A investigação conducente a estes resultados foi financiada pelo Sétimo Programa-Quadro da União Europeia (FP7/2007-2013), sob contrato de subvenção n ° 323267, da Electricité de France (EDF) e de l&#39;Agence Nationale de la Recherche - Santé-Environnement et Santé-Travail (ANR-SEST, Neurorad).

1. Procedimentos com animais Este protocolo foi desenvolvido em conformidade com as directivas da Comunidade Europeia do Conselho de 24 de novembro de 1986 (86/609/CEE) e foi aprovado pelo nosso comitê institucional sobre bem-estar animal (CETEA-CEA DSV IDF). <ol><li> Injeções com edu / BrdU e irradiação de E14.5 camundongos grávidas (Figura 2A). <ol><li> Prepara-se uma solução de EdU a 1 mg / ml e de BrdU em 5 mg / ml em PBS. Realizar uma injecção intra-peritoneal (IP) de 100 ul de solução de edu utilizando uma agulha de 25 G em ratas grávidas. 1,5 h mais tarde, os ratinhos irradiar (irradiação total do corpo), a 0 Gy, ou 2 Gy (0,6 Gy / min). Imediatamente depois, injectar (IP) de 200 ul de solução de BrdU utilizando uma agulha G 25, nas ratas grávidas. </li></ol></li><li> Preparação de cortes coronais de cérebros embrionárias. <ol><li> Em 1 hora ou 4 horas após a irradiação, eutanásia os ratos grávidas por dióxido de carbono. </li><li> Abra a cavi abdominalty mais de três centímetros por meio de tesoura. Separa-se os dois cornos uterinos do resto do útero por corte de ambas as extremidades os chifres. Transferir os cornos uterinos em uma placa de Petri contendo PBS 0,6% de glucose. Abra os cornos uterinos com a pinça, a fim de isolar os embriões. Retire o saco amniótico de cada embrião usando uma pinça. </li><li> Dissecar cabeças embrionários com fórceps e corrigi-los por imersão em 4% de paraformaldeído (PFA, pH = 7,4) O / N a 4 ° C. </li><li> Lavar cabeças embrionárias em PBS durante pelo menos uma noite a 4 ° C. Cabeças pode ser mantido em PBS, durante vários dias. </li><li> Incorporar cabeças em parafina usando um processador de infiltração de vácuo, tal como indicado na Tabela 1. Cabeças incorporação também pode ser realizada sob um capuz químico para etanol e um banho de xilema e depois num forno de 65 ° C para os banhos de parafina. </li><li> Prepare 5 mícrons cortes coronais de espessura com um micrótomo. </li><li> Montar em lâminas de microscópio revestidas com polilisina. &lt;/ Li&gt; <li> Slides pode ser mantido à temperatura ambiente (RT) durante vários meses. </li></ol></li></ol> 2. Edu / BrdU Coloração <ol><li> Desparafinização e antígeno desmascaramento <ol><li> Retire a parafina as seções do cérebro em parafina por imersão das lâminas em 3 banhos de tolueno por 5 min. </li><li> Re-hidratar durante 3 min em dois banhos de cada soluções de concentrações decrescentes de etanol, como se segue: 100% de etanol, etanol a 95%, etanol a 70%, e 5 min em H 2 O. As lâminas podem ser mantidos em água desionizada durante vários horas. </li><li> Ferver as fatias durante 10 min em solução de citrato (10 mM, pH 6), e em seguida, incuba-se água desionizada, durante 5 min. </li></ol></li><li> Coloração EdU <ol><li> Realizar a detecção EdU de acordo com o protocolo do fabricante. Permeabilizar as células com 0,5% de triton X-100 em PBS durante 10 