Abstract
Legs are locomotor appendages used by a variety of evolutionarily distant vertebrates and invertebrates. The primary biological leg function, locomotion, requires the formation of a specialised appendicular musculature. Here we report evidence that ladybird, an orthologue of the Lbx1 gene recognised as a hallmark of appendicular myogenesis in vertebrates, is expressed in leg myoblasts, and regulates the shape, ultrastructure and functional properties of leg muscles in Drosophila. ladybird expression is progressively activated in myoblasts associated with the imaginal leg disc and precedes that of the founder cell marker dumbfounded. The RNAi-mediated attenuation of ladybird expression alters properties of developing myotubes, impairing their ability to grow and interact with the internal tendons and epithelial attachment sites. It also affects sarcomeric ultrastructure, resulting in reduced leg muscle performance and impaired mobility in surviving flies. The over-expression of ladybird also results in an abnormal pattern of dorsally located leg muscles, indicating different requirements for ladybird in dorsal versus ventral muscles. This differential effect is consistent with the higher level of Ladybird in ventrally located myoblasts and with positive ladybird regulation by extrinsic Wingless signalling from the ventral epithelium. In addition, ladybird expression correlates with that of FGF receptor Heartless and the read-out of FGF signalling downstream of FGF. FGF signals regulate the number of leg disc associated myoblasts and are able to accelerate myogenic differentiation by activating ladybird, leading to ectopic muscle fibre formation. A key role for ladybird in leg myogenesis is further supported by its capacity to repress vestigial and to down-regulate the vestigial-governed flight muscle developmental programme. Thus in Drosophila like in vertebrates, appendicular muscles develop from a specialised pool of myoblasts expressing ladybird/Lbx1. The ladybird/Lbx1 gene family appears as a part of an ancient genetic circuitry determining leg-specific properties of myoblasts and making an appendage adapted for locomotion.
Highlights
Skeletal leg musculature is required for walking in all animals, but the genetic mechanisms that control its development have been analysed mainly in vertebrates [1,2,3,4,5,6]
We found that ladybird early, an orthologue of a key regulator of appendicular myogenesis in vertebrates, Lbx1 [27], is dynamically expressed in myoblasts associated with the leg imaginal disc proper (Fig. 1, Video S1 and Video S2)
The analysis of muscle phenotypes in mice lacking the functional Lbx1 gene has led to the conclusion that Lbx1 is involved in the interpretation of signals that guide appendicular muscle precursor migration [2,3,35]
Summary
Skeletal leg musculature is required for walking in all animals, but the genetic mechanisms that control its development have been analysed mainly in vertebrates [1,2,3,4,5,6]. The conserved family of Distal-less/ Dlx homeobox genes was found to be involved in outgrowth of appendages over a broad spectrum of proteostome and deuterostome phyla, suggesting the existence of ancient genetic circuitry controlling leg development [7,8,9]. This prompted us to find out whether the genetic programme governing leg muscle formation, required for the main biological leg function, which is locomotion, was under the control of conserved genes. The mechanisms governing the myogenic programme in the developing leg disc remain largely unknown
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