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1.
Methods Mol Biol ; 2490: 269-279, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35486252

RESUMO

Here we describe a method to engraft epiblast stem cells (EpiSC) into the epiblast of gastrulation-stage mouse embryo to test the lineage propensity acquired by the EpiSCs during in vitro culture under different signaling conditions. After dissection and grafting, the recipient embryos can be grown in whole-embryo culture for up to 48 h and the contribution of the EpiSC-derived cells to tissues in the recipient embryo is assessed by light sheet 3D microscopy.


Assuntos
Embrião de Mamíferos , Camadas Germinativas , Animais , Diferenciação Celular , Gastrulação , Camundongos , Células-Tronco
2.
Methods Mol Biol ; 2403: 33-42, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34913114

RESUMO

Analysis of animal models allows a deeper understanding of craniofacial development in health and diseases of humans. Wholemount in situ hybridization (WISH) is an informative technique to visualize gene expression in tissues across the developmental stages of embryos. The principle of WISH is based on the complementary binding (hybridization) of the DNA/RNA probe to the target transcript. The bound probe can then be visualized by an enzymatic color reaction to delineate the expression pattern of transcripts within a tissue. Here we describe an optimized method to perform in situ hybridization in mouse embryos.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Animais , Expressão Gênica , Hibridização In Situ , Camundongos , Sondas RNA
3.
Methods Mol Biol ; 2403: 43-50, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34913115

RESUMO

Craniofacial morphogenesis is underpinned by orchestrated growth and form-shaping activity of skeletal and soft tissues in the head and face. Disruptions during development can lead to dysmorphology of the skull, jaw, and the pharyngeal structures. Developmental disorders can be investigated in animal models to elucidate the molecular and cellular consequences of the morphogenetic defects. A first step in determining the disruption in the development of the head and face is to analyze the phenotypic features of the skeletal tissues. Examination of the anatomy of bones and cartilage over time and space will identify structural defects of head structures and guide follow-up analysis of the molecular and cellular attributes associated with the defects. Here we describe a protocol to simultaneously visualize the cartilage and bone elements by Alcian blue and Alizarin red staining, respectively, of wholemount specimens in mouse models.


Assuntos
Cartilagem , Crânio , Azul Alciano , Animais , Antraquinonas , Camundongos , Coloração e Rotulagem
4.
Development ; 142(11): 2069-79, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25977363

RESUMO

Lhx1 encodes a LIM homeobox transcription factor that is expressed in the primitive streak, mesoderm and anterior mesendoderm of the mouse embryo. Using a conditional Lhx1 flox mutation and three different Cre deleters, we demonstrated that LHX1 is required in the anterior mesendoderm, but not in the mesoderm, for formation of the head. LHX1 enables the morphogenetic movement of cells that accompanies the formation of the anterior mesendoderm, in part through regulation of Pcdh7 expression. LHX1 also regulates, in the anterior mesendoderm, the transcription of genes encoding negative regulators of WNT signalling, such as Dkk1, Hesx1, Cer1 and Gsc. Embryos carrying mutations in Pcdh7, generated using CRISPR-Cas9 technology, and embryos without Lhx1 function specifically in the anterior mesendoderm displayed head defects that partially phenocopied the truncation defects of Lhx1-null mutants. Therefore, disruption of Lhx1-dependent movement of the anterior mesendoderm cells and failure to modulate WNT signalling both resulted in the truncation of head structures. Compound mutants of Lhx1, Dkk1 and Ctnnb1 show an enhanced head truncation phenotype, pointing to a functional link between LHX1 transcriptional activity and the regulation of WNT signalling. Collectively, these results provide comprehensive insight into the context-specific function of LHX1 in head formation: LHX1 enables the formation of the anterior mesendoderm that is instrumental for mediating the inductive interaction with the anterior neuroectoderm and LHX1 also regulates the expression of factors in the signalling cascade that modulate the level of WNT activity.


Assuntos
Embrião de Mamíferos/metabolismo , Cabeça/embriologia , Proteínas com Homeodomínio LIM/metabolismo , Fatores de Transcrição/metabolismo , Animais , Caderinas/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Proteínas com Homeodomínio LIM/genética , Camundongos Knockout , Modelos Biológicos , Mutação , Fenótipo , Transdução de Sinais , Fatores de Transcrição/genética , Proteínas Wnt/metabolismo
5.
Philos Trans R Soc Lond B Biol Sci ; 369(1657)2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25349457

RESUMO

Mouse epiblast stem cells (EpiSCs) display temporal differences in the upregulation of Mixl1 expression during the initial steps of in vitro differentiation, which can be correlated with their propensity for endoderm differentiation. EpiSCs that upregulated Mixl1 rapidly during differentiation responded robustly to both Activin A and Nodal in generating foregut endoderm and precursors of pancreatic and hepatic tissues. By contrast, EpiSCs that delayed Mixl1 upregulation responded less effectively to Nodal and showed an overall suboptimal outcome of directed differentiation. The enhancement in endoderm potency in Mixl1-early cells may be accounted for by a rapid exit from the progenitor state and the efficient response to the induction of differentiation by Nodal. EpiSCs that readily differentiate into the endoderm cells are marked by a distinctive expression fingerprint of transforming growth factor (TGF)-ß signalling pathway genes and genes related to the endoderm lineage. Nodal appears to elicit responses that are associated with transition to a mesenchymal phenotype, whereas Activin A promotes gene expression associated with maintenance of an epithelial phenotype. We postulate that the formation of definitive endoderm (DE) in embryoid bodies follows a similar process to germ layer formation from the epiblast, requiring an initial de-epithelialization event and subsequent re-epithelialization. Our results show that priming EpiSCs with the appropriate form of TGF-ß signalling at the formative phase of endoderm differentiation impacts on the further progression into mature DE-derived lineages, and that this is influenced by the initial characteristics of the cell population. Our study also highlights that Activin A, which is commonly used as an in vitro surrogate for Nodal in differentiation protocols, does not elicit the same downstream effects as Nodal, and therefore may not effectively mimic events that take place in the mouse embryo.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camadas Germinativas/embriologia , Subunidades beta de Inibinas/metabolismo , Proteína Nodal/metabolismo , Animais , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Camadas Germinativas/citologia , Proteínas de Homeodomínio/metabolismo , Camundongos , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo
6.
EMBO Rep ; 15(8): 903-10, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24916387

