Euiseok J. Kim

Ascl1 defines sequentially generated lineage-restricted neuronal and oligodendrocyte precursor cells in the spinal cord

The neural basic helix-loop-helix transcription factor Ascl1 (previously Mash1) is present in ventricular zone cells in restricted domains throughout the developing nervous system. This study uses genetic fate mapping to define the stage and neural lineages in the developing spinal cord that are derived from Ascl1-expressing cells. We find that Ascl1 is present in progenitors to both neurons and oligodendrocytes, but not astrocytes. Temporal control of the fate-mapping paradigm reveals rapid cell-cycle exit and differentiation of Ascl1-expressing cells. At embryonic day 11, Ascl1 identifies neuronal-restricted precursor cells that become dorsal horn neurons in the superficial laminae. By contrast, at embryonic day 16, Ascl1 identifies oligodendrocyte-restricted precursor cells that distribute throughout the spinal cord. These data demonstrate that sequentially generated Ascl1-expressing progenitors give rise first to dorsal horn interneurons and subsequently to late-born oligodendrocytes. Furthermore, Ascl1-null cells in the spinal cord have a diminished capacity to undergo neuronal differentiation, with a subset of these cells retaining characteristics of immature glial cells.

Ascl1 (Mash1) Defines Cells with Long-Term Neurogenic Potential in Subgranular and Subventricular Zones in Adult Mouse Brain

Ascl1 (Mash1) is a bHLH transcription factor essential for neural differentiation during embryogenesis but its role in adult neurogenesis is less clear. Here we show that in the adult brain Ascl1 is dynamically expressed during neurogenesis in the dentate gyrus subgranular zone (SGZ) and more rostral subventricular zone (SVZ). Specifically, we find Ascl1 levels low in SGZ Type-1 cells and SVZ B cells but increasing as the cells transition to intermediate progenitor stages. In vivo genetic lineage tracing with a tamoxifen (TAM) inducible Ascl1CreERT2 knock-in mouse strain shows that Ascl1 lineage cells continuously generate new neurons over extended periods of time. There is a regionally-specific difference in neuron generation, with mice given TAM at postnatal day 50 showing new dentate gyrus neurons through 30 days post-TAM, but showing new olfactory bulb neurons even 180 days post-TAM. These results show that Ascl1 is not restricted to transit amplifying populations but is also found in a subset of neural stem cells with long-term neurogenic potential in the adult brain.

In vivo analysis of Ascl1 defined progenitors reveals distinct developmental dynamics during adult neurogenesis and gliogenesis.

In the adult mammalian brain, new neurons and glia are continuously generated but molecular factors regulating their differentiation and lineage relationships are largely unknown. We show that Ascl1, a bHLH (basic helix-loop-helix) transcription factor, transiently labels neuronal and oligodendrocyte precursors in the adult brain. Using in vivo lineage tracing with inducible Cre recombinase, we followed the maturation of these precursors in four distinct regions. In the hippocampus, Ascl1 mostly marks type-2a progenitor cells with some late stage type-1 stem cells. Thirty days after Ascl1 expression, although a majority of the cells matured to granule neurons, a few cells remained as immature progenitors. By 6 months, however, essentially all Ascl1 lineage cells were granule neurons. In contrast, in the olfactory bulb neuronal lineage, Ascl1 is restricted to transit amplifying cells, and by 30 d all cells matured into GABAergic interneurons. Ascl1 also broadly marks oligodendrocyte precursors in subcortical gray and white matter regions. In the corpus callosum, Ascl1 defines a ventral layer of early oligodendrocyte precursors that do not yet express other early markers of this lineage like PDGFRα and Olig2. By 30 d, most had transitioned to mature oligodendrocytes. In contrast, Ascl1 expressing oligodendrocyte precursors in gray matter already coexpressed the early oligodendrocyte markers, but by 30 d they mostly remained as precursors. Our results reveal that Ascl1 is a common molecular marker of early progenitors of both neurons and oligodendrocytes in the adult brain, and these Ascl1 defined progenitors mature with distinct dynamics in different brain regions.

Ascl1 (Mash1) lineage cells contribute to discrete cell populations in CNS architecture.

