Este trabalho foi apoiado bythe Academia Americana de Pediatria Cirúrgica Award Foundation (AG) e os Institutos Nacionais de Saúde K08DK098271 (AG).

A Universidade de Wisconsin Animal Care e do Comitê Use aprovado todos os procedimentos. 1. Preparação de Soluções <ol><li> Use fosfato 1x solução salina tamponada (PBS) como tampão de dissecção e uma solução de enxaguamento. </li><li> Prepare a 30% de sacarose por pesagem de 30 g de sacarose e em lugar de uma garrafa. Adicionar 99 ml de PBS 1x e adicionar azida de sódio a 1 ml de 10%. Misturar bem até que toda a sacarose é dissolvido. Armazenar a 4 ° C até serem necessárias. </li><li> Preparar a solução de bloqueio através da mistura de 1x PBS, 3% de albumina de soro bovino (BSA) e 0,1% de Triton-X-100. Misture bem e guarde na geladeira até ser necessário. </li><li> Preparar solução a 8% de paraformaldeído (PFA) em PBS 1x por pesagem da quantidade apropriada de PFA em PBS 1x e, em seguida, incuba-se a 65 ° C até que esteja completamente dissolvido. Armazenar em alíquotas de 25 ml no congelador a -20 ° C até ser necessário. Diluir a 4% de PFA em PBS 1x no dia da utilização. </li></ol> 2. Embryo e Gut Dissectíon <ol><li> De acordo com o Animal Care Institucional e Comitê de Uso protocolos aprovados, eutanásia rato grávida cronometrado e transferir o útero em um 60 milímetros placa de Petri contendo gelada 1x PBS. </li><li> Sob um microscópio de dissecção com uma tesoura afiada, cortar a parede uterina aberto para expor os embriões. Remova os embriões do útero e lugar em um ambiente limpo 60 milímetros placa de Petri contendo gelada 1x PBS. </li><li> Euthanize cada embrião por decapitação em 1x PBS gelado. Se você estiver usando ratinhos transgénicos que contenham proteínas fluorescentes, sob iluminação fluorescente, identificar os embriões transgênicos positivos. </li><li> Dissecar o tracto GI de cada embrião utilizando um microscópio de dissecção. Usando fórceps finos, orientar o embrião de tal modo que o lado esquerdo está virado para cima e o lado direito é contra o fundo da placa de Petri. Remover a parede superior do corpo do embrião para expor os órgãos internos. Inserir uma pinça fina entre a parede do corpo dorsal e os órgãos internos. CrOss fórceps uns contra os outros em uma acção de corte do tipo tesoura para remover os órgãos internos do embrião. </li><li> Outras sub-dissecar o tracto GI para cima dos órgãos circundantes e, em seguida, colocar cada tracto GI para um tubo de microcentrífuga de 1,5 ml contendo gelada PBS 1x. </li></ol> 3. Fixação de GI Tracts <ol><li> Lavar cada trato GI 3 vezes com 1x PBS gelado e depois substituir com PFA 4%. Fixar os tractos gastrointestinais nos tubos de microcentrífuga de 1,5 ml sobre uma plataforma de agitação à TA durante 1,5 h. Lavar os tractos gastrointestinais 3 vezes durante 5 min à temperatura ambiente e em seguida durante 1 h na plataforma de agitação. Nesta fase, armazenar os trato gastrointestinal, a 4 ° C em 30% de sacarose em 1x PBS contendo azida de sódio a 0,1% até ser necessário. NOTA: Como alternativa, armazenar os embriões em 30% de sacarose até um ano, sem qualquer efeito sobre a integridade dos