Aneuploid cells are differentially susceptible to caspase-mediated death during embryonic cerebral cortical development

Abstract

Neural progenitor cells, neurons, and glia of the normal vertebrate brain are diversely aneuploid, forming mosaics of intermixed aneuploid and euploid cells. The functional significance of neural mosaic aneuploidy is not known; however, the generation of aneuploidy during embryonic neurogenesis, coincident with caspase-dependent programmed cell death (PCD), suggests that a cell's karyotype could influence its survival within the CNS. To address this hypothesis, PCD in the mouse embryonic cerebral cortex was attenuated by global pharmacological inhibition of caspases or genetic removal of caspase-3 or caspase-9. The chromosomal repertoire of individual brain cells was then assessed by chromosome counting, spectral karyotyping, fluorescence in situ hybridization, and DNA content flow cytometry. Reducing PCD resulted in markedly enhanced mosaicism that was comprised of increased numbers of cells with the following: (1) numerical aneuploidy (chromosome losses or gains); (2) extreme forms of numerical aneuploidy (>5 chromosomes lost or gained); and (3) rare karyotypes, including those with coincident chromosome loss and gain, or absence of both members of a chromosome pair (nullisomy). Interestingly, mildly aneuploid (<5 chromosomes lost or gained) populations remained comparatively unchanged. These data demonstrate functional non-equivalence of distinguishable aneuploidies on neural cell survival, providing evidence that somatically generated, cell-autonomous genomic alterations have consequences for neural development and possibly other brain functions.

Figure 1.

Schematic for analyzing aneuploidy in mitotic and non-mitotic cortical cells. A, Cortices were dissected from embryos at E14, triturated, and placed in culture in Colcemid (100 ng/ml) to arrest cells in metaphase and obtain chromosome spreads. Each cell's chromosomal complement was analyzed in one of two ways. First, chromosomes were stained with DAPI and counted using fluorescence microscopy (bottom left). Second, metaphase chromosome spreads were processed for SKY to determine exact karyotypes (bottom right). B, E14 cortices were isolated, triturated, and fixed for staining with the DNA-intercalating dye Propidium Iodide. Dye-saturated cells were analyzed by flow cytometry, where the dominant peak on a DNA content histogram contains cells in the G₀/G₁ phase of the cell cycle. Relative DNA content was expressed as a ratio of the mean fluorescent intensity of the G₀/G₁ peak divided by the peak of an internal standard, CEN. C, For analysis of interphase or non-mitotic cells, E19 cortices were triturated, cell nuclei were isolated, applied to a slide, and hybridized with chromosome-specific FISH probes. For these experiments, nuclei were hybridized with probes for chromosomes 16 (green) and 8 (red), and stained with DAPI (blue). Normal, euploid cells would be disomic for both chromosome 8 and 16 and thus would have 2 green dots and 2 red dots. The nucleus on the left is monosomic for chromosome 16 and the nucleus on the right is trisomic for chromosome 16, meaning both nuclei are aneuploid. Both nuclei are disomic for chromosome 8.

Figure 2.

Pharmacological inhibition of caspases leads to increased aneuploidy in mitotic cells from the embryonic mouse cortex. A, Intact hemispheres of E14 cortex were treated ex vivo with vehicle control (DMSO) or the pan caspase inhibitor zVAD-fmk (100 nm). DMSO-treated sections show high levels of immunoreactivity for cleaved caspase-3 (red), while those treated with zVAD-fmk showed markedly reduced immunoreactivity (from 15.3% to 0.4%, respectively; p = 0.01, Student's t test). Tissues were counterstained with DAPI (blue). Scale bar, 20 μm. VZ, Ventricular zone. B, Histogram of the distribution of aneuploid cells identified following exposure of cortices to vehicle control (DMSO; black) and zVAD-fmk (red).

Figure 3.

Genetic ablation of caspases leads to increased aneuploidy in mitotic cells from the embryonic mouse cortex. A, Analysis of metaphase chromosome counts showed increased aneuploidy in E14 caspase-3 and caspase-9-null cortices compared with wild-type cortices from littermates (100 metaphase spreads were counted per embryo; for caspase-3, n = 3; *p = 0.02; for caspase-9, n = 3; *p = 0.04, Student's t test). B, Quantitation of DNA content by flow cytometry showed a significant 3–4% decrease in overall DNA content from cortices of E14 caspase nulls compared with sex-matched, wild-type littermates (caspase-3 wild-type and nulls, n = 4, *p = 0.002, Student's t test; caspase-9 wild-type and nulls, n = 3, *p = 0.03, Student's t test). C, Histogram of the distribution of aneuploidy in E14 wild-type (black), caspase-3-null (red), and caspase-9-null (blue) NPCs. Caspase-deficient cells showed expanded distribution of numerical aneuploidies.

Figure 4.

Caspase-attenuated mitotic cortical cells show an increase in extreme aneuploidy, while maintaining mild aneuploidy levels. A, One hundred aneuploid metaphase spreads were analyzed for each of 3 paired sets of wild-type and caspase-null littermates. Bin 1 represents mild aneuploidies where chromosome numbers flank the euploid chromosome number of 40 (light gray, 35–39 and 41–45 chromosomes). Bins 2 and 3 represent more extreme aneuploidies (bin 2: medium gray, 30–34 and 46–50 chromosomes; bin 3: black, 0–29 and 51 or more). Euploid spreads are not shown. Caspase attenuation resulted in a preferential expansion of extreme aneuploidies (bins 2 and 3, *p < 0.003, Student's t test); no significant change was observed in cells showing mild aneuploidies (bin 1). B, Pie charts of mild and extreme aneuploidies from control versus caspase-null cortices were constructed to reflect relative percentages of cells in each bin (pie slice, colors as in Fig. 4A), and percentage of total aneuploidy (pie area). Pronounced expansion of bins 2 and 3 by percentage were coincident with PCD attenuation.

Figure 5.

Caspase attenuation increases rare aneuploidies including nullisomy and coincident chromosomal gain and loss, identified by SKY. A, B, Representative metaphase spreads from E14 caspase-3-null cortices analyzed by SKY showing coincident chromosome gain and loss (A) and nullisomy (B). SKY analyses of each cell included pseudocolor chromosome spread (left), DAPI counterstain (middle), and karyotype table (right). C–E, Tabular representation of 20 aneuploid karyotypes from E14 wild-type (C), caspase-3-null (D) and caspase-9-null (E) mitotic cortical cells. Chromosome numbers are indicated across the top row. Each row below shows the karyotype of an individual aneuploid cortical cell. The rightmost column indicates the total number of chromosomes in each spread. The last row reports the number of times a chromosome is involved in a gain or loss event. Orange squares, gain of one chromosome; red squares, gain of a chromosome pair; light blue squares, loss of one chromosome; deep blue squares, loss of a chromosome pair (i.e., nullisomy). Spreads containing a nullisomy and/or coincident chromosomal gain and loss are indicated with stars and circles, respectively, along the left most column. F, Frequencies of nullisomy and coincident gain and loss of chromosomes among wild-type, caspase-3 and caspase-9-null aneuploid cortical cells. *p < 0.05, χ² for nullisomy; p < 0.03, χ² for coincident loss and gain.

Figure 6.

Model of aneuploidy-based selection in the developing cerebral cortex. The normal pathway (wild-type) is compared with experimental manipulation using inhibition of caspase activity (cell death attenuated). Both mild and extremely aneuploid cells (compare Fig. 4) are generated during neurogenesis (Before PCD) to produce a mosaic of intermixed euploid and aneuploid cortical cells. During PCD, extremely aneuploid cells are normally eliminated (red X), but when caspase-mediated PCD is attenuated, these cells are preserved (red outline). In the wild-type brain (After PCD), most cells are either euploid or mildly aneuploid. When PCD is attenuated, however, the brain becomes populated by more extremely aneuploid cells.