Clonal analysis reveals laminar fate multipotency and daughter cell apoptosis of mouse cortical intermediate progenitors

Abstract

In developing cerebral cortex, most pyramidal-projection neurons are produced by intermediate progenitors (IPs), derived in turn from radial glial progenitors. Although IPs produce neurons for all cortical layers, it is unknown whether individual IPs produce multiple or single laminar fates, and the potential of IPs for extended proliferation remains uncertain. Previously, we found that, at the population level, early IPs (present during lower-layer neurogenesis) produce lower- and upper-layer neurons, whereas late IPs produce upper-layer neurons only. Here, we employed mosaic analysis with double markers (MADM) in mice to sparsely label early IP clones. Most early IPs produced 1-2 neurons for deep layers only. Less frequently, early IPs produced larger clones (up to 12 neurons) spanning lower and upper layers, or upper layers only. The majority of IP-derived clones (∼66%) were associated with asymmetric cell death after the first division. These data demonstrate that laminar fate is not predetermined, at least in some IPs. Rather, the heterogeneous sizes and laminar fates of early IP clones are correlated with cell division/death/differentiation choices and neuron birthdays, respectively.

Keywords: Cortical development; Intermediate progenitors; Laminar fate; MADM; Mosaic analysis with double markers; Tbr2.

Fig. 1.

Early IPs generate single- and multilayered clones of neurons. (A) Experimental design timeline. (B) Diagrams of potential IP-derived clonal outcome by MADM labeling. Yellow clones were excluded (see text). (C) Confocal microscopy image of E19.5 cortex after tamoxifen injection at E11.5. A 9-neuron RFP⁺-only clone spanned layers 2-6. (D) Confocal microscopy image of E18.5 cortex after tamoxifen injection at E12.5. A 2-cell clone contained one RFP⁺ and one GFP⁺ neuron in layer 6. (E) Confocal microscopy image of E18.5 cortex after tamoxifen injection at E12.5. A 3-neuron, RFP⁺-only clone occupied upper layers. (F) Confocal microscopy image of E18.5 cortex after tamoxifen injection at E11.5. Sister cells in VZ/SVZ exhibited IP morphologies (Kowalczyk et al., 2009). (C′-F′) Schematic diagrams of the clones in C-F. (C″-F″) Higher magnification views of the clones in C-F. Scale bars: 100 µm. IZ, intermediate zone; L, layer; MZ, marginal zone; SVZ, subventricular zone; VZ, ventricular zone.

Fig. 2.

Neuronal composition of 41 IP-derived clones. (A) Schematic diagrams of all 41 evaluable neocortical clones, indicating the distribution of neurons in each layer. The ‘X’ under each diagram indicates how many clones exhibited the indicated neuron number and laminar distribution. (B) Confocal microscopy image of E19.5 cortex after tamoxifen injection at E11.5. Clone consisted of one RFP⁺ and one GFP⁺ neuron, both in layer 6a (arrows). (C) Confocal microscopy image of E19.5 cortex after tamoxifen injection at E11.5. Clone consisted of one RFP⁺ and one GFP⁺ neuron, both in layer 6b (arrowheads). (D) Confocal microscopy image of E19.5 cortex after tamoxifen injection at E11.5. Clone consisted of one RFP⁺ and one GFP⁺ neuron, in layers 6a (arrow) and 6b (arrowhead), respectively. (E) Confocal microscopy image of E18.5 cortex after tamoxifen injection on E12.5. Clone consisted of one GFP⁺ and one RFP⁺ neuron, in layers 6a (arrow) and 6b (arrowhead), respectively. (F) Plot of IP-derived clone size. (G) Pie chart depicting the percentage clonal distribution within the various layers of the 6-layer neocortex.

Fig. 3.

Inferred pathways of early IP division, death and laminar differentiation. (A) Sequence of laminar neurogenesis in mouse neocortex (Caviness et al., 2003; Hevner et al., 2003). (B) Three examples of early IP division behavior and clonal fate, inferred from lineage tracing. (1) An early IP terminal division pattern leading to a red and green L6 clone (see Fig. 1D). (2) Potential model for genesis of a multilayered red-only clone (see Fig. 1C). (3) Potential model for genesis of an upper-layer red-only clone (see Fig. 1E). h, hours; Nⁿ, neuron (superscript ‘n’ indicates the cortical layer location); gray ‘IP’ indicates the founder IP of a clone; red or green ‘N’ or ‘IP’ indicate fluorophore segregation within cells after MADM labeling.