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First published online July 19, 2004
doi: 10.1242/10.1242/dev.01249

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Founder myoblasts and fibre number during adult myogenesis in Drosophila

¹ National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bellary Road, Bangalore 560065, India
² The Salk Institute Peptide Biology Lab, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
³ Centro de Biologia Molecular "Severo Ochoa", UAM-CSIC, Madrid 28049, Spain
⁴ Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK

View larger version (43K): [in a new window]   Fig. 1. Schematic representation of the development of the indirect flight muscles (IFMs) and the abdominal muscles. The IFMs are anatomically and functionally subdivided into two distinct groups: the dorsal longitudinal muscles (DLMs) and the dorsoventral muscles (DVMs). (A) The precursor myoblasts (yellow) for the IFMs are attached to the wing disc during the larval life. At the onset of metamorphosis larval muscles begin to histolyse, with the exception of three oblique muscles. (B) By 12 hours APF, histolysis is complete and the three larval templates are clearly visible. By this stage, myoblasts migrate into the muscle-forming regions. (C) The larval templates start splitting by 13 hours APF and the process is complete by 16-17 hours APF. At the same time, the DVMs form by the de novo fusion of myoblasts. (D) By 24 hours APF, the DLMs and the DVMs are complete. Muscle formation in the abdomen occurs later than in the thorax. The abdominal muscles develop from an adult myoblast pool (yellow) associated with the segmental and intersegmental nerves (A). (A-D) During early pupal stages (from 0-24 hours APF), myoblasts proliferate and migrate out along the nerves. (E) Fusion of these myoblasts begins by 28-30 hours APF. (F) The arrangement of the DLMs, the DVMs (I, II and III) and the dorsal muscles of the first abdominal segment in an adult fly: by 50 hours APF, the pattern of the adult muscles is largely complete.

View larger version (79K): [in a new window]   Fig. 7. Effects of dominant-negative Rac1 expression in adult myoblasts. (A) Fibres of wild-type lateral muscles expressing myosin (brown) at 31 hours APF (29°C). Each fibre has a brightly stained duf-lacZ nucleus (black arrow). In some fibres, this nucleus is not present within the field of view. Other nuclei in the fibres express duf-lacZ at lower levels and are seen in a few fibres (black arrowheads). (B) 1151Gal4/UAS-Rac1N17-misexpression pupa of the same stage showing the absence of syncitial fibres. The putative founder cells (one of them indicated by the black arrow) are correctly specified and express duf-lacZ and myosin. Unfused myoblasts also express myosin and, in some cases, can be seen clustered around duflacZ-expressing founders (arrowhead). (C-E) Two lateral founders, of the same stage as shown in in B, at a higher magnification. The founder cells extending processes in an orientation similar to that of the wild-type lateral fibres. The nucleus of each cell expressing duflacZ is indicated by a black arrow. (C) Extended processes of one of the founder cells are in focus (indicated by white arrows). (D) Same preparation as in C, shown at a different plane of focus. Black asterisks mark some of the unfused myoblasts surrounding the founder cell. (E) Processes of the second founder cell are in focus in this image (white arrows). Unfused myoblasts are again seen clustered around this founder. (F-I) X-Gal and anti-MHC-stained lateral muscle fibres at 42 hours APF (in 29°C) in wild-type (F,G,H) and 1151Gal4/UAS-Rac1N17-misexpression (I) pupae. (F) Lateral muscles showing one duf-lacZ-expressing nucleus in each fibre. This nucleus corresponds to the high duf-lacZ-expressing founder nucleus observed in the fluorescent images in Fig. 3G and H. X-Gal staining cannot detect the low ß-galactosidase activity in the remaining nuclei of the syncitium. These nuclei can be detected at a higher magnification as shown in G and H. (G,H) Magnified views of the region outlined by the box in F, at different planes of focus showing the presence of multiple nuclei in each fibre. Each white asterisk is placed below a nucleus in the plane of focus. (I) In the absence of normal fusion, thin mononucleate lateral fibres span the region. (J,K) Fluorescent preparations at 36 hours APF (at 29°C). (J) Wild-type DVM II expressing myosin (red) and duf-lacZ (green). White arrows indicate the nucleus in each syncitium that expresses high levels of duf-lacZ. (K) Developing fibres of DVM II in a pupa with Rac1N17 misexpression in adult myoblasts. The DV muscle fibres are reduced in size but the pattern of one high duf-lacZ-expressing nucleus per fibre remains unaffected. (L) Wild-type pattern of DLMs, and DVMs I, II and III, in one hemisegment of an adult CS fly (grown at 29°C). Black asterisks indicate the DVM II fibres. (M) Muscles in one thoracic hemisegment of a fly after Rac1N17 misexpression in adult myoblasts. In focus are the two fibres of DVM II (black arrows). Fibre size is severely reduced but fibre number remains unchanged. In A-I, anterior is to the right, dorsal midline to the bottom; in J-K, anterior is to the top, dorsal to the right; in L-M, anterior is to the left, dorsal to the top. Scale bars: in A, 4 µm for A,B,F,I; in C, 4 µm for C,D,E; in G, 4 µm for G,H; in J, 20 µm for J,K; in L, 40 µm for L,M.

