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

This Article Summary Full Text Full Text (PDF) 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 Chartier, A. Articles by Gratecos, D. Search for Related Content PubMed PubMed Citation Articles by Chartier, A. Articles by Gratecos, D.

Pericardin, a Drosophila type IV collagen-like protein is involved in the morphogenesis and maintenance of the heart epithelium during dorsal ectoderm closure

Aymeric Chartier^*, Stéphane Zaffran, Martine Astier, Michel Sémériva and Danielle Gratecos

Laboratoire de Génétique et Physiologie du Développement, UMR 6545 CNRS-Université, IBDM-CNRS-INSERM-Université de la Méditerranée, Campus de Luminy, Case 907, 13288 Marseille Cedex 09, France
^* Present address: Institut de Génétique humaine, 142 rue de la Cardonille, 34396 Montpellier Cedex 05, France
Present address: Génétique moléculaire du développement, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France

View larger version (29K): [in a new window]   Fig. 1. Coordinated movement of the dorsal ectoderm and of the pericardial and heart cells during dorsal closure. In the schemes, the dorsal ectoderm (ect) is in white and the dorsalmost leading edge (LE) cells in blue. The ectoderm overlays the somatic mesoderm (sm, in green), the pericardial cells (pc in red) and the cardial cells (cc, in purple). as, amnioserosa. The embryos labeled with anti-Mef2 show the cardial cells (white arrows) and the amnioserosa (as, black arrows). (A) In a stage 13 embryo, the LE cells are one or two cells ahead with respect to the pericardial and cardial cells in close proximity to the ectoderm. The hypothesis of a link by the extracellular matrix (red dots and red arrows around the pc) between the two cells layers implies the existence of specific receptors localized in the dorsal ectoderm as well as on the heart cells. (B) The migration of the ectoderm in the dorsal direction (black arrow) carries along the somatic mesoderm and the heart cells. The extracellular matrix is represented as in A. (C) Dorsal ectoderm has closed. The heart tube is formed and encloses the lumen. The LE cells are now aligned with the heart cells. The extracellular matrix covers the basal face of the cardioblasts.

View larger version (96K): [in a new window]   Fig. 2. Alteration of the fate of the puc-expressing LE cells hampers the migration of the two rows of cardioblasts to form the heart tube. The embryos have been double labeled to detect the nuclear ß-gal expression (blue) of a P(lacZ) insertion in the puc gene and, with the EC11 monoclonal antibody, to detect the expression of Prc (brown), a component of the extracellular matrix outlining the periphery of the heart tube. (A) A dorsal view of an embryo (end of stage 13) during dorsal closure. puc is expressed in the dorsal ectoderm (Ring and Martinez Arias, 1993; Martin-Blanco et al., 1998) and Prc is localized at the basal surface of the cardial cells (Zaffran et al., 1995) (see Fig. 3). Migration of the two rows of cardial cells (black arrow) towards the dorsal midline is coordinated with that of LE cells (white arrow) during dorsal closure. Notice (arrowhead) the space separating the two types of cells. (B) In a lateral view of the same embryo, the pericardial cells (black arrow) coincide with ectodermal cells located in a position that appears one or two rows more lateral than LE cells (white arrow) (Ruggendorff et al., 1994), indicating a shift in the migration of the pericardial cells with respect to the LE cells. The white arrowhead indicates the distance between the two layers. (C) At the end of dorsal closure (dorsal view), LE (white arrow) and Prc-expressing cells (black arrow) are aligned (Martin-Blanco et al., 1998) and the two rows of cardial cells that are not visible have joined at the dorsal midline below the ectoderm. (D) Dorsal view of a pucE69 mutant embryo bearing a P(lacZ) insertional mutation in the puc gene. The dorsal ectoderm is properly closed but the heart tube is not closed and the two rows remain separated (black arrows). More cells express ß-gal (white arrowhead) and occupy, in the dorsal ectoderm, a domain from which the cardial cells seem to be excluded. (E) In a lateral view of the same pucE69 mutant embryo, only one side of the embryo is depicted and, consequently, only one row of cardioblasts is visible (black arrow). The labeling by anti-Prc is excluded from the puc-expressing cells territory (white arrow). (F) Dorsal view. Optical confocal sections of a fluorescently double stained pucE69 mutant embryo for -Spectrin (green) and Pericardin (red). The embryo is closed dorsally (not seen in the plane of focus) but the two rows of cardioblasts have not joined to form the heart tube. The polarity of the cardioblasts has not, however, been altered, as judged from the concentration of Prc on their basal surface (white arrow) where are attached the pericardial cells. In one exception (white arrowhead), Prc has been detected on the apical face of the cardioblasts. In all the views, anterior is towards the left and posterior is towards the right.

View larger version (121K): [in a new window]   Fig. 3. Pericardin is located around the pericardial cells and at the basal surface of the cardioblasts in close proximity to the ectoderm. Embryos were double labeled by using either anti-Prc (red) and anti-Mef2 (green) that stained the nuclei of all myogenic cells (A,B) or anti-Prc (red) and anti--Spectrin (green) that is specific for the basolateral membrane of epithelial cells (C-E). (A) Dorsal view of a stage 14 embryo during dorsal closure and (B) of a stage 15 embryo in which the heart tube is formed. At both stages, Prc is located around the periphery of the pericardial cells (white arrowhead) and at the basal surface of the cardioblasts (white arrow). Attachment sites of alary muscles are also decorated by Prc (asterisk in A). (C-E) Confocal microscope sections. (C) Transverse section of the dorsal side of an embryo that shows successively from the top to the bottom, the polarized epithelial cells of the dorsal ectoderm (ect), the pericardial cells (pc) and the cardioblasts (cc), whose basolateral surface is stained with anti--Spectrin. Prc is concentrated at the boundary between the basal surface of the ectodermal epithelial cells and that of the cardial epithelium. The yellow color (white arrow) results from the superimposition of the two markers in the thickness of the section. Prc is also expressed in two out of every six cardial cells (stars) that correspond to the seven-up (svp)-expressing cells (Gajewski et al., 2000; Lo and Frasch, 2001). (D,E) Sections in the dorsal part of an embryo in which the ectoderm but not the heart tube is closed. (D) Superficial section through the dorsal ectodermal cells (arrowhead). Prc (red) is concentrated to the basal face of the cardioblasts whose basolateral membranes labeled with anti--Spectrin (green) are weakly detectable (arrow). In several positions (yellow color), Prc is in contact with the ectodermal cells. (E) Deeper section at the level of the cardioblasts (white arrow). Prc is still located at the basal face of the polarized cardioblasts arranged as a typical epithelium whose apical surface is not stained by anti--Spectrin (arrowhead). (F-I) Immunogold staining of heart ultrathin sections with anti-Prc as probe. (F) Cross section of a stage 17 embryo treated as described in the Materials and Methods and (G) enlargement of the insert. It shows two opposing cardiomyocytes (cc) enclosing a small lumen (lu) in which no gold particles are visible in the nascent basement membrane underlining the luminal surface. Particles are present (arrows) along the basal surface of the cardial cells and around the pericardial cells (pc), indicating the presence of Prc in the extracellular space. (H) Cross section of a larval heart focused on the enlarged lumen, which is still devoid of gold particles in the luminal basement membrane. In the mature basal extracellular matrix (I, enlargement of the boxed area in H), a high concentration of gold particles (arrowheads) is detected. Scale bars: 2 µm in F; 4 µm in G; 2.5 µm in H; 1 µm in I.

View larger version (14K): [in a new window]   Fig. 4. Molecular characterization of the prc locus and schematic representation of the Prc protein. (A) The 68 E locus drawn to scale. The locus is encompassed by the DS 00169 P1 phage. prc4.5 has been prepared as described in the Materials and Methods, and this fragment contains the regulatory sequences necessary for the expression of prc. Black boxes represent the coding regions of the mRNA, while empty boxes represent untranslated parts. The translation start and stop codons are indicated by ATG (145 in the cDNA) and TAG. The intron-exon structure of prc as deduced from the Drosophila genome sequence (BDGP) reveals nine exons and eight introns. The locations of CK 02611 and CK 01593 whose sequences match to the 3'-end of the cDNA are shown above the gene. CK 01593 extends further downstream in 3' of the prcV2 cDNA. prc dsRNA indicates the position of the 1 kb sequence used to construct UAS-IR prc. (B) The Prc protein. Small black box indicates signal peptide; arrow above the black box indicates putative signal peptide cleavage site (in 21); arrowhead indicates putative furin cleavage site (in 38); stars (72 and 1570) indicate the two N(I/G)S sequences that may serve as sites for N-linked glycosylation; large black box indicates C-terminal non-collagenous region. The potential attachment site for integrin is RGD (1652-1654). NFQSTYYTK is the tryptic peptide sequence (1707-1714). The collagen-like domain (140-1267) contains 26 atypical repeats illustrated by vertical bars (140-927) and the typical (Gly-X-Y)n repeat (920-1267 moiety, gray box). The consensus sequence of one of the atypical repeats is shown below.

View larger version (75K): [in a new window]   Fig. 5. Expression of the prc transcript and of the protein. (A,B) In situ hybridization to whole-mount stage 13 (A) and stage 17 (B) embryos with prcV2 as probe. The mRNA is already detected before dorsal closure (A) in the pericardial cells. When the heart is formed (B), the mRNA is expressed in the pericardial cells and ring gland (arrows), the oenocytes (arrowhead) and in some unidentified cells in the anterior part of the embryo (star). This pattern of expression can be superimposed on that observed with the anti-Prc (EC11) monoclonal antibody (C,D,L). White arrow, pericardial cells; white arrowhead, alary muscles. (E,F) In a transformed line carrying the prc4.5 genomic fragment inserted upstream of the lacZ reporter gene, anti-ß-gal staining reflecting Prc expression is detected in the same cells as the prc mRNA (compare with A,B, same symbols). (G) Double staining of a stage 17 embryo with anti-Prc (brown) and anti-Odd (blue) showing only the heart. All the Odd-expressing cells express also Prc (arrow). The Tin- and Prc-expressing cells (arrowhead) do not express Odd (compare with H). (H) Confocal section of a stage 17 embryo fluorescently double labeled for Tin (red) and Prc (green), as monitored by anti-ß-gal staining in the prclacZ transformed line. The arrows indicate yellow pericardial cells that co-express Tin and Prc in the heart. (I,J) In I (double staining as in G), clusters of segmentally repeated cardioblasts (arrowhead) do not express Odd but they express lacZ and the prc mRNA (J, arrow) (in situ hybridization as in B); they are svp-expressing cardioblasts. (K) Double staining as in G. Prc stains the ring gland (white arrow) and Odd labels the lymph glands (black arrow). (L) In a stage 17 embryo, labeling with anti-Prc detects an epitope present in the pericardial cells (arrow), the ring gland (arrow), the oenocytes (arrowhead) and unidentified cells (asterisk). (M) Embryos in which the prc cDNA is driven under UAS control with engrailed-GAL4. EC11 immunoreactivity is detected in a pattern in stripes (arrow) in addition to the endogenous labeling of oenocytes (arrowhead). In all views, anterior is leftwards and dorsal is upwards.

View larger version (107K): [in a new window]   Fig. 6. Perturbation in prc expression results in a disorganization of the cardial epithelium. (A-D) Hairpin-loop RNA interference experiments. (A,B) Confocal microscope observations of embryos double-labeled for Mef2 (green) and Prc (red) expression. A dorsal view of a stage 16 embryo (B) shows an important decrease in Prc expression (arrowhead, compare with a wild-type embryo in A), a cluster of cardioblasts (arrow) and a hole in the cardial epithelium (asterisk). (C,D) In embryos stained for Odd (D, which is an enlargement of the insert in C), holes (arrowhead) and clusters (arrows) of cells are visible. (E,F,G-J) vin6-deficient embryos. (E and box area enlarged in F) Disorganization of the cardial epithelium, visualized using anti-Mef2, with holes in the cardial epithelium (asterisk in F) and clusters of cardioblasts (arrow in F). The cardiac phenotype is very similar to that observed in B. The number of pericardial cells stained with anti-Odd is reduced in vin6-deficient embryos (H, compare with a wild-type embryo in G). Black arrows indicate the row of pericardial cells. (I,J) Rescue of the cardiacvin6 phenotype with the minigene MG-prc. In embryos double-labeled for Mrf2 (green) and Prc (red), the staining with anti-Prc (I) shows a restoration of Prc expression (arrows in I and J). As the number of pericardial cells is still reduced, the staining with anti-Prc is not completely normal. The structure of the cardial epithelium is less disorganized (arrowhead; compare with B,F). In all views, anterior is leftwards and dorsal is upwards.

View larger version (85K): [in a new window]   Fig. 7. Cardial phenotypes in prc silenced embryos and in prc loss-of-function embryos. (A,B) Staining with anti-Mef2 and anti-Prc of prc ds RNA silenced embryos. (A) At the onset of dorsal closure, the alignment of the cardioblasts appears normal (arrows). (B) When the dorsal closure progresses, the epithelium is disorganized (arrows) and clusters of cells are shown along the row of cardioblasts (arrowheads). (C) Stage 16 ds RNA silenced embryo double stained for Mef2 (green) and Prc (red). Owing to the low level of expression of Prc, the cardial epithelium (arrows) has collapsed and displays holes (asterisk) from which the cells are absent. Clusters of cardioblasts appear along the row of cardial cells (arrowhead). The red color for Prc has been artificially increased to become visible. (D) Stage 16 vin6-deficient embryo double stained for Mef2 (green) and Nrt (red). The polarity of the cardial cells in the epithelium seems to be affected (arrow). These cells appeared to be detached from the overlying ectoderm. In all views, anterior is leftwards and dorsal is upwards.