HaloTag-based reporters for sparse labeling and cell tracking.

Multiscale analysis of morphogenesis requires to follow and measure in real-time the in vivo behaviour of large numbers of individual cells over long period of time. Despite recent progress, the large-scale automated tracking of cells in developing embryos and tissues remains a challenge. Here we describe a genetic tool for the random and sparse labelling of individual cells in developing Drosophila tissues. This tool is based on the conditional expression of a nuclear HaloTag protein that can be fluorescently labelled upon the irreversible binding of a cell permeable synthetic ligand. While the slow maturation of genetically encoded fluorescent renders the tracking of individual cells difficult in rapidly dividing tissues, nuclear HaloTag proteins allowed for rapid labelling of individual cells in cultured imaginal discs. To study cell shape changes, we also produced an HaloTag version of the actin-bound protein LifeAct. Since sparse labelling facilitates cell tracking, nuclear HaloTag reporters will be useful for the single-cell analysis of fate dynamics in Drosophila tissues cultured ex vivo.

A combination of Notch signaling, preferential adhesion and endocytosis induces a slow mode of cell intercalation in the Drosophila retina.

Movement of epithelial cells in a tissue occurs through neighbor exchange and drives tissue shape changes. It requires intercellular junction remodeling, a process typically powered by the contractile actomyosin cytoskeleton. This has been investigated mainly in homogeneous epithelia, where intercalation takes minutes. However, in some tissues, intercalation involves different cell types and can take hours. Whether slow and fast intercalation share the same mechanisms remains to be examined. To address this issue, we used the fly eye, where the cone cells exchange neighbors over ∼10 h to shape the lens. We uncovered three pathways regulating this slow mode of cell intercalation. First, we found a limited requirement for MyosinII. In this case, mathematical modeling predicts an adhesion-dominant intercalation mechanism. Genetic experiments support this prediction, revealing a role for adhesion through the Nephrin proteins Roughest and Hibris. Second, we found that cone cell intercalation is regulated by the Notch pathway. Third, we show that endocytosis is required for membrane removal and Notch activation. Taken together, our work indicates that adhesion, endocytosis and Notch can direct slow cell intercalation during tissue morphogenesis.

Tissue-wide coordination of epithelium-to-neural stem cell transition in the Drosophila optic lobe requires Neuralized.

Many tissues are produced by specialized progenitor cells emanating from epithelia via epithelial-to-mesenchymal transition (EMT). Most studies have so far focused on EMT involving single or isolated groups of cells. Here we describe an EMT-like process that requires tissue-level coordination. This EMT-like process occurs along a continuous front in the Drosophila optic lobe neuroepithelium to produce neural stem cells (NSCs). We find that emerging NSCs remain epithelial and apically constrict before dividing asymmetrically to produce neurons. Apical constriction is associated with contractile myosin pulses and involves RhoGEF3 and down-regulation of the Crumbs complex by the E3 ubiquitin ligase Neuralized. Anisotropy in Crumbs complex levels also results in accumulation of junctional myosin. Disrupting the regulation of Crumbs by Neuralized lowered junctional myosin and led to imprecision in the integration of emerging NSCs into the front. Thus, Neuralized promotes smooth progression of the differentiation front by coupling epithelium remodeling at the tissue level with NSC fate acquisition.

Optogenetic inhibition of Delta reveals digital Notch signalling output during tissue differentiation.

Spatio-temporal regulation of signalling pathways plays a key role in generating diverse responses during the development of multicellular organisms. The role of signal dynamics in transferring signalling information in vivo is incompletely understood. Here, we employ genome engineering in Drosophila melanogaster to generate a functional optogenetic allele of the Notch ligand Delta (opto-Delta), which replaces both copies of the endogenous wild-type locus. Using clonal analysis, we show that optogenetic activation blocks Notch activation through cis-inhibition in signal-receiving cells. Signal perturbation in combination with quantitative analysis of a live transcriptional reporter of Notch pathway activity reveals differential tissue- and cell-scale regulatory modes. While at the tissue-level the duration of Notch signalling determines the probability with which a cellular response will occur, in individual cells Notch activation acts through a switch-like mechanism. Thus, time confers regulatory properties to Notch signalling that exhibit integrative digital behaviours during tissue differentiation.

Regulation of Notch output dynamics via specific E(spl)-HLH factors during bristle patterning in Drosophila.

