RESUMO
How diverse cell fates and complex forms emerge and feed back to each other to sculpt functional organs remains unclear. In the developing heart, the myocardium transitions from a simple epithelium to an intricate tissue that consists of distinct layers: the outer compact and inner trabecular layers. Defects in this process, which is known as cardiac trabeculation, cause cardiomyopathies and embryonic lethality, yet how tissue symmetry is broken to specify trabecular cardiomyocytes is unknown. Here we show that local tension heterogeneity drives organ-scale patterning and cell-fate decisions during cardiac trabeculation in zebrafish. Proliferation-induced cellular crowding at the tissue scale triggers tension heterogeneity among cardiomyocytes of the compact layer and drives those with higher contractility to delaminate and seed the trabecular layer. Experimentally, increasing crowding within the compact layer cardiomyocytes augments delamination, whereas decreasing it abrogates delamination. Using genetic mosaics in trabeculation-deficient zebrafish models-that is, in the absence of critical upstream signals such as Nrg-Erbb2 or blood flow-we find that inducing actomyosin contractility rescues cardiomyocyte delamination and is sufficient to drive cardiomyocyte fate specification, as assessed by Notch reporter expression in compact layer cardiomyocytes. Furthermore, Notch signalling perturbs the actomyosin machinery in cardiomyocytes to restrict excessive delamination, thereby preserving the architecture of the myocardial wall. Thus, tissue-scale forces converge on local cellular mechanics to generate complex forms and modulate cell-fate choices, and these multiscale regulatory interactions ensure robust self-organized organ patterning.
Assuntos
Retroalimentação Fisiológica , Coração/anatomia & histologia , Coração/embriologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Organogênese , Peixe-Zebra/embriologia , Actomiosina/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Modelos Animais , Receptores Notch/metabolismo , Transdução de Sinais , Peixe-Zebra/anatomia & histologiaRESUMO
Significant efforts have advanced our understanding of foregut-derived organ development; however, little is known about the molecular mechanisms that underlie the formation of the hepatopancreatic ductal (HPD) system. Here, we report a role for the homeodomain transcription factor Hhex in directing HPD progenitor specification in zebrafish. Loss of Hhex function results in impaired HPD system formation. We found that Hhex specifies a distinct population of HPD progenitors that gives rise to the cystic duct, common bile duct, and extra-pancreatic duct. Since hhex is not uniquely expressed in the HPD region but is also expressed in endothelial cells and the yolk syncytial layer (YSL), we tested the role of blood vessels as well as the YSL in HPD formation. We found that blood vessels are required for HPD patterning, but not for HPD progenitor specification. In addition, we found that Hhex is required in both the endoderm and the YSL for HPD development. Our results shed light on the mechanisms directing endodermal progenitors towards the HPD fate and emphasize the tissue specific requirement of Hhex during development.
Assuntos
Hepatopâncreas/embriologia , Hepatopâncreas/crescimento & desenvolvimento , Proteínas Repressoras/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Padronização Corporal/fisiologia , Sistema Digestório/metabolismo , Embrião não Mamífero/metabolismo , Endoderma/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Hepatopâncreas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas Repressoras/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Cardiac trabeculation is a highly regulated process that starts with the delamination of compact layer cardiomyocytes. The Hippo signaling pathway has been implicated in cardiac development but many questions remain. We have investigated the role of Wwtr1, a nuclear effector of the Hippo pathway, in zebrafish and find that its loss leads to reduced cardiac trabeculation. However, in mosaic animals, wwtr1-/- cardiomyocytes contribute more frequently than wwtr1+/- cardiomyocytes to the trabecular layer of wild-type hearts. To investigate this paradox, we examined the myocardial wall at early stages and found that compact layer cardiomyocytes in wwtr1-/- hearts exhibit disorganized cortical actin structure and abnormal cell-cell junctions. Accordingly, wild-type cardiomyocytes in mosaic mutant hearts contribute less frequently to the trabecular layer than when present in mosaic wild-type hearts, indicating that wwtr1-/- hearts are not able to support trabeculation. We also found that Nrg/Erbb2 signaling, which is required for trabeculation, could promote Wwtr1 nuclear export in cardiomyocytes. Altogether, these data suggest that Wwtr1 establishes the compact wall architecture necessary for trabeculation, and that Nrg/Erbb2 signaling negatively regulates its nuclear localization and therefore its activity.