min. </li><li> Prepare aditivo tampão 1X EdU diluindo a solução 10X em água desionizada. Esta solução deve ser preparado e usado no mesmo day. </li><li> Prepare EdU cocktail reação, incluindo EdU Alexa Fluor 488 azida. É importante adicionar os ingredientes seguintes pela ordem indicada na Tabela 2, de outro modo, a reacção não prosseguirá optimamente. Use o cocktail reação EdU dentro de 15 minutos de preparação. </li><li> Remover o tampão de permeabilização (passo 2.1.1), em seguida, lavar duas vezes com PBS. </li><li> Adicionar 150 mL de EdU cocktail reação, colocar uma lamela e incubar por 30 min no escuro à temperatura ambiente. </li><li> Remover o cocktail reacção EdU, em seguida, lavar uma vez com PBS. </li></ol></li><li> Coloração BrdU <ol><li> Preparar tampão de saturação com 7,5% de soro de cabra e 7,5% de soro fetal bovino em PBS. Adicionar 150 ul de tampão de saturação em fatias de cérebro, colocar uma lamela, e incubar durante 1 hora à temperatura ambiente para bloquear o anticorpo não-específico de ligação. </li><li> Retirar as lamelas. Adicionar 150 mL de rato anti-BrdU anticorpo primário diluído em 1/300 em tampão de saturação, colocar uma lamela e incubar O /N a 4 ° C. </li><li> Retirar as lamelas, em seguida, lava-se 3 vezes em PBS. </li><li> Adicionar 150 ul de anticorpo de cabra anti-ratinho-Alexa594 anticorpo secundário diluído a 1/400 no tampão de saturação, colocar uma lamela, e incubar durante 1 hora à temperatura ambiente. </li><li> Retirar as lamelas, em seguida, lavar uma vez com PBS. </li><li> A coloração nuclear: adicionar 150 uL de 4 &#39;,6-diamidino-2-fenilindol (DAPI) em 1 ug / ml em PBS, colocar uma lamela e incubar durante 2 minutos à temperatura ambiente. </li><li> Monte slides em meio de montagem. </li></ol></li></ol> 3. Análise <ol><li> Examine seções do cérebro com um microscópio de fluorescência com um objetivo 20X ou um microscópio confocal e adquirir imagens em três canais (488 nm, 594 nm e UV), como separa arquivos. </li><li> Empilhe as imagens com o software Photoshop. </li><li> Analisar seções do cérebro em um setor padrão da parede cerebral dorsomedial. Esse sector é de 100 um, na sua dimensão médio-lateral e está dividido em 18 bandejas (ou mais) de 10 μm de altura na sua dimensão radial utilizando uma grelha sobreposta imagens (Figura 1) como previamente descrito 31. <ol><li> Alinhar a grade, como o primeiro compartimento está na superfície ventricular, com o seu eixo longo paralelo ao arco ventricular. Então o número da UDE rotulados, BrdU e / ou núcleos picnóticos (correspondendo a núcleos apoptóticos) em cada bin. Numero um núcleo localizado no limite de dois compartimentos do compartimento que contém a sua maior parte, ou no compartimento inferior, quando o núcleo ocupa áreas iguais dentro dos dois compartimentos. </li></ol></li><li> A análise estatística. </li></ol> Analisar, pelo menos, duas fatias corticais por embrião. Repetir experiências em, pelo menos, 3 a partir de embriões de ninhadas diferentes. Os resultados devem ser dadas como erro médio ± padrão da média (SEM). As análises estatísticas são realizadas com Graphpad Prism usando ANOVA de duas vias e vários testes de comparação posthoc Bonferroni.

