Myosin XV is a negative regulator of signaling filopodia during long-range lateral inhibition.

The self-organization of cells during development is essential for the formation of healthy tissues and requires the coordination of cell activities at local scales. Cytonemes, or signaling filopodia, are dynamic actin-based cellular protrusions that allow cells to engage in contact mediated signaling at a distance. While signaling filopodia have been shown to support several signaling paradigms during development, less is understood about how these protrusions are regulated. We investigated the role of the plus-end directed, unconventional MyTH4-FERM myosins in regulating signaling filopodia during sensory bristle patterning on the dorsal thorax of the fruit fly Drosophila melanogaster. We found that Myosin XV is required for regulating signaling filopodia dynamics and, as a consequence, lateral inhibition more broadly throughout the patterning epithelium. We found that Myosin XV is required for limiting the length and number of signaling filopodia generated by bristle precursor cells. Cells with additional and longer signaling filopodia due to loss of Myosin XV are not signaling competent, due to altered levels of Delta ligand and Notch receptor along their lengths. We conclude that Myosin XV acts to negatively regulate signaling filopodia, as well as promote the ability of signaling filopodia to engage in long-range Notch signaling. Since Myosin XV isoforms are present across several vertebrate and invertebrate systems, this may have significance for other long-range signaling mechanisms.

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.

Myosin XV is a negative regulator of signaling filopodia during long-range lateral inhibition.

The self-organization of cells during development is essential for the formation of healthy tissues, and requires the coordination of cell activities at local scales. Cytonemes, or signaling filopodia, are dynamic actin-based cellular protrusions that allow cells to engage in contact mediated signaling at a distance. While signaling filopodia have been shown to support several signaling paradigms during development, less is understood about how these protrusions are regulated. We investigated the role of the plus-end directed, unconventional MyTH4-FERM myosins in regulating signaling filopodia during sensory bristle patterning on the dorsal thorax of the fruit fly Drosophila melanogaster. We found that Myosin XV is required for regulating signaling filopodia dynamics and, as a consequence, lateral inhibition more broadly throughout the patterning epithelium. We found that Myosin XV is required for limiting the length and number of signaling filopodia generated by bristle precursor cells. Cells with additional and longer signaling filopodia due to loss of Myosin XV are not signaling competent, due to altered levels of Delta ligand and Notch receptor along their lengths. We conclude that Myosin XV acts to negatively regulate signaling filopodia, as well as promote the ability of signaling filopodia to engage in long-range Notch signaling. Since Myosin XV is present across several vertebrate and invertebrate systems, this may have significance for other long-range signaling mechanisms.

Modeling collective cell behavior in cancer: Perspectives from an interdisciplinary conversation.

Collective cell behavior contributes to all stages of cancer progression. Understanding how collective behavior emerges through cell-cell interactions and decision-making will advance our understanding of cancer biology and provide new therapeutic approaches. Here, we summarize an interdisciplinary discussion on multicellular behavior in cancer, draw lessons from other scientific disciplines, and identify future directions.

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.

Isotropic myosin-generated tissue tension is required for the dynamic orientation of the mitotic spindle.

The ability of cells to divide along their longest axis has been proposed to play an important role in maintaining epithelial tissue homeostasis in many systems. Because the division plane is largely set by the position of the anaphase spindle, it is important to understand how spindles become oriented. While several molecules have been identified that play key roles in spindle orientation across systems, most notably Mud/NuMA and cortical dynein, the precise mechanism by which spindles detect and align with the long cell axis remain poorly understood. Here, in exploring the dynamics of spindle orientation in mechanically distinct regions of the fly notum, we find that the ability of cells to properly reorient their divisions depends on local tissue tension. Thus, spindles reorient to align with the long cell axis in regions where isotropic tension is elevated, but fail to do so in elongated cells within the crowded midline, where tension is low, or in regions that have been mechanically isolated from the rest of the tissue via laser ablation. Importantly, these differences in spindle behavior outside and inside the midline can be recapitulated by corresponding changes in tension induced by perturbations that alter nonmuscle myosin II activity. These data lead us to propose that isotropic tension within an epithelium provides cells with a mechanically stable substrate upon which localized cortical motor complexes can act on astral microtubules to orient the spindle.

Phosphorylation and Proteolytic Cleavage of Notch in Canonical and Noncanonical Notch Signaling.

The Notch signaling pathway seems deceptively simple, with its key feature being a direct connection between extracellular signal and transcriptional output without the need for an extended chain of protein intermediaries as required by so many other signaling paradigms. However, this apparent simplicity hides considerable complexity. Consistent with its central role in many aspects of development, Notch signaling has an extensive collection of mechanisms that it employs alongside of its core transcriptional machinery. These so-called noncanonical Notch pathways diversify the potential outputs of Notch, and allow it to coordinate regulation of many aspects of the biology of cells. Here we will review noncanonical Notch signaling with special attention to the role of posttranslational modifications of Notch. We will also consider the importance of coordinating the activity of gene expression with regulation of cell morphology in biological processes, including axon guidance and other morphological events during embryogenesis.

A role for actomyosin contractility in Notch signaling.

BACKGROUND: Notch-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. RESULTS: To 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. CONCLUSIONS: Together 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.

A new mechanism for spatial pattern formation via lateral and protrusion-mediated lateral signalling.

