Os autores gostariam de agradecer a Irene Choi para ilustrar cabeças de embriões em Fig. 1 e os outros membros do laboratório de ajuda e discussão. Este trabalho é apoiado pelo NIH concessão DE012728 (TW)

1. Prepare Dispase II <ol><li> Preparar uma solução fresca de Hepes-solução salina tamponada (HBS) (50 mM de Hepes / KOH, pH 7,4, 150 mM NaCl). Adicionar 2 ml de 0,5 M de tampão Hepes / KOH a pH 7,4 e 1,2 ml de 2,5 M de NaCl em 16,8 ml de H 2 O. Ultrapure </li><li> Faça 10 mg / ml Dispase II. Pesar 0,2 g Dispase II (Roche, Cat. # 4942078001) e dissolver em 20 ml de HBS. Preparar 1,0 ml alíquotas em tubos de Eppendorf individuais, e armazenar a -20 ° C para armazenamento a longo prazo. </li><li> Diluir 10 mg / ml Dispase II 1:10 em PBS antes da utilização. Coloque a Dispase II diluído em gelo. </li></ol> 2. Dissecar CD-1 embriões E10.5 de mulher grávida Todos os experimentos com animais foram realizados de acordo com os protocolos aprovados pelo Animal Care Universidade do Colorado, Denver (UCD) e Comitê de uso. Ratinhos CD-1 (ICR) foram obtidos a partir de Harlan Laboratories (Indianapolis, IN). <ol><li> Limpe a área de dissecção e ferramentas com etanol 70%. </li><li> Euthanize fêmea CD-1 grávidas usando aprovaprotocolos ed. </li><li> Pulverizar o abdômen com etanol 70%, em seguida abrir o abdômen. Usar um conjunto de pinças para retirar o útero e outro para rasgar o mesométrio do útero. Remover o útero do corpo. Repita o procedimento para corno uterino no outro lado do abdômen. </li><li> Resumidamente enxaguar o útero com PBS gelado em uma placa de Petri de 10 cm, para lavar o sangue. </li><li> Transferir o útero para uma nova placa de Petri com PBS gelado. Separar em um embrião de-fragmentos cortando entre os locais de implantação. </li><li> Transferir um embrião em PBS gelado sob um estereomicroscópio. Use uma pinça fina para arrancar a parede muscular do útero a partir de um dos locais de corte para expor o embrião com o saco vitelino. Retire o saco vitelino e âmnio. A transferência de embriões dissecados para uma placa de Petri de 6 cm com PBS em gelo. Prossiga até que todos os embriões foram coletados desta forma. </li></ol> 3. Dispase II Tratamento <ol><li> Lave todos os embriões com PBS numa placa de Petri de 6 cm. SubstituirPBS com 10 ml diluídos em PBS Dispase II. Cobrir a placa de Petri e incubar a 37 ° C durante 25 minutos - uma incubadora bacteriana ou sala quente deve ser suficiente - não ter usado um tecido de CO 2 incubadora de cultura para esta etapa. </li><li> Pegue a placa de Petri para fora e observar o embrião sob estereomicroscópio. O ectoderma deve ter se soltado, caracterizado por uma diferença clara entre esta camada de tecido e no mesênquima subjacente. Se não, incubar por mais 5 min a 37 ° C. Em seguida, coloque a placa de Petri no gelo. </li></ol> 4. Dissecar os destaques intactas Facial <ol><li> Use uma pipeta de vidro descartável (VWR, Cat # 14672-380) para transferir um Dispase II tratados embrião em PBS gelado em uma placa de Petri de 6 cm sob um estereomicroscópio. </li><li> Utilizar um par de pinças para segurar o embrião. Utilizar um outro par de fórceps finos cuidadosamente para cortar os limites das proeminências faciais associadas a cabeça (Figura 1 mostra os limites daos destaques facial). Iniciar a partir de um lado da MnP, em seguida, o MxP e FNP o último. Fazer o mesmo para o outro lado para dissecar as proeminências para fora da face intacto (Figura 2A). </li></ol> 5. Ectoderma Facial separado do mesênquima <ol><li> Observar as proeminências faciais sob um estereomicroscópio com distância de trabalho de 3x. Use um longo vidro Pasteur pipeta suavemente para pipetar as proeminências faciais cima e para baixo 5 vezes. Depois disso, a ectoderme é fácil de descascar. </li><li> Use uma pinça para segurar as proeminências faciais. Usar o outro par de pinças para retirar a ectoderme muito lentamente. Figura 2B mostra as folhas separadas de ectoderma facial. </li><li> Usar uma nova longo vidro Pasteur pipeta para transferir o ectoderma facial em um tubo Eppendorf de 1,5 ml com PBS em gelo. Transferir o mesênquima para outro tubo Eppendorf de 1,5 ml com PBS em gelo. </li><li> Use o novo gelada PBS para cada dissecção. Coletar amostras. </li><li> Lave tele amostras com PBS. Centrifugar a 4 ° C, 500 xg durante 3 min. </li><li> Aspirar o PBS utilizando pipetas de vidro longos de Pasteur. Se as amostras devem ser usados ​​para o ensaio de chip, vá para o passo 6.1. Para a extração de RNA, vá para o passo 6.2. Para a extração de proteínas, vá para o passo 6.3. </li></ol> 6. Amostras de processo para novas experiências <ol><li> As amostras para ensaio ChIP <ol><li> Adicionar 1 ml de PBS com formaldeído a 1% e de reticulação. Homogeneizar as amostras. </li><li> Incubar à temperatura ambiente durante 10 minutos com rotação. </li><li> Adicionar 110 ul de 1,25 M de glicina a cada tubo para extinguir o formaldeído que não reagiu. </li><li> Misturar e incubar à temperatura ambiente durante 5 min. </li><li> Girar a 500 xg a 4 ° C durante 3 min. Remover o sobrenadante. </li><li> Adicionar 1 ml de PBS frio para lavar tecidos. </li><li> Girar a 500 xg a 4 ° C durante 3 min. </li><li> Remover PBS e PBS lavar repetir uma vez. </li><li> Remover o sobrenadante, Encaixe congelar os tecidos em azoto líquido. Armazenar a samples a -80 ° C para posterior partilha e processamento. </li></ol></li><li> As amostras para a extração de RNA <ol><li> Coloque o ectoderma facial e mesênquima em tubos contendo RNAlater (Ambion). </li><li> Incubar as amostras em RNAlater solução durante a noite a 4 ° C para permitir a penetração profunda do tecido. </li><li> Armazenar a -20 ° C para subsequente conjugação e processamento. </li></ol></li><li> As amostras para a extração de proteínas </li></ol> Adicionar tampão de lise para a amostra para o processamento subsequente. Ou tirar congelar os tecidos em nitrogênio líquido. Armazenar as amostras a -80 ° C para subsequente conjugação e processamento. Notas <ol><li> Limitações de tempo. Pensando em 4-6 horas desde o início da dissecção até o início da seção 6. </li><li> Quando proficiente, leva ~ 10-15 minutos por embrião para realizar seções 4 e 5 (dissecção destaque e separação ectoderme). </li><li> Se as amostras são para o isolamento de ARN, cada solution deve ser tratado com dietilpirocarbonato primeiro. Também limpar a pinça e da área de trabalho com RNase Fora. </li><li> Mantenha as amostras em gelo em todos os momentos, exceto para o tratamento II Dispase e dissecações. Mudar para nova gelada PBS para cada dissecção para garantir que o PBS utilizado para dissecção é frio. </li><li> Pinças cortantes são cruciais para obter a dissecção precisa. Limpe a pinça completamente para evitar qualquer contaminação. </li><li> Para a coleta de amostras de proeminências diferentes, dissecar cada proeminência separadamente após Dispase tratamento II. Em seguida, separar o ectoderma e mesênquima. </li><li> Após o tratamento com Dispase II, o ectoderma facial é fácil de descascar. Preste atenção especial à contaminação por ectoderma outro. Descartar todo o tecido potencialmente contaminadas. </li><li> Depois de começar, não pare até que as amostras atingir o ponto adequado na Secção 6. Concluir separação de ectoderma facial e mesênquima para todos os embriões tão rapidamente como você pode. </li><li> O e-atividades nzyme de Dispase II pode ser diferente entre os lotes e marcas. É necessário regular o tempo de tratamento para as diferentes marcas e lotes de Dispase II. </li></ol>