View larger version (125K): [in a new window]   Fig. 2. Expression of duf-lacZ in DVMs. (A) Diagram showing the position of the three DVM bundles (blue) relative to the DLM fibres (white) in the adult fly. (B) actin-lacZ pupal preparation (24-hour APF) showing the DVM fibre bundles. The DVM I fibres are marked by black asterisks; fibres of DVM II are marked by red asterisks. DVM III fibres are formed beneath the DLM fibres. In this figure, one of the two DVM III fibres (black arrow) can be observed. The DLM fibre most adjacent to the DVMs is indicated by the black arrowhead. (C-E) duf-lacZ-expressing DVM nuclei assayed by lacZ staining. The nuclei corresponding to DVM I are indicated by black asterisks, to DVM II by red asterisks and to DVM III by black arrows. (C) The three duf-lacZ-expressing nuclei of DVM I, and the two of DVM II, at 7 hours APF. The linear arrays of nuclei, indicated by an arrowhead, are duf-lacZ-expressing nuclei in the adjacent larval templates and are described in more detail in Fig. 4. (D,E) Twelve-hour APF pupae showing duf-lacZ-expressing nuclei of DVM I and DVM II (D), and of DVM II and DVM III (E). (F) Forty-hour APF preparation of duf-lacZ pupa double labeled with anti-MHC (red) and anti-ß-galactosidase (green) to mark the two myofibres (of DVM II) and duf-lacZ nuclei, respectively. One nucleus in each fibre (white arrow) expresses higher levels of lacZ than the rest. Anterior, top; dorsal midline, right. Scale bar: in C, 18 µm for C-E; in F, 20 µm.

