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>

<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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+in+sea+urchin+embryos+by+using+a+CRISPR/Cas9+system.">Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.</a> Dev Biol. 409, 420-428 (2016).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 25mg/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 ...

The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the signaling pathways and the gene regulatory networks that they activate in an in vivo developmental context.Demonstrating this procedure will be Marina Martinez-Bartolom

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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

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7.8K Görüntüleme.  Mississippi State University. The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the si...

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

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

Model Baklagil Erken Embriyo Geliştirme Yönetmeliği incelenmesi için zigotik ve somatik embriyolar Elde protokolleri<em&gt; Medicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

Gelişim Biyolojisi

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

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

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

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

Timus Karakterizasyonu kullanma Fare Embriyo atalarıdır Settling<em&gt; In Vivo</em&gt; Ve<em&gt; In Vitro</em&gt; Tahliller

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

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

İletken Airways Desenlendirme sırasında Wnt Sinyalizasyonu incelenmesi

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

Starlet Deniz Anemone Aboral physa Tam Polip Regeneration uyaran<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

Kimerik Zebra balığı Embriyolar Bireysel Retina progenitörlerin Transgenik Gen İfade Vivo Görüntüleme Hücre otonom olmayan Etkiler Eğitim için

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

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 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 ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Tüm Zebrabalığı Embriyolarının Uzun Zaman Atlamalı Konfokal Mikroskobu için Katmanlı Montaj Yöntemi

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

Erken Fare Embriyolarından Sıralama için Küçük RNA Kütüphanelerinin Hazırlanması

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Nöral Kret Hücrelerindeki Mekanik Sinyalleri İncelemek İçin Optimize Edilmiş Bir O9-1/Hydrogel Sistemi

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...