Transcriptional regulation of macrophage migration in the Drosophila melanogaster embryo

Immune cells migrating to the sites of infection navigate through diverse tissue architectures and switch their migratory mechanisms upon demand. However, little is known about systemic regulators that could allow the acquisition of these mechanisms. We performed a genetic screen in Drosophila melanogaster to identify regulators of germband invasion by embryonic macrophages into the confined space between the ectoderm and mesoderm. We have found that bZIP circadian transcription factors (TFs) Kayak (dFos) and Vrille (dNFIL3) have opposite effects on macrophage germband infiltration: Kayak facilitated and Vrille inhibited it. These TFs are enriched in the macrophages during migration and genetically interact to control it. Kayak sets a less coordinated mode of migration of the macrophage group and increases the probability and length of Levy walks. Intriguingly, the motility of kayak mutant macrophages was also strongly affected during initial germband invasion but not along another less confined route. Inhibiting Rho1 signaling within the tail ectoderm partially rescued the Kayak mutant phenotype, strongly suggesting that migrating macrophages have to overcome a barrier imposed by the stiffness of the ectoderm. Also, Kayak appeared to be important for the maintenance of the round cell shape and the rear edge translocation of the macrophages invading the germband. Complementary to this, the cortical actin cytoskeleton of Kayak- deficient macrophages was strongly affected. RNA sequencing revealed the filamin Cheerio and tetraspanin TM4SF to be downstream of Kayak. Chromatin immunoprecipitation and immunostaining revealed that the formin Diaphanous is another downstream target of Kayak. Immunostaining revealed that the formin Diaphanous is another downstream target of Kayak. Indeed, Cheerio, TM4SF and Diaphanous are required within macrophages for germband invasion, and expression of constitutively active Diaphanous in macrophages was able to rescue the kayak mutant phenotype. Moreover, Cher and Diaphanous are also reduced in the macrophages overexpressing Vrille. We hypothesize that Kayak, through its targets, increases actin polymerization and cortical tension in macrophages and thus allows extra force generation necessary for macrophage dissemination and migration through confined stiff tissues, while Vrille counterbalances it.