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Os autores não têm qualquer conflito de interesses.

O delineamento experimental descrito aqui com base na incorporação de EdU 1,5 hr antes da irradiação e incorporação de BrdU imediatamente após a irradiação permitiu a demonstração de que NSPCs são capazes de ativar S e G2 / M, mas não a ponto de verificação G1 / S durante a 1 ª hora após a estresse genotóxico no cérebro fetal mouse. Realizamos outros experimentos em que EdU foi injetado em diferentes momentos após a irradiação e BrdU, apenas 1 hora antes de ratos sacrifícios, permitindo...

Durante o desenvolvimento do cérebro embrionário, de neurónios de projecção do córtex cerebral são gerados na zona ventricular, um epitélio pseudo composto de diferentes tipos de estaminais neuronais e células progenitoras (NSPC) que reveste os ventrículos laterais. Entre NSPC, as células gliais radiais (RGC), que servem como células estaminais neurais, sofrer migração nuclear interkinetic (INM): eles realizam a mitose na superfície do ventrículo e da fase S no limite basal da zona ventricular (VZ) 1, 2,3. Eles podem dividir ou simetricamente para gerar dois RGC ou assimetricamente para gerar um RGC e um neurônio ou uma célula intermediária progenitor (IPC) 4, 5. IPC migrar para uma camada sobrejacente proliferando chamada zona subventricular (SVZ), onde depois de uma última divisão simétrica geram dois neurônios de projeção imaturos 6-8. Ao contrário do RGC, o IPC não sofrem INM (revisado em 9). Neurônios recém-gerados Migrcomeu radialmente ao longo das fibras radiais através da zona intermediária (IZ) para chegar ao seu destino final na placa cortical (CP) 10, 8. O timing perfeito de todos estes eventos é essencial para um desenvolvimento cortical correta. Por exemplo, o interruptor de proliferação a diferenciação de populações progenitoras neurais é controlada pela duração da fase G1 11, 12. O prolongamento da fase G1 assim correlaciona com a diferenciação celular. Estresse genotóxico, como radiação ionizante, o desenvolvimento do cérebro severamente prejudicar (revisado em 13). Nós e outros autores mostraram que NSPC são altamente propensos a apoptose induzida por radiação 14, 15,16. As radiações ionizantes induzir rupturas dos filamentos de DNA duplo que são os danos mais graves para células em proliferação. Um componente essencial da resposta a danos do DNA (DDR) em células de ciclismo é a ativação de pontos de controle do ciclo celular nas transições G1 / S ou G2 / M ou durante Sfase (intra-S checkpoint) 17-21. Eles bloqueiam a progressão do ciclo celular para dar tempo para a reparação de danos ao DNA ou eliminação de células muito danificadas. Consequentemente, a morte celular, bem como a progressão do ciclo celular retardada pode alterar o desenvolvimento do cérebro em resposta à exposição a radiação ionizante 22-24. Era, portanto, interessante para desenvolver um método para avaliar a activação de checkpoints do ciclo celular em NSPC no cérebro do rato embrionário irradiado. A progressão do ciclo celular é normalmente seguido usando a incorporação de um análogo da timidina, 5-bromo-2&#39;-desoxiuridina (BrdU). BrdU é incorporado durante a fase S do ciclo celular, quando o ADN está a replicar. A utilização de um anticorpo contra BrdU permite posteriormente a detecção de células que estavam na fase S durante o pulso de BrdU. Um análogo da timidina romance, 5-etinil-2&#39;-desoxiuridina (EdU) é detectada por um azida fluorescente. As diferentes maneiras de detectar EdU e B<html> RDU não reagem de forma cruzada 25, permitindo a detecção simultânea de ambos os análogos de timidina, a qual é útil para o estudo da progressão do ciclo celular. Normalmente, as células são pulsadas com EdU primeiro e, em seguida, pulsadas com BrdU, em que o tempo entre as duas incorporações dura duas horas 25, 26. A adição de BrdU em meios de cultura contendo EdU resulta preferencialmente incorporação de BrdU no ADN com a exclusão de EdU, enquanto que a adição simultânea de EdU 25 com BrdU equimolar ou equimolar metade para os meios de comunicação provoca apenas a incorporação de BrdU 27. Isto simplifica o protocolo de marcação dupla, eliminando as etapas de lavagem que são normalmente necessários para a remoção da etiqueta a partir do primeiro meio de cultura antes da adição da segunda etiqueta. Este também é de um interesse especial para o estudo in vivo, em que os passos de lavagem não são possíveis, para determinar o momento preciso da entrada na fase S ou saída da população de células. t &quot;&gt; Recentemente, um método de rotulagem de duplo pulso em cérebro embrionário do rato usando EdU e BrdU 23, 24,22 foi desenvolvido para analisar a progressão do ciclo celular e INM de NSPC no útero após a irradiação. Além disso, tem sido demonstrado que, durante fase S, células de camundongo replicar primeiro as regiões eucromatina e, em seguida, a heterocromatina pericêntrica 28,29,30. Curiosamente, heterochromatin pericêntrica de diferentes cromossomos agrupados em núcleos interphasic para formar focos heterocromática também conhecido como chromocenters e facilmente detectável pela coloração DAPI como focos brilhante. Portanto, os diferenciais Edu e BrdU colorações de eucromatina e chromocenters nos ajudou a analisar com mais precisão a progressão da fase S de NSPC. Este método permitiu a demonstração da aparente falta de G1 / S checkpoint em NSCP 22-24, o que é bastante surpreendente, pois o ponto de verificação é suposto ser crítico para a estabilidade do genoma. Vários experimental desenhos com base em várias combinações de edu e BrdU pulsos foram utilizados para analisar a progressão do ciclo celular, no telencéfalo ventral e dorsal. Aqui, damos um exemplo de protocolos para permitir o estudo do dano ao DNA aguda da NSPC no primeiro 4 horas após a irradiação em utero de embriões E14.5 do mouse.

