RESUMO
Gastrulation is a fundamental phase of animal embryogenesis during which germ layers are specified, rearranged, and shaped into a body plan with organ rudiments. Gastrulation involves four evolutionarily conserved morphogenetic movements, each of which results in a specific morphologic transformation. During emboly, mesodermal and endodermal cells become internalized beneath the ectoderm. Epibolic movements spread and thin germ layers. Convergence movements narrow germ layers dorsoventrally, while concurrent extension movements elongate them anteroposteriorly. Each gastrulation movement can be achieved by single or multiple motile cell behaviors, including cell shape changes, directed migration, planar and radial intercalations, and cell divisions. Recent studies delineate cyclical and ratchet-like behaviors of the actomyosin cytoskeleton as a common mechanism underlying various gastrulation cell behaviors. Gastrulation movements are guided by differential cell adhesion, chemotaxis, chemokinesis, and planar polarity. Coordination of gastrulation movements with embryonic polarity involves regulation by anteroposterior and dorsoventral patterning systems of planar polarity signaling, expression of chemokines, and cell adhesion molecules.
Assuntos
Gastrulação , Camadas Germinativas/citologia , Animais , Padronização Corporal , Adesão Celular , Comunicação Celular , Movimento Celular , Polaridade Celular , Forma Celular , Citoesqueleto/metabolismo , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , HumanosRESUMO
Cell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo, we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants showed impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, which affected the morphogenesis of Kupffer's vesicle (KV). Mutant KVs were smaller, contained fewer cells and displayed decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This in vivo animal study of CARMIL3 reveals its new role during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry.
Assuntos
Padronização Corporal , Movimento Celular , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário , Proteínas dos Microfilamentos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteínas dos Microfilamentos/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The spine gives structural support for the adult body, protects the spinal cord, and provides muscle attachment for moving through the environment. The development and maturation of the spine and its physiology involve the integration of multiple musculoskeletal tissues including bone, cartilage, and fibrocartilaginous joints, as well as innervation and control by the nervous system. One of the most common disorders of the spine in human is adolescent idiopathic scoliosis (AIS), which is characterized by the onset of an abnormal lateral curvature of the spine of <10° around adolescence, in otherwise healthy children. The genetic basis of AIS is largely unknown. Systematic genome-wide mutagenesis screens for embryonic phenotypes in zebrafish have been instrumental in the understanding of early patterning of embryonic tissues necessary to build and pattern the embryonic spine. However, the mechanisms required for postembryonic maturation and homeostasis of the spine remain poorly understood. Here we report the results from a small-scale forward genetic screen for adult-viable recessive and dominant zebrafish mutations, leading to overt morphological abnormalities of the adult spine. Germline mutations induced with N-ethyl N-nitrosourea (ENU) were transmitted and screened for dominant phenotypes in 1229 F1 animals, and subsequently bred to homozygosity in F3 families; from these, 314 haploid genomes were screened for adult-viable recessive phenotypes affecting general body shape. We cumulatively found 40 adult-viable (3 dominant and 37 recessive) mutations each leading to a defect in the morphogenesis of the spine. The largest phenotypic group displayed larval onset axial curvatures, leading to whole-body scoliosis without vertebral dysplasia in adult fish. Pairwise complementation testing of 16 mutant lines within this phenotypic group revealed at least 9 independent mutant loci. Using massively-parallel whole genome or whole exome sequencing and meiotic mapping we defined the molecular identity of several loci for larval onset whole-body scoliosis in zebrafish. We identified a new mutation in the skolios/kinesin family member 6 (kif6) gene, causing neurodevelopmental and ependymal cilia defects in mouse and zebrafish. We also report multiple recessive alleles of the scospondin and a disintegrin and metalloproteinase with thrombospondin motifs 9 (adamts9) genes, which all display defects in spine morphogenesis. Our results provide evidence of monogenic traits that are essential for normal spine development in zebrafish, that may help to establish new candidate risk loci for spine disorders in humans.
