RESUMO
This article focuses on the roots of the organizer concept, which was developed by Hans Spemann during his studies of early embryonic development in amphibians. The fundamental properties of this axis-inducing signaling center have been elucidated through pioneering molecular research by Eddy De Robertis' laboratory and other researchers. Evolutionary comparisons have disclosed the presence of this signaling center, involving the interaction of Wnt and TGF-beta signaling pathways, existed not only in vertebrates but also in basal Metazoa such as Cnidaria. - Notably, even prior to the groundbreaking experiments conducted by Hilde Mangold and Hans Spemann, Ethel Browne conducted similar transplantation experiments on Hydra polyps. They were performed under the guidance of Thomas H Morgan and in the laboratory of Edmund B Wilson. Howard Lenhoff was the first to draw connections between Ethel Browne's transplantation experiments and those of Spemann and Mangold, igniting a vivid debate on the precedence of the organizer concept and its recognition in Nobel Prize considerations. This review critically compares the experiments conducted by Spemann and Mangold with those preceding their seminal work, concluding that the organizer concept clearly builds upon earlier research aimed at understanding developmental gradients, such as in the simple model Hydra. However, these approaches were not pursued further by Morgan, who shifted his focus towards unraveling the genetic control of development in flies, an approach that ultimately revealed the molecular identity of the Spemann organizer in vertebrates.
Assuntos
Hydra , Animais , Organizadores Embrionários/metabolismo , História do Século XX , Transdução de SinaisRESUMO
An instructive role for metabolism in embryonic patterning is emerging, although a role for mitochondria is poorly defined. We demonstrate that mitochondrial oxidative metabolism establishes the embryonic patterning center, the Spemann-Mangold Organizer, via hypoxia-inducible factor 1α (Hif-1α) in Xenopus. Hypoxia or decoupling ATP production from oxygen consumption expands the Organizer by activating Hif-1α. In addition, oxygen consumption is 20% higher in the Organizer than in the ventral mesoderm, indicating an elevation in mitochondrial respiration. To reconcile increased mitochondrial respiration with activation of Hif-1α, we discovered that the "free" c-subunit ring of the F1Fo ATP synthase creates an inner mitochondrial membrane leak, which decouples ATP production from respiration at the Organizer, driving Hif-1α activation there. Overexpression of either the c-subunit or Hif-1α is sufficient to induce Organizer cell fates even when ß-catenin is inhibited. We propose that mitochondrial leak metabolism could be a general mechanism for activating Hif-1α and Wnt signaling.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia , Mitocôndrias , Organizadores Embrionários , Animais , Trifosfato de Adenosina/metabolismo , Hipóxia , Mitocôndrias/metabolismo , Organizadores Embrionários/metabolismo , Xenopus laevisRESUMO
In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle-related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.
Assuntos
Padronização Corporal/fisiologia , Organizadores Embrionários/fisiologia , Nicho de Células-Tronco/fisiologia , Animais , Embrião de Galinha , Endoderma/embriologia , Gástrula/embriologia , Mesoderma/embriologia , Sistema Nervoso , Notocorda/embriologia , Organizadores Embrionários/metabolismo , Nicho de Células-Tronco/genética , Células-Tronco/metabolismo , Células-Tronco/fisiologiaRESUMO
BACKGROUND: Organizing centers are groups of specialized cells that secrete morphogens, thereby influencing development of their neighboring territories. Apoptosis is a form of programmed cell death reported to limit the size of organizers. Little is known about the identity of intracellular signals driving organizer cell death. Here we investigated in Xenopus the role of both the anti-apoptotic protein Myeloid-cell-leukemia 1 (Mcl1) and the cysteine proteases Caspase-3 and Caspase-7 in formation of the axial organizing center-the notochord-that derives from the Spemann organizer, and participates in the induction and patterning of the neuroepithelium. RESULTS: We confirm a role for apoptosis in establishing the axial organizer in early neurula. We show that the expression pattern of mcl1 is coherent with a role for this gene in early notochord development. Using loss of function approaches, we demonstrate that Mcl1 depletion decreases neuroepithelium width and increases notochord cells apoptosis, a process that relies on Caspase-7, and not on Caspase-3, activity. Our data provide evidence that Mcl1 protein levels physiologically control notochord cells' survival and that Caspase-7 is the executioner protease in this developmental process. CONCLUSIONS: Our study reveals new functions for Mcl1 and Caspase-7 in formation of the axial signalling center.
