Modulation of Cell Adhesion Via Cadherin Endocytosis
Total Page：16
File Type：pdf, Size：1020Kb
Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press TGF- signaling-mediated morphogenesis: modulation of cell adhesion via cadherin endocytosis Souichi Ogata,1 Junji Morokuma,2,4 Tadayoshi Hayata,1,5 Gabriel Kolle,3 Christof Niehrs,3 Naoto Ueno,2,7 and Ken W.Y. Cho1,6 1Department of Developmental and Cell Biology, Developmental Biology Center, University of California at Irvine, Irvine, California 92697, USA; 2Department of Developmental Biology, National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan; 3Division of Molecular Embryology, German Cancer Research Center (DKFZ), Heidelberg 69120, Germany The molecular mechanisms governing the cell behaviors underlying morphogenesis remain a major focus of research in both developmental biology and cancer biology. TGF- ligands control cell fate specification via Smad-mediated signaling. However, their ability to guide cellular morphogenesis in a variety of biological contexts is poorly understood. We report on the discovery of a novel TGF- signaling-mediated cellular morphogenesis occurring during vertebrate gastrulation. Activin/nodal members of the TGF- superfamily induce the expression of two genes regulating cell adhesion during gastrulation: Fibronectin Leucine-rich Repeat Transmembrane 3 (FLRT3), a type I transmembrane protein containing extracellular leucine-rich repeats, and the small GTPase Rnd1. FLRT3 and Rnd1 interact physically and modulate cell adhesion during embryogenesis by controlling cell surface levels of cadherin through a dynamin-dependent endocytosis pathway. Our model suggests that cell adhesion can be dynamically regulated by sequestering cadherin through internalization, and subsequent redeploying internalized cadherin to the cell surface as needed. As numerous studies have linked aberrant expression of small GTPases, adhesion molecules such as cadherins, and TGF- signaling to oncogenesis and metastasis, it is tempting to speculate that this FLRT3/Rnd1/cadherin pathway might also control cell behavior and morphogenesis in adult tissue homeostasis. [Keywords: GTPase; FLRT3; Rnd1; gastrulation; activin; Xenopus] Supplemental material is available at http://www.genesdev.org. Received February 13, 2007; revised version accepted June 1, 2007. During gastrulation, the three germ layers (ectoderm, another during mediolateral intercalation (Davidson et mesoderm, and endoderm) establish new contacts, per- al. 2002; Shook et al. 2004). mitting new inductive interactions to specify the devel- Distinct adhesive properties conferred by adhesion opment of organ primordia. Some of the major move- molecules constitute a key feature of cells that undergo ments that constitute gastrulation are convergence and gastrulation movements. Type I cadherins are required extension, involution, and epiboly. The dynamic nature for proper morphogenesis in sea urchin (Miller and Mc- of cell–cell contacts in tissues undergoing these move- Clay 1997), zebrafish (Montero et al. 2005; Shimizu et al. ments has been visualized using time-lapse video mi- 2005), and mouse embryos (Riethmacher et al. 1995). In- croscopy. Cells involved in convergence and extension activation of C-cadherin, the primary mediator of adhe- movements are shown to continuously break and re- sion in the Xenopus blastula, leads to both involution make local adhesive contacts, via polarized membranous and convergent extension defects during gastrulation processes (e.g., lamellipodia), as they “slide” past one (Heasman et al. 1994). By changing the functional activ- ity of C-cadherin at the cell surface, the morphogenetic elongation of “animal cap” ectodermal explants (mim- Present addresses: 4Forsyth Center for Regenerative and Developmental icking the convergence and extension movements of the Biology (FCRDB), The Forsyth Institute and Department of Developmen- tal Biology, The Harvard School of Dental Medicine, Harvard Medical embryonic mesoderm) is altered (Brieher and Gumbiner School, 140 The Fenway, Boston, MA 02115, USA; 5Department of 1994). Molecular Pharmacology, Medical Research Institute, Tokyo Medical The Wnt signaling network has been implicated in the and Dental University, 2-3-10 Kanda-surugadai Chiyoda-ku, Tokyo 101- 0062, Japan. regulation of cell polarization and mediolateral interca- Corresponding authors. lation in both Xenopus and zebrafish convergence and 6E-MAIL [email protected]; FAX (949) 824-9395. 7E-MAIL [email protected]; FAX 0564-57-7571. extension (Heisenberg et al. 2000; Sokol 2000; Walling- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1541807. ford et al. 2000). Manipulation of components of a Wnt GENES & DEVELOPMENT 21:1817–1831 © 2007 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/07; www.genesdev.org 1817 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Ogata et al. signaling pathway (e.g., Fz7, Silberblick/Wnt11, Dishev- blastula stage were treated with cycloheximide to block elled, Prickle, and Strabismus) has effects on cell polarity ongoing protein synthesis (Cho et al. 1991). These ani- and disrupts convergence and extension of the meso- mal caps were then stimulated with activin to induce derm while not perturbing cell fate. Activin/nodal sig- transcription of direct targets (without activating further naling regulates gastrulation and subsequent morpho- downstream gene expression). Total RNAs extracted genesis and cell movements. Mice deficient in the nodal from activin-treated and untreated control caps were gene arrest at early gastrulation and form no mesoderm subjected to DNA microarray analysis using our 42,000 (Zhou et al. 1993). Nodal inhibition perturbs normal gas- homemade cDNA chips (Shin et al. 2005). We found that trulation in zebrafish and Xenopus (Feldman et al. 1998), FLRT3, encoding a potential transmembrane signaling or while stimulation of activin/nodal signaling in Xeno- adhesion molecule, was reproducibly induced to high pus animal cap ectoderm leads to cell movements that levels along with several dozen other direct activin tar- mimic convergence and extension (Asashima et al. 1990; get genes (our unpublished observations). Smith et al. 1990). Previous work has shown that these Whole-mount in situ analysis showed high FLRT3 ex- ectodermal cells, when challenged with activin, down- pression in the forming dorsal blastopore lip of early gas- regulate C-cadherin “activity” (Lee and Gumbiner 1995) trulae (Fig. 1A). As gastrulation proceeds, FLRT3 expres- and affect convergence and extension movements. At sion gradually surrounds the entire equatorial zone, co- present, how activin/nodal signaling affects cadherin ac- inciding with the progression of formation of the lateral tivity remains a mystery. and ventral blastopore (Fig. 1B), and the expression is In order to better understand the mechanism underly- confined to the deep mesoderm (Fig. 1E). Expression ing TGF--regulated morphogenesis, we conducted a around the blastopore circumference persists until the DNA microarray screen to look for potential activin/ blastopore is completely closed at late neurula stage nodal target genes with key roles in gastrulation. Two (Bottcher et al. 2004; data not shown). Overall, the ex- molecules caught our attention: Rnd1, a small GTPase pression of FLRT3 appears to coincide with the move- previously identified in an expression screen as a mol- ment and rearrangement of marginal zone cells during ecule that displays strong anti-adhesive activity (Nobes gastrulation. As we identified FLRT3 in a screen for di- et al. 1998; Wunnenberg-Stapleton et al. 1999), and rect targets of activin/nodal signaling, we sought to FLRT3, a member of the Fibronectin Leucine-rich Re- verify that FLRT3 is indeed regulated by this pathway. peat Transmembrane family of proteins (Lacy et al. Overexpression of Cerberus-S, a truncated form of the 1999). FLRT3 was also recently shown to modulate cell Cerberus protein that specifically inhibits nodal signal- adhesion (Karaulanov et al. 2006) and fibroblast growth ing (Piccolo et al. 1999), inhibited FLRT3 expression in factor (FGF) signaling by physically interacting with the the equatorial zone (Fig. 1G). Furthermore, we confirmed FGF receptor via its fibronectin (FN) type III domain FLRT3’s status as an immediate early activin target gene (Bottcher et al. 2004). In the process of uncovering by RT–PCR from animal caps (Fig. 1H). Xenopus ho- FLRT3’s function, we made the following observations: mologs of the other mammalian FLRT family members First, FLRT3 and Rnd1 are coexpressed in the involuting (Lacy et al. 1999), namely FLRT1 and FLRT2, were also equatorial cells of gastrula-stage embryos, and these isolated by database homology searches (Supplementary molecules physically and functionally interact. Second, Fig. 1). Neither of these genes are direct activin targets overexpression of FLRT3 or Rnd1 reduces cadherin-me- (Supplementary Fig. 2; Bottcher et al. 2004). FLRT1 is diated cell adhesion, whereas loss of FLRT3 or Rnd1 in- maternally expressed and the expression persists during creases cadherin-mediated cell adhesion. Third, embryos early development. FLRT2 is expressed strongly at neu- depleted of Rnd1 display significant defects during gas- rula stages. trulation and Rnd1 is required for FLRT3-mediated cell adhesion effect. Lastly, the changes in cell adhesion via FLRT3 overexpression causes cell deadhesion FLRT3/Rnd1 are regulated by controlling the availability independent of FGF