Ce travail a été soutenu par theNIH grantNS042626. Nous remercions Thorn Kurt et UCSF Nikon Imaging Centre d&#39;aide à l&#39;imagerie.

1. Préparation des embryons de poissons zèbres <ol><li> Deux jours avant l&#39;électroporation, mis en place des cages d&#39;accouplement des poissons de type sauvage en utilisant des diviseurs d&#39;hommes séparés des femelles. </li><li> Un jour avant l&#39;électroporation, tirez les diviseurs de l&#39;accouplement des cages de la journée précédente. </li><li> Recueillir des embryons et incuber 50 embryons fécondés in 30 ml de milieu contenant embryonnaire phénylthiourée 0,003% (PTU) à 28,5 ° C. </li><li> Le jour de l&#39;électroporation, les embryons dechorionate manuellement avec une pince fine (Inox 5, Dumont électronique, Suisse) où les embryons atteignent le stade de développement 22hpf. Puis transfert des embryons à 3 10ml électroporation Ringer (180 mM de NaCl, KCl 5 mM, 1,8 mM de CaCl2, 5 mM pH Hepes 7,2) contenant 0,003% PTU. </li><li> Ajouter 420 ul tricaïne stock (4mg/ml) à l&#39;électroporation 10ml Ringer pour anesthésier les embryons. </li></ol> 2. Préparation pour l&#39;électroporation <ol><li> Aiguilles d&#39;injection de verre ont été retirés de capillaires (1,2 mm de diamètre extérieur, 0,9 mm ID, avec filament) sur un extracteur Flaming-Brown P897 (Sutter Instruments, Novato, CA, USA). La pointe de l&#39;aiguille a été rompu avec une pince pour obtenir une extrémité pointue, comme indiqué dans la figure 1D. Le diamètre de la pointe devrait être d&#39;environ 10 pm et l&#39;angle du cône doit être autour de 30 degrés. </li><li> Préparer 0,8% et 1% à bas point de fusion d&#39;agarose (Shelton Scientific, Inc catalogue # IB70050) dans une solution de Ringer électroporation. Aliquoter la solution d&#39;agarose à 2 ml microtubes. Gardez les aliquotes dans un bloc thermique à ~ 37 ° C. </li><li> Le système Gal4/UAS est utilisé pour piloter l&#39;expression des gènes dans les cellules progénitrices neurales des embryons électroporé. Deux plasmides sont utilisés: le plasmide EF1α-GFF-PT2KXIG, dans lequel Gal4FF est entraîné par le promoteur ubiquitaire EF1α, et le plasmide SAMU-E1B-EGFP-PT2KXIG. </li><li> Préparez-EF1α GFF-PT2KXIG et UAS-E1B-EGFP-PT2KXIG ADN plasmidique en utilisant GenEluteTM HP plasmidique Midiprep Kit (Sigma). </li><li> Les plasmides sont ensuite concentrées par acétate de sodium / éthanol précipitations à la concentration finale de 1-2 pg / pl dans 10 mM Tris · Cl (pH 8,5). </li><li> Mélanger les deux plasmides et diluer avec sans nucléase H2O à une concentration finale de 500 ng / ul pour chaque plasmide. Ajout de rouge de phénol (dilué 1 / 10 volume total) à la solution est préférée pour la visualisation de la livraison. Conserver la solution d&#39;ADN sur la glace. </li><li> Juste avant l&#39;électroporation, backloading l&#39;aiguille d&#39;injection avec 2 ul solution d&#39;ADN et d&#39;ajuster le volume d&#39;injection à 2 nl. </li></ol> 3. L&#39;électroporation <ol><li> Un microscope de dissection stero (Zeiss Stemi 2000 avec un grossissement maximal de 50x), une pression d&#39;injection d&#39;air (Narishige IM 300 microinjecteur), deux micromanipulateurs (WPI M3301R, Instruments de précision mondiale, Sarasota, Floride, USA), et un stimulateur électrique (SD9 carrés d&#39;impulsion stimulateur, Herbe Tlefactor, West Warwick, Rhode Island, USA) sont mis en place comme dans la Figure 1A. </li><li> Pour monter les embryons, immerger deux embryons dans 1% à bas point de fusion d&#39;agarose. Puis retirer immédiatement les embryons de l&#39;agarose avec une pipette en verre. </li><li> Placer les embryons sur un couvercle de plastique inversé plat de Petri en gouttes individuelles d&#39;agarose. Orienter les embryons doucement avec une sonde à fibre à une position dorsale-up avant l&#39;agarose se solidifie pour que le prosencéphale est accessible à la fois les électrodes et l&#39;aiguille de microinjection (figure 1B). Les embryons doivent être montés dans chaque agarose gouttes. </li><li> Manipuler les électrodes avec le micromanipulateur gauche. Insérer les électrodes dans l&#39;agarose comme indiqué dans la figure 1B. Personnalisé en platine iridié électrodes