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

Este trabalho foi apoiado por uma Stem Cell New York Science (NYSTEM C028107) Grant para GHT.

1. Condicionado dos Animais para temperatura, Nutrição e ciclo claro / escuro <ol><li> Obter adultos Nematostella vectensis de um dos numerosos laboratórios Nematostella em todo o mundo, ou um fornecedor sem fins lucrativos (Tabela 1) </li><li> Manter Nematostella a uma temperatura constante (tipicamente entre 18 e 21 ° C) no escuro, em &quot;1 / 3x&quot; mar artificial água (ASW) a uma salinidade de 12 partes por mil (ppt). Manter culturas simples de soda-cal placas de cultura de vidro, tipicamente 250 mL ou 1,5 L de capacidade 11. Nota: Estas condições de cultura simples são comumente usado entre os laboratórios que estudam Nematostella, mas cuidado cultura também pode ser automatizado 19. </li><li> Alimente Nematostella Artemia recém nascidos náuplios 2 - 4x por semana. Escotilha Artemia cistos em força total (36 ppt) ou 1 / 3x ASW a 30 ° C, em um prato de vidro retangular raso 20 ou emqualquer um de uma série de pequena escala, incubadoras artémia comerciais ou caseiros. Se uma incubadora não está disponível, camarão eclodem na RT, mas fazê-lo de forma mais lenta. Nota: Isso muitas vezes requer mais de 24 h para a conclusão. </li><li> Substituir a água cultura anêmona, pelo menos uma vez por semana. Para uma melhor saúde do adulto, completamente limpa (sem sabão) a cultura tigelas, uma vez por semana de secreções mucosas acumuladas, que o revestimento da bacia e pode armadilha restos de comida e resíduos, e enredar os animais. </li></ol> 2. Seleção e relaxamento dos Animais Nutricionalmente condicionado <ol><li> Seleccione pólipos pareados por tamanho de aproximadamente o mesmo comprimento (3 - 5 cm, quando naturalmente relaxado) e coloque-os em uma tigela separada da colônia durante três dias antes da amputação. Nota: O número de animais seleccionados para o corte vai ser determinada pela experiência sendo realizada, é claro, mas, em geral, nós recomendamos pelo menos cinco animais por ponto de amostragem com seis repetições. Assim,Numa experiência típica de um mínimo de 30 animais seria pré-seleccionado. Em geral, é sábio para selecionar mais do que o número mínimo (30), uma vez que são amputações irregular (veja abaixo) podem posteriormente afetar pontuação. </li><li> Remover o prato de animais seleccionados a partir do incubador em luz ambiente, pelo menos, uma hora antes da amputação. Nota: A exposição à luz ambiente e vibrações de manipulação provavelmente fará com que os animais a se contrair, então eles precisam de ser adaptados ou &quot;relaxado&quot; por incubação na bancada do laboratório. Os animais irão tornar-se refractário ao toque e a exposição à luz e em que ponto pode ser movido por pipetagem suave. </li><li> Opcional: Anestesiar os animais através da adição de 7,5% de MgCl 2 (em 1 / 3x ASW). Delicadamente adicionar a solução de MgCl2 para a tigela com um padrão de plástico de 5 mL pipeta. Nota: Embora os animais acabará por se tornar habituados à luz e à manipulação física, pode ser vantajoso para anestesiar animais para manter ou &quot;consertar&quot; tele relaxou estado depois que eles se tornaram 16,21,22 alongado. </li><li> Utilizar um grande furo (&gt; 0,5 cm) de pipeta de plástico para transferir (em 1 / 3x ASW) cinco animais da piscina para ser amputado, no fundo de uma placa de corte de vidro estéril de 100 mm de diâmetro contendo 12 ppt ASW. Coloque o prato no palco de um microscópio estereoscópico com ampliação variável entre 10 - 40X. Nota: Se os animais não foram anestesiadas e relaxado para o corte, eles ainda podem responder ao toque e iluminação estereoscópio e, portanto, pode precisar de alguns minutos para se tornar relaxado novamente. </li></ol> 3. A amputação <ol><li> Usando um bisturi estéril, amputa o physa aboral de cada pólipo, com o objetivo de se obter uma secção da physa que é aproximadamente do mesmo comprimento como é largo e não contendo mesentério. Nota: O site corte ideal é apenas aboral à rescisão do mesentério. No plano de corte existe uma transição de mesentério adequada para diluir lines correspondente a cada inserção do mesentério (ver Figura 1, as setas). Ausência de mesentério é crítica porque produz muco que podem facilitar &#39;ligar&#39; o buraco 17,30. <ol><li> Colocar a lâmina de bisturi em contacto com o animal no local desejado de amputação. Fazer isso sem ajuda (mão livre), ou agarrando suavemente o corpo do animal com um # 5 fórceps (estilo Dumont ou similar). </li><li> Cortar o tecido, aproveitando a lâmina curva do bisturi em um movimento de &quot;balanço&quot; em todo o corpo. Nota: O tecido deve cortar limpa quanto o bisturi é abalada e libertar a seção desejada da physa do doador. No entanto, se um pequeno pedaço de tecido ainda liga o corpo e o physa, cortá-lo com o bisturi. Não tente separar as peças conectadas puxando, pois isso pode danificar o physa. </li></ol></li><li> Remover cada pólipo amputada &#39;doador&#39; do