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.

Gliogenic Potential of Single Pallial Radial Glial Cells in Lower Cortical Layers

“…We reported uniform clones composed of either astrocytes, oligodendrocytes, or NG2glia. These results match with previous studies using StarTrack and Mosaic Analysis with Double Markers (MADM) from embryonic progenitors that showed clones composed exclusively of astrocytes [30,45], oligodendrocytes [38], or NG2-glia [31]. However, other studies with MADM did not report any cortical clones containing exclusively oligodendrocytes [45], because RGCs generate diverse cell fate ratios during different embryonic temporal windows.…”

“…Moreover, although it is known how cortical progenitors give rise to neurons and some macroglial cell types, the clonal relationships between glial cell types remain partially unknown. Some studies have focused on clonal aspects of astrocyte generation [30,38,[42][43][44][45][46][47][48] and few of them have focused on oligodendrocytes [38,48,49] or NG2-glia [47]. Recent single-cell analysis revealed that glial cells in different layers exhibit distinct transcriptional profiles [36,37].…”

Detection and classification of neurons and glial cells in the MADM mouse brain using RetinaNet

“…MADM-11 mice (The Jackson Laboratory, Bar Harbor, USA; #013751, #013749) were crossed to Nestin-cre mice (bred from MADM-11 mice) using the breeding scheme described previously [11] and harvested at the age of one month under the regulations and approval from the Institutional Animal Care and Use Committee at North Carolina State University. Mice were…”

“…MADM allows for simultaneous labeling and genetic manipulation in developmentally derived clones of somatic cells [6]. We and others have extensively used MADM alleles in developmental studies on the roles of various genetic factors, which are involved with neurogenesis [7][8][9] and gliogenesis [10,11]. An advantage of MADM is that neurons and glia with distinct genotypes are permanently labeled by expression of two fluorescent proteins.…”

Potential of Multiscale Astrocyte Imaging for Revealing Mechanisms Underlying Neurodevelopmental Disorders

“…The transition from neurogenesis to gliogenesis is one of the key events during brain development, and several molecular signaling pathways regulating this process have been identified, such as janus kinase signal transducer and activator of transcription (JAK-STAT), phosphatidylinositol-3 kinase (PI3K), Notch, and Smad pathways [ 38 , 39 , 40 , 41 ], while extrinsic signals required for induction of astrocytic genes include epidermal growth factor, bone morphogenetic proteins, leukemia inhibitory factor, and ciliary neurotrophic factor (CNTF) [ 42 , 43 , 44 , 45 , 46 ]. In cultures of single neural progenitor cells, transition was associated with activation of chicken ovalbumin upstream promoter transcription factors I and II (COUP-TFI/II) [ 47 ].…”