We gratefully acknowledge Dr. Deborah L. Gumucio as our advisor and for her invaluable support in defining the culture and imaging methods. We also thank Dr. Jim Brodie, Dr. Hong-Xiang Lu, Dr. Charlotte Mistretta, and Dr. Ann Grosse for their contributions to the development of the whole intestine organ culture system. Helpful discussions on imaging provided excellent advice from Dr. Chip Montrose, Michael Czerwinski and Sasha Meshinchi. All imaging was performed in the Microscopy and Image Analysis Laboratory at the University of Michigan. Funding support was provided by NIH R01 DK065850.

NOTA: Todos os camundongos foram tratados com humanidade usando protocolos aprovados pela Universidade de Michigan Medical School Unidade de Medicina Laboratorial Animal e de acordo com as orientações do Comitê da Universidade de uso e cuidados dos animais. 1. Whole sistemas de órgãos Cultura <ol><li> Preparação de meios de comunicação e placas de cultura <ol><li> Em uma cultura capuz tecido, remover 5 ml de meio BGJb da garrafa estoque e adicionar 5 ml de Pen / Strep. Prepare um estoque de trabalho de mídia em um tubo de 50 ml, adicionando 1 ml de 5 mg / ml de ácido ascórbico a 49 ml de meio BGJb (com Pen / Strep). </li><li> Preparar uma placa de cultura de 6 poços por adição de 1 ml por baixo de cada um dos transwells. NOTA: Se todos os 6 transwells não são utilizados, mova transwells extras em uma nova estéril placa de 6 poços para uso posterior. Adicione 3 mL de meios de trabalho BGJb para cada uma de três placas de Petri de 10 centímetros estéreis. </li></ol></li><li> Preparação para dissecção <ol><li> Mergulhe ferramentas de dissecação em 70% ethanol e não se esqueça de manter as ferramentas limpas, enquanto trabalhava. </li><li> Configure a área de dissecação com um grande balde de gelo e coloque a placa de cultura de 6 poços e 10 centímetros de Petri com meio BGJb no gelo. Trabalhar no gelo, tanto quanto possível, enquanto dissecar e preparar o intestino para a cultura. </li><li> Anestesiar ratos fêmea adulta em uma câmara com isofluorano (100 l) antes de eutanásia por deslocamento cervical para a exploração dos intestinos fetais. </li><li> Após a colheita os fetos de sua mãe, encenar cada feto cuidadosamente de acordo com o gráfico 13 Theiler encenação. Não confie nos dias pós-coito para determinar a idade de cada feto como variações de até 24 horas, em fase de desenvolvimento são rotineiramente observado em uma única ninhada. </li></ol></li><li> Dissecção da intestinos fetais <ol><li> Eutanásia fetos por decapitação antes da remoção do intestino. </li><li> Dissecar cada intestino em 1x de Dulbecco Phosphate-Buffered Saline (DPBS) e place-lo em uma placa de petri 10 centímetros com o trabalho de mídia BGJb. </li><li> Cuidadosamente remover o baço do estômago e do pâncreas do estômago e duodeno superior. </li><li> Suavemente separar o omento tendo o cuidado de deixar o omento ligado, tanto quanto possível, e que separa as ligações apenas o suficiente para permitir que o intestino para ser esticado. NOTA: Para o intestino mais velhos do que os que são ou E14.5 cultivadas mais do que 24 horas, cuidadosamente separar o intestino em três segmentos iguais para permitir conteúdo luminal para fluir através do intestino e ser expelido das extremidades cortadas. No entanto, para as culturas iniciadas antes E13.5, esperar até que após 24 horas de cultura antes de se separar os três segmentos do intestino para permitir que a serosa