prato e devolvê-lo a um sepabowl taxa rotulados &#39;amputados comuns »; datar a tigela e devolvê-lo à cultura estoque. Nota: pólipos amputados irá curar a ferida aboral dentro de um dia e em seguida, podem ser alimentados normalmente. Eles vão regenerar uma physa aparência normal dentro de duas semanas em que ponto o physa pode ser amputados novamente se desejar. </li><li> Lavar o physa excisado que permanecem no prato de corte de 12 ppt ASW, em seguida, transferir cada physa a um poço separado estéril numa placa de cultura de células multi-poços que já tem 10 mL de 12 ppt ASW em cada poço. Nota: Este exemplo usa uma placa de seis, com cada poço de detenção de 10 ml de água do mar e cinco physa excisada. Na água do mar em geral devem cobrir o physa suficiente para evitar a exposição ao ar devido ao movimento na manipulação e evaporação potencial. A placa de poços ou deve ter uma tampa. </li><li> Repita os passos de 3,1-3,3 para recolher, pelo menos, 5 physa em cada poço reservado para cada tratamento experimental. </li><li> Incubar a physa a uma temperatura que vai provide a melhor taxa de regeneração para os interrogatórios experimentais planejadas. Colocar a placa contendo o physa em uma incubadora de temperatura regulada, a uma temperatura fixa, determinada pela desejada taxa de regeneração. Nota: O physa irá regenerar tecidos em falta e formar um pólipo completa quando incubados a temperaturas entre 15 e 27 ° C. A taxa de regeneração é dependente da temperatura, excepto para as primeiras duas fases. O dia médio para atingir Fase 4 para todas as temperaturas é 7 d após o corte e isso também coincide com a regeneração a 21 ° C. A 27 ° C, Fase 4 é atingido cerca de 3 dias mais cedo e a 15 ° C, Fase 4 é retardada por cerca de 3 d em comparação com a regeneração a 21 ° C (ver também referência 10). </li></ol> 4. Avaliação de regeneração com o sistema de estadiamento Nematostella Regeneração (ENSR) <ol><li> Marcar o physa usando um microscópio estéreo de composto com magnific variávelção (10 - 80X). Marcar o physa Nematostella recém-cortada como estágio 0 e continuar marcando, ao mesmo tempo, cada amputação dia pós (dpa) usando a ENSR 10. Nota: Para os critérios de paragem-chave e detalhes, consulte Referência 10. <ol><li> Pontuação physa como Estágio 0 (ferida aberta) se um physa recém-cortada aparece como uma massa em forma Cup- semelhante a um balão flácido, com um local de ferida aberta é provável visível. Nota: As bordas da ferida também pode ficar juntos desde o início, mas o tecido ainda vai ser recolhido e falta de rigidez. As bordas da ferida aberta pode ser observado a sofrer contracção radial, a ferida cicatriza. </li><li> Pontuação physa como Fase 1 (ferida fechada) se a ferida da amputação parece fechada. Nota: local da ferida irá corresponder ao futuro pólo oral. A superfície exterior em torno do futuro pólo oral podem começar a exibir arcos distintos correspondentes aos subjacentes endodérmicas radialmente simétrica inserções mesentéricos. </li><li> ph pontuaçãoysa como Fase 2 (radial dos arcos) se a superfície do pólo oral parece inflado, revelando oito arcos dispostos em relevo um padrão radialmente simétrico e separados por ranhuras. Observar pequenas bolhas, hemisféricas no vértice dos arcos. Eles serão tão alto quanto ampla transitória, provável, e, inicialmente composta por uma camada de células ectodérmica única. Nota: Em alguns casos bolhas duas camadas pode estabilizar. Nota: Neste ou mais tarde encena uma camada de muco podem aparecer para encapsular o physa (Figura 2) em uma &quot;bainha&quot; membranoso. Este material de encapsulação deve ser removido para facilitar a pontuação. </li><li> Pontuação physa como Stage 3 (Tentáculo), se as papilas dos tentáculos contendo endoderme e ectodérmicas camadas de tecido são formadas de forma estável no final oral de pelo menos alguns arcos radiais. Nota: Os tentáculos são mais longos do que eles são largas e são minimamente móveis. O physa vai mostram aumento, mas a inflação variável para que rudimentos mesentério pode tornar-se visvel que se prolonga a partir da inserção do mesentério na cavidade do corpo (coelenteron). </li><li> Pontuação physa como Fase 4 (mesentérios linear) se o physa contém oito distintos, mesentérios visíveis que se estendem para o coelenteron de inserções na parede do corpo, com comprimentos orais-aboral que são mais do que duas vezes a sua largura radial medida a partir de onde eles parecem ligar para a faringe, na sua extremidade aboral (enterostome). Nota: Quatro ou menos mesentérios têm bordas livres internos &quot;pregas&quot;. A faringe é visível. Mais de oito tentáculos são visíveis, móveis e às vezes eles contrair no corpo. </li><li> Pontuação physa como Stage 5 (predominantemente plissadas mesentérios) se o physa tem mais de quatro mesentérios com pregas, eo pregas é mais completo e sinuosa do que no estágio 4. O animal tem uma aparência adulta quase &quot;normal&quot;, mas não há nenhum visível células gonadais. </li></ol></li></ol>

