https://arep.med.harvard.edu/pdf/Wichterle02.pdf

Directed Differentiation of Embryonic Stem Cells into Motor Neurons

https://www.cell.com/cell/pdf/S0092-8674(02)00677-3.pdf

The bHLH Transcription Factors OLIG2 and OLIG1 Couple Neuronal and Glial Subtype Specification

The developmental origin of oligodendrocyte progenitors (OLPs) in the forebrain has been controversial. We now show, by Cre-lox fate mapping in transgenic mice, that the first OLPs originate in the medial ganglionic eminence (MGE) and anterior entopeduncular area (AEP) in the ventral forebrain. From there, they populate the entire embryonic telencephalon including the cerebral cortex before being joined by a second wave of OLPs from the lateral and/or caudal ganglionic eminences (LGE and CGE). Finally, a third wave arises within the postnatal cortex. When any one population is destroyed at source by the targeted expression of diphtheria toxin, the remaining cells take over and the mice survive and behave normally, with a normal complement of oligodendrocytes and myelin. Thus, functionally redundant populations of OLPs compete for space in the developing brain. Notably, the embryonic MGE- and AEP-derived population is eliminated during postnatal life, raising questions about the nature and purpose of the competition.

/pdf/competing-waves-of-oligodendrocytes-in-the-forebrain-and-1djj8du1m3.pdf

Competing waves of oligodendrocytes in the forebrain and postnatal elimination of an embryonic lineage.

Glial fibrillary acidic protein (GFAP)-positive astrocytes (type B cells) in the subventricular zone (SVZ) generate large numbers of new neurons in the adult brain. SVZ stem cells can also generate oligodendrocytes in vitro, but it is not known whether these adult primary progenitors generate oligodendrocytes in vivo. Myelin repair and oligodendrocyte formation in the adult brain is instead associated with glial-restricted progenitors cells, known as oligodendrocyte progenitor cells (OPCs). Here we show that type B cells also generate a small number of nonmyelinating NG2-positive OPCs and mature myelinating oligodendrocytes. Some type B cells and a small subpopulation of actively dividing type C (transit-amplifying) cells expressed oligodendrocyte lineage transcription factor 2 (Olig2), suggesting that oligodendrocyte differentiation in the SVZ begins early in the lineage. Olig2-positive, polysialylated neural cell adhesion molecule-positive, PDGF receptor α-positive, and β-tubulin-negative cells originating in the SVZ migrated into corpus callosum, striatum, and fimbria fornix to differentiate into the NG2-positive nonmyelinating and mature myelinating oligodendrocytes. Furthermore, primary clonal cultures of type B cells gave rise to oligodendrocytes alone or oligodendrocytes and neurons. Importantly, the number of oligodendrocytes derived from type B cells in vivo increased fourfold after a demyelinating lesion in corpus callosum, indicating that SVZ astrocytes participate in myelin repair in the adult brain. Our work identifies SVZ type B cells as progenitors of oligodendrocytes in normal and injured adult brain.

https://www.jneurosci.org/content/jneuro/26/30/7907.full.pdf

Origin of Oligodendrocytes in the Subventricular Zone of the Adult Brain

GABA-containing interneurons are crucial to both the development and function of the cerebral cortex. Unlike cortical projection neurons, which have a relatively conserved set of characteristics, interneurons include multiple phenotypes that vary on morphological, physiological and neurochemical axes. This diversity, and the relatively late, context-dependent maturation of defining features, has challenged efforts to uncover the transcriptional control of cortical interneuron development. Here, we discuss recent data that are beginning to illuminate the origins and specification of distinct subgroups of cortical interneurons.

The origin and specification of cortical interneurons

Multiple Dorsoventral Origins of Oligodendrocyte Generation in the Spinal Cord and Hindbrain

Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification.

