S843: LI1 ve Tg (EGFP kdrl): Biz Tg (GFP wt1b) hediye Prof. Christoph Englert ve Prof. Didier Stainier teşekkür suşu, sırasıyla, Tg (kdrl: mCherry) sağlanması Tayvan balığı Çekirdek Tesis ci5. Bu çalışma 102-, Bilim ve Teknoloji (96-2628-B-029-002-My3, 101-2313-B-029-001, 102-2628-B-029-002-My3 Tayvan Bakanlığı hibe ile desteklenmiştir 2321-B-400-018).

Etik Beyanı: zebrabalıkları tüm deneysel prosedürleri Tunghai Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi (KİK Onay NO 101-12.) Ve onaylı kurallarına uygun olarak gerçekleştirdi. 3β-Hsd enzimatik Aktivite Boyama için 1. Stok Çözümleri <ol><li> trans-dehidroepiandrosteron [dimetil sülfoksit (DMSO) içinde 10 mg / ml] hazırlayın. </li><li> β-nikotinamid adenin dinükleotid hidrat (0.1 M fosfat tampon maddesi içinde 1.2 mg / ml, pH 7.2) hazırlayın. </li><li> Nikotinamid hazırlanması (B3 vitamini, 50 mg / ml H2O). </li><li> 4-nitro mavi tetrazolyum (50 mg / ml% 70 dimetilformamid) hazırlayın. </li><li> -20 ° C&#39;de mikrofüj tüpleri ve mağaza içine kısım tüm bu bileşenler. NOT: bizim deneyim, tekrarlanan donma ve tüm yukarıda belirtilen çözümlerin hacimde çözülme ise 3β-Hsd etkinlik boyama yoğunluğunu etkilemez. </li></ol> 2. Wdelik montaj 3β-Hsd Etkinlik Boyama Not: 3β-HSD &#39;enzimatik aktivitesi erken 28 saat sonra döllenme (HPF) halinde kültürlendiğinde hsd3b1 2,7 mRNA ifadesinin başlangıcı ile tutarlıdır 28.5 ° C, zebra balığı embriyolar tespit edilebilir. Bu çalışmada, tüm montaj 3β-HSD &#39;aktivitesi boyama protokolü daha önce tarif edilen yönteme 8 dayanır ve 7 gün sonra gübreleme 9 gelişmekte zebrabalıkları başarılı olduğu saptanmıştır. Interrenal steroidojenik doku damar mikro-analizi için, 3β-HSD &#39;aktivitesi boyama tüm montaj gibi Tg olarak endotel spesifik floresan ifade eden transgenik kuşaklar (kdrl: EGFP) uygulanması gerekir: ci5 15 14 ve TG (MCherry kdrl) s843 . Zebra balığı böbrek gelişimini göz önünde bulundurarak interrenal steroidogenik doku analiz etmek, 3β-Hsd etkinlik boyama başvurunun olabilirLI1 balık 16 glomerülleri ve Pronephric tübüller oluşumu tarif edilmektedir: Tg (GFP wt1b) ED. <ol><li> Pigment oluşumunu inhibe etmek için, 12 ve 24 HPF arasındaki aşamasından, yumurta suda% 0.03 feniltiyoüre bölgesindeki (deiyonize su içinde 0.06 mg / L resif tuzu) onları kuluçka geliştirme zebrafish tedavi edin. </li><li> gece boyunca, oda sıcaklığında (RT) ya da (B),% 2 PFAT de 1 saat süre ile,% 0.1 Tween 20 (PFAT) ile desteklenmiş fosfat tamponlu tuz (PBS) içinde (A)% 4 paraformaldehit (PFA) dechorionated embriyolar veya larvaları saptamak 4 ° C ve oda sıcaklığında 4 saat ve ardından 4 ° C&#39;de 2 saat için. Dikkat: PFA Solunduğunda ve cilt ile temasında toksiktir. Bu cilt, mukoza zarları, gözler ve üst solunum yollarında yıkıcıdır. </li><li> PBS oda sıcaklığında,% 0.1 Tween 20 (PBST) ile desteklenmiş olan 10 dakika için embriyo 4x yıkayın. NOT: yıkama solüsyonları değiştirirken örnekleri kuru değil için çok önemlidir. </li><li> Taze sta