Notch-mediated Lateral Inhibition Research Articles

Scabrous is distributed via signaling filopodia to modulate Notch response during bristle patterning in Drosophila.

During development, cells in tissues must be patterned correctly in order to support tissue function and shape. The sensory bristles of the peripheral nervous system on the thorax of Drosophila melanogaster self-organizes from a unpatterned epithelial tissue to a regular spot pattern during pupal stages. Wild type patterning requires Notch-mediated lateral inhibition. Scabrous is a protein that can bind to and modify Notch receptor activity. Scabrous can be secreted, but it is also known to be localized to basal signaling filopodia, or cytonemes, that play a role in long-range Notch signaling. Here we show that Scabrous is primarily distributed basally, within the range of signaling filopodia extension. We show that filamentous actin dynamics are required for the distribution of Scabrous protein during sensory bristle patterning stages. We show that the Notch response of epithelial cells is sensitive to the level of Scabrous protein being expressed by the sensory bristle precursor cell. Our findings at the cell-level suggest a model for how epithelial cells engaged in lateral inhibition at a distance are sensitive local levels of Scabrous protein.

Cis-inhibition suppresses basal Notch signaling during sensory organ precursor selection

The emergence of the sensory organ precursor (SOP) from an equivalence group in Drosophila is a paradigm for studying single-cell fate specification through Notch-mediated lateral inhibition. Yet, it remains unclear how only a single SOP is selected from a relatively large group of cells. We show here that a critical aspect of SOP selection is controlled by cis-inhibition (CI), whereby the Notch ligands, Delta (Dl), cis-inhibit Notch receptors in the same cell. Based on the observation that the mammalian ligand Dl-like 1 cannot cis-inhibit Notch in Drosophila, we probe the role of CI in vivo. We develop a mathematical model for SOP selection where Dl activity is independently regulated by the ubiquitin ligases Neuralized and Mindbomb1. We show theoretically and experimentally that Mindbomb1 induces basal Notch activity, which is suppressed by CI. Our results highlight the trade-off between basal Notch activity and CI as a mechanism for singling out a SOP from a large equivalence group.

Precision patterning: How inner hair cells "hop" to it.

A combination of Notch-mediated lateral inhibition, mechanical forces, and differential adhesion generates a single row of alternating inner hair and supporting cells.

Talking to your neighbors across scales: Long-distance Notch signaling during patterning.

Tissue patterning is a critical part of animal development. Here we review the role that length- and timescales play in shaping patterns during development, focusing on the mechanisms by which Notch-mediated lateral inhibition signaling generates periodic tissue patterns. Because Notch ligands and receptors are membrane bound, the signaling that underlies lateral inhibition depends on direct cell-cell contacts. Nevertheless, there are many biological examples where effective Notch signaling occurs over distances larger than adjacent cells. Here, we summarize the theoretical and experimental evidence for mechanisms that modify the scale of Notch-mediated lateral inhibition. We focus on how cell protrusions, in addition to other cell behaviors like proliferation and neighbor exchange, allow for Notch signaling to both extend lateral inhibition beyond nearest neighbors and impact the timescale of patterning. Using recent examples, we examine how dynamic cell behaviors like the formation of protrusions affect the timing of Notch-mediated lateral inhibition as well as the density of the final tissue pattern. We suggest that mechanisms that affect the length and timescale of Notch signaling may have key implications for the evolution of patterns. This review highlights the role of cell behaviors in controlling the temporal and spatial dynamics of pattern formation across scales.

Enhancer decommissioning imposes an epigenetic barrier to sensory hair cell regeneration.

