DOCSLIB.ORG

Interference with Brachyury Function Inhibits Convergent Extension

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Interference with Brachyury Function Inhibits Convergent Extension Developmental Biology 213, 85–100 (1999) Article ID dbio.1999.9330, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE Interference with Brachyury Function Inhibits provided by Elsevier - Publisher Connector Convergent Extension, Causes Apoptosis, and Reveals Separate Requirements in the FGF and Activin Signalling Pathways Frank L. Conlon and J. C. Smith Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom Brachyury plays a key role in mesoderm formation during vertebrate development. Absence of the gene results in loss of posterior mesoderm and failure of the notochord to differentiate, while misexpression of Brachyury in the prospective ectoderm of Xenopus results in ectopic mesoderm formation. Brachyury is therefore both necessary and sufficient for posterior mesoderm formation. Here we present a detailed cellular and molecular analysis of the consequences of inhibiting Brachyury function during Xenopus development. Our results show that Brachyury is required for the convergent extension movements of gastrulation, for mesoderm differentiation in response to FGF, and for the survival of posterior mesodermal cells in both Xenopus and mouse. © 1999 Academic Press INTRODUCTION tion is essential for its biological function (Conlon et al., 1996). In order to understand how the gene exerts its effects Brachyury, or T, is required for the formation of posterior during early development, it is necessary to identify targets mesoderm and notochord in mouse (Herrmann et al., 1990), of Brachyury (Casey et al., 1998; Tada et al., 1998) as well as zebrafish (Halpern et al., 1993; Schulte-Merker et al., 1994), to understand how its expression is controlled (Clements et and Xenopus (Conlon et al., 1996) embryos. In all these al., 1996; Latinkic et al., 1997). In addition it is important to species, and in the chick (Kispert et al., 1995b), the gene is define the role of Brachyury at the molecular and cell expressed transiently throughout nascent mesoderm and biological levels, and these are the issues addressed in this transcripts persist in tailbud and notochord; Brachyury is paper. therefore essential for formation of the mesodermal tissues Our experiments concentrate on Xenopus laevis, because in which it is expressed. The importance of Brachyury in more is known about mesoderm formation in this species vertebrate mesoderm formation is emphasised by experi- than in any other vertebrate (Slack, 1994). The function of ments in which the gene is misexpressed in prospective Xenopus Brachyury (Xbra) can be inhibited by expressing a ectodermal cells of Xenopus; this causes the formation of construct in which the activation domain of Xbra is re- ectopic mesoderm, with low concentrations of Brachyury placed by the repressor domain from Drosophila engrailed inducing ventral cell types and high concentrations induc- (EnR), thus creating the fusion protein Xbra-EnR (Conlon et ing dorsal tissues (Cunliffe and Smith, 1992). Expression of al., 1996). Embryos injected with RNA encoding Xbra-EnR Brachyury alone is not sufficient to induce notochord, but do not gastrulate normally and fail to form tail structures coexpression of Brachyury with noggin (Smith and Harland, and (in many cases) a notochord (Conlon et al., 1996). They 1992) or with Pintallavis (Ruiz i Altaba and Jessell, 1992) therefore resemble mouse and zebrafish embryos which does cause notochord to form (Cunliffe and Smith, 1994; carry mutations in the Brachyury gene (Chesley, 1935; O’Reilly et al., 1995). Halpern et al., 1993; Herrmann et al., 1990; Schulte-Merker Brachyury encodes a sequence-specific DNA-binding pro- et al., 1994). The inhibitory effects of Xbra-EnR are specific tein which functions as a transcription activator (Conlon et to Xbra, because the phenotypes obtained when EnR is al., 1996; Kispert and Herrmann, 1993; Kispert et al., fused to the DNA-binding domains of the T-box proteins 1995a), and the ability of Brachyury to activate transcrip- eomesodermin and Brat differ from those obtained with 0012-1606/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 85 86 Conlon and Smith TABLE 1 Molecular Markers Used in This Study Stage(s) of Gene examination Expression pattern Reference Xbra 10, 13, 17 Pan mesodermal and axial mesoderm Smith et al., 1991 Mix.1 10, 13, 17 Pan mesodermal and endoderm Rosa, 1989 Goosecoid 10, 13, 17 Organiser Cho et al., 1991 Noggin 10, 13, 17 Organiser and axial mesoderm Smith and Harland, 1992 Chordin 10, 13, 17 Organiser, axial mesoderm, dorsal endoderm Sasai et al., 1994, 1996 Pintallavis 10, 13, 17 Dorsal and axial mesoderm Ruiz i Altaba and Jessell, 1992 Xnot 10, 13, 17 Dorsal and axial mesoderm von Dassow et al., 1993 Xwnt-8 10, 13, 17 Ventral and lateral mesoderm Christian et al., 1991; Smith and