Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 411
Filtrar
1.
Nature ; 574(7776): 112-116, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554966

RESUMO

Organogenesis is a complex and interconnected process that is orchestrated by multiple boundary tissue interactions1-7. However, it remains unclear how individual, neighbouring components coordinate to establish an integral multi-organ structure. Here we report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cells. The boundary interactions between anterior and posterior gut spheroids differentiated from human pluripotent stem cells enables retinoic acid-dependent emergence of hepato-biliary-pancreatic organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factors. Whereas transplant-derived tissues are dominated by midgut derivatives, long-term-cultured microdissected hepato-biliary-pancreatic organoids develop into segregated multi-organ anlages, which then recapitulate early morphogenetic events including the invagination and branching of three different and interconnected organ structures, reminiscent of tissues derived from mouse explanted foregut-midgut culture. Mis-segregation of multi-organ domains caused by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo8,9. In sum, we demonstrate that the experimental multi-organ integrated model can be established by the juxtapositioning of foregut and midgut tissues, and potentially serves as a tractable, manipulatable and easily accessible model for the study of complex human endoderm organogenesis.


Assuntos
Sistema Biliar/embriologia , Intestinos/embriologia , Fígado/embriologia , Modelos Biológicos , Morfogênese , Pâncreas/embriologia , Animais , Sistema Biliar/citologia , Biomarcadores/análise , Biomarcadores/metabolismo , Padronização Corporal , Endoderma/citologia , Endoderma/embriologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Intestinos/citologia , Fígado/citologia , Masculino , Camundongos , Organoides/citologia , Organoides/embriologia , Pâncreas/citologia , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Esferoides Celulares/transplante , Fatores de Transcrição HES-1/análise , Fatores de Transcrição HES-1/metabolismo
2.
PLoS Genet ; 15(8): e1008301, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31412026

RESUMO

We investigated whether Tbx1, the gene for 22q11.2 deletion syndrome (22q11.2DS) and Foxi3, both required for segmentation of the pharyngeal apparatus (PA) to individual arches, genetically interact. We found that all Tbx1+/-;Foxi3+/- double heterozygous mouse embryos had thymus and parathyroid gland defects, similar to those in 22q11.2DS patients. We then examined Tbx1 and Foxi3 heterozygous, null as well as conditional Tbx1Cre and Sox172A-iCre/+ null mutant embryos. While Tbx1Cre/+;Foxi3f/f embryos had absent thymus and parathyroid glands, Foxi3-/- and Sox172A-iCre/+;Foxi3f/f endoderm conditional mutant embryos had in addition, interrupted aortic arch type B and retroesophageal origin of the right subclavian artery, which are all features of 22q11.2DS. Tbx1Cre/+;Foxi3f/f embryos had failed invagination of the third pharyngeal pouch with greatly reduced Gcm2 and Foxn1 expression, thereby explaining the absence of thymus and parathyroid glands. Immunofluorescence on tissue sections with E-cadherin and ZO-1 antibodies in wildtype mouse embryos at E8.5-E10.5, revealed that multilayers of epithelial cells form where cells are invaginating as a normal process. We noted that excessive multilayers formed in Foxi3-/-, Sox172A-iCre/+;Foxi3f/f as well as Tbx1 null mutant embryos where invagination should have occurred. Several genes expressed in the PA epithelia were downregulated in both Tbx1 and Foxi3 null mutant embryos including Notch pathway genes Jag1, Hes1, and Hey1, suggesting that they may, along with other genes, act downstream to explain the observed genetic interaction. We found Alcam and Fibronectin extracellular matrix proteins were reduced in expression in Foxi3 null but not Tbx1 null embryos, suggesting that some, but not all of the downstream mechanisms are shared.


