Questo lavoro è stato sostenuto da theNIH grantNS042626. Ringraziamo Thorn Kurt e UCSF Nikon Imaging centro per l&#39;assistenza con l&#39;imaging.

1. Preparazione di embrioni di zebrafish <ol><li> Due giorni prima elettroporazione, istituito gabbie di accoppiamento dei pesci di tipo selvatico con divisori per separare i maschi dalle femmine. </li><li> Un giorno prima elettroporazione, tirare i divisori in accoppiamento gabbie a partire dal giorno precedente. </li><li> Raccogliere embrioni e incubare 50 embrioni fecondati in 30 ml di mezzo embrionale contenente 0,003% phenylthiourea (PTU) a 28.5 ° C. </li><li> Il giorno della elettroporazione, dechorionate gli embrioni manualmente con una pinza sottile (Inox 5, Dumont elettronico, Svizzera), quando gli embrioni raggiungono lo stadio di sviluppo di 22hpf. Poi il trasferimento degli embrioni a 3 10ml elettroporazione Ringer (180 mM NaCl, KCl 5mM, 1.8 mM CaCl2, 5 mM pH Hepes 7,2) contenente 0,003% PTU. </li><li> Aggiungere 420 microlitri tricaine stock (4mg/ml) per l&#39;elettroporazione 10ml suoneria per anestetizzare gli embrioni. </li></ol> 2. Preparazione per elettroporazione <ol><li> Aghi da iniezione di vetro sono stati estratti dai capillari (1,2 mm di diametro, 0,9 mm ID, con filamento) su un Flaming-Brown P897 estrattore (Sutter Instruments, Novato, CA, USA). La punta dell&#39;ago è stata interrotta con una pinza per ottenere un fine tagliente come mostrato nella figura 1D. Il diametro della punta dovrebbe essere di circa 10 micron e l&#39;angolo di rastremazione dovrebbe essere di circa 30 gradi. </li><li> Preparare 0,8% e 1% a basso punto di fusione agarosio (Shelton Scientific, Inc. catalogo # IB70050) in soluzione di Ringer elettroporazione. Aliquota della soluzione di agarosio in tubi di ml 2 microcentrifuga. Mantenere le aliquote in un blocco di calore a ~ 37 ° C. </li><li> Il sistema Gal4/UAS è usato per guidare l&#39;espressione genica nelle cellule progenitrici neurali di embrioni elettroporate. Due plasmidi sono utilizzati: plasmide EF1α-GFF-PT2KXIG, in cui è guidato Gal4FF dal EF1α promotore onnipresente, e plasmide UAS-E1B-EGFP-PT2KXIG. </li><li> Preparare EF1α-GFF-PT2KXIG e UAS-E1B-EGFP-PT2KXIG DNA plasmidico utilizzando GenEluteTM HP plasmidi Midiprep Kit (Sigma). </li><li> I plasmidi vengono poi concentrati da sodio acetato / etanolo precipitazione alla concentrazione finale di 1-2 mg / mL in 10 mM Tris Cl ¯ (PH 8,5). </li><li> Mescolare le due plasmidi e diluire con nucleasi-free H2O ad una concentrazione finale di 500 ng / mL per ogni plasmide. L&#39;aggiunta di rosso fenolo (diluito volume totale 1 / 10) per la soluzione è preferita per la visualizzazione di consegna. Tenere la soluzione di DNA su ghiaccio. </li><li> Poco prima elettroporazione, ritardare l&#39;ago per l&#39;iniezione con 2 microlitri soluzione di DNA e regolare il volume di iniezione a 2 nl. </li></ol> 3. Elettroporazione <ol><li> Un microscopio a dissezione stero (Zeiss Stemi 2000 con un ingrandimento massimo di 50x), una pressione di iniezione (Narishige IM 300 microinjector), due micromanipolatori (WPI M3301R, strumenti di precisione mondiale, Sarasota, FL, USA), e uno stimolatore elettrico (SD9 piazza impulso stimolatore, Erba Tlefactor, West Warwick, Rhode Island, USA) sono impostati come mostrato nella Figura 1A. </li><li> Per montare gli embrioni, immergere 2 embrioni di 1% a basso punto di fusione agarosio. Poi rimuovere immediatamente gli embrioni dal agarosio con una pipetta di vetro. </li><li> Mettere gli embrioni in un coperchio di plastica rovesciata piastra Petri in singole gocce di agarosio. Orientare gli embrioni delicatamente con una sonda ad una dorsale in fibra-up prima posizione l&#39;agarosio si solidifica in modo che il prosencefalo è accessibile sia per gli elettrodi e l&#39;ago microiniezione (Figura 1B). Gli embrioni devono essere montate nei singoli agarosio gocce. </li><li> Manipolare gli elettrodi con il micromanipolatore sinistra. Inserire gli elettrodi nella agarosio come mostrato nella Figura 1B. Personalizzati platino