. Ventricular (apical) surface is down (dashed lines); PP, preplate; VZ, ventricular zone. Asterisks indicate neurons in the VZ. (G) GFP immunogold (10 nm) electron microscopy of a neuron in the dorsal telencephalon of an E11.5 Tubb3-mGFP mouse; Mit, mitochondrion. (G–K) Note the association of Tubb3-mGFP with the plasma membrane (arrowheads) and its concentration in the Golgi complex (arrows). Scale bars: A–F, 20 µm; G, 100 nm; H and I–K, 10 µm.

Figure 3. Distinct subcellular localization of nucGFP and mGFP allows distinction between neuronal progenitors and neurons in the double transgenic Tis21-nucGFP/Tubb3-mGFP mouse line.

(A–C) Confocal scanning photomicrographs (stacks of 6–10 optical sections, 1-µm each) of 20 µm-thick cryosections through the dorsal telencephalon of E11.5 (A), E12.5 (B) and E13.5 (C) Tis21-nucGFP/Tubb3-mGFP double transgenic mouse embryos. (B') Single optical section (1 µm) of B. Green, intrinsic GFP fluorescence; blue, Hoechst staining of nuclei. Scale bar: A–C, 5 µm. (D and E) Cartoon illustrating presumptive cell lineages and the observed distinct subcellular localization of nucGFP versus mGFP; AP, apical progenitor; BP, basal progenitor; N, neuron; G1 and G2, phases of progenitor cell cycle. (A–E) Solid arrows, nuclei of neuronal progenitors showing Tis21-nucGFP fluorescence; open arrows, neuronal cell bodies showing Tis21-nucGFP plus Tubb3-mGFP fluorescence; open arrows with asterisk, neuronal cell bodies showing only Tubb3-mGFP fluorescence; arrowheads, neuronal processes showing Tubb3-mGFP fluorescence (see key in top left panel). Ventricular (apical) surface is down (dashed lines); PP, preplate; CP, cortical plate; VZ, ventricular zone.

Figure 4. Two-photon imaging of apical and basal progenitor cell divisions and analysis of daughter cell fate in the double transgenic Tis21-nucGFP/Tubb3-mGFP mouse line.

Two-photon time-lapse video microscopy of acute 500 µm-thick slice cultures prepared from dorsal telencephalon of E10.5 (A, B) and E12.5 (C) Tis21-nucGFP/Tubb3-mGFP double transgenic mouse embryos. (A) A neurogenic progenitor (solid arrows with asterisk), identified by Tis21-nucGFP expression, divides at the basal side of the ventricular zone (8–16 min). Both daughter cells (open arrows) are neurons as they show Tubb3-mGFP fluorescence in their processes (arrowheads), albeit with a different onset (left daughter, 24–216 min; right daughter, 72–216 min), and enter the neuronal layer (right daughter, 64–216 min; left daughter, 152–216 min). Note the transient apical migration of the left daughter (24–80 min). (B) A neurogenic progenitor (solid arrow with asterisk), identified by Tis21-nucGFP expression, divides in a subapical location (0–10 min). The nucleus of one of the daughter cells (open arrows) migrates rapidly towards the basal side (10–160 min) whereas that of the sibling daughter (solid arrows) migrates first to the apical surface (10–100 min) and then slowly basally (140–250 min). The cell with the leading nucleus shows Tubb3-mGFP fluorescence in its process (detectable as of 140 min, arrowheads), indicative of it being a neuron; this cell subsequently migrated perpendicular to the X-Y plane of imaging and could not be tracked further (170–250 min). The white lines at 150, 160 and 170 min were added to indicate that the upper and lower portions of the panels represent different sets of optical sections from the z-stack. Note that the cytoplasmic GFP fluorescence observed at 20 min reflects residual Tis21-nucGFP that is not yet re-sequestered into the daughter nuclei, rather than Tubb3-mGFP. (C) A neurogenic progenitor (solid arrows with asterisk), identified by Tis21-nucGFP expression, migrates rapidly from the basal boundary of the ventricular zone to the apical surface (0–24 min) and divides (24–48 min). The nuclei of the daughter cells (solid arrows) migrate towards the basal side (60–336 min), with one nucleus leading (right solid arrows). The leading nucleus was tracked until it entered the neuronal layer (after 372 min), whereas the trailing nucleus was tracked until it disappeared due to photobleaching (336–372 min). (A–C) Intrinsic GFP fluorescence is white. Each image is a maximum intensity projection of the single focal planes (2.7-µm steps) that showed the cells of interest. Numbers indicate tracking time (min). Time-lapse intervals: 8 min (A), 10 min (B), 12 min (C). Apical surface is down (dashed lines). Scale bars, 5 µm. The cartoons on the left summarize the observations; PP, preplate; CP, cortical plate; the boundary between the VZ and the preplate or cortical plate in the images is indicated by the line between VZ and PP or CP, respectively.