Adult mammalian tissues such as heart, brain, retina, and the sensory structures of the inner ear do not effectively regenerate, although a latent capacity for regeneration exists at embryonic and perinatal times. We explored the epigenetic basis for this latent regenerative potential in the mouse inner ear and its rapid loss during maturation. In perinatal supporting cells, whose fate is maintained by Notch-mediated lateral inhibition, the hair cell enhancer network is epigenetically primed (H3K4me1) but silenced (active H3K27 de-acetylation and trimethylation). Blocking Notch signaling during the perinatal period of plasticity rapidly eliminates epigenetic silencing and allows supporting cells to transdifferentiate into hair cells. Importantly, H3K4me1 priming of the hair cell enhancers in supporting cells is removed during the first post-natal week, coinciding with the loss of transdifferentiation potential. We hypothesize that enhancer decommissioning during cochlear maturation contributes to the failure of hair cell regeneration in the mature organ of Corti.

Regulation of Proneural Wave Propagation Through a Combination of Notch-Mediated Lateral Inhibition and EGF-Mediated Reaction Diffusion.

Notch-mediated lateral inhibition regulates binary cell fate choice, resulting in salt-and-pepper pattern formation during various biological processes. In many cases, Notch signaling acts together with other signaling systems. However, it is not clear what happens when Notch signaling is combined with other signaling systems. Mathematical modeling and the use of a simple biological model system will be essential to address this uncertainty. A wave of differentiation in the Drosophila visual center, the "proneural wave," accompanies the activity of the Notch and EGF signaling pathways. Although all of the Notch signaling components required for lateral inhibition are involved in the proneural wave, no salt-and-pepper pattern is found during the progression of the proneural wave. Instead, Notch is activated along the wave front and regulates proneural wave progression. How does Notch signaling control wave propagation without forming a salt-and-pepper pattern? A mathematical model of the proneural wave, based on biological evidence, has demonstrated that Notch-mediated lateral inhibition is implemented within the proneural wave and that the diffusible action of EGF cancels salt-and-pepper pattern formation. The results from numerical simulation have been confirmed by genetic experiments in vivo and suggest that the combination of Notch-mediated lateral inhibition and EGF-mediated reaction diffusion enables a novel function of Notch signaling that regulates propagation of the proneural wave. Similar mechanisms may play important roles in diverse biological processes found in animal development and cancer pathogenesis.

The Orphan Nuclear Receptor TLX Represses Hes1 Expression, Thereby Affecting NOTCH Signaling and Lineage Progression in the Adult SEZ.

SummaryIn the adult subependymal zone (SEZ), neural stem cells (NSCs) apically contacting the lateral ventricle on activation generate progenitors proliferating at the niche basal side. We here show that Tailless (TLX) coordinates NSC activation and basal progenitor proliferation by repressing the NOTCH effector Hes1. Consistent with this, besides quiescence Hes1 expression also increases on Tlx mutation. Since HES1 levels are higher at the apical SEZ, NOTCH activation is increased in Tlx−/− NSCs, but not in surrounding basal progenitors. Underscoring the causative relationship between higher HES1/NOTCH and increased quiescence, downregulation of Hes1 only in mutant NSCs normalizes NOTCH activation and resumes proliferation and neurogenesis not only in NSCs, but especially in basal progenitors. Since pharmacological blockade of NOTCH signaling also promotes proliferation of basal progenitors, we conclude that TLX, by repressing Hes1 expression, counteracts quiescence and NOTCH activation in NSCs, thereby relieving NOTCH-mediated lateral inhibition of proliferation in basal progenitors.

Division of Labor: Roles of Groucho and CtBP in Notch-Mediated Lateral Inhibition that Controls Intestinal Stem Cell Differentiation in Drosophila.

SummaryIntestinal stem cell (ISC) differentiation in the Drosophila midgut requires Delta/Notch-mediated lateral inhibition, which separates the fate of ISCs from differentiating enteroblasts (EBs). Although a canonical Notch signaling cascade is involved in the lateral inhibition, its regulation at the transcriptional level is still unclear. Here we show that the establishment of lateral inhibition between ISC-EB requires two evolutionarily conserved transcriptional co-repressors Groucho (Gro) and C-terminal binding protein (CtBP) that act differently. Gro functions in EBs with E(spl)-C proteins to suppress Delta expression, inhibit cell-cycle re-entry, and promote cell differentiation, whereas CtBP functions specifically in ISCs to mediate transcriptional repression of Su(H) targets and maintain ISC fate. Interestingly, several E(spl)-C genes are also expressed in ISCs that cooperate with Gro to inhibit cell proliferation. Collectively, our study demonstrates separable and cell-type-specific functions of Gro and CtBP in a lateral inhibition process that controls the proliferation and differentiation of tissue stem cells.

