Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis.

The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program.

Oligodendroglial maturation is dependent on intracellular protein shuttling.

Multiple sclerosis is an autoimmune disease of the CNS resulting in degeneration of myelin sheaths and loss of oligodendrocytes, which means that protection and electrical insulation of axons and rapid signal propagation are impaired, leading to axonal damage and permanent disabilities. Partial replacement of lost oligodendrocytes and remyelination can occur as a result of activation and recruitment of resident oligodendroglial precursor cells. However, the overall remyelination capacity remains inefficient because precursor cells often fail to generate new oligodendrocytes. Increasing evidence points to the existence of several molecular inhibitors that act on these cells and interfere with their cellular maturation. The p57kip2 gene encodes one such potent inhibitor of oligodendroglial differentiation and this study sheds light on the underlying mode of action. We found that subcellular distribution of the p57kip2 protein changed during differentiation of rat, mouse, and human oligodendroglial cells both in vivo and in vitro. Nuclear export of p57kip2 was correlated with promoted myelin expression, higher morphological phenotypes, and enhanced myelination in vitro. In contrast, nuclear accumulation of p57kip2 resulted in blocked oligodendroglial differentiation. Experimental evidence suggests that the inhibitory role of p57kip2 depends on specific interactions with binding proteins such as LIMK-1, CDK2, Mash1, and Hes5 either by controlling their site of action or their activity. Because functional restoration in demyelinating diseases critically depends on the successful generation of oligodendroglial cells, a therapeutic need that is currently unmet, the regulatory mechanism described here might be of particular interest for identifying suitable drug targets and devising novel therapeutic approaches.

Ascl1 controls the number and distribution of astrocytes and oligodendrocytes in the gray matter and white matter of the spinal cord.

Glia constitute the majority of cells in the mammalian central nervous system and are crucial for neurological function. However, there is an incomplete understanding of the molecular control of glial cell development. We find that the transcription factor Ascl1 (Mash1), which is best known for its role in neurogenesis, also functions in both astrocyte and oligodendrocyte lineages arising in the mouse spinal cord at late embryonic stages. Clonal fate mapping in vivo reveals heterogeneity in Ascl1-expressing glial progenitors and shows that Ascl1 defines cells that are restricted to either gray matter (GM) or white matter (WM) as astrocytes or oligodendrocytes. Conditional deletion of Ascl1 post-neurogenesis shows that Ascl1 is required during oligodendrogenesis for generating the correct numbers of WM but not GM oligodendrocyte precursor cells, whereas during astrocytogenesis Ascl1 functions in balancing the number of dorsal GM protoplasmic astrocytes with dorsal WM fibrous astrocytes. Thus, in addition to its function in neurogenesis, Ascl1 marks glial progenitors and controls the number and distribution of astrocytes and oligodendrocytes in the GM and WM of the spinal cord.

Development of the prethalamus is crucial for thalamocortical projection formation and is regulated by Olig2.

Thalamocortical axons (TCAs) pass through the prethalamus in the first step of their neural circuit formation. Although it has been supposed that the prethalamus is an intermediate target for thalamocortical projection formation, much less is known about the molecular mechanisms of this targeting. Here, we demonstrated the functional implications of the prethalamus in the formation of this neural circuit. We show that Olig2 transcription factor, which is expressed in the ventricular zone (VZ) of prosomere 3, regulates prethalamus formation, and loss of Olig2 results in reduced prethalamus size in early development, which is accompanied by expansion of the thalamic eminence (TE). Extension of TCAs is disorganized in the Olig2-KO dorsal thalamus, and initial elongation of TCAs is retarded in the Olig2-KO forebrain. Microarray analysis demonstrated upregulation of several axon guidance molecules, including Epha3 and Epha5, in the Olig2-KO basal forebrain. In situ hybridization showed that the prethalamus in the wild type excluded the expression of Epha3 and Epha5, whereas loss of Olig2 resulted in reduction of this Ephas-negative area and the corresponding expansion of the Ephas-positive TE. Dissociated cultures of thalamic progenitor cells demonstrated that substrate-bound EphA3 suppresses neurite extension from dorsal thalamic neurons. These results indicate that Olig2 is involved in correct formation of the prethalamus, which leads to exclusion of the EphA3-expressing region and is crucial for proper TCA formation. Our observation is the first report showing the molecular mechanisms underlying how the prethalamus acts on initial thalamocortical projection formation.

