Name: inducing complete polyp regeneration from the aboral physa of the starlet sea anemone nematostella vectensis
Uploaded: 2017-01-14T13:00:00.000+00:00
Duration: 8 min 17 s
Description: Watch this Scientific Journal Video about Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis at JoVE.com

Bu çalışma GHT bir New York Kök Hücre Bilim (NYSTEM C028107) Hibe tarafından desteklenmiştir.

Sıcaklık, Beslenme ve aydınlık / karanlık döngüsü için Animals 1. Klima <ol><li> Dünya çapında sayısız Nematostella laboratuarlarından biri, ya da kar amacı gütmeyen bir tedarikçiden Nematostella vectensis yetişkin elde (Tablo 1) </li><li> Olarak, karanlıkta sabit sıcaklıkta (tipik olarak 18 ve 21 ° C) Nematostella korumak &quot;/ 3x 1&quot; yapay deniz Suda bin (PPT) başına 12 parça bir tuzlulukta (ASW). Genellikle basit soda-kireç camı kültür tabaklarında 250 mL veya 1.5 L kapasiteli 11 kültürleri korumak. Not: Bu basit kültür koşulları yaygın Nematostella çalışma laboratuvarlar arasında kullanılır, ancak kültür bakımı da 19 otomatik hale getirilebilir. </li><li> Haftada 4x - 2 nauplii Nematostella taze yumurtadan Artemia besleyin. Sığ bir dikdörtgen cam tabak 20 veya 30 ° C&#39;de tam güçte (36 ppt) veya 1 / 3x ASW Hatch Artemia kistleri,küçük ölçekli, ticari veya ev yapımı tuzlu su karidesi kuluçkahanelerin herhangi biri. Bir inkübatör mevcut değilse, karides, oda sıcaklığında yumurtadan ama çok daha yavaş yapacaktır. Not: Bu genellikle tamamlanması için 24 saatten fazla gerektirir. </li><li> Haftada en az bir kez anemon kültür suyu değiştirin. en iyi yetişkin sağlığı için, (sabun olmadan) iyice temizleyin kültür tuzak meyvelerin gıda ve atık ve hayvan dolaştırmak için ceket kase ve birikmiş mukus salgıları haftada bir kez kase. </li></ol> Beslenme Klima Animals 2. Seçme ve Dinlenme <ol><li> yaklaşık aynı uzunlukta seçin boyutu uyumlu polipler (3-5 cm, ne zaman doğal olarak rahat) ve önceki amputasyon üç gün boyunca koloniden ayrılan bir kapta koyun. Not: kesim için seçilen hayvan sayısı deney tarafından belirlenecek elbette yürütülen, ancak genel olarak altı tekrarlamalı örnek noktası başına en az beş hayvan öneriyoruz. Böylece,Tipik bir deneyde, 30 hayvan minimum önceden seçilmiş olur. Genel olarak, daha sonra puanlama etkileyebilir (aşağıya bakınız) düzensiz amputasyon beri az sayıda (30) daha fazla seçmek akıllıca olacaktır. </li><li> önce amputasyon oda ışığa inkübatör en az bir saat, seçilen hayvanların çanak çıkarın. oda ışık ve taşıma titreşimleri maruz kalma olasılığı hayvanların büzülmesine neden olur, bu yüzden uyarlanmış veya laboratuarlarında inkübasyon tarafından &quot;rahat&quot; gerekir: unutmayın. Hayvanlar dokunmak refrakter ve ışığa maruz kalma olacak ve bu noktada Nazik pipetleme ile hareket ettirilebilir. </li><li> İsteğe bağlı: (1 / 3x ASW olarak)% 7.5 MgCl 2 ekleyerek hayvanların anestezisi. Yavaşça bir standart plastik 5 ml pipet ile kase MgCl2 çözüm ekleyin. hayvanlar sonunda ışığa ve fiziksel manipülasyon için alışkanlık haline gelecek olsa da, t &quot;düzeltmek&quot; korumak veya hayvanların uyutmak için avantajlı olabilir Not:Onlar uzun 16,21,22 olduktan sonra o devlet rahat. </li><li> 12 ppt ASW içeren 100 mm çapında bir steril bir cam kesme çanak tabanına, kesilmiş olan havuz beş hayvan (1 / 3x ASW) aktarmak için bir çok deliğe (&gt; 0.5 cm) plastik pipet kullanın. 