A fluorescent toolkit for spatiotemporal tracking of apoptotic cells in living Drosophila tissues.

Far from being passive, apoptotic cells influence their environment. For example, they promote tissue folding, myoblast fusion and modulate tumor growth. Understanding the role of apoptotic cells necessitates their efficient tracking within living tissues, a task that is currently challenging. In order to easily spot apoptotic cells in developing Drosophila tissues, we generated a series of fly lines expressing different fluorescent sensors of caspase activity. We show that three of these reporters (GFP-, Cerulean- and Venus-derived molecules) are detected specifically in apoptotic cells and throughout the whole process of programmed cell death. These reporters allow the specific visualization of apoptotic cells directly within living tissues, without any post-acquisition processing. They overcome the limitations of other apoptosis detection methods developed so far and, notably, they can be combined with any kind of fluorophore.

The Hox gene Dfd controls organogenesis by shaping territorial border through regulation of basal DE-Cadherin distribution.

Hox genes are highly conserved selector genes controlling tissue identity and organogenesis. Recent work indicates that Hox genes also controls cell segregation and segmental boundary in various species, however the underlying cellular mechanisms involved in this function are poorly understood. In Drosophila melanogaster, the Hox gene Deformed (Dfd) is required for specification and organogenesis of the adult Maxillary (Mx) palp. Here, we demonstrate that differential Dfd expression control Mx morphogenesis through the formation of a physical boundary separating the Mx field and the Peripodial Epithelium (PE). We show that this boundary relies on DE-cadherin (DE-cad) basal accumulation in Mx cells controlled by differential Dfd expression. Indeed, Dfd controls boundary formation through cell autonomous basal redistribution of DE-cad which leads to subsequent fold at the Dfd expression border. Finally, the loss of Mx DE-cad basal accumulation and hence of Mx-PE folding is sufficient to prevent Mx organogenesis thus revealing the crucial role of boundaries in organ differentiation. Altogether, these results reveal that Hox coordination of tissue morphogenesis relies on boundary fold formation through the modulation of DE-cad positioning.

Dissecting morphogenetic apoptosis through a genetic screen in Drosophila.

Apoptosis is an essential cellular process both in normal development and pathological contexts. Screens performed to date have focused on the cell autonomous aspect of the process, deciphering the apoptotic cascade leading to cell destruction through the activation of caspases. However, the nonautonomous aspect of the apoptotic pathway, including signals regulating the apoptotic pattern or those sent by the apoptotic cell to its surroundings, is still poorly understood. Here, we describe an unbiased RNAi-based genetic screen whose goal is to identify elements of the "morphogenetic apoptosis pathway" in an integrated model system, the Drosophila leg. We screened about 1,400 candidates, using adult joint morphology, morphogenetic fold formation, and apoptotic pattern as readouts for the identification of potential apoptosis-related genes. We identified 41 genes potentially involved in specific aspects of morphogenetic apoptosis: (1) regulation of the apoptotic process; (2) formation, extrusion, and elimination of apoptotic bodies; and (3) contribution to morphogenesis downstream of apoptosis.

Getting started for migration: A focus on EMT cellular dynamics and mechanics in developmental models.

The conversion of epithelial cells into mesenchymal ones, through a process known as epithelial-mesenchymal transition (or EMT) is a reversible process involved in critical steps of animal development as early as gastrulation and throughout organogenesis. In pathological conditions such as aggressive cancers, EMT is often associated with increased drug resistance, motility and invasiveness. The characterisation of the upstream signals and main decision takers, such as the EMT-transcription factors, has led to the identification of a core molecular machinery controlling the specification towards EMT. However, the cellular execution steps of this fundamental shift are poorly described, especially in cancerous cells. Here we review our current knowledge regarding the stepwise nature of EMT in model organisms as diverse as sea urchin, Drosophila, zebrafish, mouse or chicken. We focus on the cellular dynamics and mechanics of the transitional stages by which epithelial cells progressively become mesenchymal and leave the epithelium. We gather the currently available pieces of the puzzle, including the overlooked property of EMT cells to produce mechanical forces along their apico-basal axis before detaching from their neighbours. We discuss the interplay between EMT and the surrounding tissue. Finally, we propose a conceptual framework of EMT cell dynamics from the very first hint of epithelial cell reorganisation to the successful exit from the epithelial sheet.

Mechanical impact of epithelial-mesenchymal transition on epithelial morphogenesis in Drosophila.

Epithelial-mesenchymal transition (EMT) is an essential process both in physiological and pathological contexts. Intriguingly, EMT is often associated with tissue invagination during development; however, the impact of EMT on tissue remodeling remain unexplored. Here, we show that at the initiation of the EMT process, cells produce an apico-basal force, orthogonal to the surface of the epithelium, that constitutes an important driving force for tissue invagination in Drosophila. When EMT is ectopically induced, cells starting their delamination generate an orthogonal force and induce ectopic folding. Similarly, during mesoderm invagination, cells undergoing EMT generate an apico-basal force through the formation of apico-basal structures of myosin II. Using both laser microdissection and in silico physical modelling, we show that mesoderm invagination does not proceed if apico-basal forces are impaired, indicating that they constitute driving forces in the folding process. Altogether, these data reveal the mechanical impact of EMT on morphogenesis.

