Jacek Topczewski

Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements.

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/β-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.

The Zebrafish Glypican Knypek Controls Cell Polarity during Gastrulation Movements of Convergent Extension

Mutations in the zebrafish knypek locus impair gastrulation movements of convergent extension that narrow embryonic body and elongate it from head to tail. We demonstrate that knypek regulates cellular movements but not cell fate specification. Convergent extension movement defects in knypek are associated with abnormal cell polarity, as mutant cells fail to elongate and align medio-laterally. Positional cloning reveals that knypek encodes a member of the glypican family of heparan sulfate proteoglycans. Double mutant and overexpression analyses show that Knypek potentiates Wnt11 signaling, mediating convergent extension. These studies provide experimental and genetic evidence that glypican Knypek acts during vertebrate gastrulation as a positive modulator of noncanonical Wnt signaling to establish polarized cell behaviors underlying convergent extension movements.

Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness

Bidirectional cellular communication is integral to both cancer progression and embryological development. In addition, aggressive tumor cells are phenotypically plastic, sharing many properties with embryonic cells. Owing to the similarities between these two types of cells, the developing zebrafish can be used as a biosensor for tumor-derived signals. Using this system, we show that aggressive melanoma cells secrete Nodal (a potent embryonic morphogen) and consequently can induce ectopic formation of the embryonic axis. We further show that Nodal is present in human metastatic tumors, but not in normal skin, and thus may be involved in melanoma pathogenesis. Inhibition of Nodal signaling reduces melanoma cell invasiveness, colony formation and tumorigenicity. Nodal inhibition also promotes the reversion of melanoma cells toward a melanocytic phenotype. These data suggest that Nodal signaling has a key role in melanoma cell plasticity and tumorigenicity, thereby providing a previously unknown molecular target for regulating tumor progression.

Zebrafish Rho Kinase 2 Acts Downstream of Wnt11 to Mediate Cell Polarity and Effective Convergence and Extension Movements

BACKGROUND During vertebrate gastrulation convergence and extension (CE), movements narrow and lengthen embryonic tissues. In Xenopus and zebrafish, a noncanonical Wnt signaling pathway constitutes the vertebrate counterpart to the Drosophila planar cell polarity pathway and regulates mediolateral cell polarization underlying CE. Despite the identification of several signaling molecules required for normal CE, the downstream transducers regulating individual cell behaviors driving CE are only beginning to be elucidated. Moreover, how defective mediolateral cell polarity impacts CE is not understood. RESULTS Here, we show that overexpression of zebrafish dominant-negative Rho kinase 2 (dnRok2) disrupts CE without altering cell fates, phenocopying noncanonical Wnt signaling mutants. Moreover, Rho kinase 2 (Rok2) overexpression partially suppresses the slb/wnt11 gastrulation phenotype, and ectopic expression of noncanonical Wnts modulates Rok2 intracellular distribution. In addition, time-lapse analyses associate defective dorsal convergence movements with impaired cell elongation, mediolateral orientation, and consequently failure to migrate along straight paths. Transplantation experiments reveal that dnRok2 cells in wild-type hosts neither elongate nor orient their axes. In contrast, wild-type cells are able to elongate their cell bodies in dnRok2 hosts, even though they fail to orient their axes. CONCLUSIONS During zebrafish gastrulation, Rok2 acts downstream of noncanonical Wnt11 signaling to mediate mediolateral cell elongation required for dorsal cell movement along straight paths. Furthermore, elongation and orientation of the cell body are independent properties that require both cell-autonomous and nonautonomous Rok2 function.

Role of the zebrafish trilobite locus in gastrulation movements of convergence and extension

Summary: Convergence and extension are gastrulation movements that participate in the establishment of the vertebrate body plan. Using new methods for quantifying convergence and extension movements of cell groups, we demonstrate that in wild‐type embryos, dorsal convergence of lateral cells is initially slow, but speeds up between the end of the gastrula period and early segmentation. Convergence and extension movements of lateral cells in trilobite mutants are normal during the gastrula period but reduced by early segmentation. Morphometric studies revealed that during epiboly wild‐type gastrulae become ovoid, whereas trilobite embryos remain rounder. By segmentation, trilobite embryos exhibit shorter, broader embryonic axes. The timing of these morphological defects correlates well with impaired cell movements, suggesting reduced convergence and extension are the main defects underlying the trilobite phenotype. Our gene expression, genetic, and fate mapping analyses show the trilobite mutation affects movements without altering dorsoventral patterning or cell fates. We propose that trilobite function is required for cell properties that promote increased speed of converging cells and extension movements in the dorsal regions of the zebrafish gastrula. genesis 27:159–173, 2000.

Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP.

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP‐labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual‐channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual‐channel, three‐dimensional (3D) and four‐dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer‐assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos. Developmental Dynamics 232:359–368, 2005.

Essential roles of Gα12/13 signaling in distinct cell behaviors driving zebrafish convergence and extension gastrulation movements

Gα12/13 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 Gα12 and Gα13 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 Gα12/13 are required for cell elongation and efficient dorsalward migration during convergence independent of noncanonical Wnt signaling. Furthermore, Gα12/13 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 Gα12 and Gα13 have overlapping and essential roles in distinct cell behaviors that drive vertebrate gastrulation.

miR-27b controls venous specification and tip cell fate

We discovered that miR-27b controls 2 critical vascular functions: it turns the angiogenic switch on by promoting endothelial tip cell fate and sprouting and it promotes venous differentiation. We have identified its targets, a Notch ligand Delta-like ligand 4 (Dll4) and Sprouty homologue 2 (Spry2). miR-27b knockdown in zebrafish and mouse tissues severely impaired vessel sprouting and filopodia formation. Moreover, miR-27b was necessary for the formation of the first embryonic vein in fish and controlled the expression of arterial and venous markers in human endothelium, including Ephrin B2 (EphB2), EphB4, FMS-related tyrosine kinase 1 (Flt1), and Flt4. In zebrafish, Dll4 inhibition caused increased sprouting and longer intersegmental vessels and exacerbated tip cell migration. Blocking Spry2 caused premature vessel branching. In contrast, Spry2 overexpression eliminated the tip cell branching in the intersegmental vessels. Blockade of Dll4 and Spry2 disrupted arterial specification and augmented the expression of venous markers. Blocking either Spry2 or Dll4 rescued the miR-27b knockdown phenotype in zebrafish and in mouse vascular explants, pointing to essential roles of these targets downstream of miR-27b. Our study identifies critical role of miR-27b in the control of endothelial tip cell fate, branching, and venous specification and determines Spry2 and Dll4 as its essential targets.

Zebrafish integrin-linked kinase is required in skeletal muscles for strengthening the integrin-ECM adhesion complex

Mechanical instability of skeletal muscle cells is the major cause of congenital muscular dystrophy. Here we show that the zebrafish lost-contact mutant, that lacks a functional integrin-linked kinase (ilk) gene, suffers from mechanical instability of skeletal muscle fibres. With genetic and morpholino knock-down experiments we demonstrate that: 1) laminin, itgalpha7, Ilk and beta-parvin are all critical for mechanical stability in skeletal muscles. 2) Ilk acts redundantly with the dystrophin/dystroglycan adhesion complex in maintaining mechanical stability of skeletal muscles. 3) Ilk protein is recruited to the myotendinous junctions, which requires the ECM component laminin and the presence of itgalpha7 in the sarcolemma. 4) Ilk, unexpectedly, is dispensable for formation of the adhesion complex. Ilk, however, is required for strengthening the adhesion of the muscle fibre with the ECM and this activity requires the presence of a functional kinase domain in Ilk. 5) We identified a novel interaction between Ilk and the mechanical stretch sensor protein MLP. Thus, Ilk is an essential intracellular component downstream of laminin and itgalpha7, providing strengthening of skeletal muscle fibre adhesion with the ECM and therefore qualified as a novel candidate gene for congenital muscular dystrophy.

Sec24D-Dependent Transport of Extracellular Matrix Proteins Is Required for Zebrafish Skeletal Morphogenesis

Protein transport from endoplasmic reticulum (ER) to Golgi is primarily conducted by coated vesicular carriers such as COPII. Here, we describe zebrafish bulldog mutations that disrupt the function of the cargo adaptor Sec24D, an integral component of the COPII complex. We show that Sec24D is essential for secretion of cartilage matrix proteins, whereas the preceding development of craniofacial primordia and pre-chondrogenic condensations does not depend on this isoform. Bulldog chondrocytes fail to secrete type II collagen and matrilin to extracellular matrix (ECM), but membrane bound receptor β1-Integrin and Cadherins appear to leave ER in Sec24D-independent fashion. Consequently, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes as visualized by electron microscopy and NBD C6-ceramide staining appear functional. Consistent with the backlog of proteins in the ER, chondrocytes activate the ER stress response machinery and significantly upregulate BiP transcription. Failure of ECM secretion hinders chondroblast intercalations thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. This defect is specific to Sec24D mutants since knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial cartilage dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than in bulldog/sec24d, suggesting that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. Our study presents the first developmental perspective on Sec24D function and establishes Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects.