Name: the power of simplicity: sea urchin embryos as in vivo developmental models for studying complex cell-to-cell signaling network interactions
Uploaded: 2017-02-16T14:00:00.000+00:00
Duration: 7 min 34 s
Description: Watch this Scientific Journal Video about The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions at JoVE.com

We would like to thank Dr. Robert Angerer for his careful reading and editing of the manuscript. NIH R15HD088272-01 as well as the Office of Research and Development, and Department of Biological Sciences at Mississippi State University provided support for this project to RCR.

1. Yüksek Verimli Morfolino Tasarım Stratejisi <ol><li> Bir ilgi gen (ler) (örneğin aday gen yaklaşımı, cis-düzenleyici analiz, RNAseq ve / veya proteomik diferansiyel ekranları) belirleyin. </li><li> Sık sık güncellenen web sitelerinde mevcut genomik, transkriptomik ve gen ifadesi verileri kullanın (örn SpBase http://www.echinobase.org 20 ve S. purpuratus Genom Arama http: ///urchin.nidcr.nih.gov/blast/index .html) uzaysal ekspresyon profili söz konusu gelişimsel mekanizma ile örtüşmektedir belirlemek için. Resim sentezleme verileri yoksa, o zaman qPCR primerler oluşturur ve / ya da in situ probu bir antisens geni sentezleme modelini değerlendirmek. </li><li> uzaysal ve geçici ekspresyon modelini tespit edildikten sonra, genomik web sitelerinden DNA dizisini elde etmek. <ol><li> çeviri engelleme morfolino oligonükleotitler üretebilmek için, o dizisini eldef 5 &#39;un çevrilmiş bölge (5&#39; UTR) doğrudan yukarı gelen SpBase veya S. purpuratus Genom Arama web sitelerinde mevcut sentezlenmiş dizi etiketi (EST) veritabanlarından başlangıç kodonu. Bu bilgiler ilgi gen için mevcut değilse, o zaman 5 &#39;RACE gerçekleştirin. </li><li> , Bir bağlayıcı bloke morfolino tasarım ekson ve SpBase veya S purpuratus Genom ara aracında iskele BLASTN alet intronlar içeren genomik sekans arayın. </li></ol></li><li> istenen 25 baz çifti morfolino dizisini tasarlamak amacıyla, oligonükleotid tasarımı web sitesi http://oligodesign.gene-tools.com kullanın. <ol><li> bir çeviri-bloke morfolino, translasyon hedef dizinin kaynağı olarak 3 &#39;5&#39; mRNA dizisini kullanır. 5 &#39;UTR sekansını (kopyalama başlangıç ​​sitesinin, akış yukarı yaklaşık olarak 70 nükleotid) artı belirgin wi kodon başlaması ile (başlangıç ​​sitesinin alt) kodlama bölgesi 25 bazlarinci parantez (ATG). </li><li> bir bağlayıcı bloke morfolino, intron-ekson veya ekson-intron sınır dizisini ve sınır bölgeleri yaklaşık 50 baz (ekson sekansı 25 bazlar ve intron sekansı 25 bazları) içerir. dizi benzersiz olduğunu doğrulamak için morfolino dizisi ile genom tarayın. </li></ol></li></ol> Morfolino oligonükleotidleri 2. Mikroenjeksiyon <ol><li> 300 nmol morfolino şişenin içine nükleaz içermeyen suyun 100 uL eklenmesiyle morfolino oligonükleotid 3 mM stok çözelti hazırlayın. NOT: dietil pyrocarbonate morfolino zarar verebilir, çünkü yeniden süspansiyon için DEPC arıtılmış su kullanmayın. </li><li> tam hızda 30 sn için stok oligonükleotid çözeltisi içeren flakon aşağı ilk sulandırma spin için - 5 ila 10 dakika, kısaca vorteks için 65 ° C&#39;de (14,000 16,000 xg), kısaca girdap, ısı ve oda morfolino stok tutmak en az 1 saat boyunca sıcaklığı. Morfolino stok solüsyon<html> s + 4 ° C ile -20 ° C arasında saklanır. </li><li> istenilen konsantrasyonda morfolino oligonükleotitler içeren enjeksiyon çözeltisi hazırlayın. Bu çözelti, genellikle% 20 gliserol ya da bir taşıyıcı olarak 125 mM KCI ve 15% (fluorescein isothiocyanate dekstran 10,000 MA 2.5 mg / ml stok çözelti) FITC Dekstran içerir. FITC Dextran ve diğer floresan dekstran konjugatlar rutin