RESUMO

Cytidine (C) to Uridine (U) RNA editing is a post-transcriptional modification that is accomplished by the deaminase APOBEC1 and its partnership with the RNA-binding protein A1CF. We identify and characterise here a novel RNA-binding protein, RBM47, that interacts with APOBEC1 and A1CF and is expressed in tissues where C to U RNA editing occurs. RBM47 can substitute for A1CF and is necessary and sufficient for APOBEC1-mediated editing in vitro. Editing is further impaired in Rbm47-deficient mutant mice. These findings suggest that RBM47 and APOBEC1 constitute the basic machinery for C to U RNA editing.


Assuntos
Citidina Desaminase/fisiologia , Edição de RNA , Proteínas de Ligação a RNA/genética , Desaminase APOBEC-1 , Animais , Células CACO-2 , Núcleo Celular/metabolismo , Citidina/metabolismo , Expressão Gênica , Humanos , Camundongos Transgênicos , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Uridina/metabolismo
7.
Methods Mol Biol ; 1092: 119-42, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24318817

RESUMO

We are using knockdown of gene expression in mouse embryos by constitutive expression of small hairpin (sh)RNAs as a means of observing loss-of-function phenotypes more rapidly than gene targeting. Plasmid constructs that direct shRNA expression via an RNA pol III promoter are introduced into embryonic stem (ES) cells by electroporation and drug selection. Clones are propagated and the degree of knockdown assessed by quantitative protein or RNA methods. Selected ES cell clones are used to generate embryos by tetraploid complementation. Blastomeres of two cell embryos are electrofused to generate tetraploid embryos. Chimeric embryos are produced by injection of ES cells into blastocysts or aggregation with morulae. In these embryos, the tetraploid cells become excluded from the fetal tissues, resulting in ES cell-derived embryos harboring the shRNA knockdown construct. Embryos can be collected and their phenotype assessed by appropriate means.


Assuntos
Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , RNA Interferente Pequeno/genética , Animais , Blastômeros/citologia , Técnicas de Silenciamento de Genes , Marcação de Genes , Camundongos , Biologia Molecular/métodos , Tetraploidia
8.
Cell Stem Cell ; 14(1): 107-20, 2014 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-24139757

RESUMO

Mouse epiblast stem cells (EpiSCs) can be derived from a wide range of developmental stages. To characterize and compare EpiSCs with different origins, we derived a series of EpiSC lines from pregastrula stage to late-bud-stage mouse embryos. We found that the transcriptomes of these cells are hierarchically distinct from those of the embryonic stem cells, induced pluripotent stem cells (iPSCs), and epiblast/ectoderm. The EpiSCs display globally similar gene expression profiles irrespective of the original developmental stage of the source tissue. They are developmentally similar to the ectoderm of the late-gastrula-stage embryo and behave like anterior primitive streak cells when differentiated in vitro and in vivo. The EpiSC lines that we derived can also be categorized based on a correlation between gene expression signature and predisposition to differentiate into particular germ-layer derivatives. Our findings therefore highlight distinct identifying characteristics of EpiSCs and provide a foundation for further examination of EpiSC properties and potential.


Assuntos
Diferenciação Celular , Linhagem da Célula , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/citologia , Linha Primitiva/citologia , Animais , Biomarcadores/metabolismo , Western Blotting , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Gastrulação , Perfilação da Expressão Gênica , Camadas Germinativas/metabolismo , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Development ; 138(20): 4511-22, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21903671

RESUMO

Rhou encodes a Cdc42-related atypical Rho GTPase that influences actin organization in cultured cells. In mouse embryos at early-somite to early-organogenesis stages, Rhou is expressed in the columnar endoderm epithelium lining the lateral and ventral wall of the anterior intestinal portal. During foregut development, Rhou is downregulated in regions where the epithelium acquires a multilayered morphology heralding the budding of organ primordia. In embryos generated from Rhou knockdown embryonic stem (ES) cells, the embryonic foregut displays an abnormally flattened shape. The epithelial architecture of the endoderm is disrupted, the cells are depleted of microvilli and the phalloidin-stained F-actin content of their sub-apical cortical domain is reduced. Rhou-deficient cells in ES cell-derived embryos and embryoid bodies are less efficient in endoderm differentiation. Impaired endoderm differentiation of Rhou-deficient ES cells is accompanied by reduced expression of c-Jun/AP-1 target genes, consistent with a role for Rhou in regulating JNK activity. Downregulation of Rhou in individual endoderm cells results in a reduced ability of these cells to occupy the apical territory of the epithelium. Our findings highlight epithelial morphogenesis as a required intermediate step in the differentiation of endoderm progenitors. In vivo, Rhou activity maintains the epithelial architecture of the endoderm progenitors, and its downregulation accompanies the transition of the columnar epithelium in the embryonic foregut to a multilayered cell sheet during organ formation.


Assuntos
Sistema Digestório/embriologia , Sistema Digestório/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Junções Intercelulares/metabolismo , Junções Intercelulares/ultraestrutura , Camundongos , Camundongos Knockout , Células NIH 3T3 , RNA Interferente Pequeno/genética , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/genética
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