Assuntos
Coração/embriologia , Coração/crescimento & desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miócitos Cardíacos/citologia , Organogênese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Proliferação de Células/fisiologia , Junções Intercelulares/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Morfolinos/genética , Cadeias Pesadas de Miosina/genética , Neurregulinas/metabolismo , Organogênese/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor ErbB-2/metabolismo , Serina-Treonina Quinase 3 , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Troponina T/genética , Proteínas de Sinalização YAP , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The development of the pigmentation pattern in zebrafish is a tightly regulated process that depends on both the self-organizing properties of pigment cells and extrinsic cues from other tissues. Many of the known mutations that alter the pattern act cell-autonomously in pigment cells, and our knowledge about external regulators is limited. Here, we describe novel zebrafish mau mutants, which encompass several dominant missense mutations in Aquaporin 3a (Aqp3a) that lead to broken stripes and short fins. A loss-of-function aqp3a allele, generated by CRISPR-Cas9, has no phenotypic consequences, demonstrating that Aqp3a is dispensable for normal development. Strikingly, the pigment cells from dominant mau mutants are capable of forming a wild-type pattern when developing in a wild-type environment, but the surrounding tissues in the mutants influence pigment cell behaviour and interfere with the patterning process. The mutated amino acid residues in the dominant alleles line the pore surface of Aqp3a and influence pore permeability. These results demonstrate an important effect of the tissue environment on pigment cell behaviour and, thereby, on pattern formation.
Assuntos
Aquaporina 3/genética , Mutação/genética , Pigmentação , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Nadadeiras de Animais/anatomia & histologia , Nadadeiras de Animais/citologia , Animais , Aquaporina 3/química , Aquaporina 3/metabolismo , Cromatóforos/metabolismo , Genes Dominantes , Proteínas de Fluorescência Verde/metabolismo , Mutação de Sentido Incorreto/genética , Permeabilidade , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
During cardiac trabeculation, cardiomyocytes delaminate from the outermost (compact) layer to form complex muscular structures known as trabeculae. As these cardiomyocytes delaminate, the remodeling of adhesion junctions must be tightly coordinated so cells can extrude from the compact layer while remaining in tight contact with their neighbors. In this study, we examined the distribution of N-cadherin (Cdh2) during cardiac trabeculation in zebrafish. By analyzing the localization of a Cdh2-EGFP fusion protein expressed under the control of the zebrafish cdh2 promoter, we initially observed Cdh2-EGFP expression along the lateral sides of embryonic cardiomyocytes, in an evenly distributed pattern, and with the occasional appearance of punctae. Within a few hours, Cdh2-EGFP distribution on the lateral sides of cardiomyocytes evolves into a clear punctate pattern as Cdh2-EGFP molecules outside the punctae cluster to increase the size of these aggregates. In addition, Cdh2-EGFP molecules also appear on the basal side of cardiomyocytes that remain in the compact layer. Delaminating cardiomyocytes accumulate Cdh2-EGFP on the surface facing the basal side of compact layer cardiomyocytes, thereby allowing tight adhesion between these layers. Importantly, we find that blood flow/cardiac contractility is required for the transition from an even distribution of Cdh2-EGFP to the formation of punctae. Furthermore, using time-lapse imaging of beating hearts in conjunction with a Cdh2 tandem fluorescent protein timer transgenic line, we observed that Cdh2-EGFP molecules appear to move from the lateral to the basal side of cardiomyocytes along the cell membrane, and that Erb-b2 receptor tyrosine kinase 2 (Erbb2) function is required for this relocalization.
Assuntos
Caderinas/metabolismo , Coração/embriologia , Miócitos Cardíacos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Circulação Coronária , Proteínas de Fluorescência Verde , Contração Miocárdica , Receptor ErbB-2/metabolismo , Peixe-ZebraRESUMO
Colour patterns of adult fish are produced by several types of pigment cells that distribute in the dermis during juvenile development. The zebrafish, Danio rerio, displays a striking pattern of dark stripes of melanophores interspersed by light stripes of xanthophores. Mutants lacking either cell type do not form proper stripes, indicating that interactions between these two chromatophore types are required for stripe formation. A third cell type, silvery iridophores, participates to render a shiny appearance to the pattern, but its role in stripe formation has been unclear. Mutations in rose (rse) or shady (shd) cause a lack or strong reduction of iridophores in adult fish; in addition, the melanophore number is drastically reduced and stripes are broken up into spots. We show that rse and shd are autonomously required in iridophores, as mutant melanophores form normal sized stripes when confronted with wild-type iridophores in chimeric animals. We describe stripe formation in mutants missing one or two of the three chromatophore types. None of the chromatophore types alone is able to create a pattern but residual stripe formation occurs with two cell types. Our analysis shows that iridophores promote and sustain melanophores. Furthermore, iridophores attract xanthophores, whereas xanthophores repel melanophores. We present a model for the interactions between the three chromatophore types underlying stripe formation. Stripe formation is initiated by iridophores appearing at the horizontal myoseptum, which serves as a morphological landmark for stripe orientation, but is subsequently a self-organising process.