Assuntos
Síndrome de DiGeorge/patologia , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio T/metabolismo , Animais , Região Branquial/embriologia , Síndrome de DiGeorge/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Endoderma/embriologia , Feminino , Fatores de Transcrição Forkhead/genética , Coração/embriologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Miocárdio/patologia , Proteínas com Domínio T/genética
3.
Int J Dev Biol ; 63(3-4-5): 171-186, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058295

RESUMO

The integration of extracellular signals and lineage-specific transcription factors allows cells to react flexibly to their environment, thus endowing the mammalian embryo with the capacity of regulative development. The combination of genetic and pharmacological tools allowing disruption of the fibroblast growth factor / extracellular signal-regulated kinase (FGF/ERK) pathway, together with animal models expressing lineage-specific reporters provided new insights into the role of this signaling cascade during mammalian development, as well as in embryo-derived stem cells. Here, we combine current knowledge acquired from different mammalian models to consider the universality of this cascade in specifying cellular fate across mammalian species.


Assuntos
Blastocisto/metabolismo , Células-Tronco Embrionárias/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Sistema de Sinalização das MAP Quinases , Animais , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Mamíferos
4.
Nature ; 570(7759): 77-82, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086336

RESUMO

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes.


Assuntos
Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Feminino , Fertilização , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Especificidade de Órgãos/genética , Fenótipo , Análise de Sequência de RNA , Análise de Célula Única
5.
Nat Genet ; 51(6): 999-1010, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110351

RESUMO

Human embryonic stem cells (ESCs) and human induced pluripotent stem cells hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five Jun N-terminal kinase (JNK)-JUN family genes as key barriers of DE differentiation. The JNK-JUN pathway does not act through directly inhibiting the DE enhancers. Instead, JUN co-occupies ESC enhancers with OCT4, NANOG, SMAD2 and SMAD3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2 and SMAD3 chromatin binding from ESC to DE enhancers. Therefore, the JNK-JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine.


Assuntos
Diferenciação Celular/genética , Endoderma/embriologia , Endoderma/metabolismo , Genoma , Genômica , Sistema de Sinalização das MAP Quinases , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Expressão Gênica , Técnicas de Inativação de Genes , Genes Reporter , Genômica/métodos , Humanos , Células-Tronco Pluripotentes Induzidas , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Reprodutibilidade dos Testes , Proteínas Smad
6.
Cell Prolif ; 52(3): e12591, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896067

RESUMO

OBJECTIVES: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. MATERIALS AND METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.


Assuntos
Endoderma/citologia , Endoderma/embriologia , Suínos/embriologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Técnicas de Cocultura , Cães , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Expressão Gênica , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Suínos/genética , Suínos/metabolismo , Quimeras de Transplante
7.
Dev Cell ; 48(3): 361-370.e3, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30753836

RESUMO

The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? We combine optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. We find that Erk activity is sufficient to "posteriorize" 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1 h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. We find that the endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes.


Assuntos
Ectoderma/citologia , Embrião de Mamíferos/metabolismo , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Drosophila/embriologia , Ectoderma/embriologia , Embrião não Mamífero/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Morfogênese/genética
8.
Nature ; 566(7745): 490-495, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30787436

RESUMO

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.


Assuntos
Diferenciação Celular/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Gastrulação , Organogênese , Análise de Célula Única , Animais , Linhagem da Célula/genética , Quimera/embriologia , Quimera/genética , Quimera/metabolismo , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Endotélio/citologia , Endotélio/embriologia , Endotélio/metabolismo , Feminino , Gastrulação/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , Masculino , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Mutação/genética , Células Mieloides/citologia , Organogênese/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linha Primitiva/citologia , Linha Primitiva/embriologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T/deficiência , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética
9.
PLoS Genet ; 15(2): e1007996, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30763319