Neural progenitor cells often produce distinct types of neurons in a specific order, but the determinants that control the sequential generation of distinct neuronal subclasses in the vertebrate CNS remain poorly defined. We examined the sequential generation of visceral motor neurons and serotonergic neurons from a common pool of neural progenitors located in the ventral hindbrain. We found that the temporal specification of these neurons varies along the anterior-posterior axis of the hindbrain, and that the timing of their generation critically depends on the integrated activities of Nkx- and Hox-class homeodomain proteins. A primary function of these proteins is to coordinate the spatial and temporal activation of the homeodomain protein Phox2b, which in turn acts as a binary switch in the selection of motor neuron or serotonergic neuronal fate. These findings assign new roles for Nkx, Hox, and Phox2 proteins in the control of temporal neuronal fate determination, and link spatial and temporal patterning of CNS neuronal fates.

/pdf/coordinated-temporal-and-spatial-control-of-motor-neuron-and-4i3se09u0q.pdf

Coordinated temporal and spatial control of motor neuron and serotonergic neuron generation from a common pool of CNS progenitors

The genetic program that underlies the generation of visceral motoneurons in the developing hindbrain remains poorly defined. We have examined the role of Nkx6 and Nkx2 class homeodomain proteins in this process, and provide evidence that these proteins mediate complementary roles in the specification of visceral motoneuron fate. The expression of Nkx2.2 in hindbrain progenitor cells is sufficient to mediate the activation of Phox2b, a homeodomain protein required for the generation of hindbrain visceral motoneurons. The redundant activities of Nkx6.1 and Nkx6.2, in turn, are dispensable for visceral motoneuron generation but are necessary to prevent these cells from adopting a parallel program of interneuron differentiation. The expression of Nkx6.1 and Nkx6.2 is further maintained in differentiating visceral motoneurons, and consistent with this the migration and axonal projection properties of visceral motoneurons are impaired in mice lacking Nkx6.1 and/or Nkx6.2 function. Our analysis provides insight also into the role of Nkx6 proteins in the generation of somatic motoneurons. Studies in the spinal cord have shown that Nkx6.1 and Nkx6.2 are required for the generation of somatic motoneurons, and that the loss of motoneurons at this level correlates with the extinguished expression of the motoneuron determinant Olig2. Unexpectedly, we find that the initial expression of Olig2 is left intact in the caudal hindbrain of Nkx6.1/Nkx6.2 compound mutants, and despite this, all somatic motoneurons are missing. These data argue against models in which Nkx6 proteins and Olig2 operate in a linear pathway, and instead indicate a parallel requirement for these proteins in the progression of somatic motoneuron differentiation. Thus, both visceral and somatic motoneuron differentiation appear to rely on the combined activity of cell intrinsic determinants, rather than on a single key determinant of neuronal cell fate.

Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain

During development, different classes of neurons and glia are generated from proliferative progenitor cells lining the ventricles of the brain and the lumen of the spinal cord. A central issue in developmental neuroscience is to understand the mechanisms by which these cells are generated in space and over time. In the ventral spinal cord, the expression profile of homeodomain (HD) proteins defines five progenitor domains that each will give rise to a distinct type of neuron. Two closely related HD transcriptional repressors, Nkx6.1 and Nkx6.2 (Nkx6), are expressed by progenitors of the ventral spinal cord. We provide evidence that different levels of Nkx6 repressor activity in progenitor cells are a critical determinant of ventral neuronal fate, assigning both redundant and specific roles for these proteins in neuronal specification. A reduction in Nkx6 activity further permits V0 interneurons to be generated from progenitors that lack HD proteins normally required for their generation, providing direct evidence for a model where HD proteins direct specific cell fates by actively repressing the expression of transcription factors that direct alternative fates. In the ventral spinal cord, sMNs and oligodendrocyte precursors (OLPs) are sequentially generated from a domain defined by the expression of Olig2. We show that the generation of sMNs and OLPs in the ventral spinal cord is essentially missing in mice lacking Nkx6 function. In contrast, the same HD proteins instead act to suppress OLP specification in the ventral hindbrain. The divergent roles for Nkx6 proteins seem to reflect that OLPs in the spinal cord and hindbrain are produced by distinct ventral progenitor domains. While a ventral specification of OLPs is well established, it has remained unclear whether also more dorsal progenitor cells give rise to oligodendrocytes. We provide in vivo and in vitro evidence that oligodendrocytes are produced also by dorsal progenitors in the spinal cord and hindbrain and that the specification of these cells may result from the progressive evasion of dorsal BMP signalling over time. Together, our data suggest that oligodendrocytes are generated from multiple dorsoventral origins in the spinal cord and hindbrain, and indicate that the activation of Olig2 at different positions is controlled by distinct genetic programs. The observation that the loss of sMNs in the spinal cord of Nkx6 mutant mice correlates with an extinguished expression of the sMN determinant Olig2 has led to a model where Nkx6 proteins act strictly upstream of Olig2. However, in the hindbrain of Nkx6 mutant mice the initial expression of Olig2 is intact and despite this all sMNs are missing, indicating a parallel requirement for Nkx6 and Olig2 proteins in the generation of sMNs. Visceral motor neurons (vMNs) are generated immediately ventral to sMNs. The transcription factor Phox2b has been found to be an important determinant of these cells, but other factors involved have not been identified. We show that the HD protein Nkx2.2 is sufficient to mediate the expression of Phox2b. Furthermore, while the activities of Nkx6.1 and Nkx6.2 are dispensable for the initial generation of vMNs, they are required to prevent a parallel program of more dorsal interneuronal differentiation and to ensure a proper migration and axonal projection formation of vMNs. Thus, Nkx2 and Nkx6 proteins appear to have complementary roles in the establishment of vMN identity in the hindbrain. Taken together, our data suggest that both visceral and somatic motor neuron differentiation rely on the combined activity of cell intrinsic determinants, rather than on a singe key determinant of neuronal cell fate. Neuronal diversity is established by mechanisms that operate in space and over time. Advances have been made in regard to the mechanisms that restrict and direct neuronal generation in space, but less is known about the mechanisms that underlie how neural progenitors produce distinct types of neurons in a specific temporal order. We addressed this issue by studying a population of progenitor cells in the ventral hindbrain that gives rise to vMNs and serotonergic (S) neurons. Each hindbrain segment, or rhombomere (r), initially generates vMNs, but all the rhombomeres except for r4 switch to producing S neurons at a defined time point. We found that the temporal and spatial generation of vMNs and S neurons critically relies on the integrated activity of Nkxand Hox-class HD proteins. A primary function of these proteins is to coordinate the activation of Phox2b in space and time. Phox2b, in turn, functions as a binary switch in deciding whether progenitors differentiate into vMNs or serotonergic neurons. Taken together, these data indicate that determinants that control spatial patterning may be associated also with temporal patterning and require that expression patterns are dynamic and modulated over time. LIST OF PUBLICATIONS This thesis is based on the following papers, which will be referred to in the text by their roman numbers: I Vallstedt, A., Muhr, J., Pattyn, A., Pierani, A., Mendelsohn, M., Sander, M., Jessell, T. M., and Ericson, J. (2001). Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification. Neuron 31, 743-755. II. Pattyn, A*., Vallstedt, A*., Dias, J. M., Sander, M., and Ericson, J. (2003). Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain. Development 130, 4149-4159. III. Pattyn, A., Vallstedt, A., Dias, J. M., Samad, O. A., Krumlauf, R., Rijli, F. M., Brunet, J. F., and Ericson, J. (2003). Coordinated temporal and spatial control of motor neuron and serotonergic neuron generation from a common pool of CNS progenitors. Genes Dev 17, 729-737. IV. Vallstedt, A*., Klos, J*., Ericson, J. (2004). Multiple Dorsoventral Origins of Oligdendrocyte Generation in the Spinal Cord and Hindbrain. Submitted. * These authors contributed equally to this work.

/pdf/the-role-of-nkx-proteins-neuronal-and-glial-specification-fv3f2s31kj.pdf

Anna Vallstedt

Papers

Multiple Dorsoventral Origins of Oligodendrocyte Generation in the Spinal Cord and Hindbrain

Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification.

Coordinated temporal and spatial control of motor neuron and serotonergic neuron generation from a common pool of CNS progenitors

Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain

The role of Nkx proteins neuronal and glial specification