Zebrafish Klf4 maintains the ionocyte progenitor population by regulating epidermal stem cell proliferation and lateral inhibition.

In the skin and gill epidermis of fish, ionocytes develop alongside keratinocytes and maintain body fluid ionic homeostasis that is essential for adaptation to environmental fluctuations. It is known that ionocyte progenitors in zebrafish embryos are specified from p63+ epidermal stem cells through a patterning process involving DeltaC (Dlc)-Notch-mediated lateral inhibition, which selects scattered dlc+ cells into the ionocyte progenitor fate. However, mechanisms by which the ionocyte progenitor population is modulated remain unclear. Krüppel-like factor 4 (Klf4) transcription factor was previously implicated in the terminal differentiation of mammalian skin epidermis and is known for its bifunctional regulation of cell proliferation in a tissue context-dependent manner. Here, we report novel roles for zebrafish Klf4 in the ventral ectoderm during embryonic skin development. We found that Klf4 was expressed in p63+ epidermal stem cells of the ventral ectoderm from 90% epiboly onward. Knockdown or knockout of klf4 expression reduced the proliferation rate of p63+ stem cells, resulting in decreased numbers of p63+ stem cells, dlc-p63+ keratinocyte progenitors and dlc+ p63+ ionocyte progenitor cells. These reductions subsequently led to diminished keratinocyte and ionocyte densities and resulted from upregulation of the well-known cell cycle regulators, p53 and cdkn1a/p21. Moreover, mutation analyses of the KLF motif in the dlc promoter, combined with VP16-klf4 or engrailed-klf4 mRNA overexpression analyses, showed that Klf4 can bind the dlc promoter and modulate lateral inhibition by directly repressing dlc expression. This idea was further supported by observing the lateral inhibition outcomes in klf4-overexpressing or knockdown embryos. Overall, our experiments delineate novel roles for zebrafish Klf4 in regulating the ionocyte progenitor population throughout early stem cell stage to initiation of terminal differentiation, which is dependent on Dlc-Notch-mediated lateral inhibition.

A role for actomyosin contractility in Notch signaling

BackgroundNotch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition. Unlike many other signaling systems, however, since both the ligand and receptor are transmembrane proteins, the activation of Notch by Delta depends strictly on cell-cell contact. Furthermore, the binding of the ligand to the receptor may not be sufficient to induce signaling, since recent work in cell culture suggests that ligand-induced Notch signaling also requires a mechanical pulling force. This tension exposes a cleavage site in Notch that, when cut, activates signaling. Although it is not known if mechanical tension contributes to signaling in vivo, others have suggested that this is how endocytosis of the receptor-ligand complex contributes to the cleavage and activation of Notch. In a similar way, since Notch-mediated lateral inhibition at a distance in the dorsal thorax of the pupal fly is mediated via actin-rich protrusions, it is possible that cytoskeletal forces generated by networks of filamentous actin and non-muscle myosin during cycles of protrusion extension and retraction also contribute to Notch signaling.ResultsTo test this hypothesis, we carried out a detailed analysis of the role of myosin II-dependent tension in Notch signaling in the developing fly and in cell culture. Using dynamic fluorescence-based reporters of Notch, we found that myosin II is important for signaling in signal sending and receiving cells in both systems—as expected if myosin II-dependent tension across the Notch-Delta complex contributes to Notch activation. While myosin II was found to contribute most to signaling at a distance, it was also required for maximal signaling between adjacent cells that share lateral contacts and for signaling between cells in culture.ConclusionsTogether these results reveal a previously unappreciated role for non-muscle myosin II contractility in Notch signaling, providing further support for the idea that force contributes to the cleavage and activation of Notch in the context of ligand-dependent signaling, and a new paradigm for actomyosin-based mechanosensation.

