Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis.

Interneurons navigate along multiple tangential paths to settle into appropriate cortical layers. They undergo a saltatory migration paced by intermittent nuclear jumps whose regulation relies on interplay between extracellular cues and genetic-encoded information. It remains unclear how cycles of pause and movement are coordinated at the molecular level. Post-translational modification of proteins contributes to cell migration regulation. The present study uncovers that carboxypeptidase 1, which promotes post-translational protein deglutamylation, controls the pausing of migrating cortical interneurons. Moreover, we demonstrate that pausing during migration attenuates movement simultaneity at the population level, thereby controlling the flow of interneurons invading the cortex. Interfering with the regulation of pausing not only affects the size of the cortical interneuron cohort but also impairs the generation of age-matched projection neurons of the upper layers.

Classes and continua of hippocampal CA1 inhibitory neurons revealed by single-cell transcriptomics.

Understanding any brain circuit will require a categorization of its constituent neurons. In hippocampal area CA1, at least 23 classes of GABAergic neuron have been proposed to date. However, this list may be incomplete; additionally, it is unclear whether discrete classes are sufficient to describe the diversity of cortical inhibitory neurons or whether continuous modes of variability are also required. We studied the transcriptomes of 3,663 CA1 inhibitory cells, revealing 10 major GABAergic groups that divided into 49 fine-scale clusters. All previously described and several novel cell classes were identified, with three previously described classes unexpectedly found to be identical. A division into discrete classes, however, was not sufficient to describe the diversity of these cells, as continuous variation also occurred between and within classes. Latent factor analysis revealed that a single continuous variable could predict the expression levels of several genes, which correlated similarly with it across multiple cell types. Analysis of the genes correlating with this variable suggested it reflects a range from metabolically highly active faster-spiking cells that proximally target pyramidal cells to slower-spiking cells targeting distal dendrites or interneurons. These results elucidate the complexity of inhibitory neurons in one of the simplest cortical structures and show that characterizing these cells requires continuous modes of variation as well as discrete cell classes.

Regulation of embryonic neurogenesis by germinal zone vasculature.

In the adult rodent brain, new neurons are born in two germinal regions that are associated with blood vessels, and blood vessels and vessel-derived factors are thought to regulate the activity of adult neural stem cells. Recently, it has been proposed that a vascular niche also regulates prenatal neurogenesis. Here we identify the mouse embryo hindbrain as a powerful model to study embryonic neurogenesis and define the relationship between neural progenitor cell (NPC) behavior and vessel growth. Using this model, we show that a subventricular vascular plexus (SVP) extends through a hindbrain germinal zone populated by NPCs whose peak mitotic activity follows a surge in SVP growth. Hindbrains genetically defective in SVP formation owing to constitutive NRP1 loss showed a premature decline in both NPC activity and hindbrain growth downstream of precocious cell cycle exit, premature neuronal differentiation, and abnormal mitosis patterns. Defective regulation of NPC activity was not observed in mice lacking NRP1 expression by NPCs, but instead in mice lacking NRP1 selectively in endothelial cells, yet was independent of vascular roles in hindbrain oxygenation. Therefore, germinal zone vascularization sustains NPC proliferation in the prenatal brain.

Vascular endothelial growth factor receptor 3 directly regulates murine neurogenesis.

Neural stem cells (NSCs) are slowly dividing astrocytes that are intimately associated with capillary endothelial cells in the subventricular zone (SVZ) of the brain. Functionally, members of the vascular endothelial growth factor (VEGF) family can stimulate neurogenesis as well as angiogenesis, but it has been unclear whether they act directly via VEGF receptors (VEGFRs) expressed by neural cells, or indirectly via the release of growth factors from angiogenic capillaries. Here, we show that VEGFR-3, a receptor required for lymphangiogenesis, is expressed by NSCs and is directly required for neurogenesis. Vegfr3:YFP reporter mice show VEGFR-3 expression in multipotent NSCs, which are capable of self-renewal and are activated by the VEGFR-3 ligand VEGF-C in vitro. Overexpression of VEGF-C stimulates VEGFR-3-expressing NSCs and neurogenesis in the SVZ without affecting angiogenesis. Conversely, conditional deletion of Vegfr3 in neural cells, inducible deletion in subventricular astrocytes, and blocking of VEGFR-3 signaling with antibodies reduce SVZ neurogenesis. Therefore, VEGF-C/VEGFR-3 signaling acts directly on NSCs and regulates adult neurogenesis, opening potential approaches for treatment of neurodegenerative diseases.

