Region-specific gene expression shared with neurons imparts to astrocytes competence for region-specific neuronal reprogramming.

A broad molecular framework of how neural stem cells are specified toward astrocyte fate during brain development has proven elusive. Here we perform comprehensive and integrated transcriptomic and epigenomic analyses to delineate gene regulatory programs that drive the developmental trajectory from mouse embryonic stem cells to astrocytes. We report molecularly distinct phases of astrogliogenesis that exhibit stage- and lineage-specific transcriptomic and epigenetic signatures with unique primed and active chromatin regions, thereby revealing regulatory elements and transcriptional programs underlying astrocyte generation and maturation. By searching for transcription factors that function at these elements, we identified NFIA and ATF3 as drivers of astrocyte differentiation from neural precursor cells while RUNX2 promotes astrocyte maturation. These transcription factors facilitate stage-specific gene expression programs by switching the chromatin state of their target regulatory elements from primed to active. Altogether, these findings provide integrated insights into the genetic and epigenetic mechanisms steering the trajectory of astrogliogenesis.

Cerebellar GABAergic interneurons in mouse comprise multiple subsets of morphologically and neurochemically distinct phenotypes located at strategic nodes of cerebellar local circuits. These cells are produced by common progenitors deriving from the ventricular epithelium during embryogenesis and from the prospective white matter (PWM) during postnatal development. However, it is not clear whether these progenitors are also shared by other cerebellar lineages and whether germinative sites different from the PWM originate inhibitory interneurons. Indeed, the postnatal cerebellum hosts another germinal site along the Purkinje cell layer (PCL), in which Bergmann glia are generated up to first the postnatal weeks, which was proposed to be neurogenic. Both PCL and PWM comprise precursors displaying traits of juvenile astroglia and neural stem cell markers. First, we examine the proliferative and fate potential of these niches, showing that different proliferative dynamics regulate progenitor amplification at these sites. In addition, PCL and PWM differ in the generated progeny. GABAergic interneurons are produced exclusively by PWM astroglial-like progenitors, whereas PCL precursors produce only astrocytes. Finally, throughin vitro,ex vivo, andin vivoclonal analyses we provide evidence that the postnatal PWM hosts a bipotent progenitor that gives rise to both interneurons and white matter astrocytes.

The morphological, molecular, and functional heterogeneity of astrocytes is under intense scrutiny, but how this diversity is ontogenetically achieved remains largely unknown. Here, by quantitative in vivo clonal analyses and proliferation studies, we demonstrate that the major cerebellar astrocyte types emerge according to an unprecedented and remarkably orderly developmental program comprising (i) a time-dependent decline in both clone size and progenitor multipotency, associated with clone allocation first to the hemispheres and then to the vermis(ii) distinctive clonal relationships among astrocyte types, revealing diverse lineage potentials of embryonic and postnatal progenitors; and (iii) stereotyped clone architectures and recurrent modularities that correlate to layer-specific dynamics of postnatal proliferation/differentiation. In silico simulations indicate that the sole presence of a unique multipotent progenitor at the source of the whole astrogliogenic program is unlikely and rather suggest the involvement of additional committed components. Astrocytes are abundant cells of the brain essential to support and shape neuronal activity. They can be grouped in different subclasses based on their remarkable variety of morphologies, molecular profiles, and specialized functions. Although different astrocyte types likely display specialized interactions with distinct neuron categories, the different classes of astrocytes have only partially been unmasked. How astrocyte heterogeneity is ontogenetically achieved remains largely unknown. Here we approached this question by studying the development of the main astrocyte types of the cerebellum. The reconstruction of developmental lineages in the mouse embryo combined with proliferation studies and computational modeling demonstrate that cerebellar astrocyte types emerge according to an unprecedented and remarkably orderly developmental program. Embryonic progenitor cells produce either only a single astrocyte type or more types. These distinct astrocyte lineages display stereotyped architectures and recurrent modularities. Moreover, the generation of astrocytes follows a well-defined spatiotemporal pattern, defined by a time-dependent allocation of astrocytes to distinct cerebellar territories and an inside-out sequence of differentiation, coupled with a decline over time in both progenitor amplification and capability to produce distinct astrocyte types. These results provide the first evidence that an ontogenetic program, tightly regulated in space and time, determines astrocyte heterogeneity.

Clonal cell analysis defines the potential of single cells and the diversity they can produce. To achieve this, we have developed a novel adaptation of the genetic tracing strategy, UbC-StarTrack, which attributes a specific and unique color-code to single neural precursors, allowing all their progeny to be tracked. We used integrable fluorescent reporters driven by a ubiquitous promoter in PiggyBac-based vectors to achieve inheritable and stable clonal cell labeling. In addition, coupling this to an inducible Cre-LoxP system avoids the expression of non-integrated reporters. To assess the utility of this system, we first analyzed images of combinatorial expression of fluorescent reporters in transfected cells and their progeny. We also validated the efficiency of the UbC-StarTrack to trace cell lineages through in vivo, in vitro and ex vivo strategies. Finally, progenitors located in the lateral ventricles were targeted at embryonic or postnatal stages to determine the diversity of neurons and glia they produce, and their clonal relationships. In this way we demonstrate that UbC-StarTrack can be used to identify all the progeny of a single cell and that it can be employed in a wide range of contexts.