A–L Confocal images of third instar leg discs stained for Twi, Lbe and Duf-lacZ. Leg disc A–D Control duf lacZ, and Leg disc E–H with attenuated Htl signalling htl RNAi, I–L with increased Htl signalling htl-ca, in the myoblasts. Attenuation of Htl expression leads to reduced number of myoblasts associated with the leg disc compare A and E. This is particularly obvious when comparing the number of Twi/Lbe/Duf expressing cells within the outlined area in the dorsal femur. In contrast, forced Htl expression leads to significantly increased number of leg myoblasts outlined area 1 in I–L. Moreover, Htl, when overexpressed, induces Lbe prematurely in all myoblasts in the leg disc proper and promotes differentiation of dorsally located myoblasts into Duf-positive cells K,L, outlined areas 2,3,4. Note that the only leg disc associated Duf-lacZ myoblasts that do not express Lbe are in area 4, corresponding to the proximal part of the leg disc that contributes to the ventral thorax. Surprisingly, Htlca also induces Lbe/Duf-lacZ expression in tarsal segments outlined area 3. These cells give rise to ectopic muscles in tarsal segments N,P otherwise devoid of muscles. M A wild type adult muscle pattern revealed in the leg expressing muscle-specific MHC-tauGFP green and tendon-specific 1151-DsRed red. Note that no muscles are detected in the tarsus. Arrowhead points to the dorsal tibia muscle, talm. N Gain of Htl signalling in leg myoblasts leads to the formation of supernumerary muscle fibres arrowhead in N. Ectopic muscles form in the tarsus arrow in N. O RNAi-mediated attenuation of Htl expression leads to the reduced number of muscle fibres arrowhead in O. P An enlarged view of tarsal segments shown in N. Ectopic muscle fibres align along the long tendon but most of them are not attached to the epithelium. Q An enlarged view of the distal tibia muscles in wild type R; in Htl gain-of-function and after RNAi-based Htl attenuation S The number of muscle fibres in the talm muscle indicated by asterisk is significantly increased in R and reduced in S.