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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.

Uso de Nematostella como um modelo de cura de feridas e a regeneração está a tornar-se cada vez mais popular. Assim, é importante ser capaz de visualizar os padrões morfológicos de um protocolo em particular antes de análises celulares e moleculares eficazes podem ser atribuídas e comparados. Nematostella têm um elevado grau de &quot;flexibilidade&quot; regenerativa, ser capaz de mudar a quase qualquer estrutura que falta amputado em qualquer local, em fases pós-Planula de vida. Assim, vário...

A observação de regeneração em um único hidra foi o evento seminal na advento da biologia como um 1,2 ciência experimental. Regeneração continua a ser um fenômeno da extraordinariamente amplo apelo para biólogo e leigo alike. O potencial para os biólogos do desenvolvimento, médicos, cientistas biomédicos e engenheiros de tecido para entender e superar os limites sobre a regeneração humana faz biologia regeneração mais de intrinsecamente interessante. Agora, com o uso de tecnologias emergentes, como a sequenciação do genoma e ganho e perda de ferramentas de função, o campo está pronta para provocar mecanismos além regenerativos e compreender, finalmente, como espécies diferentes podem se regenerar enquanto outros não podem. O grau de comunalidade nas respostas moleculares, celulares e morfológicas continua a ser elucidado, mas até agora parece que as respostas básicas entre os animais que podem regenerar é mais similar do que teria sido imagiNED há apenas uma década 3. Cnidários em particular, são facile na regeneração quase todas as suas partes do corpo em meio a um amplo espectro de diversidade morfológica. A partir do solitário pólipo de água doce, Hidra, juntamente com as pequenas pólipos marinhos que constroem imensos recifes de coral, para os Siphonophores coloniais complexos, tais como o Português Man-O-War, a regeneração é muitas vezes um modo de reprodução, além de um mecanismo de reparar ou reformar partes do corpo danificadas ou perdidas resultantes de lesões e predação. Se as diversas espécies de Cnidaria utilizar mecanismos semelhantes ou diferentes para a regeneração é uma questão fundamentalmente interessante 4-6. Nós, e outros têm vindo a desenvolver o anthozoan, Nematostella vectensis como um modelo para a regeneração 7-17. Recentemente, desenvolveu um sistema de estadiamento para descrever a regeneração de um órgão inteiro a partir de um pedaço morfologicamente uniforme de tecido cortada a partir das aboral final do pólipo 10. Enquanto pólipos Nematostella pode regenerar quando cortada em qualquer nível, nós escolhemos para cortar adultos em uma posição aboral na região mais morfologicamente simples, o physa, em parte porque este é perto do plano normal da fissão assexual natural, 18, e também porque licenças de observação e análise molecular de como um corpo inteiro é remontado a partir dos componentes morfológicos mais simples. A encenação Sistema Nematostella Regeneração (ENSR) fornece um conjunto relativamente simples de pontos de referência morfológicas que poderiam ser usados para marcar o progresso de qualquer aspecto da regeneração por um physa amputada, em condições de cultura normais ou experimentalmente perturbado situações como pequenos tratamentos molécula, a manipulação genética ou alteração ambiental. Como antecipado, a NRSS está sendo adotado como um andaime morfológico em que os eventos celulares e moleculares de regeneração pode ser referenciado10. Finalmente o nosso método de corte produz uma Gaping buracos várias ordens de magnitude maior do que a punção ponto pinos usado em um estudo recente 17, mas ambas as feridas cicatrizam em torno de 6 horas. Documentar as fases visualmente distintas de prender e de encerramento de feridas deve sugerir abordagens experimentais para explicar a aparente independência do tamanho de uma ferida e o tempo que leva para a fechar. Assim, uma compreensão visual mais profunda do processo de amputação aboral, fornecida por este protocolo, irá auxiliar investigações ulteriores sobre esta sistema de regeneração de modelo e ampliar a aplicação deste sistema de estadiamento usando Nematostella vectensis.

