γ-TuRCs and the augmin complex are required for the development of highly branched dendritic arbors in Drosophila.

Microtubules are nucleated by γ-tubulin ring complexes (γ-TuRCs) and are essential for neuronal development. Nevertheless, γ-TuRC depletion has been reported to perturb only higher-order branching in elaborated Drosophila larval class IV dendritic arborization (da) neurons. This relatively mild phenotype has been attributed to defects in microtubule nucleation from Golgi outposts, yet most Golgi outposts lack associated γ-TuRCs. By analyzing dendritic arbor regrowth in pupae, we show that γ-TuRCs are also required for the growth and branching of primary and secondary dendrites, as well as for higher-order branching. Moreover, we identify the augmin complex (hereafter augmin), which recruits γ-TuRCs to the sides of pre-existing microtubules, as being required predominantly for higher-order branching. Augmin strongly promotes the anterograde growth of microtubules in terminal dendrites and thus terminal dendrite stability. Consistent with a specific role in higher-order branching, we find that augmin is expressed less strongly and is largely dispensable in larval class I da neurons, which exhibit few higher-order dendrites. Thus, γ-TuRCs are essential for various aspects of complex dendritic arbor development, and they appear to function in higher-order branching via the augmin pathway, which promotes the elaboration of dendritic arbors to help define neuronal morphology.

Multifaceted modes of γ-tubulin complex recruitment and microtubule nucleation at mitotic centrosomes.

Microtubule nucleation is mediated by γ-tubulin ring complexes (γ-TuRCs). In most eukaryotes, a GCP4/5/4/6 "core" complex promotes γ-tubulin small complex (γ-TuSC) association to generate cytosolic γ-TuRCs. Unlike γ-TuSCs, however, this core complex is non-essential in various species and absent from budding yeasts. In Drosophila, Spindle defective-2 (Spd-2) and Centrosomin (Cnn) redundantly recruit γ-tubulin complexes to mitotic centrosomes. Here, we show that Spd-2 recruits γ-TuRCs formed via the GCP4/5/4/6 core, but Cnn can recruit γ-TuSCs directly via its well-conserved CM1 domain, similar to its homologs in budding yeast. When centrosomes fail to recruit γ-tubulin complexes, they still nucleate microtubules via the TOG domain protein Mini-spindles (Msps), but these microtubules have different dynamic properties. Our data, therefore, help explain the dispensability of the GCP4/5/4/6 core and highlight the robustness of centrosomes as microtubule organizing centers. They also suggest that the dynamic properties of microtubules are influenced by how they are nucleated.

High hedgehog signaling is transduced by a multikinase-dependent switch controlling the apico-basal distribution of the GPCR smoothened.

The oncogenic G-protein-coupled receptor (GPCR) Smoothened (SMO) is a key transducer of the hedgehog (HH) morphogen, which plays an essential role in the patterning of epithelial structures. Here, we examine how HH controls SMO subcellular localization and activity in a polarized epithelium using the Drosophila wing imaginal disc as a model. We provide evidence that HH promotes the stabilization of SMO by switching its fate after endocytosis toward recycling. This effect involves the sequential and additive action of protein kinase A, casein kinase I, and the Fused (FU) kinase. Moreover, in the presence of very high levels of HH, the second effect of FU leads to the local enrichment of SMO in the most basal domain of the cell membrane. Together, these results link the morphogenetic effects of HH to the apico-basal distribution of SMO and provide a novel mechanism for the regulation of a GPCR.

Engrailed, Suppressor of fused and Roadkill modulate the Drosophila GLI transcription factor Cubitus interruptus at multiple levels.

Morphogen gradients need to be robust, but may also need to be tailored for specific tissues. Often this type of regulation is carried out by negative regulators and negative feedback loops. In the Hedgehog (Hh) pathway, activation of patched (ptc) in response to Hh is part of a negative feedback loop limiting the range of the Hh morphogen. Here, we show that in the Drosophila wing imaginal disc two other known Hh targets genes feed back to modulate Hh signaling. First, anterior expression of the transcriptional repressor Engrailed modifies the Hh gradient by attenuating the expression of the Hh pathway transcription factor cubitus interruptus (ci), leading to lower levels of ptc expression. Second, the E-3 ligase Roadkill shifts the competition between the full-length activator and truncated repressor forms of Ci by preferentially targeting full-length Ci for degradation. Finally, we provide evidence that Suppressor of fused, a negative regulator of Hh signaling, has an unexpected positive role, specifically protecting full-length Ci but not the Ci repressor from Roadkill.

Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused.

From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.

Dose-dependent transduction of Hedgehog relies on phosphorylation-based feedback between the G-protein-coupled receptor Smoothened and the kinase Fused.

Smoothened (SMO) is a G-protein-coupled receptor-related protein required for the transduction of Hedgehog (HH). The HH gradient leads to graded phosphorylation of SMO, mainly by the PKA and CKI kinases. How thresholds in HH morphogen regulate SMO to promote switch-like transcriptional responses is a central unsolved issue. Using the wing imaginal disc model in Drosophila, we identified new SMO phosphosites that enhance the effects of the PKA/CKI kinases on SMO accumulation, its localization at the plasma membrane and its activity. Surprisingly, phosphorylation at these sites is induced by the kinase Fused (FU), a known downstream effector of SMO. In turn, activation of SMO induces FU to act on its downstream targets. Overall, our data provide evidence for a SMO/FU positive regulatory loop nested within a multikinase phosphorylation cascade. We propose that this complex interplay amplifies signaling above a threshold that allows high HH signaling.

Notch-mediated repression of bantam miRNA contributes to boundary formation in the Drosophila wing.

Subdivision of proliferating tissues into adjacent compartments that do not mix plays a key role in animal development. The Actin cytoskeleton has recently been shown to mediate cell sorting at compartment boundaries, and reduced cell proliferation in boundary cells has been proposed as a way of stabilizing compartment boundaries. Cell interactions mediated by the receptor Notch have been implicated in the specification of compartment boundaries in vertebrates and in Drosophila, but the molecular effectors remain largely unidentified. Here, we present evidence that Notch mediates boundary formation in the Drosophila wing in part through repression of bantam miRNA. bantam induces cell proliferation and we have identified the Actin regulator Enabled as a new target of bantam. Increased levels of Enabled and reduced proliferation rates contribute to the maintenance of the dorsal-ventral affinity boundary. The activity of Notch also defines, through the homeobox-containing gene cut, a distinct population of boundary cells at the dorsal-ventral (DV) interface that helps to segregate boundary from non-boundary cells and contributes to the maintenance of the DV affinity boundary.

A role of receptor Notch in ligand cis-inhibition in Drosophila.

Notch and its ligands mediate short-range cell interactions that play a conserved role in inducing cell fate specification. Several regulatory mechanisms have been described to ensure robust polarized signaling from signal-sending to signal-receiving cells. High levels of ligand expression activate Notch in nearby cells and exert a cell-autonomous dominant-negative effect on Notch activity. This regulatory process is called cis-inhibition and helps to restrict Notch activation to signal-receiving cells. By combining genetic mosaics in the Drosophila wing primordium with cell culture assays, we present evidence here that Notch promotes the clearance of Serrate ligand from the cell surface and exerts an inhibitory effect on the activity of Serrate expressed in the same cell. These regulatory mechanisms are independent of Notch-mediated transcription and are executed by the extracellular domain of Notch. We show that this process is required to block Serrate-mediated activation of Notch in the signal-sending cell population and helps to restrict Notch activation to the signal-receiving cells. Altogether, our results, in concert with previous results on ligand-mediated Notch cis-inhibition, indicate that mutual inhibition between ligand and receptor in signal-sending cells helps to block Notch activity in these cells and to restrict receptor activation in signal-receiving cells.

A permissive role of Notch in maintaining the DV affinity boundary of the Drosophila wing.

Cell affinities can contribute to organizing cells into tissues and organs. Drosophila limbs and the vertebrate central nervous system are subdivided into adjacent populations that do not mix. These cell populations are called compartments. Cell interactions mediated by receptor Notch have been implicated in the specification of compartment boundaries. However, the contribution of Notch in this process remains controversial. The instructive role of Notch and the transcriptional requirement of the pathway have been questioned in the last few years. Due to its central role in making developmental boundaries in vertebrates and invertebrates, we have reevaluated the contribution of Notch and its signaling pathway in the maintenance of an affinity difference between dorsal (D) and ventral (V) compartments in the Drosophila wing. Here we present evidence that unrestricted, low levels of Notch are sufficient for the formation of a stable DV affinity boundary. Cleavage of the Notch protein, release of the intracellular domain and a transcriptional function of Notch via the Suppressor of Hairless transcription factor are required and sufficient in this process. Our data support a permissive role of Notch in maintaining the DV affinity boundary. This contrasts with the instructive role of Notch in executing the organizing activity of the DV boundary.

[Genetic control of intercellular adhesion or how cadherins shape the fruitfly Drosophila melanogaster]. / Régulation génétique de l'adhérence intercellulaire ou comment les cadhérines sculptent la drosophile.

The beauty and diversity of cell shapes have always fascinated both biologists and physicists. In the early 1950, J. Holtfreter coined the term "tissue affinities" to describe the forces behind the spontaneous shaping of groups of cells. These tissue affinites were later on related to adhesive properties of cell membranes. In the 1960, Malcom Steinberg proposed the differential adhesion hypothesis (DAH) as a physical explanation of the liquid-like behaviour of tissues and cells during morphogenesis. However, the link between the cellular properties of adhesion molecules, such as the cadherins, and the physical rules that shape the body, has remained unclear. Recent in vitro studies have now shown that surface tensions, which drive the spontaneous liquid-like behaviour of cell rearrangements, are a direct and linear function of cadherin expression levels. Tissue surface tensions thus arise from differences in intercellular adhesiveness, which validates the DAH in vitro. The DAH was also vindicated in vivo by stunning experiments in Drosophila. The powerful genetic tools available in Drosophila allow to manipulate the levels and patterns of expression of several cadherins and to create artificially differences in intercellular adhesiveness. The results showed that simple laws of thermodynamics, as well as quantitative and qualitative differences in cadherins expression were sufficient to explain processes as complex as the establishment of the anterior-posterior axis and the formation of the compound eye in Drosophila.

Control of G2/M transition by Drosophila Fos.

The transcription factors of the Fos family have long been associated with the control of cell proliferation, although the molecular and cellular mechanisms that mediate this function are poorly understood. We investigated the contributions of Fos to the cell cycle and cell growth control using Drosophila imaginal discs as a genetically accessible system. The RNA interference-mediated inhibition of Fos in proliferating cells of the wing and eye discs resulted in a specific defect in the G2-to-M-phase transition, while cell growth remained unimpaired, resulting in a marked reduction in organ size. Consistent with the conclusion that Fos is required for mitosis, we identified cyclin B as a direct transcriptional target of Fos in Drosophila melanogaster, with Fos binding to a region upstream of the cyclin B gene in vivo and cyclin B mRNA being specifically reduced under Fos loss-of-function conditions.

Integrin-independent repression of cadherin transcription by talin during axis formation in Drosophila.

The Drosophila melanogaster anterior-posterior axis becomes polarized early during oogenesis by the posterior localization of the oocyte within the egg chamber. The invariant position of the oocyte is thought to be driven by an upregulation of the adhesion molecule DE-cadherin in the oocyte and the posterior somatic follicle cells, providing the first in vivo example of cell sorting that is specified by quantitative differences in cell-cell adhesion. However, it has remained unclear how DE-cadherin levels are regulated. Here, we show that talin, known for its role in linking integrins to the actin cytoskeleton, has the unexpected function of specifically inhibiting DE-cadherin transcription. Follicle cells that are mutant for talin show a strikingly high level of DE-cadherin, due to elevated transcription of DE-cadherin. We demonstrate that this deregulation of DE-cadherin is sufficient to attract the oocyte to lateral and anterior positions. Surprisingly, this function of talin is independent of integrins. These results uncover a new role for talin in regulating cadherin-mediated cell adhesion.