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SUMMARYBrain neurons arise from relatively few progenitors capable of giving rise to an enormous diversity of neuronal types. Nonetheless, a cardinal feature of mammalian brain neurogenesis in both the cortex and the cerebellum is that excitatory neurons and inhibitory neurons derive from separate, spatially segregated, progenitors. Whether bi-potential progenitors with an intrinsic capacity to generate both excitatory and inhibitory lineages exist and how such a fate decision may be regulated is unknown. Using cerebellar development as a model, we discover that individual embryonic cerebellar progenitors give rise to both inhibitory and excitatory lineages. We find that gradations of Notch activity levels determine the fates of the progenitors and their daughters. Daughters with the highest levels of Notch activity retain the progenitor fate. Daughters with intermediate levels of Notch activity become fate restricted to generate inhibitory neurons, while daughters with very low levels of Notch signaling adopt the excitatory fate. Therefore, Notch mediated binary cell fate choice is a mechanism for regulating the ratio of excitatory to inhibitory neurons from common progenitors.Graphical summary

SUMMARY: Mosaic analysis with double markers (MADM) offers one approach to visualize and concomitantly manipulate genetically defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage, single-cell morphology and physiology, genomic imprinting phenotypes, and dissection of cell-autonomous gene functions in vivo in health and disease. Yet, MADM can only be applied to 96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond a proof of principle, we apply our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We find striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division. IN BRIEF: Contreras et al. generate a resource and suite of transgenic MADM mice for genetic mosaic analysis with double markers of >96% of the entire mouse genome. In addition to providing a proof of principle, they find non-random mitotic sister chromatid segregation in distinct somatic cell lineages in vivo.

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

The cerebral cortex contains multiple areas with distinctive cytoarchitectonic patterns, but the cellular mechanisms underlying the emergence of this diversity remain unclear. Here, we have investigated the neuronal output of individual progenitor cells in the developing mouse neocortex using a combination of methods that together circumvent the biases and limitations of individual approaches. Our experimental results indicate that progenitor cells generate pyramidal cell lineages with a wide range of sizes and laminar configurations. Mathematical modeling indicates that these outcomes are compatible with a stochastic model of cortical neurogenesis in which progenitor cells undergo a series of probabilistic decisions that lead to the specification of very heterogeneous progenies. Our findings support a mechanism for cortical neurogenesis whose flexibility would make it capable to generate the diverse cytoarchitectures that characterize distinct neocortical areas., eLife digest Recognizable by its deep outer folds in humans, the cerebral cortex is a region of the mammalian brain which handles complex processes such as conscious perception or decision-making. It is organized in several layers that contain different types of ‘excitatory’ neurons which can activate other cells. The various areas of the cortex have different characteristics as they contain various proportions of each kind of neurons. Stem cells are cells capable to divide and create various types of specialized cells. The excitatory neurons in the cortex are created during development by stem cells known as radial glial cells. These cells divide several times, giving rise to different types of neurons in sucessive divisions, presumably thanks to internal molecular clocks. In the cortex, it is generally assumed that an individual radial glial cell produces all the different types of excitatory neurons. However, studies have suggested that certain cells could be specialized in creating specific types of neurons. To explore this question, Llorca et al. used three complementary approaches to follow individual radial glial cells and track the neurons they created in mouse embryos. This helped to understand how groups of stem cells work together to build the cortex. The experiments revealed that radial glial cells differ more than anticipated in the number and the types of neurons they generate, and rarely produce all types of excitatory neurons. In other words, the output of individual radial glial cells is not always the same. The results by Llorca et al. suggest that as radial glial cells divide, they undergo a series of probabilistic decisions – that is, in each division the cells have a certain probability to generate a specific type of neuron. Consequently, the resulting lineages are rarely identical or contain all types of excitatory neurons, but collectively they generate the full diversity of excitatory neurons in the cortex. Ultimately, new insights into how excitatory neurons form and connect in the brain may be used to help understand psychiatric conditions where circuits in the cortex might be impaired, such as in autism spectrum disorders.

Studying the progression of the proliferative and differentiative patterns of neural stem cells at the individual cell level is crucial to the understanding of cortex development and how the disruption of such patterns can lead to malformations and neurodevelopmental diseases. However, our understanding of the precise lineage progression programme at single‐cell resolution is still incomplete due to the technical variations in lineage‐tracing approaches. One of the key challenges involves developing a robust theoretical framework in which we can integrate experimental observations and introduce correction factors to obtain a reliable and representative description of the temporal modulation of proliferation and differentiation. In order to obtain more conclusive insights, we carry out virtual clonal analysis using mathematical modelling and compare our results against experimental data. Using a dataset obtained with Mosaic Analysis with Double Markers, we illustrate how the theoretical description can be exploited to interpret and reconcile the disparity between virtual and experimental results.

SUMMARYThe cerebral cortex contains multiple hierarchically organized areas with distinctive cytoarchitectonical patterns, but the cellular mechanisms underlying the emergence of this diversity remain unclear. Here, we have quantitatively investigated the neuronal output of individual progenitor cells in the ventricular zone of the developing mouse neocortex using a combination of methods that together circumvent the biases and limitations of individual approaches. We found that individual cortical progenitor cells show a high degree of stochasticity and generate pyramidal cell lineages that adopt a wide range of laminar configurations. Mathematical modelling these lineage data suggests that a small number of progenitor cell populations, each generating pyramidal cells following different stochastic developmental programs, suffice to generate the heterogenous complement of pyramidal cell lineages that collectively build the complex cytoarchitecture of the neocortex.

In mammalian genomes, a subset of genes is regulated by genomic imprinting, resulting in silencing of one parental allele. Imprinting is essential for cerebral cortex development, but prevalence and functional impact in individual cells is unclear. Here, we determined allelic expression in cortical cell types and established a quantitative platform to interrogate imprinting in single cells. We created cells with uniparental chromosome disomy (UPD) containing two copies of either the maternal or the paternal chromosome; hence, imprinted genes will be 2-fold overexpressed or not expressed. By genetic labeling of UPD, we determined cellular phenotypes and transcriptional responses to deregulated imprinted gene expression at unprecedented single-cell resolution. We discovered an unexpected degree of cell-type specificity and a novel function of imprinting in the regulation of cortical astrocyte survival. More generally, our results suggest functional relevance of imprinted gene expression in glial astrocyte lineage and thus for generating cortical cell-type diversity.

SummaryThe medial ganglionic eminence (MGE) gives rise to the majority of mouse forebrain interneurons. Here, we examine the lineage relationship among MGE-derived interneurons using a replication-defective retroviral library containing a highly diverse set of DNA barcodes. Recovering the barcodes from the mature progeny of infected progenitor cells enabled us to unambiguously determine their respective lineal relationship. We found that clonal dispersion occurs across large areas of the brain and is not restricted by anatomical divisions. As such, sibling interneurons can populate the cortex, hippocampus striatum, and globus pallidus. The majority of interneurons appeared to be generated from asymmetric divisions of MGE progenitor cells, followed by symmetric divisions within the subventricular zone. Altogether, our findings uncover that lineage relationships do not appear to determine interneuron allocation to particular regions. As such, it is likely that clonally related interneurons have considerable flexibility as to the particular forebrain circuits to which they can contribute.