View larger version (120K): [in a new window]   Fig. 3. Expression of duf-lacZ in abdominal muscles. (A-E) duf-lacZ expression in developing dorsal muscles of the abdomen. (A-C) One hemisegment of a 24-hour APF duf-lacZ pupa double labeled with anti-ß-galactosidase (green) and 22C10 (red). (A) Single cells with duf-lacZ expression in their nuclei. (B) The same preparation as shown in A showing the duf-lacZ-expressing cells stained by 22C10. The nerve to which the myoblasts remain attached initially is also labeled by 22C10 and is indicated by the white arrowhead. (C) Merged image of A and B. (D) A 28-hour APF duf-lacZ pupa similarly double labeled as in C, showing the duf-lacZ-expressing cells stretching out at positions where future muscle fibres will form. (E) A 50-hour APF duf-lacZ pupa double labeled with anti-MHC (red) and anti-ß-galactosidase (green), showing a subset of dorsal muscles. By this stage, formation of the multi-nucleate fibres is largely complete. Each fibre has one prominent duf-lacZ-expressing nucleus (white arrows), presumably belonging to the single cell that preceded the multi-nucleate fibre. The remaining nuclei express duf-lacZ at a lower level. (F-H) duf-lacZ expression in developing lateral muscles of the abdomen. (F,G) duf-lacZ pupa double labeled with anti-ß-galactosidase (green) and 22C10 (red). (F) Twenty-four hours APF. Single cells expressing duf-lacZ are present in the region where the future lateral muscles will form. These cells, like the cells in the dorsal region, are labeled by 22C10. (G) The developing lateral myotubes at 36 hours APF. The fibres, labeled by 22C10, are now multi-nucleate. (H) Lateral muscles at 41 hours APF, labeled by anti-ß-galactosidase (green) and anti-MHC (red). In both G and H, each fibre contains a single nucleus with high levels of duf-lacZ expression (white arrows), against a backdrop of fainter expression in the other nuclei. For some fibres in H, nuclei with higher duflacZ expression are not present in the field of view. In A-E, anterior is to the top, dorsal midline to the right; in F-H, anterior is to the right, dorsal midline to the bottom. Scale bar: 50 µm.

View larger version (77K): [in a new window]   Fig. 4. duf-lacZ expression in developing DLMs. (A) The position of the larval templates and the six DLM fibres with respect to the fly notum. (B) The three larval templates at 12 hours APF; (C) the six DLM fibres at 26 hours APF, as revealed by histochemical staining in Mhc-lacZ pupae. (D-G) duf-lacZ expression followed by assaying for ß-galactosidase activity (D) and anti-ß-galactosidase labeling (E-G). (D) A 6.5-hour APF duf-lacZ pupal preparation showing ß-galactosidase histochemical activity in the three larval templates. The three templates have been labeled as LOM I, II and III. Each template has 8-13 duf-lacZ-expressing nuclei aligned along their length. Some nuclei in LOM I and LOM II are out of focus. (E-G) duf-lacZ expression at 12 hours APF (E), 14 hours APF (F) and 24 hours APF (G), when the DLMs are in the six fibre stage. (H-J) Confocal images of a 12-hour APF duf-lacZ pupa stained with anti-ß-galactosidase (green) and anti-Ewg (red) antibodies. (H) The three larval templates expressing duf-lacZ. (I) Ewg expression in larval nuclei, and in the nuclei of incoming adult myoblasts. White arrowhead points to a larval nucleus expressing Ewg in the template fibre; arrows indicate the adult nuclei. Note the difference in size between the nuclei of the two different origins. (J) Merged image showing the co-localization of ß-galactosidase (green) with Ewg (red), expressed in the larval nuclei. Anterior is to the top; dorsal midline to the right. Scale bar 20 µm for D-J.

View larger version (82K): [in a new window]   Fig. 5. Expression of duf-lacZ during abdominal myogenesis. Preparations of duflacZ pupae double labeled with anti-ß-galactosidase (green) and anti-Twist (red). Panels A and B are of the same preparations shown separately in the red and green channels. (C-F) Merged images. (A) Twist-expressing nerve-associated myoblasts in the dorsal hemisegment of A3 in a late third instar larva. (B) duf-lacZ expression in the same cells as in A. (C) A 13-hour APF pupa, again showing co-localization of duf-lacZ with Twist in almost all nerve-associated myoblasts. (D) By 20 hours APF, lacZ expression has faded from many of the myoblasts, and only a subset of myoblasts shows co-localization. (E) At 24 hours APF, the number of Twist and duf-lacZ co-localizing myoblasts is further reduced. By this stage, individual myoblasts expressing high levels of duf-lacZ are seen. (F) Twenty-eight hours APF. Single duf-lacZ-expressing myoblasts are observed at positions corresponding to future fibre forming sites. In all figures anterior is to the top, dorsal to the right. Scale bar: 20 µm.

