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Jean-Paul Vincent

Publications and source records attributed to Jean-Paul Vincent.

11 recordsLinked to original sources
PubMed2006

An efficient promoter trap for detection of patterned gene expression and subsequent functional analysis in Drosophila.

Transposable elements have been used in Drosophila to detect gene expression, inactivate gene function, and induce ectopic expression or overexpression. We have combined all of these features in a single construct. A promoterless GAL4 cDNA is expressed when the construct inserts within a transcriptional unit, and GAL4 activates a GFP-encoding gene present in the same transposon. In a primary screen, patterned gene expression is detected as GFP fluorescence in the live progeny of dysgenic males. Many animals expressing GFP in distinct patterns can be recovered with relatively little effort. As expected, many insertions cause loss of function. After insertion at a genomic location, specific parts of the transposon can be excised by FLP recombinase, thus allowing it to induce conditional misexpression of the tagged gene. Therefore, both gain- and loss-of-function studies can be carried out with a single insertion in a gene identified by virtue of its expression pattern. Using this promoter trap approach, we have identified a group of cells that innervate the calyx of the mushroom body and could thus define a previously unrecognized memory circuit.

Animals↗
PubMed2006

How does cholesterol affect the way Hedgehog works?

Members of the Hedgehog (Hh) family of proteins are conserved morphogens that spread and modulate cell fates in target tissue. Mature Hh carries two lipid adducts, a palmitoyl group at the N terminus and cholesterol at the C terminus. Recent findings have addressed how these lipid modifications affect the function and transport of Hh in Drosophila. In contrast to the palmitoyl adduct, cholesterol appears not to be essential for signalling. However, the absence of the cholesterol adduct affects the spread of Hh within tissues. As we discuss here, the exact nature of this effect is controversial.

Animals↗
PubMed2005

Arrow (LRP6) and Frizzled2 cooperate to degrade Wingless in Drosophila imaginal discs.

Lysosome-mediated ligand degradation is known to shape morphogen gradients and modulate the activity of various signalling pathways. We have investigated the degradation of Wingless, a Drosophila member of the Wnt family of secreted growth factors. We find that one of its signalling receptors, Frizzled2, stimulates Wingless internalization both in wing imaginal discs and cultured cells. However, this is not sufficient for degradation. Indeed, as shown previously, overexpression of Frizzled2 leads to Wingless stabilization in wing imaginal discs. We show that Arrow (the Drosophila homologue of LRP5/6), another receptor involved in signal transduction, abrogates such stabilization. We provide evidence that Arrow stimulates the targeting of Frizzled2-Wingless (but not Dally-like-Wingless) complexes to a degradative compartment. Thus, Frizzled2 alone cannot lead Wingless all the way from the plasma membrane to a degradative compartment. Overall, Frizzled2 achieves ligand capture and internalization, whereas Arrow, and perhaps downstream signalling, are essential for lysosomal targeting.

Animals↗
PubMed2005

Glypicans shunt the Wingless signal between local signalling and further transport.

The two glypicans Dally and Dally-like have been implicated in modulating the activity of Wingless, a member of the Wnt family of secreted glycoprotein. So far, the lack of null mutants has prevented a rigorous assessment of their roles. We have created a small deletion in the two loci. Our analysis of single and double mutant embryos suggests that both glypicans participate in normal Wingless function, although embryos lacking maternal and zygotic activity of both genes are still capable of transducing the signal from overexpressed Wingless. Genetic analysis of dally-like in wing imaginal discs leads us to a model whereby, at the surface of any given cell of the epithelium, Dally-like captures Wingless but instead of presenting it to signalling receptors expressed in this cell, it passes it on to neighbouring cells, either for paracrine signalling or for further transport. In the absence of dally-like, short-range signalling is increased at the expense of long-range signalling (reported by the expression of the target gene distalless) while the reverse is caused by Dally-like overexpression. Thus, Dally-like act as a gatekeeper, ensuring the sharing of Wingless among cells along the dorsoventral axis. Our analysis suggests that the other glypican, Dally, could act as a classical co-receptor.

Animals↗
PubMed2003

Segment boundary formation in Drosophila embryos.

In Drosophila embryos, segment boundaries form at the posterior edge of each stripe of engrailed expression. We have used an HRP-CD2 transgene to follow by transmission electron microscopy the cell shape changes that accompany boundary formation. The first change is a loosening of cell contact at the apical side of cells on either side of the incipient boundary. Then, the engrailed-expressing cells flanking the boundary undergo apical constriction, move inwards and adopt a bottle morphology. Eventually, grooves regress, first on the ventral side, then laterally. We noted that groove formation and regression are contemporaneous with germ band retraction and shortening, respectively, suggesting that these rearrangements could also contribute to groove morphology. The cellular changes accompanying groove formation require that Hedgehog signalling be activated, and, as a result, a target of Ci expressed, at the posterior of each boundary (obvious targets like stripe and rhomboid appear not to be involved). In addition, Engrailed must be expressed at the anterior side of each boundary, even if Hedgehog signalling is artificially maintained. Thus, there are distinct genetic requirements on either side of the boundary. In addition, Wingless signalling at the anterior of the domains of engrailed (and hedgehog) expression represses groove formation and thus ensures that segment boundaries form only at the posterior.

