Stéphane Noselli

Type ID unconventional myosin controls left–right asymmetry in Drosophila

Breaking left–right symmetry in Bilateria embryos is a major event in body plan organization that leads to polarized adult morphology, directional organ looping, and heart and brain function. However, the molecular nature of the determinant(s) responsible for the invariant orientation of the left–right axis (situs choice) remains largely unknown. Mutations producing a complete reversal of left–right asymmetry (situs inversus) are instrumental for identifying mechanisms controlling handedness, yet only one such mutation has been found in mice (inversin) and snails. Here we identify the conserved type ID unconventional myosin 31DF gene (Myo31DF) as a unique situs inversus locus in Drosophila. Myo31DF mutations reverse the dextral looping of genitalia, a prominent left–right marker in adult flies. Genetic mosaic analysis pinpoints the A8 segment of the genital disc as a left–right organizer and reveals an anterior–posterior compartmentalization of Myo31DF function that directs dextral development and represses a sinistral default state. As expected of a determinant, Myo31DF has a trigger-like function and is expressed symmetrically in the organizer, and its symmetrical overexpression does not impair left–right asymmetry. Thus Myo31DF is a dextral gene with actin-based motor activity controlling situs choice. Like mouse inversin, Myo31DF interacts and colocalizes with β-catenin, suggesting that situs inversus genes can direct left–right development through the adherens junction.

An unconventional myosin in Drosophila reverses the default handedness in visceral organs

The internal organs of animals often have left–right asymmetry. Although the formation of the anterior–posterior and dorsal–ventral axes in Drosophila is well understood, left–right asymmetry has not been extensively studied. Here we find that the handedness of the embryonic gut and the adult gut and testes is reversed (not randomized) in viable and fertile homozygous Myo31DF mutants. Myo31DF encodes an unconventional myosin, Drosophila MyoIA (also referred to as MyoID in mammals; refs 3, 4), and is the first actin-based motor protein to be implicated in left–right patterning. We find that Myo31DF is required in the hindgut epithelium for normal embryonic handedness. Disruption of actin filaments in the hindgut epithelium randomizes the handedness of the embryonic gut, suggesting that Myo31DF function requires the actin cytoskeleton. Consistent with this, we find that Myo31DF colocalizes with the cytoskeleton. Overexpression of Myo61F, another myosin I (ref. 4), reverses the handedness of the embryonic gut, and its knockdown also causes a left–right patterning defect. These two unconventional myosin I proteins may have antagonistic functions in left–right patterning. We suggest that the actin cytoskeleton and myosin I proteins may be crucial for generating left–right asymmetry in invertebrates.

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.

The retinoic-like juvenile hormone controls the looping of left-right asymmetric organs in Drosophila

In vertebrate development, the establishment of left-right asymmetry is essential for sidedness and the directional looping of organs like the heart. Both the nodal pathway and retinoic acid play major and conserved regulatory roles in these processes. We carried out a novel screen in Drosophila to identify mutants that specifically affect the looping of left-right asymmetric organs. We report the isolation of spin, a novel mutant in which the looping of the genitalia and spermiduct are incomplete; under-rotation of the genitalia indicates that spin controls looping morphogenesis but not direction, thus uncoupling left-right asymmetry and looping morphogenesis. spin is a novel, rotation-specific allele of the fasciclin2 (Fas2) gene, which encodes a cell-adhesion protein involved in several aspects of neurogenesis. In spin mutants, the synapses connecting specific neurosecretory cells to the corpora allata are affected. The corpus allatum is part of the ring gland and is involved in the control of juvenile hormone titers during development. Our genetic and pharmacological results indicate that Fas2spin rotation defects are linked to an abnormal endocrine function and an elevated level of juvenile hormone. As juvenile hormone is an insect sesquiterpenoid related to retinoic acid, these results establish a new genetic model for studying organ looping and demonstrate an evolutionarily conserved role for terpenoids in this process.

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.

