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At least 19 recordsLinked to original sources
PubMed2004

Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation.

Telencephalic inhibitory neurons originate in the ganglionic eminences and migrate to the cerebral cortex following a tangential trajectory, before they differentiate and integrate within the local circuitry. Current studies of interneuron development and function benefit from the use of knock-out and transgenic mice, whereas none take advantage of the versatility of in utero electroporation. Here, we show how in utero electroporation can be directed to the ganglionic eminences to specifically target gene expression to interneurons. Electroporation of GFP-encoding plasmids into the ganglionic eminences results in selective labeling of migrating interneurons during development. In the adult brain of electroporated animals, a wide variety of cortical, hippocampal and olfactory bulb interneurons are labeled. We also show that GFP-expressing interneurons can be visualized in living slices of adult cerebral cortex, where they display normal electrophysiological properties. Photostimulation studies using acute slices show that cortical GFP+ interneurons receive normal, layer-specific synaptic input, indicating that these neurons integrate within the local cortical circuitry. Ganglionic eminence-directed in utero electroporation is therefore an effective, rapid, and versatile method of selectively transfecting telencephalic interneurons, optimal for both developmental studies and adult functional studies.

Animals↗
PubMed2006

Migration and nucleogenesis of mouse precerebellar neurons visualized by in utero electroporation of a green fluorescent protein gene.

Neural migration is a critical step for accurate CNS development, but the molecular mechanisms that regulate migration, settlement and nucleogenesis remain largely unknown. The precerebellar neurons (PCNs), generated in the lower rhombic lip (LRL), migrate towards their destinations: some neurons form the pontine gray nucleus (PGN) and reticulotegmental nucleus (RTN) in the ipsilateral pons, while others form the lateral reticular and external cuneate nuclei in the contralateral medulla after crossing the midline. Here, by introducing an EGFP gene into a unilateral LRL of mouse embryos by in utero electroporation, we specifically labeled and tracked the PCNs in vivo. We found that a substantial number of the labeled neurons crossed the midline and formed PGN/RTN on the contralateral side. In addition, we found that a subpopulation of the interpolar subnucleus of the spinal trigeminal nucleus, which projects the axons to the cerebellum, was one of the PCNs derived from the LRL. Furthermore, because the electroporated mice were born and grew up healthy, we could visualize the PCNs and their mossy fibers in the adult brain. Therefore, the EGFP labeling of PCNs can be applied to studying the physiology of the mossy fiber system as well as PCN development in embryos.

Amino Acids↗
PubMed2005

Neuronal generation, migration, and differentiation in the mouse hippocampal primoridium as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation.

Neuronal migration defects in the hippocampus during development are thought to be involved in various mental disorders. Studies of neural cell migration in the developing cerebrum have focused mainly on the neocortex, but those that have been performed on the developing hippocampal formation have not been adequately carried out. In the present study, the morphological differentiation of immature neurons that form the laminar structure of the hippocampus was investigated by labeling ventricular surface cells with the expression vector of the enhanced-green-fluorescent-protein (EGFP) gene. Vector DNA was transfected into spatially and temporally restricted neuroepithelium of the hippocampal primordium by in utero electroporation, and the morphology of EGFP-labeled migratory neurons and their interrelationships with the radial glial arrangement were observed. Pyramidal cells of Ammon's horn began to migrate radially along glial processes from a broad area of neuroepithelium on embryonic day (E)14. Large numbers of multipolar cells were found in the intermediate zone in the initial stage and stratified pyramidal cells appeared later. Dentate granule cells were labeled later than (E)16 and originated from a restricted area of neuroepithelium adjacent to the fimbria. Their initial migration was rapid and independent of radial glial fibers. Subsequent tangential migration in the subpial space and their ultimate settling into the forming dentate gyrus were closely associated with the radial glia. These findings indicate that distinct cellular mechanisms are involved in the development of the cortical layer of Ammon's horn and dentate gyrus.

Age Factors↗
PubMed2025

Multicolor Cell Lineage Tracing Using MAGIC Markers Strategies.

Multicolor MAGIC Markers strategies are useful lineage tracing tools to study brain development at a multicellular scale. In this chapter, we describe an in utero electroporation method to simultaneously label multiple neighboring progenitors and their respective progeny using these multicolor reporters. In utero electroporation enables the introduction of any gene of interest into embryonic neural progenitors lining the brain ventricles through a simple pipeline consisting of a micro-injection followed by the application of electrical pulses. Successful in utero electroporation requires a concise yet complete understanding of each step of the surgical protocol, spanning from the preoperative preparation to the postoperative care, as well as the MAGIC Markers tool outlined in this study. Besides a detailed protocol, we present non-integrative and integrative approaches to demonstrate the range of cell and lineage tracking possibilities of multicolored progenitors and their descent over time.

