Name: development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos
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Description: Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

Ulusal Sağlık Enstitüleri tarafından sağlanan finansman (NIH: K01-DK087814-01A1 D.L.S. için), Kaliforniya Devlet Üniversitesi programı için eğitim ve araştırma biyoteknoloji (CSUPERB: omurgalı hematopoetik niş D.L.S. için moleküler kontrolü) ve California State Üniversitesi Chico (için A.C.B.) lisansüstü ofisinden.

Chico, California eyalet Üniversitesi'nde Kurumsal hayvan bakım ve kullanım Komitesi (IACUC) Danışma Kurulu aşağıda açıklanan tüm yöntemleri onayladı.
1. çamaşır suyu 48 hpf Zebra balığı embriyolar
<ol><li>15 cm (w) x 15 cm (l) x 7 cm (h) plastik kaplar oluşturmak 3 yıkama istasyonları: 1) ile 1 mL % 5 çamaşır suyu 1000 mL embriyo orta (E3; bkz: Tablo 1), 2) ile steril E3 ve 3) ile steril E3. Her kapsayıcı embriyo daldırın çözeltinin 500 mL olduğundan emin olun. Not: en az 10 embriyo test her koşul için gerekli olacaktır. Bu çamaşır yordamı 200 embriyo ağırlayacak.</li>
	<li>Transfer pipet ile embriyo bir çay süzgeci yerleştirin ve 5 dk. (ile embriyo içinde) çay süzgeci kaldırmak ve yıkama istasyonu #2 5 min için içine koyun yumurta yıkama istasyonu #1 daldırın; son 5 min için yıkama istasyonu #3 için yineleyin.</li>
	<li>Son yıkama sonra durulama embriyo çay süzgeci kısıtlayarak bu baş aşağı bir temiz 10 cm Petri kabına yavaşça embriyo durulama üzerinden kapalı steril E3 ile çay süzgeci ve transfer pipet.</li>
</ol>2. Dechorionate embriyo
<ol><li>Bir aktarım pipet ile kaldırın ve Petri kabına üzerinden mümkün olduğu kadar E3 atmak ve dechorionation proteaz (10 mg/mL) 500 μL embriyolar için ekleyin. 5 dk. hafifçe dokunun için tamamen chorions kaldırmak için Petri kabına yan oda sıcaklığında kuluçkaya.</li>
	<li>Serolojik pipet ile 20 mL steril E3 proteaz sulandırmak için ekleyin. Embriyo yerleşmek izin ve bir transfer pipet ile E3 kaldırın. Bu yıkama 3 kez dechorionation proteaz bütün izlerini kaldırmak için adımları yineleyin.</li>
</ol>3. embriyo örnekleri hazırlama
<ol><li>P1000 pipet kullanarak, 10 embriyo bir tek steril 1.5 mL microcentrifuge tüp içine yerleştirin.</li>
	<li>Bir pipet ile E3 kaldırmak ve atın. Dikkat: embriyo kolayca yerinden ve yıkama aşağıdakileri sırasında atılır gibi özenle pipet.</li>
</ol>4. embriyo ayrılma için hazırlanması
<ol><li>Laminar akış Hood'a örnekleri aktarmak ve yıkama embriyo 1 mL ekleyerek steril E3. Embriyo tüp altına yerleşmek ve süpernatant P1000 pipet ile kaldırmak izin verir. Toplam 3 yıkama için işlemi yineleyin.</li>
	<li>Son yıkama sonra E3 kaldırmak ve atın. 10 mM dithiothreitol (DTT) 1 mL mukus kaplama (ve herhangi bir sporlar, mantar veya bakteri içinde sıkışmış) embriyonik Zebra balığı çevreleyen kaldırmak için E3 içinde ekleyin. Microcentrifuge tüp yatay olarak yatıyordu ve laminar akış başlıklı 25 min için oda sıcaklığında kuluçkaya.</li>
</ol>5. embriyo ayrılma
<ol><li>Örnek 3 kez ile 1 mL steril DPBS (Ca2 + ve Mg2 +ile) yıkayın. Sonra son yıkama DPBS 500 μL (ile Ca2 + ve Mg2 +) ekleyin ve 5 mg/mL (26 U/mL) ayrılma proteaz 5 μL ekleyin. Dikkat: Bu enzim Ca2 + ve Mg2 +ihtiyacı var, bu yüzden DPBS Bu iyonlar içerdiğinden emin olun.</li>
