이 작품은 theNIH grantNS042626에 의해 지원되었다. 우리는 커트 손 및 이미징과 함께 도움을 UCSF 니콘 이미징 센터를 주셔서 감사합니다.

1. Zebrafish 태아의 준비 <ol><li> 이틀 electroporation 전에, 여성에서 남성으로 별도의 디바이더를 사용하여 야생 유형 물고기의 교배 새장을 설정합니다. </li><li> electroporation 전 어느 날, 전날의 새장을 짝짓기에 디바이더를 가져옵니다. </li><li> 배아를 수집하고 28.5에서 0.003 %의 phenylthiourea (PTU)를 포함하는 30 ML 배아 중간 ° C. 50 수정된 배아를 품어 </li><li> electroporation의 날, dechorionate 수동으로 배아는 22hpf의 발달 단계에 도달 벌금 포셉 (Inox 5 더몬트 전자, 스위스)와 배아. 다음 PTU 0.003 %를 포함 10ml electroporation 대타의 3 (180 MM NaCl, 5mM KCl, 1.8 MM CaCl2, 5 MM Hepes의 산도 7.2)로 배아를 전송합니다. </li><li> 링어이 배아를 마취 위하여 10ml의 electroporation을 420 μl tricaine 주식 (4mg/ml)를 추가합니다. </li></ol> 2. Electroporation 준비 <ol><li> 유리 사출 니들은 플라밍 - 브라운 P897 풀러 (셔터 인 스트 루먼트, 노바토, CA, 미국)에서 모세 혈관 (필라멘트와 1.2 mm OD, 0.9 mm ID가)에서 가져온되었습니다. 바늘 끝이 그림 1D와 같이 날카로운 끝을 얻으려면 포셉와 부러했다. 팁의 직경 10 μm의 주위해야하며 테이퍼 각도는 30도 근처에있을 것입니다. </li><li> 0.8 %을 준비하고 electroporation 대​​타의 솔루션에 1퍼센트 낮은 용융 점 아가로 오스 (셸턴 과학 주식 회사 카탈로그 # IB70050). 2 ML의 microcentrifuge 튜브에서 나누어지는 아가로 오스 솔루션입니다. ~ 37 ° C.에서 열 블록에 aliquots 보관 </li><li> Gal4/UAS 시스템 electroporated 태아의 신경 전구 세포의 유전자 발현을 유발하는 데 사용됩니다. 두 plasmids이 사용됩니다 : Gal4FF는 유비 쿼터스 모터의 EF1α에 의해 구동되는 플라스미드 EF1α - GFF - PT2KXIG, 그리고 플라스미드 UAS - E1B - EGFP - PT2KXIG합니다. </li><li> EF1α - GFF - PT2KXIG 및 GenEluteTM HP 플라스미드 Midiprep 키트 (시그마)를 사용하여 UAS - E1B - EGFP - PT2KXIG 플라스미드 DNA를 준비합니다. </li><li> plasmids은 다음 10 MM 트리스 · Club 호텔에서 1-2 μg / μl의 최종 농도 (PH 8.5)에 아세트산 나트륨 / 에탄올 침전에 의해 집중되고있다. </li><li> 두 plasmids를 섞어 각 플라스미드 500 NG / μl의 최종 농도 nuclease 무료 H2O로 희석. 솔루션에 페놀 레드 (10분의 1 총 볼륨을 희석)의 추가는 전달의 시각화를 선호합니다. 얼음의 DNA 솔루션을 유지. </li><li> 오른쪽 2 μl DNA 솔루션과 electroporation, backload 주사 바늘 이전 2 선배님은 사출 볼륨을 조정합니다. </li></ol> 3. Electroporation <ol><li> stero의 해부 현미경 (50x 최대 배율로 자이스 혈구 Stemi 2000), 공기 압력 주입기 (Narishige IM 300 microinjector) 두 micromanipulators (WPI M3301R, 세계 정밀 계측기, 사라소타, 플로리다, 미국), 그리고 전기 자극기 (SD9 그림 1A와 같이 사각형 펄스 자극기, 그라스 Tlefactor, 웨스트 워릭,로드 아일랜드, 미국)이 설정됩니다. </li><li> 1퍼센트 낮은 용융 점 아가로 오스의 배아, 잠그다이 배아를 마운트하려면 다음과 같이하십시오. 그런 다음 즉시 유리 피펫으로 아가로 오스의 배아를 제거합니다. </li><li> 아가로 오스의 개별 방울에 거꾸로 플라스틱 페트리 접시의 뚜껑에있는 배아를 놓습니다. 아가로 오스 너무 forebrain은 전극과 microinjection 니들 (그림 1B) 모두에 액세스할 수 굳은 전에 등의 접속 위치로 광섬유 프로브와 함께 부드럽게 배아를 동쪽을 향하게하다. 배아는 개인 아가로 오스 방울에 마운트해야합니다. </li><li> 왼쪽 micromanipulator로 전극을 조작. 