RESUMO
The Hippo-Yap pathway regulates multiple cellular processes in response to mechanical and other stimuli. In Drosophila, the polarity protein Lethal (2) giant larvae [L(2)gl], negatively regulates Hippo-mediated transcriptional output. However, in vertebrates, little is known about its homolog Llgl1. Here, we define a novel role for vertebrate Llgl1 in regulating Yap stability in cardiomyocytes, which impacts heart development. In contrast to the role of Drosophila L(2)gl, Llgl1 depletion in cultured rat cardiomyocytes decreased Yap protein levels and blunted target gene transcription without affecting Yap transcript abundance. Llgl1 depletion in zebrafish resulted in larger and dysmorphic cardiomyocytes, pericardial effusion, impaired blood flow and aberrant valvulogenesis. Cardiomyocyte Yap protein levels were decreased in llgl1 morphants, whereas Notch, which is regulated by hemodynamic forces and participates in valvulogenesis, was more broadly activated. Consistent with the role of Llgl1 in regulating Yap stability, cardiomyocyte-specific overexpression of Yap in Llgl1-depleted embryos ameliorated pericardial effusion and restored blood flow velocity. Altogether, our data reveal that vertebrate Llgl1 is crucial for Yap stability in cardiomyocytes and its absence impairs cardiac development.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Coração/embriologia , Miócitos Cardíacos/metabolismo , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteínas de Ciclo Celular/genética , Estabilidade Proteica , Transativadores/genética , Proteínas de Sinalização YAP , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
RATIONALE: Pathological biomechanical signaling induces vascular anomalies including cerebral cavernous malformations (CCM), which are caused by a clonal loss of CCM1/KRIT1 (Krev interaction trapped protein 1), CCM2/MGC4607, or CCM3/PDCD10. Why patients typically experience lesions only in lowly perfused venous capillaries of the cerebrovasculature is completely unknown. OBJECTIVE: In contrast, animal models with a complete loss of CCM proteins lack a functional heart and blood flow and exhibit vascular anomalies within major blood vessels as well. This finding raises the possibility that hemodynamics may play a role in the context of this vascular pathology. METHODS AND RESULTS: Here, we used a genetic approach to restore cardiac function and blood flow in a zebrafish model of CCM1. We find that blood flow prevents cardiovascular anomalies including a hyperplastic expansion within a large Ccm1-deficient vascular bed, the lateral dorsal aorta. CONCLUSIONS: This study identifies blood flow as an important physiological factor that is protective in the cause of this devastating vascular pathology.
Assuntos
Velocidade do Fluxo Sanguíneo/fisiologia , Neoplasias do Sistema Nervoso Central/diagnóstico por imagem , Modelos Animais de Doenças , Hemangioma Cavernoso do Sistema Nervoso Central/diagnóstico por imagem , Animais , Animais Geneticamente Modificados , Neoplasias do Sistema Nervoso Central/fisiopatologia , Angiografia Cerebral/métodos , Hemangioma Cavernoso do Sistema Nervoso Central/fisiopatologia , Peixe-ZebraRESUMO
Background and Purpose- Cerebral cavernous malformations (CCMs) are vascular malformations of the brain that lead to cerebral hemorrhages. A pharmacological treatment is needed especially for patients with nonoperable deep-seated lesions. We and others obtained CCM mouse models that were useful for mechanistic studies and rapid trials testing the preventive effects of candidate drugs. The shortened lifespan of acute mouse models hampered evaluation of compounds that would not only prevent lesion appearance but also cure preexisting lesions. Indirubin-3'-monoxime previously demonstrated its efficacy to reverse the cardiac phenotype of ccm2m201 zebrafish mutants and to prevent lesion development in an acute CCM2 mouse model. In the present article, we developed and characterized a novel chronic CCM2 mouse model and evaluated the curative therapeutic effect of indirubin-3'-monoxime after CCM lesion development. Methods- The chronic mouse model was obtained by a postnatal induction of brain-endothelial-cell-specific ablation of the Ccm2 gene using the inducible Slco1c1-CreERT2 mouse line. Results- We obtained a fully penetrant novel CCM chronic mouse model without any obvious off-target phenotypes and compatible with long-term survival. By 3 months of age, CCM lesions ranging in size from small isolated lesions to multiple caverns developed throughout the brain. Lesion burden was quantified in animals from 1 week to 5 months of age. Clear signs of intracerebral hemorrhages were noticed in brain-endothelial-cell-specific ablation of the Ccm2 gene. In contrast with its preventive effect in the acute CCM2 mouse model, a 20 mg/kg indirubin-3'-monoxime treatment for 3 weeks in 3-month old animals neither had any beneficial effect on the lesion burden nor alleviated cerebral hemorrhages. Conclusions- The brain-endothelial-cell-specific ablation of the Ccm2 gene chronic model is a strongly improved disease model for the CCM community whose challenge today is to decipher which candidate drugs might have a curative effect on patients' preexisting lesions. Visual Overview- An online visual overview is available for this article.
Assuntos
Encéfalo/patologia , Neoplasias do Sistema Nervoso Central/genética , Neoplasias do Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Proteínas dos Microfilamentos/genética , Animais , Neoplasias do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/deficiênciaRESUMO
Lumen expansion driven by hydrostatic pressure occurs during many morphogenetic processes. Although it is well established that members of the Claudin family of transmembrane tight junction proteins determine paracellular tightness within epithelial/endothelial barrier systems, functional evidence for their role in the morphogenesis of lumenized organs has been scarce. Here, we identify Claudin5a as a core component of an early cerebral-ventricular barrier system that is required for ventricular lumen expansion in the zebrafish embryonic brain before the establishment of the embryonic blood-brain barrier. Loss of Claudin5a or expression of a tight junction-opening Claudin5a mutant reduces brain ventricular volume expansion without disrupting the polarized organization of the neuroepithelium. Perfusion experiments with the electron-dense small molecule lanthanum nitrate reveal that paracellular tightness of the cerebral-ventricular barrier decreases upon loss of Claudin5a. Genetic analyses show that the apical neuroepithelial localization of Claudin5a depends on epithelial cell polarity and provide evidence for concerted activities between Claudin5a and Na(+),K(+)-ATPase during luminal expansion of brain ventricles. These data establish an essential role of a barrier-forming Claudin in ventricular lumen expansion, thereby contributing to brain morphogenesis.
Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas de Membrana/metabolismo , Células Neuroepiteliais/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Barreira Hematoencefálica , Encéfalo/citologia , Linhagem Celular , Permeabilidade da Membrana Celular , Polaridade Celular , Claudina-5 , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Camundongos , Microscopia Eletrônica , Mutação , Células Neuroepiteliais/citologia , Junções Íntimas/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
DNA-protein crosslinks (DPCs) are frequent and damaging DNA lesions that affect all DNA transactions, which in turn can lead to the formation of double-strand breaks, genomic instability and cell death. At the organismal level, impaired DPC repair (DPCR) is associated with cancer, ageing and neurodegeneration. Despite the severe consequences of DPCs, little is known about the processes underlying repair pathways at the organism level. SPRTN is a protease that removes most cellular DPCs during replication, whereas tyrosyl-DNA phosphodiesterase 1 repairs one of the most abundant enzymatic DPCs, topoisomerase 1-DPC (TOP1-DPC). How these two enzymes repair DPCs at the organism level is currently unknown. We perform phylogenetic, syntenic, structural and expression analysis to compare tyrosyl-DNA phosphodiesterase 1 (TDP1) orthologues between human, mouse and zebrafish. Using the zebrafish animal model and human cells, we demonstrate that TDP1 and SPRTN repair endogenous, camptothecin- and formaldehyde-induced DPCs, including histone H3- and TOP1-DPCs. We show that resolution of H3-DNA crosslinks depends on upstream proteolysis by SPRTN and subsequent peptide removal by TDP1 in RPE1 cells and zebrafish embryos, whereas SPRTN and TDP1 function in different pathways in the repair of endogenous TOP1-DPCs and total DPCs. Furthermore, we have found increased TDP2 expression in TDP1-deficient cells and embryos. Understanding the role of TDP1 in DPCR at the cellular and organismal levels could provide an impetus for the development of new drugs and combination therapies with TOP1-DPC inducing drugs.
Assuntos
Histonas , Peixe-Zebra , Humanos , Animais , Camundongos , Histonas/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Peptídeo Hidrolases/genética , Reparo do DNA , Filogenia , Dano ao DNA , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , DNA/química , Proteínas de Ligação a DNA/metabolismoRESUMO
The increasing use of the zebrafish model in biomedical and (eco)toxicological studies aimed at understanding the function of various proteins highlight the importance of optimizing existing methods to study gene and protein expression and localization in this model. In this context, zebrafish cryosections are still underutilized compared with whole-mount preparations. In this study, we used zebrafish embryos (24-120 hpf) to determine key factors for the preparation of high-quality zebrafish cryosections and to determine the optimal protocol for (immuno)fluorescence analyses of Na+ /K+ -ATPase and F-actin, across developmental stages from 1 to 5 dpf. The results showed that the highest quality zebrafish cryosections were obtained after the samples were fixed in 4% paraformaldehyde (PFA) for 1 h, incubated in 2.5% bovine gelatin/25% sucrose mixture, embedded in OCT, and then sectioned to 8 µm thickness at -20°C. Fluorescence microscopy analysis of phalloidin-labeled zebrafish skeletal muscle revealed that 1-h-4% PFA-fixed samples allowed optimal binding of phalloidin to F-actin. Further immunofluorescence analyses revealed detailed localization of F-actin and Na+ /K+ -ATPase in various tissues of the zebrafish and a stage-dependent increase in their respective expression in the somitic muscles and pronephros. Finally, staining of zebrafish cryosections and whole-mount samples revealed organ-specific and zone-dependent localizations of the Na+ /K+ -ATPase α1-subunit. RESEARCH HIGHLIGHTS: This study brings optimization of existing protocols for preparation and use of zebrafish embryos cryosections in (immuno)histological analyses. It reveals stage-dependent localization/expression of F-actin and Na+ /K+ -ATPase in zebrafish embryos.
Assuntos
Actinas , Peixe-Zebra , Animais , Bovinos , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Faloidina/metabolismo , CrioultramicrotomiaRESUMO
The capillary-venous pathology cerebral cavernous malformation (CCM) is caused by loss of CCM1/Krev interaction trapped protein 1 (KRIT1), CCM2/MGC4607, or CCM3/PDCD10 in some endothelial cells. Mutations of CCM genes within the brain vasculature can lead to recurrent cerebral hemorrhages. Pharmacological treatment options are urgently needed when lesions are located in deeply-seated and in-operable regions of the central nervous system. Previous pharmacological suppression screens in disease models of CCM led to the discovery that treatment with retinoic acid improved CCM phenotypes. This finding raised a need to investigate the involvement of retinoic acid in CCM and test whether it has a curative effect in preclinical mouse models. Here, we show that components of the retinoic acid synthesis and degradation pathway are transcriptionally misregulated across disease models of CCM. We complemented this analysis by pharmacologically modifying retinoic acid levels in zebrafish and human endothelial cell models of CCM, and in acute and chronic mouse models of CCM. Our pharmacological intervention studies in CCM2-depleted human umbilical vein endothelial cells (HUVECs) and krit1 mutant zebrafish showed positive effects when retinoic acid levels were increased. However, therapeutic approaches to prevent the development of vascular lesions in adult chronic murine models of CCM were drug regiment-sensitive, possibly due to adverse developmental effects of this hormone. A treatment with high doses of retinoic acid even worsened CCM lesions in an adult chronic murine model of CCM. This study provides evidence that retinoic acid signaling is impaired in the CCM pathophysiology and suggests that modification of retinoic acid levels can alleviate CCM phenotypes.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central , Adulto , Humanos , Animais , Camundongos , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Peixe-Zebra/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Encéfalo/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Many vertebrate organs are derived from monolayered epithelia that undergo morphogenesis to acquire their shape. Whereas asymmetric left/right gene expression within the zebrafish heart field has been well documented, little is known about the tissue movements and cellular changes underlying early cardiac morphogenesis. Here, we demonstrate that asymmetric involution of the myocardium of the right-posterior heart field generates the ventral floor, whereas the noninvoluting left heart field gives rise to the dorsal roof of the primary heart tube. During heart tube formation, asymmetric left/right gene expression within the myocardium correlates with asymmetric tissue morphogenesis. Disruption of left/right gene expression causes randomized myocardial tissue involution. Time-lapse analysis combined with genetic analyses reveals that motility of the myocardial epithelium is a tissue migration process. Our results demonstrate that asymmetric morphogenetic movements of the 2 bilateral myocardial cell populations generate different dorsoventral regions of the zebrafish heart tube. Failure to generate a heart tube does not affect the acquisition of atrial versus ventricular cardiac cell shapes. Therefore, establishment of basic cardiac cell shapes precedes cardiac function. Together, these results provide the framework for the integration of single cell behaviors during the formation of the vertebrate primary heart tube.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Morfogênese , Animais , Movimento Celular , Embrião não Mamífero , Células Epiteliais , Coração/crescimento & desenvolvimento , Miocárdio/citologia , Peixe-ZebraRESUMO
Endothelial cells respond to different levels of fluid shear stress through adaptations of their mechanosensitivity. Currently, we lack a good understanding of how this contributes to sculpting of the cardiovascular system. Cerebral cavernous malformation (CCM) is an inherited vascular disease that occurs when a second somatic mutation causes a loss of CCM1/KRIT1, CCM2, or CCM3 proteins. Here, we demonstrate that zebrafish Krit1 regulates the formation of cardiac valves. Expression of heg1, which encodes a binding partner of Krit1, is positively regulated by blood-flow. In turn, Heg1 stabilizes levels of Krit1 protein, and both Heg1 and Krit1 dampen expression levels of klf2a, a major mechanosensitive gene. Conversely, loss of Krit1 results in increased expression of klf2a and notch1b throughout the endocardium and prevents cardiac valve leaflet formation. Hence, the correct balance of blood-flow-dependent induction and Krit1 protein-mediated repression of klf2a and notch1b ultimately shapes cardiac valve leaflet morphology.