	<li>Örnekleri üzerinde 60 dk. yer örnekleri laminar akış Hood için bir yatay orbital Çalkalayıcı-180 rpm'de 37 ° C'de kuluçkaya ve örnekleri tam ayrışmış kadar embriyo bir P1000 ile triturate. Dikkat: embriyo düzenli olarak ne zaman onlar ayrışmış haline belirlemek için denetleyin. Embriyo çözüm bir miktar doku mevcut olmalıdır; tamamen homojen olmaması gerekir. Mümkündür (bkz. ek şekil 1) HSPCs yok edecek aşırı Özet.</li>
</ol>6. Disosiye embriyo hazırlanması
<ol><li>Ayrışmış pipet 10 embriyo 5 mL polistiren üst rezervuarı üzerine alt tüp 35 mikron hücre süzgeç kap ile yuvarlak.</li>
	<li>Bir pipet ile durulama Petri tüp (Ca2 + veya olmayan Mg2 +) steril PBS ve 5 mL polistren tüp içeren transfer çözüm adım 6.1 hücrelerden filtre. Sıvı 4 mL 5 mL polistren tüp mevcuttur kadar yineleyin. Not: Ca2 + veya Mg2 + içerdikleri değil sürece PBS, DPBS veya diğer izotonik tamponlu çözümleri bu aşamada kullanılabilir
</li>
	<li>4 ° C ve 300 x g homojenize hücre örnek cips 5 min için tüpler santrifüj kapasitesi. Bir pipet ile kaldırmak ve yuvarlak alt tüp süpernatant atın. Tüp alt kısmında pelleted hücreleri bozmak değil için dikkat ediniz. PBS 100 μL hücrelerde resuspend.</li>
</ol>7. Methylcellulose hazırlanması
<ol><li>1 x tam methylcellulose (Tablo 1) hisse senedi çözüm üretmek ve 2.5 mL steril yuvarlak alt 14 mL tüpler 3 mL şırınga ve 16 G iğne ile ekleyin. Bir tüp test her koşul için kullanın.</li>
	<li>
Sitokinler, küçük moleküller veya diğer ajanlar her örnek için soruşturma olması için ekleyin.
	<ol>
<li>Myeloid farklılaşma için uyarıcı faktör (Gcsf)34 methylcellulose orta % 1 sazan serum ve 0.3 mg/mL rekombinant Zebra balığı granülosit koloni ekleyin. Bkz: Svoboda vd. 37 sazan serum ve rekombinant sitokinler oluşturmak nasıl tam açıklama için.</li>
		<li>Erythroid farklılaşma için % 1 sazan serum ve 0.1 mg/mL rekombinant Zebra balığı Eritropoietin (Epo)38 methylcellulose Orta olarak ekleyin.</li>
		<li>Multilineage ataları incelemek için Epo ve Gcsf ekleyin. Dikkat: Orta bireysel kolonileri ayırt etmek viskoz olmayacak gibi sitokinler ve katkı maddeleri toplam hacminin % 10 daha fazla toplam değil.</li>
	</ol>
</li>
</ol>8. Methylcellulose için Disosiye embriyo eklenmesi
<ol><li>Bir pipet kullanarak 100 μL Disosiye 10 embriyoların rekombinant sitokinler ve sazan serum ile hazırlanmış methylcellulose yüzeyine ekleyin. Cap kapak ve yavaşça tam örnek homojenize girdap. Her tüp Şimdi 10 embriyo Disosiye dokusunun içermelidir.</li>
	<li>3 mL şırınga ve 16 G iğneler, aliquot 1 mL örnek içine 2 ayrı 35 mm Petri yemekler kullanarak. Örnek tamamen Petri kabına dağınık olduğunu emin olun. Her örnek için yineleyin.</li>
	<li>35 mm Petri yemekler hücreleri ile methylcellulose içinde bir 15 cm Petri kabına yerleştirin. Her 15 cm yemek için bir 35 mm Petri tabağı ile 5 mL steril su örnekleri nemlendirmek için ekleyin. 32 ° C ve % 5 CO2 ' de 7-10 gün kuluçkaya.</li>
</ol>9. görselleştirme birimleri (CFUs) oluşturan Colony HSPC elde edilen
<ol><li>7 gün sonra kuluçka, 40-100 X görselleştirme ve numaralandırma örnekleri ters bir mikroskop üzerine yerleştirin. Bu noktada, bireysel kolonileri bir pipet ile izole ve boyama ve/veya gen analize tabi.</li>
</ol>