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	<li>Wilkie, A. O., Morriss-Kay, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+craniofacial+development+and+malformation.">Genetics of craniofacial development and malformation.</a> Nat. Rev. Genet. 2, 458-468 (2001).</li><li>Gritli-Linde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2+-+The+Etiopathogenesis+of+cleft+lip+and+cleft+palate:+usefulness+and+caveats+of+mouse+models.">Chapter 2 - The Etiopathogenesis of cleft lip and cleft palate: usefulness and caveats of mouse models.</a> Current Topics in Developmental Biology. 84, 37-138 (2008).</li><li>Schutte, B. C., Murray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+faces+and+factors+of+orofacial+clefts.">The many faces and factors of orofacial clefts.</a> Human Molecular Genetics. 8, 1-7 (1999).</li><li>Chai, Y., Maxson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+craniofacial+morphogenesis.">Recent advances in craniofacial morphogenesis.</a> Dev. Dyn. 235, 2353-2375 (2006).</li><li>Jiang, R., Bush, J. O., Lidral, A. C. <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+the+upper+lip:+Morphogenetic+and+molecular+mechanisms.">Development of the upper lip: Morphogenetic and molecular mechanisms.</a> Dev. Dyn. 235, 1152-1166 (2006).</li><li>Reid, B. S., Yang, H., Melvin, V. S., Taketo, M. M., Williams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectodermal+WNT/&#946;-catenin+signaling+shapes+the+mouse+face.">Ectodermal WNT/&#946;-catenin signaling shapes the mouse face.</a> Developmental Biology. 349, 261-269 (2011).</li><li>Feng, W., 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=Spatial+and+temporal+analysis+of+gene+expression+during+growth+and+fusion+of+the+mouse+facial+prominences.">Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences.</a> PLoS ONE. 4, e8066 (2009).</li><li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+uses+of+mouse+outbred+stocks.">The origins and uses of mouse outbred stocks.</a> Nat. Genet. 37 (11), 1181-1186 (2005).</li><li>Nagai, T., Aruga, J., Takada, 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=The+expression+of+the+mouse+Zic1,+Zic2,+and+Zic3+gene+suggests+an+essential+role+for+Zic+genes+in+body+pattern+formation.">The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation.</a> Developmental Biology. 182, 299-313 (1997).</li><li>Britsch, 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=The+transcription+factor+Sox10+is+a+key+regulator+of+peripheral+glial+development.">The transcription factor Sox10 is a key regulator of peripheral glial development.</a> Genes Dev. 15 (1), 66-78 (2001).</li></ol>