EMX1 regulates NRP1-mediated wiring of the mouse anterior cingulate cortex.

Transcription factors act during cortical development as master regulatory genes that specify cortical arealization and cellular identities. Although numerous transcription factors have been identified as being crucial for cortical development, little is known about their downstream targets and how they mediate the emergence of specific neuronal connections via selective axon guidance. The EMX transcription factors are essential for early patterning of the cerebral cortex, but whether EMX1 mediates interhemispheric connectivity by controlling corpus callosum formation remains unclear. Here, we demonstrate that in mice on the C57Bl/6 background EMX1 plays an essential role in the midline crossing of an axonal subpopulation of the corpus callosum derived from the anterior cingulate cortex. In the absence of EMX1, cingulate axons display reduced expression of the axon guidance receptor NRP1 and form aberrant axonal bundles within the rostral corpus callosum. EMX1 also functions as a transcriptional activator of Nrp1 expression in vitro, and overexpression of this protein in Emx1 knockout mice rescues the midline-crossing phenotype. These findings reveal a novel role for the EMX1 transcription factor in establishing cortical connectivity by regulating the interhemispheric wiring of a subpopulation of neurons within the mouse anterior cingulate cortex.

Tumor suppressor role of phospholipase C epsilon in Ras-triggered cancers.

Phospholipase Cε (PLCε) has been characterized as a direct effector of Ras in vitro and in cellular systems; however, the role of PLCε in tumorigenesis and its link to Ras in this context remain unclear. To assess the role of PLCε in Ras-driven cancers, we generated two new mouse strains: one carrying a targeted deletion of Plce (Plce(-/-)) and the other carrying mutant alleles of Plce unable to bind to Ras (Plce(RAm/RAm)). The Plce(-/-) and, to a lesser degree, Plce(RAm/RAm) transgenic mice exhibited increased susceptibility to tumor formation in the two-stage skin carcinogenesis protocol, revealing a tumor suppressor function for this PLC. This result also suggests that in this context Ras binding in part regulates functions of PLCε. Although significant differences were not seen in the LSL-Kras(G12D) nonsmall cell lung carcinoma model, down-regulation of PLCε was found in animal tumors and in cellular systems following expression of the oncogenic Ras. An inhibitory impact of PLCε on cell growth requires intact lipase activity and is likely mediated by protein kinase C enzymes. Further cellular studies suggest involvement of histone deacetylase in the mechanism of PLCε down-regulation. Taken together, our results show a previously unidentified tumor suppressor role for this PLC in animal models and, together with observations of marked down-regulation in colorectal, lung, and skin tumors, suggest its use as a biological marker in cancer.

Rac-GTPases Regulate Microtubule Stability and Axon Growth of Cortical GABAergic Interneurons.

Cortical interneurons are characterized by extraordinary functional and morphological diversity. Although tremendous progress has been made in uncovering molecular and cellular mechanisms implicated in interneuron generation and function, several questions still remain open. Rho-GTPases have been implicated as intracellular mediators of numerous developmental processes such as cytoskeleton organization, vesicle trafficking, transcription, cell cycle progression, and apoptosis. Specifically in cortical interneurons, we have recently shown a cell-autonomous and stage-specific requirement for Rac1 activity within proliferating interneuron precursors. Conditional ablation of Rac1 in the medial ganglionic eminence leads to a 50% reduction of GABAergic interneurons in the postnatal cortex. Here we examine the additional role of Rac3 by analyzing Rac1/Rac3 double-mutant mice. We show that in the absence of both Rac proteins, the embryonic migration of medial ganglionic eminence-derived interneurons is further impaired. Postnatally, double-mutant mice display a dramatic loss of cortical interneurons. In addition, Rac1/Rac3-deficient interneurons show gross cytoskeletal defects in vitro, with the length of their leading processes significantly reduced and a clear multipolar morphology. We propose that in the absence of Rac1/Rac3, cortical interneurons fail to migrate tangentially towards the pallium due to defects in actin and microtubule cytoskeletal dynamics.

