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

This Article Summary Full Text Full Text (PDF) Movies 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 Ponzielli, R. Articles by Sémériva, M. Search for Related Content PubMed PubMed Citation Articles by Ponzielli, R. Articles by Sémériva, M.

Heart tube patterning in Drosophila requires integration of axial and segmental information provided by the Bithorax Complex genes and hedgehog signaling

Romina Ponzielli, Martine Astier, Aymeric Chartier^*, Armel Gallet, Pascal Thérond and Michel Sémériva

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: Génétique du Développement de la Drosophile, Institut de Génétique Humaine,141, rue de la Cardonille, 34396 MONTPELLIER Cedex 5, France
Present address: Centre de Biochimie, ISBDC UMR CNRS 6543, Parc Valrose, 06108 NICE Cedex 2, France
Both authors contributed equally to the work

View larger version (16K): [in a new window]   Fig. 1. Schematic representation of the larval cardiac tube. Only cardiac cells are shown in this scheme. The cardiac tube is formed by two rows of cardiac cells which are both epithelial and muscular cells. There are six pairs of cardiac cells per segment from T3 to A7 and only two pairs in T2 and A8. The boundary between aorta and heart is situated within segment A5. The cells shaded grey express tin, ß-3 Tub and D-sur. The cells colored yellow express svp and Tb66F. It has been proposed by Molina and Cripps (Molina and Cripps, 2001) that the svp-expressing cells in the heart form the larval ostiae. This scheme has been drawn according to the actual knowledge on cardiac tube differentiation. cvv, cardiovascular valve; am, alary muscles.

View larger version (126K): [in a new window]   Fig. 2. Differentiation of the cardiac tube during embryogenesis. Dorsal confocal images of the embryonic cardiac tube double labeled with (A-G) anti-Prc (red) and anti--Spectrin (green); (H-N) anti-Prc (red) and anti-DMef2 (green); (O) anti-- Spectrin (green) and anti-ß-gal in svpAE127P-line (red); (P) anti-Tin (green) and anti-ß-gal in svpAE127P-line (red). (A,H) Whole cardiac tube; (B,D,F,I,K,M) part of the aorta; and (C,E,G,J,L,N) part of the heart. (A,H) At late stage 16, the cardiomyocytes in the cardiac tube display signs of morphological heterogeneity. The cells in the aorta (open arrowheads) and in the heart (arrowheads) with morphological features that distinguish them from the other cardioblasts situated between them are the cells that express svp (O,P). The other cardioblasts are the tin-expressing cells (P). Their nuclei in (H) have an egg-like shape as compared to the nuclei of the other cardioblasts. These morphological features are more pronounced in the heart than in the aorta. Note the larger size of the heart cardioblasts compared to the aorta cardioblasts. (B,C,I,J) At the onset of dorsal closure (stage 13), all cardioblasts along the whole length of the cardiac tube appear similar (arrows). The svp-expressing cardioblasts, however, which will differentiate into the second ostia in the heart, have initiated a slight change in their shape (arrowheads). Only one row of cardioblasts is shown. (D,E,K,L) At late stage 14, the cardiac tube is not yet closed but the cardioblasts in the heart are already larger than in the aorta and the svp- expressing cells (arrowheads) have acquired their distinctive morphology. (F,G,M,N) At stage 16, the difference in size between heart and aorta cardiomyocytes is accentuated. The specific morphology of the svp-expressing cells is clearly visible in the aorta (open arrowheads) as well as in the heart (arrowheads). The asterisks in A,G,H,J,L indicate the alary muscles. In all views, anterior is to the left.

View larger version (128K): [in a new window]   Fig. 3. Differentiation of the larval ostiae. Dissected third instar larval cardiac tube stained with anti-DMef2 (A,B) or with mCD8GFP (C,D) whose expression is driven in the myogenic lineage by 24B-GAL4. (A,B) The large nuclei labeled by anti-DMef2 are tin-expressing cells nuclei whereas the small nuclei are svp-expressing cell nuclei (Molina and Cripps, 2001). In the aorta, these latter cells (arrows) form a butterfly-like structure in which the striated muscle fibers from each cell converge in a central position (red arrowhead). No sign of opening is apparent in the aorta while, in the heart, the cardiomyocytes with small nuclei form genuine ostiae (large open arrow). (C,D) mCD8GFP labels the membranes of all the cardiomyocytes. The two cells with small nuclei (arrows) in the aorta are flanked on either side by two cells with larger nuclei (arrowheads) and they do not form a functional ostia. Genuine opening is visible in the heart between the first pair of ostiae cells (long open arrow). The aorta is separated from the heart by a non muscular cardiovascular valve (cvv). The double headed arrow points to the very thin cytoplasm in the heart. lu, lumen.

