この作品はアメリカの小児外科協会財団賞（AG）と健康K08DK098271（AG）の国立研究所メートがサポートされていました。

ウィスコンシン大学の動物実験委員会は、すべての手順を承認しました。 ソリューションの調製<ol><li>解剖緩衝液およびリンス液として1×リン酸緩衝生理食塩水（PBS）を使用します。 </li><li>ボトルにスクロースおよび場所の30グラムを計量することにより、30％スクロースを準備します。 1×PBS 99 mlを加え、1mlの10％アジ化ナトリウムを追加します。ショ糖の全てが溶解するまで十分に混合します。 4℃で保存、必要になるまで。 </li><li> 1×PBS、3％ウシ血清アルブミン（BSA）および0.1％トリトンX-100を混合して溶液を遮断準備。完全に混合し、必要になるまで冷蔵庫で保管してください。 </li><li> 1×PBS中にPFAの適切な量を秤量することによって1×PBS中の8％パラホルムアルデヒド（PFA）溶液を調製し、それが完全に溶解するまで65℃でインキュベートします。 -20℃の冷凍庫で25ミリリットルの分量を保存が必要になるまで。使用日の1×PBS中の4％PFAに希釈します。 </li></ol> 2.胚とガット解剖イオン<ol><li>制度動物実験委員会によると、タイミング妊娠マウスを安楽死させるし、PBS 1X氷冷を含む60ミリメートルペトリ皿に子宮を転送プロトコルを承認しました。 </li><li>鋭いハサミを使用して、解剖顕微鏡下で、胚を露出させるオープン子宮壁をカット。子宮から胚を除去し、PBS 1X氷冷を含むクリーンな60ミリメートルペトリ皿に置きます。 </li><li>氷冷1×PBS中で断頭により各胚を安楽死させます。あなたは、蛍光タンパク質を含むトランスジェニックマウスを使用している場合は、蛍光照明の下で、正のトランスジェニック胚を識別します。 </li><li>解剖顕微鏡を使用して、各胚から胃腸管を解剖。微細鉗子の使用、左側が上向きにされ、右側には、ペトリ皿の底に反するような胚を方向付けます。内臓を露出させるために胚から上半身の壁を削除します。背側体壁と内臓の間に微細な鉗子を挿入します。クロム胚から内臓を除去するために、はさみ状の切断動作で互いに対して鉗子をOSS。 </li><li>さらに離れて周囲の器官から胃腸管をサブ解剖した後、氷冷1×PBSを含む1.5 mlマイクロチューブに各GI管を配置します。 </li></ol> GI地帯の3固定<ol><li>氷冷1×PBSで各GI管を3回すすぎ、次いで4％PFAと交換してください。 1.5時間室温でロッキングプラットフォーム上で1.5mlの微小管における消化管を修正しました。 RTで消化管を5分間3回すすぎ、その後、ロッキングプラットフォーム上で1時間。この段階で、必要になるまで、0.1％アジ化ナトリウムを含有する1×PBS中30％スクロースに4℃でGIトラクトを格納します。 注：別の方法として、組織の完全性に影響することなく、1年まで、30％スクロース中の胚を格納します。 30％スクロース中のサンプルの保管は、免疫染色のために、またはクライオsectioniのため10月にいずれかのサンプルの後の処理を可能にしますNG。あるいは、以下に詳述免疫染色プロトコルを進めます。 </li></ol> 4.免疫染色プロトコル<ol><li>サンプルは30％スクロース中で保存されている場合は、ロッキングプラットフォーム上で、それらを1×PBSで20分間3回洗浄します。 </li><li> RTで1時間のためのロッキングプラットフォーム上でブロッキング溶液中に消化管を配置します。 </li><li>ブロッキング溶液を除去し、ロッキングプラットフォーム上で4℃でRTまたはO / Nで4時間のいずれかのためのブロッキング溶液中で希釈した一次抗体の適切な量の消化管をインキュベートします。 （患者から得られた血清）ヒト抗胡抗体1000希釈、1：ニワトリ抗緑色蛍光タンパク質（GFP）抗体1000希釈および1：ヤギ抗ChATの抗体の100倍希釈1を使用してください。 注：私たちは、患者から局所的に得られたヒト抗胡抗体を利用したが、抗胡抗体は、例えば、市販されており、マウス抗胡、（1での使用：500希釈）。 </li><li> 1×PBS中の消化管をすすぎます3回5分間、その後、ロッキングプラットフォーム上で室温で1時間。 </li><li> 4℃でのRTまたはO / Nで4時間のいずれかのためのロッキングプラットフォーム上のブロッキング溶液中で1：500に希釈した二次抗体を用いて1×PBSを交換してください。このようなDylight、ロバ抗ニワトリCy2とし、ロバ抗ヤギCy5のようなロバ抗ヒトアミン反応性染料を使用してください。ロバ抗マウスアミン反応性染料405：500希釈のマウス抗胡抗体を用いた場合は、1を使用します。 注：内因性GFPとtdTomato発現および発達年齢とともに増加して免疫染色を明確にする強さ。我々は、一般的に使用される抗体の量を低減し、また、組織の効率的な染色を確実にするために、染色溶液の150μlを、0.2 mlチューブ中に個別に胚勇気を免疫染色します。 </li><li> 1×PBS中の消化管に5分間、3回洗浄し、その後、ロッキングプラットフォーム上で室温で1時間。 </li><li>スライドガラス上にDAPIで蛍光封入剤を数滴を置きます。 GI TRAを浸します蛍光マウンティング培地中にCTと組織の上に直接カバーガラスを追加します。媒体-Gを取り付ける蛍光は半永久的なシールを提供する水溶性の化合物です。 注：このようなベクタシールドなどを使用し、蛍光封入フェージングおよび抗体の光退色防止媒体を装着するベースグリセロールです。これらの製品はいずれも、組織は4℃で長期間保存することを可能にします。 </li><li>共焦点顕微鏡を用いて蛍光体のそれぞれの画像をキャプチャします。 表3に示す蛍光団および使用するフィルタの励起波長を使用してください。 </li><li>画像をキャプチャするために使用される共焦点の種類に応じて、適切なソフトウェアを使用して、コンピュータ支援画像解析を実行します。 </li></ol>

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The authors have nothing to disclose.

