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First published online 25 February 2009
doi: 10.1242/dev.031567

Development 136, 1105-1113 (2009)
Published by The Company of Biologists 2009
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Crossveinless and the TGFβ pathway regulate fiber number in the Drosophila adult jump muscle

MaryAnn S. Jaramillo, Candice V. Lovato, Erica M. Baca and Richard M. Cripps*

Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA.


Figure 1
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  		Fig. 1. Characterization and analysis of TDT fiber number in laboratory strains. (A) In Oregon R wild type, the jump muscle comprises four small cells (arrowheads) and ~26 large cells (arrow), as visualized in paraffin wax-embedded sections stained with Hematoxylin and Eosin. (B) In y w, this number is reduced to four small cells and ~19 large cells, visualized here using: anti-β-integrin (red), which outlines individual fibers; phalloidin (green), to visualize F-actin; and DAPI (blue), to visualize nuclei. (C) The TDT can also be identified based upon its accumulation of the Z(210) Z-disc associated protein (red). This protein is detected at high levels in the indirect flight muscles (single asterisks) and in the large cells of the TDT (arrow), and at very low levels in the small cells of the TDT (arrowhead); however, it is not detected in other tubular muscles of the thorax (double asterisk). (D-F) Comparable images of the TDT in y cv1 v f car mutants. The number of large fibers is always reduced and the number of small cells is occasionally reduced. Frequently, the fibers fail to organize properly into the rosette characteristic of the wild-type TDT. Scale bar: 50 µm.

 

Figure 2
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  		Fig. 2. Aneuploid mapping of the TDT fiber phenotype on the X chromosome. (A,B) In females, a deficiency adjacent to the cv gene did not uncover the defect in fiber number when heterozygous with y cv1 v f car (A; there are 27 large fibers in this example), but a deficiency that also uncovered cv did uncover the TDT fiber defect (B; there are only 14 large fibers in this example). (C) Furthermore, a duplication for the cv region rescued the y cv1 v f car TDT defect in otherwise hemizygous males (21 large fibers). (D,E) To determine whether the fiber defect arose from mutation of cv, we tested a different cv allele, cv43. (D) Females homozygous for cv43 showed severe TDT defects (D; nine large fibers), as did cv1/cv43 trans-heterozygotes (E; approximately nine large fibers). Arrows indicate TDT. Scale bar: 50 µm.

 

Figure 3
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  		Fig. 3. Rescue of the posterior crossvein and TDT phenotypes of cv mutants. (A) Wild type. The posterior crossvein is present in the wing (arrow in left panel) and the TDT is normal (arrow, right panel). (B) In cv43 mutants, the posterior crossvein is absent (arrow in left panel) and the TDT phenotype is apparent (arrow in right panel. (C,D) In cv43; UAS-cv homozygotes, there is partial rescue of the posterior crossvein (C; arrow in left panel), and essentially complete rescue of the TDT (arrow in right panel). This effect probably arises from slight leakiness of the UAS-cv transgene, as one copy of the UAS-cv is not sufficient to rescue either phenotype (D). (E,F) Both the wing and the TDT phenotypes can be rescued using drivers that are predominantly ectodermal (ptc-Gal4; E) or mesodermal (24B-Gal4, F) (arrows). Scale bar: 400 µm for wing images; 50 µm for paraffin sections.

 

Figure 4
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  		Fig. 4. The TGFβ pathway controls TDT fiber number. TDT fiber number and identity were assayed in both paraffin (A,D,G) and frozen sections (B,C,E,F,H,I) of wild type and mutant combinations. Stains are as in Fig. 1. (A-C) Wild-type, showing the numbers of large TDT cells (arrows). (D-F) In 1151-Gal4/+; UAS-tkv*/+ adults, the number of TDT fibers (arrows) was increased significantly. This could be observed in paraffin sections (D), as well as in the cryosections, where the β-integrin stain outlined a very large number of individual fibers (E). The identity of this muscle as the TDT was confirmed by anti-Z(210) reactivity (F). (G-I) In 1151-Gal4/+; UAS-Dad/+ adults, the number of TDT fibers was drastically reduced (arrows), although the identity of the fibers was retained, visualized using anti-Z(210) reactivity (I). Scale bar: 50 µm.

 

Figure 5
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  		Fig. 5. Specification of founder cells during development of the TDT. In order to follow TDT specification, we studied duf-lacZ expression in wild type during late larval to early pupal development. Founders for the TDT can be first observed in the T2 leg imaginal disc at the late third instar (L3) larval stage (A), identified as being positive for both β-Gal (green) and MEF2 (red). At this stage, the number of specified founders is ~12. During early pupal development, these cells spread laterally and dorsally across the developing ectoderm. Two hours after puparium formation (APF), there were 12-15 founders (B), and this number increased to approximately 21 founder cells by 8 hours APF in wild-type (C). (D) By 16 hours APF, the number of founders in wild type was roughly 25, comparable with the final number of TDT fibers. Arrows indicate TDT founder cells. Scale bar: 50 µm.

 

Figure 6
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  		Fig. 6. The TGFβ pathway regulates TDT founder cell specification. (A-H) Control and TGFβ pathway mutants were analyzed for TDT founder cell specification 16 hours APF using a duf-lacZ marker (A,C,E,G) and for TDT fiber formation 24 hours APF (B,D,F,H). Arrows indicate the developing TDT. Owing to the thickness of the samples at this stage, not all founder cells nor all fibers are apparent in a single focal plane; thus, representative planes are shown. In duf-lacZ controls, founder cells were apparent at the location of the developing TDT (A), and these subsequently gave rise to clearly defined fibers by 24 hours APF (B). In cv43 mutants, founder cells were still present (C), although significantly reduced in number compared with wild type (see text for details). The fibers that were subsequently formed (D) were fewer in number than in wild type and somewhat disorganized. Upon activation of the TGFβ pathway (1151>tkv*), large numbers of founder cells were observed at 16 hours APF (E), and a concomitant increase in the number of nascent fibers was observed at 24 hours APF (F). Upon repression of the TGFβ pathway (1151>Dad), there were very few founder cells observed at 16 hours APF (G). In addition, based upon the accumulation of MEF2 at this stage, there were significantly fewer myoblasts present to form the TDT. By 24 hours APF in these mutants, the TDT was barely visible and only comprised a handful of fibers (H). Scale bar: 50 µm.

 

Figure 7
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  		Fig. 7. A role for Dpp and Gbb in TDT fiber number specification. (A,B) Combination of cv43 with mutants or deficiencies for dpp (A) or gbb (B) resulted in significant reductions in TDT fiber number, observed in double-heterozygotes compared with any single heterozygotes. (C-F) Evaluation of cv expression during development via in situ hybridization. (C,D) In stage 11 embryos, a cv antisense probe revealed significant expression (arrow) around the tracheal pits (C) (Vilmos et al., 2005Go), whereas a control sense probe showed no specific signal (D). Embryos are oriented with anterior towards the left and dorsal side uppermost. (E) In 16-hour APF pupae, the antisense probe showed significant hybridization to a region of the cuticle (arrow) located just lateral to the lumen of the developing second leg (marked with an asterisk). (F) In control pupae, the sense probe did not show hybridization to this region. The ventral midline is indicated by paired arrowheads in E,F, and anterior is towards the left. Scale bar: 100 µm for C,D; 25 µm for E,F.

 

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