Symmetry breaking and fate divergence during lateral inhibition in Drosophila.

Lateral inhibition mediates alternative cell fate decision and produces regular cell fate patterns with fate symmetry breaking (SB) relying on the amplification of small stochastic differences in Notch activity via an intercellular negative feedback loop. Here, we used quantitative live imaging of endogenous Scute (Sc), a proneural factor, and of a Notch activity reporter to study the emergence of Sensory Organ Precursor cells (SOPs) in the pupal abdomen of Drosophila. SB was observed at low Sc levels and was not preceded by a phase of intermediate Sc expression and Notch activity. Thus, mutual inhibition may only be transient in this context. In support of the intercellular feedback loop model, cell-to-cell variations in Sc levels promoted fate divergence. The size of the apical area of competing cells did not detectably bias this fate choice. Surprisingly, cells that were in direct contact at the time of SB could adopt the SOP fate, albeit at low frequency (10%). These lateral inhibition defects were corrected by cellular rearrangements, not cell fate change, highlighting the role of cell-cell intercalation in pattern refinement.

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.

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.

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.

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.

Cell Polarity and Notch Signaling: Linked by the E3 Ubiquitin Ligase Neuralized?

Notch is a mechanosensitive receptor that requires direct cell-cell contact for its activation. Both the strength and the range of notch signaling depend on the size and geometry of the contact sites between cells. These properties of cell-cell contacts in turn depend on cell shape and polarity. At the molecular level, the E3 ubiquitin ligase Neuralized (Neur) links receptor activation with epithelial cell remodeling. Neur regulates the endocytosis of the Notch ligand Delta (Dl), hence Notch activation. It also targets the apical polarity protein Stardust (Sdt) to promote the endocytosis of the Crumbs complex, thereby contributing to epithelium remodeling. Here, we review the interplay between Notch signaling and cell polarity and discuss the possible significance of linking Notch signaling with epithelial cell polarity via a common regulator.

Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling.

The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems.

Imogene: identification of motifs and cis-regulatory modules underlying gene co-regulation.

Cis-regulatory modules (CRMs) and motifs play a central role in tissue and condition-specific gene expression. Here we present Imogene, an ensemble of statistical tools that we have developed to facilitate their identification and implemented in a publicly available software. Starting from a small training set of mammalian or fly CRMs that drive similar gene expression profiles, Imogene determines de novo cis-regulatory motifs that underlie this co-expression. It can then predict on a genome-wide scale other CRMs with a regulatory potential similar to the training set. Imogene bypasses the need of large datasets for statistical analyses by making central use of the information provided by the sequenced genomes of multiple species, based on the developed statistical tools and explicit models for transcription factor binding site evolution. We test Imogene on characterized tissue-specific mouse developmental CRMs. Its ability to identify CRMs with the same specificity based on its de novo created motifs is comparable to that of previously evaluated 'motif-blind' methods. We further show, both in flies and in mammals, that Imogene de novo generated motifs are sufficient to discriminate CRMs related to different developmental programs. Notably, purely relying on sequence data, Imogene performs as well in this discrimination task as a previously reported learning algorithm based on Chromatin Immunoprecipitation (ChIP) data for multiple transcription factors at multiple developmental stages.

Distinct levels of Notch activity for commitment and terminal differentiation of stem cells in the adult fly intestine.

Tight regulation of self-renewal and differentiation of adult stem cells ensures that tissues are properly maintained. In the Drosophila intestine, both commitment, i.e. exit from self-renewal, and terminal differentiation are controlled by Notch signaling. Here, we show that distinct requirements for Notch activity exist: commitment requires high Notch activity, whereas terminal differentiation can occur with lower Notch activity. We identified the gene GDP-mannose 4,6-dehydratase (Gmd), a modulator of Notch signaling, as being required for commitment but dispensable for terminal differentiation. Gmd loss resulted in aberrant, self-renewing stem cell divisions that generated extra ISC-like cells defective in Notch reporter activation, as well as wild-type-like cell divisions that produced properly terminally differentiated cells. Lowering Notch signaling using additional genetic means, we provided further evidence that commitment has a higher Notch signaling requirement than terminal differentiation. Our work suggests that a commitment requirement for high-level Notch activity safeguards the stem cells from loss through differentiation, revealing a novel role for the importance of Notch signaling levels in this system.

Transcriptional control of stem cell maintenance in the Drosophila intestine.

Adult stem cells maintain tissue homeostasis by controlling the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs) that self-renew and produce differentiated progeny. Control of ISC identity and maintenance is poorly understood. Here we find that transcriptional repression of Notch target genes by a Hairless-Suppressor of Hairless complex is required for ISC maintenance, and identify genes of the Enhancer of split complex [E(spl)-C] as the major targets of this repression. In addition, we find that the bHLH transcription factor Daughterless is essential to maintain ISC identity and that bHLH binding sites promote ISC-specific enhancer activity. We propose that Daughterless-dependent bHLH activity is important for the ISC fate and that E(spl)-C factors inhibit this activity to promote differentiation.

Genome-wide identification of cis-regulatory motifs and modules underlying gene coregulation using statistics and phylogeny.

