Bu çalışma theNIH grantNS042626 tarafından desteklenmiştir. Biz Kurt Thorn UCSF Nikon Görüntüleme merkezi ve görüntüleme ile yardım için teşekkür ederim.

1. Zebra balığı Embriyolar hazırlanması <ol><li> Elektroporasyon İki gün önce, çiftleşme kafesleri ayrı kadınlarda erkeklere bölücüler kullanarak vahşi tip balık kurdu. </li><li> Elektroporasyon bir gün önce, önceki günden itibaren kafeslerde çiftleşme bölücüler çekin. </li><li> Embriyolar toplayın ve 28.5% 0.003 phenylthiourea (PTU) içeren 30 ml embriyonik orta ° C 50 döllenmiş embriyolar inkübe </li><li> Elektroporasyon gününde, dechorionate elle embriyoların 22hpf gelişimsel aşaması ulaşmak ince forseps (Inox 5, Dumont Elektronik, İsviçre) ile embriyoların. Sonra PTU% 0.003 içeren 10ml elektroporasyon ringer 3 (180 mM NaCl, 5mM KCl, 1.8 mM CaCl2, 5 mM HEPES pH 7.2) embriyolar transfer. </li><li> Zil sesi embriyoların uyutmak için 10ml elektroporasyon 420 ul tricaine stoku (4mg/ml) ekleyin. </li></ol> 2. Elektroporasyon için hazırlanması <ol><li> Cam enjeksiyon iğneleri Flaming-Brown P897 çektirmesi (Sutter Instruments, Novato, CA, USA) kılcal damarlar (filament 1.2 mm OD, 0.9 mm ID) çekilmiştir. Rakam 1D gösterildiği gibi iğne ucu keskin bir ucu almak için forseps ile kırıldı. Ucunun çapı yaklaşık 10 mikron olmalı ve konik açısı 30 derece civarında olmalıdır. </li><li> % 0.8 &#39;hazırlayın ve elektroporasyon ringer solüsyonu% 1 düşük erime noktası agaroz (Shelton Scientific, Inc. Katalog # IB70050). Kısım 2 ml mikrosantrifüj tüpler agaroz çözüm. ~ 37 ° C&#39;de ısı bloğu alikotları tutun </li><li> Gal4/UAS sistemi, electroporated embriyoların nöral progenitör hücrelerin gen ekspresyonu sürücü kullanılır. İki plazmid kullanılır: Gal4FF her yerde organizatörü EF1α tarafından tahrik edildiği plazmid EF1α GFF-PT2KXIG, ve plazmid UAS-E1B-EGFP-PT2KXIG. </li><li> EF1α-GFF-PT2KXIG ve UAS-E1B-EGFP-PT2KXIG plazmid DNA GenEluteTM HP Plazmid Midiprep Kiti (Sigma) kullanarak hazırlayın. </li><li> Plazmid sonra 1-2 mg / ml 10 mM Tris · Cl final konsantrasyonu (pH 8.5) sodyum asetat / etanol yağış yoğunlaşmıştır. </li><li> Iki plazmid karıştırın ve her plazmid için 500 ng / ml bir final konsantrasyon nükleaz ücretsiz H2O ile seyreltilir. Çözümü için fenol kırmızısı (1 / 10 toplam hacmi seyreltilmiş) eklenmesi teslimat görünüm için tercih edilir. DNA çözümü buz üzerinde tutun. </li><li> 2 ul DNA çözümü ile elektroporasyon, backload bir iğne sağ öncesi ve enjeksiyon hacmi 2 nl ayarlayabilirsiniz. </li></ol> 3. Elektroporasyon <ol><li> A Stero diseksiyon mikroskobu (Zeiss 50x maksimum büyütme STEMI 2000), bir hava basıncı enjektör (Narishige IM 300 mikroenjektör), iki mikromanipülatörler (TEFE M3301R, Dünya Hassas Aletler, Sarasota, FL, ABD), ve elektrik stimülatörü (SD9 Şekil 1A gösterildiği gibi kare dalga Stimülatörü, Çim Tlefactor, West Warwick, Rhode Island, ABD) kurulur. </li><li> % 1 düşük erime noktası agaroz embriyolar batırmayın 2 embriyolar monte etmek için. Sonra hemen bir cam pipet ile agaroz embriyolar kaldırmak. </li><li> Embriyolar agaroz bireysel damla ters bir plastik Petri kabı kapağının üzerine koyun. Agaroz önbeyin elektrotlar ve mikroenjeksiyon iğnesi (Şekil 1B) erişilebilir olduğunu katılaşır önce lifli bir prob ile dorsal pozisyonda yavaşça embriyolar yolunuzu. Embriyolar bireysel agaroz damla monte edilmelidir. </li><li> Sol mikromanipülatör ile elektrotlar işleyin. Agaroz içine elektrotlar Şekil 1B gösterildiği