Magali Suzanne

Shaping organisms with apoptosis

Programmed cell death is an important process during development that serves to remove superfluous cells and tissues, such as larval organs during metamorphosis, supernumerary cells during nervous system development, muscle patterning and cardiac morphogenesis. Different kinds of cell death have been observed and were originally classified based on distinct morphological features: (1) type I programmed cell death (PCD) or apoptosis is recognized by cell rounding, DNA fragmentation, externalization of phosphatidyl serine, caspase activation and the absence of inflammatory reaction, (2) type II PCD or autophagy is characterized by the presence of large vacuoles and the fact that cells can recover until very late in the process and (3) necrosis is associated with an uncontrolled release of the intracellular content after cell swelling and rupture of the membrane, which commonly induces an inflammatory response. In this review, we will focus exclusively on developmental cell death by apoptosis and its role in tissue remodeling.

Apico-basal forces exerted by apoptotic cells drive epithelium folding

Epithelium folding is a basic morphogenetic event that is essential in transforming simple two-dimensional epithelial sheets into three-dimensional structures in both vertebrates and invertebrates. Folding has been shown to rely on apical constriction. The resulting cell-shape changes depend either on adherens junction basal shift or on a redistribution of myosin II, which could be driven by mechanical signals. Yet the initial cellular mechanisms that trigger and coordinate cell remodelling remain largely unknown. Here we unravel the active role of apoptotic cells in initiating morphogenesis, thus revealing a novel mechanism of epithelium folding. We show that, in a live developing tissue, apoptotic cells exert a transient pulling force upon the apical surface of the epithelium through a highly dynamic apico-basal myosin II cable. The apoptotic cells then induce a non-autonomous increase in tissue tension together with cortical myosin II apical stabilization in the surrounding tissue, eventually resulting in epithelium folding. Together our results, supported by a theoretical biophysical three-dimensional model, identify an apoptotic myosin-II-dependent signal as the initial signal leading to cell reorganization and tissue folding. This work further reveals that, far from being passively eliminated as generally assumed (for example, during digit individualization), apoptotic cells actively influence their surroundings and trigger tissue remodelling through regulation of tissue tension.

Sharp boundaries of Dpp signalling trigger local cell death required for Drosophila leg morphogenesis

Morphogens are secreted signalling molecules that govern many developmental processes. In the Drosophila wing disc, the transforming growth factor β (TGFβ) homologue Decapentaplegic (Dpp) forms a smooth gradient and specifies cell fate by conferring a defined value of morphogen activity. Thus, neighbouring cells have similar amounts of Dpp protein, and if a sharp discontinuity in Dpp activity is generated between these cells, Jun kinase (JNK)-dependent apoptosis is triggered to restore graded positional information. To date, it has been assumed that this apoptotic process is only activated when normal signalling is distorted. However, we now show that a similar process occurs during normal development: rupture in Dpp activity occurs during normal segmentation of the distal legs of Drosophila. This sharp boundary of Dpp signalling, independently of the absolute level of Dpp activity, induces a JNK—reaper-dependent apoptosis required for the morphogenesis of a particular structure of the leg, the joint. Our results show that Dpp could induce a developmental programme not only in a concentration dependent manner, but also by the creation of a sharp boundary of Dpp activity. Furthermore, the same process could be used either to restore a normal pattern in response to artificial disturbance or to direct a morphogenetic process.

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.

Survival of Escherichia coli during long-term starvation: effects of aeration, NaCl, and the rpoS and osmC gene products

The survival of Escherichia coli was investigated during long-term starvation in rich media. In aerated cultures, E. coli lost the ability to form colonies earlier in NaCl-free Luria broth than in LB medium containing NaCl. Improved survival at low aeration and the sensitivity to hydrogen peroxide in aging cultures indicated a major role for oxidative stress in cell mortality. Mutants in rpoS, lacking the sigmaS subunit of RNA polymerase, showed altered survival in salt-containing media. However, in the absence of NaCl, although these mutants exhibited a massive loss of viability during the first 2 days, this was followed by a stabilization of the number of survivors. The starved culture contained survivors until at least day 9, long after a wild-type strain had completely lost viability. This peculiar behavior suggests that, in rich media of low osmotic pressure, sigmaS helps in short-term survival but hampers long-term survival. Mutants in osmC, a member of the rpoS regulon, also exhibited reduced survival and increased sensitivity to oxidative stress. The biochemical function of the envelope protein OsmC remains unknown, but present data indicated that it participates, directly or indirectly, in the defense against oxidative compounds.

