Synchrony of clonal cell proliferation and contiguity of clonally related cells: production of mosaicism in the ventricular zone of developing mouse neocortex

Abstract

We have analyzed clonal cell proliferation in the ventricular zone (VZ) of the early developing mouse neocortex with a replication-incompetent retrovirus encoding human placental alkaline phosphatase (AP). The retrovirus was injected into the lateral ventricles on embryonic day 11 (E11), i.e., at the onset of neuronogenesis. Three days postinjection, on E14, a total of 259 AP-labeled clones of various sizes were found in 7 fetal brains. There are approximately 7 cell cycles between E11 and E14 (), and there is a 1-2 cell cycle delay between retroviral injection and the production of a retrovirally labeled "founder" cell; thus, we estimate that the "age" of the clones was about 5-6 cell cycles. Almost one-half of the clones (48.3%) identified were pure proliferating clones containing cells only in the VZ. Another 18.5% contained both proliferating and postproliferative cells, and 33.2% contained only postproliferative cells. It was striking that over 90% of the clonally related proliferating cells occurred in clusters of two or more apparently contiguous cells, and about 73% of the proliferating cells occurred in clusters of three or more cells. Regardless of the number of cells in the clone, these clusters were tightly packed and confined to a single level of the VZ. This clustering of proliferating cells indicates that clonally related cells maintain neighbor-neighbor relationships as they undergo interkinetic nuclear migration and progress through several cell cycles, and, as a result, the ventricular zone is a mosaic of small clusters of clonally related and synchronously cycling cells. In addition, cells in the intermediate zone and the cortical plate were also frequently clustered, indicating that they became postproliferative at a similar time and that the output of the VZ is influenced by its mosaic structure.

Fig. 1.

Frequency histograms showing the distribution of cells and clones found in the mouse neocortex at E14, 3 d after injections of DAP retrovirus at E11. A, The number of clones as a function of clone size is plotted as a percentage of all 259 AP-labeled clones. A majority of clones were small, containing only 1–3 cells, and only a small proportion of clones were large, containing >11 cells. The mean size of the clones (3.7 cells/clone) is indicated by an arrow. B, The number of VZ cells (proliferating cells) per clone is plotted as a percentage of the 173 clones that contained VZ cells. The largest cluster in the VZ contains 15 cells as shown at the right.C, The cellular composition of the VZ at E14 in terms of the percentage of cells residing in clones containing only a single VZ cell (white wedge) versus the percentage of cells residing in clones containing 2 or more cells (gray wedges). The percentages along the perimeter of the pie chart indicate the percentage of AP-labeled VZ cells found in clones containing 1, 2, 3, 4, and 5 or more VZ cells. This chart shows that only ∼9% of the total number of AP-labeled VZ cells (498) belonged to clones that contained only a single VZ cell and that a vast majority of the AP-labeled VZ cells (91%) were from clones that contained multiple cells clustered in the VZ.

Fig. 2.

Examples of AP-labeled clones in mouse neocortex at E14, 3 d after DAP retrovirus injection at E11.A, B, P clones in which all AP-labeled cells are restricted to the VZ. The clone shown in A has 8 cells that all seem to be in contact and are located near the ventricular surface. The clone shown in B also has 8 cells in a tight cluster, but it is located in the outer one-half of the VZ near the VZ/IZ border. C, D, Q clones in which no cells were found in the VZ. The clone shown inC consists of four cells in the middle of the IZ. One cell has a process that extends toward the CP (arrowhead). The clone shown in Dconsists of one cell in the middle of the CP. No cells were found in the VZ and/or IZ or in the adjacent sections spanning 150 μm in any direction. E, F, PQ clones that contain VZ cells and cells in at least one other stratum. The clone shown inE contains 5 VZ cells and 1 IZ cell; no cells were found in the CP. The VZ cells (lower arrow) are tightly clustered in the middle of the VZ. The IZ cell (upper arrow) is located close to the VZ/IZ border, indicating that it recently left the VZ and started to migrate. A bundle of stained processes (arrowheads) can be seen extending radially from the ventricular surface to the middle of the IZ, and all of the cells of this clone are located along this radial bundle. The clone shown in F contains cells in all three developing cortical strata, i.e., 5 VZ cells, 2 IZ cells, and 2 CP cells. The cells are radially aligned at strikingly evenly spaced intervals, suggesting that they were produced at consecutive generations. Radial processes can be seen intertwining among the clusters of cells throughout the whole thickness of the developing cortical strata. The 2 VZ cells are located side-by-side at the ventricular surface, as if they had recently completed anaphase. An additional VZ cell is in the middle of the VZ, as if it were in G₁ or G₂, and 2 more VZ cells are located in the outer one-third of the VZ close to the VZ/IZ border. The 2 IZ cells were clustered near the VZ cells at the border as if they had left the VZ only recently. The 2 CP cells were also closely spaced, indicating that they also may be cousins that had left the VZ simultaneously but before the exit time of the 2 IZ cells. V, Lateral ventricle. Scale bars, 20 μm.

Fig. 4.