gibi yerleştirin. Özel platin iridyum 125 mm çap ve 500 mm aralıklı paralel bipolar elektrotlar dışında elektroporasyon (FHC, katalog # PBSA0575) için kullanılır. </li><li> Kırmızı sıvı akan ve ventrikül şişme görülebilir, ön beyin ventrikül içine mikroenjeksiyon iğnesi takın ve 4NL DNA karışımı enjekte edilir. </li><li> Hemen bir 28.5 V darbe ömürlü 1 milisaniyelik elle uygulandı. Sonra ters polarite ve birden fazla 28.5 V darbe kalıcı 1 milisaniye geçerlidir. Bu ikili mozaik etiketleme önbeyin nöral progenitör hücrelerin neden olacaktır. </li><li> 10 embriyoların elektroporasyon sonra, embriyolar serbest bırakmak için bir mikrocerrahi bıçakla embriyolar kapak ve agaroz soyma 5ml embriyonik orta ekleyin. </li><li> 28,5 az% 0.003 PTU ve inkübe içeren 30 ml embriyonik orta taze bir çanağı ° C electroporated embriyolar transfer </li></ol> 4. Montaj ve zaman atlamalı Canlı Görüntüleme <ol><li> Elektroporasyon sonra yaklaşık 4 saat, eğer bir epifluorescent diseksiyon mikroskobu altında incelendi EGFP sinyal gözlemlenebilir. </li><li> Elektroporasyon beş saat sonra, son derece mozaik değil, ön beyin bölgede güçlü EGFP ifade ile embriyolar seçin. </li><li> % 0.003 PTU içeren 10ml embriyonik orta seçilen embriyolar transfer. </li><li> 10ml embriyonik orta t 420 ul tricaine stoku (4mg/ml)o embriyoların uyutmak. </li><li> Embriyoların monte etmek için,% 0.8 düşük erime noktası agaroz 1 embriyo batırmayın. Sonra hemen bir cam pipet ile agaroz embriyo kaldırmak ve 35mm cam alt kültür çanak merkezi (Mattek şirketi, Part # P35GC-1.5-10-C, embriyo <a href="http://www.glass-bottom-dishes.com/">www.glass-alt-dishes.com</a> ) . </li><li> Agaroz katılaşır önce dorsal-yukarı konumda lifli bir prob hafifçe embriyolar yolunuzu. </li><li> Konfokal mikroskop sıcaklık kontrollü sahnede çanak düzgün şekil 1C (sağ-üst hedefleri ile Nikon C1 spektral konfokal mikroskop kullanımı) gösterildiği gibi yerleştirin. 28.5 için sıcaklığını ayarlamak ° C </li><li> Ekle 3ml 28.5 ° C embriyo kapsayacak şekilde PTU% 0.003 içeren embriyonik önceden ısıtılmış orta. Embriyo artık görüntüleme için hazırdır. </li><li> Su daldırma amacı kullanarak sabit bir aralığı ile zaman atlamalı canlı görüntüleme gerçekleştirin. </li></ol> 5. Temsilcisi Sonuçlar Şekil 2&#39;de gösterildiği gibi 22 hpf EF1α-GFF + UAS-E1B-EGFP electroporated embriyonun bir zaman atlamalı konfokal canlı görüntüleme kare seçilir. <img src="/files/ftp_upload/2594/2594fig1.jpg" alt="Şekil 1" /> Şekil 1. Aparatı set-up. (A) elektroporasyon ekipmanların montaj: Çim SD9 kare dalga Stimülatörü (i), (ii) sol mikromanipülatör, elektrotlar (iii), (iv), (v) bir iğne, sağ mikromanipülatör Zeiss diseksiyon mikroskobu (vi) ve Narishige IM 300 mikroenjektör (vii). (B) elektrotlar (i), ön beyin ventrikül (ii) ve enjeksiyon iğnesi (iii) göreli konumunu. Kırmızı renkli DNA çözüm, ön beyin ventrikül içine enjekte edilmiştir. (C) Nikon C1 konfokal mikroskop canlı görüntüleme montaj: sıcaklık kontrolörü (i) su objektif len (ii) ve cam bir alt kültür çanak (iii) (D düşük erime noktası agaroz gömülü electroporated embriyo daldırma DNA mikroenjeksiyon için çekti ve kesilmiş iğne). <img src="/files/ftp_upload/2594/2594fig2.jpg" alt="Şekil 2" /> Şekil 2 22 hpf EF1α-GFF + UAS-E1B-EGFP electroporated embriyonun 18 saatlik zaman atlamalı konfokal canlı görüntüleme Seçilmiş kareleri. Ventrikül yüzeyi her görüntü çerçevesinin alt tarafta. Ölçek çubuğu 10 mm temsil eder.