Coupling of Apoptosis and L/R Patterning Controls Stepwise Organ Looping

Handed asymmetry in organ shape and positioning is a common feature among bilateria, yet little is known about the morphogenetic mechanisms underlying left-right (LR) organogenesis. We utilize the directional 360° clockwise rotation of genitalia in Drosophila to study LR-dependent organ looping. Using time-lapse imaging, we show that rotation of genitalia by 360° results from an additive process involving two ring-shaped domains, each undergoing 180° rotation. Our results show that the direction of rotation for each ring is autonomous and strictly depends on the LR determinant myosin ID (MyoID). Specific inactivation of MyoID in one domain causes rings to rotate in opposite directions and thereby cancels out the overall movement. We further reveal a specific pattern of apoptosis at the ring boundaries and show that local cell death is required for the movement of each domain, acting as a brake-releaser. These data indicate that organ looping can proceed through an incremental mechanism coupling LR determination and apoptosis. Furthermore, they suggest a model for the stepwise evolution of genitalia posture in Diptera, through the emergence and duplication of a 180° LR module.

Dynamics of the basement membrane in invasive epithelial clusters in Drosophila

The basement membrane (BM) represents a barrier to cell migration, which has to be degraded to promote invasion. However, the role and behaviour of the BM during the development of pre-invasive cells is only poorly understood. Drosophila border cells (BCs) provide an attractive genetic model in which to study the cellular mechanisms underlying the migration of mixed cohorts of epithelial cells. BCs are made of two different epithelial cell types appearing sequentially during oogenesis: the polar cells and the outer BCs. Here, we show that the pre-invasive polar cells undergo an unusual and asymmetrical apical capping with major basement membrane proteins, including the two Drosophila Collagen IV α chains, Laminin A and Perlecan. Capping of polar cells proceeds through a novel, basal-to-apical transcytosis mechanism that involves the small GTPase Drab5. Apical capping is transient and is followed by rapid shedding prior to the initiation of BC migration, suggesting that the apical cap blocks migration. Consistently, non-migratory polar cells remain capped. We further show that JAK/STAT signalling and recruitment of outer BCs are required for correct shedding and migration. The dynamics of the BM represents a marker of migratory BC, revealing a novel developmentally regulated behaviour of BM coupled to epithelial cell invasiveness.

JNK Signalling Controls Remodelling of the Segment Boundary through Cell Reprogramming during Drosophila Morphogenesis

Reprogramming of a specific group of Drosophila epidermal cells allows the mixing of normally segregated populations and the release of mechanical tension that arises during morphogenesis.

JNK signalling influences intracellular trafficking during Drosophila morphogenesis through regulation of the novel target gene Rab30

JNK-mediated closure of the Drosophila dorsal epidermis during embryogenesis is a well-characterised model for morphogenesis. However, little is known about how JNK signalling modifies particular cellular behaviours such as intracellular transport. Here we demonstrate that the gene encoding the small GTPase Rab30 is a new JNK transcriptional target whose function is required during embryonic and adult morphogenesis including JNK-dependent dorsal closure, embryonic head involution and thorax closure. Using immuno-fluorescence and live imaging, we show that EGFP-Rab30 localises to trans-Golgi in addition to small unidentified vesicles, and moves in a microtubule-dependent, polarised dorso-ventral manner in the leading edge during dorsal closure. We propose that JNK activity upregulates genes involved in intracellular transport in order to provide an increased level of trafficking activity in cells undergoing complex morphogenetic arrangements such as dorsal closure.

Left-right asymmetry in Drosophila.

Seminal studies of left-right (L/R) patterning in vertebrate models have led to the discovery of roles for the nodal pathway, ion flows and cilia in this process. Although the molecular mechanisms underlying L/R asymmetries seen in protostomes are less well understood, recent work using Drosophila melanogaster as a novel genetic model system to study this process has identified a number of mutations affecting directional organ looping. The genetic analysis of this, the most evolutionary conserved feature of L/R patterning, revealed the existence of a L/R pathway that involves the actin cytoskeleton and an associated type I myosin. In this review, we describe this work in the context of Drosophila development, and discuss the implications of these results for our understanding of L/R patterning in general.