Temporal control of neural crest lineage generation by Wnt/ß-catenin signaling.

Wnt/ß-catenin signaling controls multiple steps of neural crest development, ranging from neural crest induction, lineage decisions, to differentiation. In mice, conditional ß-catenin inactivation in premigratory neural crest cells abolishes both sensory neuron and melanocyte formation. Intriguingly, the generation of melanocytes is also prevented by activation of ß-catenin in the premigratory neural crest, which promotes sensory neurogenesis at the expense of other neural crest derivatives. This raises the question of how Wnt/ß-catenin signaling regulates the formation of distinct lineages from the neural crest. Using various Cre lines to conditionally activate ß-catenin in neural crest cells at different developmental stages, we show that neural crest cell fate decisions in vivo are subject to temporal control by Wnt/ß-catenin. Unlike in premigratory neural crest, ß-catenin activation in migratory neural crest cells promotes the formation of ectopic melanoblasts, while the production of most other lineages is suppressed. Ectopic melanoblasts emerge at sites of neural crest target structures and in many tissues usually devoid of neural crest-derived cells. ß-catenin activation at later stages in glial progenitors or in melanoblasts does not lead to surplus melanoblasts, indicating a narrow time window of Wnt/ß-catenin responsiveness during neural crest cell migration. Thus, neural crest cells appear to be multipotent in vivo both before and after emigration from the neural tube but adapt their response to extracellular signals in a temporally controlled manner.

N-cadherin sustains motility and polarity of future cortical interneurons during tangential migration.

In the developing brain, cortical GABAergic interneurons migrate long distances from the medial ganglionic eminence (MGE) in which they are generated, to the cortex in which they settle. MGE cells express the cell adhesion molecule N-cadherin, a homophilic cell-cell adhesion molecule that regulates numerous steps of brain development, from neuroepithelium morphogenesis to synapse formation. N-cadherin is also expressed in embryonic territories crossed by MGE cells during their migration. In this study, we demonstrate that N-cadherin is a key player in the long-distance migration of future cortical interneurons. Using N-cadherin-coated substrate, we show that N-cadherin-dependent adhesion promotes the migration of mouse MGE cells in vitro. Conversely, mouse MGE cells electroporated with a construct interfering with cadherin function show reduced cell motility, leading process instability, and impaired polarization associated with abnormal myosin IIB dynamics. In vivo, the capability of electroporated MGE cells to invade the developing cortical plate is altered. Using genetic ablation of N-cadherin in mouse embryos, we show that N-cadherin-depleted MGEs are severely disorganized. MGE cells hardly exit the disorganized proliferative area. N-cadherin ablation at the postmitotic stage, which does not affect MGE morphogenesis, alters MGE cell motility and directionality. The tangential migration to the cortex of N-cadherin ablated MGE cells is delayed, and their radial migration within the cortical plate is perturbed. Altogether, these results identify N-cadherin as a pivotal adhesion substrate that activates cell motility in future cortical interneurons and maintains cell polarity over their long-distance migration to the developing cortex.

Gli3 is required in Emx1+ progenitors for the development of the corpus callosum.

The corpus callosum (CC) is the largest commissure in the forebrain and mediates the transfer of sensory, motor and cognitive information between the cerebral hemispheres. During CC development, a number of strategically located glial and neuronal guidepost structures serve to guide callosal axons across the midline at the corticoseptal boundary (CSB). Correct positioning of these guideposts requires the Gli3 gene, mutations of which result in callosal defects in humans and mice. However, as Gli3 is widely expressed during critical stages of forebrain development, the precise temporal and spatial requirements for Gli3 function in callosal development remain unclear. Here, we used a conditional mouse mutant approach to inactivate Gli3 in specific regions of the developing telencephalon in order to delineate the domain(s) in which Gli3 is required for normal development of the corpus callosum. Inactivation of Gli3 in the septum or in the medial ganglionic eminence had no effect on CC formation, however Gli3 inactivation in the developing cerebral cortex led to the formation of a severely hypoplastic CC at E18.5 due to a severe disorganization of midline guideposts. Glial wedge cells translocate prematurely and Slit1/2 are ectopically expressed in the septum. These changes coincide with altered Fgf and Wnt/ß-catenin signalling during CSB formation. Collectively, these data demonstrate a crucial role for Gli3 in cortical progenitors to control CC formation and indicate how defects in CSB formation affect the positioning of callosal guidepost cells.

