A research project* at the IBDM (Marseille Developmental Biology Institute, CNRS – AMU), led by Clément Rodier, has recently challenged long-standing scientific hypotheses about how muscles elongate during development, thanks to advanced microscopy. Let’s take a closer look!
Sarcomeres, the “bricks” of muscles
Striated muscles are made up of muscle fibers, themselves composed of myofibrils, chains of thousands of contractile “bricks” called sarcomeres. In mammals, a sarcomere measures between 2 and 3 micrometers.
As an organism grows, its muscles must expand to keep pace with skeletal development. Yet sarcomeres maintain roughly the same size. For decades, the prevailing hypothesis held that new bricks, sarcomeres, were added exclusively at the ends of myofibril chains. However, it had never been directly observed. Clément Rodier’s goal was to visualize, using both light and electron microscopy**, how new sarcomeres are inserted during muscle growth, with the fly Drosophila as a model system.
A scientific hypothesis challenged
The research team analyzed Drosophila flight muscles between 32 and 40 hours after puparium formation, the developmental stage following the larval phase. This model has a key advantage: its growth is extremely fast (around 100 new sarcomeres are added in just 4 hours!) making the mechanism easier to capture.
Contrary to the initial hypothesis, microscopy images showed that sarcomere insertion was not restricted to the ends of the myofibrillar chain. Each brick is capped by terminal structures called Z-discs, which should have shown changes if new sarcomeres were only added there.
(B) Schematic of a Drosophila hemithorax at pupal stages with the six dorsal longitudinal flight muscles (DLMs) in magenta and the tendon cell epithelium in green. DLM4 is highlighted and was used for quantifications in (E). The stable connection of the flight muscles to the tendon cell extensions (green lines) during the growth phase from 32 to 40 hours after puparium formation (APF). h, hours. (C and D) Confocal images of pupae of the indicated stages displaying the six flight muscles stained for actin (phalloidin in red) and Shot (anti-Shortstop in gray, also labeling the tendon extensions) in (C). High magnifications displaying the myofibrils stained for actin (red) and myosin [anti–Mhc (myosin heavy chain) in blue] in (D). Scale bars, 100 μm in (C) and 5 μm in (D). Note the marked muscle length increases after 32 hours APF.
(B and C) About 34 hours APF, a pupa expressing Sls-GFP with a focus on the anterior flight muscle end. Stills from movie S1 are shown (B), and a kymograph of a selected region is shown (C). The myofibril ends, marked with yellow arrowheads, move to the left, while muscle fiber length increases and thus moves away from the reference point marked with the orange arrowhead. No obvious sarcomere addition is seen at the ends.
So how do muscles really grow?
To probe this mechanism further, the team developed a computational tool capable of analyzing the length of thousands of sarcomeres and identifying those with abnormal size.
Using this tool combined with high-resolution microscopy techniques (sush as in vivo bi-photon imaging), the researchers observed that some sarcomeres gradually elongated and then split into two daughter sarcomeres of normal length. The essential proteins needed for proper sarcomere function were also restored.
Unlike the long-accepted end hypothesis, this division mechanism allows new sarcomeres to be inserted at multiple points along the myofibril.
Fig. 7. Sarcomere division model. Working model of the tension-induced sarcomere division in Drosophila flight muscle sarcomeres. The growth of the skeleton, marked by integrin-based attachments, induces high tension, which triggers the division of the highlighted middle sarcomere. Thus, the sarcomere number increases from three to four. For details, see Discussion.
Does this mechanism also occur in mammals?
The researchers next turned to 3D electron microscopy and FRAP to test whether this mechanism also applied to other muscle types, namely Drosophila larval muscles, which are similar to mammalian skeletal muscles.
These imaging techniques revealed a zipper-like division process of sarcomeres during larval development. Further analyses confirmed the recruitment of new proteins that reestablish proper function in the daughter sarcomeres.
Looking ahead
Thanks to the combination of light and electron microscopy, both realized on France-BioImaging’s core facilities*, the team was able to put a long-standing hypothesis to the test… and overturn it! This work sheds new light on developmental mechanisms in insects, and possibly in mammals, including humans. Better understanding how muscles grow at the microscopic level could one day pave the way for novel therapies to fight muscle degenerative diseases.
* Clement Rodier et al., Muscle growth by sarcomere divisions. Sci. Adv.11, eadw9445 (2025). DOI:10.1126/sciadv.adw9445
** Electronic microscopy (3D EM) was performed on a PICsL core facility and advanced photonic microscopy (FRAP imaging, in vivo bi-photon imaging) on IBDM core facility.
