Slit cleavage is essential for producing an active, stable, non-diffusible short-range signal that guides muscle migration

Abstract

During organogenesis, secreted signaling proteins direct cell migration towards their target tissue. In Drosophila embryos, developing muscles are guided by signals produced by tendons to promote the proper attachment of muscles to tendons, essential for proper locomotion. Previously, the repulsive protein Slit, secreted by tendon cells, has been proposed to be an attractant for muscle migration. However, our findings demonstrate that through tight control of its distribution, Slit repulsion is used for both directing and arresting muscle migration. We show that Slit cleavage restricts its distribution to tendon cells, allowing it to function as a short-range repellent that directs muscle migration and patterning, and promotes their halt upon reaching the target site. Mechanistically, we show that Slit processing produces a rapidly degraded C-terminal fragment and an active, stable N-terminal polypeptide that is tethered to the tendon cell membrane, which further protects it from degradation. Consistently, the requirement for Slit processing can be bypassed by providing an uncleavable, membrane-bound form of Slit that is stable and is retained on expressing tendon cells. Moreover, muscle elongation appears to be extremely sensitive to Slit levels, as replacing the entire full-length Slit with the stable Slit-N-polypeptide results in excessive repulsion, which leads to a defective muscle pattern. These findings reveal a novel cleavage-dependent regulatory mechanism controlling Slit spatial distribution, which may operate in other Slit-dependent processes.

A non-signaling role of Robo2 in tendons is essential for Slit processing and muscle patterning

“…In slit mutant embryos, the LT muscles are closer to the posterior segment border and are not spaced correctly, and the DA3 muscle does not extend its leading edge in an orthogonal fashion (Fig. 1A-B′) (Ordan et al, 2015).…”

“…Active cross-talk between tendons and elongating muscles has been demonstrated in Drosophila, where an important tendonspecific guidance signal is the secreted protein Slit (Kramer et al, 2001;Ordan et al, 2015;Wayburn and Volk, 2009). The contribution of the different Slit receptors, namely Robo1 (also known as Robo), Robo2 and Robo3, to muscle patterning remains elusive.…”

“…Recently, we showed that, in tendons, fulllength Slit (Slit-FL) undergoes cleavage, creating an active, stable N-terminal polypeptide (Slit-N), which associates with the tendon cell surface, thus limiting the range of Slit signaling. Moreover, Slit cleavage at the surface of tendon cells was found to be crucial for short-range muscle repulsion, as well as for providing a stop signal for LT and DA3 muscles (Ordan et al, 2015); however, the molecular mechanism promoting localized Slit cleavage has yet to be elucidated. Here, we show that, for muscle guidance, Robo2 activity is required in tendon cells and not in muscles.…”

Cleaved Slit directs embryonic muscles

“…The immobilized cleaved Slit induces short-range repulsion that directs the elongating muscles to their final destination, and provides a stop signal once the entire muscle leading edge is in contact with Slit on the tendon cell membrane. 10 Moreover, we propose that, whereas Robo and Robo3 cooperate to respond to Slit signaling at the muscle leading edge, Robo2 is uniquely expressed by the tendon cells; its activity on these cells contributes to Slit cleavage and does not require its signaling cytoplasmic tail.…”

“…7 Alternatively, our recent work Keywords: muscle, myotendinous jucntion, Robo, Slit, Slit cleavage suggests that Slit represents a short-range repellent and provides a stop signal for the approaching elongating muscle, and thus mechanistically contributes to the collapse of leading edge filopodia, similar to its function in neurons. 10 Slit contains a highly conserved cleavage site and undergoes cleavage in vivo in both vertebrates and invertebrates. Slit cleavage produces 2 polypeptides; the N-terminal (Slit-N) contains 4 Leucine-Rich-Repeats (LRR), which includes the Robo binding domain, and 5 EGF repeats; the C-terminal polypeptide includes a laminin G domain and additional 4 EGF repeats.…”