Assuntos
Células Endoteliais/fisiologia , Valvas Cardíacas/embriologia , Fatores de Transcrição Kruppel-Like/metabolismo , Mecanotransdução Celular , Glicoproteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteínas MuscularesRESUMO
Cerebral cavernous malformations (CCMs) are vascular lesions in the central nervous system causing strokes and seizures which currently can only be treated through neurosurgery. The disease arises through changes in the regulatory networks of endothelial cells that must be comprehensively understood to develop alternative, non-invasive pharmacological therapies. Here, we present the results of several unbiased small-molecule suppression screens in which we applied a total of 5,268 unique substances to CCM mutant worm, zebrafish, mouse, or human endothelial cells. We used a systems biology-based target prediction tool to integrate the results with the whole-transcriptome profile of zebrafish CCM2 mutants, revealing signaling pathways relevant to the disease and potential targets for small-molecule-based therapies. We found indirubin-3-monoxime to alleviate the lesion burden in murine preclinical models of CCM2 and CCM3 and suppress the loss-of-CCM phenotypes in human endothelial cells. Our multi-organism-based approach reveals new components of the CCM regulatory network and foreshadows novel small-molecule-based therapeutic applications for suppressing this devastating disease in patients.
Assuntos
Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Hemangioma Cavernoso do Sistema Nervoso Central/fisiopatologia , Animais , Caenorhabditis elegans , Técnicas Citológicas/métodos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Indóis/metabolismo , Camundongos , Oximas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Biologia de Sistemas/métodos , Peixe-ZebraRESUMO
The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.
Assuntos
Procedimentos Cirúrgicos Cardíacos/veterinária , Perfilação da Expressão Gênica/métodos , Coração/embriologia , Miocárdio/química , RNA Mensageiro/isolamento & purificação , Peixe-Zebra/embriologia , Peixe-Zebra/cirurgia , Animais , Animais Geneticamente Modificados , Procedimentos Cirúrgicos Cardíacos/métodos , Dissecação/métodos , Dissecação/veterinária , Reação em Cadeia da Polimerase em Tempo Real , Proteínas de Peixe-Zebra/genéticaRESUMO
Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor ß1 integrin and occurs in the absence of blood flow. Downregulation of ß1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a ß1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells.
Assuntos
Movimento Celular/fisiologia , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Integrina beta1/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Neovascularização Patológica/metabolismo , Proteínas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Adesão Celular/fisiologia , Movimento Celular/genética , Neoplasias do Sistema Nervoso Central/metabolismo , Família de Proteínas EGF , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Mecanotransdução Celular/fisiologia , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Peixe-ZebraRESUMO
Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel or sapje) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells. Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses. Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish.
Assuntos
Lasers , Músculo Esquelético/lesões , Peixe-Zebra/embriologia , Animais , Modelos Animais , Músculo Esquelético/embriologia , Músculo Esquelético/fisiologia , RegeneraçãoRESUMO
Signaling by Nodal and Bmp is essential for cardiac laterality. How activities of these pathways translate into left-right asymmetric organ morphogenesis is largely unknown. We show that, in zebrafish, Nodal locally reduces Bmp activity on the left side of the cardiac field. This effect is mediated by the extracellular matrix enzyme Hyaluronan synthase 2, expression of which is induced by Nodal. Unilateral reduction of Bmp signaling results in lower expression of nonmuscle myosin II and higher cell motility on the left, driving asymmetric displacement of the entire cardiac field. In silico modeling shows that left-right differences in cell motility are sufficient to induce a robust, directional migration of cardiac tissue. Thus, the mechanism underlying the formation of cardiac left-right asymmetry involves Nodal modulating an antimotogenic Bmp activity.
Assuntos
Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Coração/embriologia , Fatores de Determinação Direita-Esquerda , Proteína Nodal/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Movimento Celular , Regulação da Expressão Gênica no Desenvolvimento , Glucuronosiltransferase/metabolismo , Hialuronan Sintases , Miosina não Muscular Tipo IIA , Miosina não Muscular Tipo IIB , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Endosomal membrane traffic serves crucial roles in cell physiology, signaling, and development. Sorting between endosomes and the trans-Golgi network (TGN) is regulated among other factors by the adaptor AP-1, an essential component of multicellular organisms. Membrane recruitment of AP-1 requires phosphatidylinositol 4-phosphate [PI(4)P], though the precise mechanisms and PI4 kinase isozyme (or isozymes) involved in generation of this PI(4)P pool remain unclear. The Wnt pathway is a major developmental signaling cascade and depends on endosomal sorting in Wnt-sending cells. Whether TGN/endosomal sorting modulates signaling downstream of Frizzled (Fz) receptors in Wnt-receiving cells is unknown. Here, we identify PI4-kinase type 2ß (PI4K2ß) as a regulator of TGN/endosomal sorting and Wnt signaling. PI4K2ß and AP-1 interact directly and are required for efficient sorting between endosomes and the TGN. Zebrafish embryos depleted of PI4K2ß or AP-1 lack pectoral fins due to defective Wnt signaling. Rescue experiments demonstrate requirements for PI4K2ß-AP-1 complex formation and PI4K2ß-mediated PI(4)P synthesis. Furthermore, PI4K2ß binds to the Fz-associated component Dishevelled (Dvl) and regulates endosomal recycling of Fz receptors and Wnt target gene expression. These data reveal an evolutionarily conserved role for PI4K2ß and AP-1 in coupling phosphoinositide metabolism to AP-1-mediated sorting and Wnt signaling.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fator de Transcrição AP-1/genética , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/genética , Animais , Linhagem Celular , Endossomos/metabolismo , Receptores Frizzled/metabolismo , Humanos , Camundongos , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transporte Proteico , Ratos , Fator de Transcrição AP-1/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Myocellular regeneration in vertebrates involves the proliferation of activated progenitor or dedifferentiated myogenic cells that have the potential to replenish lost tissue. In comparison little is known about cellular repair mechanisms within myocellular tissue in response to small injuries caused by biomechanical or cellular stress. Using a microarray analysis for genes upregulated upon myocellular injury, we identified zebrafish Xin-actin-binding repeat-containing protein1 (Xirp1) as a marker for wounded skeletal muscle cells. By combining laser-induced micro-injury with proliferation analyses, we found that Xirp1 and Xirp2a localize to nascent myofibrils within wounded skeletal muscle cells and that the repair of injuries does not involve cell proliferation or Pax7(+) cells. Through the use of Xirp1 and Xirp2a as markers, myocellular injury can now be detected, even though functional studies indicate that these proteins are not essential in this process. Previous work in chicken has implicated Xirps in cardiac looping morphogenesis. However, we found that zebrafish cardiac morphogenesis is normal in the absence of Xirp expression, and animals deficient for cardiac Xirp expression are adult viable. Although the functional involvement of Xirps in developmental and repair processes currently remains enigmatic, our findings demonstrate that skeletal muscle harbours a rapid, cell-proliferation-independent response to injury which has now become accessible to detailed molecular and cellular characterizations.
Assuntos
Proliferação de Células , Coração/embriologia , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Coração/fisiopatologia , Imunoprecipitação , Hibridização In Situ , Morfogênese/genética , Músculo Esquelético/citologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cicatrização , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Retinal lamination is known to depend on cell polarity and localized signaling. In vertebrates, atypical protein kinase C proteins, aPKClambda/iota and aPKCzeta, are essential for apical-basal cell polarity. However, it is not known to what extent functional redundancy has precluded a comprehensive functional characterization of aPKC signaling during vertebrate retinogenesis. Here, we show that aPKCs lambda and zeta are functionally redundant for multiple aspects of retinogenesis including mitotic division location and orientation, cell-type positioning, and retinal pigment epithelial (RPE) and photoreceptor cell morphogenesis. Genetic mosaic analyses demonstrate a cell-autonomous requirement of aPKCs for RPE and photoreceptor development, and a cell-non-cell-autonomous function that is intrinsic to the neural retina for cell-type positioning. Our observations uncover a previously unappreciated involvement of aPKCzeta during zebrafish retinogenesis and suggest that aPKC signaling primes the retinal environment for appropriate cell migration of post-mitotic progenitor cells but is not essential for correct cell-type specification.