Epifloresans mikroskobu ile enjekte edilen embriyolar tanımlamak için kullanılır. -20 ° C&#39;de saklayın enjeksiyon çözümleri. </li><li> 5 dakika - en az 2 bir ısı blok ya da su banyosu içinde 65 ° C sıcaklıkta morfolino çözeltisi ısıtılır. </li><li> En az 10 dakika boyunca - (16,000 xg 14,000) tam hızda 1 dakika santrifüj için - (16,000 xg 14,000), girdap Kısaca morfolino tam hızda 30 sn için çözüm dönerler. </li><li> morfolino çözümü ile enjeksiyon iğneleri yükleyin. Ayrıntılı bir mikroenjeksiyon protokolü için Stepicheva ve Song 2014 21 ve alkış ve Ettensohn, 2004 bakınız&quot;&gt; 22. </li></ol> S. purpuratus Embriyolar 24 saat sonrası fertilizasyon (hpf) 3. Sabitleme ve Yerinde Protokol NOT: Bu protokol Arena-Mena ark değiştirilir. 2000 23 ve Sethi ve arkadaşları. 2014 24. <ol><li> tespit <ol><li> , Embriyoları içeren oyuklara (aşağıya bakınız) tespit edici birkaç damla pipetleme yavaşça karıştırın ve bunları oturmasına izin verin. fiksatif çözüm çıkarın ve daha sonra sabitleştirici iki ek 180 uL yıkama ile embriyolar karıştırın. NOT: iyice nazik pipetleme yukarı ve aşağı birkaç kez yıkama sırasında embriyoların karıştırmak için önemlidir. Bunu yapmamak sinyal gürültü oranı düşürebilirsiniz. </li><li> pH 7.0, 10 mM MOPS oluşan% 4 elektron mikroskobu sınıf paraformaldehid içinde oda sıcaklığında 50 dakika 1 saat süresince ikinci sabitleyici yıkaması (RT) &#39;de düzeltmek için embriyolar boş,% 0.1 Tween-20 ve yapay seawater (ASW). En iyi sonuçlar için her zaman bu çözüm taze olun. Buna ek olarak, kullanım kolaylığı ve kullanışlılık embriyolar için 96-yuvalı plakalar içinde sabitlenir. <ol><li> 20 ml sabitleyici kullanım 5 mL% 16 paraformaldehit, 15 ml ASW 200 uL 1 M MOPS pH 7.0, 20 uL Tween-20. </li></ol></li><li> MOPS, 180 uL, oda sıcaklığında 5 kez yıkayın 0.1 M MOPS pH 7.0, 0.5 M NaCI ve Tween-20, en az 5 dakika boyunca ya da embriyolar kadar kuyunun dibine% 0.1 aşağıdakilerden oluşan yıkama tamponu. Yine, iyice hafifçe yukarı pipetleme ve birkaç kez aşağı yıkama tamponu içine embriyolar karıştırmak için önemlidir. 4 ° C&#39;de depolandığında, MOPS yıkama tampon çözeltisi 2 gün için kullanılabilir ° C. <ol><li> 40 ml MOPS tamponu için 4 mL 1 M MOPS pH 7.0, 4 ml 5 M NaCI, 32 mL DH 2 O ve 40 uL Tween-20 yıkanır. </li><li> Sabit embriyolar, 2 gün süreyle 4 o C&#39;de saklanabilir. Not: uzun sabit embriyolar depolanması, daha sonra bakteriyel büyümeyi önlemek için yıkama tamponu MOPS,% 0.2 sodyum azit ilave edin.</li></ol></li></ol></li><li> Ön-hibritleme <ol><li> MOPS tampon yıkama aspire ve 2 180 mcL: hibridizasyon tampon tampon hafifçe pipetleme birkaç kat çözeltisi içine embriyolar karıştırın ve oda sıcaklığında en az 20 dakika süreyle inkübe yıkama MOPS 1 oranında. Hibridizasyon tamponu% 70 formamit, 0.1 M MOPS pH 7.0, 0.5 M NaCI oluşur, 1 mg / ml BSA ve% 0.1 Tween-20. <ol><li> 40 ml melezleme tamponu kullanılması 4 mL 1 M MOPS, pH 7.0, 4 ml 5 M NaCI, 4 mL DH 2 O, 0.04 g BSA ve 40 uL Tween-20. , Vorteks ile iyice karıştırın tekrar 28 ml formamid ve vorteks ekleyin. </li></ol></li><li> MOPS yıkama ve melezleştirme tamponları 1 oranında ve 1: 180 uL ekleyin: 2 çıkarın hibridizasyon tampon tampon çözeltisi içine embriyolar karıştırın ve oda sıcaklığında en az 20 dakika süreyle inkübe yıkama MOPS 2 oranında. </li><li> hibridizasyon tamponu 150 ul - prob hibridizasyon nazikçe 100 içine embriyolar karıştırın önce. en az 1, 50 ° C de inkübe embriyolarh. NOT: gecede embriyolar Hazırlanıyor kabul edilebilir. saatliğine inkübe edilmeden önce, buharlaşmayı önlemek için, bir yapışkan tabaka ile kuyu mühür. </li></ol></li><li> melezleme <ol><li> prob çözeltisi oluşturmak için yavaşça, hibridizasyon tamponu içinde girdap 0.3 prob ng / ml ve 500 ug / ml maya tRNA - Ayrı bir tüp içinde 0.1 ekleyin. Maya tRNA, anti-sens probunun özel olmayan bağlanmayı azaltmak için prob çözeltisi ilave edilir. Bu, 50 ° C&#39;ye kadar çözeltisi ve aspire hibridizasyon tamponu önceden ısıtın. </li><li> Prob çözeltisi 100 uL içine önceden hibridize embriyolar karıştırın bir yapışkan tabaka ile sızdırmaz ve prob bağlı olarak 2 ile 7 gün boyunca 50 ° C&#39;de hibridize (foxq2 prob 2-gün inkübe edilebilir). NOT: İnkübasyon süreleri sonda kapalı göre değişir. düşük seviyelerde sentezlendiği bazı genler 7 günlük bir inkübasyon gerektirmez. 96 oyuklu plakalar ADHES halinde buharlaştırma karşı sigorta bir nem kutu yerleştirilebilirive sayfası düzgün mühür başarısız olur. </li><li> taze MOPS 180 uL, 50 ° C de 5 kez yıkayın 3 saatlik bir toplam yıkama tamponu. Daha sonra, MOPS uL oda sıcaklığında yıkama tamponu 180 ile 3 kez (15 dakika) yıkanır. yıkama içine embriyolar hafifçe birkaç kez pipetleme her zaman tampon karıştırmak unutmayın. </li></ol></li><li> Antikor Kuluçka <ol><li> MOPS tampon yıkama aspire ve% 10 normal koyun serumunda ve 5 mg içeren bloke edici tampon içinde 180 uL embriyolar karıştırın / tampon ve gece boyunca oda sıcaklığında en az 45 dakika veya 4 ° C&#39;de inkübe edin yıkama MOPS mL BSA. </li><li> bloklama tamponunu çıkarın ve sonra 1 içeren bloke edici tampon içinde embriyolar karıştırın blokaj tamponunda seyreltilmiş alkalin fosfataz-konjuge edilmiş anti-digoksijenin antikoru ile 1500 seyreltme. Buharlaşmayı önlemek için sızdırmaz kılınmış bir plaka, oda sıcaklığında gece boyunca inkübe edilir. NOT: gecede daha uzun üzerinde antikor bırakmayın. </li><li> embriyolar 6 kez yıkayın (5 dk ya da embriyolar damla kadar) MOPS oda sıcaklığında tamponu yıkayın. embriyolar can4 ° C&#39;de gece boyunca saklanır. </li></ol></li><li> In situ gelişmede <ol><li> 0.1 M Tris pH 9.5, 100 mM NaCl, 50 mM MgCl2, 1 mM Levamizol oluşan taze pH 9.5 tamponu ile oda sıcaklığında embriyolar 3 kere (10 dakika) yıkanır ve% 0.1 Tween-20. <ol><li> 20 ml pH 9.5 tampon kullanım için 2 mL 1 M Tris pH 9.5, 400 uL 5 M NaCl, 1 mL, 1 M MgCl2, 20 uL Tween-20, 16.6 mi DH 2 O ve 0.0048 gr Levamisole. </li></ol></li><li> ışıktan koruyarak lekeleme tampon maddesi, 37 ° C &#39;de embriyolar inkübe edin. Lekeleme tampon maddesi,% 10, dimetil formamid oluşur, 4.5 ul / ml 4-Nitro Mavi tetrazolyum klorid (NBT) ve taze yapılmış, pH 9.5 tampon maddesi içinde 3.5 ul / ml 5-bromo-4-kloro-3-indolil-fosfat (BCIP) . <ol><li> lekeleme tampon maddesi ile 1 mL, 100 uL, dimetil formamid, 4.5 mL NBT ve 3.5 uL BCIP kullanın. </li></ol></li><li> MOPS 3 ile 5 kez yıkanarak alkalin fosfataz reaksiyonu durdurun yıkama tamponu. reaksiyon wifoxq2 probu tipik olarak 1 saat 30 dakika sürer inci. Bazı Probes RT ya da 4 ° C&#39;de ya da gece boyunca inkübasyon gerekebilir. </li><li> % 70 MOPS yıkama ve% 30 gliserol çözeltisi içine embriyolar karıştırın. gliserol mikroskopi için gerekli bir kırılma indeksine sağlar. Embriyolar birkaç hafta boyunca bu çözelti içinde saklanabilir. buharlaşmasını önlemek için plastik parafin ile plakaları Seal. </li></ol></li><li> Slayt hazırlama ve görüntü yakalama <ol><li> Bir slayt üzerine şeritler arasındaki küçük boşlukları olan bir üçgen içine çift taraflı bant üç küçük şeritler düzenleyerek bir slayt hazırlayın. </li><li> üçgenin merkezinde% 70 MOPS yıkama ve% 30 gliserol çözeltisi içinde embriyo transferine ve bir lamel ile kaplayın. </li><li> Örtücü camın kenarları, yaklaşık oje tabakası uygulayarak lamel mühür. </li><li> 20X objektif ve ekli kamera ile bir bileşik ışık mikroskobu kullanarak görüntüleri yakalayabilir. </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Wnt-FoxQ2-nodal+pathway+links+primary+and+secondary+axis+specification+in+sea+urchin+embryos.">A Wnt-FoxQ2-nodal pathway links primary and secondary axis specification in sea urchin embryos.</a> Dev Cell. 14, 97-107 (2008).</li><li>Molina, M. D., de Croze, N., Haillot, E., Lepage, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal:+master+and+commander+of+the+dorsal-ventral+and+left-right+axes+in+the+sea+urchin+embryo.">Nodal: master and commander of the dorsal-ventral and left-right axes in the sea urchin embryo.</a> Curr Opin Genet Dev. 23, 445-453 (2013).</li><li>Range, R. C., Glenn, T. D., Miranda, E., McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LvNumb+works+synergistically+with+Notch+signaling+to+specify+non-skeletal+mesoderm+cells+in+the+sea+urchin+embryo.">LvNumb works synergistically with Notch signaling to specify non-skeletal mesoderm cells in the sea urchin embryo.</a> Development. 135, 2445-2454 (2008).</li><li>Range, R., 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=Cis-regulatory+analysis+of+nodal+and+maternal+control+of+dorsal-ventral+axis+formation+by+Univin,+a+TGF-beta+related+to+Vg1.">Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-beta related to Vg1.</a> Development. 134, 3649-3664 (2007).</li><li>Warner, J. F., Miranda, E. L., McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+hedgehog+signaling+to+the+establishment+of+left-right+asymmetry+in+the+sea+urchin.">Contribution of hedgehog signaling to the establishment of left-right asymmetry in the sea urchin.</a> Dev Biol. 411, 314-324 (2016).</li><li>Rottinger, E., 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=FGF+signals+guide+migration+of+mesenchymal+cells,+control+skeletal+morphogenesis+[corrected]+and+regulate+gastrulation+during+sea+urchin+development.">FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis [corrected] and regulate gastrulation during sea urchin development.</a> Development. 135, 353-365 (2008).</li><li>Warner, J. F., McCarthy, A. M., Morris, R. L., McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+requires+motile+cilia+in+the+sea+urchin.">Hedgehog signaling requires motile cilia in the sea urchin.</a> Mol Biol Evol. 31, 18-22 (2014).</li><li>Technau, U., Steele, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology.">Evolutionary crossroads in developmental biology.</a> Cnidaria. Development. 138, 1447-1458 (2011).</li><li>Yaguchi, J., Takeda, N., Inaba, K., Yaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+Wnt-Nodal+Signals+Regulate+the+Patterning+of+Anterior+Neuroectoderm.">Cooperative Wnt-Nodal Signals Regulate the Patterning of Anterior Neuroectoderm.</a> PLoS Genet. 12, e1006001 (2016).</li><li>Duboc, V., Rottinger, E., Besnardeau, L., Lepage, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+and+BMP2/4+signaling+organizes+the+oral-aboral+axis+of+the+sea+urchin+embryo.">Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo.</a> Dev Cell. 6, 397-410 (2004).</li><li>Bradham, C. A., 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=Chordin+is+required+for+neural+but+not+axial+development+in+sea+urchin+embryos.">Chordin is required for neural but not axial development in sea urchin embryos.</a> Dev Biol. 328, 221-233 (2009).</li><li>Su, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+for+ectoderm+specification+in+sea+urchin+embryos.">Gene regulatory networks for ectoderm specification in sea urchin embryos.</a> Biochimica et biophysica acta. 1789, 261-267 (2009).</li><li>Lin, C. Y., Su, Y. 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The authors have nothing to disclose.

Burada sunulan metodoloji .. Birçok laboratuvar incelemek için erken deniz kestanesi gelişimi sırasında benzer deneyleri kullanan temel gelişimsel mekanizmaları düzenleyen sinyalizasyon iletim yollarını ve GRNs anlamak için omurgalılar daha az genomik ve morfolojik karmaşıklığı ile embriyoları kullanılarak gücünü gösteren bir örnektir diğer hücre kaderi şartname olaylarda yer alan sinyal yolları (örn Notch, Kirpi, TGF β, ve FGF sinyal) 27, <sup cl...

Gen düzenleyici ağları (GRNs) ve sinyal iletim yolları yetişkin hayvan vücut planı oluşturmak için kullanılan embriyonik gelişim sırasında genlerin mekansal ve zamansal ifade kurmak. Hücre-hücre sinyal iletim yolları hücrelerin iletişim hangi araçları sağlayan bu düzenleme ağların temel bileşenleri vardır. Bu hücresel etkileşimler kurmak ve embriyogenez 1, 2 sırasında çeşitli bölgelerde ve arasında düzenleyici ve farklılaşma genlerinin ekspresyonunu rafine. salgılanmış hücre dışı modülatörlerinin (ligandlar, antagonistler), reseptör ve ko-reseptörleri arasındaki etkileşimi sinyal transdüksiyon yollarının aktivitelerini kontrol eder. hücre içi moleküller bir yelpazesine bir hücrenin gen ekspresyonu değişimlerinin, bölme ve / veya şekli ile sonuçlanan, bu giriş çeviren. ana yoldan büyük hücre dışı ve hücre içi seviyede kullanılan anahtar moleküllerinin çoğu, birliktebilinen, tek tek sinyal yollarının karmaşıklığı nedeniyle büyük ölçüde eksik bilgidir. Buna ek olarak, farklı sinyal yolları çoğunlukla dışı, hücre içi de olumlu veya olumsuz ya birbiri ile etkileşim ve transkripsiyon seviyeleri 3, 4, 5, 6. Önemli olarak, sinyal transdüksiyon yollarının ana bileşenleri çok tüm metazoan türlerinde korunan ve özellikle yakından ilgili filumlar organizmaların karşılaştırırken, son derece önemli sinyal yollarının en sık birçok türün içindeki gelişim işlevleri yerine 7, 8, 9, 10, 11. gelişimi sırasında sinyal çalışma herhangi bir organizmada zor bir görev olduğunu ve oradaomurgalılarda büyük olası ligand ve reseptör / ko-modülatör etkileşimlerinin sayılar, hücre içi iletim molekülleri, hem de orada 1): en ikincil ağızlılar modelleri (omurgalıların omurgasız omurgalılar, yarı sırtipliler ve derisidikenlilerde) &#39;de sinyal yolları okuyan bazı önemli zorluklar nedeniyle genom 12, 13, 14 karmaşıklığı farklı sinyaller arasındaki etkileşimler; 2) omurgalıların karmaşık morfolojisi ve morfogenetik hareketler genellikle daha zor ve sinyal iletim yollarının arasında fonksiyonel etkileşimleri yorumlamak yapmak; 3) En dışı derisi dikenliler omurgasız ikincil ağızlılar modeli türlerinde Analizleri bazı tunikat türlerinin 15, 16 hariç gravida kısa pencereler ile sınırlıdır. Deniz kestanesi embriyo yukarıda belirtilen sınırlamaları birkaç vardır ve in vivo sinyal iletim yollarının ayrıntılı bir analiz yapmak için birçok benzersiz niteliklere sahiptir. Bunlar aşağıdakileri içerir: 1), deniz kestanesi genomunun uygulama kolaylığı önemli ölçüde mümkündür ligand, reseptör / co-reseptör ve hücre içi iletim molekülü sayısını azaltır 17 etkileşimi; 2) germ ve büyük embriyonik eksenlerin özellikleri ve desenlendirme kontrol GRNs iyi sinyaller 18, 19 alıcı hücre / topraklarının düzenleyici bağlamında anlamada yardımcı, deniz kestanesi embriyolarda kurulur; Embriyolar olan morfoloji analiz etmek kolay bir tek katmanlı epiteli oluşur zaman 3) Birçok sinyal iletim yolları erken bölünme ve gastrula aşamaları arasında ele alınabilir; 4) molekülleri içerenkolayca manipüle edilir deniz kestanesi sinyal yolları d; 5) Birçok deniz kestanesi 10 ila 11 ay bir yıl (örneğin Strongylocentrotus purpuratus ve Lytechinus variegatus) için gebe edilir. Burada, sistematik ve verimli bir şekilde belirtmek ve deniz kestanesi embriyolarda desen bölgeleri birkaç omurgasız model sistemler karmaşık moleküler mekanizmaların çalışmada sunduğumuz avantajları göstermek için sinyal yollarının bileşenlerini karakterize bir yöntem mevcut.

<table><tbody><tr><td>Translational-blocking morpholino and/or splice-blocking morpholino</td><td>Gene Tools LLC</td><td>Customized</td><td>More information at www.gene-tools.com</td></tr><tr><td>Glycerol</td><td>Invitrogen</td><td>15514-011</td><td></td></tr><tr><td>FITC (dextran fluorescein isothiocyanate)</td><td>Invitrogen, Life Technologies</td><td>D1821</td><td>Make 25 mg/mL stock solution</td></tr><tr><td>Paraformaldehyde 16% solution EM Grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>MOPS</td><td>Sigma Aldrich</td><td>M1254-250G</td><td></td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>23336-0010</td><td></td></tr><tr><td>Formamide</td><td>Sigma Aldrich</td><td>47671-1L-F</td><td></td></tr><tr><td>Yeast tRNA</td><td>Invitrogen</td><td>15401-029</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Sigma Aldrich</td><td>G9023-10mL</td><td></td></tr><tr><td>Alkaline Phosphatase-conjugated anti-digoxigenin antibody</td><td>Roche</td><td>11 093 274 910</td><td></td></tr><tr><td>Tetramisole hydrochloride (levamisole)</td><td>Sigma Aldrich</td><td>L9756-5G</td><td></td></tr><tr><td>Tris Base UltraPure</td><td>Research Products Internationall Corp</td><td>56-40-6</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358-10</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma Aldrich</td><td>7786-30-3</td><td></td></tr><tr><td>BCIP (5-Bromo-4-Chloro-3-indolyl-phosphate</td><td>Roche</td><td>11 383 221 001</td><td></td></tr><tr><td>4 Nitro blue tetrazolium chloride (NBT)</td><td>Roche</td><td>11 383 213 001</td><td></td></tr><tr><td>Dimethyl Formamide</td><td>Sigma Aldrich</td><td>D4551-500mL</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma Aldrich</td><td>P9541-5KG</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>S5761-500G</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma Aldrich</td><td>M7506-2KG</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma Aldrich</td><td>C1016-500G</td><td></td></tr></tbody></table>