Harland, 1991 Endodermin 10, 13, 17 Notochord, prechordal plate, and endoderm Sasai et al., 1996 Otx-2 20 Anterior mesendoderm and neurectoderm Kablar et al., 1996; Pannese et al., 1995 HoxB9 20 Posterior mesoderm and neurectoderm Sharpe et al., 1987 Keratin 40 Epidermis Snape et al., 1990 Cardiac actin 40 Skeletal and cardiac muscle Mohun et al., 1984 aT4 globin 40 Blood Walmsley et al., 1994 N-CAM 40 Neural tissue Kintner and Melton, 1987 EF-1a All Ubiquitous Sargent and Bennett, 1990 Xbra-EnR (Horb and Thomsen, 1997; Ryan et al., 1996). tiation in response to FGF but not in response to activin. Furthermore, the effects of Brat-EnR can be rescued by However, the ability of activin to induce convergent exten- coexpression of Brat, but not by Xbra (Horb and Thomsen, sion movements in prospective ectodermal tissue (Symes 1997), and vice-versa (F.L.C. and J.C.S., unpublished). and Smith, 1987) is inhibited by Xbra-EnR, suggesting that In this paper we first present a detailed analysis of the Brachyury is required for normal gastrulation movements phenotype of Xenopus embryos in which Xbra function is to occur (see also Wilson et al., 1995). inhibited. Our results indicate that Xbra-EnR causes a decrease in expression of molecular markers associated with ventral and posterior tissue types and that after MATERIALS AND METHODS gastrulation cells expressing these genes are lost through programmed cell death. This suggests that Brachyury has a Xenopus Embryos, Microinjection, and Dissection trophic activity for posterior mesodermal cells, a conclu- Xenopus embryos were obtained by in vitro fertilisation (Smith sion strengthened by the observation that mouse embryos and Slack, 1983). They were maintained in 10% Normal Amphib- mutant for Brachyury also undergo apoptosis in posterior ian Medium (NAM; Slack, 1984) and staged according to Nieuw- structures. koop and Faber (1975). Xenopus embryos at the 1- to 2-cell stage or We have inferred additional functions for Brachyury in at the 32-cell stage were injected with RNA in 10 or 1 nl water, cell differentiation and cell movement from studies in respectively, as described (Smith, 1993). For animal cap assays, animal caps. The mesoderm of Xenopus is formed through embryos were dissected in 75% NAM, and caps were cultured in the same medium. Xenopus FGF-2 was prepared using an expres- an inductive interaction in which cells of the vegetal sion plasmid provided by David Kimelman and Marc Kirschner. hemisphere act on overlying equatorial cells (Sudarwati and Partially purified human activin A was prepared from the condi- Nieuwkoop, 1971). Two families of signalling molecules tioned medium of COS cells transfected with a human inhibin bA are thought to be involved in this interaction: members of cDNA. The cells were a gift from Dr. Gordon Wong (Genetics the fibroblast growth factor (FGF) family, such as FGF-2 Institute, Inc.). (Amaya et al., 1991; Isaacs et al., 1992; Kimelman and Kirschner, 1987; Slack et al., 1987), and members of the RNA Isolation and RNase Protection Assays transforming growth factor type b family, such as activin and Vg1 (Dale et al., 1993; Dyson and Gurdon, 1996; Smith RNase protection analysis was carried out as described (Jones et et al., 1990a; Thomsen et al., 1990; Thomsen and Melton, al., 1995). Probes used are listed in Table 1. The endodermin probe was constructed by excising an XhoI/SspI fragment corresponding 1993). Both FGF and activin induce expression of Xbra in to nucleotides 1928–2207 from a 5-kb endodermin cDNA kindly prospective ectodermal tissue in an immediate-early fash- provided by Dr. E. M. De Robertis. The fragment was cloned into ion (Smith et al., 1991), and in the second part of this paper pBSKS1. To make an RNase protection probe, the plasmid was R we use Xbra-En to investigate the role of Xbra in the digested with XhoI and transcribed with T7 RNA polymerase. response to these two factors. Our results indicate that RNase protection data shown in Figs. 2 and 7 are representative of Xbra function is essential for terminal mesoderm differen- at least two independent experiments. Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved. Inhibition of Brachyury during Xenopus Development 87 RNA Synthesis RNA encoding Xbra-EnR was prepared as described (Conlon et al., 1996). RNA synthesised from the following constructs was used as controls in various experiments; all gave results identical to those obtained with uninjected embryos (not shown). XbraDBD encodes the DNA-binding domain of Xbra (Cunliffe and Smith, 1992). XbraAD comprises amino acids 301 to 432 of Xbra preceded by an initiator methionine; it comprises the transcription activa- tion domain of the protein (Conlon et al., 1996). EnR encodes the R repressor domain of the Drosophila engrailed protein preceded by FIG. 1. Injection of RNA encoding Xbra-En inhibits posterior an initiator methionine (Conlon et al., 1996). Xbra-EnR(Mut) is mesoderm development.
