RESUMO
Breast cancer is one of the most common invasive cancers among women. The leading cause of difficulty in treating breast cancer patients is metastasis. Because cell migration is closely related to breast cancer metastasis, elucidating the detailed mechanism by which breast cancer cells promote their migration is crucial for improving the prognosis of patients. In this study, we investigated the relationship between breast cancer cell migration and Mind bomb1 (MIB1), an E3 ubiquitin ligase. We found that the downregulation of MIB1 promotes the cell migration of MCF7, a breast cancer-derived cell line. Furthermore, knockdown of MIB1 caused a reduction in CTNND1 and thereby impaired E-cadherin membrane localization in the cell boundary region. Taken together, our data suggest that MIB1 might play a role in suppressing breast cancer cell migration.
Assuntos
Neoplasias da Mama , Ubiquitina-Proteína Ligases , Feminino , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas , Linhagem Celular Tumoral , Movimento Celular/fisiologia , delta Catenina , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In the vertebrate ventral spinal cord, p2 progenitors give rise to two interneuron subtypes: excitatory V2a interneurons and inhibitory V2b interneurons. In the differentiation of V2a and V2b cells, Notch signaling promotes V2b fate at the expense of V2a fate. Later, V2b cells extend axons along the ipsilateral side of the spinal cord and express the inhibitory transmitter GABA. Notch signaling has been reported to inhibit the axonal outgrowth of mature neurons of the central nervous system; however, it remains unknown how Notch signaling modulates V2b neurite outgrowth and maturation into GABAergic neurons. Here, we have investigated neuron-specific Notch functions regarding V2b axon growth and maturation into zebrafish GABAergic neurons. We found that continuous neuron-specific Notch activation enhanced V2b fate determination but inhibited V2b axonal outgrowth and maturation into GABAergic neurons. These results suggest that Notch signaling activation is required for V2b fate determination, whereas its downregulation at a later stage is essential for V2b maturation. Accordingly, we found that a Notch signaling downstream gene, her15.1, showed biased expression in V2 linage cells and downregulated expression during the maturation of V2b cells, and continuous expression of her15.1 repressed V2b axogenesis. Our data suggest that spatiotemporal control of Notch signaling activity is required for V2b fate determination, maturation and axogenesis.
Assuntos
Axônios/metabolismo , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Receptores Notch/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BMP signaling is critical for many biological processes. Therefore, small molecules that modulate BMP signaling are useful for elucidating the function of BMP signaling and treating BMP signaling-related diseases. Here, we performed a phenotypic screening in zebrafish to examine the in vivo effects of N-substituted-2-amino-benzoic acid analogs NPL1010 and NPL3008 and found that they affect BMP signaling-dependent dorsal-ventral (D-V) patterning and bone formation in zebrafish embryos. Furthermore, NPL1010 and NPL3008 suppressed BMP signaling upstream of BMP receptors. BMP1 cleaves Chordin, an antagonist of BMP, and negatively regulates BMP signaling. Docking simulations demonstrated that NPL1010 and NPL3008 bind BMP1. We found that NPL1010 and NPL3008 partially rescued the disruptions in the D-V phenotype caused by bmp1 overexpression and selectively inhibited BMP1-dependent Chordin cleavage. Therefore, NPL1010 and NPL3008 are potentially valuable inhibitors of BMP signaling that act through selective inhibition of Chordin cleavage.