Notch-mediated lateral inhibition is an evolutionarily conserved mechanism patterning the ectoderm in echinoids.

Notch signaling is a crucial cog in early development of euechinoid sea urchins, specifying both non-skeletogenic mesodermal lineages and serotonergic neurons in the apical neuroectoderm. Here, the spatial distributions and function of delta, gcm, and hesc, three genes critical to these processes in euechinoids, are examined in the distantly related cidaroid sea urchin Eucidaris tribuloides. Spatial distribution and experimental perturbation of delta and hesc suggest that the function of Notch signaling in ectodermal patterning in early development of E. tr ibuloides is consistent with canonical lateral inhibition. Delta transcripts were observed in t he archenteron, apical ectoderm, and lateral ectoderm in gastrulating e mbryos of E. tribuloides. Perturbation of Notch signaling by either delta morpholino or treatment of DAPT downregulated hesc and upregulated delta and gcm, resulting in ectopic expression of delta and gcm. Similarly, hesc perturbation mirrored the effects of delta perturbation. Interestingly, perturbation of delta or hesc resulted in more cells expressing gcm and supernumerary pigment cells, suggesting that pigment cell proliferation is regulated by Notch in E. tribuloides. These results are consistent with an evolutionary scenario whereby, in the echinoid ancestor, Notch signaling was deployed in the ectoderm to specify neurogenic progenitors and controlled pigment cell proliferation in the dorsal ectoderm.

Cell-Cell Contact Area Affects Notch Signaling and Notch-Dependent Patterning

SummaryDuring development, cells undergo dramatic changes in their morphology. By affecting contact geometry, these morphological changes could influence cellular communication. However, it has remained unclear whether and how signaling depends on contact geometry. This question is particularly relevant for Notch signaling, which coordinates neighboring cell fates through direct cell-cell signaling. Using micropatterning with a receptor trans-endocytosis assay, we show that signaling between pairs of cells correlates with their contact area. This relationship extends across contact diameters ranging from microns to tens of microns. Mathematical modeling predicts that dependence of signaling on contact area can bias cellular differentiation in Notch-mediated lateral inhibition processes, such that smaller cells are more likely to differentiate into signal-producing cells. Consistent with this prediction, analysis of developing chick inner ear revealed that ligand-producing hair cell precursors have smaller apical footprints than non-hair cells. Together, these results highlight the influence of cell morphology on fate determination processes.

Notch-mediated lateral inhibition regulates proneural wave propagation when combined with EGF-mediated reaction diffusion

Notch-mediated lateral inhibition regulates binary cell fate choice, resulting in salt and pepper patterns during various developmental processes. However, how Notch signaling behaves in combination with other signaling systems remains elusive. The wave of differentiation in the Drosophila visual center or "proneural wave" accompanies Notch activity that is propagated without the formation of a salt and pepper pattern, implying that Notch does not form a feedback loop of lateral inhibition during this process. However, mathematical modeling and genetic analysis clearly showed that Notch-mediated lateral inhibition is implemented within the proneural wave. Because partial reduction in EGF signaling causes the formation of the salt and pepper pattern, it is most likely that EGF diffusion cancels salt and pepper pattern formation in silico and in vivo. Moreover, the combination of Notch-mediated lateral inhibition and EGF-mediated reaction diffusion enables a function of Notch signaling that regulates propagation of the wave of differentiation.

Coordinated control of Notch/Delta signalling and cell cycle progression drives lateral inhibition-mediated tissue patterning.

Coordinating cell differentiation with cell growth and division is crucial for the successful development, homeostasis and regeneration of multicellular tissues. Here, we use bristle patterning in the fly notum as a model system to explore the regulatory and functional coupling of cell cycle progression and cell fate decision-making. The pattern of bristles and intervening epithelial cells (ECs) becomes established through Notch-mediated lateral inhibition during G2 phase of the cell cycle, as neighbouring cells physically interact with each other via lateral contacts and/or basal protrusions. Since Notch signalling controls cell division timing downstream of Cdc25, ECs in lateral contact with a Delta-expressing cell experience higher levels of Notch signalling and divide first, followed by more distant neighbours, and lastly Delta-expressing cells. Conversely, mitotic entry and cell division makes ECs refractory to lateral inhibition signalling, fixing their fate. Using a combination of experiments and computational modelling, we show that this reciprocal relationship between Notch signalling and cell cycle progression acts like a developmental clock, providing a delimited window of time during which cells decide their fate, ensuring efficient and orderly bristle patterning.