New Olig1 null mice confirm a non-essential role for Olig1 in oligodendrocyte development.

BACKGROUND: Olig1 and Olig2, encoding closely related basic helix-loop-helix transcription factors, were originally identified in screens for glial-specific genes. Olig1 and Olig2 are both expressed in restricted parts of the neuroepithelium of the embryonic spinal cord and telencephalon and subsequently in oligodendrocyte lineage cells throughout life. In the spinal cord, Olig2 plays a crucial role in the development of oligodendrocytes and motor neurons, and both cell types are lost from Olig2 null mutant mice. The role of Olig1 has been more cryptic. It was initially reported that Olig1 null mice (with a Cre-Pgk-Neo cassette at the Olig1 locus) have a mild developmental phenotype characterized by a slight delay in oligodendrocyte differentiation. However, a subsequent study of the same line following removal of Pgk-Neo (leaving Olig1-Cre) found severe disruption of oligodendrocyte production, myelination failure and early postnatal lethality. A plausible explanation was proposed, that the highly expressed Pgk-Neo cassette in the original line might have up-regulated the neighbouring Olig2 gene, compensating for loss of Olig1. However, this was not tested, so the importance of Olig1 for oligodendrocyte development has remained unclear. RESULTS: We generated two independent lines of Olig1 null mice. Both lines had a mild phenotype featuring slightly delayed oligodendrocyte differentiation and maturation but no long-term effect. In addition, we found that Olig2 transcripts were not up-regulated in our Olig1 null mice. CONCLUSIONS: Our findings support the original conclusion that Olig1 plays a minor and non-essential role in oligodendrocyte development and have implications for the interpretation of studies based on Olig1 deficient mice (and perhaps Olig1-Cre mice) from different sources.

Developmental origin of oligodendrocytes determines their function in the adult brain.

In the mouse embryonic forebrain, developmentally distinct oligodendrocyte progenitor cell populations and their progeny, oligodendrocytes, emerge from three distinct regions in a spatiotemporal gradient from ventral to dorsal. However, the functional importance of this oligodendrocyte developmental heterogeneity is unknown. Using a genetic strategy to ablate dorsally derived oligodendrocyte lineage cells (OLCs), we show here that the areas in which dorsally derived OLCs normally reside in the adult central nervous system become populated and myelinated by OLCs of ventral origin. These ectopic oligodendrocytes (eOLs) have a distinctive gene expression profile as well as subtle myelination abnormalities. The failure of eOLs to fully assume the role of the original dorsally derived cells results in locomotor and cognitive deficits in the adult animal. This study reveals the importance of developmental heterogeneity within the oligodendrocyte lineage and its importance for homeostatic brain function.

Rac1-dependent cell cycle exit of MGE precursors and GABAergic interneuron migration to the cortex.

Cortical γ-aminobutyric acid (GABA)ergic interneurons are characterized by extraordinary neurochemical and functional diversity. Although recent studies have uncovered some of the molecular components underlying interneuron development, including the cellular and molecular mechanisms guiding their migration to the cortex, the intracellular components involved are still unknown. Rac1, a member of the Rac subfamily of Rho-GTPases, has been implicated in various cellular processes such as cell cycle dynamics, axonogenesis, and migration. In this study, we have addressed the specific role of Rac1 in interneuron progenitors originating in the medial ganglionic eminence, via Cre/loxP technology. We show that ablation of Rac1 from Nkx2.1-positive progenitors, results in a migratory impairment. As a consequence, only half of GABAergic interneurons are found in the postnatal cortex. The rest remain aggregated in the ventral telencephalon and show morphological defects in their growing processes in vitro. Ablation of Rac1 from postmitotic progenitors does not result in similar defects, thus underlying a novel cell autonomous and stage-specific requirement for Rac1 activity, within proliferating progenitors of cortical interneurons. Rac1 is necessary for their transition from G1 to S phase, at least in part by regulating cyclin D levels and retinoblastoma protein phosphorylation.