Assuntos
Proteínas Morfogenéticas Ósseas , Peixe-Zebra , Animais , Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Peixe-Zebra/genéticaRESUMO
Persistent directional cell migration is involved in animal development and diseases. The small GTPase Rac1 is involved in F-actin and focal adhesion dynamics. Local Rac1 activity is required for persistent directional migration, whereas global, hyperactivated Rac1 enhances random cell migration. Therefore, precise control of Rac1 activity is important for proper directional cell migration. However, the molecular mechanism underlying the regulation of Rac1 activity in persistent directional cell migration is not fully understood. Here, we show that the ubiquitin ligase mind bomb 1 (Mib1) is involved in persistent directional cell migration. We found that knockdown of MIB1 led to an increase in random cell migration in HeLa cells in a wound-closure assay. Furthermore, we explored novel Mib1 substrates for cell migration and found that Mib1 ubiquitinates Ctnnd1. Mib1-mediated ubiquitination of Ctnnd1 K547 attenuated Rac1 activation in cultured cells. In addition, we found that posterior lateral line primordium cells in the zebrafish mib1ta52b mutant showed increased random migration and loss of directional F-actin-based protrusion formation. Knockdown of Ctnnd1 partially rescued posterior lateral line primordium cell migration defects in the mib1ta52b mutant. Taken together, our data suggest that Mib1 plays an important role in cell migration and that persistent directional cell migration is regulated, at least in part, by the Mib1-Ctnnd1-Rac1 pathway.
Assuntos
Movimento Celular/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Adesões Focais/metabolismo , Adesões Focais/fisiologia , Células HeLa , Humanos , Ubiquitinação/fisiologia , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologiaRESUMO
Actin filaments and microtubules are principal components of the cytoskeleton that regulate the basic cellular phenomena underlying many fundamental cellular processes. Therefore, analyzing their dynamics in living cells is important for understanding cellular events more precisely. In this article, we report two novel transgenic zebrafish lines expressing red fluorescent proteins tagged with Lifeact or EB1 that interact with actin filaments and microtubule plus ends, respectively, under the control of the GAL4-UAS system. Using these transgenic lines, we could detect F-actin and microtubule plus end dynamics in specific tissues of living zebrafish embryos by crossing with GAL4 driver lines. In addition, we could achieve multi-color imaging using these transgenic lines with GFP-expressing transgenic lines. Therefore, our transgenic lines that carry UAS-driven red fluorescent cytoskeletal probes are useful tools for analyzing spatiotemporal changes of the cytoskeletal elements using multicolor live imaging.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Luminescentes/metabolismo , Regiões Promotoras Genéticas , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados/genética , Proteínas Luminescentes/genética , Proteína Vermelha FluorescenteRESUMO
The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-interneuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Here, using zebrafish embryos with altered Notch signaling, we show that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD, and three receptors, Notch1a, Notch1b, and Notch3 redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib, which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand-receptor combinations that occur concurrently during development in zebrafish.
Assuntos
Interneurônios/citologia , Células-Tronco Neurais/citologia , Neurogênese/genética , Receptores Notch/genética , Medula Espinal/embriologia , Peixe-Zebra/embriologia , Animais , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes/veterinária , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Morfolinos/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Receptor Notch1/metabolismo , Receptor Notch3 , Receptores Notch/metabolismo , Transdução de Sinais/genética , Medula Espinal/citologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Wnt signaling regulates various processes such as cell proliferation, differentiation, and embryo development. However, numerous diseases have been attributed to the aberrant transduction of Wnt signaling. We screened a plant extract library targeting TCF/ß-catenin transcriptional modulating activity with a cell-based luciferase assay. Activity-guided fractionation of the MeOH extract of the E. longifolia root led to the isolation of 9-hydroxycanthin-6-one (1). Compound 1 exhibited TCF/ß-catenin inhibitory activity. Compound 1 decreased the expression of Wnt signal target genes, mitf and zic2a, in zebrafish embryos. Treatment of SW480 cells with 1 decreased ß-catenin and increased phosphorylated ß-catenin (Ser 33, 37, Tyr 41) protein levels. The degradation of ß-catenin by 1 was suppressed by GSK3ß-siRNA, while compound 1 decreased ß-catenin even in the presence of CK1α-siRNA. These results suggest that 1 inhibits Wnt signaling through the activation of GSK3ß independent of CK1α.