iridio elettrodi bipolari parallelo 125 micron di diametro e distanziati 500 micron a parte sono utilizzati per l&#39;elettroporazione (FHC, catalogo PBSA0575). </li><li> Inserire l&#39;ago microiniezione nel ventricolo proencefalo e iniettare mix 4NL DNA, come fluire di liquido rosso e il gonfiore del ventricolo può essere osservato. </li><li> Immediatamente, una 28,5 V impulso della durata di 1 millisecondo è stato applicato manualmente. Poi invertire la polarità e applicare un altro 28,5 V impulso della durata di 1 millisecondo. Questo si tradurrà in etichettatura mosaico bilaterale del proencefalo cellule progenitrici neurali. </li><li> Dopo l&#39;elettroporazione di 10 embrioni, aggiungere 5 ml di media embrionale per coprire gli embrioni e la buccia di agarosio con un coltello microchirurgica per liberare gli embrioni. </li><li> Trasferire gli embrioni elettroporate per un piatto fresco di 30 ml di mezzo embrionale contenente 0,003% PTU e incubare a 28.5 ° C. </li></ol> 4. Montaggio e Time-lapse dell&#39;immagine in tempo reale <ol><li> Circa 4 ore dopo l&#39;elettroporazione, il segnale EGFP è osservabile se osservato al microscopio epifluorescente dissezione. </li><li> Cinque ore dopo l&#39;elettroporazione, selezionare gli embrioni con il mosaico, ma espressione di EGFP molto forte nella regione proencefalo. </li><li> Trasferire gli embrioni selezionati per media 10ml embrionale contenente 0,003% PTU. </li><li> Aggiungere 420 microlitri tricaine stock (4mg/ml) al embrionale 10ml medio to anestetizzare gli embrioni. </li><li> Per montare gli embrioni, immergere 1 embrione nello 0,8% di agarosio a basso punto di fusione. Poi rimuovere immediatamente l&#39;embrione dal agarosio con una pipetta di vetro e posto l&#39;embrione al centro di un piatto di 35 millimetri cultura fondo di vetro (MatTek società, parte # P35GC-1.5-10-C, <a href="http://www.glass-bottom-dishes.com/">www.glass-bottom-dishes.com</a> ) . </li><li> Orientare gli embrioni delicatamente con una sonda ad una dorsale in fibra-up prima posizione l&#39;agarosio si solidifica. </li><li> Porre la capsula correttamente sul palcoscenico temperatura controllata del microscopio confocale come mostrato in figura 1C (Usiamo una Nikon C1 microscopio confocale spettrale con in alto a destra obiettivi). Regolare la temperatura a 28,5 ° C. </li><li> Aggiungere 3 ml 28,5 ° C preriscaldato medio embrionale contenente 0,003% PTU per coprire l&#39;embrione. L&#39;embrione è ora pronto per l&#39;imaging. </li><li> Eseguire time-lapse imaging dal vivo con un intervallo fisso con un obiettivo d&#39;acqua ad immersione. </li></ol> 5. Rappresentante Risultati Mostrato nella Figura 2 sono selezionati fotogrammi di un time-lapse confocale vivo dell&#39;embrione elettroporate di EF1α-GFF + UAS-E1B-EGFP a 22 HPF. <img src="/files/ftp_upload/2594/2594fig1.jpg" alt="Figura 1" /> Figura 1. Apparatus set-up. (A) L&#39;assemblea di attrezzature elettroporazione: l&#39;erba SD9 piazza impulso stimolatore (i), la micromanipolatore sinistra (ii), gli elettrodi (iii), la dissezione microscopio Zeiss (iv), l&#39;ago di iniezione (v), il micromanipolatore destra (vi) e la Narishige IM 300 microinjector (vii). (B) La posizione relativa degli elettrodi (i), il ventricolo proencefalo (ii) e l&#39;ago per iniezione (iii). Notare il colore rosso soluzione di DNA è stato iniettato nel ventricolo proencefalo. (C) Il montaggio delle immagini in diretta su microscopio confocale Nikon C1: il regolatore di temperatura (i), l&#39;acqua immergendo obiettivo len (ii) e l&#39;embrione elettroporate incorporato in agarosio a basso punto di fusione in un piatto di cultura fondo di vetro (iii) (D ) Immagine di un ago tirato e tagliato per microiniezione di DNA. <img src="/files/ftp_upload/2594/2594fig2.jpg" alt="Figura 2" /> Figura 2. Fotogrammi selezionati di 18 ore di time-lapse confocale vivo dell&#39;embrione elettroporate con EF1α-GFF + UAS-E1B-EGFP a 22 HPF. Superficie ventricolo si trova sul lato inferiore di ogni telaio immagine. Barra di scala rappresenta 10 micron.