Neurogenin 2 has an essential role in development of the dentate gyrus.

The dentate gyrus (DG) of the hippocampus has a central role in learning and memory in adult rodents. The DG is generated soon after birth, although new neurons continue to be generated in the DG throughout life. The proneural factors Mash1 (Ascl1) and neurogenin 2 (Ngn2) are expressed during formation of the DG but their role in the development of this structure has not yet been addressed. Here, we show that Ngn2 is essential for the development of the DG. Ngn2 mutant mice have fewer DG progenitors and these cells present defects in neuronal differentiation. By contrast, the DG is normal in Mash1 mutant mice at birth, and loss of both Mash1 and Ngn2 does not aggravate the defect observed in Ngn2 single mutants. These data establish a unique role of Ngn2 in DG neurogenesis during development and raise the possibility that Ngn2 has a similar function in adult neurogenesis.

Ascl1 is required for oligodendrocyte development in the spinal cord.

Development of oligodendrocytes, myelin-forming glia in the central nervous system (CNS), proceeds on a protracted schedule. Specification of oligodendrocyte progenitors (OLPs) begins early in development, whereas their terminal differentiation occurs at late embryonic and postnatal periods. How these distinct steps are controlled remains unclear. Our previous study demonstrated an important role of the helix-loop-helix (HLH) transcription factor Ascl1 in early generation of OLPs in the developing spinal cord. Here, we show that Ascl1 is also involved in terminal differentiation of oligodendrocytes late in development. Ascl1-/- mutant mice showed a deficiency in differentiation of myelin-expressing oligodendrocytes at birth. In vitro culture studies demonstrate that the induction and maintenance of co-expression of Olig2 and Nkx2-2 in OLPs, and thyroid hormone-responsive induction of myelin proteins are impaired in Ascl1-/- mutants. Gain-of-function studies further showed that Ascl1 collaborates with Olig2 and Nkx2-2 in promoting differentiation of OLPs into oligodendrocytes in vitro. Overexpression of Ascl1, Olig2 and Nkx2-2 alone stimulated the specification of OLPs, but the combinatorial action of Ascl1 and Olig2 or Nkx2-2 was required for further promoting their differentiation into oligodendrocytes. Thus, Ascl1 regulates multiple aspects of oligodendrocyte development in the spinal cord.

The proneural gene Mash1 specifies an early population of telencephalic oligodendrocytes.

The bHLH (basic helix-loop-helix) transcription factor Mash1 is best known for its role in the regulation of neurogenesis. However, Mash1 is also expressed in oligodendrocyte precursors and has recently been shown to promote the generation of oligodendrocytes in cell culture, suggesting that it may regulate oligodendrogenesis as well. Here, we show that in the developing ventral forebrain, Mash1 is expressed by a subset of oligodendrocyte precursors (OPCs) as soon as they are generated in the ventricular zone. Using reporter mice, we demonstrate that a subset of OPCs in both the embryonic and postnatal forebrain originate from Mash1-positive progenitors, including a large fraction of adult NG2-positive OPCs. Using Mash1 null mutant mice, we show that Mash1 is required for the generation of an early population of OPCs in the ventral forebrain between embryonic day 11.5 (E11.5) and E13.5, whereas OPCs generated later in embryonic development are not affected. Overexpression of Mash1 in the dorsal telencephalon induces expression of PDGFRalpha (platelet-derived growth factor receptor alpha) but not other OPC markers, suggesting that Mash1 specifies oligodendrogenesis in cooperation with other factors. Analysis of double-mutant mice suggests that Olig2 is one of the factors that cooperate with Mash1 for generation of OPCs. Together, our results show for the first time that Mash1 cooperates in vivo with Olig2 in oligodendrocyte specification, demonstrating an essential role for Mash1 in the generation of a subset of oligodendrocytes and revealing a genetic heterogeneity of oligodendrocyte lineages in the mouse forebrain.