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L., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+dissection+of+zebrafish+hematopoiesis.">Cellular dissection of zebrafish hematopoiesis.</a> Methods Cell Biol. 133, 11-53 (2016).</li><li>Traver, D., 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=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat Immunol. 4, 1238-1246 (2003).</li><li>Kobayashi, I., 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=Characterization+and+localization+of+side+population+(SP)+cells+in+zebrafish+kidney+hematopoietic+tissue.">Characterization and localization of side population (SP) cells in zebrafish kidney hematopoietic tissue.</a> Blood. 111, 1131-1137 (2008).</li><li>Henninger, J., 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=Clonal+fate+mapping+quantifies+the+number+of+haematopoietic+stem+cells+that+arise+during+development.">Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development.</a> Nat Cell Biol. 19, 17-27 (2017).</li><li>Tamplin, O. J., 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=Hematopoietic+stem+cell+arrival+triggers+dynamic+remodeling+of+the+perivascular+niche.">Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.</a> Cell. 160, 241-252 (2015).</li></ol>

Yazarlar ifşa gerek yok.

Zebra balığı modeli sistemi ilkel ve kesin omurgalı hematopoiesis çalışmak için verimli, etkili ve ucuz bir model haline gelmiştir. Hızlı, ucuz ve az teknik zorluklar deneyleri nesil küçük moleküller testi, mutant embriyo çözümlemek ve moleküler pathways HSPC Biyoloji için önemli elucidating için yararlı olabilir. HSPCs in vitro kaplama--dan yetişkin Zebra balığı mutagenesis, sitokinler ve hematopoetik hataları incelemek için etkili bir yöntemdir. Bu iletişim kuralı bu çalışmal...

