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1.  Quantitative differences in tissue surface tension influence zebrafish germ layer positioning 
HFSP Journal  2008;2(1):42-56.
This study provides direct functional evidence that differential adhesion, measurable as quantitative differences in tissue surface tension, influences spatial positioning between zebrafish germ layer tissues. We show that embryonic ectodermal and mesendodermal tissues generated by mRNA-overexpression behave on long-time scales like immiscible fluids. When mixed in hanging drop culture, their cells segregate into discrete phases with ectoderm adopting an internal position relative to the mesendoderm. The position adopted directly correlates with differences in tissue surface tension. We also show that germ layer tissues from untreated embryos, when extirpated and placed in culture, adopt a configuration similar to those of their mRNA-overexpressing counterparts. Down-regulating E-cadherin expression in the ectoderm leads to reduced surface tension and results in phase reversal with E-cadherin-depleted ectoderm cells now adopting an external position relative to the mesendoderm. These results show that in vitro cell sorting of zebrafish mesendoderm and ectoderm tissues is specified by tissue interfacial tensions. We perform a mathematical analysis indicating that tissue interfacial tension between actively motile cells contributes to the spatial organization and dynamics of these zebrafish germ layers in vivo.
PMCID: PMC2640996  PMID: 19404452
2.  Wnt11 controls cell contact persistence by local accumulation of Frizzled 7 at the plasma membrane 
The Journal of Cell Biology  2006;175(5):791-802.
Wnt11 is a key signal, determining cell polarization and migration during vertebrate gastrulation. It is known that Wnt11 functionally interacts with several signaling components, the homologues of which control planar cell polarity in Drosophila melanogaster. Although in D. melanogaster these components are thought to polarize cells by asymmetrically localizing at the plasma membrane, it is not yet clear whether their subcellular localization plays a similarly important role in vertebrates. We show that in zebrafish embryonic cells, Wnt11 locally functions at the plasma membrane by accumulating its receptor, Frizzled 7, on adjacent sites of cell contacts. Wnt11-induced Frizzled 7 accumulations recruit the intracellular Wnt signaling mediator Dishevelled, as well as Wnt11 itself, and locally increase cell contact persistence. This increase in cell contact persistence is mediated by the local interaction of Wnt11, Frizzled 7, and the atypical cadherin Flamingo at the plasma membrane, and it does not require the activity of further downstream effectors of Wnt11 signaling, such as RhoA and Rok2. We propose that Wnt11, by interacting with Frizzled 7 and Flamingo, modulates local cell contact persistence to coordinate cell movements during gastrulation.
PMCID: PMC2064678  PMID: 17130287
3.  Three Functions of Cadherins in Cell Adhesion 
Current Biology  2013;23(14):R626-R633.
Cadherins are transmembrane proteins that mediate cell–cell adhesion in animals. By regulating contact formation and stability, cadherins play a crucial role in tissue morphogenesis and homeostasis. Here, we review the three major functions of cadherins in cell–cell contact formation and stability. Two of those functions lead to a decrease in interfacial tension at the forming cell–cell contact, thereby promoting contact expansion — first, by providing adhesion tension that lowers interfacial tension at the cell–cell contact, and second, by signaling to the actomyosin cytoskeleton in order to reduce cortex tension and thus interfacial tension at the contact. The third function of cadherins in cell–cell contact formation is to stabilize the contact by resisting mechanical forces that pull on the contact.
PMCID: PMC3722483  PMID: 23885883
4.  Completion of the epithelial to mesenchymal transition in zebrafish mesoderm requires Spadetail 
Developmental biology  2011;354(1):102-110.