Cell Lineage↗
PubMed2002

Neurons tend to stop migration and differentiate along the cortical internal plexiform zones in the Reelin signal-deficient mice.

The Reelin molecule plays a fundamental role in corticogenesis. After Reelin binds to its receptors, the Reelin signal is transduced through tyrosine phosphorylation of the intracellular adaptor protein disabled 1 (Dab1). The reelin-gene-deficient mouse, reeler, and Dab1-deficient mouse, yotari, show disrupted positioning of neurons. Several molecules have been identified recently as being involved in Reelin signaling, however, the biological function of Reelin during cortical plate development was still unknown. We observed the migrating behavior of neurons during development in Reelin-signal-deficient mice. To visualize the migrating neurons directly, we introduced green fluorescent protein (GFP)-expression vectors into the ventricular zone with an in utero electroporation system and allowed the embryos to develop in utero until they were analyzed. The result showed that the migrating cells in the mutants were morphologically indistinguishable from those of normal mice. At the stage when the GFP-expressing cells reached the marginal zone near the pial surface and began dendrite formation in normal mice, the GFP-positive cells were found at various deeper positions in the mutant cortex. They had the morphology of migrating cells extending leading processes toward the pial surface. By contrast, in the mutants these cells tended to stop migration along the borders of the internal plexiform zone, the irregular structure consisting mainly of dendrites in the mutant cortex. Postnatally, these neurons began to develop dendrites later than the cells in the normal cortex. During this process, some neurons above the internal plexiform zone extended and developed dendrites in the opposite direction into the internal plexiform zone. These results suggest that the abnormal positioning of neurons in the Reelin-signal-deficient mice is caused, at least in part, by abnormal formation of the internal plexiform zone in the mutant cortex.

Animals↗
PubMed2004

Distinct migratory behavior of early- and late-born neurons derived from the cortical ventricular zone.

Time-lapse studies indicate that ventricular zone (VZ)-derived cells show two migratory modes in the cerebral cortex at different stages of mammalian embryogenesis: somal translocation and locomotion. We carried out a systematic analysis to examine whether the migratory behavior of cortical neurons derived from the cortical VZ is stage-dependent. We labeled VZ cells of mouse embryos with green fluorescent protein (gfp) -encoding plasmids by in utero electroporation and evaluated the labeled cells after appropriate survival periods. After electroporation at either embryonic day (E) 12.5 or E15.5, GFP+ VZ cells were initially spindle-shaped and radially oriented. After leaving the VZ, they transformed into round or horizontally oriented fusiform neurons with many short processes. They then seemed to gradually change into radially oriented bipolar cells as they moved upward. Whereas the earliest emigrants from the VZ labeled at E12.5 (early-born neurons) reached the top of the cortical plate (CP) after these changes, VZ cells labeled at E15.5 (late-born neurons) further migrated along the length of radial fibers to reach the top of the CP. A dominant negative form of the gene for cyclin-dependent kinase 5 (Cdk5DN) was then introduced into VZ cells. Transfection of E12.5 VZ with cdk5dn did not disrupt the migration of the early-born neurons. However, this caused a failure in migration of the late-born neurons, although they transformed into bipolar shapes in the intermediate zone. Thus, there appear to be at least two distinct migratory phases of cortical neurons: one common to the early- and late-born neurons, and the other specific to late-born neurons and Cdk5-dependent.

Animals↗
PubMed2002

Pak1 is involved in dendrite initiation as a downstream effector of Rac1 in cortical neurons.

Dendrite development in neurons is one of the bases for the formation of a complex neuronal network in the nervous system, and involvement of the Rho family GTPases, including Rac1, Cdc42, and RhoA, in dendrite formation has been demonstrated. One of the effectors of Rac1 and Cdc42, p21-activated kinase 1 (Pak1), is abundant in the brain; however, the function of Pak1 in neurons remains unknown. In order to clarify the roles of Pak1 in neurons, we introduced mutant Pak1 into immature neurons by a novel gene transfer technique using in utero electroporation. Introduction of constitutive active (CA)-Pak1 led to increase the number of dendrites, whereas introduction of dominant negative (DN)-Pak1 caused a reduction, and coexpression of CA-Rac1 and DN-Pak1 also resulted in a reduction in the number of dendrites. These results suggest that Pak1 regulates dendrite initiation as a downstream effector of Rac1.