Assuntos
Caspase 7/biossíntese , Sobrevivência Celular , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Animais , Apoptose , Padronização Corporal/fisiologia , Caspase 3/biossíntese , Epitélio/metabolismo , Fertilização in vitro , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Humanos , Neurônios/metabolismo , Notocorda/metabolismo , Organizadores Embrionários/metabolismo , Biossíntese de Proteínas , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
A hallmark of Spemann organizer function is its expression of Wnt antagonists that regulate axial embryonic patterning. Here we identify the tumor suppressor Protein tyrosine phosphatase receptor-type kappa (PTPRK), as a Wnt inhibitor in human cancer cells and in the Spemann organizer of Xenopus embryos. We show that PTPRK acts via the transmembrane E3 ubiquitin ligase ZNRF3, a negative regulator of Wnt signaling promoting Wnt receptor degradation, which is also expressed in the organizer. Deficiency of Xenopus Ptprk increases Wnt signaling, leading to reduced expression of Spemann organizer effector genes and inducing head and axial defects. We identify a '4Y' endocytic signal in ZNRF3, which PTPRK maintains unphosphorylated to promote Wnt receptor depletion. Our discovery of PTPRK as a negative regulator of Wnt receptor turnover provides a rationale for its tumor suppressive function and reveals that in PTPRK-RSPO3 recurrent cancer fusions both fusion partners, in fact, encode ZNRF3 regulators.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Wnt/antagonistas & inibidores , Animais , Padronização Corporal/genética , Endocitose , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Organizadores Embrionários/metabolismo , Transdução de Sinais , Proteínas de Xenopus/metabolismo , Xenopus laevis , beta Catenina/metabolismoRESUMO
Existence and biomedical relevance of the neurenteric canal, a transient midline structure during early neurulation in the human embryo, have been controversially discussed for more than a century by embryologists and clinicians alike. In this study, the authors address the long-standing enigma by high-resolution histology and three-dimensional reconstruction using new and historic histological sections of 5 human 17- to 21-day-old embryos and of 2 marmoset monkey embryos of the species Callithrix jacchus at corresponding stages. The neurenteric canal presents itself as the classical vertical connection between the amniotic cavity and the yolk sac cavity and is lined (a) craniolaterally by a horseshoe-shaped "hinge of involuting notochordal cells" within Hensen's node and (b) caudally by the receding primitive streak epiblast dorsally and by notochordal plate epithelium ventrally, the latter of which covered the (longitudinal) notochordal canal on its ventral side at the preceding stage. Furthermore, asymmetric parachordal nodal expression in Callithrix and morphological asymmetries within the nodes of the other specimens suggest an early non-cilium-dependent left-right symmetry breaking mode previously postulated for other mammals. We conclude that structure and position of the mammalian neurenteric canal support the notion of its homology with the reptilian blastopore as a whole and with a dorsal segment of the blastopore in amphibia. These new features of the neurenteric canal may further clarify the aetiology of foetal malformations such as junctional neurulation defects, neuroendodermal cysts, and the split notochord syndrome.