Assuntos
Carbolinas/isolamento & purificação , Carbolinas/farmacologia , Caseína Quinase Ialfa/metabolismo , Eurycoma/química , Quinase 3 da Glicogênio Sintase/metabolismo , Alcaloides Indólicos/isolamento & purificação , Alcaloides Indólicos/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Western Blotting , Carbolinas/química , Quinase 3 da Glicogênio Sintase/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta , Células HCT116 , Humanos , Alcaloides Indólicos/química , Luciferases/metabolismo , Raízes de Plantas/química , Tailândia , Peixe-Zebra , beta Catenina/análise , beta Catenina/efeitos dos fármacosRESUMO
Wnt signaling plays important roles in proliferation, differentiation, development of cells, and various diseases. Activity-guided fractionation of the MeOH extract of the Ricinus communis stem led to the isolation of four compounds (1-4). The TCF/ß-catenin transcription activities of 1 and 3 were 2.2 and 2.5 fold higher at 20 and 30µM, respectively. Cells treated with ricinine (1) had higher ß-catenin and lower of p-ß-catenin (ser 33, 37, 45, Thr 41) protein levels, whereas glycogen synthase kinase 3ß (GSK3ß) and casein kinase 1α (CK1α) protein levels remained unchanged. Cells treated with pyrvinium, an activator of CK1α, had lower ß-catenin levels. However, the combined treatment of pyrvinium and 1 led to higher ß-catenin levels than those in cells treated with pyrvinium alone, which suggested that 1 inhibited CK1α activity. Furthermore, 1 increased ß-catenin protein levels in zebrafish embryos. These results indicated that 1 activated the Wnt signaling pathway by inhibiting CK1α.
Assuntos
Alcaloides/farmacologia , Caseína Quinase Ialfa/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridonas/farmacologia , Ricinus/química , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Alcaloides/química , Alcaloides/isolamento & purificação , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Estrutura Molecular , Caules de Planta/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/isolamento & purificação , Piridonas/química , Piridonas/isolamento & purificação , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Peixe-Zebra/embriologiaRESUMO
Many age-related neurological diseases still lack effective treatments, making their understanding a critical and urgent issue in the globally aging society. To overcome this challenge, an animal model that accurately mimics these diseases is essential. To date, many mouse models have been developed to induce age-related neurological diseases through genetic manipulation or drug administration. These models help in understanding disease mechanisms and finding potential therapeutic targets. However, some age-related neurological diseases cannot be fully replicated in human pathology due to the different aspects between humans and mice. Although zebrafish has recently come into focus as a promising model for studying aging, there are few genetic zebrafish models of the age-related neurological disease. This review compares the aging phenotypes of humans, mice, and zebrafish, and provides an overview of age-related neurological diseases that can be mimicked in mouse models and those that cannot. We presented the possibility that reproducing human cerebral small vessel diseases during aging might be difficult in mice, and zebrafish has potential to be another animal model of such diseases due to their similarity of aging phenotype to humans.
RESUMO
In the developing embryo, cell-cell signalling is necessary for tissue patterning and structural organization. During midline development, the notochord plays roles in the patterning of its surrounding tissues while forming the axial structure; however, how these patterning and structural roles are coordinated remains elusive. Here, we identify a mechanism by which Notch signalling regulates the patterning activities and structural integrity of the notochord. We found that Mind bomb (Mib) ubiquitylates Jagged 1 (Jag1) and is essential in the signal-emitting cells for Jag1 to activate Notch signalling. In zebrafish, loss- and gain-of-function analyses showed that Mib-Jag1-Notch signalling favours the development of non-vacuolated cells at the expense of vacuolated cells in the notochord. This leads to changes in the peri-notochordal basement membrane formation and patterning surrounding the muscle pioneer cells. These data reveal a previously unrecognized mechanism regulating the patterning and structural roles of the notochord by Mib-Jag1-Notch signalling-mediated cell-fate determination.