Assuntos
Mutação em Linhagem Germinativa , Coluna Vertebral/crescimento & desenvolvimento , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Embrião não Mamífero/embriologia , Genoma , Humanos , Neurogênese/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Vertebrate heart development requires spatiotemporal regulation of gene expression to specify cardiomyocytes, increase the cardiomyocyte population through proliferation, and to establish and maintain atrial and ventricular cardiac chamber identities. The evolutionarily conserved chromatin factor Gon4-like (Gon4l), encoded by the zebrafish ugly duckling (udu) locus, has previously been implicated in cell proliferation, cell survival, and specification of mesoderm-derived tissues including blood and somites, but its role in heart formation has not been studied. Here we report two distinct roles of Gon4l/Udu in heart development: regulation of cell proliferation and maintenance of ventricular identity. We show that zygotic loss of udu expression causes a significant reduction in cardiomyocyte number at one day post fertilization that becomes exacerbated during later development. We present evidence that the cardiomyocyte deficiency in udu mutants results from reduced cell proliferation, unlike hematopoietic deficiencies attributed to TP53-dependent apoptosis. We also demonstrate that expression of the G1/S-phase cell cycle regulator, cyclin E2 (ccne2), is reduced in udu mutant hearts, and that the Gon4l protein associates with regulatory regions of the ccne2 gene during early embryogenesis. Furthermore, udu mutant hearts exhibit a decrease in the proportion of ventricular cardiomyocytes compared to atrial cardiomyocytes, concomitant with progressive reduction of nkx2.5 expression. We further demonstrate that udu and nkx2.5 interact to maintain the proportion of ventricular cardiomyocytes during development. However, we find that ectopic expression of nkx2.5 is not sufficient to restore ventricular chamber identity suggesting that Gon4l regulates cardiac chamber patterning via multiple pathways. Together, our findings define a novel role for zygotically-expressed Gon4l in coordinating cardiomyocyte proliferation and chamber identity maintenance during cardiac development.
Assuntos
Fatores de Ligação de DNA Eritroide Específicos/metabolismo , Coração/embriologia , Miócitos Cardíacos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Cromatina/metabolismo , Fatores de Ligação de DNA Eritroide Específicos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Átrios do Coração/embriologia , Átrios do Coração/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Fase S/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Cell proliferation has generally been considered dispensable for anteroposterior extension of embryonic axis during vertebrate gastrulation. Signal transducer and activator of transcription 3 (Stat3), a conserved controller of cell proliferation, survival and regeneration, is associated with human scoliosis, cancer and Hyper IgE Syndrome. Zebrafish Stat3 was proposed to govern convergence and extension gastrulation movements in part by promoting Wnt/Planar Cell Polarity (PCP) signaling, a conserved regulator of mediolaterally polarized cell behaviors. Here, using zebrafish stat3 null mutants and pharmacological tools, we demonstrate that cell proliferation contributes to anteroposterior embryonic axis extension. Zebrafish embryos lacking maternal and zygotic Stat3 expression exhibit normal convergence movements and planar cell polarity signaling, but transient axis elongation defect due to insufficient number of cells resulting largely from reduced cell proliferation and increased apoptosis. Pharmacologic inhibition of cell proliferation during gastrulation phenocopied axis elongation defects. Stat3 regulates cell proliferation and axis extension in part via upregulation of Cdc25a expression during oogenesis. Accordingly, restoring Cdc25a expression in stat3 mutants partially suppressed cell proliferation and gastrulation defects. During later development, stat3 mutant zebrafish exhibit stunted growth, scoliosis, excessive inflammation, and fail to thrive, affording a genetic tool to study Stat3 function in vertebrate development, regeneration, and disease.