Os autores não têm interesses conflitantes ou outros conflitos associados com o conteúdo deste artigo.

Este protocolo fornece um método simples para separar ectoderma rato embrionário mesênquima facial e base em uma primeira etapa de tratamento Dispase II. Em estudos anteriores, temos realizado dissecção das proeminências faciais antes Dispase tratamento II, mas temos de forma consistente que o mesênquima se torna &quot;pegajoso&quot; e mais difícil de manipular. Nosso novo protocolo evita esse problema. Combinação Dispase II tratamento com força física suave pipetando para cima e para baixo faz a separação...

<table><tbody><tr><td>Dispase II (neutral protease, grade II)</td><td>Roche</td><td>4942078001</td><td>Make 10 mg/ml Dispase II stock solution in HBS. Aliquot and store at -20 &amp;deg;C . </td></tr><tr><td>RNAlater</td><td>Ambion</td><td>AM7021</td><td></td></tr></tbody></table>

separation of mouse embryonic facial ectoderm and mesenchyme

Fendas orofaciais são os defeitos mais freqüentes craniofaciais, que afetam 1,5 em 1000 1,2 recém-nascidos em todo o mundo. Fissuras orofaciais é causada por três desenvolvimento anormal facial. No ser humano e do rato, o crescimento inicial e padronização da cara depende de várias papilas pequenas de tecido, as proeminências faciais 4,5. O rosto é derivado de seis principais destaques: emparelhados processos frontais nasais (FNP), proeminências maxilares (MXP) e proeminências mandibulares (MDP). Estes consistem de proeminências inchaços de mesênquima que estão incorporados em um epitélio de revestimento. Estudos realizados em várias espécies têm mostrado que a sinalização crosstalk entre ectoderma facial e mesênquima é crítico para a formação da face 6. No entanto, os detalhes mecanísticos sobre os genes envolvidos nestes relés de sinalização são escassos. Uma maneira de obter uma compreensão abrangente da expressão do gene, fator de transcrição de ligação, e marcas de cromatina associados ao desenectoderme pingue facial e mesênquima é isolar e caracterizar os compartimentos dos tecidos separados. Aqui apresentamos um método para separar ectoderma facial e mesênquima no dia embrionário (E) 10,5, uma fase crítica de desenvolvimento na formação do mouse facial que precede a fusão dos destaques. O nosso método é adaptado a partir do método já anteriormente utilizado para dissecar proeminências faciais 7. Neste estudo anterior havia sido empregado C57BL inato / 6 ratos como esta cepa se tornou um padrão para a genética, morfologia facial genômica e 8. Aqui, no entanto, devido às quantidades mais limitadas de tecido disponível, nós utilizamos a estirpe CD-1 outbred que é mais barata para comprar, mais robusto para a criação, e que tende a produzir mais embriões (12-18) por ninhada do que qualquer inbred rato tensão 8. Seguindo o isolamento do embrião, neutro protease Dispase II foi usada para tratar o embrião inteiro. Em seguida, as proeminências faciais foram dissecared para fora, e a ectoderme facial foi separado do mesênquima. Este método mantém tanto ectoderme facial e mesênquima intacta. As amostras obtidas utilizando este método pode ser usado para a detecção de proteínas, incluindo as técnicas, a imunoprecipitação da cromatina (ChIP) ensaio, os estudos de microarranjos e RNA-seq.