The process of gastrulation is highly conserved across vertebrates on both the genetic and morphological levels, despite great variety in embryonic shape and speed of development. This mechanism spatially separates the germ layers and establishes the organizational foundation for future development. Mesodermal identity is specified in a superficial layer of cells, the epiblast, where cells maintain an epithelioid morphology. These cells involute to join the deeper hypoblast layer where they adopt a migratory, mesenchymal morphology. Expression of a cascade of related transcription factors orchestrates the parallel genetic transition from primitive to mature mesoderm. Although the early and late stages of this process are increasingly well understood, the transition between them has remained largely mysterious. We present here the first high resolution in vivo observations of the blebby transitional morphology of involuting mesodermal cells in a vertebrate embryo. We further demonstrate that the zebrafish spadetail mutation creates a reversible block in the maturation program, stalling cells in the transition state. This mutation creates an ideal system for dissecting the specific properties of cells undergoing the morphological transition of maturing mesoderm, as we demonstrate with a direct measurement of cell-cell adhesion.
PMCID: PMC3090540  PMID: 21463614
Mesoderm; Gastrulation; Involution; Adhesion; Bleb; spadetail; Cadherin; Myosin
5.  The role of adhesion energy in controlling cell–cell contacts 
Current Opinion in Cell Biology  2011;23(5):508-514.
► In this review, we describe the role of adhesion energy in cell–cell adhesion. ► We review recent advances in elucidating the molecular and cellular basis of adhesion energy. ► We propose both experimental and theoretical strategies to determine adhesion energy between cells in vitro and in vivo.
Recent advances in microscopy techniques and biophysical measurements have provided novel insight into the molecular, cellular and biophysical basis of cell adhesion. However, comparably little is known about a core element of cell–cell adhesion—the energy of adhesion at the cell–cell contact. In this review, we discuss approaches to understand the nature and regulation of adhesion energy, and propose strategies to determine adhesion energy between cells in vitro and in vivo.
PMCID: PMC3188705  PMID: 21807491
6.  Control of Directed Cell Migration In Vivo by Membrane-to-Cortex Attachment 
PLoS Biology  2010;8(11):e1000544.
Analysis of cell migration in vivo combined with biophysical measurements reveals how membrane-to-cortex attachment fine-tunes the type of protrusions formed by cells and, as a consequence, controls directed migration during zebrafish gastrulation.
Cell shape and motility are primarily controlled by cellular mechanics. The attachment of the plasma membrane to the underlying actomyosin cortex has been proposed to be important for cellular processes involving membrane deformation. However, little is known about the actual function of membrane-to-cortex attachment (MCA) in cell protrusion formation and migration, in particular in the context of the developing embryo. Here, we use a multidisciplinary approach to study MCA in zebrafish mesoderm and endoderm (mesendoderm) germ layer progenitor cells, which migrate using a combination of different protrusion types, namely, lamellipodia, filopodia, and blebs, during zebrafish gastrulation. By interfering with the activity of molecules linking the cortex to the membrane and measuring resulting changes in MCA by atomic force microscopy, we show that reducing MCA in mesendoderm progenitors increases the proportion of cellular blebs and reduces the directionality of cell migration. We propose that MCA is a key parameter controlling the relative proportions of different cell protrusion types in mesendoderm progenitors, and thus is key in controlling directed migration during gastrulation.
Author Summary
Cell migration, like any event involving shape changes, is a mechanical process controlled by complex biochemical pathways. Here, we examine cell migration in developing embryos with a combination of cell biological tools and atomic force microscopy, so as to investigate how cellular mechanical properties control migration. A fundamental step during migration is the formation of a protrusion at the leading edge of the cell. In three-dimensional environments, and particularly in vivo, cells use different protrusion types: spike-like filopodia and flattened lamellipodia, whose growth is driven by actin polymerization, and spherical blebs, which grow because of intracellular pressure pushing on the membrane. It is important to understand how the formation of different protrusion types is mechanically and molecularly controlled, and how the different protrusions specifically contribute to migration. We have addressed this using the zebrafish embryo as a model system. We show that reducing the strength of the attachment between the plasma membrane and the underlying cortical network of actin filaments, or increasing intracellular pressure, increases the proportion of cellular blebs and reduces the directionality of cell migration. Our work reveals that blebs, lamellipodia, and filopodia are not interchangeable and that the relative proportion of each type of protrusion, under the control of mechanical parameters, determines migration directionality during zebrafish gastrulation.
