This study was supported by the Astellas Foundation for Research on Metabolic Disorders (HT), Takeda Science Foundation (HT), and MEXT-Supported Program for the Strategic Research Foundation at Private Universities (HT).

참고 : DE-CAD :: GFP 10 : 본 연구에 사용 된 형질 전환 초파리는 다음과 같습니다. 애플 플레이트 1. 준비 <ol><li> 마이크로파 12.5 g 한천, 125 mL의 100 % 시판 사과 주스, 12.5 g의 글루코스, 및 375 mL의 H 2 O 열 혼합물의 혼합물을 제조하고, 3cm 세포 배양 접시에 부어. 나중에 사용하기 위해 4 ° C에서 혼합물을 저장합니다. </li><li> 사과 플레이트를 제조 한 후, 파리가 알을 배치 할 수 있도록 그 위에 반죽 효모 붙여 넣기의 얇은 층을 추가합니다. </li></ol> 2. 배아 준비 및 라이브 영상 프로토콜 <ol><li> 짝이어서 반죽 효모에 덮여 사과 접시에 하룻밤 알을 낳기 위해 병에 약 50 파리 (25 명의 남성과 25 명의 여성)을 놓습니다. 병의 입에 사과 주스 플레이트를 설정합니다. 거꾸로 인큐베이터에서 병을 유지합니다. 제 프롬프트 알루미늄 호일 플라스틱 초파리 재고 병 포장전자는 더 많은 알을 낳기 위해 날아갑니다. </li><li> 다음날 아침, 새와 오래된 사과 주스 플레이트를 교체합니다. </li><li> 파리 알루미늄 호일로 병을 포장하여 3 ~ 4 시간 동안 알을 낳기 수 있습니다. </li><li> 세 4 시간 후, 브러시 또는면 나쁜를 사용하여 사과 주스 플레이트에서 배아 체에서 배아를 수집하고 PBS로 씻는다. 12 웰 배양 접시에 배아를 PBS로 2 ~ 3 회 이상 세척 할 것. </li><li> 12 웰 배양 접시에 5 분 동안 50 % 표백제와 배아를 Dechorionate. </li><li> dechorionation 과정에 따라, PBS와 배아를 2 ~ 3 번 더 씻는다. </li><li> PBS 선택 단계 그들의 경계에서의 투명도 수준에 기초하여 현미경 5 배아 함유 3cm 세포 배양 접시에 배아를 옮긴다. 200 μL 피펫 팁을 사용하여 준비된 배아 피펫. </li><li> 다음으로, 유리 커버 슬립에 선택한 두 배아를 배치하고 가늘게 꼰 티슈로 여분의 PBS를 제거합니다. 최대의 배아에게 지느러미 쪽의 방향미세 트위스트 티슈 페이퍼를 이용하여 커버 슬립. 그 후, 미세 바늘과 트위스트 티슈 페이퍼를 사용하여 실리콘 그리스와 유리 커버 슬립에 배아를 연결합니다. </li><li> 슬라이드의 하단에 공간을두고, 배아에 할로 카본 오일 (700)의 작은 방울을 추가하고 거꾸로 들여 쓰기 슬라이드에서 배아를 포함 coverslip에 배치합니다. </li><li> 공 초점 영상 시스템 아르곤 / 488 레이저 및 오일 액침 대물 (63X)를 이용하여 태아를 조사한다. 주 : 해당 발달 단계에서 배아를 식별은 낭 배기 이벤트의 이미지를 캡처하는 것이 중요합니다. 이러한 조건에서, 거의 모든 배아는 배아 분석 성인 개발 배양 될 수있는 적어도 14 단계까지 정상적으로 발전 관찰한다. </li></ol>

