The Notch receptor and its ligands are key components in a core metazoan signaling pathway that regulates the spatial patterning, timing and outcome of many cell-fate decisions. Ligands contain a disulfide-rich Delta/Serrate/LAG-2 (DSL) domain required for Notch trans-activation or cis-inhibition. Here we report the X-ray structure of a receptor binding region of a Notch ligand, the DSL-EGF3 domains of human Jagged-1 (J-1(DSL-EGF3)). The structure reveals a highly conserved face of the DSL domain, and we show, by functional analysis of Drosophila melanogster ligand mutants, that this surface is required for both cis- and trans-regulatory interactions with Notch. We also identify, using NMR, a surface of Notch-1 involved in J-1(DSL-EGF3) binding. Our data imply that cis- and trans-regulation may occur through the formation of structurally distinct complexes that, unexpectedly, involve the same surfaces on both ligand and receptor.

Background: The site of Jagged/Serrate ligand recognition by Notch is unknown.Results: Two critical residues involved in an intramolecular hydrophobic interaction across the central β-sheet of EGF12 form a ligand-binding platform.Conclusion: The ligand-binding region is adjacent to a Fringe-sensitive residue involved in modulating Notch activity.Significance: The results have implications for understanding receptor/ligand recognition, Notch regulation by O-glycosylation, and the development of paralogue-specific antibodies.

Epithelial morphogenesis generates the shape of tissues, organs and embryos and is fundamental for their proper function. It is a dynamic process that occurs at multiple spatial scales from macromolecular dynamics, to cell deformations, mitosis and apoptosis, to coordinated cell rearrangements that lead to global changes of tissue shape. Using time lapse imaging, it is possible to observe these events at a system level. However, to investigate morphogenetic events it is necessary to develop computational tools to extract quantitative information from the time lapse data. Toward this goal, we developed an image-based computational pipeline to preprocess, segment and track epithelial cells in 4D confocal microscopy data. The computational pipeline we developed, for the first time, detects the adherens junctions of epithelial cells in 3D, without the need to first detect cell nuclei. We accentuate and detect cell outlines in a series of steps, symbolically describe the cells and their connectivity, and employ this information to track the cells. We validated the performance of the pipeline for its ability to detect vertices and cell-cell contacts, track cells, and identify mitosis and apoptosis in surface epithelia of Drosophila imaginal discs. We demonstrate the utility of the pipeline to extract key quantitative features of cell behavior with which to elucidate the dynamics and biomechanical control of epithelial tissue morphogenesis. We have made our methods and data available as an open-source multiplatform software tool called TTT (http://github.com/morganrcu/TTT)

SUMMARY Elongation and invagination of epithelial tissues are fundamental developmental processes that contribute to the morphogenesis of embryonic and adult structures and are dependent on coordinated remodeling of cell-cell contacts. The morphogenesis of Drosophila leg imaginal discs depends on extensive remodeling of cell contacts and thus provides a useful system with which to investigate the underlying mechanisms. The small Rho GTPase regulator RhoGAP68F has been previously implicated in leg morphogenesis. It consists of an N-terminal Sec14 domain and a C-terminal GAP domain. Here we examined the molecular function and role of RhoGAP68F in epithelial remodeling. We find that depletion of RhoGAP68F impairs epithelial remodeling from a pseudostratified to simple, while overexpression of RhoGAP68F causes tears of lateral cell-cell contacts and thus impairs epithelial integrity. We show that the RhoGAP68F protein localizes to Rab4 recycling endosomes and forms a complex with the Rab4 protein. The Sec14 domain is sufficient for localizing to Rab4 endosomes, while the activity of the GAP domain is dispensable. RhoGAP68F, in turn, inhibits the scission and movement of Rab4 endosomes involved in transport the adhesion proteins Fasciclin3 and E-cadherin back to cell-cell contacts. Expression of RhoGAP68F is upregulated during prepupal development suggesting that RhoGAP68F decreases the transport of key adhesion proteins to the cell surface during this developmental stage to decrease the strength of adhesive cell-cell contacts and thereby facilitate epithelial remodeling and leg morphogenesis.

The Drosophila GATA factor gene serpent (srp) is required for the early differentiation of the anterior and posterior midgut primordia. In particular, srp is sufficient and necessary for the primordial gut cells to undertake an epithelial-to-mesenchimal transition (EMT). Two other GATA factor genes, dGATAe and grain (grn), are also specifically expressed in the midgut. On the one hand, dGATAe expression is activated by srp. Embryos homozygous for a deficiency uncovering dGATAe were shown to lack the expression of some differentiated midgut genes. Moreover, ectopic expression of dGATAe was sufficient to drive the expression of some of these differentiation marker genes, thus establishing the role of dGATAe in the regulation of their expression. However, due to the gross abnormalities associated with this deficiency, it was not possible to assess whether, similarly to srp, dGATAe might play a role in setting the midgut morphology. To further investigate this role we decided to generate a dGATAe mutant. On the other hand, grn is expressed in the midgut primordia around stage 11 and remains expressed until the end of embryogenesis. Yet, no midgut function has been described for grn. First, here we report that, as for dGATAe, midgut grn expression is dependent on srp; conversely, dGATAe and grn expression are independent of each other. Our results also indicate that, unlike srp, dGATAe and grn are not responsible for setting the general embryonic midgut morphology. We also show that the analysed midgut genes whose expression is lacking in embryos homozygous for a deficiency uncovering dGATAe are indeed dGATAe-dependent genes. Conversely, we do not find any midgut gene to be grn-dependent, with the exception of midgut repression of the proventriculus iroquois (iro) gene. In conclusion, our results clarify the expression patterns and function of the GATA factor genes expressed in the embryonic midgut.