Groucho restricts rhomboid expression and couples EGFR activation with R8 selection during Drosophila photoreceptor differentiation

Notch and EGFR signaling pathways play important roles in photoreceptor differentiation during Drosophila eye development. Notch signaling induces Enhancer of Split (E(spl)) proteins to repress atonal (ato) expression and restrict R8 photoreceptor cell fate. The R8 precursors express rhomboid (rho), which is required for the release of active EGFR ligand to activate EGFR signaling in surrounding cells for the subsequent stepwise recruitment. However, it is not clear about the mechanisms of transcriptional regulation of rho and how the lateral inhibition of Notch signaling and rho expression are coordinated. In this study, we show that inactivation of Groucho (Gro), an evolutionally conserved transcriptional corepressor, inhibits Ato upregulation, delays R8 determination, and promotes differentiation of R2-5 type of neurons. We demonstrate that these phenotypes are caused by a combination of the loss of Notch-mediated lateral inhibition and the precocious activation of EGFR signaling due to deregulated rho expression. Blocking EGFR signaling by Pnt-RNAi in conjunction with Gro-inactivation leads to lateral inhibition defects with deregulated Ato expression and R8 differentiation. We further show that inactivation of E(spl), which are the Gro binding transcription factors, causes deregulated rho expression and extra R8 cells within and posterior to the morphogenetic furrow (MF), and that E(spl) mediates the binding of Gro to the regulatory regions of both rho and ato genes in eye disc cells. Our results suggest that Gro inhibits rho expression in undifferentiated cells and represses the expression of both ato and rho in non-R8 precursors during initiation of photoreceptor differentiation in an E(spl)-dependent manner. The latter function of Gro provides novel insights into the mechanism that coordinates R8 specification with the restriction of initial rho expression to developing R8 cells.

Identification of the Mind Bomb1 Interaction Domain in Zebrafish DeltaD.

Ubiquitylation promotes endocytosis of the Notch ligands like Delta and Serrate and is essential for them to effectively activate Notch in a neighboring cell. The RING E3 ligase Mind bomb1 (Mib1) ubiquitylates DeltaD to facilitate Notch signaling in zebrafish. We have identified a domain in the intracellular part of the zebrafish Notch ligand DeltaD that is essential for effective interactions with Mib1. We show that elimination of the Mind bomb1 Interaction Domain (MID) or mutation of specific conserved motifs in this domain prevents effective Mib1-mediated ubiquitylation and internalization of DeltaD. Lateral inhibition mediated by Notch signaling regulates early neurogenesis in zebrafish. In this context, Notch activation suppresses neurogenesis, while loss of Notch-mediated lateral inhibition results in a neurogenic phenotype, where too many cells are allowed to become neurons. While Mib1-mediated endocytosis of DeltaD is essential for effective activation of Notch in a neighboring cell (in trans) it is not required for DeltaD to inhibit function of Notch receptors in the same cell (in cis). As a result, forms of DeltaD that have the MID can activate Notch in trans and suppress early neurogenesis when mRNA encoding it is ectopically expressed in zebrafish embryos. On the other hand, when the MID is eliminated/mutated in DeltaD, its ability to activate Notch in trans fails but ability to inhibit in cis is retained. As a result, ectopic expression of DeltaD lacking an effective MID results in a failure of Notch-mediated lateral inhibition and a neurogenic phenotype.