tecidos. Armazenamento das amostras em 30% de sacarose permite o processamento posterior das amostras ou para immunostaining ou em outubro para crio-sectioning. Como alternativa, prosseguir com o protocolo immunostaining detalhado abaixo. </li></ol> 4. Protocolo Immunostaining <ol><li> Se as amostras foram armazenadas em 30% de sacarose, enxaguar três vezes durante 20 minutos em 1x PBS numa plataforma oscilante. </li><li> Colocar os tractos gastrointestinais em solução numa plataforma oscilante durante 1 h à TA bloqueio. </li><li> Remover a solução de bloqueio e incubar os tratos GI, com a quantidade adequada de anticorpos primários diluídos em solução, quer por 4 horas à temperatura ambiente ou O / N a 4 ° C numa plataforma oscilante bloqueio. Use 1: 1000 de diluição de anticorpo anti-Hu humana (soro obtido a partir do paciente), 1: 1000 de diluição de frango anticorpo anti-proteína verde fluorescente (GFP) e diluição 1: 100 de anticorpo de cabra anti-ChAT. NOTA: Nós utilizamos um anticorpo anti-Hu humano que foi obtido localmente a partir de um paciente, no entanto, os anticorpos anti-Hu estão disponíveis comercialmente, por exemplo, de ratinho anti-Hu, (utilização a diluição 1: 500). </li><li> Lavar as trato gastrointestinal em 1x PBS3 vezes durante 5 min e depois durante 1 hora à temperatura ambiente numa plataforma oscilante. </li><li> Substituir o 1x PBS com anticorpos secundários diluídos 1: 500 em solução numa plataforma oscilante ou 4 horas para o bloqueio à TA ou O / N a 4 ° C. Use corante amino-reactivo anti-humana de burro como DyLight, burro Cy2 anti-frango e de burro anti-cabra Cy5. Se o rato utilizando anticorpo anti-Hu, em seguida, usar uma diluição de 1: burro anti-rato de amina de corante reactivo 405 500. NOTA: A intensidade da GFP e expressão endógena tdTomato e a clareza de imunocoloração aumento com a idade de desenvolvimento. Nós normalmente imunocoloração tripas embrionárias individualmente em tubos de 0,2 ml com 150 ul de solução de coloração, a fim de reduzir o volume de anticorpo utilizado e também para assegurar a coloração eficaz do tecido. </li><li> Lavar os tractos gastrointestinais em 1X PBS 3 vezes durante 5 min e depois durante 1 h à TA em plataforma oscilante. </li><li> Coloque algumas gotas de fluorescência meio de montagem com DAPI numa lâmina de vidro. Mergulhe os tra GICT para o meio de montagem de fluorescência e adicionar uma tampa de vidro directamente em cima do tecido. A fluorescência média de montagem -G é um composto solúvel em água que proporciona uma vedação semi-permanente. NOTA: Use meio de montagem de fluorescência, como Vectashield que é uma base de glicerol meio que evita o desbotamento e fotodegradação de anticorpos de montagem. Ambos os produtos permitem tecidos para ser armazenado por longos períodos de tempo, a 4 ° C. </li><li> Captar imagens de cada um fluoróforo utilizando um microscópio confocal. Use comprimentos de onda de excitação de fluoróforos e filtros empregues apresentados na Tabela 3. </li><li> Executar análise de imagem assistida por computador, utilizando software apropriado, dependendo do tipo de confocal usado para capturar imagens. </li></ol>