Assuntos
Embrião de Mamíferos/embriologia , Embrião de Mamíferos/ultraestrutura , Notocorda/embriologia , Organizadores Embrionários/embriologia , Animais , Callithrix/embriologia , Callithrix/genética , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteína Nodal/análise , Proteína Nodal/genética , Notocorda/metabolismo , Notocorda/ultraestrutura , Organizadores Embrionários/metabolismo , Organizadores Embrionários/ultraestruturaRESUMO
Wdr5 is an essential component of SET/MLL methylase complexes that catalyze histone H3 lysine 4 trimethylation. The maternal Wnt/ß-catenin signaling is necessary for the H3K4me3 deposition at organizer genes in early Xenopus embryos. However, it remains unknown whether any component of SET/MLL methylase complex is required for Wnt signaling to establish H3K4me3 at its targets during the organizer induction. Here, we provide evidence that Wdr5 is required for dorsal axis development and organizer gene activation in Xenopus. Depletion of maternal Wdr5 resulted in ventralized development, phenocopying depletion of maternal ß-catenin. Depletion of maternal Wdr5 also drastically reduced the ability of ß-catenin to activate organizer genes. Siamois, a direct target of maternal Wnt/ß-catenin signaling, was able to reinitiate dorsal axis formation when Wdr5 was depleted. Importantly, we demonstrate that Wdr5 is required for H3K4me3 establishment at the promoter region of siamois. Moreover, we found evidence that Setd1b, a maternally provided methyltransferase, is required for organizer gene expression. Our findings indicate that Wdr5-mediated H3K4 trimethylation plays a part in the organizer formation and dorsal axis development that are controlled by the maternal Wnt/ß-catenin pathway.
Assuntos
Histona-Lisina N-Metiltransferase/genética , Metiltransferases/genética , Proteína de Leucina Linfoide-Mieloide/genética , Ativação Transcricional/genética , Proteínas de Xenopus/genética , beta Catenina/genética , Animais , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Organizadores Embrionários/crescimento & desenvolvimento , Organizadores Embrionários/metabolismo , Regiões Promotoras Genéticas , Quercetina/análogos & derivados , Quercetina/genética , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimentoRESUMO
The Kupffer's vesicle (KV) is the so-called left-right organizer in teleost fishes. KV is formed from dorsal forerunner cells (DFCs) and generates asymmetrical signals for breaking symmetry of embryos. It is unclear how DFCs or KV cells are prevented from intermingling with adjacent cells. In this study, we show that the Eph receptor gene ephb4b is highly expressed in DFCs whereas ephrin ligand genes, including efnb2b, are expressed in cells next to the DFC cluster during zebrafish gastrulation. ephb4b knockdown or mutation and efnb2b knockdown cause dispersal of DFCs, a smaller KV and randomization of laterality organs. DFCs often dynamically form lamellipodium-like, bleb-like and filopodium-like membrane protrusions at the interface, which attempt to invade but are bounced back by adjacent non-DFC cells during gastrulation. Upon inhibition of Eph/ephrin signaling, however, the repulsion between DFCs and non-DFC cells is weakened or lost, allowing DFCs to migrate away. Ephb4b/Efnb2b signaling by activating RhoA activity mediates contact and repulsion between DFCs and neighboring cells during gastrulation, preventing intermingling of different cell populations. Therefore, our data uncover an important role of Eph/ephrin signaling in maintaining DFC cluster boundary and KV boundary for normal left-right asymmetrical development.