hazırlamakreaksiyonlar daha önce ining çözeltisi (Tablo 1). Hazırlamak girdap ve ayrı tüplerde santrifüj parçaları A ve B. NOT: Düz tek tüp içine tüm bileşenleri ekleme şiddetli yağış neden olur. </li><li> , B kısmının tamamı çözeltiye A kısmının tüm solüsyonu ilave boyama solüsyonu hazırlamak için iyice karıştırın ve hemen sabit ve yıkandı embriyoları içeren her bir mikrofüj tüpüne 1 mi dağıtın. </li><li> rotator veya oda sıcaklığında nazik çalkalama için sallanan bir platform da ışık ve yerine korumak için alüminyum folyo ile mikrofüj tüpleri sarın. </li><li> Ampirik mikroskobu ile kromojenik sinyalleri izleyerek reaksiyon süresini belirler. zamanla gelişecektir hafif yağışlar; Bununla birlikte, bu reaksiyon işlemi engel olmaz. Not: Embriyolar 4 saat 25 ° C&#39;de 28 HPF boyama sabit için interrenal doku net bir sinyal üretir. </li><li> PBST içinde 10 dakika için 4x yıkama ederek reaksiyonu durdurun. benf başka immünohistokimyasal (IHC) analizi gereklidir sonrası tamir oda sıcaklığında 60 dakika boyunca% 4 PFAT ile boyanmış embriyolar. Aksi takdirde, bir sonraki kullanıma kadar 4 ° C &#39;de yıkandı, embriyolar saklayın. </li><li> tüm montaj mikroskopi için, PBS içinde% 50 gliserol ile lekeli sonradan tespit ve yıkanmış embriyolar temizlemek bir slayt yukarı bakacak şekilde sırt tarafında onları yönlendirmek ve parlak saha aydınlatma altında dik bir mikroskop kullanılarak gözlemlemek. NOT: tavsiye edilir interrenal doku morfolojisi bir yüksek çözünürlüklü görüntü, 3β-Hsd aktivite lekeli ve deyolked embriyo analizi, düz monte yapmak için. Interrenal steroidogenik doku sağ yumurta sarısının üzerine konumlandırılmış olarak, interrenal doku ventral düz montaj analizi interrenal morfolojisi 17 doğrudan ve net gözlem sağlar. </li></ol> 3β-Hsd Etkinlik lekeli Embriyolar 3. IHK Analizi NOT: steroi vasküler mikro analiz içindogenic dokusu, bir transgenik endotel spesifik flüoresan raportör 3β-HSD &#39;aktivitesi lekeli embriyolar vibratome kesilmesi ve daha sonra IHC analizi 12,18 enine tabi tutulur. <ol><li> Post-fiksasyon <ol><li> 4 ° C &#39;de 1 saat süre ile% 1 Triton X-100 ile desteklenen% 2 PFA 3β-HSD&#39; aktivitesi-boyanmış ve yıkandı embriyolar sonrası düzeltmek ve% 1 Triton X-100 içeren PBS içinde 10 dakika boyunca 4x yıkayın (PBSTx) oda sıcaklığında karıştırıldı. </li></ol></li><li> katıştırma <ol><li> % 4, 95 ° C, 4-8 ° C de mikrofüj tüplerine kısım ve mağazada PBS içinde agaroz eritilerek agaroz erime noktası düşük hazırlayın. </li><li> 10 dakika süre ile 70 ° C &#39;de agaroz% 4 düşük erime bir kısım eritin ve sonra 47 ° C&#39;de kısım tutun. Her embriyo yerleştirme eriyik% 4 bir kalıp olarak hizmet veren bir 1.5 mi mikrofüj tüpün müstakil bir kap içinde agaroz, erime noktası düşük, ve katı kadar soğumaya bırakın. </li></ol></li><li> vibratome Kesit <ol><li> Kuru p kağıt şerit bir parça sopavibratome latform. </li><li> bir jilet ile kalıp sertleştirilmiş agaroz blok çıkarın. platformda kağıt şerit superglue uygulayın