RESUMO

Pharyngeal pouches, a series of outpocketings that bud from the foregut endoderm, are essential to the formation of craniofacial skeleton as well as several important structures like parathyroid and thymus. However, whether pharyngeal pouch progenitors exist in the developing gut tube remains unknown. Here, taking advantage of cell lineage tracing and transgenic ablation technologies, we identified a population of nkx2.3+ pouch progenitors in zebrafish embryos and demonstrated an essential requirement of ectodermal BMP2b for their specification. At early somite stages, nkx2.3+ cells located at lateral region of pharyngeal endoderm give rise to the pouch epithelium except a subpopulation expressing pdgfαa rather than nkx2.3. A small-scale screen of chemical inhibitors reveals that BMP signaling is necessary to specify these progenitors. Loss-of-function analyses show that BMP2b, expressed in the pharyngeal ectoderm, actives Smad effectors in endodermal cells to induce nkx2.3+ progenitors. Collectively, our study provides in vivo evidence for the existence of pouch progenitors and highlights the importance of BMP2b signaling in progenitor specification.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Padronização Corporal/fisiologia , Proteína Morfogenética Óssea 2/genética , Linhagem da Célula/genética , Linhagem da Célula/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Proteínas de Homeodomínio/genética , Mutação , Faringe/embriologia , Faringe/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Somitos/embriologia , Somitos/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
10.
Nature ; 565(7740): 480-484, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30651642

RESUMO

The embryonic gut tube is a cylindrical structure from which the respiratory and gastrointestinal tracts develop1. Although the early emergence of the endoderm as an epithelial sheet2,3 and later morphogenesis of the definitive digestive and respiratory organs4-6 have been investigated, the intervening process of gut tube formation remains relatively understudied7,8. Here we investigate the molecular control of macroscopic forces underlying early morphogenesis of the gut tube in the chick embryo. The gut tube has been described as forming from two endodermal invaginations-the anterior intestinal portal (AIP) towards the rostral end of the embryo and the caudal intestinal portal (CIP) at the caudal end-that migrate towards one another, internalizing the endoderm until they meet at the yolk stalk (umbilicus in mammals)1,6. Migration of the AIP to form foregut has been descriptively characterized8,9, but the hindgut is likely to form by a distinct mechanism that has not been fully explained10. We find that the hindgut is formed by collective cell movements through a stationary CIP, rather than by movement of the CIP itself. Further, combining in vivo imaging, biophysics and mathematical modelling with molecular and embryological approaches, we identify a contractile force gradient that drives cell movements in the hindgut-forming endoderm, enabling tissue-scale posterior extension of the forming hindgut tube. The force gradient, in turn, is established in response to a morphogenic gradient of fibroblast growth factor signalling. As a result, we propose that an important positive feedback arises, whereby contracting cells draw passive cells from low to high fibroblast growth factor levels, recruiting them to contract and pull more cells into the elongating hindgut. In addition to providing insight into the early gut development, these findings illustrate how large-scale tissue level forces can be traced to developmental signals during vertebrate morphogenesis.


Assuntos
Trato Gastrointestinal/embriologia , Morfogênese , Animais , Padronização Corporal , Movimento Celular , Embrião de Galinha , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Trato Gastrointestinal/citologia , Trato Gastrointestinal/metabolismo , Transdução de Sinais
11.
Development ; 146(2)2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30630826

RESUMO

Defects in the middle ear ossicles - malleus, incus and stapes - can lead to conductive hearing loss. During development, neural crest cells (NCCs) migrate from the dorsal hindbrain to specific locations in pharyngeal arch (PA) 1 and 2, to form the malleus-incus and stapes, respectively. It is unclear how migratory NCCs reach their proper destination in the PA and initiate mesenchymal condensation to form specific ossicles. We show that secreted molecules sonic hedgehog (SHH) and bone morphogenetic protein 4 (BMP4) emanating from the pharyngeal endoderm are important in instructing region-specific NCC condensation to form malleus-incus and stapes, respectively, in mouse. Tissue-specific knockout of Shh in the pharyngeal endoderm or Smo (a transducer of SHH signaling) in NCCs causes the loss of malleus-incus condensation in PA1 but only affects the maintenance of stapes condensation in PA2. By contrast, knockout of Bmp4 in the pharyngeal endoderm or Smad4 (a transducer of TGFß/BMP signaling) in the NCCs disrupts NCC migration into the stapes region in PA2, affecting stapes formation. These results indicate that region-specific endodermal signals direct formation of specific middle ear ossicles.