Ascl1 (previously Mash1) is a bHLH transcription factor essential for neuronal differentiation and specification in the nervous system. Although it has been studied for its role in several neural lineages, the full complement of lineages arising from Ascl1 progenitor cells remains unknown. Using an inducible Cre-flox genetic fate-mapping strategy, Ascl1 lineages were determined throughout the brain. Ascl1 is present in proliferating progenitor cells but these cells are actively differentiating as evidenced by rapid migration out of germinal zones. Ascl1 lineage cells contribute to distinct cell types in each major brain division: the forebrain including the cerebral cortex, olfactory bulb, hippocampus, striatum, hypothalamus, and thalamic nuclei, the midbrain including superior and inferior colliculi, and the hindbrain including Purkinje and deep cerebellar nuclei cells and cells in the trigeminal sensory system. Ascl1 progenitor cells at early stages in each CNS region preferentially become neurons, and at late stages they become oligodendrocytes. In conclusion, Ascl1-expressing progenitor cells in the brain give rise to multiple, but not all, neuronal subtypes and oligodendrocytes depending on the temporal and spatial context, consistent with a broad role in neural differentiation with some subtype specification.

Ascl1 Genetics Reveals Insights into Cerebellum Local Circuit Assembly

Two recently generated targeted mouse alleles of the neurogenic gene Ascl1 were used to characterize cerebellum circuit formation. First, genetic inducible fate mapping (GIFM) with an Ascl1CreER allele was found to specifically mark all glial and neuron cell types that arise from the ventricular zone (vz). Moreover, each cell type has a unique temporal profile of marking with Ascl1CreER GIFM. Of great utility, Purkinje cells (Pcs), an early cohort of Bergmann glia, and four classes of GABAergic interneurons can be genetically birth dated during embryogenesis using Ascl1CreER GIFM. Astrocytes and oligodendrocytes, in contrast, express Ascl1CreER throughout their proliferative phase in the white matter. Interestingly, the final position each neuron type acquires differs depending on when it expresses Ascl1. Interneurons (including candelabrum) attain a more outside position the later they express Ascl1, whereas Pcs have distinct settling patterns each day they express Ascl1. Second, using a conditional Ascl1 allele, we discovered that Ascl1 is differentially required for generation of most vz-derived cells. Mice lacking Ascl1 in the cerebellum have a major decrease in three types of interneurons with a tendency toward a loss of later-born interneurons, as well as an imbalance of oligodendrocytes and astrocytes. Double-mutant analysis indicates that a related helix-loop-helix protein, Ptf1a, functions with Ascl1 in generating interneurons and Pcs. By fate mapping vz-derived cells in Ascl1 mutants, we further discovered that Ascl1 plays a specific role during the time period when Pcs are generated in restricting vz progenitors from becoming rhombic lip progenitors.

Gate control of mechanical itch by a subpopulation of spinal cord interneurons

A circuit controlling mechanical itch Considerable progress has been made in understanding and treating chemically induced itch. However, little is known about the mechanisms underlying mechanically evoked itch. Bourane et al. produced a model of mechanical itch by reducing the number of neuropeptide Y–expressing inhibitory spinal interneurons. This led to a selective increase in mechanically evoked itch-like behavior in mice. In contrast, chemically evoked itch or pain behavior remained unaffected. Science, this issue p. 550 Neuropeptide Y–expressing inhibitory neurons selectively gate mechanical itch sensations, but not chemical itch. Light mechanical stimulation of hairy skin can induce a form of itch known as mechanical itch. This itch sensation is normally suppressed by inputs from mechanoreceptors; however, in many forms of chronic itch, including alloknesis, this gating mechanism is lost. Here we demonstrate that a population of spinal inhibitory interneurons that are defined by the expression of neuropeptide Y::Cre (NPY::Cre) act to gate mechanical itch. Mice in which dorsal NPY::Cre-derived neurons are selectively ablated or silenced develop mechanical itch without an increase in sensitivity to chemical itch or pain. This chronic itch state is histamine-independent and is transmitted independently of neurons that express the gastrin-releasing peptide receptor. Thus, our studies reveal a dedicated spinal cord inhibitory pathway that gates the transmission of mechanical itch.

Adult Lineage-Restricted CNS Progenitors Specify Distinct Glioblastoma Subtypes

A central question in glioblastoma multiforme (GBM) research is the identity of the tumor-initiating cell, and its contribution to the malignant phenotype and genomic state. We examine the potential of adult lineage-restricted progenitors to induce fully penetrant GBM using CNS progenitor-specific inducible Cre mice to mutate Nf1, Trp53, and Pten. We identify two phenotypically and molecularly distinct GBM subtypes governed by identical driver mutations. We demonstrate that the two subtypes arise from functionally independent pools of adult CNS progenitors. Despite histologic identity as GBM, these tumor types are separable based on the lineage of the tumor-initiating cell. These studies point to the cell of origin as a major determinant of GBM subtype diversity.

Activin and GDF11 collaborate in feedback control of neuroepithelial stem cell proliferation and fate

Studies of the olfactory epithelium model system have demonstrated that production of neurons is regulated by negative feedback. Previously, we showed that a locally produced signal, the TGFβ superfamily ligand GDF11, regulates the genesis of olfactory receptor neurons by inhibiting proliferation of the immediate neuronal precursors (INPs) that give rise to them. GDF11 is antagonized by follistatin (FST), which is also produced locally. Here, we show that Fst–/– mice exhibit dramatically decreased neurogenesis, a phenotype that can only be partially explained by increased GDF11 activity. Instead, a second FST-binding factor, activin βB (ACTβB), inhibits neurogenesis by a distinct mechanism: whereas GDF11 inhibits expansion of INPs, ACTβB inhibits expansion of stem and early progenitor cells. We present data supporting the concept that these latter cells, previously considered two distinct types, constitute a dynamic stem/progenitor population in which individual cells alternate expression of Sox2 and/or Ascl1. In addition, we demonstrate that interplay between ACTβB and GDF11 determines whether stem/progenitor cells adopt a glial versus neuronal fate. Altogether, the data indicate that the transition between stem cells and committed progenitors is neither sharp nor irreversible and that GDF11, ACTβB and FST are crucial components of a circuit that controls both total cell number and the ratio of neuronal versus glial cells in this system. Thus, our findings demonstrate a close connection between the signals involved in the control of tissue size and those that regulate the proportions of different cell types.

Ascl1 expression defines a subpopulation of lineage-restricted progenitors in the mammalian retina

The mechanisms of cell fate diversification in the retina are not fully understood. The seven principal cell types of the neural retina derive from a population of multipotent progenitors during development. These progenitors give rise to multiple cell types concurrently, suggesting that progenitors are a heterogeneous population. It is thought that differences in progenitor gene expression are responsible for differences in progenitor competence (i.e. potential) and, subsequently, fate diversification. To elucidate further the mechanisms of fate diversification, we assayed the expression of three transcription factors made by retinal progenitors: Ascl1 (Mash1), Ngn2 (Neurog2) and Olig2. We observed that progenitors were heterogeneous, expressing every possible combination of these transcription factors. To determine whether this progenitor heterogeneity correlated with different cell fate outcomes, we conducted Ascl1- and Ngn2-inducible expression fate mapping using the CreER™/LoxP system. We found that these two factors gave rise to markedly different distributions of cells. The Ngn2 lineage comprised all cell types, but retinal ganglion cells (RGCs) were exceedingly rare in the Ascl1 lineage. We next determined whether Ascl1 prevented RGC development. Ascl1-null mice had normal numbers of RGCs and, interestingly, we observed that a subset of Ascl1+ cells could give rise to cells expressing Math5 (Atoh7), a transcription factor required for RGC competence. Our results link progenitor heterogeneity to different fate outcomes. We show that Ascl1 expression defines a competence-restricted progenitor lineage in the retina, providing a new mechanism to explain fate diversification.

Spatiotemporal fate map of neurogenin1 (Neurog1) lineages in the mouse central nervous system

Neurog1 (Ngn1, Neurod3, neurogenin1) is a basic helix‐loop‐helix (bHLH) transcription factor essential for neuronal differentiation and subtype specification during embryogenesis. Due to the transient expression of Neurog1 and extensive migration of neuronal precursors, it has been challenging to understand the full complement of Neurog1 lineage cells throughout the central nervous system (CNS). Here we labeled and followed Neurog1 lineages using inducible Cre‐flox recombination systems with Neurog1‐Cre and Neurog1‐CreERT2 BAC (bacterial artificial chromosome) transgenic mice. Neurog1 lineage cells are restricted to neuronal fates and contribute to diverse but discrete populations in each brain region. In the forebrain, Neurog1 lineages include mitral cells and glutamatergic interneurons in the olfactory bulb, pyramidal and granule neurons in the hippocampus, and pyramidal cells in the cortex. In addition, most of the thalamus, but not the hypothalamus, arises from Neurog1 progenitors. Although Neurog1 lineages are largely restricted to glutamatergic neurons, there are multiple exceptions including Purkinje cells and other GABAergic neurons in the cerebellum. This study provides the first overview of the spatiotemporal fate map of Neurog1 lineages in the CNS. J. Comp. Neurol. 519:1355–1370, 2011.