Figure 5. Behavior of daughter cells arising from apical and basal progenitors.

Slice cultures prepared from dorsal telencephalon of E10.5–E12.5 Tis21-nucGFP/Tubb3-mGFP double transgenic mouse embryos were analyzed by two-photon time-lapse video microscopy for the behavior of daughter cells arising from Tis21-nucGFP–expressing APs (79 mitoses) and BPs (83 mitoses) in a total of 23 independent experiments. (A) Proportion of Tis21-nucGFP–positive AP and BP daughter cells that show discernible Tubb3-mGFP expression. Numerals in the sectors refer to the number of Tis21-nucGFP–expressing mother cells observed; white, no detectable (or discernible) Tubb3-mGFP expression in daughter cells; gray, discernible Tubb3-mGFP expression in one of the daughter cells; black, discernible Tubb3-mGFP expression in both daughter cells. (B) Onset of Tubb3-mGFP expression in daughter cells derived from Tis21-nucGFP–expressing APs and BPs. Open circles indicate individual daughters; bars, mean±S.D.; triple asterisk, p <0.001 (ANOVA) AP versus BP daughters. (C) Proportion of AP and BP daughter cells that show migration of Tis21-nucGFP–positive nuclei beyond the basal boundary of the ventricular zone (VZ exit). Only cases in which both daughter cells lacked Tubb3-mGFP expression were scored. Numerals in the sectors refer to the number of Tis21-nucGFP–expressing mother cells observed; black, both daughter cell nuclei remain in the VZ; gray, VZ exit of one daughter cell nucleus; white, VZ exit of both daughter cell nuclei. (D) Time point of VZ exit of the Tis21-nucGFP–positive nucleus of Tubb3-mGFP–negative daughter cells derived from APs and BPs. Note that for single daughters exiting the VZ, only cases in which the other daughter cell lacked Tubb3-mGFP expression were scored. Open circles indicate individual daughters; bars, mean±S.D.; triple asterisk, p <0.0001 (t-test) AP versus BP daughters. (E and F) Fate of daughter cells arising from APs (E, 18 cases) and BPs (F, 73 cases). Width of the sectors reflects the number of daughter cell pairs observed. The circle lines indicate the major classes of behavior of daughter cell pairs. (E) Green/black, one daughter expresses Tubb3-mGFP and one daughter nucleus remains in the VZ; green/white, one daughter expresses Tubb3-mGFP and one daughter nucleus exits the VZ; black/red, one daughter nucleus remains in the VZ and one exits and can be tracked in the NL, with the time of observation exceeding the mean length of one cell cycle ; black/white, one daughter nucleus remains in the VZ and one exits; solid white, both daughter nuclei exit the VZ. (F) Solid green, both daughters express Tubb3-mGFP; green/white, one daughter expresses Tubb3-mGFP and one daughter nucleus exits the VZ; solid white, both daughter nuclei exit the VZ; solid black, both daughter nuclei remain in the VZ longer than 100 min; green/black, one daughter expresses Tubb3-mGFP and one daughter nucleus remains in the VZ; white/black, one daughter nucleus exits, and the other remains in, the VZ.

Figure 6. Cleavage plane orientation in apical and basal progenitors.