ve yukarı bakacak şekilde numunenin ön tarafı, kağıt şerit üzerine agaroz blok düzeltmek. sırasıyla, yaklaşık olarak 4 mm, üst ve alt yüzlerin her bir tarafında 8 mm olan, bir yamuk prizma şekline agaroz blok kesin. </li><li> Bir damlalık kullanılarak,% 0.5 Triton X-100 (% 0.5 PBSTx) ile desteklenmiş, soğuk PBS ile numune içeren agaroz blok durulayın. </li><li> vibratome kullanım kılavuzuna göre 100 mikron bölüme agaroz blok dilimleyin. Sürekli kurumasını önlemek için agaroz blok durulayın. Her bir dilim kesme gibi, bir 26 G iğne kullanılarak şırınga vibratome çıkarın ve soğuk% 0.5 PBSTx (500 ul / oyuk) ihtiva eden bir nokta plakaya aktarılır. </li><li> Diseksiyon mikroskobu altında kontrol ederek 3β-Hsd aktivite pozitif doku bölümleri toplayın ve bir pi ile bölümleri aktarmakSoğuk% 0.5 PBSTx (150 ul / oyuk) ihtiva eden bir 96-çukurlu plaka uç kesme (açıklık yaklaşık 1 mm iç çap) sahip Pette ucu. doku örnekleri genellikle bu aşamadan sonra agaroz blok ayırmak. </li></ol></li><li> Permeabilization ve Yıkama <ol><li> PBS içinde 10 dakika% 1 sığır serum albümini /% 1 DMSO /% 0.1 Triton X-100 (PBDTx) ile desteklenmiş dilimler, oda sıcaklığında,% 0.5 PBSTx 15 dakika PBS içinde 10 dakika ve 4 kere 6x yıkayın. </li></ol></li><li> bloke etme <ol><li> Oda sıcaklığında PBDTx 1 saat,% 2 fetal sığır serumu (FCS), dilimler inkübe edin. </li></ol></li><li> Primer antikor reaksiyonu <ol><li> Birincil antikor içeren PBDTx 1.5 gün inkübe dilimleri (örneğin, tavşan poliklonal anti-insan fibronektin ve fare anti-insan fokal yapışma kinaz) 4 ° C&#39;de ilave edildi. </li></ol></li><li> Yıkama <ol><li> Dilimler oda sıcaklığında PBDTx 10 dakika boyunca 6 kat yıkayın. </li></ol></li><li> bloke etme <ol><li> PB içinde% 2 FCS içinde 1 saat boyunca inkübe dilimleriOda sıcaklığında DTx. </li></ol></li><li> İkincil antikor reaksiyonu <ol><li> 4 ° C &#39;de ikinci bir antikor (florofor-konjuge keçi anti-tavşan veya anti-fare IgG&#39;si) ihtiva eden PBDTx 1.5 gün inkübe dilimleri. </li></ol></li><li> Yıkama <ol><li> Dilimler oda sıcaklığında PBST 10 dakika süreyle 10 oda sıcaklığında PBDTx içinde min ve 4x için 6x yıkayın. , Odaklı mikroskopik analizi için PBS içinde% 50 gliserol ile örnekleri temizlemek: NOT. </li></ol></li></ol> 4. Konfokal Analizi <ol><li> tüm montaj embriyo ve 10X / 0.5 ve 20X / 0.75 objektif lens ile donatılmış bir konfokal mikroskop ile bölümüne dikkat ediniz. </li><li> aşağıdaki gibi floresans ve 3β-HSD &#39;boyama birinin eş zamanlı saptanması için, çok kanallı ayar modu: 488 nm argon lazeri ve GFP tespiti için 505-530 nm bant geçişi filtresi; 543 nm Helyum-Neon lazer ve mCherry tespiti için 560-615 nm bant geçiş filtresi; ve 3β-Hsd boyama tespiti için ışık kanalı iletti.iğne deliği boyutu 1 Airey birimi ve çözünürlüğü 1024 x 1024 piksel olduğunu. </li><li> 3D projeksiyon olarak (6 mikron aralığı ile) z yığınları birleştirin ve kullanarak, projeksiyon ve parlak bir alan birleştirme yerleşik konfokal yazılımı. </li></ol>

