Introduction In recent years significant progress has been made in understanding how different sarcomeric proteins interact during development, myofibril assembly and muscle contraction. Formation of the highly ordered repetitive muscle cytoarchitecture is a complex process and genetic defects produce different forms of myopathy (reviewed in Clark et al., 2002; Redwood et al., 1999). It is well established that muscle contraction is regulated by changes in the concentration of Ca 2+ . The troponin-tropomyosin (Tn-Tm) complex, which is formed by three different troponin polypeptides, T, I and C (TnT, TnI and TnC), together with tropomyosin (Tm), regulates acto-myosin interactions in response to neurally stimulated intracellular release of Ca 2+ ions. In the absence of Ca 2+ , TnI inhibits the generation of acto-myosin forces during muscle contraction (reviewed in Clark et al., 2002; Geeves and Holmes, 1999; Gordon et al., 2000). Although the role of TnI during muscle contraction is well documented, its role during muscle development has not been studied in detail. Two major reasons for this are that loss of function mutations are lethal to individuals (see nemaline TnI nulls) and in vertebrate models expression of alternate isoforms compensate during early developmental stages. For example, cardiac TnI gene knockout mice develop apparently normal hearts owing to the compensatory fetal TnI isoform but eventually acute heart failure occurs in later stages (Huang et al., 1999). In Drosophila melanogaster the IFMs develop by fusion of myoblasts following their migration from the notum region of the wing imaginal discs during early stages of pupation. One group of IFMs, the dorsal longitudinal muscles (DLM), develop by fusion of these myoblasts to the remnants of larval oblique muscles (LOM, Fig. 1A) in the thorax region that escape complete histolysis at metamorphosis and serve as templates (TEM, Fig. 1B), whereas the other IFM group, the dorso ventral muscles (DVM), develop by de novo fusion of the myoblasts. During fusion the IFMs elongate (Fig. 1C) to span completely the developing thorax, attach to the tendon cells and, following a fibre shortening to one-third of the original size (Fig. 1D), begin to undergo myofibrillogenesis, a stage that involves high-level expression of the adult-specific structural sarcomeric proteins. Initial sarcomere organization occurs during this stage and the muscles elongate again. The tendon cells retract as the muscles increase in length and size until functional myofibres are formed (Fernandes et al., 1991; Fernandes et al., 1996; Reedy and Beall, 1993) (Fig. 1E,F). As Drosophila express many IFM-specific sarcomeric protein 1795 Myofibrillar proteins assemble to form the highly ordered repetitive contractile structural unit known as a sarcomere. Studies of myogenesis in vertebrate cell culture and embryonic developmental systems have identified some of the processes involved during sarcomere formation. However, isoform changes during vertebrate muscle development and a lack of mutants have made it difficult to determine how these proteins assemble to form sarcomeres. The indirect flight muscles (IFMs) of Drosophila provide a unique genetic system with which to study myofibrillogenesis in vivo. We show in this paper that neither sarcomeric myosin nor actin are required for myoblast fusion or the subsequent morphogenesis of muscle fibres, i.e. fibre morphogenesis does not depend on myofibrillogenesis. However, fibre formation and myofibrillogenesis are very sensitive to the interactions between the sarcomeric proteins. A troponin I (TnI) mutation, hdp 3 , leads to an absence of TnI in the IFMs and tergal depressor of trochanter (TDT) muscles due to a transcript-splicing defect. Sarcomeres do not form and the muscles degenerate. TnI is part of the thin filament troponin complex which regulates muscle contraction. The effects of the hdp 3 mutation are probably caused by unregulated acto-myosin interactions between the thin and thick filaments as they assemble. We have tested this proposal by using a transgenic myosin construct to remove the force-producing myosin heads. The defects in sarcomeric organisation and fibre degeneration in hdp 3 IFMs are suppressed, although not completely, indicating the need for inhibition of muscle contraction during muscle development. We show that mRNA and translated protein products of all the major thin filament proteins are reduced in hdp 3 muscles and discuss how this and previous studies of thin filament protein mutants indicate a common co- ordinated control mechanism that may be the primary cause of the muscle defects. Key words: Myogenesis, Myofibrillogenesis, Mutant, Troponin I, Acto-myosin Summary Troponin I is required for myofibrillogenesis and sarcomere formation in Drosophila flight muscle Upendra Nongthomba, Sam Clark, Mark Cummins, Maqsood Ansari, Meg Stark and John C. Sparrow* Department of Biology, University of York, York, YO10 5DD, UK *Author for correspondence (e-mail: jcs1@york.ac.uk) Accepted 27 November 2003 Journal of Cell Science 117, 1795-1805 Published by The Company of Biologists 2004 doi:10.1242/jcs.01024 Research Article