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The Hox gene abdominal-A specifies heart cell fate in the Drosophila dorsal vessel

TyAnna L. Lovato, Thiennga P. Nguyen, Marco R. Molina and Richard M. Cripps*

Department of Biology, University of New Mexico, Albuquerque, NM 87131-1091, USA



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  		Fig. 1. The Drosophila dorsal vessel consists of a posterior heart and an anterior aorta, which can be distinguished on the basis of morphological and molecular criteria. (A,C,E,G) Low-magnification views of stage 16 embryos stained with various markers. (B,D,F,H) High-magnification views of same embryos. (A,B) MEF2 protein accumulation in the muscle cells of the embryo. The dorsal vessel consisted of two parallel rows of cells at the dorsal midline. Note that the distance between the two rows of MEF2-positive nuclei is greater in the heart (Ht) compared to the aorta (Ao). The pattern of Hand transcripts (C,D) and muscle MHC protein (E,F) in the cardial cells of the dorsal vessel can also be used to distinguish heart from aorta, and to visualize the heart lumen. Numbered body segments are indicated. (G,H) Tina-1 transcripts in the dorsal vessel accumulated only in the heart. Scale bars: 50 µm for A,C,E,G; 25 µm for B,D,F,H. All pictures are dorsal views oriented with anterior towards the left. In C,E,G, the location of the heart is bracketed.

 


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  		Fig. 2. Expression of Bithorax-Complex genes in the dorsal vessel. For each BX-C gene product, single-channel views of an embryo stained with an antibody to the BX-C product (A,E,I, top row) and the same embryo stained with an antibody to MEF2 (B,F,J, second row) are shown. These images are merged in the third row (C,G,K). The top three rows of micrographs represent combination of several individual z-series images. This is necessary to show the arrangement of the entire dorsal vessel. To confirm co-expression in the mesoderm, the lower row shows the combined images for only one or two optical sections (differing by 2 µm). (A) UBX accumulation. (B) MEF2 accumulation in the same embryo. (C) Merged image of A and B (UBX in green, MEF2 in red). (D) Merged image at limited optical sections to show co-expression in the mesoderm. UBX was detected broadly albeit at low levels in the dorsal vessel. (E) ABDA accumulation. (F) MEF2 accumulation in the same embryo. (G) Merged image of E and F (ABDA in green, MEF2 in red). (H) Merged image at limited optical sections to show co-expression in the mesoderm. ABDA was detected in cardial (yellow) and pericardial (green) cells of the heart region only. (I) ABDB accumulation. (J) MEF2 accumulation in the same embryo. (K) Merged image of I and J (ABDA in green, MEF2 in red). (L) Merged image at limited optical sections to show co-expression in the mesoderm. ABDB was detected only in the posterior four nuclei of the dorsal vessel. Scale bars: 50 µm for A-C,E-G,I-K; 8 µm for D,H,L. All micrographs are dorsal views of stage 16 embryos with the anterior towards the left. The location of the heart (Ht) is bracketed in the lower magnification images.

 


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  		Fig. 3. Ectopic expression of abd-A in the mesoderm resulted in an aorta-to-heart transformation. (A-D) Ectopic expression of abd-A in the ectoderm did not affect heart cell identity. (A) MEF2 staining revealed that the dorsal vessel did not form normally; however, expression of Hand in the dorsal vessel was detectable (B, inset). (B) Closer examination of Hand-expression cells revealed that those at the posterior (Ht, the presumptive heart region) were larger than those more anterior (Ao, the presumptive aorta region), suggesting that heart cell identity had been retained. This was confirmed by staining for MHC (C), which showed stronger staining in the presumptive heart region. Heart identity at the molecular level was also retained, as determined by expression of Tina-1 (D), which was restricted to the posterior of the dorsal vessel. Ectopic expression of abd-A in the mesoderm using the 24B-gal4 driver (E-H) or the twi-gal4 driver (I-L) transformed the aorta into a heart. (E,I) MEF2 staining revealed a gap between the two rows of cardial cells throughout the length of the dorsal vessel suggesting the formation of a larger lumen than wild type. This was confirmed by examination of Hand expression in embryos (F,J), and by accumulation of muscle MHC (G,K). (H,L) Tina-1, the marker of heart cells in wild-type embryos showed an expansion of its expression domain throughout the dorsal vessel. Scale bars: 50 µm for all panels except B (15 µm). All micrographs are dorsal views of stage 16 embryos with anterior towards the left. The location of the heart is bracketed.