Assuntos
Proliferação de Células/genética , Desenvolvimento Embrionário/genética , Fator de Transcrição STAT3/genética , Proteínas de Peixe-Zebra/genética , Fosfatases cdc25/genética , Animais , Polaridade Celular/genética , Gástrula/crescimento & desenvolvimento , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Morfogênese/genética , Proteínas Mutantes/genética , Fator de Transcrição STAT3/biossíntese , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/biossíntese , Fosfatases cdc25/biossínteseRESUMO
During vertebrate gastrulation, convergence and extension movements elongate embryonic tissues anteroposteriorly and narrow them mediolaterally. Planar cell polarity (PCP) signaling is essential for mediolateral cell elongation underlying these movements, but how this polarity arises is poorly understood. We analyzed the elongation, orientation and migration behaviors of lateral mesodermal cells undergoing convergence and extension movements in wild-type zebrafish embryos and mutants for the Wnt/PCP core component Vangl2 (Trilobite). We demonstrate that Vangl2 function is required at the time when cells transition to a highly elongated and mediolaterally aligned body. vangl2 mutant cells fail to undergo this transition and to migrate along a straight path with high net speed towards the dorsal midline. Instead, vangl2 mutant cells exhibit an anterior/animal pole bias in cell body alignment and movement direction, suggesting that PCP signaling promotes effective dorsal migration in part by suppressing anterior/animalward cell polarity and movement. Endogenous Vangl2 protein accumulates at the plasma membrane of mesenchymal converging cells at the time its function is required for mediolaterally polarized cell behavior. Heterochronic cell transplantations demonstrated that Vangl2 cell membrane accumulation is stage dependent and regulated by both intrinsic factors and an extracellular signal, which is distinct from PCP signaling or other gastrulation regulators, including BMP and Nodals. Moreover, mosaic expression of fusion proteins revealed enrichment of Vangl2 at the anterior cell edges of highly mediolaterally elongated cells. These results demonstrate that the dynamic Vangl2 intracellular distribution is coordinated with and necessary for the changes in convergence and extension cell behaviors during gastrulation.
Assuntos
Gástrula/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Padronização Corporal , Linhagem da Célula , Membrana Celular/metabolismo , Movimento Celular , Polaridade Celular/fisiologia , Citoplasma/metabolismo , Embrião não Mamífero/metabolismo , Feminino , Gastrulação , Proteínas de Membrana/metabolismo , Mesoderma/metabolismo , Mutação , Placa Neural/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Formation of the heart tube requires synchronized migration of endocardial and myocardial precursors. Our previous studies indicated that in S1pr2/Gα13-deficient embryos, impaired endoderm convergence disrupted the medial migration of myocardial precursors, resulting in the formation of two myocardial populations. Here we show that endoderm convergence also regulates endocardial migration. In embryos defective for S1pr2/Gα13 signaling, endocardial precursors failed to migrate towards the midline, and the presumptive endocardium surrounded the bilaterally-located myocardial cells rather than being encompassed by them. In vivo imaging of control embryos revealed that, like their myocardial counterparts, endocardial precursors migrated with the converging endoderm, though from a more anterior point, then moved from the dorsal to the ventral side of the endoderm (subduction), and finally migrated posteriorly towards myocardial precursors, ultimately forming the inner layer of the heart tube. In embryos defective for endoderm convergence due to an S1pr2/Gα13 deficiency, both the medial migration and the subduction of endocardial precursors were impaired, and their posterior migration towards the myocardial precursors was premature. This placed them medial to the myocardial populations, physically blocking the medial migration of the myocardial precursors. Furthermore, contact between the endocardial and myocardial precursor populations disrupted the epithelial architecture of the myocardial precursors, and thus their medial migration; in embryos depleted of endocardial cells, the myocardial migration defect was partially rescued. Our data indicate that endoderm convergence regulates the medial migration of endocardial precursors, and that premature association of the endocardial and myocardial populations contributes to myocardial migration defects observed in S1pr2/Gα13-deficient embryos. The demonstration that endoderm convergence regulates the synchronized migration of endocardial and myocardial precursors reveals a new role of the endoderm in heart development.
Assuntos
Padronização Corporal/fisiologia , Endocárdio/embriologia , Endoderma/embriologia , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Padronização Corporal/genética , Movimento Celular , Embrião não Mamífero/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Humanos , Proteínas Luminescentes/análise , Morfolinos/genética , Morfolinos/farmacologia , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: Cell polarity is essential for directed migration of mesenchymal cells and morphogenesis of epithelial tissues. Studies in cultured cells indicate that a condensed Golgi Complex (GC) is essential for directed protein trafficking to establish cell polarity underlying directed cell migration. Dynamic changes of the GC intracellular organization during early vertebrate development remain to be investigated. RESULTS: We used antibody labeling and fusion proteins in vivo to study the organization and intracellular placement of the GC during early zebrafish embryogenesis. We found that the GC was dispersed into several puncta containing cis- and trans-Golgi Complex proteins, presumably ministacks, until the end of the gastrula period. By early segmentation stages, the GC condensed in cells of the notochord, adaxial mesoderm, and neural plate, and its intracellular position became markedly polarized away from borders between these tissues. CONCLUSIONS: We find that GC is dispersed in early zebrafish cells, even when cells are engaged in massive gastrulation movements. The GC accumulates into patches in a stage and cell-type specific manner, and becomes polarized away from borders between the embryonic tissues. With respect to tissue borders, intracellular GC polarity in notochord is independent of mature apical/basal polarity, Wnt/PCP, or signals from adaxial mesoderm. Developmental Dynamics 245:678-691, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Complexo de Golgi/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Polaridade Celular/genética , Polaridade Celular/fisiologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Gastrulação/genética , Gastrulação/fisiologia , Complexo de Golgi/genética , Notocorda/embriologia , Notocorda/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
During vertebrate gastrulation, Wnt/planar cell polarity (PCP) signaling orchestrates polarized cell behaviors underlying convergence and extension (C&E) movements to narrow embryonic tissues mediolaterally and lengthen them anteroposteriorly. Here, we have identified Gpr125, an adhesion G protein-coupled receptor, as a novel modulator of the Wnt/PCP signaling system. Excess Gpr125 impaired C&E movements and the underlying cell and molecular polarities. Reduced Gpr125 function exacerbated the C&E and facial branchiomotor neuron (FBMN) migration defects of embryos with reduced Wnt/PCP signaling. At the molecular level, Gpr125 recruited Dishevelled to the cell membrane, a prerequisite for Wnt/PCP activation. Moreover, Gpr125 and Dvl mutually clustered one another to form discrete membrane subdomains, and the Gpr125 intracellular domain directly interacted with Dvl in pull-down assays. Intriguingly, Dvl and Gpr125 were able to recruit a subset of PCP components into membrane subdomains, suggesting that Gpr125 may modulate the composition of Wnt/PCP membrane complexes. Our study reveals a role for Gpr125 in PCP-mediated processes and provides mechanistic insight into Wnt/PCP signaling.
Assuntos
Movimento Celular , Polaridade Celular , Receptores Acoplados a Proteínas G/metabolismo , Via de Sinalização Wnt , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Desgrenhadas , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Mutação , Fosfoproteínas/metabolismo , Receptores Acoplados a Proteínas G/genética , Asas de Animais/citologia , Asas de Animais/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
αE-catenin is an actin-binding protein associated with the E-cadherin-based adherens junction that regulates cell-cell adhesion. Recent studies identified additional E-cadherin-independent roles of αE-catenin in regulating plasma membrane dynamics and cell migration. However, little is known about the roles of αE-catenin in these different cellular processes in vivo during early vertebrate development. Here, we examined the functions of αE-catenin in cell-cell adhesion, cell migration and plasma membrane dynamics during morphogenetic processes that drive epiboly in early Danio rerio (zebrafish) development. We show that depletion of αE-catenin caused a defect in radial intercalation that was associated with decreased cell-cell adhesion, in a similar manner to E-cadherin depletion. Depletion of αE-catenin also caused deep cells to have protracted plasma membrane blebbing, and a defect in plasma membrane recruitment of ERM proteins that are involved in controlling membrane-to-cortex attachment and membrane blebbing. Significantly, depletion of both E-cadherin and αE-catenin suppressed plasma membrane blebbing. We suggest that during radial intercalation the activities of E-cadherin and αE-catenin in the maintenance of membrane-to-cortex attachment are balanced, resulting in stabilization of cell-cell adhesion and suppression of membrane blebbing, thereby enabling proper radial intercalation.