Combinatorial actions of patterning and HLH transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing spinal cord.

During development, the three major neural cell lineages, neurons, oligodendrocytes and astrocytes, differentiate in specific temporal orders at topologically defined positions. How the timing and position of their generation are coordinately regulated remains poorly understood. Here, we provide evidence that the transcription factors Pax6, Olig2 and Nkx2.2 (Nkx2-2), which define the positional identity of multipotent progenitors early in development, also play crucial roles in controlling the timing of neurogenesis and gliogenesis in the developing ventral spinal cord. We show that each of these factors has a unique ability to either enhance or inhibit the activities of the proneural helix-loop-helix (HLH) factors Ngn1 (Neurog1), Ngn2 (Neurog2), Ngn3 (Neurog3) and Mash1 (Ascl1), and the inhibitory HLH factors Id1 and Hes1, thereby regulating both the timing of differentiation of multipotent progenitors and their fate. Consistent with this, dynamic changes in their co-expression pattern in vivo are closely correlated to stage- and domain-specific generation of three neural cell lineages. We also show that genetic manipulations of their temporal expression patterns in mice alter the timing of differentiation of neurons and glia. We propose a molecular code model whereby the combinatorial actions of two classes of transcription factors coordinately regulate the domain-specific temporal sequence of neurogenesis and gliogenesis in the developing spinal cord.

Mash1 specifies neurons and oligodendrocytes in the postnatal brain.

Progenitors in the telencephalic subventricular zone (SVZ) remain mitotically active throughout life, and produce different cell types at embryonic, postnatal and adult stages. Here we show that Mash1, an important proneural gene in the embryonic telencephalon, is broadly expressed in the postnatal SVZ, in progenitors for both neuronal and oligodendrocyte lineages. Moreover, Mash1 is required at birth for the generation of a large fraction of neuronal and oligodendrocyte precursors from the olfactory bulb. Clonal analysis in culture and transplantation experiments in postnatal brain demonstrate that this phenotype reflects a cell-autonomous function of Mash1 in specification of these two lineages. The conservation of Mash1 function in the postnatal SVZ suggests that the same transcription mechanisms operate throughout life to specify cell fates in this structure, and that the profound changes in the cell types produced reflect changes in the signalling environment of the SVZ.

Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity.

The neural bHLH genes Mash1 and Ngn2 are expressed in complementary populations of neural progenitors in the central and peripheral nervous systems. Here, we have systematically compared the activities of the two genes during neural development by generating replacement mutations in mice in which the coding sequences of Mash1 and Ngn2 were swapped. Using this approach, we demonstrate that Mash1 has the capacity to respecify the identity of neuronal populations normally derived from Ngn2-expressing progenitors in the dorsal telencephalon and ventral spinal cord. In contrast, misexpression of Ngn2 in Mash1-expressing progenitors does not result in any overt change in neuronal phenotype. Taken together, these results demonstrate that Mash1 and Ngn2 have divergent functions in specification of neuronal subtype identity, with Mash1 having the characteristics of an instructive determinant whereas Ngn2 functions as a permissive factor that must act in combination with other factors to specify neuronal phenotypes. Moreover, the ectopic expression of Ngn2 can rescue the neurogenesis defects of Mash1 null mutants in the ventral telencephalon and sympathetic ganglia but not in the ventral spinal cord and the locus coeruleus, indicating that Mash1 contribution to the specification of neuronal fates varies greatly in different lineages, presumably depending on the presence of other determinants of neuronal identity.