Animals↗
PubMed2003

Membranes, trafficking, and signaling during animal development.

Molecular genetics has been key in allowing developmental biologists to uncover many of the molecules that participate in pattern formation. Cell biology is now beginning to help developmental biologists in their quest to understand how these molecules interact within cells to direct tissue behavior. This is particularly true in the areas of membrane trafficking and cell motility. Recent work has shown that various trafficking events such as secretion, endocytosis, segregation in membrane microdomains, intracellular transport, and targeting to lysosomes regulate various signaling pathways. It is likely that within the context of an embryo, these trafficking events are integrated such that secreted factors reliably orchestrate many developmental decisions.

Animals↗
PubMed2003

Modulation of developmental signals by endocytosis: different means and many ends.

Recent advances have highlighted the importance of endocytic processes in regulating the activity and distribution of developmental signals. Classically, signalling is downregulated by endocytosis and subsequent trafficking to lysosomes (e.g. Notch, Hedgehog, Roundabout). However, endocytosis can also have a positive role in signalling. For example, endocytosis of Delta, the ligand of Notch, is needed for activation of the signal. In the case of signalling by Hedgehog, endocytic trafficking segregates an inhibitory receptor (Patched) from the positive effector (Smoothened). Endosomes could also be the site where signalling is activated (e.g. transforming growth factor beta). Finally, endocytosis could power the transport of morphogens along epithelia.

Animals↗
PubMed2003

Requirements for transcriptional repression and activation by Engrailed in Drosophila embryos.

Genetic analysis shows that Engrailed (En), a homeodomain-containing transcription factor, has both negative and positive targets. Negative regulation is expected from a factor that has a well-defined repressor domain but activation is harder to comprehend. We used VP16En, a form of En that had its repressor domain replaced by the activation domain of VP16, to show that En activates targets using two parallel routes, by repressing a repressor and by being a bona fide activator. We identified the intermediate repressor activity as being encoded by sloppy paired 1 and 2 and showed that bona fide activation is dramatically enhanced by Wingless signaling. Thus, En is a bifunctional transcription factor and the recruitment of additional cofactors presumably specifies which function prevails on an individual promoter. Extradenticle (Exd) is a cofactor thought to be required for activation by Hox proteins. However, in thoracic segments, Exd is required for repression (as well as activation) by En. This is consistent with in vitro results showing that Exd is involved in recognition of positive and negative targets. Moreover, we provide genetic evidence that, in abdominal segments, Ubx and Abd-A, two homeotic proteins not previously thought to participate in the segmentation cascade, are also involved in the repression of target genes by En. We suggest that, like Exd, Ubx and Abd-A could help En recognize target genes or activate the expression of factors that do so.

Abdomen↗
PubMed2002

Producing cells retain and recycle Wingless in Drosophila embryos.

There is considerable interest in the mechanisms that drive and control the spread of morphogens in developing animals. Although much attention is given to events occurring after release from expressing cells, release itself could be an important modulator of range. Indeed, a dedicated protein, Dispatched, is needed to release Hedgehog from the surface of expressing cells. We find that, in Drosophila embryos, much Wingless (as well as a GFP-Wingless fusion protein) remains tightly associated with secreting cells. Retention occurs both within the secretory pathway and at the cell surface and requires functional heparan sulfate proteoglycans. As a further means of retention, secreting cells readily endocytose Wingless protein that does reach the cell surface. Such endocytosed Wingless can in turn be sent back to the cell surface (the first direct observation of ligand recycling in live embryos). Recycling may serve to sustain high-level signaling in this region of the epidermis.

Animals↗
PubMed2002

Morphogen transport along epithelia, an integrated trafficking problem.

Graded signals are an important component of current models of pattern formation. Typically, a group of cells produces a signal that decays as it spreads through neighboring tissue. By contrast with endocrine signals, which spread systemically, patterning signals or morphogens have a restricted zone of influence, an area classically known as a field. The widely accepted model is that graded distribution of such signals allow cells to measure their position relative to the source. Although it provides a framework for understanding pattern formation, the concept of the morphogen raises many mechanistic issues. For example, how the distribution of a morphogen is established and maintained remains an outstanding issue. There is no doubt that signals are transported over distances of tens of cell diameters and that stable gradients do form. The question of how this is achieved has aroused the interest of many cell biologically minded developmental biologists.

Animals↗