bipolaires en parallèle 125 mm de diamètre et espacés de 500 um d&#39;intervalle sont utilisés pour l&#39;électroporation (FHC, catalogue # PBSA0575). </li><li> Insérez l&#39;aiguille de microinjection dans le ventricule du cerveau antérieur et injecter mélange d&#39;ADN 4NL, comme découlant de liquide rouge et le gonflement du ventricule peuvent être observés. </li><li> Immédiatement, une impulsion de 28,5 V à 1 milliseconde durables a été appliquée manuellement. Ensuite, on inverse la polarité et d&#39;appliquer une plus 28,5 V impulsion de durée de 1 milliseconde. Cela se traduira par l&#39;étiquetage de mosaïque de cellules du cerveau antérieur bilatérale du progénitrices neurales. </li><li> Après électroporation de 10 embryons, ajouter 5ml moyenne embryonnaires pour couvrir les embryons et le zeste de l&#39;agarose avec un couteau de microchirurgie pour libérer les embryons. </li><li> Transfert des embryons électroporées à un plat frais de 30 ml de milieu embryonnaire contenant 0,003% et PTU incuber à 28,5 ° C. </li></ol> 4. Montage et imagerie time-lapse en direct <ol><li> Environ 4 heures après l&#39;électroporation, le signal de l&#39;EGFP est observable si vu sous un microscope à épifluorescence dissection. </li><li> Cinq heures après l&#39;électroporation, sélectionner les embryons à la mosaïque, mais très forte expression de l&#39;EGFP dans la région du cerveau antérieur. </li><li> Transfert des embryons sélectionnés pour 10ml embryonnaire milieu contenant PTU 0,003%. </li><li> Ajouter 420 ul tricaïne stock (4mg/ml) à l&#39;embryon t 10ml moyenneso anesthésier les embryons. </li><li> Pour monter les embryons, immerger une embryon dans 0,8% d&#39;agarose à faible point de fusion. Puis retirer immédiatement l&#39;embryon de l&#39;agarose avec une pipette en verre et le lieu de l&#39;embryon au centre d&#39;une boîte de culture à fond de verre 35mm (MatTek Corporation, partie # P35GC-1.5-10-C, <a href="http://www.glass-bottom-dishes.com/">www.glass-bas dishes.com</a> ) . </li><li> Orienter les embryons doucement avec une sonde à fibre à une position dorsale-up avant l&#39;agarose se solidifie. </li><li> Placer le plat bien sur la scène à température contrôlée du microscope confocal, comme indiqué dans la figure 1C (Nous utilisons un microscope Nikon C1 spectrale confocale avec haut-droite objectifs). Réglez la température à 28,5 ° C. </li><li> Ajouter 3ml 28,5 ° C préchauffé moyenne embryonnaire contenant 0,003% PTU pour couvrir l&#39;embryon. L&#39;embryon est maintenant prêt pour l&#39;imagerie. </li><li> Effectuer imagerie time-lapse vivre avec un intervalle fixe en utilisant un objectif de l&#39;eau de trempage. </li></ol> 5. Les résultats représentatifs Montre la figure 2 sont sélectionnés les cadres d&#39;une imagerie time-lapse direct confocale de l&#39;embryon électroporées des EF1α-GFF + SAMU-E1B-EGFP à 22 HPF. <img src="/files/ftp_upload/2594/2594fig1.jpg" alt="Figure 1" /> Figure 1. Appareillage set-up. (A) L&#39;assemblée des équipements d&#39;électroporation: l&#39;herbe SD9 carrée pouls stimulateur (i), le micromanipulateur gauche (ii), les électrodes (iii), le Zeiss loupe binoculaire (iv), l&#39;aiguille d&#39;injection (v), le droit de micromanipulateur (vi) et l&#39;IM 300 Narishige microinjecteur (vii). (B) La position relative des électrodes (i), le ventricule du cerveau antérieur (ii) et l&#39;aiguille d&#39;injection (iii). Notez la solution d&#39;ADN de couleur rouge a été injecté dans le ventricule du cerveau antérieur. (C) L&#39;Assemblée de l&#39;imagerie en direct sur Nikon microscope confocal C1: le régulateur de température (i), l&#39;eau plongeant objectif len (ii) et l&#39;embryon électroporées intégré dans agarose bas point de fusion dans une boîte de culture à fond de verre (iii) (D ) Image tirée d&#39;une aiguille et de coupe pour micro-injection d&#39;ADN. <img src="/files/ftp_upload/2594/2594fig2.jpg" alt="Figure 2" /> Figure 2. Cadres sélectionnés d&#39;un 18-heures imagerie time-lapse direct confocale de l&#39;embryon électroporation avec EF1α-GFF + SAMU-E1B-EGFP à 22 HPF. La surface du ventricule est sur le côté bas de chaque trame d&#39;image. La barre d&#39;échelle représente 10 um.

<ol>
	<li>Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+neural+stem+cells.">The development of neural stem cells.</a> Nature. 414, 112-117 (2001).</li><li>G#246;tz, M., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+neurogenesis.">The cell biology of neurogenesis.</a> Nat. Rev. Mol. Cell Biol. 6, 777-788 (2005).</li><li>Cerda, G. A., Thomas, J. E., Allende, M. L., Karlstrom, R. O., Palma, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+DNA,+RNA,+and+morpholinos+into+zebrafish+embryos.">Electroporation of DNA, RNA, and morpholinos into zebrafish embryos.</a> Methods. 39, 207-211 (2006).</li><li>Hendricks, M., Jesuthasan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-based+methods+for+in+vivo,+whole+mount+and+primary+culture+analysis+of+zebrafish+brain+development.">Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development.</a> Neural. Dev. 2, 6-6 (2007).</li><li>Tawk, M., Bianco, I. H., Clarke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+electroporation+in+zebrafish+embryos+and+larvae.">Focal electroporation in zebrafish embryos and larvae.</a> Methods Mol. Biol. 546, 145-151 (2009).</li><li>Tawk, M., Tuil, D., Torrente, Y., Vriz, S., Paulin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+gene+transfer+into+adult+fish:+a+new+tool+to+study+fin+regeneration.">High-efficiency gene transfer into adult fish: a new tool to study fin regeneration.</a> Genesis. 32, 27-31 (2002).</li></ol>

Dans cette vidéo, nous démontrons une méthode pour imagerie time-lapse en direct de cellules progénitrices neurales au niveau clonal dans le prosencéphale poisson zèbre en développement. Nous avons testé et modifié les protocoles existants électroporation 4-6 pour génétiquement et par fluorescence étiquette individuelle des cellules progénitrices neurales. Un arbre lignée composée de cellules par clonage progéniture connexes peuvent être mis en place depuis seulement quelques cellules ont é...

time-lapse live imaging of clonally related neural progenitor cells in the developing zebrafish forebrain