Tissue organization and patterning are critical during development when genetically identical cells take on different fates. Lateral signalling plays an important role in this process by helping to generate self-organized spatial patterns in an otherwise uniform collection of cells. Recent data suggest that lateral signalling can be mediated both by junctional contacts between neighbouring cells and via cellular protrusions that allow non-neighbouring cells to interact with one another at a distance. However, it remains unclear precisely how signalling mediated by these distinct types of cell-cell contact can physically contribute to the generation of complex patterns without the assistance of diffusible morphogens or pre-patterns. To explore this question, in this work we develop a model of lateral signalling based on a single receptor/ligand pair as exemplified by Notch and Delta. We show that allowing the signalling kinetics to differ at junctional versus protrusion-mediated contacts, an assumption inspired by recent data which show that the cleavage of Notch in several systems requires both Delta binding and the application of mechanical force, permits individual cells to act to promote both lateral activation and lateral inhibition. Strikingly, under this model, in which Delta can sequester Notch, a variety of patterns resembling those typical of reaction-diffusion systems is observed, together with more unusual patterns that arise when we consider changes in signalling kinetics, and in the length and distribution of protrusions. Importantly, these patterns are self-organizing-so that local interactions drive tissue-scale patterning. Together, these data show that protrusions can, in principle, generate different types of patterns in addition to contributing to long-range signalling and to pattern refinement.

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.

An absolute interval scale of order for point patterns.

Human observers readily make judgements about the degree of order in planar arrangements of points (point patterns). Here, based on pairwise ranking of 20 point patterns by degree of order, we have been able to show that judgements of order are highly consistent across individuals and the dimension of order has an interval scale structure spanning roughly 10 just-notable-differences (jnd) between disorder and order. We describe a geometric algorithm that estimates order to an accuracy of half a jnd by quantifying the variability of the size and shape of spaces between points. The algorithm is 70% more accurate than the best available measures. By anchoring the output of the algorithm so that Poisson point processes score on average 0, perfect lattices score 10 and unit steps correspond closely to jnds, we construct an absolute interval scale of order. We demonstrate its utility in biology by using this scale to quantify order during the development of the pattern of bristles on the dorsal thorax of the fruit fly.

Ion channels contribute to the regulation of cell sheet forces during Drosophila dorsal closure.

We demonstrate that ion channels contribute to the regulation of dorsal closure in Drosophila, a model system for cell sheet morphogenesis. We find that Ca(2+) is sufficient to cause cell contraction in dorsal closure tissues, as UV-mediated release of caged Ca(2+) leads to cell contraction. Furthermore, endogenous Ca(2+) fluxes correlate with cell contraction in the amnioserosa during closure, whereas the chelation of Ca(2+) slows closure. Microinjection of high concentrations of the peptide GsMTx4, which is a specific modulator of mechanically gated ion channel function, causes increases in cytoplasmic free Ca(2+) and actomyosin contractility and, in the long term, blocks closure in a dose-dependent manner. We identify two channel subunits, ripped pocket and dtrpA1 (TrpA1), that play a role in closure and other morphogenetic events. Blocking channels leads to defects in force generation via failure of actomyosin structures, and impairs the ability of tissues to regulate forces in response to laser microsurgery. Our results point to a key role for ion channels in closure, and suggest a mechanism for the coordination of force-producing cell behaviors across the embryo.

PCB126 exposure disrupts zebrafish ventricular and branchial but not early neural crest development.

We have used zebrafish and 3,3',4,4',5-pentachlorobiphenyl (PCB126) to investigate the developmental toxicity of polychlorinated biphenyls (PCBs) that exert their effects through the aryl hydrocarbon receptor (AHR). We found that cardiac and neural crest (NC)-derived jaw and branchial cartilages are specifically targeted early in development. The suite of malformations, which ultimately leads to circulatory failure, includes a severely dysmorphic heart with a reduced bulbus arteriosus and abnormal atrioventricular and outflow valve formation. Early NC migration and patterning of the jaw and branchial cartilages was normal. However, the jaw and branchial cartilages failed to grow to normal size. In the heart, the ventricular myocardium showed a reduction in cell number and size. The heart and jaw/branchial phenotype could be rescued by pifithrin-alpha, a blocker of p53. However, the function of pifithrin-alpha in this model may act as a competitive inhibitor of PCB at the AHR and is likely independent of p53. Morpholinos against p53 did not rescue the phenotype, nor were zebrafish with a mutant p53-null allele resistant to PCB126 toxicity. Morpholino knockdown of cardiac troponin T, which blocks the onset of cardiac function, prevented the PCB126-induced cardiac dysmorphogenesis but not the jaw/branchial phenotype. The cardiovascular characteristics appear to be similar to hypoplastic left heart syndrome (HLHS) and introduce the potential of zebrafish as a model to study this environmentally induced cardiovascular malformation. HLHS is a severe congenital cardiovascular malformation that has previously been linked to industrial releases of dioxins and PCBs.

Suppressors of zyg-1 define regulators of centrosome duplication and nuclear association in Caenorhabditis elegans.

In Caenorhabditis elegans, the kinase ZYG-1 is required for centrosome duplication. To identify factors that interact with ZYG-1, we used a classical genetic approach and identified 21 szy (suppressor of zyg-1) genes that when mutated restore partial viability to a zyg-1 mutant. None of the suppressors render animals completely independent of zyg-1 activity and analysis of a subset of the suppressors indicates that all restore the normal process of centrosome duplication to zyg-1 mutants. Thirteen of these suppressor mutations confer phenotypes of their own and cytological examination reveals that these genes function in a variety of cellular processes including cell cycle timing, microtubule organization, cytokinesis, chromosome segregation, and centrosome morphology. Interestingly, several of the szy genes play a role in attaching the centrosome to the nuclear envelope. We have found that one such szy gene is sun-1, a gene encoding a nuclear envelope component. We further show that the role of SUN-1 in centrosome duplication is distinct from its role in attachment. Our approach has thus identified numerous candidate regulators of centrosome duplication and uncovered an unanticipated regulatory mechanism involving factors that tether the centrosome to the nucleus.