<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>

Bu video, zaman atlamalı, gelişmekte olan zebrafish önbeyin bir klonal düzeyde nöral progenitör hücrelerin canlı görüntüleme için bir yöntem ortaya koymaktadır. Biz genetik ve floresan etiket bireysel nöral progenitör hücrelerin test edilmiş ve mevcut elektroporasyon protokolleri 4-6 değiştirilmiş. Klonal olarak ilişkilidir döl hücrelerinin oluşan bir soy ağacı sadece birkaç hücre seyrek elektroporasyon göreceli düşük voltaj ile etiketlenmiş beri kurulabilir. EGFP ek olarak, ...

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

Bölünme, göç ve nöral progenitör hücrelerin farklılaşma hassas kalıpları, uygun bir beyin gelişimi ve işlevi 1,2 için çok önemlidir. In vivo ortamda karmaşık nöral progenitör hücrelerin davranışını anlamak için, sağlam bir beyin nöral progenitör hücrelerin zaman atlamalı canlı görüntüleme eleştirel gereklidir . Bu video, in vivo önbeyin nöral progenitör hücrelerin gelişimini görselleştirmek için zebrafish embriyoların benzersiz özellikleri istifade eder. Biz genetik ve seyrek etiket bireysel nöral progenitör hücrelerin elektroporasyon kullanın. Kısacası, DNA floresan belirteçler için kodlama yapıları, 22 saat sonra döllenme (hpf) zebrafish embriyolar ve elektrik bakliyat hemen teslim edildi önbeyin ventrikül içine enjekte edildi. Altı saat sonra, electroporated zebrafish embriyoların cam alt kültür yemekleri, düşük erime noktası agaroz ile monte edilmiştir. Floresan ile işaretlenmiş nöral progenitör hücreler daha sonra suya daldırma objektif lens kullanarak konfokal mikroskop altında sabit aralıklarla 36hours için görüntülenmiştir. Bu yöntemi, ön beyin nöral progenitör hücrelerin in vivo gelişiminde içgörüler kazanmak için bir yol sağlar ve Zebra balığı embriyonun merkezi sinir sisteminin diğer bölgelerinde uygulanabilir .

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

Mevcut video, gelişmekte olan zebrafish önbeyin bireysel nöral progenitör hücrelerin canlı görüntüleme gerçekleştirmek için elektroporasyon ve konfokal mikroskopi kombinasyonu yararlanır bir yöntem gösteriyor.<em&gt; In vivo</em&gt; Önbeyin klonal bir seviyede nöral progenitör hücrelerin gelişimini analiz bu şekilde elde edilebilir.

Gelişmekte olan Zebra balığı önbeyin klonal olarak ilişkilidir Sinir Progenitör Hücreler Zaman atlamalı Canlı Görüntüleme

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

10.2K Görüntüleme.  University of California San Francisco. Mevcut video, gelişmekte olan zebrafish önbeyin bireysel nöral progenitör hücrelerin canlı görüntüleme gerçekleştirmek için elektroporasyon ve konfokal mikroskopi kombinasyonu yararlanır bir yöntem gösteriyor. In vivo Önbeyin klonal bir seviyede nöral progenitör hücrelerin gelişimini analiz bu şekilde elde edilebilir.

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

Bozulmamış Duyu Organ Öncü Hücreler Canlı hücre Görüntüleme Drosophila Pupa

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 ...

Nörobilim

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.

Gelişmekte Neuroepithelium Hücre Davranış Yüksek çözünürlüklü Canlı Görüntüleme

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.

Yetişkin Fare önbeyin akut dilimleri içinde Neuroblast Göç Time-lapse Görüntüleme

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

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

Larva Zebra balığı olarak önbeyin Elektrofizyolojik Kaydı

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

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 Sinir transeksiyonu ve Canlı Zebra balığı Glial Hücre Davranışlarının Time-lapse Görüntüleme

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.

Fare Akut Beyin Dilimleri Neuroblast Göç vivo Doğum Sonrası Elektroporasyonla ve Time-lapse Görüntüleme

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.

Genotoksik Stres sonrası nöral kök ve Fare Gelişmekte Beyin Progenitör Hücrelerinin Değerlendirilmesi Hücre Döngüsü İlerlemesi

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.

Gelişmekte Fare Embriyonik Cortex mitoz Canlı Görüntüleme

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.

İnsan Pluripotent dan Sinir Crest Progenitör Hücreler besleyici ücretsiz Derivasyon Kök Hücre

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.

Hücre Yetişkin Fare subventricular Bölgesinden Nöral Kök ve Progenitör Hücreler Sıralama ve Hücre Döngüsü Dynamics Canlı görüntüleme

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