We thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital for their technical support.

すべての手順は、眼科と視覚研究における動物の使用のためのビジョンと眼科計算書の研究のための協会に従う本研究で続き、国立台湾大学病院の施設内動物管理使用委員会によって承認されました。 1.単離、初代培養の準備、およびウシのCECの免疫染色<ol><li>地元の食肉処理場から新鮮な牛の目を獲得。 </li><li> 3分間10％w / vのポビドンヨード溶液中で目を消毒。リン酸緩衝生理食塩水（PBS）溶液でそれらを洗浄します。 </li><li>無菌条件下でのメスやハサミで角膜ボタンを収穫。ピール解剖顕微鏡下でピンセットでデスメ膜は、（本研究では、ウシ目は地元の食肉処理場により摘出していることに注意してください。したがって、最初の研究手順は研究室でpreenucleated目の消毒ありました）。 </li><li>トライの1ミリリットルでデスメ膜をインキュベート37℃で30分間PSIN。 5分間112×gで遠心分離することによってウシのCECを収集します。補充ホルモン上皮培地（SHEM）HEPES緩衝ダルベッコ改変イーグル培地、5％ウシ胎児血清を補充したハムF12培地、0.5％のジメチルスルホキシド、人間の表皮の2 ng / mlでの等量を含有する1mlの細胞を再懸濁成長因子、インスリンの5 mg / mlで、トランスフェリンの5 mg / mlで、セレン5ngの/ mlのコレラ毒素の1ナノモル/ Lのゲンタマイシンを50mg / mlであり、アンフォテリシンBの1.25 mg / mlで</li><li> 6センチメートル皿に細胞（眼あたり約1×10 5細胞）をシード。 SHEM文化それらを。空気中5％のCO 2の雰囲気中、37℃で皿をインキュベートします。 3日ごとに培地を変更します。 </li><li>細胞がコンフルエントに達したとき、PBSでそれらを洗浄し、5分間37℃でトリプシン1mlにそれらをインキュベートします。 5分間、112×gで遠心分離することによって、それらを収集します。 SHEMの1ミリリットル中に細胞ペレットを再懸濁します。 <ol&gt; <li>血球計で細胞をカウントします。 24ウェルプレート中のウェル当たり1×10 4の密度、および文化SHEM中の細胞でカバースライド上の細胞をシード。マリマスタットのEnMT抑制効果を調査するために、培養培地に添加しマリマスタットの10μMとSHEM中で細胞をインキュベートし、3日ごとに培地を変更します。 </li></ol></li><li>室温で30分間、4％パラホルムアルデヒドを250μl、pH7.4で指示された時点で細胞を固定してください。 5分間、0.5％トリトンX-100250μlの透過性。 30分間、10％のウシ血清アルブミンでブロックします。 </li><li> 4℃で一晩アクティブβ-カテニン（ABC）、カタツムリ、およびナメクジに対する一次抗体で細胞をインキュベートします。用いた一次抗体の希釈は1：200。抗体を10mMのPBS、1％w / vのウシ血清アルブミン、および0.09％のw / vアジ化ナトリウムからなる抗体希釈液で希釈します。 </li><li> 15分間、PBSで2回細胞を洗浄し、インキュベート室温で1時間：アレクサフルオロ結合二次抗体（抗体希釈液で100 1）を持つメートル。 </li><li> 4 &#39;、6-ジアミジノ-2-フェニルインドールを2μg/ mlの細胞をカバーすることにより細胞核対比、15分間、PBSで2回細胞を洗浄し、取り付け溶液抗退色でそれらをマウント。 </li><li> 20X対物倍率でレーザ走査共焦点顕微鏡を用いて405および488nmでの励起波長での免疫蛍光画像を得ます。 </li></ol> 2.ラット角膜内皮Cryoinjuryモデルと房内注入<ol><li> 2％キシラジン（5.6ミリグラム/キログラム）とチレタミンプラスゾラゼパム（18ミリグラム/キログラム）の筋肉内注射で12週齢の雄性Sprague-Dawleyラットを麻酔。優しく肌のけいれんの不在下で適切な麻酔を確認するために、動物の皮膚をつまみます。 </li><li>眼の痛みや瞬目反射を最小限に抑えるために、各ラットの右眼に0.5％塩酸プロパラカインを一滴を植え付けます。植え付けます角膜の乾燥を防ぐために、左眼にテトラサイクリン軟膏。 </li><li>液体窒素中でステンレススチールプローブ（直径= 3 mm）を冷却します。 30秒間、右眼の角膜中央にステンレス製のプローブを適用します。頻繁に角膜の乾燥を防ぐための処置中に右眼にPBSを植え付けます。 </li><li>すぐcryoinjuryた後、0.1％アトロピンおよび0.3％の硫酸ゲンタマイシンを植え付けると、一日一回毛様体のけいれんに起因する眼の痛みを和らげるために、感染を防ぐために。 <ol><li>手順の後、ヒートランプを使用して温め、そして彼らは、モータ制御を取り戻すまで、15分ごとに、麻酔後にそれらの回復を観察したラットを保ちます。また、回復期間中に角膜の乾燥を防ぐために、右目にテトラサイクリン軟膏を適用します。 </li></ol></li><li> 3日間連続して角膜cryoinjuryを繰り返します。 </li><li>前述のようにラットの眼の前房内にマリマスタットまたはbFGFのを提供するために、ラットを麻酔。一滴を植え付けます眼の痛みや瞬目反射を最小限に抑えるために、右眼に0.5％プロパラカイン塩酸塩。 </li><li>滅菌PBSで眼表面を灌漑。アイリスに上記と平行面でparalimbal透明な角膜で1mlシリンジに取り付けた30 G針を挿入することにより、手術用顕微鏡下で前房穿刺を行います。 </li><li>針のベベルアップを回し、少しいくつかの房水を排出し、眼圧を低下させるために角膜の傷を押し下げます。房内薬剤の0.02ミリリットルを注入します。静かに針引き抜きの間綿棒で針管を圧縮します。 </li><li>手術用顕微鏡下で指示された時点で、外部の目を撮影します。 </li></ol> 3.収穫ラット角膜ボタンと免疫染色<ol><li>ラットを安楽死させるために、安楽死室でそれらを配置し、毎分室容積の10から30パーセントの充填率で100％のCO 2を吹き込みます。以下のためのCO 2注入を維持します呼吸と色あせた目の色の欠如後の追加分。 </li><li>鋭利な刃で角膜輪部でのラットの目を貫通します。角膜輪部に沿って角膜ハサミで角膜をカットします。スライド上の角膜を平ら。角膜が丸まる場合には、追加の半径方向の切開を行います。 </li><li>室温で30分間、4％パラホルムアルデヒドを250μl、pH7.4で角膜を修正しました。 5分間、0.5％トリトンX-100250μlの透過性、及び30分間、10％のウシ血清アルブミンでブロックします。 </li><li> 1の希釈で4℃で一晩ABCに対する一次抗体を用いて角膜をインキュベート：抗体希釈液で200。 15分間、PBSで2回角膜を洗浄し、二次抗体でそれらをインキュベート：1時間室温で（1抗体希釈液中の100）。 4を2μg/ mlの細胞を覆うことによって細胞核を対比染色 &#39;、6-ジアミジノ-2-フェニルインドール、及び15分間、PBSで細胞を2回洗浄します。 </li><li>退色防止マウントソリューションで角膜をマウントします。オビ20X対物倍率でのレーザー走査型共焦点顕微鏡を用いて405と561 nmでの励起波長での免疫蛍光画像をテイン。 </li></ol>

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The authors have no competing financial interests to declare.

CECは、細胞増殖中EnMTを受けるそれらの傾向のために知られています。治療目的のためにEnMTプロセスを抑制するための戦略を開発するには、EnMTメカニズムの完全な理解が必要です。我々はEnMT、 インビトロ培養モデルおよびラット角膜内皮cryoinjuryモデルで 、すなわちウシCECを調査するために2モデルを説明しました。我々の結果は、両方のモデルでEnMTプロセスを実証しました。...

Corneal endothelial cells (CECs) play a vital role in maintaining corneal clarity and thus visual acuity by regulating the hydration status of the corneal stroma through active pumping1. Because of the limited proliferative potential of human CECs, the cell number decreases with age, and the repair of corneal endothelial wounds following injury is usually achieved through cell enlargement and migration, rather than cell mitosis2. When the CEC count decreases below a threshold of approximately 500 cells/mm2, the dehydration status of the corneal stroma cannot be maintained, leading to bullous keratopathy and vision impairment3,4.
The limited proliferative potential of human CECs has been attributed to several factors, including reduced expression of the epidermal growth factor and its receptor in aging cells5, antiproliferative TGF&#946;2 in the aqueous humor6, and contact inhibition2,7. Although some growth factors, such as basic fibroblast growth factor (bFGF), can increase proliferation in a cultured human corneal endothelium, the culture efficiency remains limited8,9. Furthermore, CECs may undergo a phenotypic change during ex vivo expansion, resembling epithelial-mesenchymal transition (EMT)10-13. Endothelial-mesenchymal transition (EnMT) is characterized by cell junction destabilization, apical-basal polarity loss, cytoskeletal rearrangement, alpha smooth muscle actin expression, and type I collagen secretion14. All of these characteristics may abrogate the normal function of CECs, hampering the use of ex vivo cultured CECs in tissue engineering. Moreover, EnMT has been associated with the pathogenesis of several corneal endothelial diseases, including Fuchs endothelial corneal dystrophy and retrocorneal membrane formation15,16. Therefore, understanding the mechanism of EnMT may aid in manipulating the EnMT process and facilitate the regeneration of CECs to enable competent function. 
In this study, we described a method for isolating bovine CECs from the corneal button. In the primary culture in vitro, the EnMT process, including a phenotypic change, the nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, was observed. We further described a method for demonstrating EnMT in vivo by using a rat corneal endothelium cryoinjury model. Using these 2 models, we demonstrated that marimastat, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, can suppress the EnMT process. The described protocols facilitate the detailed analysis of the EnMT mechanism and the development of strategies for manipulating the EnMT process for further clinical application.

<table border="0" cellpadding="0" cellspacing="0" width="1447">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">trypsin</td>
			<td style="width:269px;">ThermoFisher Scientific</td>
			<td style="width:143px;">12604-013</td>
			<td style="width:340px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s modified Eagle medium and Ham&#39;s F12 medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal bovine serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human epidermal growth factor</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">insulin, transferrin, selenium&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cholera toxin</td>
			<td>Sigma</td>
			<td>C8052-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentamicin</td>
			<td>ThermoFisher Scientific</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">amphotericin B</td>
			<td>ThermoFisher Scientific</td>
			<td>15290-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>111219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">marimastat</td>
			<td>Sigma</td>
			<td>M2699-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-active beta-catenin antibody</td>
			<td>Millpore</td>
			<td>05-665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-snail antibody</td>
			<td>Santa cruz</td>
			<td>sc28199</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-slug antibody</td>
			<td>Santa cruz</td>
			<td>sc15391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11001</td>
			<td>for staining of ABC of bovine CECs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11003</td>
			<td>for staining of ABC of rat corneal endothelium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-rabbit IgG (H+L) secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11008</td>
			<td>for staining of snail and slug of bovine CECs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">antibody diluent</td>
			<td>Genemed Biotechnologies</td>
			<td>10-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#39;,6-diamidino-2-phenylindole</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">laser scanning confocal microscope</td>
			<td>ZEISS</td>
			<td>LSM510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">xylazine&#160;</td>
			<td>Bayer</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tiletamine plus zolazepam</td>
			<td>Virbac</td>
			<td>N/A</td>
			<td>veterinary drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">proparacaine hydrochloride ophthalmic solution</td>
			<td>Alcon</td>
			<td>N/A</td>
			<td>veterinary drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1% atropine</td>
			<td>Wu-Fu Laboratories Co., Ltd</td>
			<td>N/A</td>
			<td>clinical drug&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.3% gentamicin sulfate</td>
			<td>Sinphar Group</td>
			<td>N/A</td>
			<td>clinical drug&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">basic fibroblast growth factor</td>
			<td>ThermoFisher Scientific</td>
			<td>PHG0024</td>
			<td>clinical drug&#160;</td>
		</tr>
	</tbody>
</table>

in vitro and in vivo models to study corneal endothelial-mesenchymal transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

ウシのCECの単離後、細胞をインビトロで培養した。 図1は、ウシのCECの位相コントラスト画像を提示します。コンフルエンスの細胞の六角形は、細胞が細胞単離の間、角膜間質線維芽細胞によって汚染されていないことを示した。 図2に示された時点でABC、カタツムリ及びナメクジに対する抗体を用いて行った免疫染色を示します。 ...

Watch this Scientific Journal Video about In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition at JoVE.com

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

ウシ角膜内皮細胞の初代培養は、角膜内皮間葉移行のメカニズムを調べるために使用しました。さらに、ラット角膜内皮cryoinjuryモデルは、in vivoで角膜内皮間葉移行を示すために使用されました。

in vitroおよびin vivoモデルで角膜内皮間葉転換を研究します

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

in-vitro-vivo-models-to-study-corneal-endothelial-mesenchymal

Research

JoVE Journal

Developmental Biology

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

7.9K ビュー.  Far Eastern Memorial Hospital. ウシ角膜内皮細胞の初代培養は、角膜内皮間葉移行のメカニズムを調べるために使用しました。さらに、ラット角膜内皮cryoinjuryモデルは、in vivoで角膜内皮間葉移行を示すために使用されました。 

ビデオ: In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺の特性を用いてマウス胚における前駆細胞をセトリング<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;アッセイ

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

発生生物学

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

幹細胞様<em&gt;アフリカツメガエル</em&gt;胚外植片を早期神経発達の特徴を研究するために<em&gt;インビトロ</em&gt;と<em&gt;インビボ</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

開発<em&gt;インビトロ</em&gt;アッセイは、間葉細胞受けた上皮間葉移行の収縮機能を評価します

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

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.

マウス二次口蓋融合のライブイメージング

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 Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

郭清ときのこ体の免疫蛍光染色と大人のショウジョウバエ脳光受容器ニューロン

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

線虫のニューロンに生殖細胞を再プログラムする RNAi と熱衝撃による転写因子の発現のアプリケーション

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

ノードと走査型電子顕微鏡と蛍光抗体法マウント全体を使用して Gastrulating のマウス胚における脊索板を可視化

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

骨組織の再生にプレドニゾロンの効果を研究する大人のゼブラフィッシュ傷害モデル

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

移植組織の細菌付着を勉強する平行平板灌流システムのIn Vitroモデル

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

マウス血液精巣関門の整合性を研究するIn Vivo法

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...