View larger version (89K): [in a new window]   Fig. 4. Observation of the cardiac tube in wild-type embryos. A schematic drawing of the cardiac cells in the cardiac tube is presented for each photograph. Double-headed arrows delineate the internal limit of the cardiac tube, small arrows show the ostiae cells and large arrowheads, the hemocytes. (A-H) Progression of the development of the aorta (A,B,E,F) and of the heart (C,D,G,H). The cardiomyocytes enlarge with the maturation of the cardiac tube. Ostiae are fully functional at 22 hours. (I-L) Recordings of heart beat at 22 hours allow measurement of the end-diastolic (dd) and end-systolic (ds) diameters. (M-V) Entry of hemolymph through the ostiae into the cardiac cavity is apparent from the passage of a large hemocyte (large arrowhead). The ostiae cells and the hemocytes adjust their shapes to allow transport of the hemocytes. Note the filamentous material (dotted lines) linking the two pairs of ostiae on either side of the cardiac tube.

View larger version (67K): [in a new window]   Fig. 5. Expression of Ubx, Abd-A, Abd-B in the cardiac tube. Immunostaining with anti-Ubx (A-C; red), anti-Abd-A (D-F; red), anti-Abd-B (G-I; red) or double labeling with anti-Tin (A, green), anti--Spectrin (B,C,E,F,H,I; green) and anti-ß-gal (D,G; green). Open arrowheads indicate svp-expressing cardioblasts in the aorta; solid arrowheads indicate svp-expressing cardioblasts in the heart. (A,D,G) Wild-type embryos. Ubx (in A) is expressed in all the cardioblasts (arrow) in the A2 to A5 segments (yellow) including the svp-expressing cells (red) that do not express Tin and a subset of pericardiac cells (asterisks). Abd-A (in D) is expressed in the cardioblasts (arrow) in the A5 to A7 segments, including the svp-expressing cells (yellow). Abd-B (in G) is expressed in the four posterior most cardioblasts (arrows) and, to a lesser extent, in the svp-expressing cells in segment A7. (B,H) abd-Am4 mutant embryos. Ubx (in B) is ectopically expressed in cardioblasts in the posterior A5 to A7 segments (arrows), including the svp-expressing cells. Abd-B (in H) expression is as in wild-type embryos (arrows). (C) UAS-abd-A, twist-GAL4 embryos. Ubx expression in the aorta (A1-A5 segments) is considerably less than in wild-type embryos. tin-expressing cardioblasts (arrows) and svp-expressing cardioblasts. (E) Ubx9.22 mutant embryos. The expression of Abd-A is as in wild-type embryos (arrows). (F) UAS-Ubx, twist-GAL4 embryos. Abd-A expression is as in wild-type embryos (arrows). (I) Ubx, abd-A double mutant embryos. Abd-B expression is as in wild-type embryos (arrows). The large arrow points to the ectodermal cells expressing Abd-B.

