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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 The bewildering diversity of brain neurons arises from relatively few pluripotent progenitors through poorly understood mechanisms. The cerebellum is an attractive model to investigate mechanisms of neuronal diversification because the different subtypes of excitatory and inhibitory neurons are well described 1,2 . The cerebellum is a hub for control of motor function and contributes to a number of higher brain functions such as reward-related cognitive processes 3 . Deficits in cerebellar development lead to severe neurological disorders such as cerebellar ataxias 4 and medulloblastomas 5 , a heterogeneous and severe groups of childhood brain tumors, thus underlying the importance of understanding the cellular and molecular control of cerebellar development. In contrast to text book models, we report that excitatory and inhibitory cerebellar neurons derive from the same pluripotent embryonic cerebellar stem cells (eCSC). We find that the excitatory versus inhibitory fate decision of a progenitor is regulated by Notch signaling, whereby the cell with lower Notch activity adopts the excitatory fate, while the cell with higher Notch activity adopts the inhibitory fate. Thus, Notch-mediated binary cell fate choice is a conserved strategy for generating neuronal diversity from common progenitors that is deployed at different developmental time points in a context specific manner.

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.

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.