para crescer adequadamente ao longo do comprimento do intestino. </li><li> Usar uma pipeta de boca para transferir as peças intestinais para os transwells da placa de cultura (1.1.2). </li><li> Gentilmente reposicionar os intestinos como necessário para que eles fiquem em frente a Transwell. NOTA: Uma vez que os intestinos começam a se mover conteúdo luminal através do tubo, todas as torções no intestino fará uma cópia de segurança e danos ao intestino. </li></ol></li><li> Cultura e tratamento <ol><li> Remova a mídia sob o transwell, adicionar 700 mL de mídia (media de tratamento que trabalham com proteínas recombinantes adicionados ou inibidores farmacológicos), sob o transwell. </li><li> Adicionar 300 mL de meio de tratamento gota a gota, em cima dos intestinos e deixe de molho por meio da transwell. Reposicionar os intestinos, se necessário. </li><li> Mudar meio de tratamento, pelo menos, uma vez por dia ou mais frequentemente, dependendo da meia-vida do reagente de tratamento. </li></ol></li><li> Preparação de esferas de agarose de proteína embebido por uma fonte localizada de tratamento reagente <ol><li> Ressuspender as esferas de agarose em seu recipiente de armazenagem e transferência de 100 ul de esferas de agarose para um tubo de microcentrífuga de 1,5 ml. </li><li> Encher o volume restante do tubo estéril com 1x Phosphate-Buffered Saline (PBS) e misturar as contas para lavá-los. Colocar o tubo de microcentrífuga num bastidor durante alguns minutos para permitir que os grânulos se depositem no fundo e, em seguida, para fora da pipeta a partir de PBS no topo dos grânulos. Tornar a encher o tubo de microcentrífuga estéril com 1x PBS e repetir o processo de lavagem mais duas vezes. </li><li> Prepare a proteína de interesse em duas vezes a concentração desejada para o tratamento. </li><li> Misturar 5 mL de pérolas de agarose lavados com 5 l de estoque a proteína 2x e agitar suavemente as pérolas em proteína. Colocar o tubo em gelo durante 1 hora, misturando suavemente cada 10-15 min. </li><li> Lavar as contas brevemente em estéril 1x PBS, antes de colocar as contas no intestino. </li></ol></li><li> Colocação de pérolas de agarose <ol><li> Coloque suavemente as esferas de agarose no topo do intestino utilizando uma pipeta de boca com agulhas capilares puxados. Coloque as contas com extrema cautela, como manuseio descuidado pode danificar o intestino e prevenir o desenvolvimento de vilosidades no local da lesão. </li><li> Inserir o dobro de grânulos como são necessários para a análise uma vez que os intestinos irá sofrer movimentos peristálticos e cerca de metade das pérolas colocadas sairão dos intestinos mais o tempo de cultura. </li></ol></li><li> Fixação de intestinos cultivadas <ol><li> Retire cuidadosamente a mídia de tratamento abaixo do transwell e permitir que os intestinos para o ar seco por 3 min. </li><li> Adicionar 1 ml de fixador sob a transwell durante 2 min, em seguida cobrir a parte superior do intestino com 2 ml de fixador para o restante do tempo de fixação. Fixar com paraformaldeído a 4% O / N a 4 ° C ou durante 2 horas à temperatura ambiente. </li></ol></li></ol> 2 Imagem de intestinos fixas <ol><li> Imaging Wholemount <ol><li> Coloque os intestinos inteiros (com fluorescência endógena) em um slide com 100 ml de 1x DPBS. Use uma pinça para organizar cuidadosamente os intestinos. </li><li> Use um lenço de laboratório para remover cuidadosamente os DPBS e imediatamente adicione 100 ml de glicerol