Load more

Recommended publications
  • NIH Public Access Author Manuscript Dev Dyn
    NIH Public Access Author Manuscript Dev Dyn. Author manuscript; available in PMC 2006 November 13. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Dev Dyn. 2004 January ; 229(1): 201±218. T-box Genes in Early Embryogenesis Chris Showell, Olav Binder, and Frank L. Conlon* Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Abstract The T-box gene family, encoding related DNA-binding transcriptional regulators, plays an essential role in controlling many aspects of embryogenesis in a wide variety of organisms. The T-box genes exhibit diverse patterns of spatial and temporal expression in the developing embryo, and both genetic and molecular embryological studies have demonstrated their importance in regulating cell fate decisions that establish the early body plan, and in later processes underlying organogenesis. Despite these studies, little is known of either the regulation of the T-box genes or the identities of their transcriptional targets. The aim of this review is to examine the diverse yet conserved roles of several T-box genes in regulating early patterning in chordates and to discuss possible mechanisms through which this functional diversity might arise. Keywords T-box; T-domain; Brachyury; Eomesodermin; VegT; spadetail; no tail; tbx6; mesoderm; transcription INTRODUCTION Adult multicellular organisms typically contain a variety of different specialized cell types, their cooperative activity underpinning the function of the organism as a whole. The cellular diversity in the adult arises during embryogenesis, and one of the aims of modern developmental biology is to gain an understanding of the molecular mechanisms through which this process occurs.
    [Show full text]
  • The Xenopus Brachyury Promoter Is Activated by FGF and Low Concentrations of Activin and Suppressed by High Concentrations of Ac
    Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press The Xenopus Brachyury promoter is activated by FGF and low concentrations of activin and suppressed by high concentrations of activin and by paired-type homeodomain proteins Brancko V. Latinkic´, Muriel Umbhauer,1 Kathy A. Neal, Walter Lerchner, James C. Smith,3 and Vincent Cunliffe2 Division of Developmental Biology, National Institute for Medical Research (NIMR), The Ridgeway, London NW7 1AA, UK The mesoderm of Xenopus laevis arises through an inductive interaction in which signals from the vegetal hemisphere of the embryo act on overlying equatorial cells. One candidate for an endogenous mesoderm-inducing factor is activin, a member of the TGFb superfamily. Activin is of particular interest because it induces different mesodermal cell types in a concentration-dependent manner, suggesting that it acts as a morphogen. These concentration-dependent effects are exemplified by the response of Xbra, expression of which is induced in ectodermal tissue by low concentrations of activin but not by high concentrations. Xbra therefore offers an excellent paradigm for studying the way in which a morphogen gradient is interpreted in vertebrate embryos. In this paper we examine the trancriptional regulation of Xbra2, a pseudoallele of Xbra that shows an identical response to activin. Our results indicate that 381 bp 5* of the Xbra2 transcription start site are sufficient to confer responsiveness both to FGF and, in a concentration-dependent manner, to activin. We present evidence that the suppression of Xbra expression at high concentrations of activin is mediated by paired-type homeobox genes such as goosecoid, Mix.1, and Xotx2.