私たちの研究室等はHSCR腸内の欠陥があっても神経節の小腸5,15,16に、無神経節結腸に限らず、近位に延長されていないことを示しています。これらの変化はENS神経密度と神経伝達物質表現型の変化を含み、HSCRを有する患者において観察された運動障害を説明することができます。私たちは、ENS形成の決定要因を理解するための努力で上記の技術を利用しています。具体的には、これ?...

運動、栄養吸収、およびローカルの血流を制御する機能腸管神経系（ENS）は、生命1に不可欠です。 ENSは、それらはニューロンおよびグリア細胞を含む神経節に分化腸を移動し、コロニー形成、増殖、神経堤細胞（NCC）によって形成されます。ヒルシュスプルング病（HSCR、男性でのオンラインメンデル遺伝）、1 4,000で出生の発生率とmultigeneic先天性障害は、破壊されたENSの形成を研究するためのプロトタイプの病気と考えることができます。 HSCRでは、NCCはに移動し、遠位後腸2の変数の長さを植民地化することができません。また、他の一般的な胃腸（GI）は、肛門直腸奇形、腸atresias、および運動障害などの小児集団における発達障害は、基本的なENS機能の障害と関連しており、おそらく微妙な、過小評価、解剖学的変化とで機能的変化と関連していますENS 3-6。そのため、私たちはENS形成の発達決定要因を理解することを可能にする技術は、病因および小児消化管障害の治療の可能性に光を当てることができます。 移行と植民地化の後、NCCは、それらの神経伝達物質の表現型に特異的なマーカーでニューロンに分化します。コリン作動性ニューロンは、腸溶性ニューロン7の約60％を含み、かつコリンアセチルトランスフェラーゼ（チャット）、興奮性神経伝達物質アセチルコリンのための合成酵素を染色することによって検出することができます。歴史的には、コリン作動性ニューロンを可視化する試みは、中枢神経系に対して向けられた抗体の抗原特異性（CNS）は、末梢神経系（PNS）のChAT 8-10に対してチャット異なるによって混乱しました。しかし、胎盤のChATに対する抗体は、両方の中央と周辺のChAT 11-13を認識し、我々は最近、visuaを可能にする技術が記載されています高感度のENSのコリン作動性ニューロンのlization以前の開発でチャットレポーター線14で達成されたよりも。 ここでは、ニューロンにおけるENSの神経伝達物質の発現を可視化するマウス胚GI管の、固定および免疫染色を解剖するための手法を提示します。これらの研究のために、我々は、 チャット-Creマウスを利用しているR26Rと交配：floxSTOP：チャットのCreを生成するためにtdTomato動物を、R26R：floxSTOP：tdTomatoマウス （原稿全体のChAT-CreをtdTomatoと定義されます）。これらの動物は、その後のChAT発現14を検出する蛍光レポーターの両方を発現するマウスを得るために、ホモ接合のChAT-GFPレポーターマウスと交配させました。これら二つのレポーター動物は、C57BL / 6Jバックグラウンド上にあり、（ジャクソン·ラボラトリーズ、バーハーバー、ME）は、市販されています。

<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.

我々は以前のChAT発現14を検出GFPおよびtdTomato蛍光レポーターの両方を発現するマウスの生成を記載しています。簡単に説明すると、 チャットCreマウスは、R26Rと交配させた：floxSTOP：チャットのCreを生成するためにtdTomato動物; R26R：floxSTOP：（チャット-CreをtdTomatoと呼ばれる）tdTomatoマウスを。これらの動物は、その後、ホモ接合のChAT-GFPレ...

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マウスの腸管神経系の発達を可視化するための免疫染色 |発生生物学 |ビデオ

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.

マウス腸神経系の開発を可視化するために免疫染色

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 ビュー.  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.

ビデオ: 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).

最適化された<em&gt;元OVO</em&gt;開発の進んだ段階にニワトリ胚の培養

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

発生生物学

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.

イメージゼブラフィッシュアイ開発にライトシート蛍光顕微鏡を用いて、

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.

血管内皮幹細胞の血管形成を視覚化するための新規乳腺脂肪移植技術

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.

リアルタイムで開発を可視化する多光子タイムラプス イメージング: ゼブラフィッシュ胚の神経堤細胞の可視化

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.

ニワトリ胚組織の開発培養法

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.

核の分布、蛋白質および細胞骨格を研究する線虫生殖細胞の解析

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.

「マウス卵巣における現象を可視化する卵巣の組織培養

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.

大人のキューティクルをショウジョウバエ脚運動ニューロン軸索を可視化します。

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.

内皮コロニー形成細胞と間葉系幹細胞によって形成された共培養スフェロイドを用いた3次元血管新生アッセイ系

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.

iPSC-ICにおけるゼブラフィッシュ胚の全山ハイブリダイゼーションとチューブ形成アッセイは血管開発におけるエンドリンの役割を研究する

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