<ol>
	<li>Haruta, T., Warrior, R., Yonemura, S., Oda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proximal+half+of+the+Drosophila+E-cadherin+extracellular+region+is+dispensable+for+many+cadherin-dependent+events+but+required+for+ventral+furrow+formation.">The proximal half of the Drosophila E-cadherin extracellular region is dispensable for many cadherin-dependent events but required for ventral furrow formation.</a> Genes Cells. 15 (3), 193-208 (2010).</li><li>Oda, H., Takeichi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution:+structural+and+functional+diversity+of+cadherin+at+the+adherens+junction.">Evolution: structural and functional diversity of cadherin at the adherens junction.</a> J. Cell Biol. 193 (7), 1137-1146 (2011).</li><li>Fernandez-Sanchez, M. E., Serman, F., Ahmadi, P., Farge, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+induction+in+embryonic+development+and+tumor+growth+integrative+cues+through+molecular+to+multicellular+interplay+and+evolutionary+perspectives.">Mechanical induction in embryonic development and tumor growth integrative cues through molecular to multicellular interplay and evolutionary perspectives.</a> Methods Cell Biol. 98, 295-321 (2010).</li><li>Alderton, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+Epithelial+to+mesenchymal+and+back+again.">Metastasis: Epithelial to mesenchymal and back again.</a> Nat. Rev. Cancer. 13 (1), 3 (2013).</li><li>Fabregat, I., Malfettone, A., Soukupova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Insights+into+the+Crossroads+between+EMT+and+Stemness+in+the+Context+of+Cancer.">New Insights into the Crossroads between EMT and Stemness in the Context of Cancer.</a> J. Clin. Med. 5 (3), (2016).</li><li>Serrano-Gomez, S. J., Maziveyi, M., Alahari, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+epithelial-mesenchymal+transition+through+epigenetic+and+post-translational+modifications.">Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications.</a> Mol. Cancer. 15, (2016).</li><li>Yu, A. Q., Ding, Y., Li, C. L., Yang, Y., Yan, S. R., Li, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALEN-induced+disruption+of+Nanog+expression+results+in+reduced+proliferation,+invasiveness+and+migration,+increased+chemosensitivity+and+reversal+of+EMT+in+HepG2+cells.">TALEN-induced disruption of Nanog expression results in reduced proliferation, invasiveness and migration, increased chemosensitivity and reversal of EMT in HepG2 cells.</a> Oncol. Rep. 35 (3), 1657-1663 (2016).</li><li>Zheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+is+dispensable+for+metastasis+but+induces+chemoresistance+in+pancreatic+cancer.">Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer.</a> Nature. 527 (7579), 525-530 (2015).</li><li>Kalluri, R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basics+of+epithelial-mesenchymal+transition.">The basics of epithelial-mesenchymal transition.</a> J. Clin. Invest. 119 (6), 1420-1428 (2009).</li><li>Huang, J., Zhou, W., Dong, W., Watson, A. M., Hong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed,+efficient,+and+versatile+modifications+of+the+Drosophila+genome+by+genomic+engineering.">Directed, efficient, and versatile modifications of the Drosophila genome by genomic engineering.</a> Proc. Natl. Acad. Sci. U.S.A. 106 (20), 8284-8289 (2009).</li></ol>

The authors have nothing to disclose.

Although we have previously reported a similar procedure to capture live images of the gastrulation process in Drosophilla1, the method we describe here is detailed and easy to trace endogenous cadherin expression and thus is quite useful for genetic screening of key factors involved in gastrulation. To maximize success with this imaging procedure, it is essential to use an indented slide. Mechanical pressure sometimes causes embryonic death. Therefore, it is also important to handle the embryos as ge...

낭 배기는 초파리 1, 2 등의 다세포 동물의 배아 개발하는 동안 발생하는 형태 학적으로 동적 이벤트의 첫 번째 집합입니다. 흥미롭게도, 증거 신흥 것은이 과정이 기계 및 분자 메커니즘 (3) 사이의 상호 작용을 통해 조절되는 것이 좋습니다. 또한, 낭 배기에 중요한 과정입니다 중간 엽 전이 (EMT)로 상피는 또한 암 전이 4-8로 인간의 질병 과정에 연루되어있다. 혀끝의 수축을 제어 등, 많은 유전자가 또한 알려진 바와 같이 암 전이 9에서 관찰 된 EMT의 핵심 요소가 될 수 있습니다. 따라서, 낭배시의 정점 수축은 전술 한 조절 메커니즘을 조사하는 암 전이에 대한 이해를 향상시키는 우수한 모델이다. 이 기술의 이점은 우리는 따라서 실시간에서 낭배시 세포의 움직임을 관찰 할 수 있다는 우리 것암 전이뿐만 아니라 낭 배기에 관여하는 유전자를 선별 할 수. 비교적 알려지지 않지만, 세포 간 접착 정점 수축 1에서 중심 역할을하는 것으로 생각된다. 초파리 유전학은 잘 규제 분자 메커니즘을 탐구 단일 세포 수준의 연구에 적합합니다. 이 모델은 낭배 동안 혀끝의 수축의 중요성을 발견 할 수있게됩니다. 또한,이 방법은 암의 전이에 관여하는 유전자를 선별 할 수있다. 초파리의 낭배의 캡처 라이브 이미지는 더욱 더 상세하게 조직의 재 배열을 관리하는 분자 메커니즘을 이해 할 수있게했다. 여기서, 우리는이를 달성하기 위해 간단한 방법의 포괄적 인 설명을 제공한다.