Assuntos
Membrana Celular/metabolismo , Movimento Celular , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , alfa Catenina/metabolismo , Animais , Caderinas/metabolismo , Adesão Celular , Proteínas de Ligação a DNA/metabolismo , Morfogênese , Transdução de Sinais , Fatores de Transcrição/metabolismo , Peixe-Zebra/metabolismoRESUMO
Six3 exerts multiple functions in the development of anterior neural tissue of vertebrate embryos. Whereas complete loss of Six3 function in the mouse results in failure of forebrain formation, its hypomorphic mutations in human and mouse can promote holoprosencephaly (HPE), a forebrain malformation that results, at least in part, from abnormal telencephalon development. However, the roles of Six3 in telencephalon patterning and differentiation are not well understood. To address the role of Six3 in telencephalon development, we analyzed zebrafish embryos deficient in two out of three Six3-related genes, six3b and six7, representing a partial loss of Six3 function. We found that telencephalon forms in six3b;six7-deficient embryos; however, ventral telencephalic domains are smaller and dorsal domains are larger. Decreased cell proliferation or excess apoptosis cannot account for the ventral deficiency. Instead, six3b and six7 are required during early segmentation for specification of ventral progenitors, similar to the role of Hedgehog (Hh) signaling in telencephalon development. Unlike in mice, we observe that Hh signaling is not disrupted in embryos with reduced Six3 function. Furthermore, six3b overexpression is sufficient to compensate for loss of Hh signaling in isl1- but not nkx2.1b-positive cells, suggesting a novel Hh-independent role for Six3 in telencephalon patterning. We further find that Six3 promotes ventral telencephalic fates through transient regulation of foxg1a expression and repression of the Wnt/ß-catenin pathway.
Assuntos
Embrião não Mamífero/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Telencéfalo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteína Homeobox SIX3RESUMO
Embryonic axis formation in vertebrates is initiated by the establishment of the dorsal Nieuwkoop blastula organizer, marked by the nuclear accumulation of maternal ß-catenin, a transcriptional effector of canonical Wnt signaling. Known regulators of axis specification include the canonical Wnt pathway components that positively or negatively affect ß-catenin. An involvement of G-protein coupled receptors (GPCRs) was hypothesized from experiments implicating G proteins and intracellular calcium in axis formation, but such GPCRs have not been identified. Mobilization of intracellular Ca(2+) stores generates Ca(2+) transients in the superficial blastomeres of zebrafish blastulae when the nuclear accumulation of maternal ß-catenin marks the formation of the Nieuwkoop organizer. Moreover, intracellular Ca(2+) downstream of non-canonical Wnt ligands was proposed to inhibit ß-catenin and axis formation, but mechanisms remain unclear. Here we report a novel function of Ccr7 GPCR and its chemokine ligand Ccl19.1, previously implicated in chemotaxis and other responses of dendritic cells in mammals, as negative regulators of ß-catenin and axis formation in zebrafish. We show that interference with the maternally and ubiquitously expressed zebrafish Ccr7 or Ccl19.1 expands the blastula organizer and the dorsoanterior tissues at the expense of the ventroposterior ones. Conversely, Ccr7 or Ccl19.1 overexpression limits axis formation. Epistatic analyses demonstrate that Ccr7 acts downstream of Ccl19.1 ligand and upstream of ß-catenin transcriptional targets. Moreover, Ccl19/Ccr7 signaling reduces the level and nuclear accumulation of maternal ß-catenin and its axis-inducing activity and can also inhibit the Gsk3ß -insensitive form of ß-catenin. Mutational and pharmacologic experiments reveal that Ccr7 functions during axis formation as a GPCR to inhibit ß-catenin, likely by promoting Ca(2+) transients throughout the blastula. Our study delineates a novel negative, Gsk3ß-independent control mechanism of ß-catenin and implicates Ccr7 as a long-hypothesized GPCR regulating vertebrate axis formation.