The stereotyped arrangement of sensory bristles on the adult fly thorax arises from a self-organized process, in which inhibitory Notch signaling both delimits proneural stripes and singles out sensory organ precursor cells (SOPs). A dynamic balance between proneural factors and Enhancer of split-HLH (E(spl)-HLH) Notch targets underlies patterning, but how this is regulated is unclear. Here, were identify two classes of E(spl)-HLH factors, whose expression both precedes and delimits proneural activity, and is dependent on proneural activity and required for proper SOP spacing within the stripes, respectively. These two classes are partially redundant, since a member of the second class, that is normally cross-repressed by members of the first class, can functionally compensate for their absence. The regulation of specific E(spl)-HLH genes by proneural factors amplifies the response to Notch as SOPs are being selected, contributing to patterning dynamics in the notum, and likely operates in other developmental contexts.

Activation of Arp2/3 by WASp Is Essential for the Endocytosis of Delta Only during Cytokinesis in Drosophila.

The actin nucleator Arp2/3 generates pushing forces in response to signals integrated by SCAR and WASp. In Drosophila, the activation of Arp2/3 by WASp is specifically required for Notch signaling following asymmetric cell division. How WASp and Arp2/3 regulate Notch activity and why receptor activation requires WASp and Arp2/3 only in the context of intra-lineage fate decisions are unclear. Here, we find that WASp, but not SCAR, is required for Notch activation soon after division of the sensory organ precursor cell. Conversely, SCAR, but not WASp, is required to expand the cell-cell contact between the two SOP daughters. Thus, these two activities of Arp2/3 can be uncoupled. Using a time-resolved endocytosis assay, we show that WASp and Arp2/3 are required for the endocytosis of Dl only during cytokinesis. We propose that WASp-Arp2/3 provides an extra pushing force that is specifically required for the efficient endocytosis of Dl during cytokinesis.

Self-Organization in Pattern Formation.

Self-organization is pervasive in development, from symmetry breaking in the early embryo to tissue patterning and morphogenesis. For a few model systems, the underlying molecular and cellular processes are now sufficiently characterized that mathematical models can be confronted with experiments, to explore the dynamics of pattern formation. Here, we review selected systems, ranging from cyanobacteria to mammals, where different forms of cell-cell communication, acting alone or together with positional cues, drive the patterning of cell fates, highlighting the insights that even very simple models can provide as well as the challenges on the path to a predictive understanding of development.

Stem Cell Proliferation Is Kept in Check by the Chromatin Regulators Kismet/CHD7/CHD8 and Trr/MLL3/4.

Chromatin remodeling accompanies differentiation, however, its role in self-renewal is less well understood. We report that in Drosophila, the chromatin remodeler Kismet/CHD7/CHD8 limits intestinal stem cell (ISC) number and proliferation without affecting differentiation. Stem-cell-specific whole-genome profiling of Kismet revealed its enrichment at transcriptionally active regions bound by RNA polymerase II and Brahma, its recruitment to the transcription start site of activated genes and developmental enhancers and its depletion from regions bound by Polycomb, Histone H1, and heterochromatin Protein 1. We demonstrate that the Trithorax-related/MLL3/4 chromatin modifier regulates ISC proliferation, colocalizes extensively with Kismet throughout the ISC genome, and co-regulates genes in ISCs, including Cbl, a negative regulator of Epidermal Growth Factor Receptor (EGFR). Loss of kismet or trr leads to elevated levels of EGFR protein and signaling, thereby promoting ISC self-renewal. We propose that Kismet with Trr establishes a chromatin state that limits EGFR proliferative signaling, preventing tumor-like stem cell overgrowths.

Mechanisms of Notch signaling: a simple logic deployed in time and space.

Most cells in our body communicate during development and throughout life via Notch receptors and their ligands. Notch receptors relay information from the cell surface to the genome via a very simple mechanism, yet Notch plays multiple roles in development and disease. Recent studies suggest that this versatility in Notch function may not necessarily arise from complex and context-dependent integration of Notch signaling with other developmental signals, but instead arises, in part, from signaling dynamics. Here, we review recent findings on the core Notch signaling mechanism and discuss how spatial-temporal dynamics contribute to Notch signaling output.

Spen limits intestinal stem cell self-renewal.

Precise regulation of stem cell self-renewal and differentiation properties is essential for tissue homeostasis. Using the adult Drosophila intestine to study molecular mechanisms controlling stem cell properties, we identify the gene split-ends (spen) in a genetic screen as a novel regulator of intestinal stem cell fate (ISC). Spen family genes encode conserved RNA recognition motif-containing proteins that are reported to have roles in RNA splicing and transcriptional regulation. We demonstrate that spen acts at multiple points in the ISC lineage with an ISC-intrinsic function in controlling early commitment events of the stem cells and functions in terminally differentiated cells to further limit the proliferation of ISCs. Using two-color cell sorting of stem cells and their daughters, we characterize spen-dependent changes in RNA abundance and exon usage and find potential key regulators downstream of spen. Our work identifies spen as an important regulator of adult stem cells in the Drosophila intestine, provides new insight to Spen-family protein functions, and may also shed light on Spen's mode of action in other developmental contexts.