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First published online 15 December 2008
doi: 10.1242/dev.022533

Development 136, 317-326 (2009)
Published by The Company of Biologists 2009
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The Drosophila homolog of vertebrate Islet1 is a key component in early cardiogenesis

Tabea Mann1, Rolf Bodmer2 and Petra Pandur1,*

1 Institute for Biochemistry and Molecular Biology, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany.
2 Burnham Institute for Medical Research, Center for Neuroscience, Aging and Stem Cell Research, Development and Aging Program, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.


Figure 1
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  		Fig. 1. Tup expression during cardiogenesis in wild-type Drosophila embryos. (A) At stage 10, Tup is expressed in a broad domain in the dorsal ectoderm (arrowhead). The expression in the amnioserosa (as) persists throughout embryogenesis. (B) Double labeling for Wg protein and tup RNA confirms the ectodermal expression of tup. (C) At mid-stage 11, Tup starts to be expressed in the cardiac mesoderm in ~10 small clusters of cells (arrowheads). (D) These clusters are also positive for Eve (arrowheads). (E) By late stage 11, Tup is co-expressed with Tin throughout the cardiac mesoderm (arrowheads). (F-H) Tup is expressed in all six myocardial cells (arrowheads) and in the Tin-positive pericardial cells (arrows in H). Arrowheads in H point to the two Tin-negative, Tup-positive myocardial cells. (I) Tup is expressed in all Odd-positive pericardial cells (arrows) and in a subset of Odd-expressing cells of the lymph glands (lg). (J) tup RNA expression in myocardial Dmef2-expressing cells matches Tup protein localization (arrowheads), as seen in G. Except for H and I, which are dorsal views of stage 15 embryos, all images are lateral views. Anterior is to the left. WT, wild type.

 

Figure 2
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  		Fig. 2. Heart phenotypes in tupisl-1 mutants. (A,B,D-J) Compared with wild-type Drosophila embryos, tupisl-1 mutants are characterized by gaps in expression of all examined myocardial (Dmef2 and Tin) and all pericardial (Pc, Odd and Eve) cell markers. (C,K) Embryos that are transheterozygotic for tupisl-1 and a deficiency that includes the tup locus, Df(2L)OD15, also show gaps in Dmef2 expression at stage 14 (arrows in C) and show a strong reduction of Tin-expressing cardiac cells at late stage 11 (asterisk in K). Arrowheads in K point to Tin-positive visceral mesodermal cells. as, amnioserosa; rg, ring glands.

 

Figure 3
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  		Fig. 3. tup is required for the normal expression of early cardiac transcription factors. (A,B) Drosophila stage 11 tupisl-1 mutants are characterized by a reduction in Tin-expressing cells (arrows). (C,D) The Pnr expression domain is strongly reduced in tupisl-1 mutants (arrows). (E,F) Double fluorescence labeling for Dmef2 protein and pnr RNA shows the mesodermal reduction of pnr expression in tupisl-1 mutants (arrows). (G,H) Reduced pnr expression (arrows) in the ectoderm is demonstrated by co-staining for Wg protein. (I,J) Stage 11 tupisl-1 mutants lack cardiac Doc2-positive cells (arrows). Arrowheads indicate missing Eve-expressing cells.

 

Figure 4
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  		Fig. 4. Tup expression requires the presence of early cardiac transcription factors and depends on wg and dpp signaling. (A-D) Tup expression is initiated in the cell clusters in Drosophila tin346 mutants (arrowheads in B) but is not maintained at later stages (compare C with D). (E) Myocardial Tup and Dmef2 expression is absent in Df(3L)DocA mutants. Since Doc mutants have been shown to also lack pericardial cells, the remaining Tup-expressing cells (green) are unlikely to be cardiac-related cells. (F) pnrVX6 mutants also show a dramatic reduction in myocardial Tup- and Dmef2-expressing cells. (G,H) Tup expression at stage 13/14 depends on Wg (G) and Dpp (H) signaling. Arrowheads in all images point to Dmef2/Tup co-expressing cells, which appear yellow in the merged optical sections. Asterisks are placed in the region of the myocardial cell row, which has defects to various degrees in all mutants shown.

 

Figure 5
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  		Fig. 5. Genetic interactions between tup, tin, pnr and Doc. The cardiac phenotypes in transheterozygotic Drosophila embryos demonstrate that tup interacts genetically with all three factors. The phenotypes were compared with those of the cardiac markers in single heterozygotes, and were evaluated statistically for Dmef2 (see Tables 1 and 2). (A-D) Dmef2 expression in the wild type (A) and in embryos transheterozygotic for tupisl-1 and pnrVX6 (B), tupisl-1 and tin346 (C), tupisl-1 and Df(3L)DocA (D). Dorsal views of embryos at stage 15/16 are shown. Arrows point to gaps in the myocardial rows of the dorsal vessel. (E,F) Pnr is reduced in tup/tin transheterozygotic embryos (arrows in F). A lateral view of a stage 11 embryo is shown. (G-I) Tin expression in the wild type (G), and in embryos transheterozygotic for tup and pnr (H), and tup and DocA (I). Reduced Tin expression is seen in both cases (arrows in H,I). Dorsal views of stage 14 embryos are shown.