<table border="0" cellpadding="0" cellspacing="0" width="727">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:228px;">EdU</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">A10044</td>
			<td style="width:163px;">1 mg/ml</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">BrdU</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">B5002</td>
			<td style="width:163px;">5 mg/ml</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:228px;">IBL 637</td>
			<td style="width:212px;">CIS BIO International</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">Tissu Tek VIP</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Microtome</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;">RM 2125 RT</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Triton X-100</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td align="right" style="width:125px;">93443</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:228px;">Click-iT EdU Alexa 488 imaging kit</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">C10083</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:228px;">Anti-BrdU</td>
			<td style="width:212px;">GE Healthcare</td>
			<td style="width:125px;">RPN202</td>
			<td style="width:163px;">1/300</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:228px;">Goat anti mouse-Alex594</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">A11001</td>
			<td style="width:163px;">1/400</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Fluoromount</td>
			<td style="width:212px;">SouthernBiotech</td>
			<td style="width:125px;">0100-01</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:228px;">Polysine slide</td>
			<td style="width:212px;">Thermo scientific</td>
			<td style="width:125px;">J2800AMNZ</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:228px;">Paraformaldehyde</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:125px;">P6148</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:228px;">PBS</td>
			<td style="width:212px;">Life Technologies</td>
			<td style="width:125px;">20012-068</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:228px;">DAPI</td>
			<td style="width:212px;">Sigma-Aldrich</td>
			<td style="width:125px;">D9542</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:228px;">Microscope BX51</td>
			<td style="width:212px;">Olympus</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:228px;">Confocal microscope SPE</td>
			<td style="width:212px;">Leica</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:228px;">Prism software</td>
			<td style="width:212px;">Graphpad</td>
			<td style="width:125px;"></td>
			<td style="width:163px;">Version 5.0c</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:228px;">Photoshop software</td>
			<td style="width:212px;">Adobe</td>
			<td style="width:125px;"></td>
			<td style="width:163px;"></td>
		</tr>
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assessing cell cycle progression of neural stem and progenitor cells in the mouse developing brain after genotoxic stress