Assuntos
Padronização Corporal , Embrião não Mamífero/citologia , Efrinas/metabolismo , Morfogênese , Organizadores Embrionários/citologia , Receptor EphB4/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Agregação Celular , Comunicação Celular , Movimento Celular , Embrião não Mamífero/metabolismo , Lateralidade Funcional , Técnicas de Inativação de Genes , Mesoderma/citologia , Mutação/genética , Organizadores Embrionários/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
A vertebrate signaling center, known in zebrafish as the organizer, is essential for axis patterning and formation and is regulated by multiple cell signaling pathways, including Wnt, Nodal, and Bmp. Organizer-specific Bmp2b plays important roles in the maintenance of the Bmp activity gradient and dorsal-ventral patterning. However, it is unknown how transcription of bmp2b in the organizer is regulated. In this study, we generated a bmp2b transgenic line Tsg(-2.272bmp2b:gfp) that reproduced organizer-specific bmp2b expression. Dissection analysis revealed that a 0.273-kb minimal promoter was indispensable for bmp2b expression in the dorsal organizer. Reporter assays showed that organizer-specific bmp2b is negatively regulated by the Nodal signal and positively regulated by the Wnt signal in both embryos and cell lines. Promoter analysis and chromatin-immunoprecipitation (ChIP) indicated that one consensus Smad-binding element (SBE) (CAGAC) and one Lef/Tcf-binding element (LBE) (AGATAA) were present in the 0.273-kb promoter, and could be directly bound by Smad2 and ß-catenin proteins. Together, these results suggest that maintenance of organizer-specific bmp2b expression involves opposite and concerted regulation by Nodal and Wnt signaling.
Assuntos
Proteína Morfogenética Óssea 2/genética , Proteína Nodal/genética , Organizadores Embrionários/metabolismo , Via de Sinalização Wnt/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Padronização Corporal/genética , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular Tumoral , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Hibridização In Situ , Proteína Nodal/metabolismo , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética , Proteína Smad2/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , beta Catenina/metabolismoRESUMO
Tiki1 is a Wnt protease and antagonist specifically expressed in the Spemann-Mangold Organizer and is required for head formation in Xenopus embryos. Here we report neighbor-joining phylogenetic analysis of vertebrate Tiki genes and their mRNA expression patterns in chick, mouse, and rabbit embryos. Tiki1 and Tiki2 orthologues are highly conserved, and exhibit similar but also different developmental expression patterns among the vertebrate/mammalian species analyzed. The Tiki1 gene is noticeably absent in the rodent lineage, but is present in lagomorphs and all other vertebrate/mammalian species examined. Expression in Hensen's node, the equivalent of the Xenopus Organizer, was observed for Chick Tiki2 and Rabbit Tiki1 and Tiki2. Mouse Tiki2 was detected at low levels at gastrulation and head fold stages, but not in the node. Mouse Tiki2 and chick Tiki1 display similar expression in the dorsal spinal cord. Chick Tiki1 expression was also detected in the surface ectoderm and maxillary bud, while chick Tiki2 was found in the anterior intestinal portal, head mesenchyme and primitive atrium. Our expression analyses provide evidence that Tiki1 and Tiki2 are evolutionarily conserved among vertebrate species and their expression in the Organizer and other regions suggests contributions of these Wnt inhibitors to embryonic patterning, as well as organogenesis. Our analyses further reveal mis-regulation of TIKI1 and TIKI2 in human cancer and diseases.
Assuntos
Padronização Corporal/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Metaloproteases/genética , Filogenia , Animais , Embrião de Galinha , Proteínas de Membrana/metabolismo , Metaloendopeptidases , Metaloproteases/metabolismo , Camundongos , Organizadores Embrionários/embriologia , Organizadores Embrionários/metabolismo , CoelhosRESUMO
BACKGROUND: Activator protein-1 (AP-1) is a mediator of BMP or FGF signaling during Xenopus embryogenesis. However, specific role of AP-1 in activin signaling has not been elucidated during vertebrate development. METHODOLOGY/PRINCIPAL FINDINGS: We provide new evidence showing that overexpression of heterodimeric AP-1 comprised of c-jun and c-fos (AP-1(c-Jun/c-Fos)) induces the expression of BMP-antagonizing organizer genes (noggin, chordin and goosecoid) that were normally expressed by high dose of activin. AP-1(c-Jun/c-Fos) enhanced the promoter activities of organizer genes but reduced that of PV.1, a BMP4-response gene. A loss of function study clearly demonstrated that AP-1(c-Jun/c-Fos) is required for the activin-induced organizer and neural gene expression. Moreover, physical interaction of AP-1(c-Jun/c-Fos) and Smad3 cooperatively enhanced the transcriptional activity of goosecoid via direct binding on this promoter. Interestingly, Smad3 mutants at c-Jun binding site failed in regulation of organizer genes, indicating that these physical interactions are specifically necessary for the expression of organizer genes. CONCLUSIONS/SIGNIFICANCE: AP-1(c-Jun/c-Fos) plays a specific role in organizer gene expression in downstream of activin signal during early Xenopus embryogenesis.