A simple and efficient method to identify replacements of P-lacZ by P-Gal4 lines allows obtaining Gal4 insertions in the bithorax complex of Drosophila

The functional replacement of one gene product by another one is a powerful method to study specificity in development and evolution. In Drosophila, the Gal4/UAS method has been used to analyze in vivo such functional substitutions. To this aim, Gal4 lines that inactivate a gene and reproduce its expression pattern are required, and they can be frequently obtained by replacing pre-existing P-lacZ lines with such characteristics. We have devised a new method to quickly identify replacements of P-lacZ lines by P-Gal4 lines, and applied it successfully to obtain Gal4 insertions in the Ultrabithorax and Abdominal-B Hox genes. We have used these lines to study the functional replacement of a Hox gene by another one. Our experiments confirm that the abdominal-A gene can replace Ultrabithorax in haltere development but that it cannot substitute for Abdominal-B in the formation of the genitalia.

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.

The Drosophila serine protease homologue Scarface regulates JNK signalling in a negative-feedback loop during epithelial morphogenesis.

In Drosophila melanogaster, dorsal closure is a model of tissue morphogenesis leading to the dorsal migration and sealing of the embryonic ectoderm. The activation of the JNK signal transduction pathway, specifically in the leading edge cells, is essential to this process. In a genome-wide microarray screen, we identified new JNK target genes during dorsal closure. One of them is the gene scarface (scaf), which belongs to the large family of trypsin-like serine proteases. Some proteins of this family, like Scaf, bear an inactive catalytic site, representing a subgroup of serine protease homologues (SPH) whose functions are poorly understood. Here, we show that scaf is a general transcriptional target of the JNK pathway coding for a secreted SPH. scaf loss-of-function induces defects in JNK-controlled morphogenetic events such as embryonic dorsal closure and adult male terminalia rotation. Live imaging of the latter process reveals that, like for dorsal closure, JNK directs the dorsal fusion of two epithelial layers in the pupal genital disc. Genetic data show that scaf loss-of-function mimics JNK over-activity. Moreover, scaf ectopic expression aggravates the effect of the JNK negative regulator puc on male genitalia rotation. We finally demonstrate that scaf acts as an antagonist by negatively regulating JNK activity. Overall, our results identify the SPH-encoding gene scaf as a new transcriptional target of JNK signalling and reveal the first secreted regulator of the JNK pathway acting in a negative-feedback loop during epithelial morphogenesis.

Drosophila Left/Right Asymmetry Establishment Is Controlled by the Hox Gene Abdominal-B

In Drosophila, left/right (LR) asymmetry is apparent in the directional looping of the gut and male genitalia. The dextral orientation of the organs depends on the activity of a single gene, MyosinID (myoID), whose mutation leads to a fully inverted LR axis, thus revealing the activity of a recessive sinistral pathway. Here, we present the identification of the Hox gene Abdominal-B (Abd-B) as an upstream regulator of LR determination. This role appears distinct from its function in anteroposterior patterning. We show that the Abd-Bm isoform binds to regulatory sequences of myoID and controls MyoID expression in the organ LR organizer. Abd-Bm is also required for the sinistral pathway. Thus, when Abd-B activity is missing, no symmetry breaking occurs and flies develop symmetrically. These findings identify the Hox gene Abd-B as directing the earliest events of LR asymmetry establishment in Drosophila.

The Drosophila gene zfh2 is required to establish proximal-distal domains in the wing disc

Three main events characterize the development of the proximal-distal axis of the Drosophila wing disc: first, generation of nested circular domains defined by different combinations of gene expression; second, activation of wingless (wg) gene expression in a ring of cells; and third, an increase of cell number in each domain in response to Wg. The mechanisms by which these domains of gene expression are established and maintained are unknown. We have analyzed the role of the gene zinc finger homeodomain 2 (zfh2). We report that in discs lacking zfh2 the limits of the expression domains of the genes tsh, nub, rn, dve and nab coincide, and expression of wg in the wing hinge, is lost. We show that zfh2 expression is delimited distally by Vg, Nub and Dpp signalling, and proximally by Tsh and Dpp. Distal repression of zfh2 permits activation of nab in the wing blade and wg in the wing hinge. We suggest that the proximal-most wing fate, the hinge, is specified first and that later repression of zfh2 permits specification of the distal-most fate, the wing blade. We propose that proximal-distal axis development is achieved by a combination of two strategies: on one hand a process involving proximal to distal specification, with the wing hinge specified first followed later by the distal wing blade; on the other hand, early specification of the proximal-distal domains by different combinations of gene expression. The results we present here indicate that Zfh2 plays a critical role in both processes.