Chris Englund

Pax6, Tbr2, and Tbr1 Are Expressed Sequentially by Radial Glia, Intermediate Progenitor Cells, and Postmitotic Neurons in Developing Neocortex

The developing neocortex contains two types of progenitor cells for glutamatergic, pyramidal-projection neurons. The first type, radial glia, produce neurons and glia, divide at the ventricular surface, and express Pax6, a homeodomain transcription factor. The second type, intermediate progenitor cells, are derived from radial glia, produce only neurons, and divide away from the ventricular surface. Here we show that the transition from radial glia to intermediate progenitor cell is associated with upregulation of Tbr2, a T-domain transcription factor, and downregulation of Pax6. Accordingly, Tbr2 expression in progenitor compartments (the subventricular zone and ventricular zone) rises and falls with cortical plate neurogenesis. The subsequent transition from intermediate progenitor cell to postmitotic neuron is marked by downregulation of Tbr2 and upregulation of Tbr1, another T-domain transcription factor. These findings delineate the transcription factor sequence Pax6 → Tbr2 → Tbr1 in the differentiation of radial glia → intermediate progenitor cell → postmitotic projection neuron. This transcription factor sequence is modified in preplate neurons, in which Tbr2 is transiently coexpressed with Tbr1, and in the direct differentiation pathway from radial glia → postmitotic projection neuron, in which Tbr2 is expressed briefly or not at all.

Role of intermediate progenitor cells in cerebral cortex development.

Intermediate progenitor cells (IPCs) are a type of neurogenic transient amplifying cells in the developing cerebral cortex. IPCs divide symmetrically at basal (abventricular) positions in the neuroepithelium to produce pairs of new neurons or, in amplifying divisions, pairs of new IPCs. In contrast, radial unit progenitors (neuroepithelial cells and radial glia) divide at the apical (ventricular) surface and produce only single neurons or single IPCs by asymmetric division, or self-amplify by symmetric division. Histologically, IPCs are most prominent during the middle and late stages of neurogenesis, when they accumulate in the subventricular zone, a progenitor compartment linked to the genesis of upper neocortical layers (II–IV). Nevertheless, IPCs are present throughout cortical neurogenesis and produce neurons for all layers. In mice, changes in the abundance of IPCs caused by mutations of Pax6, Ngn2, Id4 and other genes are associated with parallel changes in cortical thickness but not surface area. In gyrencephalic brains, IPCs may play broader roles in determining not only laminar thickness, but also cortical surface area and gyral patterns. We propose that regulation of IPC genesis and amplification across developmental stages and regional subdivisions modulates laminar neurogenesis and contributes to the cytoarchitectonic differentiation of cortical areas.

Beyond laminar fate: Toward a molecular classification of cortical projection/pyramidal neurons

Cortical projection neurons exhibit diverse morphological, physiological, and molecular phenotypes, but it is unknown how many distinct types exist. Many projection cell phenotypes are associated with laminar fate (radial position), but each layer may also contain multiple types of projection cells. We have investigated two hypotheses: (1) that different projection cell types exhibit characteristic molecular expression profiles and (2) that laminar fates are determined primarily by molecular phenotype. We found that several transcription factors were differentially expressed by projection neurons, even within the same layer: Otx1 and Er81, for example, were expressed by different neurons in layer 5. Retrograde tracing showed that Er81 was expressed in corticospinal and corticocortical neurons. In contrast, Otx1 has been detected only in corticobulbar neurons [Weimann et al., Neuron 1999;24:819–831]. Birthdating demonstrated that different molecularly defined types were produced sequentially, in overlapping waves. Cells adopted laminar fates characteristic of their molecular phenotypes, regardless of cell birthday. Molecular markers also revealed the locations of different projection cell types in the malformed cortex of reeler mice. These studies suggest that molecular profiles can be used advantageously for classifying cortical projection cells, for analyzing their neurogenesis and fate specification, and for evaluating cortical malformations.

Development of the Deep Cerebellar Nuclei: Transcription Factors and Cell Migration from the Rhombic Lip

The deep cerebellar nuclei (DCN) are the main output centers of the cerebellum, but little is known about their development. Using transcription factors as cell type-specific markers, we found that DCN neurons in mice are produced in the rhombic lip and migrate rostrally in a subpial stream to the nuclear transitory zone (NTZ). The rhombic lip-derived cells express transcription factors Pax6, Tbr2, and Tbr1 sequentially as they enter the NTZ. A subset of rhombic lip-derived cells also express reelin, a key regulator of Purkinje cell migrations. In organotypic slice cultures, the rhombic lip was necessary and sufficient to produce cells that migrate in the subpial stream, enter the NTZ, and express Pax6, Tbr2, Tbr1, and reelin. In later stages of development, the subpial stream is replaced by the external granular layer, and the NTZ organizes into distinct DCN nuclei. Tbr1 expression persists to adulthood in a subset of medial DCN projection neurons. In reeler mutant mice, which have a severe cerebellar malformation, rhombic lip-derived cells migrated to the NTZ, despite reelin deficiency. Studies in Tbr1 mutant mice suggested that Tbr1 plays a role in DCN morphogenesis but is not required for reelin expression, glutamatergic differentiation, or the initial formation of efferent axon pathways. Our findings reveal underlying similarities in the transcriptional programs for glutamatergic neuron production in the DCN and the cerebral cortex, and they support a model of cerebellar neurogenesis in which glutamatergic and GABAergic neurons are produced from separate progenitor compartments.

Postnatal shifts of interneuron position in the neocortex of normal and reeler mice: evidence for inward radial migration.

During development, interneurons migrate to precise positions in the cortex by tangential and radial migration. The objectives of this study were to characterize the net radial migrations of interneurons during the first postnatal week, and to investigate the role of reelin signaling in regulating those migrations. To observe radial migrations, we compared the laminar positions of interneurons (immunoreactive for GABA or Dlx) in mouse neocortex on postnatal days (P) 0.5 and P7.5. In addition, we used bromodeoxyuridine birthdating to reveal the migrations of different interneuron cohorts. To study the effects of reelin deficiency, experiments were performed in reeler mutant mice. In normal P0.5 cortex, interneurons were most abundant in the marginal zone and layer 5. By P7.5, interneurons were least abundant in the marginal zone, and were distributed more evenly in the cortical plate. This change was attributed mainly to inward migration of middle- to late-born interneurons (produced on embryonic days (E) 13.5 to E16.5) from the marginal zone to layers 2-5. During the same interval, late-born projection neurons (non-immunoreactive for GABA or Dlx) migrated mainly outward, from the intermediate zone to upper cortical layers. In reeler cortex, middle- and late-born interneurons migrated from the superplate on P0.5, to the deep cortical plate on P7.5. Late-born projection neurons in reeler migrated in the opposite direction, from the intermediate zone to the deep cortical plate. We conclude that many middle- and late-born interneurons migrate radially inward, from the marginal zone (or superplate) to the cortical plate, during the first postnatal week in normal and reeler mice. We propose that within the cortical plate, interneuron laminar positions may be determined in part by interactions with projection neurons born on the same day in neurogenesis.

Cajal-Retzius cells in the mouse: transcription factors, neurotransmitters, and birthdays suggest a pallial origin.

Cajal-Retzius cells are reelin-secreting neurons found in the marginal zone of the mammalian cortex during development. Recently, it has been proposed that Cajal-Retzius cells may be generated both early and late in corticogenesis, and may migrate into the cortex from proliferative zones in the subpallium (lateral ganglionic eminence and medial ganglionic eminence) or cortical hem. In the present study, we used reelin as a marker to study the properties of Cajal-Retzius cells, including their likely origins, neurotransmitters, and birthdates. In double labeling experiments, Cajal-Retzius cells (reelin(+)) expressed transcription factors characteristic of pallial neurons (Tbr1 and Emx2), contained high levels of glutamate, were usually calretinin(+), and were born early in corticogenesis, on embryonic days (E)10.5 and E11.5. Tbr1(+) cells in the marginal zone were almost always reelin(+). The first Cajal-Retzius cells (Tbr1(+)/reelin(+)) appeared in the preplate on E10.5. In contrast, interneurons expressed a subpallial transcription factor (Dlx), contained high levels of GABA, were frequently calbindin(+), and were born throughout corticogenesis (from E10.5 to E16.5). Interneurons (Dlx(+)) first appeared in the cortex on E12.5. Our results suggest that the marginal zone contains two main types of neurons: Cajal-Retzius cells derived from the pallium, and migrating interneurons derived from the subpallium.

Unipolar Brush Cells of the Cerebellum Are Produced in the Rhombic Lip and Migrate through Developing White Matter

Unipolar brush cells (UBCs) are glutamatergic interneurons in the cerebellar cortex and dorsal cochlear nucleus. We studied the development of UBCs, using transcription factor Tbr2/Eomes as a marker for UBCs and their progenitors in embryonic and postnatal mouse cerebellum. Tbr2+ UBCs appeared to migrate out of the upper rhombic lip via two cellular streams: a dorsal pathway into developing cerebellar white matter, where the migrating cells dispersed widely before entering the internal granular layer, and a rostral pathway along the cerebellar ventricular zone toward the brainstem. Ablation of the rhombic lip in organotypic slice cultures substantially reduced the production of Tbr2+ UBCs. In coculture experiments, Tbr2+ UBCs migrated from rhombic lip explants directly into the developing white matter of adjacent cerebellar slices. The origin of Tbr2+ UBCs was confirmed by colocalization with β-galactosidase expressed from the Math1 locus, a molecular marker of rhombic lip lineages. Moreover, the production of Tbr2+ UBCs was Math1 dependent, as Tbr2+ UBCs were severely reduced in Math1-null cerebellum. In reeler mutant mice, Tbr2+ UBCs accumulated near the rhombic lip, consistent with impaired migration through developing white matter. Our results suggest that UBCs arise from the rhombic lip and migrate via novel pathways to their final destinations in the cerebellum and dorsal cochlear nucleus. Our findings support a model of cerebellar neurogenesis, in which glutamatergic and GABAergic neurons are produced from separate progenitor pools located mainly in the rhombic lip and the cerebellar ventricular zone, respectively.

Aberrant neuronal-glial differentiation in Taylor-type focal cortical dysplasia (type IIA/B)

Focal cortical dysplasia (FCD) type IIA/B (Taylor type) is a malformation of cortical development characterized by laminar disorganization and dysplastic neurons. FCD IIA and FCD IIB denote subtypes in which balloon cells are absent or present, respectively. The etiology of FCD IIA/B is unknown, but previous studies suggest that its pathogenesis may involve aberrant, mixed neuronal-glial differentiation. To investigate whether aberrant differentiation is a consistent phenotype in FCD IIA/B, we studied a panel of neuronal and glial marker antigens in a series of 15 FCD IIB cases, and 2 FCD IIA cases. Double-labeling immunofluorescence and confocal imaging revealed that different combinations of neuronal and glial antigens were co-expressed by individual cells in all cases of FCD IIA/B, but not in control cases of epilepsy due to other causes. Co-expression of neuronal and glial markers was most common in balloon cells, but was also observed in dysplastic neurons. The relative expression of neuronal and glial antigens varied over a broad range. Microtubule-associated protein 1B, an immature neuronal marker, was more frequently co-expressed with glial antigens than were mature neuronal markers, such as neuronal nuclear antigen. Our results indicate that aberrant neuronal-glial differentiation is a consistent and robust phenotype in FCD IIA/B, and support the hypothesis that developmental defects of neuronal and glial fate specification play an important role in its pathogenesis.

ERK5 MAP kinase regulates neurogenin1 during cortical neurogenesis

The commitment of multi-potent cortical progenitors to a neuronal fate depends on the transient induction of the basic-helix-loop-helix (bHLH) family of transcription factors including Neurogenin 1 (Neurog1). Previous studies have focused on mechanisms that control the expression of these proteins while little is known about whether their pro-neural activities can be regulated by kinase signaling pathways. Using primary cultures and ex vivo slice cultures, here we report that both the transcriptional and pro-neural activities of Neurog1 are regulated by extracellular signal-regulated kinase (ERK) 5 signaling in cortical progenitors. Activation of ERK5 potentiated, while blocking ERK5 inhibited Neurog1-induced neurogenesis. Furthermore, endogenous ERK5 activity was required for Neurog1-initiated transcription. Interestingly, ERK5 activation was sufficient to induce Neurog1 phosphorylation and ERK5 directly phosphorylated Neurog1 in vitro. We identified S179/S208 as putative ERK5 phosphorylation sites in Neurog1. Mutations of S179/S208 to alanines inhibited the transcriptional and pro-neural activities of Neurog1. Our data identify ERK5 phosphorylation of Neurog1 as a novel mechanism regulating neuronal fate commitment of cortical progenitors.

NeuN expression correlates with reduced mitotic index of neoplastic cells in central neurocytomas

In the developing brain, neuronal differentiation is associated with permanent exit from the mitotic cycle. This raises the possibility that neuronal differentiation may suppress proliferative activity, even in neoplastic cells. As a first step towards understanding the relation between neuronal differentiation and mitotic cycling in brain tumours, we studied the expression of NeuN (a neuronal marker) and Ki‐67 (a mitotic marker) by double‐labelling immuno‐fluorescence in 16 brain tumours with neuronal differentiation. The tumours included a series of 11 central neurocytomas, and five single cases of other tumour types. In the central neurocytomas, NeuN+ cells had a 15‐fold lower Ki‐67 labelling index, on average, than did NeuN– cells (P < 0.01). In the other tumours (one extraventricular neurocytoma, one desmoplastic medulloblastoma, one olfactory neuroblastoma, one ganglioglioma and one anaplastic ganglioglioma), the Ki‐67 labelling index was always at least fourfold lower in NeuN+ cells than in NeuN– cells. These results indicate that neuronal differentiation is associated with a substantial decrease of proliferative activity in neoplastic cells of central neurocytomas, and suggest that the same may be true across diverse types of brain tumours. However, tumours with extensive neuronal differentiation may nevertheless have a high overall Ki‐67 labelling index, if the mitotic activity of NeuN– cells is high. The correlation between NeuN expression and reduced mitotic activity in neurocytoma cells is consistent with the hypothesis that neuronal differentiation suppresses proliferation, but further studies will be necessary to determine causality and investigate underlying mechanisms.