a 70% para fazer a mo tecidore transparente e possível aumentar a profundidade da imagem. NOTA: Se a penetração do intestino é desejado, em vez de montagem no intestino em 70% de glicerol, embeber o intestino em algumas gotas de solução de focagem claro para 10-30 min. Pipeta off a solução foco claro e montar o intestino com o Monte Clear. </li><li> Mergulhe cada um dos quatro cantos de uma lamela em massa de modelar e pegar uma pequena quantidade de argila para usar como espaçadores entre lâmina e lamela para manter a lamela de achatamento do intestino. </li><li> Selar a lamela sobre a lâmina com valap derretida aplicado com um cotonete. </li><li> Imagem do intestino de cada um microscópio confocal vertical ou invertida. Consulte a discussão para mais detalhes. </li></ol></li><li> Vibratome corte de intestino fixos (para corte transversal das estruturas intestinais) <ol><li> Intestinos inserir em 7% agarose em pequenos moldes de plástico (10 x 10 x 15 mm) e permitir que os blocos de agarose paraesfriar e solidificar. </li><li> Remova os blocos de agarose a partir dos moldes de plástico e cola blocos de agarose para a etapa de coleta vibratome com cola instantânea. </li><li> Preencha a etapa de coleta com o frio do gelo 1x DPBS. </li><li> Cortar secções com uma espessura entre 100-150 m. Para seções mais grossas usar soluções de compensação (descritos no ponto 2.1.2) para visualizar mais profundamente na seção transversal. </li><li> Despeje vibratome secções a partir da fase de recolha para um poço de uma placa de 6 cavidades, para armazenamento a 4 ° C. </li></ol></li><li> Coloração de imunofluorescência, tecidos seccionados vibratome fixos <ol><li> Colocar 5-12 secções vibratome por cavidade em uma placa de 24 poços com 1 x DPBS. </li><li> Remover 1x DPBS e adicione 500 ml de solução de permeabilização por poço. Agite delicadamente as amostras por 25 min em temperatura ambiente. </li><li> Depois de permeabilização, lavar as amostras 3 vezes com PBS 1x. </li><li> Bloquear as amostras de, pelo menos, 30 minutos em 500 ul de solução de bloqueio. </li><li> Após o bloqueio, exchange a solução de bloqueio com 500 ul de anticorpos primários diluídos em solução de bloqueio e a manter as amostras a 4 ° CO / N. NOTA: As diluições de anticorpos primários que funcionam bem em cortes congelados e parafina geralmente funcionam bem em vibratome seções também, no entanto os anticorpos que exige recuperação do antígeno nuclear geralmente não funcionam bem com este protocolo (por exemplo, BrdU). </li><li> No dia seguinte, as amostras de lavar três vezes durante 15 min de cada vez com solução de bloqueio. </li><li> Após a lavagem, aplicam-se 500 ul do anticorpo secundário diluído em solução de bloqueio. Envolva a placa de 24 poços com folha de alumínio para proteger os fluoróforos de luz e balançar as amostras durante 45 minutos a 2 horas à temperatura ambiente. </li><li> Lave as amostras 3 vezes com PBS 1x por 15 minutos cada e montar as seções vibratome em slides como descrito na Seção 2.4. </li></ol></li><li> Montagem vibratome seções <ol><li> Preparar lâminas para vibratome montagem cortando fatias finas de double-stick fita e colocá-los ao longo dos lados longos dos slides. </li><li> Com cuidado, coloque 5-10 seções vibratome entre a fita dupla-stick nos slides em uma gota de 100 l de PBS 1x. </li><li> Desdobrar todas as seções e espalhá-los em uma única camada em todo o slide. </li><li> Remova qualquer excesso de agarose