40X - 10 arasında değişen büyütme ile bir stereomikroskopta sahneye çanak yerleştirin. Not: hayvanlar anestezi ve kesmek için rahat değil, onlar hala tekrar rahat olmak için birkaç dakika gerekebilir böylece dokunun ve stereoskop aydınlatma cevap olabilir ve. </li></ol> 3. Amputasyon <ol><li> Steril bir neşter kullanılarak, yaklaşık sürece geniş ve hiçbir mezenter içeren olduğu gibidir physa bir bölümünü elde etmek için amacı ile, her polip gelen aboral physa kesmek. Not: İdeal kesim sitesi mezentere sona ermesi sadece aboral olduğunu. kesme düzleminde ince l uygun mezenterinde bir geçiş vardanielaesquivel Her mesenterial ekleme (bakınız Şekil 1, oklar) karşılık gelir. O deliği 17,30 &#39;takmayı&#39; kolaylaştırabilir mukus üretir çünkü mezenter bulunmaması önemlidir. <ol><li> amputasyon arzulanan yere hayvan ile temas neşter bıçak yerleştirin. Bu, ya yardımsız (freehand) yapmak, ya da hafifçe # 5 forseps (Dumont tarzı veya benzeri) ile hayvanın vücudunu tutarak. </li><li> vücudun genelinde bir &#39;sallanan&#39; hareket neşter kavisli bıçak yararlanarak dokusu kesti. Not: Doku neşter sarsan olarak temiz sever ve donörden physa istenilen bölümünü serbest olmalıdır. doku küçük bir parça hala vücudu ve physa bağlayan Ancak, neşter ile kesmek. Bu physa zarar verebilir, çekerek bağlı parçalarını ayırmak için çalışmayın. </li></ol></li><li> Çanak her kesilmiş &#39;donör&#39; polip çıkarın ve ÖÇK iade&#39;Toplanmış amputees&#39; etiketli oran kase; kase tarih ve stok kültürüne geri. Not: Ampute polipler normal beslenebilir sonra bir gün içinde aboral yara iyileşmesi ve olacaktır. Onlar istenirse physa tekrar ampute edilebilir hangi noktada iki hafta içinde normal görünümlü physa yeniden oluşturulur. </li><li> daha sonra adı her bir 12 ppt ASW 10 mL olan bir çok-yuvalı hücre kültürü plakasının ayrı bir steril oyuğuna her physa transfer 12 ppt ASW kesme çanak kalır kesilmiş physa durulayın. Not: Bu örnekte, her deniz suyu de tutarak, 10 ml beş eksize physa ile altı plaka kullanır. Genel deniz suyu nedeniyle kullanım ve potansiyel buharlaşma harekete havaya maruz kalmasını önlemek için yeterince physa kapsamalıdır. plakası veya kuyu bir kapak olmalıdır. </li><li> 3.3 Her deneysel tedavi için her bir kuyunun ayrılmış en az 5 physa toplamak için - Tekrar 3.1 adımları. </li><li> prov bir sıcaklıkta physa inkübePlanlanan deneysel sorgulamaları için yenilenme en iyi oranı ide. yenilenme istenen oran olarak belirlenen, sabit bir sıcaklıkta, bir ısı ayarlı inkübatör içine physa içeren plaka koyun. Not: physa eksik dokuların yeniden 15 ve 27 ° C arasındaki sıcaklıklarda inkübe tam polip oluşturacaktır. yenilenme hızı ilk iki aşamada dışında sıcaklığa bağlıdır. Tüm sıcaklıklar için Aşama 4 ulaşmak için ortalama gün kestikten sonra 7 D, ve bu da 21 ° C &#39;de rejenerasyon ile çakışmaktadır. 