Assuntos
Padronização Corporal , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Notocorda/fisiologia , Receptores Notch/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Células 3T3 , Animais , Células COS , Chlorocebus aethiops , Endocitose , Proteína Jagged-1 , Camundongos , Modelos Biológicos , Proteínas Serrate-Jagged , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Peixe-ZebraRESUMO
The brain is an essential organ that controls various biological activities via the nervous system. The cerebral blood vessels supply oxygen and nutrients to neuronal cells and carry away waste products, which is essential in maintaining brain functions. Aging affects cerebral vascular function and decreases brain function. However, the physiological process of age-dependent cerebral vascular dysfunction is not fully understood. In this study, we examined aging effects on cerebral vascular patterning, vascular function, and learning ability in adult zebrafish. We found that the tortuosity of the blood vessels was increased, and the blood flow rate was reduced with aging in the zebrafish dorsal telencephalon. Moreover, we found cerebral blood flow positively correlated with learning ability in middle-old-aged zebrafish, as in aged humans. In addition, we also found that the elastin fiber decreased in the middle-old-aged fish brain vessel, suggesting a possible molecular mechanism underlying vessel dysfunction. Therefore, adult zebrafish may serve as a useful model for studying the aging-dependent decline in vascular function and human diseases such as vascular dementia.
Assuntos
Encéfalo , Peixe-Zebra , Animais , Humanos , Pessoa de Meia-Idade , Idoso , Telencéfalo , Envelhecimento/fisiologia , Circulação CerebrovascularRESUMO
Sensory systems are specialized to recognize environmental changes. Sensory organs are complex structures composed of different cell types, including neurons and sensory receptor cells, and how these organs are generated is an important question in developmental neurobiology. The posterior lateral line (pLL) is a simple sensory system in fish and amphibians that detects changes in water motion. It consists of neurons and sensory receptor hair cells, both of which are derived from the cranial ectoderm preplacodal region. However, it is not clearly understood how neurons and the sensory epithelium develop separately from the same preplacodal progenitors. We found that the numbers of posterior lateral line ganglion (pLLG) neurons, which are marked by neurod expression, increased in embryos with reduced Notch activity, but the forced activation of Notch reduced their number, suggesting that Notch-mediated lateral inhibition regulates the pLLG cell fate in zebrafish. By fate-mapping analysis, we found that cells adjacent to the pLLG neurons in the pre-pLL placodal region gave rise to the anterior part of the pLL primordium (i.e., sensory epithelial progenitor cells), and that the choice of cell fate between pLLG neuron or pLL primordium was regulated by Notch signaling. Since Notch signaling also affects hair cell fate determination at a later stage, our study suggests that Notch signaling has dual, time-dependent roles in specifying multiple cell types during pLL development.
Assuntos
Células Epiteliais/metabolismo , Sistema da Linha Lateral/citologia , Sistema da Linha Lateral/embriologia , Neurônios/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Homeodomínio/metabolismo , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Receptores Notch/classificação , Receptores Notch/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Tempo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The control of organ size and position relies, at least in part, upon appropriate regulation of the signals that specify organ progenitor fields. Pancreatic cell fates are specified by retinoic acid (RA), and proper size and localization of the pancreatic field are dependent on tight control of RA signaling. Here we show that the RA-degrading Cyp26 enzymes play a critical role in defining the normal anterior limit of the pancreatic field. Disruption of Cyp26 function causes a dramatic expansion of pancreatic cell types toward the anterior of the embryo. The cyp26a1 gene is expressed in the anterior trunk endoderm at developmental stages when RA is signaling to specify pancreas, and analysis of cyp26a1/giraffe (gir) mutant zebrafish embryos confirms that cyp26a1 plays the primary role in setting the anterior limit of the pancreas. Analysis of the gir mutants further reveals that cyp26b1 and cyp26c1 function redundantly to partially compensate for loss of Cyp26a1 function. We used cell transplantation to determine that Cyp26a1 functions directly in endoderm to modulate RA signaling and limit the pancreatic field. Taken together with our finding that endodermal expression of cyp26 genes is subject to positive regulation by RA, our data reveal a feedback loop within the endoderm. Such feedback can maintain consistent levels of RA signaling, despite environmental fluctuations in RA concentration, thus ensuring a consistent size and location of the pancreatic field.
Assuntos
Sistema Enzimático do Citocromo P-450/fisiologia , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Pâncreas/metabolismo , Animais , Linhagem da Célula , Transplante de Células , Hibridização In Situ , Modelos Biológicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Pâncreas/citologia , Pâncreas/embriologia , Ácido Retinoico 4 Hidroxilase , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-ZebraRESUMO
Activation of Notch signaling requires physical interaction between ligand- and receptor-expressing cells and pulling force to release the Notch intracellular domain. Therefore, the soluble recombinant ligand protein is not suitable for the activation of Notch signaling in a cell culture system. Here, we describe an efficient method for transient activation of Notch signaling using immobilized ligand beads. Using this method, the timing of Notch signaling can be efficiently controlled.