Slice cultures prepared from dorsal telencephalon of E10.5-E12.5 Tis21-nucGFP/Tubb3-mGFP double transgenic mouse embryos were analyzed by two-photon video microscopy for the cleavage plane orientation of mitotic APs and BPs. (A) Diagram showing the four classes of cleavage planes; 0° corresponds to parallel (horizontal cleavage plane), and 90° to perpendicular (vertical cleavage plane), to the ventricular surface (dashed lines in (C). (B, C) Examples of a horizontal cleavage plane (solid lines) in a BP (B, arrow) and a vertical cleavage plane (solid lines) in an AP (C, arrow); time intervals were 10 min (B) and 8 min (C) (see numbers in the lower left corner of the panels). Pie charts show the distribution of cleavage plane orientation between the four classes defined in (A) for 33 mitoses of BPs (B) and 42 APs (C). Only cases in which cytoplasmic Tis21-nucGFP could be observed (i.e. the cell being in anaphase; B, 10 min; C, 8 min) and which therefore allowed the deduction of cleavage plane orientation were included (75 of 184); white triangles indicate the position of daughter cell chromosomes/nuclei. Scale bar, 5 µm.

Figure 7. Lack of apical-basal polarity in mitotic basal progenitors.

Confocal scanning photomicrographs of 20 µm-thick fixed cryosections through the dorsal telencephalon of heterozygous E10.5 (A–B'), E13.5 (C–D'), E10.5 (E–H) and E11.5 (I–L) Tis21-nucGFP knock-in mouse embryos. (A–D') Stack of 7–10 optical sections (1 µm each) showing Tis21-nucGFP–negative (A, A', C, C') and Tis21-nucGFP–positive (B, B', D, D') mitotic BPs (identified by Hoechst staining and phosphohistone 3 immunofluorescence, not shown); red, MPM-2 immunofluorescence (A, B) or intrinsic mRFP fluorescence after in utero electroporation at E12.5 (C, D); green, intrinsic GFP fluorescence (A–D'). Note the absence of apically-directed processes in both Tis21-nucGFP–negative and –positive mitotic BPs. Arrowheads, short basal extension from the cell body that did not exceed one cell body diameter. Scale bar in D', 5 µm. (E–P) Single optical sections (1 µm) showing Tis21-nucGFP–negative (E–H, P) and Tis21-nucGFP–positive (I–O) mitotic BPs (arrows) and APs (arrowheads); white, prominin-1 (E, H, I, L), megalin (M), ZO-1 (N) or aPKC (O–P) immunofluorescence; red, MPM-2 immunofluorescence (F, H, J, L); green, intrinsic GFP fluorescence (G, H, K, L, M–P); blue, Hoechst staining (H, L). Note the absence of prominin-1, megalin, ZO-1 and aPKC immunoreactivity in both Tis21-nucGFP–negative and –positive mitotic BPs, but its presence in mitotic APs and on the apical surface. The prominin-1 immunoreactive dots in the VZ (E–H, I–L) are extracellular particles known to be present within the neuroepithelium , ; note that these are spatially distinct from the mitotic BPs. Dashed lines, apical plasma membrane; dashed circle in (P) outlines the soma of a Tis21-nucGFP–negative mitotic BP (as determined by Hoechst staining, data not shown). PP, preplate. Scale bar in P, 10 µm. (A–P) Ventricular (apical) surface is down.

Figure 8. Proposed relationship between Tis21-nucGFP–positive and –negative apical progenitors, basal progenitors and neurons in the dorsal telencephalon.

(A) Asymmetrically dividing Tis21-nucGFP–positive APs. (B) Asymmetrically dividing Tis21-nucGFP–negative AP. Tis21-nucGFP and Tubb3-mGFP are indicated in green; red and dashed line, apical plasma membrane and ventricular surface, respectively; dotted lines, cleavage plane; N, neuron; PP, preplate; VZ, ventricular zone. Note that the proposed model is confined to asymmetrically dividing APs and incorporates observations of the present and previous studies , .