View larger version (127K): [in a new window]   Fig. 6. Expression of duf-lacZ in the adult thoracic myoblast pool. (A-C) Expression of duf-lacZ in the wing disc-associated myoblasts. (A) Faint duf-lacZ expression in the myoblasts that adhere to the notal region of the wing disc. The region outlined by the black box is magnified in B and C. (B,C) Twist-expressing myoblasts (red; B) that also express duf-lacZ (green; C). (D-F) Co-localization of duf-lacZ with 1151Gal4-expressing myoblasts. (D) Confocal image of a 12-hour APF hemithorax showing the 1151-GAL4 expressing myoblasts of the developing DVM II fibres. 1151Gal4-driven expression is revealed by UAS-nls-GFP, with duf-lacZ in the background. Each arrowhead indicates a nucleus, which is larger in size than the other nuclei within the syncitium. (E) The duf-lacZ-expressing myoblasts of DVM II, assayed by anti-ß-galactosidase labeling. These two nuclei are the same as those indicated by arrowheads (D). (F) Merged image showing the co-localization of duf-lacZ (red) with 1151GAL4 (green). (G-I) Co-localization of duf-lacZ with Vg. (G) Confocal image showing Vg-expressing myoblasts that have fused to, or have swarmed over, the DVM II syncitium. Vg expression is observed by anti-Vg labeling on a 12-hour APF duflacZ pupa. Amidst the cluster of myoblasts, arrowheads indicate the myoblasts that also express duf-lacZ. (H) Anti-ß-galactosidase labeling reveals the duf-lacZ-expressing myoblasts of DVM II in the same preparation. (I) Merged image showing the co-localization of Vg with duf-lacZ. Scale bar: 50 µm for B,C; 30 µm for D-I.

View larger version (69K): [in a new window]   Fig. 8. Notch signalling and founder cell numbers. (A) Wild-type preparation of 24-hour APF duf-lacZ pupa (grown at 29°C). The box outlines duf-lacZ-expressing founders of DVM I fibres. (B) The duflacZ founders of DVM I (present within the outlined region in A) shown at a higher magnification. (C,D) duf-lacZ-expressing DVM I founders in animals expressing dnNotch (C) and MamN (D). The number of founders is unchanged. The relative orientation of the three duf-lacZ-expressing cells with respect to each other varies slightly in B, C and D. Such variation is also observed in wild-type preparations. (E,F) 22C10 staining in the dorsal A4 hemisegment of a Nts1pupa grown entirely at permissive temperatures (E) and of a heat-pulsed Nts1 pupa (F), grown for 24 hours at 22°C and then pulsed for 4 hours and 36 minutes at non-permissive temperature (see Materials and methods for details). The small arrows indicate the 22C10-stained cells, whose number (20 in this case) remains unchanged despite reduced Notch function in Nts1 pupae. (G) A 12-hour APF preparation from a duf-lacZ-expressing pupa (grown at 29°C), showing the wild-type pattern of founders in DVMs and DLMs. The three black asterisks indicate the three DVM I founders, while the red asterisks show the two founders corresponding to DVM II. The arrow indicates duf-lacZ nuclei belonging to the larval template adjacent to the DVMs. (H) duf-lacZ expression in a pupa (similar age to that in G) in which Nintra has been expressed in all myoblasts. DVM founders are missing in these pupae. Bold asterisks mark the region where the founders are expected. (I) duf-lacZ expression in a 1151Gal4/UAS-Twi pupa. Constitutive expression of Twist in the myoblasts results in an absence of DVM founders. (J-L) Complementary expression pattern of duf-lacZ and Twist in the myoblasts. (J) Confocal section of Twist-expressing myoblasts in the presumptive region of formation of DVM II. (K) Same image as that in J showing duf-lacZ-expressing founders of DVM II. (L) Merged image showing the absence of Twist immunoreactivity in the duflacZ-expressing nuclei. Anterior is to the top, dorsal midline to the right. Scale bars: in A, 9 µm; in B, 9 µm for B-D: in E, 25 µm for E,F; in I, 20 µm for G-I; in J, 15 µm for J-L.

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