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First published online 10 November 2004
doi: 10.1242/dev.01527

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Coordinated development of muscles and tendons of the Drosophila leg

¹ INSERM U.384, Faculté de Médecine, 28 Place Henri Dunant, 63001 Clermont Ferrand, France
² Department of General Zoology, Wroclaw University, 21 Sienkiewicza Street, 50-335 Wroclaw, Poland

View larger version (69K): [in a new window]   Fig. 1. Appendicular myoblasts in second and third instar larvae. (A,B) Leg discs from a second larval instar. (A) All the Twi-positive myoblasts co-express the 1151-GFP marker. (B) All second instar appendicular myoblasts, except those located in the stalk (arrowhead), express a mitosis marker (red), indicating that they are proliferating. (C,D) Leg discs from late third instar larvae. (C) Several hundreds of appendicular myoblasts co-expressing 1151-GFP and Twi are detected. (D) In the third larval instar, the majority of leg disc myoblasts do not display mitotic activity. Proliferating epithelial cells are indicated by arrows. (E,F) A prepupal leg disc stained for 1151-GFP and Twi, showing a subset of non-mesodermal (Twi-negative) 1151-GFP cells. These cells are seen within the outlined area and along the double-headed arrow. (G,H) Non-mesodermal 1151-GFP cells co-express Sr, indicating that they correspond to tendon precursors. Presumptive tarsal (double-headed arrow) and femur tendon precursors (arrow) are indicated. Asterisks in G point to the background staining resulting from the weak signal to background ratio seen when using the available anti-Sr antibody.

View larger version (71K): [in a new window]   Fig. 2. Spatial distribution of appendicular myoblasts and tendon precursors during larval and early pupal stages. (A-D) Tendon precursor position revealed by Stripe-Gal4/UAS-GFP with respect to the Wg-positive anteroventral domain (A,B) and the Dl-labelled proximodistal axis (C,D). (A,C) In the third instar leg disc, five GFP patches are observed. They are designated a in dorsal, b and c in ventral, e in posteroventral, and d in the distal part of the leg epithelium. (B,D) At 0 hours APF, tendon precursors invaginate and become extended. The proximodistal positions of tendon precursors are as follows: the a and b patches lie within the femur, c lies within the tibia and d crosses the tarsus, tibia and femur. At the onset of eversion (D) two supplementary tendon precursors, e in the proximal region (coxa, trochanter) and g in the dorsal tibia, are detected. (E-H) Merged images showing myoblast (blue) and tendon precursor (green) organisation in relation to the dorsoventral and proximodistal axes. Note that at 0 hours APF (F,H), myoblasts are excluded from the most distal part of the disc (double-headed arrows). Disc orientation is noted by: A, anterior; P, posterior; V, ventral; D, dorsal; Pr, proximal; and Di, distal.

View larger version (115K): [in a new window]   Fig. 4. Development of internal tendons and late steps of appendicular myogenesis. Tendons are visualised in the leg discs of (A) 1151-Gal4/UAS-GFP and (B) Stripe-Gal4/UAS-GFP pupae at 8 hours APF. Tendon precursors are annotated as in Fig. 2. Two tendon precursors, a and b, are located within the femur, two, g and c, are located in the tibia, and two others, e and f, in the proximal part of the leg. At this stage we are unable to define precisely the identity of proximal tendon precursors annotated e and f. (A) In addition to tendon precursors, 1151-Gal4/UAS-GFP also marks myoblasts, showing that they are deployed in all leg segments. A few individual 1151-GFP-positive cells, corresponding most probably to myoblasts, are detected at 8 hours APF in the tarsus. These cells do not express twi (see Fig. 7) and are no longer detected in later stages of development (see D). (C-E) General views and (F-H) enlarged views of dissected femur, tibia and tarsus from 1151-Gal4/UAS-GFP pupae at three different times during metamorphosis. In enlarged views asterisks mark the tendons. (C) General organisation of tendons and myoblasts at 20 hours APF. (F) Enlarged view of the region indicated in C, showing a proximal part of the femur a and b tendons. Myoblasts (arrows) lie tightly around the tendons. Note that only very rare myoblasts (arrowheads in F) appear not to be associated with tendons. (D) At 25 hours APF, the number of non-associated myoblasts increases (arrowheads). (G) Enlarged area corresponding to that shown in D. The arrangement of myoblasts associated with tendons has changed so that they form syncytial muscle fibre precursors composed of 5 to 10 nuclei (arrows). (E) At 35 hours APF, the precursors of the muscle fibres elongate. Myoblasts not associated with tendons are no longer detected. (H) Enlarged view of the region indicated in E, showing an increased number of myoblast nuclei in the muscle fibres (arrows). (I,J) Proximal part of the ventral tibia dissected from the Stripe-Gal4/UAS-GFP pupae, showing differentiation of the epithelial muscle attachment sites, the apodemes (arrows). Asterisks indicate the ventral internal tibia tendon, previously annotated c. Muscle fibres are visualised by their autofluorescence. (I) At 40 hours APF, Stripe-GFP expression appears in the nuclei of some epithelial cells (arrows). At this time, muscle fibres seem not to be attached to the epithelium. (J) At 55 hours APF, the Stripe-GFP cells are easy to detect. They have delaminated from the leg epithelium to form muscle attachment sites called apodemes (arrows). Muscle fibres attach to the apodemes. Note that a morphologically distinct area forms at the junction between the muscle fibres and the internal tendon (arrowhead). (G,H) Ventral tibia regions from MHC-tauGFP pupae, showing Myosin Heavy Chain (MHC-tauGFP) expression in differentiating myotubes. (G) At 40 hours APF, low levels MHC-tauGFP expression can be detected in appendicular tibia fibres. (H) About 10 hours later, MHC-tauGFP levels increase.