Os neurónios do córtex cerebral são gerados durante o desenvolvimento do cérebro a partir de diferentes tipos de células estaminais e progenitoras neurais (NSPC), que formam um epitélio pseudo alinhando os ventrículos laterais do cérebro embrionário. Estresses genotóxicos, como a radiação ionizante, têm efeitos altamente deletérios sobre o cérebro em desenvolvimento relacionado com a alta sensibilidade do NSPC. A elucidação dos mecanismos celulares e moleculares envolvidas depende da caracterização da resposta a danos no ADN destes tipos particulares de células, o que requer um método preciso para determinar a progressão NSPC longo do ciclo celular no tecido danificado. Aqui é mostrado um método baseado em sucessivas injeções intraperitoneais Edu e BrdU em camundongos grávidas e ainda a detecção destes dois análogos da timidina em cortes coronais do cérebro embrionário. EdU BrdU e ambos são incorporados no ADN das células em replicação durante a fase S e são detectados através de duas técnicas diferentes (ou azidaum anticorpo específico, respectivamente), que facilitam a sua detecção simultânea. EdU e coloração BrdU são então determinados para cada núcleo NSPC em função da sua distância a partir da margem do ventrículo numa região padrão do telencéfalo dorsal. Assim, esta técnica de marcação dupla permite distinguir células que progrediu ao longo do ciclo celular das que ter activado um checkpoint do ciclo celular, levando à paragem do ciclo celular em resposta ao dano do DNA. Um exemplo de experimento é apresentado, no qual EdU foi injetado antes da irradiação e BrdU imediatamente após e análises realizadas no âmbito da 4 h após a irradiação. Este protocolo apresenta uma análise precisa da resposta de dano de DNA aguda de NSPC em função da fase do ciclo celular em que eles foram irradiadas. Este método é facilmente transponível para muitos outros sistemas, a fim de determinar os efeitos de um tratamento específico sobre a progressão do ciclo celular em tecidos vivos.

Na experiência descrita na Figura 2, EdU foi administrado 1,5 horas antes da irradiação e BrdU logo após a irradiação. Quatro tipos de células foram então distinguidos nas fatias corticais preparadas a 1 ou 4 horas após a irradiação, de acordo com a incorporação de BrdU quer EdU ou, ambos ou nenhum (Figuras 2A e 2B). Importante, nem EdU nem a incorporação de BrdU alterou o nível de apoptose induzida por radiação (dados não mostrados). Além disso, os ...

Watch this Scientific Journal Video about Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress at JoVE.com

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

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

A administração de dois análogos de timidina, EdU e BrdU, em ratinhas grávidas permite a análise da progressão do ciclo celular em células progenitoras neurais e no cérebro do rato embrionário. Este método é útil para determinar os efeitos do stress genotóxico, incluindo a radiação ionizante, durante o desenvolvimento do cérebro.

Avaliando a progressão do ciclo celular de tronco neurais e as células progenitoras no desenvolvimento do cérebro do rato após estresse genotóxico

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

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15.2K visualizações.  CEA DSV iRCM SCSR.  A administração de dois análogos de timidina, EdU e BrdU, em ratinhas grávidas permite a análise da progressão do ciclo celular em células progenitoras neurais e no cérebro do rato embrionário. Este método é útil para determinar os efeitos do stress genotóxico, incluindo a radiação ionizante, durante o desenvolvimento do cérebro. 

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

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

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Lapso de tempo de imagens ao vivo de relação clonal células progenitoras neurais no desenvolvimento Zebrafish Encéfalo anterior

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

Neurociência

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neurais Colony-Forming Assay Cell: um ensaio para Discriminar Bona Fide células-tronco neurais de células progenitoras neurais

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

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

Em células vivas, imagens de células precursoras de órgãos sensoriais na Intact Drosophila As pupas

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 ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

O Ensaio neuroblasto: um ensaio para a Geração e Enriquecimento de células progenitoras neuronais de Diferenciando Progeny Neural Stem Cell utilizando citometria de fluxo

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolamento e cultivo de células da crista neural do tubo neural embrionário murino

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

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.

Imagens ao vivo da mitose no desenvolvimento Cortex embrionárias de camundongos

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.

Derivação sem alimentador de células da crista neural progenitoras pluripotentes humanas de células-tronco

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.

Seleção de células-tronco neurais de células progenitoras e do rato adulto subventricular Zone e Live-imagem de seus Dynamics com o ciclo celular

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

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

Modelagem de infecção de vírus Zika do desenvolvimento cérebro humano In Vitro usando células-tronco derivadas de Organoids Cerebral

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.