Assuntos
Ativinas/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica , Organizadores Embrionários/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Ativinas/genética , Animais , Western Blotting , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Imunoprecipitação da Cromatina , Embrião não Mamífero/citologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteína Goosecoid/genética , Proteína Goosecoid/metabolismo , Imunoprecipitação , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Luciferases/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização Proteica , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fator de Transcrição AP-1/genética , Ativação Transcricional , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.
Assuntos
Células-Tronco Embrionárias/citologia , Notocorda/citologia , Organizadores Embrionários/citologia , Animais , Antígenos de Diferenciação/metabolismo , Benzamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células , Embrião de Galinha , Dioxóis/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Proteínas de Fluorescência Verde/biossíntese , Fator 3-beta Nuclear de Hepatócito/farmacologia , Fator 3-beta Nuclear de Hepatócito/fisiologia , Proteínas de Homeodomínio/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Camundongos , Organizadores Embrionários/metabolismo , Hormônios Peptídicos/farmacologia , Hormônios Peptídicos/fisiologia , Pirróis/farmacologia , Receptores de Fatores de Crescimento/agonistas , Receptores de Fatores de Crescimento/antagonistas & inibidores , Proteínas Recombinantes de Fusão/biossíntese , Transdução de Sinais , Técnicas de Cultura de Tecidos , Transplante HeterólogoRESUMO
The Spemann-Mangold organizer is the structure that provides the signals, which initiate pattern formation in the developing vertebrate embryo, affecting the main body axes. Very little is known about axial induction in the gastrulating human embryo, as research is hindered by obvious ethical restrictions. Human embryonic stem cells (hESCs) are pluripotent cells derived from the pregastrula embryo that can differentiate in culture following a program similar to normal embryonic development but without pattern formation. Here, we show that in hESC-derived embryoid bodies, we can induce differentiation of cells that harbor markers and characteristics of the gastrula-organizer. Moreover, genetic labeling of these cells enabled their purification, and the discovery of a comprehensive set of their secreted proteins, cell surface receptors, and nuclear factors characteristic of the organizer. Remarkably, transplantation of cell populations enriched for the putative human organizer into frog embryos induced a secondary axis. Our research demonstrates that the human organizer can be induced in vitro and paves the way for the study of pattern formation and the initial regulation of body axis establishment in humans.
Assuntos
Células-Tronco Embrionárias/metabolismo , Gastrulação , Organizadores Embrionários/metabolismo , Padronização Corporal , Diferenciação Celular/fisiologia , Corpos Embrioides/metabolismo , Indução Embrionária , Regulação da Expressão Gênica no Desenvolvimento , Proteína Goosecoid/biossíntese , Humanos , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismoRESUMO
BACKGROUND: In vertebrates, the primordium of the brain is subdivided by the expression of Otx genes (forebrain/anterior midbrain), Hox genes (posterior hindbrain), and the genes Pax2, Pax5 and Pax8 (intervening region). The latter includes the midbrain/hindbrain boundary (MHB), which acts as a key organizer during brain patterning. Recent studies in Drosophila revealed that orthologous sets of genes are expressed in a similar tripartite pattern in the late embryonic brain, which suggested correspondence between the Drosophila deutocerebral/tritocerebral boundary region and the vertebrate MHB. To gain more insight into the evolution of brain regions, and particularly the MHB, I examined the expression of a comprehensive array of MHB-specific gene orthologs in the procephalic neuroectoderm and in individually identified neuroblasts during early embryonic stages 8-11, at which the segmental organization of the brain is most clearly displayed. RESULTS AND CONCLUSION: I show that the early embryonic brain exhibits an anterior Otx/otd domain and a posterior Hox1/lab domain, but that Pax2/5/8 orthologs are not expressed in the neuroectoderm and neuroblasts of the intervening territory. Furthermore, the expression domains of Otx/otd and Gbx/unpg exhibit a small common interface within the anterior deutocerebrum. In contrast to vertebrates, Fgf8-related genes are not expressed posterior to the otd/unpg interface. However, at the otd/unpg interface the early expression of other MHB-specific genes (including btd, wg, en), and of dorsoventral patterning genes, closely resembles the situation at the vertebrate MHB. Altogether, these results suggest the existence of an ancestral territory within the primordium of the deutocerebrum and adjacent protocerebrum, which might be the evolutionary equivalent of the region of the vertebrate MHB. However, lack of expression of Pax2/5/8 and Fgf8-related genes, and significant differences in the expression onset of other key regulators at the otd/unpg interface, imply that genetic interactions crucial for the vertebrate organizer activity are absent in the early embryonic brain of Drosophila.
Assuntos
Drosophila melanogaster/embriologia , Proteínas de Homeodomínio/metabolismo , Mesencéfalo/embriologia , Prosencéfalo/embriologia , Rombencéfalo/embriologia , Vertebrados/embriologia , Animais , Aves/embriologia , Aves/genética , Padronização Corporal/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Evolução Molecular , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Organizadores Embrionários/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Rombencéfalo/citologia , Rombencéfalo/metabolismo , Roedores/embriologia , Roedores/genética , Especificidade da Espécie , Células-Tronco/citologia , Células-Tronco/metabolismo , Vertebrados/metabolismoRESUMO
The process of invasion and metastasis during tumor progression is often reminiscent of cell migration events occurring during embryonic development. We hypothesized that genes controlling cellular changes in the Spemann organizer at gastrulation might be reactivated in tumors. The Goosecoid homeobox transcription factor is a known executer of cell migration from the Spemann organizer. We found that indeed Goosecoid is overexpressed in a majority of human breast tumors. Ectopic expression of Goosecoid in human breast cells generated invasion-associated cellular changes, including an epithelial-mesenchymal transition. TGF-beta signaling, known to promote metastasis, induced Goosecoid expression in human breast cells. Moreover, Goosecoid significantly enhanced the ability of breast cancer cells to form pulmonary metastases in mice. These results demonstrate that Goosecoid promotes tumor cell malignancy and suggest that other conserved organizer genes may function similarly in human cancer.
Assuntos
Proteína Goosecoid/metabolismo , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Organizadores Embrionários/metabolismo , Envelhecimento/fisiologia , Animais , Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína Goosecoid/genética , Humanos , Camundongos , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
The transcription factor Otx2 is required to determine mesencephalic versus metencephalic (cerebellum/pons) territory during embryogenesis. This function of Otx2 primarily involves positioning and maintaining the mid-hindbrain organizer at the border between midbrain and anterior hindbrain. Otx2 expression is maintained long after this organizer is established. We therefore generated conditional mutants of Otx2 using the Cre/loxP system to study later roles during rostral brain development. For inactivation of Otx2 in neuronal progenitor cells, we crossed Otx2(flox/flox) animals with Nestin-Cre transgenic animals. In Nestin-Cre/+; Otx2(flox/flox) embryos, Otx2 activity was lost from the ventral midbrain starting at embryonic day 10.5 (E10.5). In these mutant embryos, the mid-hindbrain organizer was properly positioned at E12.5, although Otx2 is absent from the midbrain. Hence, the Nestin-Cre/+; Otx2(flox/flox) animals represent a novel mouse model for studying the role of Otx2 in the midbrain, independently of abnormal development of the mid-hindbrain organizer. Our data demonstrate that Otx2 controls the development of several neuronal populations in the midbrain by regulating progenitor identity and neurogenesis. Dorsal midbrain progenitors ectopically expressed Math1 and generate an ectopic cerebellar-like structure. Similarly, Nkx2.2 ectopic expression ventrally into tegmentum progenitors is responsible for the formation of serotonergic neurons and hypoplasia of the red nucleus in the midbrain. In addition, we discovered a novel role for Otx2 in regulating neurogenesis of dopaminergic neurons. Altogether, these results demonstrate that Otx2 is required from E10.5 onward to regulate neuronal subtype identity and neurogenesis in the midbrain.
Assuntos
Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mesencéfalo/citologia , Neurônios/fisiologia , Fatores de Transcrição Otx/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bromodesoxiuridina/metabolismo , Contagem de Células/métodos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Dopamina/metabolismo , Embrião de Mamíferos , Indução Embrionária/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Proteínas de Filamentos Intermediários/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Membrana/metabolismo , Mesencéfalo/embriologia , Mesencéfalo/crescimento & desenvolvimento , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Nestina , Organizadores Embrionários/metabolismo , Fatores de Transcrição Otx/deficiência , Receptores Patched , Receptores de Superfície Celular , Serotonina/genética , Serotonina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Wnt1/metabolismo , Proteínas de Peixe-Zebra , Proteína GLI1 em Dedos de ZincoRESUMO
The mouse homeobox gene Gbx2 is first expressed throughout the posterior region of the embryo during gastrulation, and becomes restricted to rhombomeres 1-3 (r1-3) by embryonic day 8.5 (E8.5). Previous studies have shown that r1-3 do not develop in Gbx2 mutants and that there is an early caudal expansion of the midbrain gene Otx2 to the anterior border of r4. Furthermore, expression of Wnt1 and Fgf8, two crucial components of the isthmic organizer, is no longer segregated to adjacent domains in Gbx2 mutants. In this study, we extend the phenotypic analysis of Gbx2 mutants by showing that Gbx2 is not only required for development of r1-3, but also for normal gene expression in r4-6. To determine whether Gbx2 can alter hindbrain development, we generated Hoxb1-Gbx2 (HG) transgenic mice in which Gbx2 is ectopically expressed in r4. We show that Gbx2 is not sufficient to induce r1-3 development in r4. To test whether an Otx2/Gbx2 interface can induce r1-3 development, we introduced the HG transgene onto a Gbx2-null mutant background and recreated a new Otx2/Gbx2 border in the anterior hindbrain. Development of r3, but not r1 and r2, is rescued in Gbx2-/-; HG embryos. In addition, the normal spatial relationship of Wnt1 and Fgf8 is established at the new Otx2/Gbx2 border, demonstrating that an interaction between Otx2 and Gbx2 is sufficient to produce the normal pattern of Wnt1 and Fgf8 expression. However, the expression domains of Fgf8 and Spry1, a downstream target of Fgf8, are greatly reduced in mid/hindbrain junction area of Gbx2-/-; HG embryos and the posterior midbrain is truncated because of abnormal cell death. Interestingly, we show that increased cell death and a partial loss of the midbrain are associated with increased expression of Fgf8 and Spry1 in Gbx2 conditional mutants that lack Gbx2 in r1 after E9.0. These results together suggest that cell survival in the posterior midbrain is positively or negatively regulated by Fgf8, depending on Fgf8 expression level. Our studies provide new insights into the regulatory interactions that maintain isthmic organizer gene expression and the consequences of altered levels of organizer gene expression on cell survival.