<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

Cnidários e, especificamente, Hydra, foram os primeiros animais indicado para regenerar estruturas danificadas ou cortada, e na verdade esses estudos sem dúvida lançados investigação biológica moderna através do trabalho de Trembley mais de 250 anos atrás. Presentemente o estudo de regeneração tenha visto um ressurgimento usando ambos os organismos regenerativos &quot;clássicas&quot;, tais como a hidra, e Planaria Urodelos, bem como um alargamento do espectro de espécies que abrangem a gama de metazoários, a partir de esponjas através de mamíferos. Além de seu interesse intrínseco como um fenômeno biológico, a compreensão de como a regeneração funciona em uma variedade de espécies irá informar-nos sobre se a processos regenerativos compartilhar recursos e / ou espécies comuns ou mecanismos celulares e moleculares específicas ao contexto. A anêmona de mar estrela, Nematostella vectensis, é um organismo modelo emergente para a regeneração. Como Hydra, Nematostella é membro do filo antiga, cnidaria, mas dentro de tele anthozoa classe, um clado irmão do Hydrozoa que é evolutivamente mais basal. Assim, os aspectos da regeneração na Nematostella será interessante comparar e contrastar com os de Hydra e outros cnidários. Neste artigo, apresentamos um método para bissetriz, observar e classificar a regeneração do fim aboral do adulto Nematostella, que é chamado de physa. O physa naturalmente sofre fissão como um meio de reprodução assexuada, e quer fissão naturais ou amputação manual da physa desencadeia re-crescimento e reforma de morfologias complexas. Aqui temos codificada estas alterações morfológicas simples em um sistema de estadiamento Nematostella Regeneração (a ENSR). Usamos o NRSS para testar os efeitos de cloroquina, um inibidor da função lisossómica que bloqueia autofagia. Os resultados mostram que a regeneração de estruturas de pólipos, particularmente os mesentério, é anormal quando autofagia é inibida.

A progressão de eventos morfológicas durante a regeneração em physa cortada é mostrado na Figura 1A, que inclui vistas representativas de physa em cada fase NRSS. O site típico physa corte é indicado no adulto (setas). As fotografias na Figura 1A mostra a regeneração progressiva das estruturas orais e corporais a partir physa recém-cortada através pólipo completamente formado. Figura 1B, C mostram a disposição dos septos in...

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.

Induzindo completa Polyp Regeneração do aboral Physa da Anémona Starlet<em&gt; Vectensis Nematostella</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 de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

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

8.5K visualizações.  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.

Vídeo: 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.

Química amputação e regeneração da faringe nos Turbellaria mediterranea Schmidtea

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 ...

Biologia do Desenvolvimento

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.

Métodos para o estudo da regeneração em Stentor

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.

Genotyping de Anemone de mar durante o desenvolvimento adiantado

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.

Identificação da Atividade Hemolítica e Fosfolipase em Extratos Brutos de Anêmonas do Mar por Bioensaões Simples

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

Bioquímica

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.

Farmacológica e funcional Ensaios genética para manipular Regeneração da planária Dugesia japonica</em

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

Biologia

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.

Geração e Manutenção a Longo Prazo de Nerve-free<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 ...

Neurociência

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.

Imobilização planária, irradiação parcial e Transplante de Tecidos

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.

Um Método para microinjeção de<em&gt; Patiria minata</em&gt; zigotos

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.

Indução de resgate de pólipos em colônias de corais para obtenção de micropropagates individualizados para uso experimental em laboratório

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.

Induzindo completa Polyp Regeneração do aboral Physa da Anémona Starlet

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