그림 1B와 같이 아가로 오스에 전극을 삽입합니다. 사용자 플래티넘 이리듐 병렬 바이폴라 전극 직경 및 간격 500 μm의 125 μm의 간격은 electroporation (FHC, 카탈로그 # PBSA0575)에 사용됩니다. </li><li> 붉은 유체의 흐르는 및 뇌실의 팽창 관찰 될 수 있듯이, forebrain의 뇌실에 microinjection 바늘을 삽입하고 4nl DNA 믹스를 주입. </li><li> 바로 하나의 28.5 V 펄스 지속적인 1 밀리초는 수동으로 적용되었다. 다음 극성을 반대로 하나 이상 28.5 V 펄스 지속 1 밀리초를 적용합니다. 이것은 forebrain 신경 전구 세포의 양자 모자이크 라벨집니다. </li><li> 10 배아의 electroporation 후 배아를 공개 microsurgical 나이프로 배아를 커버하고 아가로 오스 껍질에 5ml 배아 매체를 추가하십시오. </li><li> 28.5에서 0.003 % PTU와 부화를 포함한 30 ML 배아 매체의 신선한 요리 ° C.에 electroporated 배아를 전송 </li></ol> 4. 장착 및 시간 경과 라이브 영상 <ol><li> epifluorescent 해부 현미경으로 볼 경우에는 electroporation 후 4시간 주변, EGFP 신호가 관찰됩니다. </li><li> 다섯 시간 electroporation 후 높은 모자이크하지만 forebrain 지역에서 강한 EGFP 표현으로 배아를 선택합니다. </li><li> 0.003 %의 PTU를 포함 10ml 배아 매체로 선택한 배아를 전송합니다. </li><li> 10ml 배아 매체 t에 420 μl tricaine 주식 (4mg/ml)를 추가O는 배아를 마취. </li><li> 배아를 탑재하기 위해 0.8 % 낮은 용융 점 아가로 오스 1 배아 젖어. 그런 다음 즉시 유리 피펫으로 아가로 오스의 배아를 제거하고 35mm 유리 바닥 문화 요리의 중심지 (MatTek 법인, 파트 # P35GC - 1.5-10 - C에 배아를 장소 <a href="http://www.glass-bottom-dishes.com/">www.glass - 하단 dishes.com</a> ) . </li><li> 아가로 오스는 굳은 전에 등의 접속 위치로 광섬유 프로브와 함께 부드럽게 배아를 동쪽을 향하게하다. </li><li> 그림 1C (우리가 오른쪽 목표와 니콘 C1 스펙트럼 공촛점 현미경을 사용하여)에 같이 공촛점 현미경의 온도 조절 무대에서 제대로 요리를 놓습니다. 28.5로 온도를 조절 ° C. </li><li> 3ml 28.5 추가 ° C 배아를 커버하는 PTU 0.003 %를 포함하는 preheated 배아 중간. 배아는 이제 이미지 준비가되었습니다. </li><li> 물이 마른 목표를 사용하여 고정 간격으로 시간이 경과 라이브 영상을 수행합니다. </li></ol> 5. 대표 결과 그림 2에 표시된 22 HPF에서 EF1α - GFF + UAS - E1B - EGFP의 electroporated 배의 시간이 경과 공촛점 라이브 영상의 프레임을 선택하고 있습니다. <img src="/files/ftp_upload/2594/2594fig1.jpg" alt="그림 1" /> 그림 1. 장치 셋업. (A) electroporation 설비의 조립 : 그라스 SD9 광장 펄스 자극기 (I), 왼쪽 micromanipulator (2), 전극 (3), 자이스 혈구가 현미경을 해부 (IV), 사출 바늘 (V)가, 오른쪽 micromanipulator (VI)과 Narishige IM 300 microinjector (VII). (B) 전극은 (i), forebrain 뇌실 (II) 및 사출 바늘 (3)의 상대 위치. 붉은 색의 DNA 솔루션 forebrain의 뇌실로 주입되어 있습니다. (C) 니콘 C1 공촛점 현미경에 살고 이미지의 조립 : 온도 컨트롤러 (I), 객관적인 렌 (2)와 유리 바닥 문화 요리 (3) (D에서 낮은 용해 지점 아가로 오스에 포함된 electroporated 배아 디핑 물 의 DNA microinjection에 뽑아 및 절단 바늘의) 이미지. <img src="/files/ftp_upload/2594/2594fig2.jpg" alt="그림 2" /> 그림 2. 22 HPF에서 EF1α - GFF + UAS - E1B - EGFP와 electroporated 배아의 18 시간 시간이 경과 공촛점 라이브 이미지의 선택된 프레임. 뇌실의 표면은 각각의 이미지 프레임의 하단 측면에 있습니다. 스케일 바는 10 μm의를 나타냅니다.