Assuntos
Ossículos da Orelha/embriologia , Endoderma/embriologia , Endoderma/metabolismo , Crista Neural/citologia , Transdução de Sinais , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Movimento Celular , Sobrevivência Celular , Deleção de Genes , Proteínas Hedgehog , Bigorna/embriologia , Bigorna/metabolismo , Martelo/embriologia , Martelo/metabolismo , Camundongos , Modelos Biológicos , Crista Neural/embriologia , Crista Neural/metabolismo , Especificidade de Órgãos , Faringe/embriologia , Fenótipo , Estribo/embriologia , Estribo/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo
12.
Dev Biol ; 447(1): 14-23, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29653088

RESUMO

The secreted TGF-ß superfamily signals Nodal and BMP coordinate the patterning of vertebrate embryos. Nodal specifies endoderm and mesoderm during germ layer formation, and BMP specifies ventral fates and patterns the dorsal/ventral axis. Five major models have been proposed to explain how the correct distributions of Nodal and BMP are achieved within tissues to orchestrate embryogenesis: source/sink, transcriptional determination, relay, self-regulation, and shuttling. Here, we discuss recent experiments probing these signal dispersal models, focusing on early zebrafish development.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Desenvolvimento Embrionário/fisiologia , Modelos Biológicos , Ligantes da Sinalização Nodal/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Endoderma/citologia , Endoderma/embriologia , Mesoderma/citologia , Mesoderma/embriologia , Ligantes da Sinalização Nodal/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
13.
Dev Biol ; 445(2): 256-270, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30472119

RESUMO

The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut.


Assuntos
Sistema Nervoso Entérico/embriologia , Animais , Linhagem da Célula/genética , Duodeno/embriologia , Duodeno/inervação , Duodeno/metabolismo , Endoderma/citologia , Endoderma/embriologia , Endoderma/metabolismo , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Transgênicos , Crista Neural/citologia , Crista Neural/embriologia , Crista Neural/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Pâncreas/embriologia , Pâncreas/inervação , Pâncreas/metabolismo , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismo
14.
Dev Biol ; 448(2): 210-225, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30365920

RESUMO

Electroporation-based assays were used to test whether the myogenic regulatory factor (MRF) of Ciona intestinalis (CiMRF) interferes with endogenous developmental programs, and to evaluate the importance of its unusual N-terminus for muscle development. We found that CiMRF suppresses both notochord and endoderm development when it is expressed in these tissues by a mechanism that may involve activation of muscle-specific microRNAs. Because these results add to a large body of evidence demonstrating the exceptionally high degree of functional conservation among MRFs, we were surprised to discover that non-ascidian MRFs were not myogenic in Ciona unless they formed part of a chimeric protein containing the CiMRF N-terminus. Equally surprising, we found that despite their widely differing primary sequences, the N-termini of MRFs of other ascidian species could form chimeric MRFs that were also myogenic in Ciona. This domain did not rescue the activity of a Brachyury protein whose transcriptional activation domain had been deleted, and so does not appear to constitute such a domain. Our results indicate that ascidians have previously unrecognized and potentially novel requirements for MRF-directed myogenesis. Moreover, they provide the first example of a domain that is essential to the core function of an important family of gene regulatory proteins, one that, to date, has been found in only a single branch of the family.


Assuntos
Ciona intestinalis/genética , Fatores de Regulação Miogênica/química , Fatores de Regulação Miogênica/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Endoderma/embriologia , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Desenvolvimento Muscular/genética , Músculos/metabolismo , Notocorda/embriologia , Notocorda/metabolismo , Regiões Promotoras Genéticas/genética , Domínios Proteicos , Relação Estrutura-Atividade
15.
Development ; 145(24)2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30470703