View larger version (111K): [in a new window]   Fig. 5. Organisation of appendicular muscles and tendons. (A,B,H,I) Computer assisted 3D reconstruction of muscles and tendons from 55-hour APF whole-mount legs expressing muscle-specific MHC-tauGFP (green) and tendon-specific 1151-driven DsRED (red). A,B and H,I show muscles and tendons of the tibia and femur, respectively. (C,E-G) Dissected adult tibia muscles from MHC-tauGFP legs. (D) A view of the tibia muscles from a whole-mounted MHC-tauGFP adult leg. In C, D and F, tendons (red) are revealed by their strong autofluorescence. (E) MHC-tauGFP depressor muscle fibres stained with propidium iodide (PI) to show the nuclei (red) within the syncytial myotubes. (F,G) A higher accumulation of MHC-tauGFP is seen in the tarsus levator (talm) compared with in the tarsus depressor (tadm) muscle. (J-P) Dissected adult femur muscles expressing MHC-tauGFP. (J) General view of muscles (green) and tendons (red) of the femur dissected from MHC-tauGFP/1151-DsRED leg stained with PI to reveal the nuclei (yellow). A neural ganglion of the femur (fng) associated with the long tendon (lt) is indicated. (K) Tibia levator muscle (tilm). (L) Long tendon (lt)-associated muscle (ltm2). (M) Proximal and (N) distal part of the tibia depressor muscle (tidm). Note that the tibia levator tendon (tilt) shown in K is much larger than the depressor tendon (tidt) shown in M. The opposite is true (C) for the tarsus levator (talt) and depressor (tadt) tendons. (O,P) Tibia reductor muscle fibres (tirm). The nuclei in K, L and N are stained with PI and appear yellow. (M) Note the alignment of sarcomeres in neighbouring tidm fibres. (P) An enlarged view of a tibia depressor fibre (tidm), showing two rows of peripherally located nuclei. All muscle and tendon names and corresponding abbreviations are defined in Table 1.

View larger version (72K): [in a new window]   Fig. 3. Appendicular duf-lacZ-expressing cells are associated with tendon precursors. (A-C) duf-lacZ-expressing cells visualised in leg discs dissected from duf-lacZ; Stripe-Gal4/UAS-GFP (A) larvae, (B) prepupae and (C) pupae. (A) About 50 duf-lacZ-labelled cells (arrows) are detected in the dorsal femur close to the sr-expressing a domain. Notice that the duf-lacZ transgene is also expressed in the epithelium of the most distal leg part. (D-F) Merged Twi/duf-lacZ views confirm the presence of presumptive founder myoblasts around the a tendon domain. (B,E) At the prepupal stage (0 hours APF), the number of duf-lacZ-expressing cells around the a domain increases (arrows, compare A with B). Some of them lose twi expression. At 0 hours APF, duf-lacZ-positive cells also appear in other regions (dotted arrow in E). (F) At 5 hours APF, the sr-expressing a domain extends to form a long structure in the femur. The associated duf-lacZ cells follow the extending tendon precursor (arrowheads in C and F). Few myoblasts co-expressing duf-lacZ and Twi are visible near the g domain in the tibia (arrows in F). (G-G''') A model schematising the invagination of the tendon precursors during the leg disc eversion, showing the associated duf-lacZ-expressing cells. (H-J) Prepupal leg disc double-stained for Stripe-GFP (H) and Odd-lacZ (I; merged image is shown in J), showing co-expression of odd with sr in invaginating tendon precursors.

View larger version (45K): [in a new window]   Fig. 6. Comparison of appendicular muscles revealed by MHC-tauGFP staining with the leg musculature described by Miller. (A) Adult leg muscles (green) and tendons (red) visualised by MHC-tauGFP and 1151-GAL4/UAS-DsRED staining, respectively. (B) Schematic representation of appendicular muscles (green) and tendons (red). Regions located between double-headed arrows, labelled 1-5, are represented in the cross-sections shown in D. (C) Adult Drosophila leg muscles identified by Miller and labelled according to Miller's description (Miller, 1950). Tarsal segments are devoid of muscles and harbour only the long tendon, 44. A comparison of B and C reveals the presence of previously undescribed muscles and tendons within the leg. These are the tarsus reductor muscles tarm1 and tarm2, the long tendon muscles ltm1 and ltm2, and a femur depressor muscle, fedm, as well as internal depressor and levator tendons in all leg segments. This implies that the levator and the depressor muscle fibres attach to the internal tendons and display an organisation that is different from that proposed by Miller. (D) Schematic representation of cross-sections through coxa (1), femur (2,3) and tibia (4,5) in the regions indicated in B. The cross-sections show the locations of muscles and tendons with respect to the dorsoventral and anteroposterior axes. Note that all of the levator muscles are located dorsally, and the depressors ventrally. All names and abbreviations of muscle and tendons are defined in Table 1.