Hematopoiesis olgun kan hücreleri bir canlının hayatta kalmak için gerekli sayıda yapma işlemidir. Bu farklılaşma hematopoetik kök hücre (HSCs) içeren bir anahtar gelişimsel olgun kan oluşturan gelişimsel olarak sınırlı hücre tipleri çeşitli içine süreçtir. Bu HSCs böylece sistem asla yorgun ve onlar ölene kadar erken embriyonik gelişme ısrar gerekir kendini yenilemesi gerekir. Omurgalılar, sürekli farklılaşma ve hematopoetik kök ve progenitör hücrelerin (HSPCs) çoğalması yeterince sonrası Mitotik kan hücrelerinin çoğunluğu doldurmak için gerekli olan ve her gün geri dönüşümlü. HSCs ilk kısıtlı progenitör hücre alt kümeleri ayırt tarafından olgun kan hücreleri üretirler; sonunda T, B ve NK hücreleri üretmek, ortak lenfoid ataları (CLPs)1ve granülosit, eritrositler, makrofajlar ve megakaryocytes ortak Miyeloid ataları (CMPs)2 . Bu ataları belirli hücre soy oluşturmak için kararlı olan ve daha fazla daha fazla gelişim kısıtlı progenitör hücreler eritrositler ve trombosit veya granülosit oluşturmak myeloid erythroid ataları (milletvekilleri) gibi içine ayırt etmek bazofil, eozinofil, nötrofiller ve makrofajlar2üreten makrofaj ataları (GMPs). Belirlenmesi ve bu ataları yalıtma önemli moleküler yolları tanımlaması hematopoetik ayrımında yer sağlar ve lösemi gibi birçok hematopoetik hastalığın ortaya bu progenitör hücrelerin düzgün başarısız ayırt etmek.
Son birkaç on yıl içinde Zebra balığı (Danio rerio) modeli sistemi embriyonik ve yetişkin hematopoetik çalışmaları için bir anahtar araştırma aracı haline gelmiştir. Zebra balığı için genetik analiz mükellef bulunmaktadır ve bir benzer damarlara ve insanlara hematopoetik sistem filogenetik en düşük omurgalı modeli türü bulunmaktadır. Zebra balığı embriyolar rahim geliştirmek ve 48 saat sonrası içinde fertilizasyon (hpf) oluşturmak HSPCs3,4,5,6,7,8. Zebra balığı da çok bereketli, tek bir debriyaj 100 yumurta üzerinde döşeme kadın ile büyük ve deneysel çoğaltma için izin. Zebra balığı embriyo optik şeffaf, hematopoetik sistem mikroskobik görselleştirme için izin vardır. Zebra balığı HSCs runx1gibi işaretleme birkaç floresan transgenik satırlık: EGFP balık9, cd41: EGFP10, Balık ve kdrl:mCherry; cmyb: GFP3 çift pozitif hayvanlar, HSC ortaya çıkması ve genişleme vivo içinde3,4,7,8, canlı, gerçek zamanlı görselleştirme için izin 9. Zebra balığı'nın hızlı nesil zaman ve geliştirme rahim mutagenesis çalışmalar11,12,13,14,15 ve uyuşturucu kullanımı yol açmıştır insan kanı bozuklukları için tedavi edici sözü tutun bileşikler için16,17,18,19,20 eleme. Genel olarak, koruma, varlığı ve kolay geliştirebilme imkanı transgenik hatları ve hızlı yenilenme zamanı hematopoetik sistem Zebra balığı hematopoetik çalışmaları için ucuz, hızlı, esnek ve ideal bir model haline getirdi.
İzole ve HSCs test birçok yöntem içinde memeli hematopoetik sistem geliştirdik. Müfettişler bir arada hücre yüzey reseptörlerinin yanı sıra HSCs21,22,23,24işaretlemek sızma boya25,26HSCs yeteneği yararlanmak için kullanabilir. Sonra onlar etiketlenir, Floresans aktif hücre (FACS) sıralama fiziksel ayırma sağlar. Bir hücreyi bir HSC kanıtlayan sözde HSCs dikim ve uzun vadeli, çok lineage sulandırma kan hücre tipleri olgun her şeyden gözlemleyerek endojen HSPCs yok etmek için bir ev sahibi hayvan irradiating gerektirir. Çok sayıda hücre yüzeyine antikorlar hematopoetik hücreler ve bağışıklık ekimi için eşleşen kolaylaştırmak doğuştan fare suşları karşı olduğundan bu deneyleri de farelerde, iş. Ancak, birkaç Zebra balığı hematopoietik hücre yüzeyine antikorlar oluşturulan kimlik ve HSCs yalıtım engelleyen27, olmuştur. Mark ve Zebra balığı HSCs yalıtmak için en yaygın bir hücre özgü organizatörü sıra floresan protein ifade sayede sürüş Transjenik hayvanlar ile yoludur. Çalışmalar görüntülenir ve HSCs dorsal aort ventral duvar bu tekniği3,4,8,9kullanan mikroskobu ile numaralandırılan. Ultraviyole Zebra balığı28,29 klonal suşları üretilip ve MHC eşlemeli hayvanlar30' u başarılı nakli gerçekleştirdik. Ancak, bu teknikler maliyet birçok laboratuvarları engelleyici vardır, teknik olarak zor ve zaman alıcı vardır. Bu sorunlara yönelik olarak laboratuvarları varlığı, nükleer silahların yayılmasına karşı oranları ve HSPCs31,32,33ayırt etme kapasitesi için test etmek için birkaç vitro deneyleri üretilip, 34,35,36. Bu deneyleri yayılması ve HSPCs vitro31,32,33,34,35,36, kurtarma farklılaşma göster hematopoetik kusurları36ve keşfetmek ve sitokinler33,34,35test için verimli bir yöntem. Onlar da HSPC Biyoloji31,32için sorumlu genlerin tanımlamak için kullanılmıştır. Bu çalışmada, bu deneyleri bir adım daha ileri, HSPCs Nefelometri gelişmekte olan bir Zebra balığı embriyo içinde izin alıyoruz. Bu deneyleri de mutant hayvanlar HSPCs sayısında quantitate için kullanılması gereken ve hayvanlar yıkıcı hematopoetik ilaçlar ile tedavi. Aslında, bu deneyleri hızlı, mevcut birkaç teknik sorunlar ve HSPC numaraları quantitate, onların yayılması incelemek ve blok ayrımında araştırmak için ucuz yolu vardır.

<table><tbody><tr><td>10% Bovine Serum Albumin in Iscove&#039;s MDM</td><td>StemCell Technologies&amp;nbsp;</td><td>9300</td><td></td></tr><tr><td>DPBS (10x) with Calcium&lt;sup&gt;2+&lt;/sup&gt; and Magnesium&lt;sup&gt;2+&lt;/sup&gt;</td><td>Life Technologies</td><td>14080-055</td><td></td></tr><tr><td>HyClone PBS (1x)</td><td>GE Healthcare Life Sciences</td><td>sh30256.01</td><td></td></tr><tr><td>&amp;nbsp;DMEM 500 mL</td><td>Corning CellGrow</td><td>10-017-CV</td><td></td></tr><tr><td>&amp;nbsp;Ham&#039;s F12 500 mL</td><td>Corning CellGrow</td><td>10-080-CV</td><td></td></tr><tr><td>FBS 500 mL</td><td>Gemini Bio-Products</td><td>100-108</td><td></td></tr><tr><td>HEPES 100 mL (1 M)</td><td>Gibco life technologies</td><td>15-630-080</td><td></td></tr><tr><td>Penicillin/streptomycin (5000 U/mL&lt;br/&gt; and 5000 mg/mL) with L-Glutamine (200 mM)</td><td>Corning Mediatech</td><td>30-009-CI</td><td></td></tr><tr><td>Gentamycin Sulfate 10 mL (50 mg/mL)</td><td>Corning Mediatech</td><td>30-005-CR</td><td></td></tr><tr><td>1.5 mL MCF tube</td><td>FisherBrand</td><td>05-408-129</td><td></td></tr><tr><td>3 mL 23gx1 injection needle with Luer lock</td><td>BD Safety Glide</td><td>305905</td><td></td></tr><tr><td>5 mL polystyrene round bottom tube with cell strainer cap</td><td>Corning Falcon</td><td>352235</td><td></td></tr><tr><td>Methocel MC</td><td>Sigma-Aldrich</td><td>64630</td><td></td></tr><tr><td>14 mL Polystyrene round bottom tube</td><td>Corning Falcon</td><td>352057</td><td></td></tr><tr><td>10 mm polystyrene easygrip Petri dish</td><td>Corning Falcon</td><td>351008</td><td></td></tr><tr><td>Librease TM</td><td>Roche Sigma-Aldrich</td><td>5401119001</td><td>dissociation protease</td></tr><tr><td>Pronase</td><td>Roche Sigma-Aldrich</td><td>11459643001</td><td>dechorionation protease</td></tr><tr><td>15 cm Petri dish</td><td>Corning Falcon</td><td>351058</td><td></td></tr><tr><td>AB</td><td>Zebrafish International Resource Center (ZIRC)</td><td>ZL1</td><td>zebrafish strain used</td></tr><tr><td>Dithiolthreitol (DTT)</td><td>Sigma-Aldrich</td><td>646563</td><td></td></tr></tbody></table>

development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos

Hematopoiesis, hematopoetik kök ve progenitör hücre (HSPCs) olgun kan oluşturan farklı hücre soy çok sayıda ayırmak önemli bir hücresel işlemidir. Yalıtım ve bu HSPCs tanımlaması zor çünkü onlar tanımlanmış ex post facto; Onlar sadece kendi farklılaşma belirli hücre soy içine sonra tanımlanabilir. Son birkaç on yıl içinde Zebra balığı (Danio rerio) hematopoiesis eğitim için bir model organizma haline gelmiştir. Zebra balığı embriyolar rahimgeliştirmek ve 48 saat sonrası döllenme (hpf) tarafından HSPC farklılaşma değerlendirmek için kesin HSPCs. deneyleri üretilip ve nükleer silahların yayılmasına karşı yetenekleri geliştirilmiştir, nakli kullanan ve sonraki sulandırma hematopoetik sisteminin yanı sıra özel transgenik hatları confocal mikroskobu ile görüntülenmesi. Ancak, bu deneyleri maliyet engelleyici, teknik olarak zor ve zaman alıcı birçok laboratuarlar için vardır. HSPCs değerlendirmek için bir vitro modeli geliştirilmesi olacak maliyet etkin, daha hızlı ve daha önce karşılaştırıldığında daha az mevcut zorluklar HSPC Biyoloji etkileyen mutagenesis ve uyuşturucu ekranlar hızlı bir şekilde değerlendirmek laboratuvarlar sağlayan yöntemlerini açıklanan. HSPCs değerlendirmek için bu roman vitro tahlil ayrışmış kaplama bütün Zebra balığı embriyo ve sadece HSPC farklılaşma ve nükleer silahların yayılmasına karşı teşvik ekleyerek eksojen faktörler tarafından gerçekleştirilir. Embriyolar tek hücrelere ayrışmış ve onları bir tek progenitör hücre ortaya koloni oluşturan birimler (CFUs) oluşturmak neden HSPC destekleyici koloni uyarıcı faktör ile kaplama. Bu deneyleri moleküler yolları daha dikkatli incelenmesi HSPC yayılması, farklılaşma ve araştırmacılar omurgalı hematopoiesis temelleri ve onun bozukluk anlamak sağlayacak yönetmelik sorumlu izin vermelidir hastalık sırasında.