RESUMO

Evolution of the animal body plan is driven by changes in developmental gene regulatory networks (GRNs), but how networks change to control novel developmental phenotypes remains, in most cases, unresolved. Here, we address GRN evolution by comparing the endomesoderm GRN in two echinoid sea urchins, Strongylocentrotus purpuratus and Eucidaris tribuloides, with at least 268 million years of independent evolution. We first analyzed the expression of twelve transcription factors and signaling molecules of the S. purpuratus GRN in E. tribuloides embryos, showing that orthologous regulatory genes are expressed in corresponding endomesodermal cell fates in the two species. However, perturbation of regulatory genes revealed that important regulatory circuits of the S. purpuratus GRN are significantly different in E. tribuloides For example, mesodermal Delta/Notch signaling controls exclusion of alternative cell fates in E. tribuloides but controls mesoderm induction and activation of a positive feedback circuit in S. purpuratus These results indicate that the architecture of the sea urchin endomesoderm GRN evolved by extensive gain and loss of regulatory interactions between a conserved set of regulatory factors that control endomesodermal cell fate specification.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genética , Animais , Linhagem da Célula , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Retroalimentação Fisiológica , Gastrulação/genética , Mesoderma/embriologia , Mesoderma/metabolismo , Ouriços-do-Mar/citologia , Transdução de Sinais
16.
Genes Dev ; 32(21-22): 1430-1442, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30366903

RESUMO

After acquiring competence for selected cell fates, embryonic primordia may remain plastic for variable periods before tissue identity is irrevocably determined (commitment). We investigated the chromatin basis for these developmental milestones in mouse endoderm, a tissue with recognizable rostro-caudal patterning and transcription factor (TF)-dependent interim plasticity. Foregut-specific enhancers are as accessible and active in early midgut as in foregut endoderm, and intestinal enhancers and identity are established only after ectopic cis-regulatory elements are decommissioned. Depletion of the intestinal TF CDX2 before this cis element transition stabilizes foregut enhancers, reinforces ectopic transcriptional programs, and hence imposes foregut identities on the midgut. Later in development, as the window of chromatin plasticity elapses, CDX2 depletion weakens intestinal, without strengthening foregut, enhancers. Thus, midgut endoderm is primed for heterologous cell fates, and TFs act on a background of shifting chromatin access to determine intestinal at the expense of foregut identity. Similar principles likely govern other fate commitments.


Assuntos
Endoderma/metabolismo , Elementos Facilitadores Genéticos , Mucosa Intestinal/metabolismo , Intestinos/embriologia , Transcrição Genética , Animais , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Cromatina/metabolismo , Endoderma/embriologia , Intestinos/anatomia & histologia , Camundongos
17.
Development ; 145(22)2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30327323

RESUMO

The early post-implantation mouse embryo changes dramatically in both size and shape. These morphological changes are based on characteristic cellular behaviors, including cell growth and allocation. To perform clonal analysis, we established a Cre/loxP-based reporter mouse line, R26R-ManGeKyou, that enables clonal labeling with multiple colors. We also developed a novel ImageJ plugin, LP-Clonal, for quantitative measurement of the tilt angle of clonal cluster shape, enabling identification of the direction of cluster expansion. We carried out long-term and short-term lineage tracking. We also performed time-lapse imaging to characterize cellular behaviors using R26-PHA7-EGFP and R26R-EGFP These images were subjected to quantitative image analyses. We found that the proximal visceral endoderm overlying the extra-embryonic ectoderm shows coherent cell growth in a proximal-anterior to distal-posterior direction. We also observed that directional cell migration is coupled with cell elongation in the anterior region. Our observations suggest that the behaviors of visceral endoderm cells vary between regions during peri-implantation stages.


Assuntos
Endoderma/citologia , Endoderma/embriologia , Processamento de Imagem Assistida por Computador , RNA não Traduzido/metabolismo , Vísceras/embriologia , Animais , Blastômeros/citologia , Forma Celular , Células Clonais , Implantação do Embrião , Embrião de Mamíferos/metabolismo , Feminino , Gastrulação , Integrases/metabolismo , Masculino , Camundongos , Especificidade de Órgãos , Imagem com Lapso de Tempo
18.
Development ; 145(17)2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30111654

RESUMO

During gastrulation, endodermal cells actively migrate to the interior of the embryo, but the signals that initiate and coordinate this migration are poorly understood. By transplanting ectopically induced endodermal cells far from the normal location of endoderm specification, we identified the inputs that drive internalization without the confounding influences of fate specification and global morphogenic movements. We find that Nodal signaling triggers an autocrine circuit for initiating endodermal internalization. Activation of the Nodal receptor directs endodermal specification through sox32 and also induces expression of more Nodal ligands. These ligands act in an autocrine fashion to initiate endodermal cell sorting. Our work defines an 'AND' gate consisting of sox32-dependent endodermal specification and Nodal ligand reception controlling endodermal cell sorting to the inner layer of the embryo at the onset of gastrulation.


