The Snail repressor positions Notch signaling in the Drosophila embryo

The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. We have investigated the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, Notch(IC), in transgenic embryos using the eve stripe2 enhancer. The Notch(IC) stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in 'na?ve' embryos that contain low uniform levels of Dorsal. We suggest that these dual activities of Snail, repression of Notch target genes and stimulation of Notch signaling, help define precise lines of sim expression within the neurogenic ectoderm.     

Involvement of co-repressors Groucho and CtBP in the regulation of single-minded in Drosophila

Dorso-ventral patterning results in the establishment of the two germ layers in the Drosophila embryo, mesoderm and mesectoderm, that are separated by a strip of cells giving rise to the mesectoderm and eventually to the ventral midline. The mesectoderm is specified by the expression of single-minded (sim) which is activated through the concerted action of Dorsal and Twist in addition to a Notch signal. In the mesoderm, sim is repressed by Snail together with the co-repressor C-terminal binding protein (CtBP). Here, we address the involvement of the two co-repressors CtBP and Groucho (Gro) in repression of sim in the neuroectoderm. It was shown earlier that sim is restricted in the neuroectoderm with help of Suppressor of Hairless [Su(H)] and Hairless. Using the female sterile technique, we generated germ line clones deficient for Gro, CtBP or Hairless and assayed sim mRNA relative to snail mRNA expression. We show that sim repression requires both co-repressors Gro and CtBP to be fully repressed in the neuroectoderm, suggesting that a repression complex is assembled including Su(H) and Hairless as was shown for other Notch target genes before. Moreover, our work implies that Gro is important for the repression of sim specifically within the mesoderm anlagen, indicating that Snail and CtBP are insufficient to entirely silence sim in this germ layer.     

Localized repressors delineate the neurogenic ectoderm in the early Drosophila embryo

The Dorsal gradient produces sequential patterns of gene expression across the dorsoventral axis of early embryos, thereby establishing the presumptive mesoderm, neuroectoderm, and dorsal ectoderm. Spatially localized repressors such as Snail and Vnd exclude the expression of neurogenic genes in the mesoderm and ventral neuroectoderm, respectively. However, no repressors have been identified that establish the dorsal limits of neurogenic gene expression. To investigate this issue, we have conducted an analysis of the ind gene, which is selectively expressed in lateral regions of the presumptive nerve cord. A novel silencer element was identified within the ind enhancer that is essential for eliminating expression in the dorsal ectoderm. Evidence is presented that the associated repressor can function over long distances to silence neighboring enhancers. The ind enhancer also contains a variety of known activator and repressor elements. We propose a model whereby Dorsal and EGF signaling, together with the localized Schnurri repressor, define a broad domain of ind expression throughout the entire presumptive neuroectoderm. The ventral limits of gene expression are defined by the Snail and Vnd repressors, while the dorsal border is established by the newly defined silencer element.     

Snail is required for Delta endocytosis and Notch-dependent activation of<Emphasis Type="Italic"> single-minded</Emphasis> expression

In the Drosophila embryo, the mesectoderm corresponds to a single row of cells abutting the mesoderm. It is specified by the expression of the single-minded (sim) gene. The information that precisely positions the sim-expressing cells along the dorso-ventral axis is incompletely understood. Previous studies have shown that Dorsal and Twist activate sim expression in a large ventral domain, while two negative regulators, Snail (Sna) and Suppressor of Hairless [Su(H)], repress sim expression in the mesoderm and neuroectoderm, respectively. Repression by Su(H) is relieved in the presumptive mesectoderm by Notch signaling. In this paper, we show that Sna also has a positive regulatory function on sim expression in the presumptive mesectoderm. This positive effect of Sna depends on the Su(H)-binding sites within the sim promoter, suggesting that Sna regulates Notch signaling. In addition, we find that Delta is endocytosed together with the extracellular domain of Notch. The endocytosis of Delta is only seen in the mesoderm and requires the activity of the sna and neuralized (neur) genes. Interestingly, the Neur-mediated endocytosis of Delta has recently been shown to be sufficient for the non-autonomous activation of Notch target genes in wing imaginal discs. We therefore propose a novel model in which Sna positions the mesectoderm via its dual regulatory activity. In this model, Sna cell-autonomously represses sim expression in the mesoderm and relieves Su(H)-dependent repression in a cell non-autonomous fashion by promoting the Neur-dependent endocytosis of Delta in the mesoderm.     

Regulation of epithelial polarity by the E3 ubiquitin ligase Neuralized and the Bearded inhibitors in Drosophila

Understanding how epithelial polarity is established and regulated during tissue morphogenesis is a major issue. Here, we identify a regulatory mechanism important for mesoderm invagination, germ-band extension and transepithelial migration in the Drosophila melanogaster embryo. This mechanism involves the inhibition of the conserved E3 ubiquitin ligase Neuralized by proteins of the Bearded family. First, Bearded mutant embryos exhibited a loss of epithelial polarity associated with an early loss of the apical domain. Bearded regulated epithelial polarity by antagonizing neuralized. Second, repression of Bearded gene expression by Snail was required for the Snail-dependent disassembly of adherens junctions in the mesoderm. Third, neuralized was strictly required to promote the downregulation of the apical domain in the midgut epithelium and to facilitate the transepithelial migration of primordial germ cells across this epithelium. This function of Neuralized was independent of its known role in Notch signalling. Thus, Neuralized has two distinct functions in epithelial cell polarity and Notch signalling.     

The Ca-dependent protease Calpain A regulates Cactus/IκB levels during Drosophila development in response to maternal Dpp signals

Regulation of NFκB activity is central to many processes during development and disease. Activation of NFκB family members depends on degradation of inhibitory IκB proteins. In Drosophila, a nuclear gradient of the NFκB/c-rel protein Dorsal subdivides the embryonic dorsal–ventral axis, defining the extent and location of mesodermal and ectodermal territories. Activation of the Toll pathway directs Dorsal nuclear translocation by inducing proteosomal degradation of the IκB homologue Cactus. Another mechanism that impacts on Dorsal activation involves the Toll-independent pathway, which regulates constitutive Cactus degradation. We have shown that the BMP protein Decapentaplegic (Dpp) inhibits Cactus degradation independent of Toll. Here we report on a novel element of this pathway: the calcium-dependent protease Calpain A. CalpainA knockdowns increase Cactus levels, shifting the Dorsal gradient and dorsal–ventral patterning. As shown for mammalian IκB, this effect requires PEST sequences in the Cactus C-terminus, implying a conserved role for calpains. Alteration of CalpainA or dpp results in similar effects on Dorsal target genes. Epistatic analysis confirms CalpainA activity is regulated by Dpp, indicating that Dpp signals increase Cactus levels through Calpain A inhibition, thereby interfering with Dorsal activation. This mechanism may allow coordination of Toll, BMP and Ca²⁺ signals, conferring precision to Dorsal-target expression domains.     

Is mechano‐sensitive expression of twist involved In mesoderm formation?

Mesoderm invagination, the first morphogenetic movement of gastrulation in the early Drososphila embryo, is controlled by the expression of the twist and snail genes. Our knowledge concerning epistatic relationships between these genes implies the existence of a poorly understood biochemical maintenance of twist expression during mesoderm invagination by the snail gene. In the light of a review detailing the role of these genes in the cell shape changes leading to invagination, and of recent findings showing the expression of twist as mechanically sensitive, we suggest that the expression of twist in the mesoderm could alternatively be maintained by mechanical strains developed during mesoderm invagination.     

Cell shape changes during gastrulation in Drosophila

The first morphogenetic movement during Drosophila development is the invagination of the mesoderm, an event that folds a one-layered epithelium into a multilayered structure. In this paper, we describe the shape changes and behaviour of the cells participating in this process and show how mutations that change cell fate affect this behaviour. We divide the formation of the mesodermal germ layer into two phases. During the first phase, the ventral epithelium folds into a tube by a series of concerted cell shape changes (ventral furrow formation). Based on the behaviour of cells in this phase, we conclude that the prospective mesoderm is not a homogeneous cell population, but consists of two subpopulations. Each subpopulation goes through a distinctive sequence of specific cell shape changes which together mediate the invagination of the ventral furrow. In the second phase, the invaginated tube of mesoderm loses its epithelial character, the mesoderm cells disperse, divide and then spread out along the ectoderm to form a single cell layer. To test how ventral furrow formation depends on cell fates in the mesoderm and in neighbouring cells we alter these fates genetically using maternal and zygotic mutations. These experiments show that some of the aspects of cell behaviour specific for ventral furrow cells are part of an autonomous differentiation programme. The force driving the invagination is generated within the region of the ventral furrow, with the lateral and dorsal cell populations contributing little or none of the force. Two known zygotic genes that are required for the formation of the mesoderm, twist and snail, are expressed in ventral furrow cells, and the correct execution of cell shape changes in the mesoderm depends on both. Finally, we show that the region where the ventral furrow forms is determined by the expression of mesoderm-specific genes, and not by mechanical or other epigenetic properties of the egg.