Assuntos
Cromatóforos/metabolismo , Melanóforos/metabolismo , Pigmentação , Peixe-Zebra/embriologia , Animais , Padronização Corporal , Modelos Biológicos , Peixe-Zebra/genéticaRESUMO
The importance of pancreatic endocrine cell activity modulation by autonomic innervation has been debated. To investigate this question, we established an in vivo imaging model that also allows chronic and acute neuromodulation with genetic and optogenetic tools. Using the GCaMP6s biosensor together with endocrine cell fluorescent reporters, we imaged calcium dynamics simultaneously in multiple pancreatic islet cell types in live animals in control states and upon changes in innervation. We find that by 4 days post fertilization in zebrafish, a stage when islet architecture is reminiscent of that in adult rodents, prominent activity coupling between beta cells is present in basal glucose conditions. Furthermore, we show that both chronic and acute loss of nerve activity result in diminished beta-beta and alpha-beta activity coupling. Pancreatic nerves are in contact with all islet cell types, but predominantly with beta and delta cells. Surprisingly, a subset of delta cells with detectable peri-islet neural activity coupling had significantly higher homotypic coupling with other delta cells suggesting that some delta cells receive innervation that coordinates their output. Overall, these data show that innervation plays a vital role in the maintenance of homotypic and heterotypic cellular connectivity in pancreatic islets, a process critical for islet function.
Assuntos
Células Endócrinas , Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Ilhotas Pancreáticas/metabolismo , Pâncreas , Peixe-ZebraRESUMO
Transgenerational epigenetic inheritance (TEI) is mostly discussed in the context of physiological or environmental factors. Here, we show intergenerational and transgenerational inheritance of transcriptional adaptation (TA), a process whereby mutant messenger RNA (mRNA) degradation affects gene expression, in nematodes and zebrafish. Wild-type offspring of animals heterozygous for mRNA-destabilizing alleles display increased expression of adapting genes. Notably, offspring of animals heterozygous for nontranscribing alleles do not display this response. Germline-specific mutations are sufficient to induce TA in wild-type offspring, indicating that, at least for some genes, mutations in somatic tissues are not necessary for this process. Microinjecting total RNA from germ cells of TA-displaying heterozygous zebrafish can trigger TA in wild-type embryos and in their progeny, suggesting a model whereby mutant mRNAs in the germline trigger a TA response that can be epigenetically inherited. In sum, this previously unidentified mode of TEI reveals a means by which parental mutations can modulate the offspring's transcriptome.
Assuntos
Aclimatação , Peixe-Zebra , Animais , Peixe-Zebra/genética , Heterozigoto , Mutação , RNA Mensageiro/genéticaRESUMO
The transcription factor Snai1, a well-known regulator of epithelial-to-mesenchymal transition, has been implicated in early cardiac morphogenesis as well as in cardiac valve formation. However, a role for Snai1 in regulating other aspects of cardiac morphogenesis has not been reported. Using genetic, transcriptomic, and chimeric analyses in zebrafish, we find that Snai1b is required in cardiomyocytes for myocardial wall integrity. Loss of snai1b increases the frequency of cardiomyocyte extrusion away from the cardiac lumen. Extruding cardiomyocytes exhibit increased actomyosin contractility basally as revealed by enrichment of p-myosin and α-catenin epitope α-18, as well as disrupted intercellular junctions. Transcriptomic analysis of wild-type and snai1b mutant hearts revealed the dysregulation of intermediate filament genes, including desmin b (desmb) upregulation. Cardiomyocyte-specific desmb overexpression caused increased cardiomyocyte extrusion, recapitulating the snai1b mutant phenotype. Altogether, these results indicate that Snai1 maintains the integrity of the myocardial epithelium, at least in part by repressing desmb expression.
