The Drosophila insulin pathway controls Profilin expression and dynamic actin-rich protrusions during collective cell migration.

Understanding how different cell types acquire their motile behaviour is central to many normal and pathological processes. Drosophila border cells represent a powerful model for addressing this issue and to specifically decipher the mechanisms controlling collective cell migration. Here, we identify the Drosophila Insulin/Insulin-like growth factor signalling (IIS) pathway as a key regulator in controlling actin dynamics in border cells, independently of its function in growth control. Loss of IIS activity blocks the formation of actin-rich long cellular extensions that are important for the delamination and the migration of the invasive cluster. We show that IIS specifically activates the expression of the actin regulator chickadee, the Drosophila homolog of Profilin, which is essential for promoting the formation of actin extensions and migration through the egg chamber. In this process, the transcription factor FoxO acts as a repressor of chickadee expression. Altogether, these results show that local activation of IIS controls collective cell migration through regulation of actin homeostasis and protrusion dynamics.

Starvation induces FoxO-dependent mitotic-to-endocycle switch pausing during Drosophila oogenesis.

When exposed to nutrient challenge, organisms have to adapt their physiology in order to balance reproduction with adult fitness. In mammals, ovarian follicles enter a massive growth phase during which they become highly dependent on gonadotrophic factors and nutrients. Somatic tissues play a crucial role in integrating these signals, controlling ovarian follicle atresia and eventually leading to the selection of a single follicle for ovulation. We used Drosophila follicles as a model to study the effect of starvation on follicle maturation. Upon starvation, Drosophila vitellogenic follicles adopt an 'atresia-like' behavior, in which some slow down their development whereas others enter degeneration. The mitotic-to-endocycle (M/E) transition is a critical step during Drosophila oogenesis, allowing the entry of egg chambers into vitellogenesis. Here, we describe a specific and transient phase during M/E switching that is paused upon starvation. The Insulin pathway induces the pausing of the M/E switch, blocking the entry of egg chambers into vitellogenesis. Pausing of the M/E switch involves a previously unknown crosstalk between FoxO, Cut and Notch that ensures full reversion of the process and rapid resumption of oogenesis upon refeeding. Our work reveals a novel genetic mechanism controlling the extent of the M/E switch upon starvation, thus integrating metabolic cues with development, growth and reproduction.

Drosophila RalA is essential for the maintenance of Jak/Stat signalling in ovarian follicles.

Small GTPases of the Ras-like (Ral) family are crucial for signalling functions in both normal and cancer cells; however, their role in a developing organism is poorly understood. Here, we identify the Drosophila Ral homologue RalA as a new key regulator of polar-cell differentiation during oogenesis. Polar cells have a crucial role in patterning the egg chamber and in recruiting border cells, which undergo collective and guided migration. We show that RalA function is essential for the maintenance of anterior and posterior polar-cell fate and survival. RalA is required cell autonomously to control the expression of polar-cell-specific markers, including the Jak/Stat ligand Unpaired. The loss of RalA also causes a cell non-autonomous phenotype owing to reduced Jak/Stat signalling in neighbouring follicle cells. As a result, border-cell assembly and migration as well as the polarization of the oocyte are defective. Thus, RalA is required in organizing centres to control proper patterning and migration in vivo.

The endocytic control of JAK/STAT signalling in Drosophila.

Domeless (Dome) is an IL-6-related cytokine receptor that activates a conserved JAK/STAT signalling pathway during Drosophila development. Despite good knowledge of the signal transduction pathway in several models, the role of receptor endocytosis in JAK/STAT activation remains poorly understood. Using both in vivo genetic analysis and cell culture assays, we show that ligand binding of Unpaired 1 (Upd1) induces clathrin-dependent endocytosis of receptor-ligand complexes and their subsequent trafficking through the endosomal compartment towards the lysosome. Surprisingly, blocking trafficking in distinct endosomal compartments using mutants affecting either Clathrin heavy chain, rab5, Hrs or deep orange led to an inhibition of the JAK/STAT pathway, whereas this pathway was unchanged when rab11 was affected. This suggests that internalization and trafficking are both required for JAK/STAT activity. The requirement for clathrin-dependent endocytosis to activate JAK/STAT signalling suggests a model in which the signalling 'on' state relies not only on ligand binding to the receptor at the cell surface, but also on the recruitment of the complex into endocytic vesicles on their way to lysozomes. Selective activation of the pool of receptors marked for degradation thus provides a way to tightly control JAK/STAT activity.