Após tratamento Dispase II, a ectoderme do embrião tende a ser solto. As proeminências faciais devem estar intacto após dissecção (Figura 2A). O ectoderma facial isolada é clara e livre de mesênquima tecido (Figura 2B). Para determinar a eficácia do protocolo e para detectar a contaminação cruzada nós ensaiadas para prosencéfalo expressaram Zic3 9, ectodérmica específico CDH1 5 e mesenquimal específico SOX10 10 utilizando transcriptase re...

Watch this Scientific Journal Video about Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme at JoVE.com

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Um protocolo para a separação da ectoderme embrionária facial e mesênquima é descrito. Usamos Dispase II para tratar embriões inteiros primeiro, dissecar todo proeminências faciais, e então separar o ectoderma facial e mesênquima.

Separação de Ectoderma embrionárias de camundongos Facial e mesênquima

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

separation-of-mouse-embryonic-facial-ectoderm-and-mesenchyme

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.9K visualizações.  University of Colorado Denver Anschutz Medical Campus. Um protocolo para a separação da ectoderme embrionária facial e mesênquima é descrito. Usamos Dispase II para tratar embriões inteiros primeiro, dissecar todo proeminências faciais, e então separar o ectoderma facial e mesênquima.

Vídeo: Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Dissecção de 6,5 embriões de camundongos dpc

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Biologia

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Imagem ao vivo do mouse Fusão do palato secundário

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Biologia do Desenvolvimento

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Dissecção, cultura e análise de células da crista neural craniana primária do mouse para o estudo de delaminação e migração de células de crista neural

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering the molecular signals that cooperatively guide neuronal migration in the embryonic brain is therefore important to understand how the complex neural networks form which later support postnatal life. Facial branchiomotor (FBM) neurons in the mouse embryo hindbrain migrate from rhombomere (r) 4 caudally to form the paired facial nuclei in the r6-derived region of the hindbrain. Here we provide a detailed protocol for wholemount ex vivo culture of mouse embryo hindbrains suitable to investigate the signaling pathways that regulate FBM migration. In this method, hindbrains of E11.5 mouse embryos are dissected and cultured in an open book preparation on cell culture inserts for 24 hr. During this time, FBM neurons migrate caudally towards r6 and can be exposed to function-blocking antibodies and small molecules in the culture media or heparin beads loaded with recombinant proteins to examine roles for signaling pathways implicated in guiding neuronal migration.

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Embryonic neurons are born in the ventricular zone of the neural tube, but migrate to reach appropriate targets. Facial branchiomotor (FBM) neurons are a useful model to study neuronal migration. This protocol describes the wholemount ex vivo culture of mouse embryo hindbrains to investigate mechanisms that regulate FBM migration.

Rato rombencéfalo<em&gt; Ex Vivo</em&gt; Cultura para estudar Facial Migração Branchiomotor Neuron

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

Neurociência

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Isolamento e imagem de lapso de tempo das células mesenquima palatinas embrionárias do camundongo primário para analisar atributos de movimento coletivo

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 &#176;C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (&#62;90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.

We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues.

Dissociação celular do Epitélio da Língua e Mesenquime/Tecido Conjuntivo de Ratos Embrionários 12,5 e 8 Semanas

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA ...

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

Isolamento de Lysatos de Proteína Celular Inteira de Processos Faciais de Camundongos e Células de Mesenchyme Palatal cultivadas para análise de fosfoproteína

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal ...

Fixation of Embryonic Mouse Tissue for Cytoneme Analysis

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (&#8804;200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.

Here, an optimized step-by-step protocol is provided for fixation, immunostaining, and sectioning of embryos to detect specialized signaling filopodia called cytonemes in developing mouse tissues.

Fixação de tecido de rato embrionário para análise de cytoneme

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

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

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

Avaliar Propriedades Sinalização de Ectodérmica Epitélio Durante o Desenvolvimento Craniofacial

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

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Monte todo In Situ Hibridação de E8.5 de Embriões E11.5 rato

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...