Assuntos
Regulação da Expressão Gênica , Coração/fisiologia , Filamentos Intermediários/genética , Fatores de Transcrição da Família Snail/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Miocárdio/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
To form new blood vessels (angiogenesis), endothelial cells (ECs) must be activated and acquire highly migratory and proliferative phenotypes. However, the molecular mechanisms that govern these processes are incompletely understood. Here, we show that Apelin signaling functions to drive ECs into such an angiogenic state. Zebrafish lacking Apelin signaling exhibit defects in endothelial tip cell morphology and sprouting. Using transplantation experiments, we find that in mosaic vessels, wild-type ECs leave the dorsal aorta (DA) and form new vessels while neighboring ECs defective in Apelin signaling remain in the DA. Mechanistically, Apelin signaling enhances glycolytic activity in ECs at least in part by increasing levels of the growth-promoting transcription factor c-Myc. Moreover, APELIN expression is regulated by Notch signaling in human ECs, and its function is required for the hypersprouting phenotype in Delta-like 4 (Dll4) knockdown zebrafish embryos. These data provide new insights into fundamental principles of blood vessel formation and Apelin signaling, enabling a better understanding of vascular growth in health and disease.
Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Quimiocinas/genética , Morfogênese/genética , Transdução de Sinais/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Vasos Sanguíneos/metabolismo , Quimiocinas/metabolismo , Células Endoteliais/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Mechanical forces regulate cell behavior and tissue morphogenesis. During cardiac development, mechanical stimuli from the heartbeat are required for cardiomyocyte maturation, but the underlying molecular mechanisms remain unclear. Here, we first show that the forces of the contracting heart regulate the localization and activation of the cytoskeletal protein vinculin (VCL), which we find to be essential for myofilament maturation. To further analyze the role of VCL in this process, we examined its interactome in contracting versus non-contracting cardiomyocytes and, in addition to several known interactors, including actin regulators, identified the slingshot protein phosphatase SSH1. We show how VCL recruits SSH1 and its effector, the actin depolymerizing factor cofilin (CFL), to regulate F-actin rearrangement and promote cardiomyocyte myofilament maturation. Overall, our results reveal that mechanical forces generated by cardiac contractility regulate cardiomyocyte maturation through the VCL-SSH1-CFL axis, providing further insight into how mechanical forces are transmitted intracellularly to regulate myofilament maturation.
Assuntos
Cofilina 1/metabolismo , Coração/embriologia , Miócitos Cardíacos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Vinculina/metabolismo , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Aminobenzoatos/farmacologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteínas dos Microfilamentos/metabolismo , Miocárdio/metabolismo , Miofibrilas/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Peixe-ZebraRESUMO
During cardiac development, cardiomyocytes form complex inner wall structures called trabeculae. Despite significant investigation into this process, the potential role of metabolism has not been addressed. Using single cell resolution imaging in zebrafish, we find that cardiomyocytes seeding the trabecular layer actively change their shape while compact layer cardiomyocytes remain static. We show that Erbb2 signaling, which is required for trabeculation, activates glycolysis to support changes in cardiomyocyte shape and behavior. Pharmacological inhibition of glycolysis impairs cardiac trabeculation, and cardiomyocyte-specific loss- and gain-of-function manipulations of glycolysis decrease and increase trabeculation, respectively. In addition, loss of the glycolytic enzyme pyruvate kinase M2 impairs trabeculation. Experiments with rat neonatal cardiomyocytes in culture further support these observations. Our findings reveal new roles for glycolysis in regulating cardiomyocyte behavior during cardiac wall morphogenesis.
Assuntos
Coração/embriologia , Coração/crescimento & desenvolvimento , Morfogênese/fisiologia , Miócitos Cardíacos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Genes erbB-2/genética , Glicólise , Coração/fisiologia , Modelos Animais , Morfogênese/genética , Organogênese/genética , Organogênese/fisiologia , Ratos , Transdução de Sinais/fisiologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Glia-axon interactions are essential for the development and function of the nervous system. We combine in vivo imaging and genetics to address the mechanism by which the migration of these cells is coordinated during embryonic development. Using stable transgenic lines, we have followed the migration of one subset of glial cells and their target axons in living zebrafish embryos. These cells coalesce at an early stage and remain coupled throughout migration, with axons apparently pathfinding for glia. Mutant analysis demonstrates that axons provide instructive cues that are sufficient to control glial guidance. Furthermore, mutations in the transcription factor Sox10/cls uncouple the migration of axons and glia. Finally, genetic ablation of this glial subtype reveals an essential role in nerve fasciculation.
Assuntos
Comunicação Celular/genética , Diferenciação Celular/genética , Movimento Celular/genética , Cones de Crescimento/metabolismo , Crista Neural/embriologia , Neuroglia/metabolismo , Sistema Nervoso Periférico/embriologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Sinais (Psicologia) , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde , Cones de Crescimento/ultraestrutura , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Indicadores e Reagentes/metabolismo , Proteínas Luminescentes/metabolismo , Crista Neural/citologia , Crista Neural/metabolismo , Neuroglia/citologia , Nervos Periféricos/anormalidades , Nervos Periféricos/citologia , Nervos Periféricos/metabolismo , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXE , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Many pathfinding axons must locate target fields that are themselves positioned by active migration. A hypothetical method for ensuring that these migrations are coordinated is towing, whereby the extension of axons is entirely dependent on the migration of their target cells. Here we combine genetics and time-lapse imaging in the zebrafish to show that towing by migrating cells is a bona fide mechanism for guiding pathfinding axons in vivo.
Assuntos
Axônios/fisiologia , Comunicação Celular/genética , Movimento Celular/fisiologia , Neurônios Aferentes/fisiologia , Animais , Animais Geneticamente Modificados , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Diagnóstico por Imagem/métodos , Embrião não Mamífero , Proteínas de Fluorescência Verde , Cones de Crescimento , Hibridização In Situ/métodos , Proteínas Luminescentes , Neurônios Aferentes/citologia , Neurônios Aferentes/transplante , Receptores CXCR4/genética , Transdução de Sinais , Fatores de Tempo , Transplante/métodos , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Pitx2c, a homeodomain transcription factor, is classically known for its left-right patterning role. However, an early wave of pitx2 expression occurs at the onset of gastrulation in several species, indicating a possible earlier role that remains relatively unexplored. Here we show that in zebrafish, maternal-zygotic (MZ) pitx2c mutants exhibit a shortened body axis indicative of convergence and extension (CE) defects. Live imaging reveals that MZpitx2c mutants display less persistent mesendodermal migration during late stages of gastrulation. Transplant data indicate that Pitx2c functions cell non-autonomously to regulate this cell behavior by modulating cell shape and protrusive activity. Using transcriptomic analyses and candidate gene approaches, we identify transcriptional changes in components of the chemokine-ECM-integrin dependent mesendodermal migration network. Together, our results define pathways downstream of Pitx2c that are required during early embryogenesis and reveal novel functions for Pitx2c as a regulator of morphogenesis.
Assuntos
Movimento Celular/genética , Desenvolvimento Embrionário/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Forma Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Embrião não Mamífero , Endoderma/citologia , Endoderma/metabolismo , Células Epiteliais/citologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Gastrulação/genética , Integrinas/genética , Integrinas/metabolismo , Mutação , Notocorda/citologia , Notocorda/metabolismo , Imagem com Lapso de Tempo , Fatores de Transcrição/metabolismo , Transcriptoma , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Zigoto/citologia , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismoRESUMO
Pathways modulating glucose homeostasis independently of insulin would open new avenues to combat insulin resistance and diabetes. Here, we report the establishment, characterization, and use of a vertebrate 'insulin-free' model to identify insulin-independent modulators of glucose metabolism. insulin knockout zebrafish recapitulate core characteristics of diabetes and survive only up to larval stages. Utilizing a highly efficient endoderm transplant technique, we generated viable chimeric adults that provide the large numbers of insulin mutant larvae required for our screening platform. Using glucose as a disease-relevant readout, we screened 2233 molecules and identified three that consistently reduced glucose levels in insulin mutants. Most significantly, we uncovered an insulin-independent beneficial role for androgen receptor antagonism in hyperglycemia, mostly by reducing fasting glucose levels. Our study proposes therapeutic roles for androgen signaling in diabetes and, more broadly, offers a novel in vivo model for rapid screening and decoupling of insulin-dependent and -independent mechanisms.