<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>

관심 없음 충돌 선언하지 않습니다.

이 비디오에서는, 우리는 개발 zebrafish의 forebrain에서 clonal 수준에서 신경 전구 세포의 시간 경과 라이브 영상에 대한 방법을 보여줍니다. 우리는 유전자와 찬란 라벨 개별 신경 전구 세포에 테스트 기존의 electroporation 프로토콜에게 4-6을 수정했습니다. 단 몇 세포가 띄엄띄엄 electroporation의 상대적인 낮은 전압으로 표시 이후 clonally 관련 자손 세포로 구성된 혈통의 나무가 설정할 수 있습?...

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

분할, 마이 그 레이션 및 신경 전구 세포의 분화의 정확한 패턴을 적절한 두뇌 개발과 기능 1,2에 중요한 있습니다. 생체내 환경에서 복잡한에서 신경 전구 세포의 동작을 이해하기 위해서는 두뇌에 손상 신경 전구 세포의 시간 경과 라이브 영상이 매우 필요합니다. 이 비디오에서는, 우리는 생체내에 forebrain 신경 전구 세포의 발전을 시각화하는 zebrafish 배아의 고유한 기능을 이용. 우리는 유전자 및 띄엄띄엄 라벨 개별 신경 전구 세포에 electroporation을 사용합니다. 간단히, DNA가 형광 표식에 대한 코딩 구조 22 시간 게시물 수정 (HPF) zebrafish 배아의 전기 펄스가 즉시 배달되었고,의 forebrain의 뇌실로 주입했다. 여섯 시간 후, electroporated zebrafish의 배아는 유리 바닥 문화 요리 낮은 용융 점 아가로 오스와 함께 장착했다. 찬란 표시 신경 전구 세포는 다음 물 수영 객관적인 렌즈를 사용하여 공촛점 현미경 고정 간격 36시간 위해 몇 군데 있었다. 현재의 방법은 forebrain 신경 전구 세포의 생체내 개발에 대한 통찰력을 얻을 수있는 방법을 제공하고 zebrafish 배아의 중추 신경 시스템의 다른 부분에 적용할 수 있습니다.

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

현재 비디오는 개발 zebrafish의 forebrain에서 개별 신경 전구 세포에 대한 라이브 영상을 수행할 수 electroporation과 공촛점 현미경의 조합을 활용하는 방법을 보여줍니다.<em&gt; 생체내에서</emclonal 수준 forebrain 신경 전구 세포의 개발&gt; 분석이 방법으로 얻을 수 있습니다.

개발 Zebrafish Forebrain에 Clonally 관련 신경 전구 세포의 시간 경과 라이브 영상

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 조회수.  University of California San Francisco. 현재 비디오는 개발 zebrafish의 forebrain에서 개별 신경 전구 세포에 대한 라이브 영상을 수행할 수 electroporation과 공촛점 현미경의 조합을 활용하는 방법을 보여줍니다. 생체내에서 분석이 방법으로 얻을 수 있습니다.

비디오: 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

손상의 감각 오르간 전구체 세포 라이브 셀 이미징 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 ...

연구

신경과학

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.

개발 Neuroepithelium의 세포 문제의 고해상도 라이브 영상

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.

성인 마우스 Forebrain의 급성 슬라이스에 Neuroblast 마이그레이션의 시간 경과 영상

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.

애벌레의 Zebrafish의 Forebrain Electrophysiological 기록

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.

모터 신경 절개와 라이브 제브라 피쉬의 아교 세포 행동의 시간 경과 영상

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.

마우스 급성 뇌 조각의 Neuroblast 마이그레이션의 생체 내 출생 일렉트로 및 시간 경과 영상

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.

유전자 독성 스트레스 후 신경 줄기 및 마우스 두뇌 개발의 전구 세포의 사정 세포주기의 진행 상황

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.

개발 마우스 배아의 피질 유사 분열의 라이브 영상

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.

인간 다 능성에서 신경 능선 세포의 전구 세포의 공급이없는 유도는 줄기 세포

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

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

마우스 인공 와우의 Explants의 장기 시간 경과 영상

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...