Modulo is a target of Myc selectively required for growth of proliferative cells in Drosophila.

In Drosophila, the homologue of the proto-oncogene Myc is a key regulator of both cell size and cell growth. The identities and roles of dMyc target genes in these processes, however, remain largely unexplored. Here, we investigate the function of the modulo (mod) gene, which encodes a nucleolus localized protein. In gain of function or loss of function experiments, we demonstrate that mod is directly controlled by dMyc. Strikingly, in proliferative imaginal cells, mod loss-of-function impairs both cell growth and cell size, whereas larval endoreplicative tissues grow normally. In contrast to dMyc, over-expressing Mod in wing imaginal discs is not sufficient to induce cell growth. Taken together, our results indicate that mod does not possess the full spectrum of dMyc activities, but is required selectively in proliferative cells to sustain their growth and to maintain their specific size.

A P-insertion screen identifying novel X-linked essential genes in Drosophila.

The recent determination and annotation of the entire euchromatic sequence of the Drosophila melanogaster genome predicted the existence of about 13600 different genes (Science 287 (2000) 2185; http://www.fruitfly.org/annot/index.html). In parallel, the Berkeley Drosophila Genome Project (BDGP) has undertaken systematic P-insertion screens, to isolate new lethals and misexpressing lines. To date, however, the genes of the X chromosome have been under-represented in the screens performed. In order both to characterize several X-linked genes of prime interest to our laboratories and contribute to the collection of lethal P-insertions available to the community, we performed a P-insertion mutagenesis of the X chromosome. Using the PlacW and PGawB P-elements as mutagens, we generated two complementary sets of enhancer-trap lines, l(1)(T)PL and l(1)(T)PG, respectively, which both contain a reporter gene whose developmental expression can be monitored when driven by nearby enhancer sequences. We report here the characterization of 260 new insertions, mapping to 133 different genes or predicted CGs. Of these, 83 correspond to genes for which no lethal mutation had yet been reported. For 64 of those, we could confirm that lethality was solely due to the P-element insertion. The primary molecular data, reporter gene expression patterns (observed in embryos, third instar larvae and adult ovaries) and proposed CG assignment for each strain can be accessed and updated on our website at the following address: http://www-cbd.ups-tlse.fr:8080/screen.

Timing of Wingless signalling distinguishes maxillary and antennal identities in Drosophila melanogaster.

The Drosophila adult head mostly derives from the composite eye-antenna imaginal disc. The antennal disc gives rise to two adult olfactory organs: the antennae and maxillary palps. Here, we have analysed the regional specification of the maxillary palp within the antennal disc. We found that a maxillary field, defined by expression of the Hox gene Deformed, is established at about the same time as the eye and antennal fields during the L2 larval stage. The genetic program leading to maxillary regionalisation and identity is very similar to the antennal one, but is distinguished primarily by delayed prepupal expression of the ventral morphogen Wingless (Wg). We find that precociously expressing Wg in the larval maxillary field suffices to transform it towards antennal identity, whereas overexpressing Wg later in prepupae does not. These results thus indicate that temporal regulation of Wg is decisive to distinguishing maxillary and antennal organs. Wg normally acts upstream of the antennal selector spineless (ss) in maxillary development. However, mis-expression of Ss can prematurely activate wg via a positive-feedback loop leading to a maxillary-to-antenna transformation. We characterised: (1) the action of Wg through ss selector function in distinguishing maxillary from antenna; and (2) its direct contribution to identity choice.

Evidence for a direct functional antagonism of the selector genes proboscipedia and eyeless in Drosophila head development.

Diversification of Drosophila segmental and cellular identities both require the combinatorial function of homeodomain-containing transcription factors. Ectopic expression of the mouthparts selector proboscipedia (pb) directs a homeotic antenna-to-maxillary palp transformation. It also induces a dosage-sensitive eye loss that we used to screen for dominant Enhancer mutations. Four such Enhancer mutations were alleles of the eyeless (ey) gene that encode truncated EY proteins. Apart from eye loss, these new eyeless alleles lead to defects in the adult olfactory appendages: the maxillary palps and antennae. In support of these observations, both ey and pb are expressed in cell subsets of the prepupal maxillary primordium of the antennal imaginal disc, beginning early in pupal development. Transient co-expression is detected early after this onset, but is apparently resolved to yield exclusive groups of cells expressing either PB or EY proteins. A combination of in vivo and in vitro approaches indicates that PB suppresses EY transactivation activity via protein-protein contacts of the PB homeodomain and EY Paired domain. The direct functional antagonism between PB and EY proteins suggests a novel crosstalk mechanism integrating known selector functions in Drosophila head morphogenesis.