Assuntos
Receptores CCR7/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , beta Catenina/antagonistas & inibidores , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Quimiocina CCL19/genética , Quimiocina CCL19/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Receptores CCR7/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
During vertebrate gastrulation, convergence and extension cell movements are coordinated with the anteroposterior and mediolateral embryonic axes. Wnt planar cell polarity (Wnt/PCP) signaling polarizes the motile behaviors of cells with respect to the anteroposterior embryonic axis. Understanding how Wnt/PCP signaling mediates convergence and extension (C&E) movements requires analysis of the mechanisms employed to alter cell morphology and behavior with respect to embryonic polarity. Here, we examine the interactions between the microtubule cytoskeleton and Wnt/PCP signaling during zebrafish gastrulation. First, we assessed the location of the centrosome/microtubule organizing center (MTOC) relative to the cell nucleus and the body axes, as a marker of cell polarity. The intracellular position of MTOCs was polarized, perpendicular to the plane of the germ layers, independently of Wnt/PCP signaling. In addition, this position became biased posteriorly and medially within the plane of the germ layers at the transition from mid- to late gastrulation and from slow to fast C&E movements. This depends on intact Wnt/PCP signaling through Knypek (Glypican4/6) and Dishevelled components. Second, we tested whether microtubules are required for planar cell polarization. Once the planar cell polarity is established, microtubules are not required for accumulation of Prickle at the anterior cell edge. However, microtubules are needed for cell-cell contacts and initiation of its anterior localization. Reciprocal interactions occur between Wnt/PCP signaling and microtubule cytoskeleton during C&E gastrulation movements. Wnt/PCP signaling influences the polarity of the microtubule cytoskeleton and, conversely, microtubules are required for the asymmetric distribution of Wnt/PCP pathway components.
Assuntos
Gastrulação/fisiologia , Centro Organizador dos Microtúbulos/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , Polaridade Celular/genética , Polaridade Celular/fisiologia , Centrossomo/metabolismo , Embrião não Mamífero/metabolismo , Gastrulação/genética , Microscopia Confocal , Microtúbulos/metabolismo , Modelos Biológicos , Proteínas Wnt/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Cell size is a key contributor to tissue morphogenesis 1 . As a notable example, growth plate hypertrophic chondrocytes use cellular biogenesis and disproportionate fluid uptake to expand 10-20 times in size to drive lengthening of endochondral bone 2,3 . Similarly, notochordal cells expand to one of the largest cell types in the developing embryo to drive axial extension 4-6 . In zebrafish, the notochord vacuolated cells undergo vacuole fusion to form a single large, fluid-filled vacuole that fills the cytoplasmic space and contributes to vacuolated cell expansion 7 . When this process goes awry, the notochord lacks sufficient hydrostatic pressure to support vertebral bone deposition resulting in adult spines with misshapen vertebral bones and scoliosis 8 . However, it remains unclear whether endochondral bone and the notochord share common genetic and cellular mechanisms for regulating cell and tissue expansion. Here, we demonstrate that the 5'-inositol phosphatase gene, inppl1a , regulates notochord expansion, spine morphogenesis, and endochondral bone lengthening in zebrafish. Furthermore, we show that inppl1a regulates notochord expansion independent of vacuole fusion, thereby genetically decoupling these processes. We demonstrate that inppl1a -dependent notochord expansion is essential to establish normal mechanical properties of the notochord to facilitate the development of a straight spine. Finally, we find that inppl1a is also important for endochondral bone lengthening in fish, as has been shown in the human INPPL1 -related endochondral bone disorder, Opsismodysplasia 9 . Overall, this work reveals a conserved mechanism of cell size regulation that influences disparate tissues critical for skeletal development and short-stature disorders.
RESUMO
Gastrulation movements form the germ layers and shape them into the vertebrate body. Gastrulation entails a variety of cell behaviors, including directed cell migration and cell delamination, which are also involved in other physiological and pathological processes, such as cancer metastasis. Decreased Prostaglandin E(2) (PGE(2)) synthesis due to interference with the Cyclooxygenase (Cox) and Prostaglandin E synthase (Ptges) enzymes halts gastrulation and limits cancer cell invasiveness, but how PGE(2) regulates cell motility remains unclear. Here we show that PGE(2)-deficient zebrafish embryos, impaired in the epiboly, internalization, convergence and extension gastrulation movements, exhibit markedly increased cell-cell adhesion, which contributes to defective cell movements in the gastrula. Our analyses reveal that PGE(2) promotes cell protrusive activity and limits cell adhesion by modulating E-cadherin transcript and protein, in part through stabilization of the Snai1a (also known as Snail1) transcriptional repressor, an evolutionarily conserved regulator of cell delamination and directed migration. We delineate a pathway whereby PGE(2) potentiates interaction between the receptor-coupled G protein betagamma subunits and Gsk3beta to inhibit proteasomal degradation of Snai1a. However, overexpression of beta-catenin cannot stabilize Snai1a in PGE(2)-deficient gastrulae. Thus, the Gsk3beta-mediated and beta-catenin-independent inhibition of cell adhesion by Prostaglandins provides an additional mechanism for the functional interactions between the PGE(2) and Wnt signaling pathways during development and disease. We propose that ubiquitously expressed PGE(2) synthesizing enzymes, by promoting the stability of Snai1a, enable the precise and rapid regulation of cell adhesion that is required for the dynamic cell behaviors that drive various gastrulation movements.
Assuntos
Gástrula/fisiologia , Prostaglandinas G/fisiologia , Peixe-Zebra/genética , Animais , Adesão Celular/fisiologia , Primers do DNA , Dinoprostona/deficiência , Dinoprostona/metabolismo , Embrião não Mamífero/fisiologia , Gastrulação/fisiologia , Hibridização In Situ , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genéticaRESUMO
The vertebrate heart arises during gastrulation as cardiac precursors converge from the lateral plate mesoderm territories toward the embryonic midline and extend rostrally to form bilateral heart fields. G protein-coupled receptors (GPCRs) mediate functions of the nervous and immune systems; however, their roles in gastrulation remain largely unexplored. Here, we show that the zebrafish homologs of the Agtrl1b receptor and its ligand, Apelin, implicated in physiology and angiogenesis, control heart field formation. Zebrafish gastrulae express agtrl1b in the lateral plate mesoderm, while apelin expression is confined to the midline. Reduced or excess Agtrl1b or Apelin function caused deficiency of cardiac precursors and, subsequently, the heart. In Apelin-deficient gastrulae, the cardiac precursors converged inefficiently to the heart fields and showed ectopic distribution, whereas cardiac precursors overexpressing Apelin exhibited abnormal morphology and rostral migration. Our results implicate GPCR signaling in movements of discrete cell populations that establish organ rudiments during vertebrate gastrulation.
Assuntos
Quimiocinas/metabolismo , Gástrula/metabolismo , Coração/embriologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mioblastos Cardíacos/metabolismo , Organogênese/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Receptores de Apelina , Padronização Corporal/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Quimiocinas/genética , Desenvolvimento Embrionário/fisiologia , Gástrula/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mesoderma/fisiologia , Mioblastos Cardíacos/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Receptores Acoplados a Proteínas G/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Embryonic morphogenesis is driven by a suite of cell behaviours, including coordinated shape changes, cellular rearrangements and individual cell migrations, whose molecular determinants are largely unknown. In the zebrafish, Dani rerio, trilobite mutant embryos have defects in gastrulation movements and posterior migration of hindbrain neurons. Here, we have used positional cloning to demonstrate that trilobite mutations disrupt the transmembrane protein Strabismus (Stbm)/Van Gogh (Vang), previously associated with planar cell polarity (PCP) in Drosophila melanogaster, and PCP and canonical Wnt/beta-catenin signalling in vertebrates. Our genetic and molecular analyses argue that during gastrulation, trilobite interacts with the PCP pathway without affecting canonical Wnt signalling. Furthermore, trilobite may regulate neuronal migration independently of PCP molecules. We show that trilobite mediates polarization of distinct movement behaviours. During gastrulation convergence and extension movements, trilobite regulates mediolateral cell polarity underlying effective intercalation and directed dorsal migration at increasing velocities. In the hindbrain, trilobite controls effective migration of branchiomotor neurons towards posterior rhombomeres. Mosaic analyses show trilobite functions cell-autonomously and non-autonomously in gastrulae and the hindbrain. We propose Trilobite/Stbm mediates cellular interactions that confer directionality on distinct movements during vertebrate embryogenesis.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Movimento Celular , Gástrula/metabolismo , Proteínas de Membrana/genética , Mutação , Neurônios/citologia , Neurônios/metabolismo , Transdução de Sinais , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Galpha(12/13) have been implicated in numerous cellular processes, however, their roles in vertebrate gastrulation are largely unknown. Here, we show that during zebrafish gastrulation, suppression of both Galpha(12) and Galpha(13) signaling by overexpressing dominant negative proteins and application of antisense morpholino-modified oligonucleotide translation interference disrupted convergence and extension without changing embryonic patterning. Analyses of mesodermal cell behaviors revealed that Galpha(12/13) are required for cell elongation and efficient dorsalward migration during convergence independent of noncanonical Wnt signaling. Furthermore, Galpha(12/13) function cell-autonomously to mediate mediolateral cell elongation underlying intercalation during notochord extension, likely acting in parallel to noncanonical Wnt signaling. These findings provide the first evidence that Galpha(12) and Galpha(13) have overlapping and essential roles in distinct cell behaviors that drive vertebrate gastrulation.
