in utero electroporation for time-lapse imaging of neuronal migration in embryonic mouse brain

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

During the development of the cerebral cortex, (apical) radial glia in the pallial ventricular zone (VZ) lining the lateral ventricle produce first neurons and then glial progenitors with some overlapping period1. Neurons are also generated from intermediate progenitors or basal radial glia in the subventricular zone (SVZ) adjacent to the VZ, both of which originate from the (apical) radial glia2,3. In mice, radial glial cells produce only neurons on embryonic day (E) 12-14, both neurons and glial progenitors on E15-16, and glial progenitors from E17 onward4. The major population of glial progenitors generated during these embryonic stages preferentially differentiates into astrocytes, although some cells also differentiate into oligodendrocytes5. Neurons and astrocyte progenitors generated at these stages migrate toward the brain surface and enter the cortical plate (future cortical gray matter). Neuronal migration from the VZ to the cortical plate occurs in multiple phases. Neurons first adopt a multipolar morphology just above the multipolar cell accumulation zone (MAZ), overlapping the SVZ or intermediate zone, where they vigorously extend and retract multiple thin processes and slowly migrate (multipolar migration)6,7. After approximately 24 h, neurons transform into a bipolar morphology, extending a thick leading process toward the brain surface and a thin trailing process backward, and migrate linearly toward the brain surface using a radial process extending from the radial glia to the pial surface as a scaffold, which is called locomotion mode2,8. Because neurons in locomotion mode always reach the outermost surface of the cortical plate, passing through their predecessors just under the marginal zone, neurons are aligned in a birthdate-dependent inside-out manner in the cortical plate9,10,11.
In contrast, astrocyte progenitors migrate rapidly to the intermediate zone and cortical plate, with frequent directional changes. This migratory behavior is completely different from neuronal migration and is called erratic migration5. Astrocyte progenitors also migrate along blood vessels in a process called blood vessel-guided migration. Astrocyte progenitors switch between these migration modes and reach the cortical plate5,12. Although the positioning of astrocytes is not strictly determined by their date of production, a mild tendency for early-born astrocytes to settle in the superficial part of the cortical plate has been observed5. Interestingly, astrocytes that settle in the cortical plate are generated in embryonic stages and eventually differentiate into protoplasmic astrocytes, whereas postnatally generated astrocytes do not migrate actively, remain in the white matter, and differentiate into fibrous astrocytes5. How this stage-dependent specification of astrocytic subtypes occurs remains unclear.
A growing number of genes involved in neuronal migration have been identified, including those involved in neurodevelopmental and psychiatric disorders13,14. Therefore, it is crucial to elucidate the effects of mutations in these genes on the behavior of migrating neurons. As previously mentioned, neuronal migration occurs in multiple phases. Time-lapse observations can directly determine the phase that is mainly affected (cell cycle exit, multipolar-bipolar transition, migration speed of locomotion, etc.). However, the molecular mechanisms underlying the specification, migration, and positioning of astrocytes remain largely unknown. Given that astrocytes play crucial roles in synaptogenesis15 and blood-brain barrier formation during brain development16, developmental defects in astrocytes may result in neurodevelopmental disorders. Time-lapse studies on astrocyte progenitors may clarify these molecular mechanisms and their relationship with mental illness.
This protocol provides a method for time-lapse observation of cortical VZ-derived cells. A similar video protocol for the observation of neuronal migration has already been published17. Here, we describe the method for both migrating neurons and astrocyte progenitors. To label these cells with fluorescent proteins, such as green and red fluorescent proteins (GFP and RFP), plasmid mixtures containing appropriate components are introduced into the cortical VZ by in utero electroporation at appropriate stages18,19,20,21. The manipulated embryos are removed at the desired stages, and the brains are sliced and used for time-lapse observations using a laser scanning microscope. The migration speed, direction, and other behaviors, which are never addressed using fixed brain samples, can be examined using this method. Using in utero electroporation, expression, and knockdown/knockout vectors can be easily transferred concomitantly with fluorescent protein vectors, enabling us to conduct gain-of-function and loss-of-function studies of specific genes.

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and gray cells are derived from the ventricular zone and migrate to other part of the brain surface. Many genes are involved in this process, including those responsible for neurodevelopmental and psychiatric disorders. We are addressing their functions on the three behaviors in this process.Recently, we have reported that astrocyte progenitors assume two distinct migration modes:Erratic and blood-vessel guided migration. These observations were made using a combination of serotype-specific labeling and time observation methods introduced in this video. To level cells, we utilize in utero electroporation system, which we developed to visualize individual cells with a high signal to noise ratio.This in vivo gene transfer system also enable us to easily perform gain of function or loss of function experiments on the given genes by electroporation of their expression or neutron vectors. Using this experimental system, we aim to observe the cell behaviors of neurons, glial, and blood vessels, and elucidates the crosstalk between them. The findings from these studies will contribute to understanding the pathogenesis of neurodevelopmental disorders.

In Utero Electroporation for Time-Lapse Imaging of Neuronal Migration in Embryonic Mouse Brain

in-utero-electroporation-for-time-lapse-imaging-neuronal-migration

Research

JoVE Journal

Neuroscience

54 vues.  Keio University School of Medicine. Pour commencer, placez la souris anesthésiée sur une plaque à 38 degrés Celsius. Après avoir pratiqué une incision cutanée médiane, placez une feuille de plastique stérile sur la peau, suivie d’un morceau de papier de soie stérile, et créez un trou au centre. Mouillez le papier avec du PBS stérile et retirez soigneusement la corne utérine à travers les trous.À l’aide d’une micropipette fixée à un tube d’aspirateur, injectez un microlitre du mélange de plasma d...

Vidéo: Électroporation in utero pour l’imagerie en accéléré de la migration neuronale dans le cerveau embryonnaire d’une souris

time-lapse imaging of migrating neurons and glial progenitors in embryonic mouse brain slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial...

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time-lapse confocal imaging of migrating neurons in organotypic slice culture of embryonic mouse brain using in utero electroporation

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

in vivo postnatal electroporation and time-lapse imaging of neuroblast migration in mouse acute brain slices

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

an organotypic slice assay for high-resolution time-lapse imaging of neuronal migration in the postnatal brain

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory...

induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors.  This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

time-lapse imaging of neuroblast migration in acute slices of the adult mouse forebrain

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of...

ex utero electroporation and organotypic slice cultures of embryonic mouse brains for live-imaging of migrating gabaergic interneurons

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

in utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic...

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

mouse in utero electroporation: controlled spatiotemporal gene transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes.  This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

in utero intraventricular injection and electroporation of e15 mouse embryos

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

in utero electroporation approaches to study the excitability of neuronal subpopulations and single-cell connectivity

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

methods for study of neuronal morphogenesis: ex vivo rnai electroporation in embryonic murine cerebral cortex

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

genetic manipulation of the mouse developing hypothalamus through in utero electroporation

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

analyzing in vivo cell migration using cell transplantations and time-lapse imaging in zebrafish embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

in utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation.  These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

inducing cre-lox recombination in mouse cerebral cortex through in utero electroporation

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

expansion of embryonic and adult neural stem cells by in utero electroporation or viral stereotaxic injection

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

time-lapse imaging of mouse macrophage chemotaxis

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune...

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...