ou pedaços irregulares que impediria uma lamela de sentar-se plana. </li><li> Com cuidado, use um tecido de laboratório para absorver o excesso de PBS 1x de todo as seções vibratome. </li><li> Adicionar uma gota de meio de montagem aquoso no topo das secções de vibratome e depois lamela. </li><li> Sele as lamelas para as lâminas com derreteu valap aplicado com um cotonete. </li><li> Imagem seções vibratome em ambos um microscópio confocal vertical ou invertida. Consulte a discussão para mais detalhes sobre a seleção de um sistema de imagem. </li></ol></li></ol> 3 Imagens ao vivo de intestinos inteiros Intestinos colhidas a partir de fetoss depois de E12.5, com o omento ligado deixado intacto, desenvolver com sucesso vilosidades em cultura usando o sistema acima. Portanto, este sistema ex vivo permite a captura de imagens de secção z vivo do intestino em desenvolvimento, que podem ser reconstituídas para uma vista tridimensional da morfogénese ao longo do tempo. <ol><li> Configurando imagens ao vivo <ol><li> Use cola instantânea para aderir uma membrana de policarbonato indivíduo a uma tela de malha fina. Isto proporciona uma estrutura de suporte de apoio para segurar o intestino em lugar abaixo da lente de imersão do microscópio confocal vertical. Por favor, consulte a seção de discussão para mais detalhes sobre a seleção de microscópios de imagem ao vivo. </li><li> Coloque a tela de malha no centro do poço de uma placa de cultura. </li><li> Inserir o intestino dissecados sobre a membrana de policarbonato e adicionar uma gota de cola instantânea em cada extremidade. Use apenas cola suficiente para apor o intestino, mas não revesti-la! </li><li> Adicionar cultura de mídia livre de fenol vermelho abaixo do policarbonatomembrana no centro de uma das cavidades da placa de cultura. </li><li> Adicionar água para a borda exterior da placa de cultura para fornecer humidade. </li><li> Uma vez que o intestino fetal sofre ondas peristálticas semelhantes de contracção da cultura, adicionar 100 ml de uma solução a 1: 5 de xilazina em 1x PBS a 3 ml de meio para controlar o movimento do intestino, enquanto imagiologia. Esta dosagem vai controlar os movimentos intestinais por cerca de 8-10 horas, sem comprometer o desenvolvimento das vilosidades. </li><li> Para um corte transversal do intestino delgado, o intestino incorporar vivos em uma solução de 3-4% de agarose e cortou secções espessas (150-500 um) sobre um vibratome. Combinar a rigidez da agarose com a rigidez do intestino que está sendo cortada e empiricamente determinada fase de desenvolvimento. Geralmente, uma solução de agarose a 3% funciona bem para os intestinos menores de E14.5 e uma solução a 4% funciona bem para os intestinos E15-E16. </li><li> Cole as seções vibratome a uma membrana de policarbonato aposta em uma tela de malha (como na Seção 3.1.1) ecultura como descrito na Seção 3.1.2-3.1.6. </li><li> Conduzir a imagens ao vivo usando um de dois fótons microscópio confocal de fluorescência (excitação laser entre 690-1,040 nm) em uma posição vertical com uma mesa ajustável. </li><li> Dois fótons de laser sintonizado 900 nm proporciona uma penetração profunda com a melhor resolução para sinais de GFP. Reduz danos aos tecidos, enquanto Imaging. Além disso, a configuração da potência do laser de emissão mais baixa possível reduzir o dano tecidual. Tipicamente, para obter uma boa excitação de GFP, usar 12% de energia em que o laser de dois fotões. </li></ol></li></ol>