View larger version (152K): [in a new window]   Fig. 6. Function of abd-A and Ubx in the differentiation and the function of the cardiac tube. Double labeling of cardiac tubes with anti--Spectrin (green) and (A,B,E,F,G,H,K) anti-Prc (red) or (N) anti-Ubx (red). (I,J) Staining with anti-D-Mef2 (red, I) and svp-RNA (green, J). (C,L,O) In vivo observations (D,M,P) and their schematic representations. In all panels, open arrowheads indicate svp-expressing cardioblasts in the aorta and arrowheads indicate svp-expressing cardioblasts in the heart. (A-D) In abd-A homozygous mutant embryos, the heart cardiomyocytes differentiate as aorta cardiomyocytes along the whole length of the cardiac tube. They are same size along the anteroposterior axis (double-headed arrow). The svp-expressing cardioblasts in the aorta and in the heart have the same morphology. As a consequence, the heart does not beat (or only very weakly) and the ostiae are not functional. (E,F) In Ubx9.22 mutant embryos (E), the aorta does not differentiate normally, particularly in the anterior region (compare to wild-type anterior aorta in F). (G,H) In Ubx, abd-A double mutant embryos the cardiac tube shows no heterogeneity along the anteroposterior axis and the aorta and heart do not differentiate properly. Cardioblasts appear smaller and less polarized than in wild-type embryos (arrow). Prc-expressing cells are disorganized (asterisk) and sometimes located in ectopic positions (dotted arrow). Non epithelial cells, which do not express Prc, form clusters along the cardiac tube (open dotted arrow). (I,J) In double mutant embryos, cardioblasts differentiate into the same number of cells expressing D-Mef2 as in wild-type embryos (arrows). Their size and shape are uniform along the whole length of the tube. (J) Svp expression is observed in the most posterior cardioblasts in each segment. (K-M) In UAS-abd-A, twist-GAL4 embryos, the aorta is transformed into heart in segments A5, A4 and part of segment A3. svp-positive cells differentiate as in a wild-type heart and functional ostiae (arrows in L,M) are visible in segments A4 and A3. (N,O,P) In UAS-Ubx, twist-GAL4, 24BGAL4 embryos, Ubx is ectopically expressed in heart region (arrows in N). Cardioblasts in the whole tube do not have a normal morphology (O,P) nor are the ostiae functional. Double-headed arrows delineate the internal limit of the cardiac tube. rg, ring gland.

View larger version (127K): [in a new window]   Fig. 7. Differentiation of ostiae in svp mutant embryos. (A-D) Confocal sections of late stage 16 embryos double labeled with anti-Prc (red) and either anti--Spectrin (green) (A,B) or anti-D-Mef2 (green) (C,D). Differentiation into aorta and heart occurs correctly but the change in morphology (A,B) or in the shape of nuclei (C,D) of the svp-expressing cells (arrowhead and open arrowhead) is not apparent, in either the heart or the aorta. The asterisks mark the alary muscles. (E) In svp mutant embryos, expression of Abd-A (red) in all cardioblasts in segments A5-A7, including svp-positive cells (arrowheads) is similar to that in wild-type embryos. (F,G) In vivo observation of the cardiac tube in svp mutant embryos. The heart starts beating normally but almost completely stops after 22 hours of development. The ostiae cells (arrows) differentiate after an initial delay; differentiation, however, remains incomplete and they do not function (or open and close). Double arrows mark the internal limit of the lumen.

View larger version (98K): [in a new window]   Fig. 8. The activity of the hh signaling pathway is required for the transcriptional activation of svp. Stage 13 embryos double stained with an anti-Engrailed (En, brown) antibody to position the segmental boundaries and either a DIG-labeled svp riboprobe (blue, A,C,E,G-J) or anti-Tin (blue, B,D,F). Anterior is to the left and dorsal is up. (A,B) Whole embryos; (C-J) enlargements showing the central abdominal region of the embryo. (A-D) Wild-type embryos. svp is expressed in seven pairs of cardioblasts per hemi-embryo in the segments A1-A7 (arrow), in the chordotonal organs (white arrowhead) and in the oenocytes (open arrowhead). Tin is expressed in four cardioblasts (arrow) per hemi-embryo in the segments T3 to A7 and in a subset of pericardiac cells (arrowhead). Tin and svp are expressed in different subsets of cardioblasts (see Fig. 2P). (E,F) In hh9K, expression of svp is abolished in svp cardioblasts (arrowhead) as well as in the chordotonal organs. More cells express Tin (arrowhead), including cardioblasts that express svp in the wild type. (G) The expression of Cirep driven by twist-GAL4/24B represses svp expression in the cardioblasts (arrowhead), although not completely (compare with E). (H) Expression of Hh in ectodermal En-expressing cells rescues the expression of svp (arrowhead). (I) Expression of a GPI membrane-bound form of Hh does not rescue svp expression (arrowhead). (J) Hh expression is maintained in the En-expressing cells in an otherwise wg mutant background. The function of wg is not required for the expression of svp (arrowhead).

View larger version (15K): [in a new window]   Fig. 9. Schematic representation of the cardiac tube patterning. The combined action of the regionalized expressions of Hox genes and of Hh provided by the ectoderm leads to segmental and axial cell diversities.