 

Figure 6
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  		Fig. 6. Germ layer- and cell-specific requirements of Tup at various stages of cardiogenesis. Drosophila embryos at stage 12 (A-F) or between stages 10 and 11 (G-I), shown from the lateral side with anterior to the left; or at stage 15/16 (J-M) or 14 (N) shown from the dorsal side. (A,B,D) Mesodermal and ectodermal inhibition of Tup function by expressing UAS-tup{Delta}HD results in a reduction of Tin-positive cells (arrows in B,D). (C,E) Full-length tup (UAS-tup) can partially restore Tin when co-expressed in the ectoderm but not in the mesoderm. (F) Mesodermal expression of a Tup construct lacking the LIM domains (UAS-tup{Delta}LIM) also affects Tin expression (arrows). (G-I) Tup is required for normal dpp expression as shown by in situ hybridization. dpp is reduced after ectodermal inhibition of Tup function (arrows in H). dpp is strongly reduced in tupisl-1 mutants (I). (J-K') Mesodermal expression of UAS-tup{Delta}HD results in a reduced number of Dmef2-positive myocardial cells. (K') An enlargement of the two segments (as delineated by the vertical lines) indicated by the brackets in K. (L-N) Inhibition of Tup function in the pericardial cell lineage results in loss of Odd-positive cells (arrow in M), including a subset of Odd-expressing lymph gland cells (arrowhead in M). Overexpression of Tup in this lineage induces additional Odd-positive pericardial cells (arrows in N). Odd expression in the lymph glands appears unaffected (arrowhead in N).

 

Figure 7
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  		Fig. 7. Mesodermal overexpression of Tup reveals different functional relationships with other cardiac transcription factors. (A-D) Overexpression of Tup leads to a moderate expansion of Tin and some ectopic Tin-expressing cells on the lateral side of the embryo (arrows in B). Overexpression the Pnr allele pnrD4 results in a strong ectopic induction of Tin across the whole lateral side of the embryo (arrows in C). Co-overexpression of Tup and PnrD4 mimics the phenotype of PnrD4 overexpression alone (arrows in D point to ectopic Tin-expressing cells). (C1-C3) The ectopic Tin-positive cell clusters induced by overexpression of PnrD4 alone are heterogenous. Some cells co-express Tin and Tup (arrow in C3), whereas others are only positive for Tup (arrowhead in C3). (E-I) Tup and Pnr counteract each other in Eve-positive pericardial cell specification. Overexpression of Tup results in additional Eve-positive cells within the clusters (arrows in F), whereas overexpression of PnrD4 leads to the complete loss of Eve-positive cell clusters (arrows in G). (H) Co-overexpression of Tup and PnrD4 can reduce the effects induced by each factor singly. (I) Pie charts showing the percentage of embryos with wild-type (WT, brown), expanded (+, yellow) or reduced (-, green) Eve-positive cell clusters. (J-M) Overexpression of Tup results in a moderate loss of Odd-positive pericardial cells (arrow in K) and to a strong reduction of Odd-positive lymph gland cells (arrowheads in J,K). Overexpression of Tin has a slightly stronger negative effect on the Odd-positive pericardial cells (arrows in L); however, the reduction of Odd-positive cells in the lymph glands appears to be less strong (arrow in L) than that caused by Tup overexpression (arrowhead in K). (M) Co-overexpression of Tup and Tin results in a similar phenotype to that seen for overexpression of Tin alone. Arrows point to the absence of Odd-positive pericardial cells; the arrowhead points to Odd-positive lymph gland cells.

 

Figure 8
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  		Fig. 8. Tup as a new component of the Drosophila early cardiac transcriptional network. At stage 10, Tup is expressed in the ectoderm and is required for normal Pnr and dpp expression. Regulation of dpp expression through Tup may be direct or indirect (dashed line). Likewise, ectodermal Tup expression may be regulated by Dpp directly or indirectly through Pnr. After Wg and Dpp have induced a cardiac fate in the dorsal mesoderm by initiating and maintaining Doc and Tin expression, respectively, Pnr and Tup start to be expressed in the cardiac mesoderm by stage 11. All four factors are required to ensure proper cardiac specification of mesodermal cells. Black arrows indicate previously characterized interactions; red arrows indicate novel interactions with Tup as proposed in this study.

 

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© The Company of Biologists Ltd 2009