Assuntos
Glucose/metabolismo , Hiperglicemia/genética , Insulina/genética , Receptores Androgênicos/genética , Antagonistas de Receptores de Andrógenos/química , Antagonistas de Receptores de Andrógenos/metabolismo , Animais , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Homeostase , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Resistência à Insulina/genética , Receptores Androgênicos/química , Transdução de Sinais/genética , Peixe-Zebra/genéticaRESUMO
Complex interplay between cardiac tissues is crucial for their integrity. The flow responsive transcription factor KLF2, which is expressed in the endocardium, is vital for cardiovascular development but its exact role remains to be defined. To this end, we mutated both klf2 paralogues in zebrafish, and while single mutants exhibit no obvious phenotype, double mutants display a novel phenotype of cardiomyocyte extrusion towards the abluminal side. This extrusion requires cardiac contractility and correlates with the mislocalization of N-cadherin from the lateral to the apical side of cardiomyocytes. Transgenic rescue data show that klf2 expression in endothelium, but not myocardium, prevents this cardiomyocyte extrusion phenotype. Transcriptome analysis of klf2 mutant hearts reveals that Fgf signaling is affected, and accordingly, we find that inhibition of Fgf signaling in wild-type animals can lead to abluminal cardiomyocyte extrusion. These studies provide new insights into how Klf2 regulates cardiovascular development and specifically myocardial wall integrity.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Miocárdio/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/metabolismo , Alelos , Animais , Sequência de Bases , Caderinas/metabolismo , Morte Celular , Polaridade Celular , Proliferação de Células , Regulação para Baixo , Embrião não Mamífero/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Ligantes , Mutação/genética , Contração Miocárdica , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fenótipo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Tretinoína/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that hhex is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian HHEX using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Linfangiogênese/genética , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Fator C de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Vasos Sanguíneos/citologia , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Linhagem Celular , Embrião de Mamíferos , Embrião não Mamífero , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Vasos Linfáticos/citologia , Vasos Linfáticos/metabolismo , Camundongos , Neovascularização Fisiológica/genética , Proteínas Repressoras/deficiência , Transdução de Sinais , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/metabolismoRESUMO
Macrophages are known to interact with endothelial cells during developmental and pathological angiogenesis but the molecular mechanisms modulating these interactions remain unclear. Here, we show a role for the Hif-1α transcription factor in this cellular communication. We generated hif-1aa;hif-1ab double mutants in zebrafish, hereafter referred to as hif-1α mutants, and find that they exhibit impaired macrophage mobilization from the aorta-gonad-mesonephros (AGM) region as well as angiogenic defects and defective vascular repair. Importantly, macrophage ablation is sufficient to recapitulate the vascular phenotypes observed in hif-1α mutants, revealing for the first time a macrophage-dependent angiogenic process during development. Further substantiating our observations of vascular repair, we find that most macrophages closely associated with ruptured blood vessels are Tnfα-positive, a key feature of classically activated macrophages. Altogether, our data provide genetic evidence that Hif-1α regulates interactions between macrophages and endothelial cells starting with the mobilization of macrophages from the AGM.
Assuntos
Vasos Sanguíneos/embriologia , Células Endoteliais/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Macrófagos/citologia , Neovascularização Patológica/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Alelos , Animais , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Microscopia Confocal , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Oxigênio/química , Fenótipo , Tamanho da Amostra , Transdução de Sinais , Peixe-Zebra/embriologiaRESUMO
Tissue integrity is critical for organ formation and function. During heart development, cardiomyocytes differentiate and integrate to form a coherent tissue that contracts synchronously. However, the molecular mechanisms regulating cardiac tissue integrity are poorly understood. Here we show that proteolysis, via the E3 ubiquitin ligase ASB2, regulates cardiomyocyte maturation and tissue integrity. Cardiomyocytes in asb2b zebrafish mutants fail to terminally differentiate, resulting in reduced cardiac contractility and output. Mosaic analyses reveal a cell-autonomous requirement for Asb2b in cardiomyocytes for their integration as asb2b mutant cardiomyocytes are unable to meld into wild-type myocardial tissue. In vitro and in vivo data indicate that ASB2 negatively regulates TCF3, a bHLH transcription factor. TCF3 must be degraded for cardiomyocyte maturation, as TCF3 gain-of-function causes a number of phenotypes associated with cardiomyocyte dedifferentiation. Overall, our results show that proteolysis has an important role in cardiomyocyte maturation and the formation of a coherent myocardial tissue.