 


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  		Fig. 4. abd-AMX1 homozygotes show no heart formation. Homozygous abd-A mutants were identified based upon a lack of reactivity to anti-ABDA antibody, or based upon abnormal morphology of the gut, and then stained for expression of the indicated genes. Small arrows bracket the cells that, in wild-type, would form the heart. (A) Anti-MEF2 staining demonstrated a lack of dorsal closure in the mutants. (B) Hand was expressed in the two rows of dorsal vessel cells; however, most cells had a uniform size and shape (compare with Fig. 3B, where the heart cells assumed an unique shape). (C,D) MHC accumulation in the dorsal vessel cells; once again, cell shape and levels of MHC accumulation along the length of the dorsal vessel were uniform. (E) Tina-1 was not expressed in the dorsal vessel (one row of dorsal vessel cells in the presumptive heart forming region is indicated by dv), although expression in the hindgut (hg) was unaffected. Scale bars: 50 µm for A-C,E; 25 µm for D. All micrographs are dorsal views of stage 16 (or equivalent) embryos with anterior towards the left.

 


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  		Fig. 5. Ectopic ostium formation occurs upon induction of ectopic heart. (A,B,E) Anti-MHC staining alone. (C,D,F) Anti-Tin staining in black, anti-MHC staining in brown. (A) In wild type, anti-MHC staining revealed the fine structure of the embryonic heart. At each segmental boundary, the heart was broader, and this structure was coincident with two pairs of cells with a unique morphology (bracketed). (B) In the aorta, dorsal vessel shape was uniform. (C) In wild type, the cells forming the broader region of the heart did not express tin and were therefore svp-expressing cells (S, bracketed), which formed the ostia. Expression of tin is indicated by T (arrows). (D) Although svp and tin expression were mutually exclusive in the aorta, svp-expressing cells were not distinguishable based upon the morphology of the vessel. (E) In twi-gal4/+; UAS-abd-A/+ embryos, variations in dorsal vessel diameter similar to that found in the heart were seen in anterior locations (indicated by asterisks). Occasionally, tiny perforations in the heart wall were observed (os, arrowhead), consistent with the interpretation that ectopic ostia were being formed. (F) These ectopic ostia form from the svp-expressing population of cells, as the widening of the dorsal vessel occurred at locations where Tin was absent (asterisks). Segment boundaries are indicated in E and F to demonstrate that the broadening of the heart occurred at ectopic locations. All panels are dorsal views of stage 16 embryos oriented with anterior towards the left. Scale bar: 20 µm.

 


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  		Fig. 6. abd-A expression induces an increase in Svp cell size in the aorta. Shown are the mean sizes (in µm) of the svp-expressing dorsal vessel cells of control y w embryos, and embryos in which abd-A is expressed throughout the mesoderm. S1 to S7 represent the segmentally repeating groups of Svp cells, from anterior to posterior. Error bars represent standard errors of the mean. Note that in y w the Svp cells that form the ostia (S5-S7) are larger than those in the aorta (S1-S4). Upon induction of ectopic heart, all Svp cells assume a larger average size.

 


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  		Fig. 7. Ubx is not required for heart specification, but can partially induce ectopic heart formation. (A) UBX levels (green) are reduced by ectopic expression of abd-A. Inset shows MEF2 accumulation (red) in the same sample at the same focal plane. (B-D) Ubx9.22 homozygotes stained for MHC accumulation (B), Tina-1 expression (C) and ABDA accumulation (D). Note that there is no effect of loss of Ubx function upon the specification of the heart, despite a slight increase in ABDA levels in the aorta. Arrow in B identifies one of the two ostia formed by S6.

(E.H) Effects of ectopic expression of Ubx upon dorsal vessel cell identity. (E) UBX accumulation is at high levels throughout the mesoderm. (F) MHC accumulation indicates that the heart region is still formed in these embryos, and that additional ostia are occasionally observed in cells which would normally form the aorta (arrow). (G) Tina-1 expression is now detected throughout the dorsal vessel. (H) abd-A expression is normal in these embryos. The normal location of the heart is bracketed in A-D. All panels are dorsal views of stage 16 embryos oriented with anterior towards the left. Scale bar: 50 µm.

 


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  		Fig. 8. Mesodermal expression of Abd-B inhibits muscle formation. (A) MHC accumulation demonstrates aberrations in the formation of skeletal muscles and a complete absence of MHC-expressing cells at the dorsal midline (between arrows). (B-D) Tin accumulation indicates that cardiac defects arise as a result of a loss of tin-expressing cardiac precursors. At stage 13 (B), the tin-expressing population is normal (arrow). By stage 14 (C), many Tin cells are absent. By stage 16 (D), very few Tin-positive cells are detectable in the embryo. (A,D) Dorsal views with anterior towards the left; (B,C) Sagittal views with anterior towards the left. Scale bar: 50 µm.

 




© The Company of Biologists Ltd 2002