Examples of AP-labeled one-, two-, and three-cell P clones in the mouse neocortex at E14, 3 d after injections of DAP retrovirus vector at E11. A–C, One-cell P clones. In general, the cells in the VZ have a large, round nucleus, abundant cytoplasm, and usually no visible processes. The three clones shown are at different levels in the VZ, at the ventricular surface (A), in the middle of the VZ (B), and in the outer one-half of the VZ near the VZ/IZ border (C). D–F, Two-cell P clones. In two of the examples (D, E), one at the ventricular surface (D) and the other in the middle of the VZ (E), the 2 cells seem to contact each other along one of their lateral borders. In the third example (F), the 2 VZ cells were in a supra/subjacent relationship and located in the outer one-half of the VZ, near the VZ/IZ border. G–I, Three-cell P clones. In all three examples, the 3 cells are clustered, but the physical arrangements vary: radially stacked at the ventricular surface (G) or in the outer one-half of the VZ (H) or forming a triangular array (I), in this case at the ventricular surface.V, Lateral ventricle. Scale bar, 20 μm.

Fig. 3.

The distribution of one-cell P clones as a function of the thickness of the VZ obtained by dividing the VZ into 10 bins (deciles) parallel to the ventricular surface. For comparison, thedotted line shows the percentage of clones/bin if they were distributed uniformly in the VZ. The distribution of one-cell P clones is not significantly different from the uniform distribution (χ² = 1.33; p = 0.998). This uniform distribution means that AP-labeled clones are also uniformly distributed in the cell cycle, which indicates that there is no preferred cell cycle phase for infection by the DAP retrovirus vector.

Fig. 5.

Large clones at E14, 3 d after injections of DAP retrovirus at E11. A, P clone containing 8 cells radially aligned in the VZ. Three cells are clustered and located near the VZ/IZ border separated from 5 cells that are tightly clustered and located at the ventricular surface. B, PQ clone containing 5 cells, 3 VZ cells and 2 IZ cells. The 3 VZ cells (larger arrow) are tightly clustered with no discernable intercellular space and are located in the outer one-half of the VZ. The 2 IZ cells (smaller arrow) are located close to each other just above the VZ/IZ border. The arrowheadindicates AP-stained radial process extending across the IZ.V, Lateral ventricle. Scale bar, 20 μm.

Fig. 6.

Photomicrographs of the two largest clones found at E14, 3 d after DAP retrovirus injection at E11.A, The largest clone contained 24 cells. There are 15 VZ cells radially split into two clusters (two larger arrows). The larger cluster contains 13 tightly spaced cells and is located in the outer one-half of the VZ. The smaller cluster contains 2 cells and is located at the ventricular surface. In the IZ (smaller arrows), 4 cells are located near the VZ/IZ border and seem to have recently entered the IZ, and 2 more cells are located at the top of the IZ close to its border with the CP. Two of the three CP cells are faintly stained and are indicated by twoarrowheads; the third CP cell is out of the plane of focus of the photomicrograph. B, The second largest clone contained 18 cells. There are 6 VZ cells (larger arrow), all located in one tight cluster in the outer one-half of the VZ. The 10 IZ cells (small arrows) are contained in three clusters, indicating perhaps that they had originated during three consecutive cell cycles. One cluster containing 5 cells is located just above the VZ/IZ border and seems to have recently left the VZ. A second cluster containing 3 IZ cells is located in the middle of the IZ. The third cluster with 2 IZ cells is located in the outer one-half of the IZ near the CP. The 2 CP cells (two arrowheads) are faintly stained. V, Lateral ventricle. Scale bar, 20 μm.

Fig. 7.

A schematic diagram of interkinetic nuclear migration and the production of mosaicism in the VZ. The to-and-fro movement of a single VZ cell and its progeny and their progression through the cell cycle are depicted over the course of two complete cell cycles and part of a third. At left, the founder cell has just completed M phase and is at the beginning of G₁ of Cell cycle 1. During G₁, its nucleus ascends to the VZ/IZ border. During S, DNA synthesis occurs in the outer one-half of the VZ. After entry into G₂, the nucleus reverses its migratory direction and descends rapidly to the ventricular surface. When Cell cycle 1 is completed, two daughter cells are formed at the beginning of G₁ inCell cycle 2. As these 2 daughter cells repeat the process, they move at approximately the same rate and, hence, maintain their proximity both in the phase of the cell cycle and in their physical location as they progress through Cell cycle 2. A transient “exception” to the maintenance of proximity can occur to produce the split clone phenotype (cells a andb in Cell cycle 2) because the synchrony in the cell cycle is not lock-step but only approximate. A slight difference in cell cycle position at the S–G₂ transition would allow the more advanced of the 2 daughter cells (cella) to enter G₂ and descend rapidly to the ventricular surface, whereas the slightly retarded daughter would be left behind (cell b) in S; the 2 daughters would be reunited later in G₂. At the end of Cell cycle 2, they divide to form 4 cousin cells in early G₁of Cell cycle 3. During G₁, the resultant cluster of cousin nuclei would move to the middle of the ventricular zone as shown at the right of the diagram; however, the fates of the cousins may not be identical. For example, some cells (cells c and d) might continue to proliferate, and they would maintain synchrony and clustering, whereas others (cell e) might leave the cell cycle to become postproliferative and migrate into the IZ. Other cells (cellf) might die (for review, see Voyvodic, 1996), lose their AP staining (Golden et al., 1995), or disperse tangentially (Fishell et al., 1993) via an unknown mechanism. As this process continues for additional cell cycles (not illustrated), the resultant cluster of clonally related cells in the VZ forms a clade that contains both siblings and cousins (both near and distant), and its composition, therefore, would be a reflection of its proliferative history.