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The authors have no competing financial interests to declare.

3β-HSD aktivitesinin sinyal gücü reaksiyon boyunca arttı. 3β-HSD aktivitesinin net sinyaller itibaren 28 HPF gelen aşamaları için reaksiyon 4 saat sonra tespit edildi. Bununla birlikte, reaksiyon süresi deneyi amacına bağlı olarak, ampirik belirlenmesini gerektirir. boyama gece boyunca işlem gerektirir durumlarda, hafif mavimsi arka örneklerinin geliştirme eğilimindedir. Bu sorun, 3β-HSD &#39;aktivitesi boyama önce sabitleme süresi (örneğin, oda sıcaklığında 4% PFAT 4 saat) arttırarak ...

böbreküstü bezi, hipotalamus-hipofiz-adrenal ekseninin önemli bir bileşeni, steroid salgılar ve steroid homeostazı ve strese bedensel yanıtını koordine eder. böbrek üstü bezi katekolamin sentezleyen bir bölge-spesifik bir şekilde steroid salgılayan dış korteks ve iç medulla içerir. Teleostlarda interrenal bezi, memelilerde adrenal bez karşılığıdır ve adrenal korteks ve medullada sırasıyla 1-3 fonksiyonel eşlenikleridir steroidojenik interrenal ve kromafin hücreleri, oluşmaktadır. Zebra balığı model kullanarak yapılan çalışmalar, hem steroidogenik ve kromaffin hücre soylar yüksek memelilerde 1,2 benzeyen moleküler ve hücresel mekanizmaları tarafından oluşturulan bildirmiştir. Bu nedenle, Zebra balığı, genetik bozukluklar, nöroendokrin kontrolü ve hipotalamus-hipofiz-adrenal (interrenal) ekseninin sistem biyolojisi çalışmak için bir potansiyel olarak güçlü bir modeldir. 4,5 ila 17α-hydroxypregnelolone gelen pregnenolon, 17a-hidroksiprogesteron progesteron dönüşümünü ve androstenedion katalize eder. 3β-HSD &#39;hormonal steroid, yani progesteron, glukokortikoid, mineralokortikoid, androjen ve östrojenlerin her sınıfları üreten mutantı için gereklidir. İki insan 3β-Hsd HSD3B1 izozimine ve HSD3B2 farklı şekilde 6 ifade edilir. HSD3B2 adrenal korteks ve gonadlar ifade edilir ise HSD3B1, plasenta ve periferik dokularda ifade edilmektedir. İnsan HSD3B1 ve HSD3B2 interrenal doku ve yetişkin gonadlar ifade edilen Zebra balığı hsd3b1, eş ortologlar vardır; Zebra balığı hsd3b2 olan transkript organogenez 7 önce kaybolur bir anne olarak ifade gendir. zebrabalıkları için tüm montaj 3β-HSD &#39;enzimatik aktivite deneyi protokol, Levy yöntemini değiştirerek geliştirilenMilano ve diğerleri tarafından tarif s. Sekiz Teleost türlerinin 8 donmuş bölümlerinde. Çünkü doku geçirgenliği ve gelişmekte olan zebrafish optik şeffaflık, 3β-Hsd histokimya başarıyla sabit Zebra balığı embriyo ve larva için kullanılabilir tüm montaj ve özellikle farklılaşmış interrenal dokuları tasvir. Bu, hassas ve hızlı bir deney interrenal dismorfogenezis farklı gösteren Çeşitli mutantlar ve morphants uygulanmıştır. Interrenal 3β-HSD &#39;aktivitesi interrenal doku dağılımı Ff1b transkripsiyon faktörünün belirli bir devirme yoluyla bozulur ve interrenal farklılaşma Ff1b coregulator Prox1 9,10 bir devirme etkilenir azaltılır embriyoda yoktur. Özellikle, 3β-Hsd faaliyeti gibi tek gözlü toplu iğne başı, şiddetli erken evre defekti olan mutantlar tespit edilebilir ve nerede 3β-H, şaşılıksd histokimyası interrenal hücre göçü 11 nasıl etkilendiğini anlatır. interrenal doku farklılaşması bile kan ve damarsal tam yokluğunda ödün verilmemiş. Bu nedenle endotelyum türevli sinyalleri şekil kadar geliştirilmesi interrenal organı 12,13 belirlenebilir. Genel olarak, bu histokimyasal deney başarıyla zebrabalıkları modelinde özellikleri, farklılaşmasını ve steroidojenik hücrelerinin göçünü incelemek için kullanılmıştır. Bu nedenle, verimli ve adrenal ve interrenal organ bozuklukları hedefleyen herhangi bir genetik veya kimyasal ekranlar için güvenilir bir aracı olmalıdır.