PMCID: PMC2994655  PMID: 21151339
7.  Monorail/Foxa2 regulates floorplate differentiation and specification of oligodendrocytes, serotonergic raphé neurones and cranial motoneurones 
Development (Cambridge, England)  2005;132(4):645-658.
In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos, we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves.
Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphé nucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins.
PMCID: PMC2790417  PMID: 15677724
Midline development; Hedgehog signalling; Zebrafish
8.  Back and Forth between Cell Fate Specification and Movement during Vertebrate Gastrulation 
Summary of recent advances
Animal body plan arises during gastrulation and organogenesis by the coordination of inductive events and cell movements. Several signaling pathways, such as BMP, FGF, Hedgehog, Nodal and Wnt, have well recognized instructive roles in cell fate specification during vertebrate embryogenesis. Growing evidence indicates that BMP, Nodal and FGF signaling also regulate cell movements, and that they do so through mechanisms distinct from those that specify cell fates. Moreover, pathways controlling cell movements can also indirectly influence cell fate specification by regulating dimensions and relative positions of interacting tissues. The current challenge is to delineate the molecular mechanisms via which the major signaling pathways regulate cell fate specification and movements, and how these two processes are coordinated to ensure normal development.
PMCID: PMC2706661  PMID: 18721878
9.  A mutation in the Gsk3–binding domain of zebrafish Masterblind/Axin1 leads to a fate transformation of telencephalon and eyes to diencephalon 
Genes & development  2001;15(11):1427-1434.
Zebrafish embryos homozygous for the masterblind (mbl) mutation exhibit a striking phenotype in which the eyes and telencephalon are reduced or absent and diencephalic fates expand to the front of the brain. Here we show that mbl−/− embryos carry an amino-acid change at a conserved site in the Wnt pathway scaffolding protein, Axin1. The amino-acid substitution present in the mbl allele abolishes the binding of Axin to Gsk3 and affects Tcf-dependent transcription. Therefore, Gsk3 activity may be decreased in mbl−/− embryos and in support of this possibility, overexpression of either wild-type Axin1 or Gsk3β can restore eye and telencephalic fates to mbl−/− embryos. Our data reveal a crucial role for Axin1-dependent inhibition of the Wnt pathway in the early regional subdivision of the anterior neural plate into telencephalic, diencephalic, and eye-forming territories.
PMCID: PMC312705  PMID: 11390362
Axin; Wnt; forebrain; zebrafish
10.  Slb/Wnt11 controls hypoblast cell migration and morphogenesis at the onset of zebrafish gastrulation 
Development (Cambridge, England)  2003;130(22):5375-5384.
During vertebrate gastrulation, highly coordinated cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. In zebrafish, silberblick (slb)/wnt11 regulates normal gastrulation movements by activating a signalling pathway similar to the Frizzled-signalling pathway, which establishes epithelial planar cell polarity (PCP) in Drosophila. However, the cellular mechanisms by which slb/wnt11 functions during zebrafish gastrulation are still unclear. Using high-resolution two-photon confocal imaging followed by computer-assisted reconstruction and motion analysis, we have analysed the movement and morphology of individual cells in three dimensions during the course of gastrulation. We show that in slb-mutant embryos, hypoblast cells within the forming germ ring have slower, less directed migratory movements at the onset of gastrulation. These aberrant cell movements are accompanied by defects in the orientation of cellular processes along the individual movement directions of these cells. We conclude that slb/wnt11-mediated orientation of cellular processes plays a role in facilitating and stabilising movements of hypoblast cells in the germ ring, thereby pointing at a novel function of the slb/wnt11 signalling pathway for the regulation of migratory cell movements at early stages of gastrulation.
PMCID: PMC1414802  PMID: 13129848
Wnt signalling; Cell migration; Gastrulation; Zebrafish
11.  Proteomics of early zebrafish embryos 
Zebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed.
As a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS), including a comparison of databases for MS identification of zebrafish proteins.
The provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis.
PMCID: PMC1363346  PMID: 16412219

Results 1-11 (11)