Assuntos
Sistema Nervoso Central/embriologia , Indução Embrionária , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Organizadores Embrionários/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose/fisiologia , Bromodesoxiuridina , Fator 8 de Crescimento de Fibroblasto , Fatores de Crescimento de Fibroblastos/metabolismo , Técnicas Histológicas , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Organizadores Embrionários/embriologia , Fosfoproteínas/metabolismo , Transgenes/genética , Proteínas Wnt , Proteína Wnt1RESUMO
Growth and patterning during Drosophila wing development are mediated by signaling from its dorso-ventral (D/V) organizer. Wingless is expressed in the D/V boundary and functions as a morphogen to activate target genes at a distance. Wingless pathway and thereby D/V signaling is negatively regulated by the homeotic gene Ultrabithorax (Ubx) to mediate haltere development. In an enhancer-trap screen to identify genes that show differential expression between wing and haltere discs, we identified CG32062, which codes for a RNA-binding protein. In wing discs, CG32062 is expressed only in non-D/V cells. CG32062 expression in non-D/V cells is dependent on Notch-mediated signaling from the D/V boundary. However, CG32062 expression is independent of Wingless function, thus providing evidence for a second long-range signaling mechanism of the D/V organizer. In haltere discs, CG32062 is negatively regulated by Ubx. The non-cell autonomous nature of Ubx-mediated repression of CG32062 expression suggests that the novel component of D/V signaling is also negatively regulated during haltere specification.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a RNA/genética , Asas de Animais/crescimento & desenvolvimento , Animais , Biomarcadores , Proteínas de Drosophila/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Organizadores Embrionários/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Asas de Animais/metabolismo , Proteína Wnt1RESUMO
Dyneins have been implicated in left-right axis determination during embryonic development and in a variety of human genetic syndromes. In this paper, we study the recently discovered mouse dynein 2 light intermediate chain (mD2LIC), which is believed to be involved in retrograde intraflagella transport and which, like left-right dynein, is expressed in the node of the mouse embryo. Cells of the ventral node of mouse embryos lacking mD2LIC have an altered morphology and lack monocilia, and expression of Foxa2 and Shh in this structure is reduced or completely absent. At later stages, consistent with the absence of nodal cilia, mD2LIC is required for the establishment of the left-right axis and for normal expression of Nodal, and the ventral neural tube fails to express Shh, Foxa2 and Ebaf. mD2LIC also functions indirectly in the survival of anterior definitive endoderm and in the maintenance of the anterior neural ridge, probably through maintenance of Foxa2/Hnf3beta expression. Together, our results indicate that mD2LIC is required to maintain or establish ventral cell fates and for correct signalling by the organiser and midline, and they identify the first embryonic function of a vertebrate cytoplasmic dynein.
Assuntos
Padronização Corporal/fisiologia , Dineínas/deficiência , Organizadores Embrionários/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Dineínas/genética , Dineínas/metabolismo , Perfilação da Expressão Gênica , Marcação de Genes , Mesoderma , Camundongos , Dados de Sequência Molecular , Mutação , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Vertebrate gastrulation is a critical step in the establishment of body plan. During gastrulation, epithelial-mesenchymal transition (EMT) occurs. EMT is one of the central events of embryonic development, organ and tissue regeneration, and cancer metastasis. Signal transducers and activators of transcription (STATs) mediate biological actions such as cell proliferation, differentiation and survival in response to cytokines and growth factors, in a variety of biological processes. STATs are also important in EMT during gastrulation, organogenesis, wound healing and cancer progression. We previously showed that STAT3 is activated in the organizer during zebrafish gastrulation and its activity is essential for gastrulation movements. The requirement for STAT3 is cell-autonomous for the anterior migration of gastrula organizer cells, and non-cell-autonomous for the convergence of neighbouring cells. The molecular mechanisms of STAT's action in EMT, however, are unknown. Here we identify LIV1, a breast-cancer-associated zinc transporter protein, as a downstream target of STAT3 that is essential and sufficient for STAT3's cell-autonomous role in the EMT of zebrafish gastrula organizer cells. Furthermore, we demonstrate that LIV1 is essential for the nuclear localization of zinc-finger protein Snail, a master regulator of EMT. These results establish a molecular link between STAT3, LIV1 and Snail in EMT.