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			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma</td>
			<td>D8418&#160;</td>
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			<td height="21" style="height:21px;">Glycerol</td>
			<td>USB</td>
			<td>US16374</td>
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			<td height="21" style="height:21px;">Hyclone Fetal Calf Serum&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30073</td>
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			<td height="21" style="height:21px;">Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#946;-Nicotinamide adenine dinucleotide hydrate</td>
			<td>Sigma</td>
			<td>N1636</td>
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			<td height="21" style="height:21px;">4-Nitro blue tetrazolium</td>
			<td>Promega</td>
			<td>S380C</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Nusieve GTG</td>
			<td>Lonza</td>
			<td>50081</td>
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			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Phenylthiourea</td>
			<td>Sigma&#160;</td>
			<td>P7629</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Sigma</td>
			<td>P4417-100Tab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PYREX Spot Plate</td>
			<td>Corning</td>
			<td>7220-85</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reef Salt</td>
			<td>AZOO</td>
			<td>AZ28001</td>
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		<tr height="21">
			<td height="21" style="height:21px;">trans-Dehydroandrosterone</td>
			<td>Sigma</td>
			<td>D4000</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td>Sigma</td>
			<td>P9416</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td>Leica</td>
			<td>VT1000M</td>
		</tr>
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visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

Pronephric böbrek glomerül ve doğmakta olan damarsal ile steroidogenik interrenal doku codevelops, 3β-Hsd enzimatik aktivite deneyi çift transgenik Tg (wt1b: GFP) üzerinde gerçekleştirildi nasıl belirlemek için LI1; Tg (kdrl: mCherry) ci5 embriyo 34 hpf (Şekil 1). Bu aşamada, 3β-Hsd aktivitesi pozitif steroidogenik doku sağ orta hatta yer alan ve bazı steroidogenik hücreler orta hatta genelinde göç başlar ve dors...

Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Zebra balığı interrenal bezi memeli adrenal bezin teleostean karşılığıdır. Enzimatik aktivite deneyi, gelişmekte olan Zebra balığı farklılaşmış steroidogenik hücreleri tespit; Bu protokol 3-β hidroksisteroid dehidrojenaz (3β-Hsd Δ 5-4 izomeraz) nasıl gerçekleştirileceği tanıtır.

Interrenal steroidogenik Doku ve Zebra balığı Onun Vasküler Mikroçevre görselleştirme

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

visualizing-interrenal-steroidogenic-tissue-its-vascular

Research

JoVE Journal

Developmental Biology

6.9K Görüntüleme.  Tunghai University.  Zebra balığı interrenal bezi memeli adrenal bezin teleostean karşılığıdır. Enzimatik aktivite deneyi, gelişmekte olan Zebra balığı farklılaşmış steroidogenik hücreleri tespit; Bu protokol 3-β hidroksisteroid dehidrojenaz (3β-Hsd Δ 5-4 izomeraz) nasıl gerçekleştirileceği tanıtır. 

Video: Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Time-lapse aydınlık stereomikroskobi ile Zebra balığı Larva Doku Onarım yakalama

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Gelişim Biyolojisi

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Evans Blue Dye ile Zebra balığı Larva İskelet Kası Bütünlüğü Analizi

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

Larva Zebra balığı Gonadal Doku Diseksiyon

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

Sineği boyanmasına ile steroidogenik organları ve İnteraktif Organlar görselleştirme için Protokoller<em&gt; Drosophila melanogaster</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

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.

Vasküler Endotelyal Kök Hücrelerin Damar Oluşumunu Görselleştirmek İçin Yeni Bir Göğüs Göğsü Transplantasyon Tekniği

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

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.

Fare İkincil Palin Füzyonunun Canlı Görüntüsü

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 ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Zebra balığı bütün Dağı Floresan Situ hibridizasyon ve ayirt kombine bir protokol aracılığıyla Multiciliated hücrelerde görüntülenmesi

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Hücre toplama Trans Drosophila çentikle bağlama değerlendirmek için deneyleri-ligandlar ve onun inhibisyon CIStarafından-ligandlar

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

Zebra balığı embriyo hücre Photoconversion ile aşağıdaki Endocardial doku hareketleri

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

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).

Düğüm ve Notochordal plaka Gastrulating fare embriyo Tarama elektron mikroskobu ve bütün Dağı ayirt kullanarak görselleştirme

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