Assuntos
Receptores Notch , Transdução de Sinais , Comunicação Celular , Ligantes , Receptores Notch/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Aging is a major risk factor for many chronic diseases, causing a general decline in physiological function and loss of homeostasis. Recently, small teleost fish have been used as animal models of aging research because their genetic structures and organs closely resemble those of humans. Guppy (Poecilia reticulata), a small teleost fish, has a shorter lifespan than zebrafish. However, the age-dependent changes in physiology and genetics in guppies are not well understood. Here, we investigated the age-associated changes in metabolic rate, physical activity, and gene expression in guppies. Our results indicated that the resting metabolic rate and spontaneous motor activity in guppies decreased from an earlier age than those in mice. Moreover, the mRNA expression level of ppargc1a and the accumulation of lipofuscin were affected by age in the guppy livers; however, these changes were species-specific. On the other hand, in aged guppy brains, the mRNA expression changes of some genes were partly consistent with aged mammals. Although the process of senescence of the liver in guppies might vary from mammals, our findings suggest that guppy could be a useful animal model for age-related changes in physiological functions.
Assuntos
Poecilia , Animais , Expressão Gênica , Camundongos , Poecilia/genética , Especificidade da Espécie , Peixe-ZebraRESUMO
Glucocorticoid-induced osteoporosis (GIOP) is a major cause of secondary osteoporosis, and the pathogenic mechanisms of GIOP remain to be elucidated. Here, we show a rapid dexamethasone-induced osteoporosis animal model using zebrafish scales. Intraperitoneal injection of dexamethasone over a 5-day period suppressed the regeneration of scales. Furthermore, the circularity of the newly formed regenerated scales was also slightly reduced compared to that of the control group on day 5. The changes in bone-related enzymes, such as cathepsin K, tartrate-resistant acid phosphatase (TRAP) for bone resorption, and alkaline phosphatase (ALP) for bone formation, provide insight into the progression of bone diseases; therefore, we further developed a method to measure the activities of cathepsin K, TRAP, and ALP using zebrafish scales. We found that a lysis buffer with detergent at neutral pH under sonication efficiently helped extract these three enzymes with high activity levels. Interestingly, treatment with a dexamethasone injection produced considerably higher levels of cathepsin K activity and a lower Ca/P ratio than those in the control group, suggesting that dexamethasone increased osteoclast activity, with no significant changes in the activities of TRAP and ALP. Our GIOP model and enzyme assay method could help to design better treatments for GIOP.
RESUMO
Polypyrrole-based polyamides are used as sequence-specific DNA probes. However, their cellular uptake and distribution are affected by several factors and have not been extensively studied in vivo. Here, we generated a series of fluorescence-conjugated polypyrrole compounds and examined their cellular distribution using live zebrafish and cultured human cells. Among the evaluated compounds, Py3-FITC was able to visualize collagen-rich tissues, such as the jaw cartilage, opercle and bulbus arteriosus, in early-stage living zebrafish embryos. Then, we stained cultured human cells with Py3-FITC and found that the staining became more intense as the amount of collagen was increased. In addition, Py3-FITC-stained HR cells, which represent a type of ionocyte on the body surface of living zebrafish embryos. Py3-FITC has low toxicity, and collagen-rich tissues and ionocytes can be visualized when soaked in Py3-FITC solution. Therefore, Py3-FITC may be a useful live imaging tool for detecting changes in collagen-rich tissue and ionocytes, including their mammalian analogues, during both normal development and disease progression.