Schémas précis de la division, la migration et la différenciation des cellules progénitrices neurales sont cruciales pour le développement du cerveau et 1,2 fonction. Pour comprendre le comportement des cellules progénitrices neurales dans le complexe dans un environnement in vivo, imagerie time-lapse en direct de cellules progénitrices neurales dans un cerveau intact est absolument nécessaire. Dans cette vidéo, nous exploitons les caractéristiques uniques des embryons de poisson zèbre pour visualiser le développement des cellules progénitrices neurales du cerveau antérieur in vivo. Nous utilisons d&#39;électroporation génétiquement et peu d&#39;étiquettes individuelles cellules progénitrices neurales. En bref, des constructions d&#39;ADN codant pour des marqueurs fluorescents ont été injectées dans le ventricule du cerveau antérieur de 22 heures après fécondation (HPF) et les embryons de poissons zèbres impulsions électriques ont été livrés immédiatement. Six heures plus tard, les embryons de poissons zèbres électroporées ont été montés avec bas point de fusion de verre dans des plats d&#39;agarose culture sur le fond. Marqués par fluorescence des cellules progénitrices neurales ont ensuite été imagés pour les 36 heures avec des intervalles fixes sous un microscope confocal à l&#39;aide d&#39;eau plongeant objectif. La méthode actuelle fournit un moyen d&#39;avoir un aperçu dans le développement in vivo des cellules progénitrices neurales du cerveau antérieur et peut être appliquée à d&#39;autres parties du système nerveux central de l&#39;embryon de poisson zèbre.

Watch this Scientific Journal Video about Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain at JoVE.com

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

La vidéo montre le présent une méthode qui prend avantage de la combinaison de l&#39;électroporation et la microscopie confocale pour l&#39;imagerie en direct sur chaque cellules progénitrices neurales dans le cerveau antérieur poisson zèbre en développement.<em&gt; In vivo</em&gt; L&#39;analyse du développement des cellules progénitrices neurales du cerveau antérieur à un niveau clonal peut être réalisé de cette façon.

Imagerie time-lapse en direct de relation clonale cellules progénitrices neurales dans le cerveau antérieur développement Zebrafish

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

time-lapse-live-imaging-clonally-related-neural-progenitor-cells

Research

JoVE Journal

Neuroscience

10.1K vues.  University of California San Francisco. La vidéo montre le présent une méthode qui prend avantage de la combinaison de l'électroporation et la microscopie confocale pour l'imagerie en direct sur chaque cellules progénitrices neurales dans le cerveau antérieur poisson zèbre en développement. In vivo L'analyse du développement des cellules progénitrices neurales du cerveau antérieur à un niveau clonal peut être réalisé de cette façon.

Vidéo: Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Imagerie des cellules vivantes des cellules sensorielles de précurseurs d&#39;organes en Intact Drosophile Les chrysalides

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Recherche

Neurosciences

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Imagerie à haute résolution en direct du comportement des cellules dans le développement neuro-épithélium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

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

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

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 migration.

Imagerie time-lapse de la migration des neuroblastes en tranches aiguës du cerveau antérieur souris adultes

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

Moteur section du nerf et imagerie time-lapse de comportements de cellules gliales chez le poisson zèbre en direct

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

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

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

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 tracking.

In vivo postnatale électroporation et imagerie time-lapse de neuroblaste migrations dans Souris aiguë tranches du cerveau

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

Évaluation de la progression du cycle cellulaire des cellules souches neurales et progénitrices dans le développement du cerveau de souris après un stress génotoxique

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imagerie en direct de la mitose dans les développement Cortex souris embryonnaires

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Dérivation sans chargeur-de cellules progénitrices de la crête neurale souches pluripotentes humaines cellules

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Tri cellulaire des neurones cellules souches et progénitrices de la souris adulte-ventriculaire Zone et Live-imagerie de la dynamique de leur cycle cellulaire

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons Time-

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...