    [Show full text]
  • Early Evolution of the T-Box Transcription Factor Family
    Early evolution of the T-box transcription factor family Arnau Sebé-Pedrósa,b,1, Ana Ariza-Cosanoc,1, Matthew T. Weirauchd, Sven Leiningere, Ally Yangf, Guifré Torruellaa, Marcin Adamskie, Maja Adamskae, Timothy R. Hughesf, José Luis Gómez-Skarmetac,2, and Iñaki Ruiz-Trilloa,b,g,2 aInstitut de Biologia Evolutiva (Consejo Superior de Investigaciones Científicas-Universitat Pompeu Fabra), 08003 Barcelona, Spain; bDepartament de Genètica, Universitat de Barcelona, 08028 Barcelona, Spain; cCentro Andaluz de Biología del Desarrollo, Consejo Superior de Investigaciones Científicas, Universidad Pablo de Olavide-Junta de Andalucía, 41013 Sevilla, Spain; dCenter for Autoimmune Genomics and Etiology and Divisions of Rheumatology and Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; eSars International Centre for Marine Molecular Biology, 5008 Bergen, Norway; fTerrence Donnelly Centre and Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 3E1; and gInstitució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain Edited by W. Ford Doolittle, Dalhousie University, Halifax, NS, Canada, and approved August 13, 2013 (received for review May 24, 2013) Developmental transcription factors are key players in animal and metazoan Brachyury genes and whether T-box genes are multicellularity, being members of the T-box family that are present in other unicellular lineages remained unclear. among the most important. Until recently, T-box transcription Here, we report a taxon-wide survey of T-box genes in several factors were thought to be exclusively present in metazoans. eukaryotic genomes and transcriptomes, including previously Here, we report the presence of T-box genes in several nonmeta- undescribed genomic data from several close relatives of meta- zoan lineages, including ichthyosporeans, filastereans, and fungi.
    [Show full text]
  • Vertebrate Ctr1 Coordinates Morphogenesis and Progenitor Cell Fate and Regulates Embryonic Stem Cell Differentiation
    Vertebrate Ctr1 coordinates morphogenesis and progenitor cell fate and regulates embryonic stem cell differentiation Tomomi Haremaki†, Stuart T. Fraser‡§, Yien-Ming Kuo¶, Margaret H. Baron‡§ʈ, and Daniel C. Weinstein†§†† Departments of †Pharmacology and Systems Therapeutics, ‡Medicine, and ʈMolecular, Cell, and Developmental Biology, Oncological Sciences, and Gene and Cell Medicine, and §The Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, NY 10029; and ¶Departments of Medicine and Pediatrics and Howard Hughes Medical Institute, University of California, San Francisco, CA 94143 Edited by Igor B. Dawid, National Institutes of Health, Bethesda, MD, and approved June 7, 2007 (received for review February 14, 2007) Embryogenesis involves two distinct processes. On the one hand, cells pus copper transporter-1 (Xctr1) was identified as a factor that must specialize, acquiring fates appropriate to their positions (differ- physically associates with the tyrosine kinase Laloo, required for entiation); on the other hand, they must physically construct the FGF-mediated mesoderm induction [supporting information (SI) embryo through coordinated mechanical activity (morphogenesis). In Fig. 5] (9, 15). Microinjection of Xctr1 cRNA at early cleavage early vertebrate development, fibroblast growth factor (FGF) regu- stages gave rise to embryos with shortened, kinked axes and lates multiple embryonic events, including germ layer differentiation reduced or absent anterior structures, including eyes and cement and morphogenesis; the cellular components that direct FGF signaling glands (93%; n ϭ 46) (Fig. 1A). Xctr1-injected embryos underwent to evoke these different responses remain largely unknown. We at least some mesodermal differentiation, as demonstrated by show here that the copper transporter 1 (Ctr1) protein is a critical staining with an antibody (12/101) against a somite-specific epitope router of FGF signals during early embryogenesis.