the power of simplicity: sea urchin embryos as in vivo developmental models for studying complex cell-to-cell signaling network interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

Deniz kestanesi embriyo biz 3 farklı Wnt sinyal dalları (Wnt / β-katenin, Wnt / JNK ve Wnt / PKC) 4, 25 etkileşim ön-arka (AP) desenlendirme yöneten bir Wnt sinyal ağı oluşturmak için olduğunu göstermiştir. Bu sinyal olayların en önemli sonuçlarından biri, ilk geniş ifade ön nöroektoderm (ANE) GRN gastrulasyon başında (S. purpuratus 24 hpf) tarafından ön direğin etrafında küçük bir bölgede s...

Watch this Scientific Journal Video about The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions at JoVE.com

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Bu video makalede sistematik ve verimli birçok omurgasız embriyolar karmaşık sinyal yolları ve düzenleyici ağları bileşenleri karakterize etmek için kullanılabilir in vivo metodolojisi basit ayrıntıları.

Sadelik Gücü: olarak deniz kestanesi Embriyolar<em&gt; Vivo</em&gt; Karmaşık Hücre-hücre Sinyal Ağ Etkileşimleri incelenmesi için Gelişim Modelleri

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

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The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Unit 1

Principles of Cell Signaling

SCIENTISTS IN ACTION

7.8K Görüntüleme.  Mississippi State University.  Bu video makalede sistematik ve verimli birçok omurgasız embriyolar karmaşık sinyal yolları ve düzenleyici ağları bileşenleri karakterize etmek için kullanılabilir in vivo metodolojisi basit ayrıntıları. 

Video: The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Zebra balığı Embriyo microbead İmplantasyonu

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Gelişim Biyolojisi

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.

Kök hücre benzeri<em&gt; Xenopus</em&gt; Embriyonik Eksplantlarındaki Erken Nöral Gelişim Özellikleri Okuyacak<em&gt; İn Vitro</em&gt; Ve<em&gt; İn Vivo</em

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

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

Kordalı embriyo Mikroenjeksiyon ve Elektroporasyon<em&gt; Ciona intestinalis</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Zebra balığı Embriyolar Bireysel Nöronlar ve Sinir Crest Hücreler Aktin Sitoiskelet Hücre Motilite ve Canlı Görüntüleme

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Biyoloji

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 ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

Model Genetik Organizma olarak kullanmak için Medaka Embriyolar, Mikroenjeksiyon

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Gelişmekte Olan (Kurbağa) Embriyoda Hücre-Soy Kılavuzlu Kütle Spektrometrisi Proteomikleri

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

Kimya

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively na&#239;ve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise na&#239;ve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Zebrafish blastoderm explants are generated by isolating embryonic cells from endogenous signaling centers within the early embryo, producing relatively na&#239;ve cell clusters easily manipulated and cultured ex vivo. This article provides instructions for making such explants and demonstrates their utility by interrogating roles for Nodal signaling during gastrulation.

Zebra Balığı Embriyolarından Naïve Blastoderm Explants Üretimi

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

Kraniofasial Geliştirme sırasında Ektodermal Epiteli Sinyal Özellikleri değerlendirilmesi

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

Deniz Kestanesi Zigotlar Yüksek Randımanlı Microinjections

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Sadelik Gücü: olarak deniz kestanesi Embriyolar

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