Cortical interneuron specification and diversification in the era of big data.

Inhibition in the mammalian cerebral cortex is mediated by a small population of highly diverse GABAergic interneurons. These largely local neurons are interspersed among excitatory projection neurons and exert pivotal regulation on the formation and function of cortical circuits. We are beginning to understand the extent of GABAergic neuron diversity and how this is generated and shaped during brain development in mice and humans. In this review, we summarise recent findings and discuss how new technologies are being used to further advance our knowledge. Understanding how inhibitory neurons are generated in the embryo is an essential pre-requisite of stem cell therapy, an evolving area of research, aimed at correcting human disorders that result in inhibitory dysfunction.

Regulation of oligodendrocyte development and myelination by glucose and lactate.

In the gray matter of the brain, astrocytes have been suggested to export lactate (derived from glucose or glycogen) to neurons to power their mitochondria. In the white matter, lactate can support axon function in conditions of energy deprivation, but it is not known whether lactate acts by preserving energy levels in axons or in oligodendrocytes, the myelinating processes of which are damaged rapidly in low energy conditions. Studies of cultured cells suggest that oligodendrocytes are the cell type in the brain that consumes lactate at the highest rate, in part to produce membrane lipids presumably for myelin. Here, we use pH imaging to show that oligodendrocytes in the white matter of the rat cerebellum and corpus callosum take up lactate via monocarboxylate transporters (MCTs), which we identify as MCT1 by confocal immunofluorescence and electron microscopy. Using cultured slices of developing cerebral cortex from mice in which oligodendrocyte lineage cells express GFP (green fluorescent protein) under the control of the Sox10 promoter, we show that a low glucose concentration reduces the number of oligodendrocyte lineage cells and myelination. Myelination is rescued when exogenous l-lactate is supplied. Thus, lactate can support oligodendrocyte development and myelination. In CNS diseases involving energy deprivation at times of myelination or remyelination, such as periventricular leukomalacia leading to cerebral palsy, stroke, and secondary ischemia after spinal cord injury, lactate transporters in oligodendrocytes may play an important role in minimizing the inhibition of myelination that occurs.

Dorsally and ventrally derived oligodendrocytes have similar electrical properties but myelinate preferred tracts.

In the developing spinal cord, most oligodendrocyte precursors (OLPs) arise from the ventral ventricular zone (VZ) under the influence of Sonic Hedgehog, but a minority are generated from the dorsal VZ in a Hedgehog-independent manner. In the developing forebrain too, OLPs arise from both the ventral and the dorsal VZ. It is not known whether dorsally and ventrally derived oligodendrocyte (OL) lineage cells have different properties. We generated a dual reporter mouse line to color code ventrally and dorsally derived OLPs (vOLPs and dOLPs) and their differentiated oligodendrocyte progeny (vOLs and dOLs) for functional studies. We found that ∼80% of OL lineage cells in the postnatal spinal cord and ∼20% in the corpus callosum are ventrally derived. In both spinal cord and corpus callosum, vOLPs and dOLPs had indistinguishable electrical properties, as did vOLs and dOLs. However, vOLPs and dOLPs had different migration and settling patterns. In the spinal cord, vOLPs appeared early and spread uniformly throughout the cord, whereas dOLPs arrived later and remained mainly in the dorsal and dorsolateral funiculi. During adulthood, corticospinal and rubrospinal tracts became myelinated mainly by dOLs, even though vOLs dominated these tracts during early postnatal life. Thus, dOLPs are electrically similar to vOLPs but appear to outcompete them for dorsal axons.

Pax6 is required at the telencephalic pallial-subpallial boundary for the generation of neuronal diversity in the postnatal limbic system.