Assuntos
Colágeno/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Corantes Fluorescentes/síntese química , Animais , Cartilagem/citologia , Cartilagem/metabolismo , Linhagem Celular , Corantes Fluorescentes/efeitos adversos , Corantes Fluorescentes/farmacocinética , Humanos , Microscopia de Fluorescência/métodos , Nylons/química , Polímeros/química , Pirróis/química , Peixe-ZebraRESUMO
The homeobox transcription factor Mtx2 is essential for epiboly, the first morphogenetic movement of gastrulation in zebrafish. Morpholino knockdown of Mtx2 results in stalling of epiboly and lysis due to yolk rupture. However, the mechanism of Mtx2 action is unknown. The role of mtx2 is surprising as most mix/bix family genes are thought to have roles in mesendoderm specification. Using a transgenic sox17-promoter driven EGFP line, we show that Mtx2 is not required for endoderm specification but is required for correct morphogenetic movements of endoderm and axial mesoderm. During normal zebrafish development, mtx2 is expressed at both the blastoderm margin and in the zebrafish equivalent of visceral endoderm, the extra-embryonic yolk syncytial layer (YSL). We show that formation of the YSL is not Mtx2 dependent, but that Mtx2 directs spatial arrangement of YSL nuclei. Furthermore, we demonstrate that Mtx2 knockdown results in loss of the YSL F-actin ring, a microfilament structure previously shown to be necessary for epiboly progression. In summary, we propose that Mtx2 acts within the YSL to regulate morphogenetic movements of both embryonic and extra-embryonic tissues, independently of cell fate specification.
Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/fisiologia , Animais , Movimento Celular/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Embrião não Mamífero , Gastrulação/fisiologia , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/fisiologia , Regiões Promotoras Genéticas , Fatores de Transcrição SOXF , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
The gastrulating vertebrate embryo develops three germlayers: ectoderm, mesoderm, and endoderm. Zebrafish endoderm differentiation starts with the activation of sox17 by casanova (cas). We report that spg (pou2/Oct4) is essential for endoderm formation. Embryos devoid of maternal and zygotic spg function (MZspg) lack endodermal precursors. Cell transplantations show that spg acts in early endodermal precursors, and cas mRNA-injection into MZspg embryos does not restore endoderm development. spg and cas together are both necessary and sufficient to activate endoderm development, and stimulate expression of a sox17 promoter-luciferase reporter. Endoderm and mesoderm derive from a common origin, mesendoderm. We propose that Spg and Cas commit mesendodermal precursors to an endodermal fate. The joint control of endoderm formation by spg and cas suggests that the endodermal germlayer may be a tissue unit with distinct genetic control, thus adding genetic support to the germlayer concept in metazoan development.
Assuntos
Embrião não Mamífero/embriologia , Endoderma/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra , Peixe-Zebra/embriologia , Animais , Padronização Corporal/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Proteínas de Ligação a DNA/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Endoderma/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Grupo de Alta Mobilidade/genética , Masculino , Fator 3 de Transcrição de Octâmero , Regiões Promotoras Genéticas/genética , RNA Mensageiro/farmacologia , Fatores de Transcrição SOX , Fatores de Transcrição SOXF , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra/metabolismo , Zigoto/citologia , Zigoto/metabolismoRESUMO
The zebrafish endoderm begins to develop at gastrulation stages as a monolayer of cells. The behaviour of the endoderm during gastrulation stages is well understood. However, knowledge of the morphogenic movements of the endoderm during somitogenesis stages, as it forms a mesenchymal rod, is lacking. Here we characterise endodermal development during somitogenesis stages, and describe the morphogenic movements as the endoderm transitions from a monolayer of cells into a mesenchymal endodermal rod. We demonstrate that, unlike the overlying mesoderm, endodermal cells are not polarised during their migration to the midline at early somitogenesis stages. Specifically, we describe the stage at which endodermal cells begin to leave the monolayer, a process we have termed 'midline aggregation'. The planar cell polarity (PCP) signalling pathway is known to regulate mesodermal and ectodermal cell convergence towards the dorsal midline. However, a role for PCP signalling in endoderm migration to the midline during somitogenesis stages has not been established. In this report, we investigate the role for PCP signalling in multiple phases of endoderm development during somitogenesis stages. Our data exclude involvement of PCP signalling in endodermal cells as they leave the monolayer.