27 ° C &#39;de, Aşama 4 3 gün önce ulaşılır ve 15 ° C sıcaklıkta, Aşama 4 21 ° C (bakınız referans 10) en rejenerasyon göre yaklaşık 3 gün kadar geciktirilir. </li></ol> 4. Nematostella Rejenerasyon Evreleme Sistemi ile Rejenerasyon değerlendirilmesi (NRSS) <ol><li> Değişken magnific ile stereo-bileşik mikroskop kullanılarak physa Puantirme (10 - 80X). Evre 0 taze kesilmiş Nematostella physa puan ve aynı zamanda NRSS 10 kullanılarak her gün sonrası amputasyon (dpa) puanlama devam ediyor. Not: Anahtar evreleme kriterleri ve ayrıntıları Referans 10 bakınız. <ol><li> Bir taze kesilmiş physa bir sarkık balon andıran bir Kupası&#39;na şeklinde bir kitle olarak belirirse bir açık yara site ile, Evre 0 (Açık Yara) olarak physa puanı olasılıkla görülebilir. Not: yara kenarları da başlangıçtan itibaren birbirine yapışıyor olabilir, ancak doku yine çöktü edilecek ve sertlik yoksundur. açık yara kenarları yara iyileşene kadar radyal daralma geçiren görülebilir. </li><li> amputasyon yara kapatılır görünüyorsa Aşama 1 (Kapalı Yara) olarak physa puan. Not: Yara yeri gelecek sözlü kutup karşılık gelecektir. Gelecekteki sözlü kutup etrafında dış yüzeyi altında yatan radyal simetrik Endodermal mesenteriumda eklemeler karşılık gelen farklı kemerler görüntülemek için başlayabilir. </li><li> skor phYSA Oral kutup yüzeyi radyal simetrik bir düzende ve oluklar ile ayrılmış sekiz kaldırdı kemerler açığa şişirilmiş görünüyorsa Aşama 2 (Radyal Kemerler) olarak. kemerlerin tepesinde küçük, yarım küre kabarcıkları dikkat edin. Onlar gibi uzun boylu kadar geniş, büyük olasılıkla geçici hakkında olacak ve başlangıçta tek bir ektodermal hücre katmanı oluşturduğu olacaktır. Not: Bazı durumlarda çift katlı kabarcıklar stabilize olabilir. Not: Bu veya daha sonraki aşamalarda bir mukoza tabakası physa bir zar &#39;kılıf&#39; (Şekil 2) saklanması görünebilir. Bu, içine alıcı malzeme puanlama kolaylaştırmak için kaldırılmalıdır. </li><li> Endodermal ve ektodermal doku katmanları içeren dokunaçlarıyla tomurcukları stabil en azından bazı radyal kemerlerin oral ucunda oluşturulan eğer Evre 3 (Tentacle) olarak physa puan. Not: dokunaçlar de geniş ve çok az hareketli olan daha uzundur. physa artış göstermektedir, ancak mezenter esasları vis değişken enflasyon haline gelebilir, böylecevücut boşluğuna (coelenteron) içine mesenteriumda ekleme uzanan ible. </li><li> physa onlar bağlamak için görünür yerden ölçülen iki kez radyal genişliğinden daha fazla olan ağız-aboral uzunlukları ile, vücut duvarı eklemeleri gelen coelenteron içine uzanan sekiz ayrı, görünür mesenteries içeriyorsa Aşama 4 (Lineer Mesenteries) olarak physa puanı onun aboral ucunda (enterostome) de yutak için. Not: Dört veya daha az mesenteries &quot;pilili&quot; iç serbest kenarları vardır. yutak görülebilir. Sekizden fazla Tentacles hareketli, görünür ve bazen vücuda sözleşme. </li><li> physa plise ile dörtten fazla mesenteries vardır ve plise Sahne hayvan neredeyse &quot;normal&quot; yetişkin bir görünüme sahiptir 4. En fazla dolu ve dolambaçlı ise Aşama 5 (Ağırlıklı Pileli Mesenteries) olarak physa puanı, ancak görünür vardır gonadal hücreleri. </li></ol></li></ol>

