Since Miller's morphological description, the Drosophila leg musculature and its formation has not been revisited. Here, using a set of GFP markers and confocal microscopy, we analyse Drosophila leg muscle development, and describe all the muscles and tendons present in the adult leg. Importantly, we provide for the first time evidence for tendons located internally within leg segments. By visualising muscle and tendon precursors,we demonstrate that leg muscle development is closely associated with the formation of internal tendons. In the third instars discs, in the vicinity of tendon progenitors, some Twist-positive myoblasts start to express the muscle founder cell marker dumbfounded (duf). Slightly later, in the early pupa, epithelial tendon precursors invaginate inside the developing leg segments, giving rise to the internal string-like tendons. The tendon-associated duf-lacZ-expressing muscle founders are distributed along the invaginating tendon precursors and then fuse with surrounding myoblasts to form syncytial myotubes. At mid-pupation, these myotubes grow towards their epithelial insertion sites, apodemes, and form links between internally located tendons and the leg epithelium. This leads to a stereotyped pattern of multifibre muscles that ensures movement of the adult leg.

SUMMARY1. The nature, magnitude and kinetics of the 4-aminopyridine-sensitive early outward current (Ito) were analysed in isolated ventricular myocytes from the septum, the apex and the left ventricular free wall of rat ventricles using the wholecell voltage clamp method. The modulatory effect of pressure overload-induced cardiac hypertrophy on the regional variations of Ito was assessed in each topographical class of cells.2. Voltage clamp experiments were performed at room temperature (20-25°C) in the absence of Na+ on both sides of the membrane and in the presence of 3 mM CoCl2.Ito was studied from a holding potential of -80 mV and determined by subtraction of total outward currents elicited by the same protocols in the presence of 3 mm 4-aminopyridine (4-AP) from those obtained in its absence.3. In normal hearts, membrane passive properties were very similar in each topographical class of cells. Our results confirmed that the predominant early outward current in rat ventricular cells was 4-AP-sensitive, time and voltage dependent, and demonstrated that the magnitude of the current varied on a regional basis: current density of Ito in left ventricular free wall cells (30-1 +9-2 pA/pF at + 60 mV) was larger than in apex cells (20-2 + 1-7 pA/pF) or in septum cells (1 1-9 + 3-3 pA/pF). We noticed a larger variability in data from left ventricular free wall compared with other regions.4 magnitude appeared not to be modified, the current density-voltage curves were slightly shifted to more positive potentials and significantly decreased as compared to normal cells (in pA/pF, at + 60 mV): 8-4 + 5-0 in the left free wall group, 1 1-6 + 2-0 in the apex group, and 3-8+ 1-5 in the septum group. Steady-state activation and inactivation parameters were not clearly modified, but kinetics were slowed down.7. We conclude, therefore, that Ito is differentially distributed among different regions of the normal rat ventricle and we propose that this regional heterogeneity may be related to different distributions of functional channel densities, rather than alterations in whole-cell kinetics or single-channel properties. Pressure overloadinduced hypertrophy reduces Ito current availability by decreasing current densities without any significant change of whole-cell kinetics, while a homogenizing tendency of the ionic profile is observed among the studied regions. One possible explanation for the hypertrophy-induced variations may be an absence of Ito channel neosynthesis, leading to a decrease of channel density per surface area unit.

SUMMARYIn Drosophila, a population of muscle-committed stem-like cells called adult muscle precursors (AMPs) keeps an undifferentiated and quiescent state during embryonic life. The embryonic AMPs are at the origin of all adult fly muscles and, as we demonstrate here, they express repressors of myogenic differentiation and targets of the Notch pathway known to be involved in muscle cell stemness. By targeting GFP to the AMP cell membranes, we show that AMPs are tightly associated with the peripheral nervous system and with a subset of differentiated muscles. They send long cellular processes running along the peripheral nerves and, by the end of embryogenesis, form a network of interconnected cells. Based on evidence from laser ablation experiments, the main role of these cellular extensions is to maintain correct spatial positioning of AMPs. To gain insights into mechanisms that lead to AMP cell specification, we performed a gain-of-function screen with a special focus on lateral AMPs expressing the homeobox gene ladybird. Our data show that the rhomboid-triggered EGF signalling pathway controls both the specification and the subsequent maintenance of AMP cells. This finding is supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Taken together, our results reveal an unsuspected capacity of embryonic AMPs to form a cell network, and shed light on the mechanisms governing their specification and maintenance.