Clonal Analysis of Gliogenesis in the Cerebral Cortex Reveals Stochastic Expansion of Glia and Cell Autonomous Responses to Egfr Dosage

Abstract

Development of the nervous system undergoes important transitions, including one from neurogenesis to gliogenesis which occurs late during embryonic gestation. Here we report on clonal analysis of gliogenesis in mice using Mosaic Analysis with Double Markers (MADM) with quantitative and computational methods. Results reveal that developmental gliogenesis in the cerebral cortex occurs in a fraction of earlier neurogenic clones, accelerating around E16.5, and giving rise to both astrocytes and oligodendrocytes. Moreover, MADM-based genetic deletion of the epidermal growth factor receptor (Egfr) in gliogenic clones revealed that Egfr is cell autonomously required for gliogenesis in the mouse dorsolateral cortices. A broad range in the proliferation capacity, symmetry of clones, and competitive advantage of MADM cells was evident in clones that contained one cellular lineage with double dosage of Egfr relative to their environment, while their sibling Egfr-null cells failed to generate glia. Remarkably, the total numbers of glia in MADM clones balance out regardless of significant alterations in clonal symmetries. The variability in glial clones shows stochastic patterns that we define mathematically, which are different from the deterministic patterns in neuronal clones. This study sets a foundation for studying the biological significance of stochastic and deterministic clonal principles underlying tissue development, and identifying mechanisms that differentiate between neurogenesis and gliogenesis.

Keywords: Egfr; astrocyte; cerebral cortex; clonal analysis; deterministic; gliogenesis; neurogenesis; oligodendrocyte MADM; stochastic.

Figure 1

Clonal Analysis of Gliogenesis using Mosaic Analysis with Double Markers (MADM). (a) Molecular basis of labeling individual progenitor clones with distinct fluorescent reporters (GFP, green and tdTomato, red). Cre mediated interchromosomal mitotic recombination is followed by two distinct segregation patterns (x-seg and z-seg) generating clonal siblings in distinct colors as indicated. In control MADM mice, all cells will be WT and referred to as +/+. When a single floxed allele for Egfr is introduced, green cells lack Egfr (Egfr-null), while the red cells are maintained as WT (F/+ mice). Background genotypes obtained from the described genetic combinations using a tamoxifen induced Nestin-creERT2 transgene result in substantial differences in the Egfr genotype in background cells (non-MADM recombination events) which exert non-autonomous effects on MADM cells. (b) Schematic of tamoxifen (TAM) induction time points during embryonic development, and processing of forebrains in serial sections. Inductions were performed in iNes:MADM:Egfr ^+/+ (+/+) and iNes:MADM:Egfr ^F/+ (F/+) mice, which result in the generation of clones containing green and red cells that become distinguished following TAM inductions. Green MADM F/+ glia (Egfr-null) fail to develop, while glial production is elevated in the red sibling’s population. (c) Confocal images of a rare symmetric “G” cortical clone in an E16.5 induced +/+ cortex. SEZ/WM, subependymal zone/white matter; DL, deep layers; UL, upper layers. Scale bar, 100 µm.

Figure 2

Glial subtypes in MADM clones. (a) Samples representing classified cell types in both red and green MADM cells. Scale bars, 10 µm. (b) Maps of cell type distributions in the cortex of +/+ and F/+ mice at different induction time points. Scale bars, 100 µm. (c) Plots of clone sizes (number of total cells) in the green and red clonal populations in the cortex at P30 under various induction time points. Each dot represents individual clones and averages are depicted ± sem. *, Student’s t-test, p < 0.05.

Figure 3

Analysis of MADM clone types. (a) Classification of clones based on their neuronal and glial compositions. (b) Percentage of all clones containing G, N or Mix types across the different induction time points and genotypes (green and red MADM cells are combined). (c) Numbers of neurons in different clone types in +/+ and F/+ cortices at different induction times. Total numbers of neurons are broken down into their green and red sibling fractions as shown. The scales for Y-axis values are not the same for the 0–20 and 20–50 ranges in order to reveal the low values for neurons in E15.5–E17.5 induced clones. (d) Numbers of glia in different clone types in +/+ and F/+ cortices at different induction times. Total numbers of glia per clone are broken down into their green and red sibling fractions as shown. Data are mean ± sem. *, rank-sum test, p < 0.05.

Figure 4

Analysis of clonal MADM data in Neuron-containing and Glia-containing clones in the dorsolateral cortices. (a) Analysis of symmetry in clones at different time points and genotypes. Data are percentages of clones that form the different categories of clones as indicated. Numbers in the bars indicate the numbers of clones that fall into each category. Charts present numbers of cells per clone for each category of neuron-containing and glia-containing clones for all time points combined. Data are mean ± sem. *, rank-sum test, p < 0.05. (b) Percentages of clones across a gradient of asymmetry depicted on the x-axes (0 = one-sided asymmetric; 50 = symmetric) in +/+ and F/+ cortices. Data are shown separately for each clone type and for all clones combined. The majority of G clones are one-sided asymmetric in both genotypes (green and red MADM cells are combined).

Figure 5

Gliogenesis exhibits a stochastic pattern of clonal expansion in the cortex. (a) Gaussian curve fitting for analysis of size distribution of clones versus their normalized frequency for early neurogenic clones (E10.5–E14.5). Values for the histogram and Gaussian curves were extracted manually using the WebPlotDigitizer (https://automeris.io/WebPlotDigitizer/citation.html) from Gao et al. [1]. (b) Gaussian curve fitting for analysis of size distribution of clones versus their normalized frequency for gliogenic clones (E15.5–E17.5). (c) Scatterplot presenting the size (number of cells) of larger versus smaller sibling subclones of individual clones during the neurogenic period at E11.5 in +/+ and F/+ cortices. (d) Scatterplots presenting the size (number of cells) of larger versus smaller sibling subclones of individual clones during the gliogenic period in +/+ and F/+ cortices. White, black, and gray dots with error bars represent mean ± sem, faded dots indicate individual clones, and the slopes indicate ratio boundaries as indicated in both (c,d).

Figure 6

Summary: MADM enabled labeling of sparse clones in dorsolateral cortices of +/+ and F/+ mice reveals switch to gliogenesis around E15 followed by seeding of glia without involvement of glia-restricted clones from early neurogenic period. Allelic Egfr dosage analysis using MADM with a conditional allele for Egfr revealed that the background cells and their genotype (Egfr-heterozygous) influence Egfr-null (green) and wild type (WT, red) MADM siblings. Comparison of cortical MADM progeny in +/+ and F/+ cortices exposed a dosage response in glial populations whereby WT MADM cells expand to compensate for loss of glia in their MADM green siblings. Neurogenic clones exhibit symmetric and deterministic patterns of expansion, whereas clonal expansion during gliogenesis is highly asymmetric and stochastic.