Assuntos
Desenvolvimento Embrionário/fisiologia , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Gástrula/metabolismo , Mesoderma/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Padronização Corporal/fisiologia , Comunicação Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Forma Celular/fisiologia , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Gástrula/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mesoderma/citologia , Dados de Sequência Molecular , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Proteínas WntRESUMO
The human spinal column is a dynamic, segmented, bony, and cartilaginous structure that protects the neurologic system and simultaneously provides balance and flexibility. Children with developmental disorders that affect the patterning or shape of the spine can be at risk of neurologic and other physiologic dysfunctions. The most common developmental disorder of the spine is scoliosis, a lateral deformity in the shape of the spinal column. Scoliosis may be part of the clinical spectrum that is observed in many developmental disorders, but typically presents as an isolated symptom in otherwise healthy adolescent children. Adolescent idiopathic scoliosis (AIS) has defied understanding in part due to its genetic complexity. Breakthroughs have come from recent genome-wide association studies (GWAS) and next generation sequencing (NGS) of human AIS cohorts, as well as investigations of animal models. These studies have identified genetic associations with determinants of cartilage biogenesis and development of the intervertebral disc (IVD). Current evidence suggests that a fraction of AIS cases may arise from variation in factors involved in the structural integrity and homeostasis of the cartilaginous extracellular matrix (ECM). Here, we review the development of the spine and spinal cartilages, the composition of the cartilage ECM, the so-called "matrisome" and its functions, and the players involved in the genetic architecture of AIS. We also propose a molecular model by which the cartilage matrisome of the IVD contributes to AIS susceptibility.
RESUMO
Distal arthrogryposis (DA) is group of syndromes characterized by congenital joint contractures. Treatment development is hindered by the lack of vertebrate models. Here, we describe a zebrafish model in which a common MYH3 missense mutation (R672H) was introduced into the orthologous zebrafish gene smyhc1 (slow myosin heavy chain 1) (R673H). We simultaneously created a smyhc1 null allele (smyhc1- ), which allowed us to compare the effects of both mutant alleles on muscle and bone development, and model the closely related disorder, spondylocarpotarsal synostosis syndrome. Heterozygous smyhc1R673H/+ embryos developed notochord kinks that progressed to scoliosis with vertebral fusions; motor deficits accompanied the disorganized and shortened slow-twitch skeletal muscle myofibers. Increased dosage of the mutant allele in both homozygous smyhc1R673H/R673H and transheterozygous smyhc1R673H/- embryos exacerbated the notochord and muscle abnormalities, causing early lethality. Treatment of smyhc1R673H/R673H embryos with the myosin ATPase inhibitor, para-aminoblebbistatin, which decreases actin-myosin affinity, normalized the notochord phenotype. Our zebrafish model of MYH3-associated DA2A provides insight into pathogenic mechanisms and suggests a beneficial therapeutic role for myosin inhibitors in treating disabling contractures.