Nemo promotes Notch-mediated lateral inhibition downstream of proneural factors

During neurogenesis, conserved tissue-specific proneural factors establish a cell׳s competence to take on neural fate from within a field of unspecified cells. Proneural genes encode basic helix-loop-helix transcription factors that promote the expression of ‘core’ and subtype-specific target genes. Target genes include both pan-neuronal genes and genes that aid in the process of refinement, known as lateral inhibition. In this process, proneural gene expression is increased in the neural progenitor while simultaneously down-regulated in the surrounding cells, in a Notch signalling-dependent manner. Here, we identify nemo (nmo) as a target of members of both Drosophila Atonal and Achaete-Scute proneural factor families and find that mammalian proneural homologs induce Nemo-like-kinase (Nlk) expression in cell culture. We find that nmo loss of function leads to reduced expression of Notch targets and to perturbations in Notch-mediated lateral inhibition. Furthermore, Notch hyperactivity can compensate for nmo loss in the Drosophila eye. Thus nmo promotes Notch-mediated lateral inhibition downstream of proneural factors during neurogenesis.

Collier/OLF/EBF-Dependent Transcriptional Dynamics Control Pharyngeal Muscle Specification from Primed Cardiopharyngeal Progenitors

In vertebrates, pluripotent pharyngeal mesoderm progenitors produce the cardiac precursors of the second heart field as well as the branchiomeric head muscles and associated stem cells. However, the mechanisms underlying the transition from multipotent progenitors to distinct muscle precursors remain obscured by the complexity of vertebrate embryos. Using Ciona intestinalis as a simple chordate model, we show that bipotent cardiopharyngeal progenitors are primed to activate both heart and pharyngeal muscle transcriptional programs, which progressively become restricted to corresponding precursors. The transcription factor COE (Collier/OLF/EBF) orchestrates the transition to pharyngeal muscle fate both by promoting an MRF-associated myogenic program in myoblasts and by maintaining an undifferentiated state in their sister cells through Notch-mediated lateral inhibition. The latter are stem cell-like muscle precursors that form most of the juvenile pharyngeal muscles. We discuss the implications of our findings for the development and evolution of the chordate cardiopharyngeal mesoderm.

Chapter Two - Proneural bHLH Genes in Development and Disease

Proneural genes encode evolutionarily conserved basic-helix-loop-helix transcription factors. In Drosophila, proneural genes are required and sufficient to confer a neural identity onto naïve ectodermal cells, inducing delamination and subsequent neuronal differentiation. In vertebrates, proneural genes are expressed in cells that already have a neural identity, but they are still required and sufficient to initiate neurogenesis. In all organisms, proneural genes control neurogenesis by regulating Notch-mediated lateral inhibition and initiating the expression of downstream differentiation genes. The general mode of proneural gene function has thus been elucidated. However, the regulatory mechanisms that spatially and temporally control proneural gene function are only beginning to be deciphered. Understanding how proneural gene function is regulated is essential, as aberrant proneural gene expression has recently been linked to a variety of human diseases-ranging from cancer to neuropsychiatric illnesses and diabetes. Recent insights into proneural gene function in development and disease are highlighted herein.

On the role of lateral stabilization during early patterning in the pancreas

The cell fate decision of multi-potent pancreatic progenitor cells between the exocrine and endocrine lineages is regulated by Notch signalling, mediated by cell-cell interactions. However, canonical models of Notch-mediated lateral inhibition cannot explain the scattered spatial distribution of endocrine cells and the cell-type ratio in the developing pancreas. Based on evidence from acinar-to-islet cell transdifferentiation in vitro, we propose that lateral stabilization, i.e. positive feedback between adjacent progenitor cells, acts in parallel with lateral inhibition to regulate pattern formation in the pancreas. A simple mathematical model of transcriptional regulation and cell-cell interaction reveals the existence of multi-stability of spatial patterns whose simultaneous occurrence causes scattering of endocrine cells in the presence of noise. The scattering pattern allows for control of the endocrine-to-exocrine cell-type ratio by modulation of lateral stabilization strength. These theoretical results suggest a previously unrecognized role for lateral stabilization in lineage specification, spatial patterning and cell-type ratio control in organ development.