Animals↗
PubMed2005

MAP1B phosphorylation is differentially regulated by Cdk5/p35, Cdk5/p25, and JNK.

Mode I phosphorylated MAP1B is observed in developing and pathogenic brains. Although Cdk5 has been believed to phosphorylate MAP1B in the developing cerebral cortex, we show that a Cdk5 inhibitor does not suppress mode I phosphorylation of MAP1B in primary and slice cultures, while a JNK inhibitor does. Coincidently, an increase in phosphorylated MAP1B was not observed in COS7 cells when Cdk5 was cotransfected with p35, but this did occur with p25 which is specifically produced in pathogenic brains. Our primary culture studies showed an involvement of Cdk5 in regulating microtubule dynamics without affecting MAP1B phosphorylation status. The importance of regulating microtubule dynamics in neuronal migration was also demonstrated by in utero electroporation experiments. These findings suggest that mode I phosphorylation of MAP1B is facilitated by JNK but not Cdk5/p35 in the developing cerebral cortex and by Cdk5/p25 in pathogenic brains, contributing to various biological events.

Animals↗
PubMed2005

In vivo function of Rnd2 in the development of neocortical pyramidal neurons.

The present study examined the in vivo role of Rnd2, a Rho family small GTPase, in brain development. Rnd2 was expressed by radially migrating cells, which primarily develop to pyramidal neurons, during their stay in the subventricular zone of embryonic cerebral cortex and hippocampus. Exogenous expression of wild-type and a constitutively active Rnd2, but not a negative mutant of Rnd2, in radially migrating cells by in utero electroporation disturbed their morphology and migration to upper layers. These results indicate that Rnd2 functions in vivo as a regulator of the migration and morphological changes associated with the development of pyramidal neurons.

Amino Acid Sequence↗
PubMed2005

Evidence that embryonic neurons regulate the onset of cortical gliogenesis via cardiotrophin-1.

Precursor cells of the embryonic cortex sequentially generate neurons and then glial cells, but the mechanisms regulating this neurogenic-to-gliogenic transition are unclear. Using cortical precursor cultures, which temporally mimic this in vivo differentiation pattern, we demonstrate that cortical neurons synthesize and secrete the neurotrophic cytokine cardiotrophin-1, which activates the gp130-JAK-STAT pathway and is essential for the timed genesis of astrocytes in vitro. Our data indicate that a similar phenomenon also occurs in vivo. In utero electroporation of neurotrophic cytokines in the environment of embryonic cortical precursors causes premature gliogenesis, while acute perturbation of gp130 in cortical precursors delays the normal timed appearance of astrocytes. Moreover, the neonatal cardiotrophin-1-/- cortex contains fewer astrocytes. Together, these results describe a neural feedback mechanism; newly born neurons produce cardiotrophin-1, which instructs multipotent cortical precursors to generate astrocytes, thereby ensuring that gliogenesis does not occur until neurogenesis is largely complete.

Analysis of Variance↗
PubMed2005

Ephrin-as guide the formation of functional maps in the visual cortex.

Ephrin-As and their receptors, EphAs, are expressed in the developing cortex where they may act to organize thalamic inputs. Here, we map the visual cortex (V1) in mice deficient for ephrin-A2, -A3, and -A5 functionally, using intrinsic signal optical imaging and microelectrode recording, and structurally, by anatomical tracing of thalamocortical projections. V1 is shifted medially, rotated, and compressed and its internal organization is degraded. Expressing ephrin-A5 ectopically by in utero electroporation in the lateral cortex shifts the map of V1 medially, and expression within V1 disrupts its internal organization. These findings indicate that interactions between gradients of EphA/ephrin-A in the cortex guide map formation, but that factors other than redundant ephrin-As are responsible for the remnant map. Together with earlier work on the retinogeniculate map, the current findings show that the same molecular interactions may operate at successive stages of the visual pathway to organize maps.

Aging↗
PubMed2006

The multipolar stage and disruptions in neuronal migration.

