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First published online 21 February 2007
doi: 10.1242/dev.000406

Development 134, 1269-1278 (2007)
Published by The Company of Biologists 2007
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Thrombospondin-mediated adhesion is essential for the formation of the myotendinous junction in Drosophila

Arul Subramanian1,*, Bess Wayburn1,*, Thomas Bunch2 and Talila Volk1,{dagger}

1 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
2 Department of Molecular/Cell Biology, University of Arizona, Tucson, AZ 85721-0001, USA.


Figure 1
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  		Fig. 1. Tsp distribution changes following the formation of the myotendinous junction. Wild-type embryos at stage 12-13 (A-D) or stage 16 (E-H) labeled for Thrombospondin (A,E, Tsp, red), Stripe (B,F, marking all tendon cells, blue) and Myosin heavy chain (C,G, MHC, marking all somatic muscles, green) are shown, together with their merged images (D,H). Arrowheads mark the distribution of Tsp prior to muscle-tendon binding, whereas arrows mark the accumulation of Tsp at the myotendinous junction following muscle binding.

 

Figure 2
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  		Fig. 2. Tsp levels are reduced in myospheroid mutant and are induced by Stripe. (A-D) Mutant embryos lacking functional ßPS (mysXG43) and labeled with Tsp (A, red), Stripe (B, blue) and Myosin heavy chain (C, MHC, green) are shown. The merged image is shown in D. Arrows indicate sites where muscles are still associated with tendon cells, and Tsp is higher. Arrowheads indicate sites where muscles have detached, and Tsp is significantly low. (E,F) stripe mutant embryos stained for Tsp (E, green) and MHC (red). F is the merged image. Tsp is still detected in these embryos. (G-I) Embryos overexpressing Stripe using the engrailed-gal4 driver, stained for Tsp (G, red) and anti-Stripe (H, green). Their merged image is shown in I. Tsp is highly induced by Stripe.

 

Figure 3
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  		Fig. 3. The CTD is deleted in tsp8R mutation. Upper panel: a scheme depicting the genomic structure of the tsp gene (upper draw) and the location of the EP element as well as the deletion obtained by its excision (lower draw). A domain map of the TSP protein as well as the truncated protein obtained after the imprecise excision lacking the conserved CTD, Tsp repeats and four out of six EGF domains is shown. Lower panel: the merged image of wild-type embryo (A), embryo homozygous for Df(2L)BSC9, which deletes tsp locus (B), and embryo homozygous for the P-excision tsp8R (C) stained for Tsp (red) and Myosin heavy chain (MHC) (green). Note the loss of Tsp staining in Df(2L)BSC9 and in tsp8R mutant embryos.

 

Figure 4
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  		Fig. 4. Muscle migration at stage 14 is normal in tsp mutant embryos. Wild-type (A-C) or tsp8R mutant (D-F) embryos at stage 14 stained for Stripe (A,D, red) and Myosin heavy chain (B,E, MHC, green). C,F are their corresponding merged images. Muscle migration and arrangement is normal in the mutant at this stage.

 

Figure 5
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  		Fig. 5. Muscle rounding and aggregation are observed in tsp8R mutant embryos at stage 16. Wild-type (A-C) or tsp8R mutant (D-F) embryos at stage 16 stained for Stripe (A,D, red) and Myosin heavy chain (B,E, MHC, green) and their corresponding merged images (C,F). The somatic muscles round up (arrowheads in E,F indicate rounding of the ventral-longitudinal muscles) and tend to form aggregates (arrows in E). (G,H) tsp8R mutant embryos overexpressing TspA driven by stripe-gal4 (G) or by mef-2-gal4 (H) double-labeled with MHC (green) and with Tsp (red).

 

Figure 6
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  		Fig. 6. tsp mutant muscles interact with neighboring muscles and not with tendon cells. Wild-type embryos (A-E) and tsp mutant embryos (F-J) labeled for ßPS integrin (A,F, red) and Myosin heavy chain (B,G, MHC, green) are shown as well as their merged images (C,H). Arrowheads in F and H indicate sites of ectopic muscle-muscle interactions. The bracket in F and H indicates the sites of attachment of the lateral transverse muscles, showing significantly reduced PSß staining at the muscle-tendon attachment sites of two lateral transverse muscles and positive PSß staining when the third lateral transverse muscle is attached to other muscles (arrowheads in F,H). (D-J) High magnification of the ventral-longitudinal muscles of embryos stained for ßPS (red, D-J), MHC (green, D,I) and Stripe (blue, D-J). Arrowhead in I and J shows ßPS staining between the ventral-longitudinal muscle that is not associated with tendons; the arrowhead in D and E shows the parallel normal association of ßPS staining with tendons in wild type.

 

Figure 7
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  		Fig. 7. Tiggrin accumulates at the muscle-muscle junction sites of tsp mutant embryos. Wild-type embryos (A-C) or tsp mutant embryos (D-F) labeled for Tiggrin (A,D, red) and Myosin heavy chain (B,E, MHC, green), and their corresponding merged images (C,F). Arrows indicate sites of muscle-muscle interactions.

 

Figure 8
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  		Fig. 8. Talin levels are significantly reduced in tsp mutant embryos. Wild-type embryos (A-C) or tsp mutant embryos (D-F) labeled for Talin (A,B, red) and Myosin heavy chain (B,E, MHC, green) are shown as well as merged images (C,F). The brackets indicate the location of the attachment sites of the three lateral transverse muscles that do not stain for Talin in the tsp mutant. Talin is still detected in sites of muscle-muscle attachments.

 

Figure 9
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  		Fig. 9. PS2 integrin-expressing S2 cells spread on purified C-terminal Tsp polypeptide. (A) S2 cells lacking or expressing PS2m8 integrins were plated on purified Tsp CTD polypeptide (CTD KGD), Tsp CTD polypeptide with a mutation in the putative integrin-binding site KGD>LGE (CTD LGE), or on no ligand (Christopherson et al., 2005Go). (B) Coomassie Blue staining of purified Tsp CTD polypeptide, wild type (CTD) and the KGD>LGE mutant protein (CTD*). (C) S2 cell spreading as seen by phase microscopy; arrowheads show non-spread (left) versus spread (right) cells. (D) Spreading of cells expressing PS2m8 integrins, PS2m8 integrins containing a mutation that severely impairs ligand binding (mutPS2m8), or PS1 integrins plated on purified Tsp CTD polypeptide or no ligand. The percentage of spread cells is shown. Numbers represent the mean and standard error of three experiments.

 

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© The Company of Biologists Ltd 2007