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S., Rentzsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+analysis+of+a+SoxB+gene+reveals+neural+progenitor+cells+in+the+cnidarian+Nematostella+vectensis.">Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis.</a> Development. 141 (24), 4681-4689 (2014).</li><li>DuBuc, T. Q., 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=In+vivo+imaging+of+Nematostella+vectensis+embryogenesis+and+late+development+using+fluorescent+probes.">In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes.</a> BMC Cell Biol. 15, (2014).</li><li>Kaur, J., Debnath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+at+the+crossroads+of+catabolism+and+anabolism.">Autophagy at the crossroads of catabolism and anabolism.</a> Nat Rev Mol Cell Biol. 16 (8), 461-472 (2015).</li><li>Carroll, B., Korolchuk, V. I., Sarkar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+and+autophagy:+cross-talk+and+co-operation+to+control+cellular+homeostasis.">Amino acids and autophagy: cross-talk and co-operation to control cellular homeostasis.</a> Amino Acids. 47 (10), 2065-2088 (2015).</li><li>Glick, D., Barth, S., Macleod, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+cellular+and+molecular+mechanisms.">Autophagy: cellular and molecular mechanisms.</a> J Pathol. 221 (1), 3-12 (2010).</li><li>Rodolfo, C., Di Bartolomeo, S., Cecconi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+stem+and+progenitor+cells.">Autophagy in stem and progenitor cells.</a> Cell Mol Life Sci. 73 (3), 475-496 (2016).</li><li>Guan, J. L., 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=Autophagy+in+stem+cells.">Autophagy in stem cells.</a> Autophagy. 9 (6), 830-849 (2013).</li><li>Phadwal, K., Watson, A. S., Simon, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tightrope+act:+autophagy+in+stem+cell+renewal,+differentiation,+proliferation,+and+aging.">Tightrope act: autophagy in stem cell renewal, differentiation, proliferation, and aging.</a> Cell Mol Life Sci. 70 (1), 89-103 (2013).</li><li>Varga, M., Fodor, E., Vellai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+zebrafish.">Autophagy in zebrafish.</a> Methods. 75, 172-180 (2015).</li><li>Varga, M., 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=Autophagy+is+required+for+zebrafish+caudal+fin+regeneration.">Autophagy is required for zebrafish caudal fin regeneration.</a> Cell Death Differ. 21 (4), 547-556 (2014).</li><li>Gonzalez-Estevez, C., Salo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+apoptosis+in+planarians.">Autophagy and apoptosis in planarians.</a> Apoptosis. 15 (3), 279-292 (2010).</li><li>Buzgariu, W., Chera, S., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+investigate+autophagy+during+starvation+and+regeneration+in+hydra.">Methods to investigate autophagy during starvation and regeneration in hydra.</a> Methods Enzymol. 451, 409-437 (2008).</li><li>Tettamanti, G., 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=Autophagy+in+invertebrates:+insights+into+development,+regeneration+and+body+remodeling.">Autophagy in invertebrates: insights into development, regeneration and body remodeling.</a> Curr Pharm Des. 14 (2), 116-125 (2008).</li></ol>