<ol>
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The authors have nothing to disclose.

O nosso laboratório e outros mostraram que defeitos intestinais em HSCR não são restritas para o cólon proximal alargada aganglionic mas, mesmo para o intestino delgado ganglionados 5,15,16. Estas alterações incluem modificações nas ENS densidade neuronal e fenótipo neurotransmissor e podem ser responsáveis ​​pela dismotilidade que tem sido observada em pacientes com HSCR. Nós utilizamos as técnicas acima em nossos esforços para entender os determinantes da formação ENS. Especificamente, es...

Um funcionamento do sistema nervoso entérico (ENS), que controla a motilidade, a absorção de nutrientes e fluxo sanguíneo local, é essencial para a vida 1. O SNE é formado por células da crista neural (NCC) que proliferam, migram e colonizar o intestino, onde se diferenciam em gânglios contendo neurónios e células gliais. Doença de Hirschsprung (HSCR, on-line Mendelian Inheritance in Man), uma desordem congênita multigeneic com uma incidência de 1 em 4.000 nascidos vivos, pode ser considerada a doença prototípica para estudar a formação ENS interrompido. Em HSCR, NCC não conseguem migrar para colonizar e comprimentos variáveis ​​do intestino posterior distal 2. Além disso, outro gastrointestinal comum (GI) defeitos de desenvolvimento na população pediátrica, como malformações anorretais, atresias intestinais e distúrbios de motilidade são associados a distúrbios nas funções básicas ENS, e estão provavelmente associados à subtis, subvalorizado, alterações anatômicas e alterações funcionaiso 3-6 ENS. Portanto, as técnicas que nos permitem entender os determinantes do desenvolvimento da formação ENS pode lançar luz sobre a patogênese e potencial tratamento de distúrbios do trato GI pediátricos. Na sequência de migração e colonização, NCC diferencia em neurônios com marcadores específicos para seu fenótipo neurotransmissor. Os neurónios colinérgicos representam cerca de 60% ​​de neurónios entéricos 7, e pode ser detectado por coloração para colina acetiltransferase (ChAT), a enzima de síntese para a acetilcolina neurotransmissor excitatório. Historicamente, as tentativas para visualizar os neurónios colinérgicos foram confundidos pela diferindo a especificidade do antigénio dos anticorpos dirigidos contra o sistema nervoso central (SNC) contra a ChAT sistema nervoso periférico (SNP) ChAT 8-10. No entanto, os anticorpos dirigidos contra placentária ChAT reconhecer tanto ChAT central e periférica 11-13, e temos técnicas recentemente descritos que permitem a visuazação da ENS neurônios colinérgicos com alta sensibilidade anteriormente no desenvolvimento do que foi alcançado com linhas de chat repórter 14. Aqui, nós apresentamos uma técnica para dissecar, fixação e imunomarcação do murino tracto GI embrionário para visualizar ENS expressão de neurotransmissores nos neurônios. Para estes estudos, nós utilizamos ratos chat-Cre acasalaram com R26R: animais tdTomato para produzir chat-Cre; R26R:: floxSTOP ratos tdTomato (definidos como chat-Cre tdTomato em todo o manuscrito): floxSTOP. Estes animais foram depois acasalados com ratinhos repórter ChAT-GFP homozigóticos, para obter ratinhos que expressam ambos os repórteres fluorescentes que detectam expressão ChAT em ​​14. Esses dois animais repórter estão em um fundo C57BL / 6J e estão comercialmente disponíveis (Jackson Laboratories, Bar Harbor, ME).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Oxoid</td>
			<td>BR0014G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Fisher</td>
			<td>BP9221</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher</td>
			<td>BP1605</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dishes</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence scope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ-18 stereoscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine science tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>SouthernBiotech, Birmingham, AL</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>16004-330</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>Nikon A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Elements</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

immunostaining to visualize murine enteric nervous system development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Descrevemos previamente a geração de ratinhos que expressam GFP e ambos tdTomato repórteres fluorescentes que detectam expressão ChAT em ​​14. Resumidamente, os ratos chat-Cre foram acasalados com R26R: floxSTOP: animais tdTomato para produzir chat-Cre; R26R: ratos tdTomato (chamados chat-Cre tdTomato): floxSTOP. Estes animais foram depois acasalados com ratinhos repórter ChAT-GFP homozigóticos. Os embriões foram isolados e os tecidos f...

Watch this Scientific Journal Video about Immunostaining to Visualize Murine Enteric Nervous System Development at JoVE.com

Imunocoloração para visualizar o desenvolvimento do sistema nervoso entérico murino | Biologia do Desenvolvimento | Vídeo

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Immunostaining para Visualize Murino desenvolvimento do sistema nervoso entérico

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

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11.3K visualizações.  University of Wisconsin School of Medicine and Public Health. The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Vídeo: Immunostaining to Visualize Murine Enteric Nervous System Development

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Otimizado<em&gt; Ex-ovo</em&gt; A cultura de Chick embriões para estágio avançado de desenvolvimento

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Biologia do Desenvolvimento

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

Usando folha de luz microscopia de fluorescência para Imagem Desenvolvimento Zebrafish Eye

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Uma nova técnica de transplante de almofada de gordura mamária para visualizar a geração de vasos de células vasculares endoteliais vasculares

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Fóton multi tempo lapso imagem para visualizar o desenvolvimento em tempo real: visualização de migrar as células da crista Neural em embriões de peixe-zebra

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Cultura de organotypic método para estudar o desenvolvimento de tecidos embrionários de galinha

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Análise computacional do Germline Caenorhabditis elegans , para estudar a distribuição de núcleos, as proteínas e o citoesqueleto

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Cultura do tecido ovariano Visualizar fenômenos no ovário de Mouse

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification&#8212;at least in terms of informing disease therapies&#8212;demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle

Here we describe a protocol to visualize the axonal targeting with a florescent protein in adult legs of Drosophila by fixation, mounting, imaging, and post-imaging steps.

Visualizar Drosophila perna neurônio Motor axônios através da cutícula adulto

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Sistema tridimensional do ensaio da angiogênese usando spheroids da co-cultura dados forma pela colônia endothelial que dá forma a pilhas e às pilhas de haste mesenchymal

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Hibridização em Embriões de Zebrafish e Ensaio de Formação de Tubos em iPSC-CEs para estudar o papel da endoglina no desenvolvimento vascular

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...