The Drosophila cytokine receptor Domeless controls border cell migration and epithelial polarization during oogenesis.

In mammals, the JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is activated in response to cytokines and growth factors to control blood cell development, proliferation and cell determination. In Drosophila, a conserved JAK/STAT signaling pathway controls segmentation in embryos, as well as blood cell development and other processes in larvae and adults. During embryogenesis, transduction of the Unpaired [Upd; also known as Outstretched (Os)] ligand through the JAK/STAT pathway requires Domeless, a putative membrane protein with distant homology to vertebrate type I cytokine receptors. We have isolated domeless (dome) in a screen to identify genes essential in epithelial morphogenesis during oogenesis. The level of dome activity is critical for proper border cell migration and is controlled in part through a negative feedback loop. In addition to its essential role in border cells, we show that dome is required in the germarium for the polarization of follicle cells during encapsulation of germline cells. In this process, dome controls the expression of the apical determinant Crumbs. In contrast to the ligand Upd, whose expression is limited to a pair of polar cells at both ends of the egg chamber, dome is expressed in all germline and follicle cells. However, the Dome protein is specifically localized at apicolateral membranes and undergoes ligand-dependent internalization in the follicle cells. dome mutations interact genetically with JAK/STAT pathway genes in border cell migration and abolish the nuclear translocation of Stat92E in vivo. We also show that dome functions downstream of upd and that both the extracellular and intracellular domains of Dome are required for JAK/STAT signaling. Altogether, our data indicate that Dome is an essential receptor molecule for Upd and JAK/STAT signaling during oogenesis.

Mechanism of activation of the Drosophila EGF Receptor by the TGFalpha ligand Gurken during oogenesis.

We have analyzed the mechanism of activation of the Epidermal growth factor receptor (Egfr) by the transforming growth factor (TGF) alpha-like molecule, Gurken (Grk). Grk is expressed in the oocyte and activates the Egfr in the surrounding follicle cells during oogenesis. We show that expression of either a membrane bound form of Grk (mbGrk), or a secreted form of Grk (secGrk), in either the follicle cells or in the germline, activates the Egfr. In tissue culture cells, both forms can bind to the Egfr; however, only the soluble form can trigger Egfr signaling, which is consistent with the observed cleavage of Grk in vivo. We find that the two transmembrane proteins Star and Brho potentiate the activity of mbGrk. These two proteins collaborate to promote an activating proteolytic cleavage and release of Grk. After cleavage, the extracellular domain of Grk is secreted from the oocyte to activate the Egfr in the follicular epithelium.

Mechanism of inhibition of the Drosophila and mammalian EGF receptors by the transmembrane protein Kekkon 1.

The transmembrane protein Kekkon 1 (Kek1) has previously been shown to act in a negative feedback loop to downregulate the Drosophila Epidermal Growth Factor Receptor (DER) during oogenesis. We show that this protein plays a similar role in other DER-mediated developmental processes. Structure-function analysis reveals that the extracellular Leucine-Rich Repeat (LRR) domains of Kek1 are critical for its function through direct association with DER, whereas its cytoplasmic domain is required for apical subcellular localization. In addition, the use of chimeric proteins between Kek1 extracellular and transmembrane domains fused to DER intracellular domain indicates that Kek1 forms an heterodimer with DER in vivo. To characterize more precisely the mechanism underlying the Kek1/DER interaction, we used mammalian ErbB/EGFR cell-based assays. We show that Kek1 is capable of physically interacting with each of the known members of the mammalian ErbB receptor family and that the Kek1/EGFR interaction inhibits growth factor binding, receptor autophosphorylation and Erk1/2 activation in response to EGF. Finally, in vivo experiments show that Kek1 expression potently suppresses the growth of mouse mammary tumor cells derived from aberrant ErbB receptors activation, but does not interfere with the growth of tumor cells derived from activated Ras. Our results underscore the possibility that Kek1 may be used experimentally to inhibit ErbB receptors and point to the possibility that, as yet uncharacterized, mammalian transmembrane LRR proteins might act as modulators of growth factor signalling.