    [Show full text]
  • Mesoderm Formation in Response to Brachyury Requires FGF Signalling S
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Mesoderm formation in response to Brachyury requires FGF signalling S. Schulte-Merker and J.C. Smith Laboratory of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Background: The Brachyury (T) gene is required for the inhibits the mesoderm-inducing activity of Xbra. We formation of posterior mesoderm and for axial develop- demonstrate that ectopic expression of Xbra activates tran- ment in both mouse and zebrafish embryos. In these scription of the embryonic FGF gene, and that embryonic species, and in Xenopus, the gene is expressed transiently FGF can induce expression of Xbra. This suggests that the throughout the presumptive mesoderm, and transcripts two genes are components of a regulatory loop. Consistent then persist in notochord and posterior tissues. In Xenopus with this idea, dissociation of Xbra-expressing cells causes a embryos, expression of the Xenopus homologue of dramatic and rapid reduction in levels of Xbra, but the Brachyury, Xbra, can be induced in presumptive ectoderm reduction can be inhibited by addition of FGF. by basic fibroblast growth factor (FGF) and activin; in the Conclusion: Formation of mesoderm tissue requires an absence of functional FGF or activin signalling pathways, intact FGF signalling pathway downstream of Brachyury. expression of the gene is severely reduced. Ectopic This requirement is due to a regulatory loop, in which expression of Xbra in presumptive ectoderm causes meso- Brachyury activates expression of a member of the FGF derm to be formed.
    [Show full text]
  • ZEB2, the Mowat-Wilson Syndrome Transcription Factor: Confirmations, Novel Functions, and Continuing Surprises
    G C A T T A C G G C A T genes Review ZEB2, the Mowat-Wilson Syndrome Transcription Factor: Confirmations, Novel Functions, and Continuing Surprises Judith C. Birkhoff 1, Danny Huylebroeck 1,2 and Andrea Conidi 1,* 1 Department of Cell Biology, Erasmus University Medical Center, 3015 CN Rotterdam, The Netherlands; [email protected] (J.C.B.); [email protected] (D.H.) 2 Department of Development and Regeneration, Unit Stem Cell and Developmental Biology, Biomedical Sciences Group, KU Leuven, 3000 Leuven, Belgium * Correspondence: [email protected]; Tel.: +31-10-7043169; Fax: +31-10-7044743 Abstract: After its publication in 1999 as a DNA-binding and SMAD-binding transcription factor (TF) that co-determines cell fate in amphibian embryos, ZEB2 was from 2003 studied by embryologists mainly by documenting the consequences of conditional, cell-type specific Zeb2 knockout (cKO) in mice. In between, it was further identified as causal gene causing Mowat-Wilson Syndrome (MOWS) and novel regulator of epithelial–mesenchymal transition (EMT). ZEB2’s functions and action mechanisms in mouse embryos were first addressed in its main sites of expression, with focus on those that helped to explain neurodevelopmental and neural crest defects seen in MOWS patients. By doing so, ZEB2 was identified in the forebrain as the first TF that determined timing of neuro- /gliogenesis, and thereby also the extent of different layers of the cortex, in a cell non-autonomous fashion, i.e., by its cell-intrinsic control within neurons of neuron-to-progenitor paracrine signaling. Transcriptomics-based phenotyping of Zeb2 mutant mouse cells have identified large sets of intact- ZEB2 dependent genes, and the cKO approaches also moved to post-natal brain development Citation: Birkhoff, J.C.; Huylebroeck, and diverse other systems in adult mice, including hematopoiesis and various cell types of the D.; Conidi, A.
    [Show full text]
  • Switching on the Notochord
    Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press PERSPECTIVE Switching on the notochord Vincent T. Cunliffe and Philip W. Ingham1 Developmental Genetics Programme, University of Sheffield, Sheffield S10 2TN, UK The notochord is a defining characteristic of the chor- ductive interactions are nevertheless critical for the es- date embryo. It is a dorsally located rod of tensile meso- tablishment of some cell lineages (Nishida 1997). In dermal tissue that lies immediately beneath the neural these organisms, notochordal fate is induced by vegetal tube. In vertebrates the notochord functions as a skeletal blastomeres in just 10 cells of the 110-cell embryo (Na- element during early embryogenesis and as a source of katani and Nishida 1994). These 10 cells then divide signals that pattern the neural tube and paraxial meso- twice to produce the entire set of 40 cells that comprises derm. Despite the recent identification of mutations af- the larval notochord. Initial expression of the Ciona in- fecting notochord development in zebrafish, the rather testinalis homolog of the vertebrate Brachyury gene, Ci- modest progress in isolating genes expressed in the no- Bra, coincides precisely with the restriction of early blas- tochord during its differentiation has limited our under- tomeres to a notochordal fate (Yasuoh and Satoh 1993). standing of the molecular mechanisms underpinning its Expression of Brachyury is then maintained exclusively structure and function. In a recent study, Takahashi et in the notochord, in contrast to the situation in verte- al. (1999), using embryos of the primitive chordate, the brate embryos, where Brachyury is expressed in both no- ascidian, have redressed the balance, identifying a large tochord and ventral–posterior mesoderm (Fig.
    [Show full text]
  • Molecule, the Xenopus Brachyury Gene, Into a Neuralizing Factor
    Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Conversion of a mesodermalizing molecule, the Xenopus Brachyury gene, into a neuralizing factor Yi Rao Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 USA It has been shown previously that a Xenopus homolog of the mouse gene Brachymy, Xbra, can initiate mesodermal differentiation. Here, I report that a Xbra mutant truncated at the carboxyl terminus, B304, has lost the mesodermalizing activity and can block the activity of the wild-type Xbra. Injection of B304 mRNA led to formation of neural structures in animal cap explants. Examination of molecular markers in B304-injected explants shows expression of anterior neural markers in the absence of mesodermal markers, indicating that B304 can cause neuralization without the mediation of mesoderm. Implications of these findings on intracellular mechanisms underlying the initiation of neural differentiation in the ectodermal cells are discussed. [Key Words: Neural induction; Brachyury; Xenopus embryos] Received January 5, 1994; revised version accepted March 4, 1994. Classic embryological studies have shown that neural obtained results that suggest intracellular molecular tissue is induced in the animal region of an amphibian mechanisms underlying the initiation of neural develop- embryo by the dorsal blastopore lip and its mesodermal ment in the animal region. derivatives (Spemann, 1938; Hamburger, 1988). The mo- The mouse gene Brachy~ry is required for the forma- lecular mechanisms for this cellular communication tion of posterior structures and the notochord (Bennett process are just beginning to be revealed. 1975; Herrmann et al. 1990). It is highly conserved in On the signal-sending side of this process, two mole- vertebrates; a Xenopus homolog, Xbra, and a zebrafish cules, noggin and follistatin, have recently been shown homolog, Zf-T, are very similar in primary sequence and to have the properties of neural inducers (Lamb et al.
    [Show full text]
  • Xwnt11 Regulates Gastrulation Movements 2229
    Development 127, 2227-2238 (2000) 2227 Printed in Great Britain © The Company of Biologists Limited 2000 DEV1490 Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway Masazumi Tada and J. C. Smith* Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK *Author for correspondence (e-mail: [email protected]) Accepted 1 March; published on WWW 18 April 2000 SUMMARY Gastrulation in the amphibian embryo is driven by cells of immediate-early fashion by Xbra and its expression in vivo the mesoderm. One of the genes that confers mesodermal is abolished by a dominant-interfering form of Xbra, identity in Xenopus is Brachyury (Xbra), which is required Xbra-EnR. Overexpression of a dominant-negative form for normal gastrulation movements and ultimately for of Xwnt11, like overexpression of Xbra-EnR, inhibits posterior mesoderm and notochord differentiation in the convergent extension movements. This inhibition can be development of all vertebrates. Xbra is a transcription rescued by Dsh, a component of the Wnt signalling activator, and interference with transcription activation pathway and also by a truncated form of Dsh which cannot leads to an inhibition of morphogenetic movements during signal through the canonical Wnt pathway involving GSK- gastrulation. To understand this process, we have screened 3 and β-catenin. Together, our results suggest that the for downstream target genes of Brachyury (Tada, M., regulation of morphogenetic movements by Xwnt11 occurs Casey, E., Fairclough, L. and Smith, J. C. (1998) through a pathway similar to that involved in planar Development 125, 3997-4006).