<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>

Nessun conflitto di interessi dichiarati.

In questo video, si dimostra un metodo per time-lapse immagini dal vivo di cellule progenitrici neurali a livello clonale nel proencefalo zebrafish via di sviluppo. Abbiamo testato e modificato i protocolli esistenti elettroporazione 4-6 a geneticamente e fluorescente etichetta singole cellule progenitrici neurali. Un albero lignaggio composto da cellule progenie clonale relativi possono essere stabiliti dal momento che solo poche cellule sono state scarsamente etichettati con un voltaggio relativamente basso...

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

Schemi precisi di divisione, migrazione e differenziazione delle cellule progenitrici neurali sono cruciali per il corretto sviluppo del cervello e 1,2 funzione. Per capire il comportamento delle cellule progenitrici neurali nel complesso in un ambiente vivo, time-lapse immagini dal vivo delle cellule progenitrici neurali in un cervello intatto è criticamente necessaria. In questo video, sfruttiamo le caratteristiche uniche di embrioni di zebrafish per visualizzare lo sviluppo delle cellule progenitrici neurali proencefalo in vivo. Noi usiamo per elettroporazione geneticamente e scarsamente etichetta singole cellule progenitrici neurali. In breve, il DNA che codifica per i costrutti marcatori fluorescenti sono stati iniettati nel ventricolo proencefalo di 22 ore dopo la fecondazione (HPF) embrioni di zebrafish e impulsi elettrici sono stati consegnati immediatamente. Sei ore più tardi, gli embrioni di zebrafish elettroporate sono stati montati con agarosio a basso punto di fusione in fondo la cultura piatti di vetro. Fluorescente cellule progenitrici neurali sono state poi ripreso per 36 ore con intervalli fissi al microscopio confocale utilizzando lenti dell&#39;obiettivo acqua immersione. L&#39;attuale metodo fornisce un modo per acquisire conoscenze nel vivo nello sviluppo delle cellule progenitrici neurali proencefalo e può essere applicato ad altre parti del sistema nervoso centrale dell&#39;embrione zebrafish.

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

Il video presente mostra un metodo che sfrutta la combinazione di elettroporazione e microscopia confocale per eseguire l&#39;imaging in diretta su singole cellule progenitrici neurali nel proencefalo zebrafish via di sviluppo.<em&gt; In vivo</em&gt; Analisi dello sviluppo delle cellule progenitrici neurali proencefalo a livello clonale può essere raggiunto in questo modo.

Time-lapse immagini dal vivo di relazione clonale cellule progenitrici neurali in via di sviluppo Zebrafish proencefalo

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 Views.  University of California San Francisco. Il video presente mostra un metodo che sfrutta la combinazione di elettroporazione e microscopia confocale per eseguire l'imaging in diretta su singole cellule progenitrici neurali nel proencefalo zebrafish via di sviluppo. In vivo Analisi dello sviluppo delle cellule progenitrici neurali proencefalo a livello clonale può essere raggiunto in questo modo.

Video: 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

Live-cellula Imaging di cellule sensoriali Precursore in Organo intatto 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 ...

Ricerca

Neuroscienze

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.

Imaging ad alta risoluzione dal vivo del comportamento delle cellule in via di sviluppo neuroepitelio

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.

Time-lapse imaging delle Migrazioni neuroblasti in fette acuta del prosencefalo topo adulto

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.

Motor Nerve transezione e Time-lapse imaging di gliali comportamenti delle cellule in vivo 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 ...

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 elettroporazione e Time-lapse imaging di migrazione neuroblasti in mouse acuta Fette cervello

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.

Valutare progressione del ciclo cellulare delle staminali e cellule progenitrici del cervello di sviluppo del mouse dopo stress genotossico

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.

Immagini dal vivo della mitosi in via di sviluppo embrionali di topo Cortex

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.

Derivazione libero-Feeder della cresta neurale progenitrici Cellule pluripotenti da cellule staminali umane

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.

Ordinamento delle cellule staminali neurali e di cellule progenitrici del topo adulto subventricolare Zone e Live-imaging loro dinamiche del ciclo cellulare

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 e quantificazione delle dinamiche del ramo dendritico veloce nello sviluppo di neuroni della Drosophila

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