<table border="0" cellpadding="0" cellspacing="0" width="694">
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		<col />
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	<tbody>
		<tr height="54">
			<td height="54" style="height:54px;width:268px;">Halocarbon oil 700</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:129px;">MKBH 5726</td>
			<td style="width:133px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:268px;">Vacuum grease Silicone</td>
			<td style="width:164px;">Beckman</td>
			<td style="width:129px;">335148</td>
			<td style="width:133px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:268px;">Glass coverslip&#160;</td>
			<td style="width:164px;">Matsunami glass</td>
			<td style="width:129px;">Thickness No1</td>
			<td style="width:133px;">24-36mm</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:268px;">Embryo stariner</td>
			<td style="width:164px;">Corning</td>
			<td style="width:129px;">Corning3477</td>
			<td style="width:133px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:268px;">Plastic Drosophilla Stock Bottles</td>
			<td style="width:164px;">Hitec</td>
			<td style="width:129px;">MKC-100</td>
			<td style="width:133px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">DE-Cadherin knock-in flies</td>
			<td></td>
			<td></td>
			<td>REF (10)</td>
		</tr>
	</tbody>
</table>

imaging of cell shape alteration and cell movement in drosophila gastrulation using de-cadherin reporter transgenic flies

낭 배기는 초파리와 같은 다세포 동물의 배아 개발하는 동안 발생하는 형태 학적으로 동적 이벤트의 첫 번째 집합입니다. 이 형태 학적 변화는 중간 엽 전이 (EMT)로 상피로 인식되고 있습니다. EMT의 조절 곤란이 섬유화와 암 전이와 관련된다. EMT 분자 메커니즘들에 의해 제어된다 신흥 증거가있다. 혀끝의 수축을 제어 등, 많은 주요 유전자도 알려진 바와 같이 암 전이에서 관찰 된 EMT 중요한 요소가 될 수 있습니다. 초파리 낭배 동안 EMT와 마찬가지로, 상피 세포는 그 모양을 변경하도록 유도 될 수 있으며, 다양한 세포 유형으로 세포의 운명을 리디렉션 재 프로그램 될 수있다. 여기에서 우리는 배아 발달의이 단계에서 형태 형성 세포 이동 및 세포 운명 식별의 개시를 분석하기 위해 초파리 낭 배기의 강력한 이미징 방법을 제공한다. 이 방법을 사용하여, 우리는 C를 식별엘의 낭배시의 재 배열 및 GFP를 이용 낭배 동안 정점 수축의 중요성을 입증은 DE-cadherin의 표지.

여기서, 우리는 초파리 배아의 낭배 이벤트 및 배아 준비 절차에 대한 일반적인 개요 (도 1)를 나타낸다. 세포막 DE 헤린 : GFP 및 세포 이동의 라이브 영상은 초파리 낭배시에 수행된다 (도 2)를 이용하여 라벨링된다. DE-cadherin의의 GFP 파리는 우리가 세포 부착 접합을 시각화 할 수 있기 때문에, 우리는 혀끝의 세포 모양이 시스템을 사용하여 ?...

Watch this Scientific Journal Video about Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies at JoVE.com

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

셀 모양 변경 허가 및 세포 운동에서의 영상<em&gt; 초파리</em&gt; 낭배 사용 DE-cadherin의 기자 형질 전환의 파리

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. ...

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Developmental Biology

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

7.0K 조회수.  Jahangirnagar University. Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

비디오: Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Photoconvertible PSmOrange 시스템을 사용하여 GFP 유전자 변형 Zebrafish의 세포를 추적

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

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발생학

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

분석<em&gt; 생체</em&gt; 셀 마이그레이션 Zebrafish의 배아에서 세포 이식 및 시간 경과 이미징을 사용하여

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

다중 광자 현미경으로 라이브 셀 이미징을위한 성인 형질 전환 Zebrafish의 망막의 Explants의 문화

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Mouse Secondary Palate Fusion의 라이브 이미징

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

해 부와 초파리 번데기 난소의 얼룩

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a G&#945;12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway.

조사 비 근육 Myosin II 수축 통해 접혀 gastrulation 신호 전달 경로 초파리 S2R + 셀에 셀 기반 분석 결과

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

배양 된 초파리 계란 챔버의 시간 경과 영화를 사용하여 로컬 세포 및 조직 저울에서 Actomyosin 역학분석

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

녹고, 경작, 그리고 초파리 셀 라인 Cryopreserving

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, and 3D reconstruction.

전체 마운트 면역 조직 화학 및 3D 재건을 사용하여 인두 아치 동맥의 시각화 및 분석

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

살아있는 열에 있는 화상 진찰 내 핵 액틴 막대는 Drosophila 배아를 강조했습니다

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...