Embriyonik Zebra balığı HSPC sayıları değerlendirmek için 48 hpf embriyo sindirilir, methylcellulose içinde eksojen destekleyici hematopoetik büyüme faktörleri ile kaplama ve 7 gün (şekil 1A) inkübe. 7 gün sonra koloni oluşturan birimler (CFUs) numaralandırılmış (şekil 1B) ve Görüntülü (şekil 1C) olduğunu. Eklenen farklı s...

Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Burada, hematopoetik kök ve progenitör hücreler (HSPCs) embriyonik Zebra balığı quantitate için basit bir yöntem mevcut. Disosiye Zebra balığı HSPCs methylcellulose içinde içine olgun kan ayırt destekleyici faktörler ile kaplama. Bu kan algılama kusurları ve kolayca yapılacak eleme uyuşturucu sağlar sağlar.

Zebra balığı embriyo geliştirilmesinde bir Vitro tahlil hematopoetik kök ve yaratıcı Quantitate gelişimi (HSPCs) hücreleri

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

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Research

JoVE Journal

Developmental Biology

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

7.9K Görüntüleme.  California State University, Chico. Burada, hematopoetik kök ve progenitör hücreler (HSPCs) embriyonik Zebra balığı quantitate için basit bir yöntem mevcut. Disosiye Zebra balığı HSPCs methylcellulose içinde içine olgun kan ayırt destekleyici faktörler ile kaplama. Bu kan algılama kusurları ve kolayca yapılacak eleme uyuşturucu sağlar sağlar.

Video: Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

Transkripsiyonal Analiz Büyük ölçekli Zebra balığı Embriyonik Kalp Diseksiyon

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Gelişim Biyolojisi

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

Yetişkin balığı beyin türevli Neurospheres İzolasyon ve Kültürü

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

İzolasyon ve Zebra balığı Embriyolar gelen Tek Hücre Karakterizasyonu

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Blastulae Gelişmekte Cerrahi Fusion tarafından Parabiyotik balığı Embriyolar Üretimi

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

Here, we present an effective, easy, and fast 3D culture protocol for the formation of spheroids of two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte).

Embriyonik ve Karaciğer Zebra Balığı Hücre Hatlarının Sferoidlerinin 3D Kültür Yöntemi

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known ...

Çevre

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

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

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

Zebrabalığı Embriyolarında Situ Hibridizasyon ve IPSC-EC'lerde Tüp Oluşumu Nda Endoglin'in Vasküler Gelişimdeki Rolünü İncelemek İçin Tam Montaj

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

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

Zebrafish kimyasal ekranlar için otomatik görüntüleme ve analiz geliştirilmesi.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Biyoloji

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

Gelişmekte Olan Zebra Balıklarında Değiştirilmiş Kan Oluşumunu Tespit Etmek ve Nicel hale Getirmek için Akış Sitometrisini Kullanma

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

Myc-induced T-cell Akut Lenfoblastik Lösemide Zebra bağıda Side Popülasyonunun İzolasyonu

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

İmmünoloji ve Enfeksiyon

<meta charset="utf8"></head><body>Rejenerasyon ve Gelişim Araştırmaları için Sağlam Zebra Balığı Hücre Nakli

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>Yazar Spotlight: Gelişim ve Rejenerasyon Sırasında Zebra Balığı Sinir Sisteminde Hücresel Bağlantının Haritalanması

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

Embriyonik ve Larva Zebra Balıklarında Yüksek Çözünürlüklü Hücre Nakli

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...