The authors have nothing to disclose.

Yara iyileşmesi ve rejenerasyon bir model olarak Nematostella kullanımı giderek daha popüler hale gelmektedir. Nedenle, atanan ve mukayese edilebilir etkili hücresel ve moleküler analizler önce belirli bir protokol morfolojik kalıplarını görselleştirmek edebilmek için önemlidir. Hayatın sonrası Planula aşamalarında, hemen hemen her yerde ampute herhangi bir eksik yapısında reform edememek, rejeneratif &quot;esneklik&quot; yüksek derecede sahip Nematostella. Bu nedenle, çeşitli ar...

Tek bir hydra rejenerasyon gözlem deneysel bilim 1,2 olarak biyoloji gelişiyle seminal olay oldu. Yenilenme biyolog olağanüstü geniş temyiz fenomen kalır ve hem kişiyi yatıyordu. İnsan rejenerasyon sınırları anlamak ve üstesinden gelmek için gelişimsel biyologlar, klinisyenler, biyomedikal bilim adamları ve doku mühendisleri için potansiyel daha içsel ilginç daha rejenerasyon biyoloji yapar. Şimdi, böyle genom dizileme ve kazanç ve fonksiyon araçları kaybı olarak ortaya çıkan teknolojilerin kullanımı ile, alan dışında rejeneratif mekanizmaların kızdırmak için hazırlanıyor ve sonuçta yeniden nasıl çeşitli türler anlamak için diğerleri olamaz. Moleküler, hücresel ve morfolojik tepkileri ortaklık derecesi aydınlatılamamıştır gerekmektedir, ancak bugüne kadar yeniden oluşturabilirsiniz hayvanlar arasında temel tepkiler olmuştur IMAGI olurdu daha benzer olduğu görülmektedirned sadece on yıl önce 3. Özellikle cnidarians morfolojik çeşitlilik geniş bir yelpazede ortasında neredeyse tüm vücut parçaları rejenere de kolay bulunmaktadır. yalnız tatlı su polipi itibaren, Hydra gibi Portekizli Man-O-War gibi karmaşık sömürge siphonophores, için, büyük mercan resifleri inşa minik deniz polip ile birlikte, rejenerasyon için bir mekanizmaya ek olarak, çoğu kez üreme şeklidir tamir veya yaralanma ve avlanma sonucu hasarlı ya da kayıp vücut parçaları reform. Cnidaria farklı türler rejenerasyonu için benzer veya farklı mekanizmaları kullanmak ister temelde ilginç bir soru 4-6. Bu ve diğer Anthozoan gelişmekte olan, Nematostella rejenerasyon 7-17 için bir model olarak vectensis. Biz son zamanlarda Abora gelen bisected doku morfolojik üniforma parça bir tüm vücudun yenilenmesini açıklayan bir evreleme sistemi geliştirmiştirpolip 10 l sonu. Herhangi bir düzeyde ikiye zaman Nematostella polipler yeniden iken, biz bu doğal aseksüel fisyon 18 normal düzlemine yakın olduğu için kısmen, en morfolojik basit bölgede, physa bir aboral pozisyonda yetişkin kesmek için seçti, ve aynı zamanda çünkü izin gözlem ve bütün vücudu basit morfolojik bileşenlerden yeniden birleştirilen nasıl moleküler analizleri. Nematostella Rejenerasyon Evreleme Sistemi (NRSS), normal kültür koşulları altında, bir kesilmiş physa rejenerasyon herhangi bir yönü ilerleme skoru kullanılabilir morfolojik kriterler nispeten basit bir dizi içerir ya da deneysel olarak, örneğin küçük molekül tedavileri, genetik manipülasyon da durumları tedirgin veya çevresel değişiklik. Beklendiği gibi, NRSS rejenerasyon hücresel ve moleküler olayların başvurulabilir hangi bir morfolojik yapı iskelesi olarak benimsenmiştir hale gelmektedir10. Nihayet kesim bizim yöntem yakın tarihli bir çalışmada 17 kullanılan pim nokta ponksiyon daha fazla büyüklükte bir gaping delik birkaç emir üretir, ancak her ikisi de yaralar 6 saat civarında iyileşir. yara kapatma görsel tutuklama ve farklı aşamalarını belgeleyen bir yaranın büyüklüğü ve kapatmak için gereken zaman belirgin bağımsızlığını açıklamaya deneysel yaklaşımlar önermek gerekir. Böylece, bu protokolü tarafından sağlanan aboral amputasyon sürecinin daha derin bir görsel anlayış, bu model rejenerasyon sistemi içine daha fazla soruşturma yardım ve Nematostella vectensis kullanarak bu evreleme sisteminin uygulanmasını genişletecek.

<table><tbody><tr><td>&lt;em&gt;Nematostella vectensis,&lt;/em&gt; adults</td><td>Marine Biological Lab (MBL)</td><td></td><td>non-profit supplier</td></tr><tr><td>Glass Culture Dish, 250 mL</td><td>Carolina Biological Supply</td><td>741004</td><td>250 mL</td></tr><tr><td>Glass Culture Dish, 1,500 mL</td><td>Carolina Biological Supply</td><td>741006</td><td>1,500 mL</td></tr><tr><td>Polyethylene transfer pipette, 5&amp;nbsp;mL</td><td>USA Scientific&amp;nbsp;</td><td>1022-2500</td><td>narrow bore, graduated</td></tr><tr><td>Polyethylene transfer pipet, tapered</td><td>Samco</td><td>202-205</td><td>cut off 1 inch of tip to make wide bore</td></tr><tr><td>Disposable Scalpel</td><td>Feather Safety Razor Co. Ltd</td><td>no. 10</td><td>blade should be curved</td></tr><tr><td>#5 Dumont Fine point tweezers</td><td>Roboz</td><td>RS5045</td><td>alternative suppliers available</td></tr><tr><td>Pyrex Petri dish, 100 mm diameter</td><td>Corning</td><td>3160</td><td>can substitute other glass Petri plates</td></tr><tr><td>Sterile 6-well plate</td><td>Corning Falcon&amp;nbsp;</td><td>353046</td><td>or similar from other manufacturer</td></tr><tr><td>Sterile 12-well plate</td><td>Nunc&amp;nbsp;</td><td>150628</td><td>or similar from other manufacturer</td></tr><tr><td>Sterile 24-well plate</td><td>Cellstar, Greiner bio-one</td><td>662-160</td><td>or similar from other manufacturer</td></tr><tr><td>Brine shrimp hathery kit</td><td>San Francisco Bay; drsfostersmith.com</td><td>CD-154005</td><td>option for growing brine shrimp</td></tr><tr><td>pyrex baking dish</td><td>common in grocery stores</td><td></td><td>option for growing brine shrimp</td></tr><tr><td>artificial seawater mix 50 gal or more&amp;nbsp;</td><td>Instant Ocean; drsfoster-smith.com</td><td>CD-116528</td><td>others brands may suffice</td></tr><tr><td>Plastic tub for stock ASW preparation</td><td>various</td><td></td><td>common 25 gallon plastic trash can OK</td></tr><tr><td>Polypropylene Carboy</td><td>Carolina Biological Supply</td><td>716391</td><td>For working stock of ASW @ 12 ppt</td></tr><tr><td>Beaker, Graduated, 4,000 mL</td><td>PhytoTechnology Laboratories</td><td>B199</td><td>For dilution of 36 ppt ASW to 12 ppt</td></tr><tr><td>Stereomicroscope and light source</td><td>various&amp;nbsp;</td><td></td><td>with continuous 1 - 40X magnification</td></tr></tbody></table>

inducing complete polyp regeneration from the aboral physa of the starlet sea anemone nematostella vectensis

Cnidarians ve özellikle Hydra, hasarlı veya kopmuş yapıları ve aslında belki 250&#39;den fazla yıl önce Trembley çalışmaları sayesinde, modern biyolojik soruşturma başlattı tür çalışmaları yeniden oluşturmak için gösterilen ilk hayvanlar vardı. Halen rejenerasyon çalışması süngerler gelen memeliler aracılığıyla bu Hydra, planaria ve Urodeles hem &quot;klasik&quot; rejeneratif organizmaları kullanarak bir diriliş yanı sıra metazoada aralığını kapsayan türlerinin genişletme spektrumunu gördü. biyolojik bir fenomen olarak içsel ilgi yanı sıra, rejenerasyon türlerinin çeşitli nasıl çalıştığını anlamak rejeneratif süreçleri ortak özellikleri ve / veya türler veya bağlam özgü hücresel ve moleküler mekanizmaları paylaşan olup olmadığı hakkında bize bilgi verecek. Starlet deniz anemon, Nematostella vectensis, rejenerasyon için gelişmekte olan bir model organizmadır. Hydra gibi, Nematostella ama t içinde, antik filum, Cnidaria üyesidirO sınıf Anthozo&#39;lar, evrimsel daha bazal olan Hydrozoa kardeş clade. Nematostella rejenerasyon Böylece yönlerini karşılaştırmak ve Hydra ve diğer cnidarians olanlar ile kontrast ilginç olacaktır. Bu yazıda, bisect gözlemlemek ve physa denir Nematostella yetişkin, bir aboral sonu yenilenmesini sınıflandırmak için bir yöntem mevcut. physa doğal eşeysiz üreme için bir araç olarak fisyon maruz ve doğal atom ya physa elle kesilmesi ya kompleks morfolojilerin yeniden büyüme ve bir reformasyon tetikler. Burada bir Nematostella Rejenerasyon Evreleme Sistemi (NRSS) bu basit morfolojik değişikliklere kodlanmıştır var. Biz klorokin etkilerini, lizozomal fonksiyonunun blokları otofaji inhibitörü test etmek için NRSS kullanın. Sonuçlar Otofaji inhibe edildiğinde, polip yapıların, özellikle mesenteries, rejenerasyon anormal olduğunu göstermektedir.

Kopmuş physa rejenerasyon sırasında morfolojik olayların ilerlemesi her NRSS aşamada physa temsili görüşlerini içeren Şekil 1A, gösterilmiştir. Tipik physa kesim sitesi yetişkin (ok başları) gösterilir. Şekil 1A fotoğraflar tamamen şekillenmiş polip ile taze kesilmiş physa sözlü ve vücut yapılarının ilerici rejenerasyon göstermektedir. Şekil 1B, sırasıyla, Sahne 4 ve 5. etapta da, iç septa, mesenteries dü...

Watch this Scientific Journal Video about Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis at JoVE.com

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 ...

inducing-complete-polyp-regeneration-from-aboral-physa-starlet-sea

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

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

8.5K Görüntüleme.  Stony Brook University. 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.

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

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call &#34;chemical amputation.&#34;&#160;Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

Kimyasal amputasyon ve yeniden oluşturulmasını Planarian Schmidtea mediterranea yutak

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to ...

Gelişim Biyolojisi

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Rejenerasyon Stentor içinde çalışma yöntemleri

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Genotyping of Sea Anemone during Early Development

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the life of the animal. Following in vitro fertilization and de-jellying, zygotes are allowed to develop for 24 h at room temperature to reach the early- to mid-gastrula stage. The gastrula embryos are then placed on an agarose gel bed in a Petri dish containing seawater. Under the dissecting microscope, a tungsten needle is used to surgically separate an aboral tissue fragment from each embryo. Post-surgery embryos are then allowed to heal and continue development. Genomic DNA is extracted from the isolated tissue fragment and used as a template for locus-specific PCR. The genotype can be determined based on the size of PCR products or presence/absence of allele-specific PCR products. Post-surgery embryos are then sorted according to the genotype. The duration of the entire genotyping process depends on the number of embryos to be screened, but it minimally requires 4&#8211;5 h. This method can be used to identify knockout mutants from a genetically heterogeneous population of embryos and enables analyses of phenotypes during development.

The goal of this protocol is to genotype the sea anemone Nematostella vectensis during gastrulation without sacrificing the embryo.

Erken gelişim sırasında deniz anemon Genotipleme

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the ...

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

Deniz Anemonlardan Elde Edilen Ham Ekstraktlarda Hemolitik ve Fosfolipaz Aktivitesinin Basit Biyotahlillerle Tanımlanması

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

Biyokimya

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Farmakolojik ve Fonksiyonel Genetik Tahliller Planarian Rejenerasyon işleyin<emJaponica> Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Biyoloji

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

Sinirsiz Kuşak ve Uzun Süreli Bakım<em&gt; Hydra</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

Nörobilim

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Planarian İmmobilizasyon, Kısmi Işınlama, ve Doku Nakli

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

Mikroenjeksiyonu Bir Yöntem<em&gt; Patiria Minata</em&gt; Zigotların

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Inducing Polyp Bail-out in Coral Colonies to Obtain Individualized Micropropagates for Laboratory Experimental Use

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to the rest of the colony and ultimately detach from the skeleton to continue living as separate individuals. Coral biologists have acknowledged the process of polyp bail-out for years, but only recently the micropropagates generated by this process have been recognized as a model system for coral biology studies. The use of polyp bail-out can create a high number of clonal units from a single coral fragment. Another benefit is that single polyps or patches of polyps can be easily visualized under a microscope and maintained in highly standardized low-cost environments such as Petri dishes, flasks, and microfluidic chips. The present protocol demonstrates reproducible methods capable of inducing coral micropropagation and different approaches for maintaining the single polyps alive in the long term. This methodology was capable of successfully cultivating polyps of the coral species Pocillopora verrucosa for up to 8 weeks after bail-out, exhibiting the practicality of using individual coral polyps for coral research.

Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.

Mercan Kolonilerinde Polip Kefalinin Laboratuvar Deneysel Kullanımı için Bireyselleştirilmiş Mikropropagatlar Elde Edilmesini İndüklemek

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of ...

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

This paper describes a method to create wounds in the epithelium of a live Clytia hemisphaerica medusa and image wound healing at a high resolution in vivo. Additionally, a technique to introduce dyes and drugs to perturb signaling processes in the epithelial cells and extracellular matrix during wound healing is presented.

Starlet Deniz Anemone Aboral physa Tam Polip Regeneration uyaran

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Kimyasal amputasyon ve yeniden oluşturulmasını Planarian Schmidtea mediterranea yutak

Rejenerasyon Stentor içinde çalışma yöntemleri

Erken gelişim sırasında deniz anemon Genotipleme

Deniz Anemonlardan Elde Edilen Ham Ekstraktlarda Hemolitik ve Fosfolipaz Aktivitesinin Basit Biyotahlillerle Tanımlanması

Farmakolojik ve Fonksiyonel Genetik Tahliller Planarian Rejenerasyon işleyin Dugesia

Sinirsiz Kuşak ve Uzun Süreli Bakım

Planarian İmmobilizasyon, Kısmi Işınlama, ve Doku Nakli

Mikroenjeksiyonu Bir Yöntem

Mercan Kolonilerinde Polip Kefalinin Laboratuvar Deneysel Kullanımı için Bireyselleştirilmiş Mikropropagatlar Elde Edilmesini İndüklemek

Cnidarian Model Organizma Clytia hemisphaerica'yı Kullanarak İn Vivo Epitelyal Yara İyileşmesini Karakterize Etmek