Fascin links Btl/FGFR signalling to the actin cytoskeleton during Drosophila tracheal morphogenesis

Abstract

A key challenge in normal development and in disease is to elucidate the mechanisms of cell migration. Here we approach this question using the tracheal system of Drosophila as a model. Tracheal cell migration requires the Breathless/FGFR pathway; however, how the pathway induces migration remains poorly understood. We find that the Breathless pathway upregulates singed at the tip of tracheal branches, and that this regulation is functionally relevant. singed encodes Drosophila Fascin, which belongs to a conserved family of actin-bundling proteins involved in cancer progression and metastasis upon misregulation. We show that singed is required for filopodia stiffness and proper morphology of tracheal tip cells, defects that correlate with an abnormal actin organisation. We propose that singed-regulated filopodia and cell fronts are required for timely and guided branch migration and for terminal branching and branch fusion. We find that singed requirements rely on its actin-bundling activity controlled by phosphorylation, and that active Singed can promote tip cell features. Furthermore, we find that singed acts in concert with forked, another actin cross-linker. The absence of both cross-linkers further stresses the relevance of tip cell morphology and filopodia for tracheal development. In summary, our results on the one hand reveal a previously undescribed role for forked in the organisation of transient actin structures such as filopodia, and on the other hand identify singed as a new target of Breathless signal, establishing a link between guidance cues, the actin cytoskeleton and tracheal morphogenesis.

Slik and the Receptor Tyrosine Kinase Breathless Mediate Localized Activation of Moesin in Terminal Tracheal Cells

“…A number of cytoskeleton-organizing molecules have been described as critical regulators of subcellular tube formation, tube stabilization and branching of terminal tracheal cells [35], [63], [76], [79]–[81].…”

“…This involves actin organization in filopodia at the migrating fronts of the embryonic terminal cells [63]. Singed is also transcriptionally upregulated by Btl in terminal cells [63].…”

“…A fibroblast growth factor signaling pathway, using the ligand Branchless (FGF) and the receptor Breathless (Btl), and operating through the canonical Ras/Raf/MEK/MAPK cascade, is used repeatedly during the different stages of tracheal development [54], [55], [57]–[59]. Several transcriptional targets of Bnl/Btl signaling have been identified in tracheal cells, including actin organizers such as Singed and Serum Response Factor [50], [55], [60]–[63].…”

Cellular and physical mechanisms of branching morphogenesis

“…Live imaging has revealed that Bnl induces tip cells to undergo cytoskeletal reorganization, enriching actin at their basal surface (Lebreton and Casanova, 2014). Bnl also induces the activation of Rac and the expression of fascin, an actin-bundling protein required for the formation of filopodia at the leading edge (Okenve-Ramos and Llimargas, 2014). By contrast, the trailing stalk cells exhibit high levels of actin at their apical surfaces (Lebreton and Casanova, 2014).…”

Origin and diversification of wings: Insights from a neopteran insect

“…Also absent from T1 EW (Table S2) was a wing-specific isoform of salimus (sls), a gene involved in muscle development and attachment (27)(28)(29) that in Drosophila is coexpressed with trh in ventral-origin cells. Another tracheal morphogenesis gene, forked (f) (30), involved in formation of mechanosensory wing bristles (31), was among the wing-specific genes missing from T1 EW. Additional trh-regulated genes (vvl, spalt, and Stat92E) (26) and the ventral-origin wing specification gene nubbin (nub) (32) were detectable but reduced in T1 EW compared with T2 wing (P = 0.0001; mean difference > twofold) (Fig.…”