apical and lateral cell protrusions interconnect ... apical and lateral cell protrusions...

Download Apical and lateral cell protrusions interconnect ... Apical and Lateral Cell Protrusions Interconnect

Post on 02-Nov-2019

0 views

Category:

Documents

0 download

Embed Size (px)

TRANSCRIPT

  • RESEARCH ARTICLE

    Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and Christian Dahmann*

    Communication among cells by means of the exchange of signaling cues is important for tissue and organ development. Recent reports indicate that one way that signaling cues can be delivered is by movement along cellular protrusions interconnecting cells. Here, by using confocal laser scanning microscopy and three-dimensional rendering, we describe in Drosophila melanogaster wing imaginal discs lateral protrusions interconnecting cells of the columnar epithelium. Moreover, we identified protrusions of the apical surface of columnar cells that reached and apparently contacted cells of the overlying squamous epithelium. Both apical and lateral protrusions could be visualized by expression of Tkv-GFP, a green fluorescent protein (GFP) -tagged version of a receptor of the Dpp/BMP4 signaling molecule, and the endosome marker GFP-Rab5. Our results demonstrate a previously unexpected richness of cellular protrusions within wing imaginal discs and support the view that cellular protrusions may provide a means for exchanging signaling cues between cells. Developmental Dynamics 236:3408–3418, 2007. © 2007 Wiley-Liss, Inc.

    Key words: Drosophila; cell protrusion; microvilli; Prominin-like; Tkv; Rab5

    Accepted 10 August 2007

    INTRODUCTION Cell protrusions are outward exten- sions of the cell plasma membrane made by many different cell types and organisms. Important cellular pro- cesses involve cell protrusions, includ- ing nutrient resorption (Louvard et al., 1992), mechanosensing (Frolenkov et al., 2004), photosensing (Corbeil et al., 2001; Pellikka et al., 2002), estab- lishment of cell adhesion (Vasioukhin et al., 2000), cell migration (Fulga and Rorth, 2002), fusion of epithelial sheets (Jacinto et al., 2000; Martin- Blanco et al., 2000), wound healing

    (Wood et al., 2002), axon guidance (Ritzenthaler et al., 2000), and cell-to- cell communication (reviewed in Rorth, 2003). Intercellular communi- cation is important for the growth and patterning of tissues and organs and cell protrusions may mediate this communication by one of several mechanisms. Signaling mediated by cell protrusions can occur by means of transport and local release of signal- ling molecules, shedding of vesicles (Marzesco et al., 2005), display of re- ceptors (Tomlinson et al., 1987; Hsi- ung et al., 2005) and membrane-teth-

    ered ligands on the protrusion (De Joussineau et al., 2003), or by estab- lishing direct organelle exchange be- tween connected cells (Rustom et al., 2004; reviewed in Demontis, 2004).

    The wing imaginal discs of Drosoph- ila melanogaster, which will give rise to the wings and parts of the body wall of adult flies, provide an attractive system for studying cell protrusions within a developing tissue. In wing imaginal discs, a monolayer of colum- nar and squamous epithelial cells is arranged in a sac-like structure with the apical membranes facing an inter-

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany The Supplementary Material referred to in this article can be found at http://www.interscience.wiley.com/jpages/1058-8388/suppmat Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: DA586/4-1. Dr. Demontis’ present address is Harvard Medical School, Department of Genetics, 77 Avenue Louis Pasteur, Boston, MA 02115. *Correspondence to: Christian Dahmann, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany. E-mail: dahmann@mpi-cbg.de

    DOI 10.1002/dvdy.21324 Published online 13 September 2007 in Wiley InterScience (www.interscience.wiley.com).

    DEVELOPMENTAL DYNAMICS 236:3408–3418, 2007

    © 2007 Wiley-Liss, Inc.

  • nal lumen (Fig. 1A; reviewed in Co- hen, 1993). Signaling among colum- nar cells as well as between columnar

    and squamous cells is important for growth and patterning of wing imagi- nal discs (reviewed in Ramirez-Weber

    and Kornberg, 2000; Gibson and Schubiger, 2001). Several protrusions have been identified in imaginal discs. The apical side of imaginal disc cells is decorated with microvilli (Poodry and Schneiderman, 1970; Ursprung, 1972) and filopodia have been described at their basal side (Eaton et al., 1995). In addition, three more kinds of protru- sions have been observed, all of which have been implicated in exchanging signaling molecules important for the growth and patterning of wing imagi- nal disc cells. Cells at the periphery of the columnar cell sheet have long, pla- nar filopodia-like protrusions, termed cytonemes, which connect to cells within the center of the columnar cell sheet (Ramirez-Weber and Kornberg, 1999). Columnar cells also display apical cell protrusions that are a few cell diameters long and that are in- volved in the process of lateral inhibi- tion (De Joussineau et al., 2003). Furthermore, a subpopulation of squamous cells forms microtubule- containing protrusions that extend through the lumen toward columnar cells (Cho et al., 2000; Gibson and Schubiger, 2000).

    A comprehensive analysis of the types of cell protrusions present in the wing imaginal disc has not been re- ported. It is therefore conceivable that we are currently underestimating the number and sort of cell protrusions present in this tissue. The identifica- tion and characterization of additional cell protrusions could be hampered by fixation of the tissue, which is known to result, at least in some cases, in the profound alteration or destruction of cell protrusions (Ramirez-Weber and Kornberg, 1999).

    Here, we have identified cellular protrusions of wing imaginal discs by expressing a membrane-bound form of green fluorescent protein (GFP) in co- lumnar cells. Live, unfixed imaginal discs were then analyzed by laser scanning microscopy and three-di- mensional (3D) rendering. We have identified and characterized two kinds of previously unreported protrusions. Protrusions of the apical plasma membrane that extended through the imaginal disc lumen and apparently contacted the squamous cells, and pro- trusions of the lateral plasma mem- brane that extended in between neighboring columnar cells. Both

    Fig. 1. Apical cell protrusions connect the columnar epithelium to the squamous epithelium in wing imaginal discs. A: Scheme of the Drosophila wing imaginal disc and location of apical cell protrusions. When viewed onto the plane of the epithelium (XY), cells of the squamous epithelium (s.e.) have a larger circumference than those of the columnar epithelium (c.e.). In cross-section (XZ), cells of the squamous and columnar epithelia are apposed with their apical membranes facing a lumen. Apical cell protrusions (Pr.) arise from the columnar epithelium and are directed toward the squamous epithelium. B,B!: XZ view of a three-dimensional (3D) rendered, living wing imaginal disc expressing CD8–green fluorescent protein (GFP; green) in the dorsal compartment (ap-GAL4, UAS-CD8-GFP) and stained with the lipophilic dye FM4-64 (red). In B!, only the CD8-GFP channel is shown. Apical cell protrusions arise from the columnar epithelium (bottom) and extend toward the squamous epithelium (top). C,D: Apical views onto the columnar epithelium of the 3D-rendered tissue show that these apical protrusions are abundant and present over the entire tissue with no apparent regional preference. E–H!: The 3D rendering of a living wing imaginal disc expressing CD8-GFP (green) in cell clones (act5c"GAL4, UAS-CD8-GFP) and stained with the lipophilic dye FM4-64 (red). Different views of the tissue are provided. E,E!: An XY view of the columnar epithelium from the basal side shows cell clones belonging to the columnar epithelium that express CD8-GFP. F,F!: An XY view of the squamous epithelium and the underlying columnar epithelium shows apical protrusions extending from the columnar cells. G–H!: Tilting of the 3D-rendered tissue shows that the apical protrusions of columnar cells extend to the level of the squamous epithelium, detected by the presence of a squamous cell expressing CD8-GFP (asterisk). I,I!: A higher magnification XY view of apical protrusions at the level of the squamous epithelium is shown. Apical protrusions display enlarged terminal tips (arrowhead), local bulges (arrows), and are sometimes branched (asterisk). In addition, some apical protrusions were bent in their terminal tract when contacting the squamous epithelium. Scale bar # 10 $m in B–D,E–H!, 5 $m in I,I!.

    PROTRUSIONS INTERCONNECT EPITHELIAL CELLS 3409

  • kinds of protrusions could be identi- fied with GFP-actin, a GFP-tagged version of the Dpp receptor Thick- veins (Tkv), and the endosomal marker GFP-Rab5. Implications for signaling among wing imaginal disc cells are discussed.

    RESULTS Apical Protrusions of the Columnar Epithelium Contact the Apposing Squamous Epithelium in Live Drosophila Wing Imaginal Discs To identify and analyze cellular pro- trusions of columnar wing imaginal disc cells, we used the ap-GAL4 driver to express CD8-GFP, a transmem- brane protein routinely used to mark plasma membranes (Lee and Luo, 1999), in columnar cells of the dorsal compartment of wing imaginal discs. ap-GAL4 is not active in the squa- mous wing imaginal disc cells, limit- ing CD8-GFP expression to the colum- nar cells. Third instar larvae were dissected, transferred to culture me- dium containing the lipophilic dye FM4-64 to stain plasma membranes of all wing imaginal discs cells, and im- aged by confocal microscopy. Stacks of XY confocal sections were collected and rendered in three dimensions (see the Experimental Procedures section). We have identified protrusions ex- tending from the apical plasma mem- brane and protrusions e