During embryogenesis, the pallial-subpallial boundary (PSB) divides the two main progenitor domains in the telencephalon: the pallium, the major source of excitatory neurons, and the subpallium, the major source of inhibitory neurons. The PSB is formed at the molecular interface between the pallial (high Pax6+) and subpallial (high Gsx2+) ventricular zone (VZ) compartments. Initially, the PSB contains cells that express both Pax6 and Gsx2, but during later stages of development this boundary is largely refined into two separate compartments. In this study we examined the developmental mechanisms underlying PSB boundary formation and the postnatal consequences of conditional loss of Pax6 function at the PSB on neuronal fate in the amygdala and olfactory bulb, two targets of PSB-derived migratory populations. Our cell fate and time-lapse imaging analyses reveal that the sorting of Pax6+ and Gsx2+ progenitors during embryogenesis is the result of a combination of changes in gene expression and cell movements. Interestingly, we find that in addition to giving rise to inhibitory neurons in the amygdala and olfactory bulb, Gsx2+ progenitors generate a subpopulation of amygdala excitatory neurons. Consistent with this finding, targeted conditional ablation of Pax6 in Gsx2+ progenitors results in discrete local embryonic patterning defects that are linked to changes in the generation of subsets of postnatal excitatory and inhibitory neurons in the amygdala and inhibitory neurons in the olfactory bulb. Thus, in PSB progenitors, Pax6 plays an important role in the generation of multiple subtypes of neurons that contribute to the amygdala and olfactory bulb.

LIM homeodomain transcription factor-dependent specification of bipotential MGE progenitors into cholinergic and GABAergic striatal interneurons.

Coordination of voluntary motor activity depends on the generation of the appropriate neuronal subtypes in the basal ganglia and their integration into functional neuronal circuits. The largest nucleus of the basal ganglia, the striatum, contains two classes of neurons: the principal population of medium-sized dense spiny neurons (MSNs; 97-98% of all striatal neurons in rodents), which project to the globus pallidus and the substantia nigra, and the locally projecting striatal interneurons (SINs; 2-3% in rodents). SINs are further subdivided into two non-overlapping groups: those producing acetylcholine (cholinergic) and those producing gamma-amino butyric acid (GABAergic). Despite the pivotal role of SINs in integrating the output of striatal circuits and the function of neuronal networks in the ventral forebrain, the lineage relationship of SIN subtypes and the molecular mechanisms that control their differentiation are currently unclear. Using genetic fate mapping, we demonstrate here that the majority of cholinergic and GABAergic SINs are derived from common precursors generated in the medial ganglionic eminence during embryogenesis. These precursors express the LIM homeodomain protein Lhx6 and have characteristics of proto-GABAergic neurons. By combining gene expression analysis with loss-of-function and misexpression experiments, we provide evidence that the differentiation of the common precursor into mature SIN subtypes is regulated by the combinatorial activity of the LIM homeodomain proteins Lhx6, Lhx7 (Lhx8) and Isl1. These studies suggest that a LIM homeodomain transcriptional code confers cell-fate specification and neurotransmitter identity in neuronal subpopulations of the ventral forebrain.

Human cortical interneuron development unraveled.

[Figure: see text].

Fate mapping reveals mixed embryonic origin and unique developmental codes of mouse forebrain septal neurons.

The septum is a key structure at the core of the forebrain that integrates inputs and relays information to other brain areas to support cognition and behaviours such as feeding and locomotion. Underlying these functions is a rich diversity of neuronal types and an intricate complexity of wiring across and within the septal region. We currently have very little understanding of how septal neuronal diversity emerges during development. Using transgenic mice expressing Cre in different subsets of telencephalic precursors we explored the origins of the three main neuronal types of the septal complex: GABAergic, cholinergic and glutamatergic neurons. We find that septal neurons originate from distinct neuroepithelial domains of the developing septum and are born at different embryonic time points. An exception to this is the GABAergic medial septal Parvalbumin-expressing population which is generated outside the septum from surrounding germinal zones. We identify the transcription factor BSX as being expressed in the developing glutamatergic neuron population. Embryonic elimination of BSX in the septum results in a reduction of septal glutamatergic cell numbers and a consequent deficit in locomotion. Further refinement of septal neuron diversity is needed to understand the multiple roles of septal neurons and their contribution to distinct behaviours.