Cell fate determination depends in part on the establishment of specific transcriptional programs of gene expression. These programs result from the interpretation of the genomic cis-regulatory information by sequence-specific factors. Decoding this information in sequenced genomes is an important issue. Here, we developed statistical analysis tools to computationally identify the cis-regulatory elements that control gene expression in a set of coregulated genes. Starting with a small number of validated and/or predicted cis-regulatory modules (CRMs) in a reference species as a training set, but with no a priori knowledge of the factors acting in trans, we computationally predicted transcription factor binding sites (TFBSs) and genomic CRMs underlying coregulation. This method was applied to the gene expression program active in Drosophila melanogaster sensory organ precursor cells (SOPs), a specific type of neural progenitor cells. Mutational analysis showed that four, including one newly characterized, out of the five top-ranked families of predicted TFBSs were required for SOP-specific gene expression. Additionaly, 19 out of the 29 top-ranked predicted CRMs directed gene expression in neural progenitor cells, i.e., SOPs or larval brain neuroblasts, with a notable fraction active in SOPs (11/29). We further identified the lola gene as the target of two SOP-specific CRMs and found that the lola gene contributed to SOP specification. The statistics and phylogeny-based tools described here can be more generally applied to identify the cis-regulatory elements of specific gene regulatory networks in any family of related species with sequenced genomes.

Glycosphingolipids in signaling and development: from liposomes to model organisms.

Since the launch of the raft hypothesis in 1997, data generated in liposomes and cultured cells have highlighted the role of glycosphingolipids (GSLs) in the dynamic organization of biological membranes and the activity of signaling complexes. In parallel studies, genetic analysis of the GSL synthetic pathway has begun to reveal some of the specific roles of GSLs in vivo. Here, we review the role of GSLs in signaling in the context of a refined raft hypothesis. Recent genetic studies in worms, flies, and mice give us the opportunity to integrate these in vivo data with earlier in vitro liposome studies.

Drosophila Ric-8 regulates Galphai cortical localization to promote Galphai-dependent planar orientation of the mitotic spindle during asymmetric cell division.

Localization and activation of heterotrimeric G proteins have a crucial role during asymmetric cell division. The asymmetric division of the Drosophila sensory precursor cell (pl) is polarized along the antero-posterior axis by Frizzled signalling and, during this division, activation of Galphai depends on Partner of Inscuteable (Pins). We establish here that Ric-8, which belongs to a family of guanine nucleotide-exchange factors for Galphai, regulates cortical localization of the subunits Galphai and Gbeta13F. Ric-8, Galphai and Pins are not necessary for the control of the anteroposterior orientation of the mitotic spindle during pl cell division downstream of Frizzled signalling, but they are required for maintainance of the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis. Thus, Frizzled and heterotrimeric G-protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis.

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.

Bearded family members inhibit Neuralized-mediated endocytosis and signaling activity of Delta in Drosophila.

Endocytosis of Notch receptor ligands in signaling cells is essential for Notch receptor activation. In Drosophila, the E3 ubiquitin ligase Neuralized (Neur) promotes the endocytosis and signaling activity of the ligand Delta (Dl). In this study, we identify proteins of the Bearded (Brd) family as interactors of Neur. We show that Tom, a prototypic Brd family member, inhibits Neur-dependent Notch signaling. Overexpression of Tom inhibits the endocytosis of Dl and interferes with the interaction of Dl with Neur. Deletion of the Brd gene complex results in ectopic endocytosis of Dl in dorsal cells of stage 5 embryos. This defect in Dl trafficking is associated with ectopic expression of the single-minded gene, a direct Notch target gene that specifies the mesectoderm. We propose that inhibition of Neur by Brd proteins is important for precise spatial regulation of Dl signaling.

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.

Formation and remodeling of epithelial polarity.

Polarity is a fundamental property of all eukaryotic cells that underlies many developmental processes. A recent EMBO workshop (March 27-31) organized by Thomas Lecuit, Norbert Perrimon, and Keith Mostov brought cell and developmental biologists together on the Mediterranean coast near Marseille, France, to share views on how epithelium polarity is established and remodeled during development and disease. Participants witnessed and celebrated the emerging convergence of intellectual and experimental approaches to address how individual cells acquire polarity and form polarized tissues in the context of developing embryos.

Unequal segregation of Neuralized biases Notch activation during asymmetric cell division.

In Drosophila, Notch signaling regulates binary fate decisions at each asymmetric division in sensory organ lineages. Following division of the sensory organ precursor cell (pI), Notch is activated in one daughter cell (pIIa) and inhibited in the other (pIIb). We report that the E3 ubiquitin ligase Neuralized localizes asymmetrically in the dividing pI cell and unequally segregates into the pIIb cell, like the Notch inhibitor Numb. Furthermore, Neuralized upregulates endocytosis of the Notch ligand Delta in the pIIb cell and acts in the pIIb cell to promote activation of Notch in the pIIa cell. Thus, Neuralized is a conserved regulator of Notch signaling that acts as a cell fate determinant. Polarization of the pI cell directs the unequal segregation of both Neuralized and Numb. We propose that coordinated upregulation of ligand activity by Neuralized and inhibition of receptor activity by Numb results in a robust bias in Notch signaling.

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.

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.