    [Show full text]
  • Inhibition of Xbra Transcription Activation Causes Defects in Mesodermal Patterning and Reveals Autoregulation of Xbra in Dorsal Mesoderm
    Development 122, 2427-2435 (1996) 2427 Printed in Great Britain © The Company of Biologists Limited 1996 DEV1070 Inhibition of Xbra transcription activation causes defects in mesodermal patterning and reveals autoregulation of Xbra in dorsal mesoderm F. L. Conlon1, S. G. Sedgwick2, K. M. Weston3 and J. C. Smith1 1Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK 2Division of Yeast Genetics, NIMR, The Ridgeway, Mill Hill, London NW7 1AA, UK 3CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK SUMMARY The Brachyury (T) gene is required for formation of which show striking similarities to genetic mutants in posterior mesoderm and for axial development in both mouse and fish. These results indicate that the function of mouse and zebrafish embryos. In this paper, we first show Brachyury during vertebrate gastrulation is to activate that the Xenopus homologue of Brachyury, Xbra, and the transcription of mesoderm-specific genes. Additional zebrafish homologue, no tail (ntl), both function as tran- experiments show that Xbra transcription activation is scription activators. The activation domains of both required for regulation of Xbra itself in dorsal, but not proteins map to their carboxy terminal regions, and we ventral, mesoderm. The approach described in this paper, note that the activation domain is absent in two zebrafish in which the DNA-binding domain of a transcription Brachyury mutations, suggesting that it is required for gene activator is fused to the engrailed repressor domain, should function. A dominant-interfering Xbra construct was assist in the analysis of other Xenopus and zebrafish tran- generated by replacing the activation domain of Xbra with scription factors.
    [Show full text]
  • Patterns and Control of Cell Motility in the Xenopus Gastrula
    Development 125, 1931-1942 (1998) 1931 Printed in Great Britain © The Company of Biologists Limited 1998 DEV6326 Patterns and control of cell motility in the Xenopus gastrula Stephan Wacker1, Anja Brodbeck1, Patrick Lemaire2, Christof Niehrs3 and Rudolf Winklbauer1,* 1Universität zu Köln, Zoologisches Institut, Weyertal 119, 50931 Köln, Germany 2Laboratoire de Génétique et Physiologie de Développement, CNRS-Université de la Méditerranée, Institut de Biologie du Développement de Marseille, Case 907, Campus de Luminy, F-13288 Marseille Cedex 09, France 3Division of Molecular Embryology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany *Author for correspondence (e-mail: [email protected]) Accepted 3 March; published on WWW 22 April 1998 SUMMARY By comparing cells with respect to several motility-related another immediate early gene, is without effect when properties and the ability to migrate on fibronectin, three expressed alone in animal cap cells, but it acts cell types can be distinguished in the Xenopus gastrula. synergistically with Mix.1 in the control of adhesion, and These occur in a distinct spatial pattern, thus defining three antagonistically in the polarization of protrusive activity. motility domains which do not correspond to the bFGF also induces migration, lamellipodia formation and prospective germ layers. Migratory behavior is confined to polarization in animal cap cells, but has no effect on a region encompassing the anterior mesoderm and adhesion. By the various treatments of animal cap cells, endoderm. When stationary animal cap cells are induced new combinations of motile properties can be generated, to migrate by treatment with activin, cells become adhesive yielding cell types which are not found in the embryo.
    [Show full text]
  • The Pitx2 Homeobox Protein Is Required Early for Endoderm
    Developmental Biology 229, 287–306 (2001) doi:10.1006/dbio.2000.9950, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE The Pitx2 Homeobox Protein Is Required Early forprovided by Elsevier - Publisher Connector Endoderm Formation and Nodal Signaling Marion Faucourt,* Evelyn Houliston,* Lydia Besnardeau,* David Kimelman,† and Thierry Lepage*,1 *Observatoire Oceanologique, UMR 7009 CNRS, Universite´de Paris VI, 06230 Villefranche- sur-Mer, France; and †Department of Biochemistry, University of Washington, Seattle, Washington 98195-7350 Nodal and Nodal-related factors play fundamental roles in a number of developmental processes, including mesoderm and endoderm formation, patterning of the anterior neural plate, and determination of bilateral asymmetry in vertebrates. pitx2, a paired-like homeobox gene, has been proposed to act downstream of Nodal in the gene cascade providing left–right cues to the developing organs. Here, we report that pitx2 is required early in the Nodal signaling pathway for specification of the endodermal and mesodermal germ layers. We found that pitx2 is expressed very early during Xenopus and zebrafish development and in many regions where Nodal signaling is required, including the presumptive mesoderm and endoderm at the blastula and gastrula stages and the prechordal mesoderm at later stages. In Xenopus embryos, overexpression of pitx2 caused ectopic expression of goosecoid and sox-17␤ and interfered with mesoderm formation. Overexpression of pitx2 in Xenopus animal cap explants partially mimics the effects of Nodal overexpression, suggesting that pitx2 is a mediator of Nodal signaling during specification of the endoderm and prechordal plate, but not during mesoderm induction.
    [Show full text]