<ol>
	<li>Grosse, A. S., 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=Cell+dynamics+in+fetal+intestinal+epithelium:+implications+for+intestinal+growth+and+morphogenesis.">Cell dynamics in fetal intestinal epithelium: implications for intestinal growth and morphogenesis.</a> Development. 138, 4423-4432 (2011).</li><li>Walton, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog-responsive+mesenchymal+clusters+direct+patterning+and+emergence+of+intestinal+villi.">Hedgehog-responsive mesenchymal clusters direct patterning and emergence of intestinal villi.</a> Proc Natl Acad Sci U S A. 109, 15817-15822 (2012).</li><li>Wells, J. M., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebrate+endoderm+development.">Vertebrate endoderm development.</a> Annu Rev Cell Dev Biol. 15, 393-410 (1999).</li><li>Hashimoto, H., Ishikawa, H., Kusakabe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+vascular+networks+during+the+morphogenesis+of+intestinal+villi+in+the+fetal+mouse.">Development of vascular networks during the morphogenesis of intestinal villi in the fetal mouse.</a> Kaibogaku zasshi Journal of anatomy. 74, 567-576 (1999).</li><li>Mathan, M., Moxey, P. C., Trier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+fetal+rat+duodenal+villi.">Morphogenesis of fetal rat duodenal villi.</a> Am J Anat. 146, 73-92 (1976).</li><li>Burns, R. C., 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=Requirement+for+fibroblast+growth+factor+10+or+fibroblast+growth+factor+receptor+2-IIIb+signaling+for+cecal+development+in+mouse.">Requirement for fibroblast growth factor 10 or fibroblast growth factor receptor 2-IIIb signaling for cecal development in mouse.</a> Dev Biol. 265, 61-74 (2004).</li><li>Pabst, O., Schneider, A., Brand, T., Arnold, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+Nkx2-3+homeodomain+gene+is+expressed+in+gut+mesenchyme+during+pre-+and+postnatal+mouse+development.">The mouse Nkx2-3 homeodomain gene is expressed in gut mesenchyme during pre- and postnatal mouse development.</a> Dev Dyn. 209, 29-35 (1997).</li><li>Pabst, O., Zweigerdt, R., Arnold, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+homeobox+transcription+factor+Nkx2-3+in+mice+results+in+postnatal+lethality+and+abnormal+development+of+small+intestine.">Targeted disruption of the homeobox transcription factor Nkx2-3 in mice results in postnatal lethality and abnormal development of small intestine.</a> Development. 126, 2215-2225 (1999).</li><li>Kaestner, K. H., Silberg, D. G., Traber, P. G., Sch&#252;tz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mesenchymal+winged+helix+transcription+factor+Fkh6+is+required+for+the+control+of+gastrointestinal+proliferation+and+differentiation.">The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation.</a> Genes &amp; Development. 11, 1583-1595 (1997).</li><li>Madison, B. B., McKenna, L. B., Dolson, D., Epstein, D. J., Kaestner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxF1+and+FoxL1+link+hedgehog+signaling+and+the+control+of+epithelial+proliferation+in+the+developing+stomach+and+intestine.">FoxF1 and FoxL1 link hedgehog signaling and the control of epithelial proliferation in the developing stomach and intestine.</a> J Biol Chem. 284, 5936-5944 (2009).</li><li>Karlsson, L., Lindahl, P., Heath, J. K., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+gastrointestinal+development+in+PDGF-A+and+PDGFR-(alpha)+deficient+mice+implicates+a+novel+mesenchymal+structure+with+putative+instructive+properties+in+villus+morphogenesis.">Abnormal gastrointestinal development in PDGF-A and PDGFR-(alpha) deficient mice implicates a novel mesenchymal structure with putative instructive properties in villus morphogenesis.</a> Development. 127, 3457-3466 (2000).</li><li>Mao, J., Kim, B., Rajurkar, M., Shivdasani, R., Mcmahon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+controls+mesenchymal+growth+in+the+developing+mammalian+digestive+tract.">Hedgehog signaling controls mesenchymal growth in the developing mammalian digestive tract.</a> Development. 137, 1721-1729 (2010).</li><li>Theiler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The house mouse. Development and normal stages from fertilization to 4 weeks of age. , (1989).</li><li>Hamilton, T. G., Klinghoffer, R. A., Corrin, P. D., Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+divergence+of+platelet-derived+growth+factor+alpha+receptor+signaling+mechanisms.">Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms.</a> Mol Cell Biol. 23, 4013-4025 (2003).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li><li>Goodrich, L. V., Milenkovic, L., Higgins, K. M., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+neural+cell+fates+and+medulloblastoma+in+mouse+patched+mutants.">Altered neural cell fates and medulloblastoma in mouse patched mutants.</a> Science. 277, 1109-1113 (1997).</li><li>Fu, Y. Y., 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=Microtome-free+3-dimensional+confocal+imaging+method+for+visualization+of+mouse+intestine+with+subcellular-level+resolution.">Microtome-free 3-dimensional confocal imaging method for visualization of mouse intestine with subcellular-level resolution.</a> Gastroenterology. 137, 453-465 (2009).</li></ol>

The authors have no financial conflicts to disclose.

A natureza dinâmica e interações complexas de tecido do intestino desenvolvimento requer visualização 3D para ter uma apreciação plena desses eventos morfogenéticos. Com a evolução da tecnologia de imagem, a capacidade de examinar vilosidades morfogênese em detalhe está a desenvolver / melhorar e, com isso, a compreensão da comunicação espacial e interação durante a organogênese é bastante reforçada. Os métodos alternativos para a cultura de intestinos inteiros foram tam...

Each intestinal villus is composed of two main tissue compartments: an epithelial surface layer and a mesenchymal core. The mouse small intestine is formed at embryonic day 10 when a sheet of endoderm closes and seals to form a tube of epithelium surrounded by mesenchymal cells3. This flat tube of epithelium undergoes rapid proliferation, growing both in length and girth and undergoes dramatic rearrangements involving dynamic cell shape changes1. At the same time, the surrounding mesenchyme also undergoes many developmental processes including the formation of the vascular plexus, differentiation of smooth muscle and recruitment of enteric neurons4. In the proximal small intestine at embryonic day 14.5, condensations (clusters) of Hedgehog- and PDGF-responsive cells begin to form adjacent to the epithelium2,5. Formation of mesenchymal clusters continues to spread along the length of the intestine so that they cover the entirety of the small intestine by embryonic day 16.52. As mesenchymal clusters form, the epithelial cells closest to the clusters begin to withdraw from the cell cycle, while the other epithelial cells continue to proliferate. Those cells directly above the mesenchymal cluster that have withdrawn from the cell cycle begin to change shape as the emerging villus buckles into the lumen. Further growth of the villus is driven in part by the continued proliferation of the epithelium between the emerging villi. The mesenchymal clusters remain tightly adhered to the epithelium of the growing villus and continue to express a variety of signaling molecules. The wave of villus emergence propagates along the length of the small intestine following the formation of mesenchymal clusters. As the intestine continues to grow and the intervillus region extends between emerging villi, new mesenchymal clusters form adjacent to the intervillus epithelium and further rounds of villus emergence and growth ensue6.
Synchronized development of the epithelium and mesenchyme is essential for villus morphogenesis. Signaling molecules are secreted from one layer to the other where receptors receive and transduce the signal message in order to coordinate development between the epithelium and mesenchyme. Mesenchymal clusters act as signaling centers and express a variety of developmental morphogens7-10. Disruption of cluster formation or pattern results in loss of villus emergence and pattern. Inhibition of PDGF signaling results in fewer clusters and fewer villi and those villi that do form are misshapen following the abnormal clusters11. Loss of Hedgehog signaling results in complete loss of cluster formation and failure of villus emergence2,12. Together, these data demonstrate that clusters coordinate development of the villus epithelium with its mesenchymal core.
Using this whole organ culture system, we are able to alter signaling involved in epithelial-mesenchymal cluster cross-talk to determine the role of those signals in villus morphogenesis. Two-photon confocal optical sectioning and reconstruction afford the ability to visualize cluster formation and villus emergence in three-dimensions and better understand the spatial relationships between the mesenchymal clusters and their overlying epithelium. Extending the culture system to four dimensions, we can capture z-stacks of developing clusters and villi over time and observe these interactions. Ultimately, the ability to follow villus development in this manner and observe changes that occur with altered signaling will revolutionize the understanding of epithelial mesenchymal interactions in villus morphogenesis.

<table border="0" cellpadding="0" cellspacing="0" width="1308">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:508px;">Fine dissecting forceps&#160;</td>
			<td style="width:223px;">Fine Science Tools</td>
			<td style="width:133px;">&#160;11254-20</td>
			<td style="width:444px;">2 pairs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Ethanol</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1x sterile Dulbecco&#39;s Phosphate-Buffered Saline (DPBS)</td>
			<td>Gibco&#160;</td>
			<td>14040-133</td>
			<td style="width:444px;">500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plates</td>
			<td>Costar&#160;</td>
			<td>3524</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 x 15 mm petri dishes</td>
			<td>Falcon</td>
			<td>&#160;451007</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transwell plates, 24 mm inserts, 8.0 mm polycarbonate membranes</td>
			<td>Corning Costar&#160;</td>
			<td>3428</td>
			<td style="width:444px;">6 inserts per plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BGJb media</td>
			<td>Invitrogen&#160;</td>
			<td>12591-038</td>
			<td style="width:444px;">500 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PenStrep (10,000U/ml Penicillin; 10,000 mg/ml Streptomycin)</td>
			<td>Gibco</td>
			<td>&#160;15140</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ascorbic Acid</td>
			<td>Sigma&#160;</td>
			<td>A0278</td>
			<td style="width:444px;">make 5 mg/ml stock, filter, aliquot and store at -20 &#176;C</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Mouth pipet (Drummond 1-15 inch aspirator tube assembly)</td>
			<td>Fisher</td>
			<td style="width:133px;">&#160;21-180-10</td>
			<td style="width:444px;">remove the aspirator assembly and replace it with a 1000 &#181;l pipet tip which acts as an adaptor to plug in a 6 inch glass Pasteur pipet.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 inch glass pasteur pipets</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose beads</td>
			<td>BioRad&#160;</td>
			<td>153-7301</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary Tubes</td>
			<td>World Precision Instruments&#160;</td>
			<td>TW100F-4</td>
			<td style="width:444px;">pull to needles</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">4% Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td style="width:444px;">made in 1 x PBS, pH to 7.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live Imaging Materials</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture plates</td>
			<td>Falcon&#160;</td>
			<td>353037</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine mesh stainless steel screen</td>
			<td>purchase at hardware store</td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Polycarbonate membranes</td>
			<td>Thomas scientific&#160;</td>
			<td>4663H25</td>
			<td style="width:444px;">alternatively, cut Corning Costar 3428 membranes off of transwell supports</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Instant glue</td>
			<td>purchase at hardware store</td>
			<td style="width:133px;">gel based preferrably</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 x 10 mm plates</td>
			<td>Falcon&#160;</td>
			<td style="width:133px;">351008</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7% agarose</td>
			<td>Sigma</td>
			<td>&#160;A9414</td>
			<td style="width:444px;">prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minutien pins</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:133px;">26002-20</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol red free media (DMEM)</td>
			<td>Gibco&#160;</td>
			<td style="width:133px;">21063-029</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine (100 mg/ml)</td>
			<td>AnaSed&#160;</td>
			<td style="width:133px;">139-236</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Matrigel</td>
			<td>BD</td>
			<td style="width:133px;">356231</td>
			<td style="width:444px;">basement membrane matrix, growth factor reduced, phenol red-free</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3-4% agarose</td>
			<td>Sigma&#160;</td>
			<td>A9414</td>
			<td style="width:444px;">prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging of fixed intestines</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vaseline</td>
			<td style="width:223px;">purchase at pharmacy</td>
			<td></td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lanolin</td>
			<td style="width:223px;">Sigma&#160;</td>
			<td>L7387</td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraffin</td>
			<td style="width:223px;">Surgipath</td>
			<td>39601006</td>
			<td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% glycerol in 1 x PBS</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Focus clear and Mount Clear</td>
			<td>CelExplorer Labs Co.</td>
			<td>&#160;F101-KIT</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modeling clay</td>
			<td>purchase at art supply store</td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">double stick tape</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cotton applicator swabs</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic molds, 10mm x 10mm x 5 mm)</td>
			<td>Tissue Tek&#160;</td>
			<td>4565</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">slides</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coverslips</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lab wipe</td>
			<td>Kimberly Clark</td>
			<td>34155</td>
			<td style="width:444px;">lint free delicate task wipe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Theiler staging chart&#160;</td>
			<td></td>
			<td></td>
			<td style="width:444px;"><a href="http://www.emouseatlas.org/emap/ema/theiler_stages/%20downloads/theiler2.pdf">http://www.emouseatlas.org/emap/ema/theiler_stages/ downloads/theiler2.pdf</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica SP5X confocal microscope&#160;</td>
			<td>Leica</td>
			<td></td>
			<td style="width:444px;">Used to conduct the live imaging&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMI 6000 stand&#160;</td>
			<td>Leica</td>
			<td></td>
			<td style="width:444px;">Used to conduct the live imaging&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Aqueous mounting medium (Prolong Gold)</td>
			<td>Molecular Probes&#160;</td>
			<td>P36930</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials for Immunofluorescence staining of fixed, vibratome sectioned intestines</td>
			<td></td>
			<td></td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td style="width:444px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plate</td>
			<td>Costar</td>
			<td>&#160;3524</td>
			<td style="width:444px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Triton X-100</td>
			<td>Sigma&#160;</td>
			<td>T-8787</td>
			<td style="width:444px;">used to make Permeabilization solution: 0.5% Triton X-100 in 1 x PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Goat serum</td>
			<td></td>
			<td></td>
			<td style="width:444px;">used to make Blocking Solution: 4% Goat serum, 0.1% Tween20 in 1x PBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tween20&#160;</td>
			<td>Sigma</td>
			<td>&#160;P9416</td>
			<td style="width:444px;"></td>
		</tr>
	</tbody>
</table>

mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development

A maioria dos processos morfogenéticos no intestino fetal têm sido inferida a partir de lâminas finas de tecidos fixados, oferecendo instantâneos de mudanças ao longo do estágio de desenvolvimento. Informação tridimensional de cortes seriados finos podem ser difíceis de interpretar por causa da dificuldade de reconstrução de cortes seriados perfeitamente e manter a orientação adequada do tecido sobre cortes seriados. Recentes descobertas por Grosse et al., 2011 destacam a importância de três informações dimensional na compreensão morfogênese das vilosidades desenvolvimento do intestino 1. A reconstrução tridimensional de células intestinais isoladamente marcadas demonstraram que a maioria das células epiteliais intestinais em contato com ambas as superfícies apicais e basais. Por outro lado, a reconstrução tridimensional do citoesqueleto de actina para a superfície apical do epitélio demonstrado que o lúmen intestinal é contínua e que lúmens secundários são um artefacto sectioning. Estes dois pontos, juntamente com a demonstração de migração nuclear interkinetic no epitélio intestinal, definido o epitélio intestinal em desenvolvimento como um epitélio pseudo estratificado e não como se pensava anteriormente 1. A capacidade de observar o epitélio tridimensionalmente foi seminal para demonstrar este ponto e redefinindo morfogênese epitelial no intestino fetal. Com a evolução da tecnologia de imagem multi-fotão e software de reconstrução tridimensional, a capacidade de visualizar intacta, o desenvolvimento de órgãos está melhorando rapidamente. Dois fótons de excitação permite a penetração mais profunda menos prejudiciais para os tecidos com alta resolução. Dois fótons e reconstrução 3D de todo o intestino do feto de rato em Walton et al., 2012 ajudou a definir o padrão de vilo conseqüência 2. Aqui nós descrevemos um sistema de cultura de órgão inteiro que permite ex vivo desenvolvimento de vilosidades e extensões desse sistema de cultura para permitiros intestinos para ser tridimensionalmente fotografada durante o seu desenvolvimento.

Culturas de explantes de intestino fetal inteiros permite a análise da localização, distribuição e duração das moléculas de sinalização que coordenam o desenvolvimento intestinal, como este sistema permite a manipulação da sinalização com reagentes farmacológicos ou proteínas recombinantes. O sistema de cultura Transwell (Figura 1A, reproduzido a partir de Walton et al., 2012) 2 fornece uma interface ar-líquido, o que faz com que seja possível colocar droga ou prote...

Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Rato Fetal Whole Intestino Sistema de Cultura para<em&gt; Ex Vivo</em&gt; Manipulação de vias de sinalização e tridimensional Imagens ao vivo de Desenvolvimento Villus

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

mouse-fetal-whole-intestine-culture-system-for-ex-vivo-manipulation

Research

JoVE Journal

Biology

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

14.8K visualizações.  University of Michigan. Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine. 

Vídeo: Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para a Cultura de Embriões início pintainho ex vivo (Nova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biologia

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Um Ex-ovo Sistema de Cultura de embriões de frango Indicado para aplicações de imagem e Microcirurgia

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Um Sistema de<em&gt; Ex vivo</em&gt; A cultura de Pâncreas embrionárias

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Duas e três dimensões imagens de células vivas de Proteínas de DNA resposta a danos

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Rato Desenvolvimento Embrionário em todo um sistema de cultura de embriões sem soro

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Ex vivo Cultura do Rato Embryonic pele e Live-imaging de Migração melanoblasto

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Uma<em&gt; Ex vivo</em&gt; Sistema Cultura para estudar o desenvolvimento da tireóide

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging no hidrogéis tridimensionais

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Um método de compensação por imagem tridimensional do tecido adiposo tecido

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Resolução unicelular imagens tridimensionais de organoides intactos

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...