Assuntos
Movimento Celular/fisiologia , Endoderma/embriologia , Gastrulação/fisiologia , Camadas Germinativas/embriologia , Proteína Nodal/metabolismo , Peixe-Zebra/embriologia , Animais , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteína Nodal/genética , Fatores de Transcrição SOX/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
19.
Development ; 145(17)2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30082271

RESUMO

During embryogenesis, the germ layers, including the endoderm, undergo convergence and extension movements to narrow and elongate the body plan. In zebrafish, the dorsal migration of endodermal cells during gastrulation is controlled by chemokine signaling, but little is known about how they migrate during segmentation. Here, we show that glypican 4 (Gpc4), a member of the heparin sulfate proteoglycan family, is required for efficient migration of anterior endodermal cells during early segmentation, regulating Rac activation to maintain polarized actin-rich lamellipodia. An endoderm transplantation assay showed that Gpc4 regulates endoderm migration in a non-cell-autonomous fashion. Further analyses revealed that the impaired endoderm migration in gpc4 mutants results from increases in the expression and assembly of fibronectin and laminin, major components of the extracellular matrix (ECM). Notably, we found that matrix metalloproteinase 14 (Mmp14a/b) is required for the control of ECM expression during endoderm migration, with Gpc4 acting through Mmp14a/b to limit ECM expression. Our results suggest that Gpc4 is crucial for generating the environment required for efficient migration of endodermal cells, uncovering a novel function of Gpc4 during development.


Assuntos
Padronização Corporal/fisiologia , Movimento Celular/fisiologia , Endoderma/embriologia , Glipicanas/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Movimento Celular/genética , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Gastrulação/fisiologia , Glipicanas/genética , Laminina/metabolismo , Metaloproteinase 14 da Matriz/genética , Pseudópodes/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo
20.
Dev Dyn ; 247(9): 1057-1069, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30016568

RESUMO

BACKGROUND: Bilaterally symmetric animals have evolved highly reproducible asymmetries between left and right. In teleosts, Kupffer's vesicle, the structure necessary for the determination of left-right asymmetry, is derived from a group of cells in the gastrula termed the dorsal forerunners. RESULTS: Wilson cells are a ring of marginal enveloping layer cells that are cytoplasmically connected to the yolk cell and thus the last blastomeres to inherit yolk cell cytoplasm. Afterward, they collapse into the yolk to form the yolk syncytial layer. Without exception, forerunner cells are the progeny of dorsal Wilson cells. At the beginning of gastrulation, these Wilson cell progeny ingress beneath the enveloping layer, transform into Kupffer's vesicle, and eventually become tail notochord and muscle. Before ingressing, the forerunner precursor cells express endodermal promoting genes and require high-levels of Nodal signaling. CONCLUSIONS: Despite a derived function of the enveloping layer as an epithelium covering the entire embryo, its dorsal margin retains many behaviors of what might be expected of the dorsal superficial layers of the ancestral fish embryo, including an early program of endodermal development, cell ingression, and an eventual contribution of cells to caudal notochord and muscle, as well as the control of laterality. Developmental Dynamics 247:1057-1069, 2018. © 2018 Wiley Periodicals, Inc.


Assuntos
Linhagem da Célula , Macrófagos do Fígado/citologia , Peixe-Zebra/embriologia , Animais , Padronização Corporal , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Endoderma/citologia , Endoderma/embriologia , Gástrula/embriologia , Músculos/citologia , Músculos/embriologia , Notocorda/citologia , Notocorda/embriologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA