QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online September 1, 2004
doi: 10.1242/10.1242/dev.01290

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Soustelle, L. Articles by Giangrande, A. Search for Related Content PubMed PubMed Citation Articles by Soustelle, L. Articles by Giangrande, A. Social Bookmarking                         What's this?

Terminal tendon cell differentiation requires the glide/gcm complex

¹ Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 10142, 67404 Illkirch Cedex, C.U. de Strasbourg, France
² Institut für Genetik, Universität Mainz, D-55099 Mainz, Germany
³ Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

View larger version (101K): [in a new window]   Fig. 1. glide and glide2 are expressed in tendon cells. (A,B) glide (A) and glide2 (B) in-situ hybridisation on wild-type Drosophila embryos, lateral views at mid-late stage 12. Unless specified, anterior is to the left in this and following figures. Both genes are expressed in epidermis (black arrows) and in glial cells of the central and peripheral nervous systems (large and small white arrowheads, respectively). Note that glide2 is expressed at lower levels compared with glide. (C-E) Anti-GFP (C) and anti-Stripe (D) immunolabelling on stage 14 glide::GFP embryos. Dorso-lateral views. GFP is expressed in epidermal cells (yellow brackets in C), peripheral glia (arrowheads in C) and hemocytes (asterisks in C), which also express glide and glide2 (Alfonso and Jones, 2002; Bernardoni et al., 1997; Kammerer and Giangrande, 2001). Tendon cells are visualised by anti-Stripe, those that are parallel to segment border cells being shown by empty lozenges (D). At this stage, intrasegmental tendon cells are organised in two clusters containing ten cells each and found at the dorsal and ventral ends of lateral transverse muscles (bracket in D indicates the dorsal cluster). (E) Merge labelling shows that epidermal glide-expressing cells at the segment border also express stripe (yellow nuclei in yellow brackets). Arrows in (D) indicate non-specific, tracheal, labelling. (F-H) Schematic representations of muscles and tendon cells in two abdominal segments of a Drosophila embryo at stage 16/17, lateral views, dorsal to the top (modified from Armand et al., 1994). Dotted and dashed lines represent dorsal and ventral midlines, respectively. Muscles are represented in white or red, tendon cells in green (segment border cells), yellow (parallel to segment border cells) or blue (intrasegmental tendon cells). Red muscles in (F,G) indicate longitudinal and ventral longitudinal muscles, respectively. Red muscles in H show lateral transverse muscles. Both ends of most longitudinal muscles (F,G) attach to segment borders, whereas a small subset displays only one attachment site at the border. Lateral transverse muscles (H) attach to intrasegmental tendon cells. The subpopulation of glide/glide2-expressing cells corresponds to green tendon cells. Black bracket in F indicates the region shown in C-E. Scale bars: 50 µm.

View larger version (88K): [in a new window]   Fig. 2. The glide complex affects muscle attachment. Anti-Myosin labelling reveals muscle organisation in early stage 17 embryos of the following genotypes: (A,C) wild type (WT); (B,D) glide-glide2 embryos (glide-glide2 lof); (E) glide embryos carrying one copy of glide2 (glide lof-glide2 +/lof); (F) glide-glide2 embryos specifically expressing glide in tendon cells (stripe-gal4 driver) (called Rescue); (G) ptc::glideDN (glideDN); and (H) ptc::glideN7-4DN (glideN7-4DN) embryos. (C,D) The regions delimited by dots in A,B, respectively. All panels show ventral views. Dashed line in C-H shows the ventral midline. In wild-type embryos (A,C), ventral longitudinal fibres attach to tendon cells at a distance from the midline. In glide-glide2 embryos (B,D), ventral longitudinal fibres bypass their target sites, attaching to others muscles at the midline (arrows in D). Note that muscle fibres normally attached to tendon cells that do not express glide are also indirectly affected. (E) Embryo carrying one copy of glide2 displays less severe phenotype than the double mutant. (F) The muscle phenotype is fully rescued by specifically expressing glide in tendon cells (n=10). (G) Inhibition of glide complex activity (ptc::glideDN) induces the midline crossing phenotype (see arrow), whereas the inactive form of the dominant negative construct (ptc::glideN7-4DN in H) does not. Scale bars: 50 µm.

