Ligand-independent traffic of notch buffers activated armadillo in <em>Drosophila</em>

Notch receptors act as ligand-dependent membrane-tethered transcription factors with a prominent role in binary cell fate decisions during development, which is conserved across species. In addition there is increasing evidence for other functions of Notch, particularly in connection with Wnt signalling: Notch is able to modulate the activity of Armadillo/ß-catenin, the effector of Wnt signalling, in a manner that is independent of its transcriptional activity. Here we explore the mechanism of this interaction in the epithelium of the Drosophila imaginal discs and find that it is mediated by the ligand-independent endocytosis and traffic of the Notch receptor. Our results show that Notch associates with Armadillo near the adherens junctions and that it is rapidly endocytosed promoting the traffic of an activated form of Armadillo into endosomal compartments, where it may be degraded. As Notch has the ability to interact with and downregulate activated forms of Armadillo, it is possible that in vivo Notch regulates the transcriptionally competent pool of Armadillo. These interactions reveal a previously unknown activity of Notch, which serves to buffer the function of activated Armadillo and might underlie some of its transcription-independent effects

Haemogenic Gastruloids Recapitulate Developmental Haematopoiesis and Provide an Ontogeny-Relevant Context to Dissect the Origins of Infant Leukemia

Meeting abstract presented at the 64th ASH Annual Meeting and Exposition, New Orleans, LA, USA, 10-13 Dec 2022..Modelling of developmental hematopoiesis has historically been challenging due to the inability to produce hematopoietic stem cells (HSC) and recapitulate microenvironment interactions ex vivo. Gastruloids are 3D aggregates of embryonic stem (ES) cells which display developmentally-specific spatial and temporal organization that recapitulate gastrulation. We adapted the gastruloid protocol to introduce hematopoietic signalling cues, and generated an in vitro model of embryonic hematopoiesis that sequentially recapitulates the formation of hemogenic endothelium, hematopoietic progenitors, and pre-HSC, over a culture period of 216 hours. Flow cytometry analysis detected the presence of c-Kit+ endothelium at 120h, followed by emergence of CD41+ hematopoietic progenitors at 144h, and the appearance of CD45+ cells from 192h. CD45+ cells were observed in small clusters adjoining endothelium-lined structures, reminiscent of developmental hemogenic-to-endothelial transition and intra-aortic clusters. Single-cell RNA sequencing revealed specification of pre-definitive and definitive waves of embryonic hematopoiesis, aligning 144h-CD41+ cells with erythro-myeloid progenitors (EMP), and late CD45+ with lympho-myeloid progenitors and pre-HSC, altogether supporting the hemogenic gastruloid as a model that is temporally and topographically congruous with the embryo. The close recapitulation of developmental ontogeny led us to explore hemogenic gastruloids to understand cell and stage-specific susceptibility to forms of Acute Myeloid Leukaemia exclusively observed in infants. The chromosomal translocation t(7;12)(q36;p13), characterized by the ectopic overexpression of the MNX1 gene, is found in up to one third of infant AML cases, but has been challenging to model using conventional strategies, largely due to the inability of MNX1 to transform adult hematopoietic cells. The age-selectivity of t(7;12) has been proposed to reflect a transient developmental window for a target cell of origin absent in adult life, but its nature is yet to be defined. In order to identify the context of MNX1-driven leukemogenesis, we produced hemogenic gastruloids using lentiviral-transduced mouse ES cells in which we overexpressed MNX1 as a proxy of t(7;12). Although MNX1 did not interfere with ES cell pluripotent cultures, it primed incipient hemogenic programmes and promoted hemogenic gastruloid formation. Critically, expression of MNX1 resulted in transformation of gastruloid-derived hematopoietic cells, as assessed by serial colony-forming cell replating, with expansion of a phenotypic myeloid cell, a phenomenon not observed in adult tissues. Detailed analysis of the cellular composition of MNX1-overexpressing hemogenic gastruloids revealed a significant effect in the output of CD41+ and c-Kit+ populations at 144h, but no effect in CD45+ cells at 192-216h, suggesting that the target of MNX1 lies within the EMP stage, an observation supported by single-cell RNA-seq analysis of MNX1 vs control gastruloids. Systematic comparison of the temporal transcriptional profiles of hemogenic gastruloids, MNX1-overexpressing gastruloids, and t(7;12) patients, pinpoints the target cell of MNX1 at the HE-to-EMP transition. In summary, we propose a novel model of embryonic hematopoiesis capable of capturing developmentally-relevant cellularity and topography of the early hematopoietic microenvironment, with the ability to mechanistically elucidate developmental associations of infant leukemia

Dynamic actin-based epithelial adhesion and cell matching during Drosophila dorsal closure

AbstractBackground: The adhesion of two epithelial sheets is a fundamental process that occurs throughout embryogenesis and during wound repair. Sealing of the dorsal epidermis along the midline of the Drosophila embryo provides a genetically tractable model to analyse the closure of such holes. Several studies indicate that the actin cytoskeleton plays a critical role in dorsal closure. Although many components of the signalling cascade directing this process have been identified, the precise cell-biological events upon which these signals act remain poorly described.Results: By confocal imaging of living fly embryos expressing green fluorescent protein (GFP)-tagged actin, we found that dorsal closure relies on the activity of dynamic filopodia and lamellipodia that extend from front-row cells to actively zipper the epithelial sheets together. As these epithelial fronts approach one another, we observed long, thin filopodia, apparently ‘sampling’ cells on the opposing face. When the assembly of these actin-based protrusions was blocked (by interfering with the activities of Cdc42 and Jun N-terminal kinase signalling), the adhesion and fusion of opposing epithelial cells was prevented and their ability to ‘sense’ correct partners was also blocked, leading to segment misalignment along the midline seam.Conclusions: Dynamic, actin-based protrusions (filopodia and lamellae) are critical, both in the mechanics of epithelial adhesion during dorsal closure and in the correct ‘matching’ of opposing cells along the fusion seam