The genetic basis is now known for several disorders of neuronal migration in the developing cerebral cortex. Identification of the cellular processes mediated by the implicated genes is revealing crucial stages of neuronal migration and has the potential to reveal common cellular causes of neuronal migration disorders. We hypothesize that a newly recognized morphological stage of neuronal migration, the multipolar stage, is vulnerable and is disrupted in several disorders of neocortical development. The multipolar stage occurs as bipolar progenitor cells become radially migrating neurons. Several studies using in utero electroporation and RNAi have revealed that transition out of the multipolar stage depends on the function of filamin A, LIS1 and DCX. Mutations in the genes encoding these proteins in humans cause distinct neuronal migration disorders, including periventricular nodular heterotopia, subcortical band heterotopia and lissencephaly. The multipolar stage therefore seems to be a critical point of migration control and a vulnerable target for disruption of neocortical development. This review is part of the INMED/TINS special issue "Nature and nurture in brain development and neurological disorders", based on presentations at the annual INMED/TINS symposium (http://inmednet.com/).

Animals↗
PubMed2005

LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages.

Mutations in the human LIS1 gene cause the smooth brain disease classical lissencephaly. To understand the underlying mechanisms, we conducted in situ live cell imaging analysis of LIS1 function throughout the entire radial migration pathway. In utero electroporation of LIS1 small interference RNA and short hairpin dominant negative LIS1 and dynactin cDNAs caused a dramatic accumulation of multipolar progenitor cells within the subventricular zone of embryonic rat brains. This effect resulted from a complete failure in progression from the multipolar to the migratory bipolar state, as revealed by time-lapse analysis of brain slices. Surprisingly, interkinetic nuclear oscillations in the radial glial progenitors were also abolished, as were cell divisions at the ventricular surface. Those few bipolar cells that reached the intermediate zone also exhibited a complete block in somal translocation, although, remarkably, process extension persisted. Finally, axonal growth also ceased. These results identify multiple distinct and novel roles for LIS1 in nucleokinesis and process dynamics and suggest that nuclear position controls neural progenitor cell division.

Animals↗
PubMed2007

Disruption of neuronal migration by RNAi of Dyx1c1 results in neocortical and hippocampal malformations.

The brains of individuals with developmental dyslexia have neocortical neuronal migration abnormalities including molecular layer heterotopias, laminar dysplasias, and periventricular nodular heterotopias (PNH). RNA interference (RNAi) of Dyx1c1, a candidate dyslexia susceptibility gene, disrupts neuronal migration in developing embryonic neocortex. Using in utero electroporation, we cotransfected cells in the rat neocortical ventricular zone (VZ) at E14/15 with short hairpin RNA vectors targeting Dyx1c1 along with either plasmids encoding enhanced green fluorescent protein or plasmids encoding monomeric red fluorescent protein only. RNAi of Dyx1c1 resulted in pockets of unmigrated neurons resembling PNH. The pattern of migration of transfected neurons was bimodal, with approximately 20% of the neurons migrating a short distance from the VZ and another 40% that migrated past their expected lamina. Approximately 25% of the transfected brains had hippocampal pyramidal cell migration anomalies. Molecular layer ectopias, which were not related to injection site artifacts, were also seen in 25% of the animals. These results support the hypothesis that targeted disruption of the candidate dyslexia susceptibility gene, Dyx1c1, results in neuronal migration disorders similar to those seen in the brains of dyslexics.

Animals↗
PubMed2003

The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration.

The coordinated migration of neurons is a pivotal step for functional architectural formation of the mammalian brain. To elucidate its molecular mechanism, gene transfer by means of in utero electroporation was applied in the developing murine brain, revealing the crucial roles of Rac1, its activators, STEF/Tiam1, and its downstream molecule, c-Jun N-terminal kinase (JNK), in the cerebral cortex. Functional repression of these molecules resulted in inhibition of radial migration of neurons without affecting their proper differentiation. Interestingly, distinct morphological phenotypes were observed; suppression of Rac1 activity caused loss of the leading process, whereas repression of JNK activity did not, suggesting the complexity of the signaling cascade. In cultured neurons from the intermediate zone, activated JNK was detected along microtubules in the processes. Application of a JNK inhibitor caused irregular morphology and increased stable microtubules in processes, and decreased phosphorylation of microtubule associated protein 1B, raising a possibility of the involvement of JNK in controlling tubulin dynamics in migrating neurons. Our data thus provide important clues for understanding the intracellullar signaling machinery for cortical neuronal migration.

Animals↗
PubMed2004

Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex.

Recent findings implicate embryonic signaling centers in patterning the mammalian cerebral cortex. We used mouse in utero electroporation and mutant analysis to test whether cortical signaling sources interact to regulate one another. We identified interactions between the cortical hem, rich in Wingless-Int (WNT) proteins and bone morphogenetic proteins (BMPs), and an anterior telencephalic source of fibroblast growth factors (FGFs). Expanding the FGF8 domain suppressed Wnt2b, Wnt3a and Wnt5a expression in the hem. Next to the hem, the hippocampus was shrunken, consistent with its dependence for growth on a hem-derived WNT signal. Maintenance of hem WNT signaling and hippocampal development thus require a constraint on the FGF8 source, which is likely to be supplied by BMP activity. When endogenous BMP signaling is inhibited by noggin, robust Fgf8 expression appears ectopically in the cortical primordium. Abnormal signaling centers were further investigated in mice lacking the transcription factor EMX2, in which FGF8 activity is increased, WNT expression reduced, and the hippocampus defective. Suggesting that these defects are causally related, sequestering FGF8 in Emx2 homozygous mutants substantially recovered WNT expression in the hem and partially rescued hippocampal development. Because noggin can induce Fgf8 expression, we examined noggin and BMP signaling in the Emx2 mutant. As the telencephalic vesicle closed, Nog expression was expanded and BMP activity reduced, potentially leading to FGF8 upregulation. Our findings point to a cross-regulation of BMP, FGF, and WNT signaling in the early telencephalon, integrated by EMX2, and required for normal cortical development.

Animals↗
PubMed2006

Direct visualization of nucleogenesis by precerebellar neurons: involvement of ventricle-directed, radial fibre-associated migration.

Nuclei are aggregates of neurons distributed in the central nervous system and are fundamental functional units that share anatomical and physiological features. Despite their importance, the cellular basis that leads to nucleogenesis is only poorly understood. Using exo utero electroporation with an enhanced yellow fluorescent protein (EYFP) gene, we show that the precerebellar neurons derived from the lower rhombic lip (lRL) undergo multiple migration steps to form nuclei. After the unilateral transfer of EYFP to the lRL of embryonic day 12.5 mice, EYFP-labelled neurons migrate tangentially from the lRL in two distinct streams, one towards the ventral metencephalon and the other towards the ventral myelencephalon. These neurons cross the ventral midline and then become radially directed. Labelled neurons in the tangential migratory streams form contralateral clusters in the external cuneate nucleus (ECN) and lateral reticular nucleus (LRN) in the myelencephalon, and bilateral clusters in the pontine grey nucleus (PGN) and reticulotegmental nucleus (RTN) in the metencephalon. Before forming the clusters, EYFP-labelled neurons begin to migrate radially towards the ventricle in close apposition to nestin-positive radial fibres, and then they aggregate as they detach from the fibres. Inhibition of cadherin function in ECN and LRN progenitors caused ipsilateral formation of the ECN and LRN, implying that the transition of their migration from tangential to radial involves a cell-intrinsic mechanism. These observations suggest that nucleogenesis of precerebellar neurons is a result of multi-phasic migration, and that ventricle-directed radial glia-guided migration is a key step for nucleogenesis.

Animals↗
PubMed2024

Bulk serum extracellular vesicles from stressed mice show a distinct proteome and induce behavioral and molecular changes in naive mice.

Chronic stress can trigger several pathologies including mood disorders for which no clear diagnostic molecular markers have been established yet. Attractive biomarker sources are extracellular vesicles (EVs). Evs are released by cells in health and disease and contain genetic material, proteins and lipids characteristic of the cell state. Here we show that Evs recovered from the blood of animals exposed to a repeated interrupted stress protocol (RIS) have a different protein profile compared to those obtained from control animals. Proteomic analysis indicated that proteins differentially present in bulk serum Evs from stressed animals were implicated in metabolic and inflammatory pathways and several of them were previously related to psychiatric disorders. Interestingly, these serum Evs carry brain-enriched proteins including the stress-responsive neuronal protein M6a. Then, we used an in-utero electroporation strategy to selectively overexpress M6a-GFP in brain neurons and found that M6a-GFP could also be detected in bulk serum Evs suggesting a neuronal origin. Finally, to determine if these Evs could have functional consequences, we administered Evs from control and RIS animals intranasally to naïve mice. Animals receiving stress EVs showed changes in behavior and brain M6a levels similar to those observed in physically stressed animals. Such changes could therefore be attributed, or at least in part, to EV protein transfer. Altogether these findings show that EVs may participate in stress signaling and propose proteins carried by EVs as a valuable source of biomarkers for stress-induced diseases.

Animals↗