View larger version (79K): [in a new window]   Fig. 4. Hemiadherens junctions are defective in embryos lacking the glide complex. (A) Schematic representation of an attachment site at late stage 16. Actin microfilaments and microtubules (MTs) are present in muscle fibres and in tendon cells, respectively, and are connected to hemiadherens junctions (HAJs). Attachment between muscle and tendon cell is formed by the adhesion of HAJs to extracellular matrix components (visualised by an electron-dense material) (modified from Subramanian et al., 2003). (B,C) Electron microscopy analysis of muscle attachment site in wild-type (B) and glide-glide2 late-stage 16 to early-stage-17 embryos (C). M and TC indicate the muscle fibre and the tendon cell, respectively. (C) Note that the electron-dense material is absent in the mutant and that muscle fibre and tendon cell membranes can be easily identified instead of being tightly interdigitated as in the wild type (B). (D-I) Anti-myosin (green) and anti-ßPS integrin (red) co-labelling reveal muscle attachment site organisation in stage-17 embryos of the following genotypes: (D-F) wild-type (WT), (G-I) glide-glide2 embryos. All panels show ventral views, anterior to the top. ßPS integrin accumulates at the ends of wild-type muscles. (G-I) Note the weak accumulation at the position of normal sites (arrowhead in G) and the ectopic accumulation at muscle terminals abutting each other at the ventral midline (arrow in G). Ventral midline is indicated by dashed line in F,I. Scale bars: 1 µm in B,C; 50 µm in D-I.

View larger version (112K): [in a new window]   Fig. 3. Tissue-specific repression of glide complex activity affects muscle organisation and locomotion. (A) Locomotion assay. Statistic evaluation of second instar larvae motility: wild-type (WT), ptc::glideN7-4DN (glideN7-4DN) and ptc::glideDN (glideDN) (n=6). Error bars indicate s.e.m.; statistical significance was calculated using Student's t-test. Significant differences versus wild-type are indicated on top of bar (***P<0.0001). Control (glideN7-4DN) and wild-type (WT) larvae do not show any difference, whereas ptc::glideDN larvae (glideDN) display reduced motility. (B-E) Polarised light micrographs showing muscle organisation in ptc::glideN7-4DN (B,D) and ptc::glideDN (C,E) second instar larvae. Dorsal to the top. Muscle attachment sites are severely altered in ptc::glideDN larvae. (D,E) Regions delimited by dashed boxes in B,C, respectively. (F-K) Muscle F-actin organisation (visualised by phalloidin labelling) on ptc::glideN7-4DN (F-H) and ptc::glideDN (I-K) late-stage 17 embryos. Dorsal to the top. (G,J) show the regions delimited by dashed boxes in F,I, respectively. Lateral (F,G,I,J) or dorsal views (H,K). Dorsal midline is indicated by dashed line in (H,K). Scale bars: 1 mm in B,C; 0.1 mm in D,E; 50 µm in F-K.

View larger version (85K): [in a new window]   Fig. 5. Epistatic relation between the glide complex and stripe. (A-F) In-situ hybridisation on wild-type and mutant embryos. (A,B) stripe a expression in wild-type (A) or en::glide embryos (B, glide gof). (C,D) stripe a/b expression on wild-type (C) or en::glide embryos (D, glide gof). Note that glide induces stripe b (large brackets in D), but not stripe a (small brackets in B) expression. (E,F) glide expression in wild-type (E) or en::stripe b (F, stripe b gof) embryos. Dorsal (A-D) and lateral (E-H) views of embryos at stage 14. (G,H) The rA87 line (glide-lacZ) is used to follow glide expression in wild-type (G) and in stripe (H, stripe lof) embryos. Scale bars: 50 µm.

View larger version (138K): [in a new window]   Fig. 6. Epistatic relationships between the glide complex and stripe reveal a novel molecular tendon cell-specific pathway. (A-F) Anti-Stripe or anti-Alien immunolabelling on wild-type (A,D, respectively) and on either en::glide (B,E, respectively) or en::glide2 (C,F, respectively) stage-15 embryos. Dorsal views, dorsal midline is represented by a dotted line. (B) stripe ectopic expression is indicated by arrowhead. Note that ectopic glide2 does not induce stripe expression (C). (D-F) Alien ectopic expression induced by either glide (E) or glide2 (F) is indicated by arrowheads. (G-I) Ent2 in-situ hybridisation on wild-type (G), en::glide (H, glide gof) and en::stripe b (I, stripe b gof) stage-14 embryos. Lateral views, dorsal to the top. Ent2 is expressed in segment border cells (bracket in G). Note that glide, but not stripe b, ectopic expression induces Ent2 expression (compare brackets in H,G and I,G, respectively). Scale bars: 50 µm.

View larger version (71K): [in a new window]   Fig. 7. glide regulation in the embryonic epidermis. (A,F,K) Schematic representation of epidermal cells in a segment of wild-type (A), patched (F) and wingless (K) stage-13 embryos. Black vertical lines indicate segment borders. The single row of border cells (red cell) is posterior to each segment border (A). Ectopic border cells are present in (F) and are absent in (K). (B,C,G,H,L,M) glide in-situ hybridisation on wild-type (B,C), patched (G,H) and wingless (L,M) embryos. (D,E,I, J,N,O) Stripe expression revealed by immunolabelling on wild-type (D,E), patched (I,J) and wingless (N,O) embryos. (C,E,H,J,M,O) Regions delimited by dots in B,D,G,I,L,N, respectively. Note that in patched embryos, glide and stripe are ectopically expressed (compare G,H with B,C and I,J with D,E, respectively) whereas, in wingless embryos, neither glide nor stripe are expressed in the epidermis (compare L,M with B,C and N,O with D,E, respectively). Red bracket in C and white bracket in E show wild-type expression of glide mRNA and Stripe protein, respectively. Red and white brackets in H,J indicate ectopic expression of glide and stripe, respectively. Red and white asterisks in M,O indicate the loss of glide and stripe expression, respectively. Scale bars: 50 µm.

View larger version (62K): [in a new window]   Fig. 8. Cell-specific role of glide. Repo and Stripe co-immunolabelling on wild-type (A-C), ptc::glide (D-F) and en::glide (G-I) embryos, lateral views at stage 15. (J) Anti-Repo labelling on en::glide2 embryos. (B) Central and peripheral glia are indicated by yellow and white brackets, respectively. In wild-type embryos, Stripe (A) and Repo (B) are expressed in distinct cells (C). In ptc::glide embryos, ectopic glide only induces Repo expression (arrows in E) whereas en::glide embryos show ectopic expression of both Repo (arrows in H) and Stripe (bracket in G). Note that only few cells coexpress the two markers (yellow nuclei shown by arrowheads in I). (J) Ectopic expression of glide2 induces Repo (arrow in J). Dorsal to the top. (K) Summary of glide and glide2 gain-of-function (gof) phenotypes in the epidermis. +++ and + symbols indicate, respectively, high or medium potential to induce tendon cell or glial markers. Arrows in (A) indicate non-specific, tracheal, labelling. Scale bar: 50 µm.

View larger version (19K): [in a new window]   Fig. 9. Model for glide pathway in tendon cell development. While Hh and Wg signaling directly control stripe expression in border cells, they act indirectly (dotted arrow) on glide. stripe b controls the expression of several genes involved in early tendon cell differentiation (left panel) as well as its own expression (Vorbruggen and Jackle, 1997); glide-glide2 have no influence on their expression (left panel). glide, glide2, and stripe b, are able to induce alien expression, indicating that the two pathways have common targets. Whether this regulation is direct or indirect remains to be determined (broken arrows). The glide complex directly activates its own pathway (Ent2). glide participates in stripe b regulation during late tendon cell differentiation. Finally, unknown glide, glide2, stripe b, alien and Ent2 targets are also indicated by a question mark.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter