Cody Narciso

Patterning of wound-induced intercellular Ca2+ flashes in a developing epithelium

Differential mechanical force distributions are increasingly recognized to provide important feedback into the control of an organs final size and shape. As a second messenger that integrates and relays mechanical information to the cell, calcium ions (Ca(2+)) are a prime candidate for providing important information on both the overall mechanical state of the tissue and resulting behavior at the individual-cell level during development. Still, how the spatiotemporal properties of Ca(2+) transients reflect the underlying mechanical characteristics of tissues is still poorly understood. Here we use an established model system of an epithelial tissue, the Drosophila wing imaginal disc, to investigate how tissue properties impact the propagation of Ca(2+) transients induced by laser ablation. The resulting intercellular Ca(2+) flash is found to be mediated by inositol 1,4,5-trisphosphate and depends on gap junction communication. Further, we find that intercellular Ca(2+) transients show spatially non-uniform characteristics across the proximal-distal axis of the larval wing imaginal disc, which exhibit a gradient in cell size and anisotropy. A computational model of Ca(2+) transients is employed to identify the principle factors explaining the spatiotemporal patterning dynamics of intercellular Ca(2+) flashes. The relative Ca(2+) flash anisotropy is principally explained by local cell shape anisotropy. Further, Ca(2+) velocities are relatively uniform throughout the wing disc, irrespective of cell size or anisotropy. This can be explained by the opposing effects of cell diameter and cell elongation on intercellular Ca(2+) propagation. Thus, intercellular Ca(2+) transients follow lines of mechanical tension at velocities that are largely independent of tissue heterogeneity and reflect the mechanical state of the underlying tissue.

Reverse-engineering organogenesis through feedback loops between model systems

Biological complexity and ethical limitations necessitate models of human development. Traditionally, genetic model systems have provided inexpensive routes to define mechanisms governing organ development. Recent progress has led to 3D human organoid models of development and disease. However, robust methods to control the size and morphology of organoids for high throughput studies need to be developed. Additionally, insights from multiple developmental contexts are required to reveal conserved genes and processes regulating organ growth and development. Positive feedback between quantitative studies using mammalian organoids and insect micro-organs enable identification of underlying principles for organ size and shape control. Advances in the field of multicellular systems engineering are enabling unprecedented high-content studies in developmental biology and disease modeling. These will lead to fundamental advances in regenerative medicine and tissue-engineered soft robotics.

Morphogen signalling patterns calcium waves in the Drosophila wing disc

Organ development is driven by a set of patterned inductive signals. However, how these signals are integrated to coordinate tissue patterning is still poorly understood. Calcium ions (Ca 2+ ) are critical signaling components involved in signal integration and are regulated by a core Ca 2+ signaling toolkit. Ca 2+ signaling encodes a significant fraction of information in cells through both amplitude and frequency-dependent regulation of transcription factors and key regulatory enzymes. A range of intercellular Ca 2+ transients, including coordinated oscillations, recently have been reported in Drosophila wing discs. In an accompanying paper, we show that impaired Ca 2+ signaling impacts the final size and shape of the wing. Here, we discover specific spatiotemporal signatures of Ca 2+ transients during wing disc development. To do so, we developed a new neural-network-based approach for registration of oscillatory signals in organs that frequently move during imaging, and a pipeline for spatiotemporal analysis of intercellular Ca 2+ oscillations. As a specific test case, we further demonstrated that the morphogen pathway, Hedgehog, controls frequencies of Ca 2+ oscillations uniformly in the tissue and is required for spatial patterning of oscillation amplitudes. Thus, the time-averaged dynamics of spontaneous intercellular Ca 2+ transients reflect the morphogenetic signaling state of the tissue during development. This suggests a general mechanism of physiological signaling that provides a memory of morphogenetic patterns. Additionally, our study provides a powerful approach for registering and quantifying oscillatory dynamics in developing organs.Spontaneous and dramatic intercellular calcium waves are frequently observed during organ development, but are poorly understood. Calcium ions are ubiquitous second messengers that carry out a wide-range of functions, including the regulation of cell proliferation, metabolism and death. Consequently, regulation of calcium signaling encodes a significant portion of the cellular decision making state of cells through both amplitude and frequency-dependent regulation of transcription factors and key regulatory enzymes. Here we report that intercellular calcium waves exhibit spatiotemporal patterns at the organ-level using a quantitative image analysis pipeline. Intercellular calcium waves in the Drosophila wing disc require a specific phospholipase C, Plc21C. Further, we demonstrate that the morphogen signaling pathway, Hedgehog, controls frequencies of calcium oscillations uniformly in the tissue and is required for non-uniform spatial patterning of oscillation amplitudes. Thus, the dynamics of spontaneous intercellular calcium waves are regulated by morphogenetic signaling. Intercellular calcium waves propagate information at the organ-scale that reflects the differentiation state of the developing wing disc.

Multi-scale computational study of the mechanical regulation of cell mitotic rounding in epithelia

Mitotic rounding during cell division is critical for preventing daughter cells from inheriting an abnormal number of chromosomes, a condition that occurs frequently in cancer cells. Cells must significantly expand their apical area and transition from a polygonal to circular apical shape to achieve robust mitotic rounding in epithelial tissues, which is where most cancers initiate. However, how cells mechanically regulate robust mitotic rounding within packed tissues is unknown. Here, we analyze mitotic rounding using a newly developed multi-scale subcellular element computational model that is calibrated using experimental data. Novel biologically relevant features of the model include separate representations of the sub-cellular components including the apical membrane and cytoplasm of the cell at the tissue scale level as well as detailed description of cell properties during mitotic rounding. Regression analysis of predictive model simulation results reveals the relative contributions of osmotic pressure, cell-cell adhesion and cortical stiffness to mitotic rounding. Mitotic area expansion is largely driven by regulation of cytoplasmic pressure. Surprisingly, mitotic shape roundness within physiological ranges is most sensitive to variation in cell-cell adhesivity and stiffness. An understanding of how perturbed mechanical properties impact mitotic rounding has important potential implications on, amongst others, how tumors progressively become more genetically unstable due to increased chromosomal aneuploidy and more aggressive.

Whole blood clot optical clearing for nondestructive 3D imaging and quantitative analysis

A technological revolution in both light and electron microscopy imaging now allows unprecedented views of clotting, especially in animal models of hemostasis and thrombosis. However, our understanding of three-dimensional high-resolution clot structure remains incomplete since most of our recent knowledge has come from studies of relatively small clots or thrombi, due to the optical impenetrability of clots beyond a few cell layers in depth. Here, we developed an optimized optical clearing method termed cCLOT that renders large whole blood clots transparent and allows confocal imaging as deep as one millimeter inside the clot. We have tested this method by investigating the 3D structure of clots made from reconstituted pre-labeled blood components yielding new information about the effects of clot contraction on erythrocytes. Although it has been shown recently that erythrocytes are compressed to form polyhedrocytes during clot contraction, observations of this phenomenon have been impeded by the inability to easily image inside clots. As an efficient and non-destructive method, cCLOT represents a powerful research tool in studying blood clot structure and mechanisms controlling clot morphology. Additionally, cCLOT optical clearing has the potential to facilitate imaging of ex vivo clots and thrombi derived from healthy or pathological conditions.

A regulated environment for micro-organs defines essential conditions for intercellular Ca2+ waves

The mechanical stress state of an organ is a critical, but still poorly understood, driver of organogenesis and regeneration. Here we report a chip-based regulated environment for micro-organs (REM-Chip) that enables systematic investigations of the crosstalk between an organ’s mechanical stress environment and biochemical signaling under a multitude of genetic and chemical perturbations. This method has enabled us to identify essential conditions for generating organ-scale intercellular calcium (Ca2+) waves (ICWs) in Drosophila wing imaginal discs that are also observed in vivo. Spontaneous ICWs require the presence of components in fly extract-based growth serum (FEX). Using the REM-Chip, we demonstrate that the release and not the initial application of mechanical compression is sufficient but not necessary to initiate ICWs. Further, the extent of the Ca2+ response is heterogeneous between discs and correlates with the degree of spontaneous ICWs activity in the pre-stress state. This system and method enable detailed examinations of the interplay between mechanical stress state, biochemical regulatory networks, and physiology in complex, hierarchically organized organ cultures.

On-chip three-dimensional tissue histology for microbiopsies

We report a novel approach that generates a high resolution, three-dimensional (3D) fluorescent staining atlas of tissue microbiopsies in a microfluidic device without destroying the tissue. We demonstrate that this method preserves tissue architecture for multiple murine organs by comparing traditional 2D slices to an optically sectioned 3D H&E-mimic. The H&E-mimic slices show a close qualitative match to traditional H&E. The 3D spatial and molecular information obtainable from this method significantly increases the amount of data available for evaluating both tissue morphology and specific biomarkers in a wide range of both research and clinically driven applications and is amenable to automation.

In vivo relevance of intercellular calcium signaling in Drosophila wing development

Recently, organ-scale intercellular Ca2+ transients (ICTs) were reported in the Drosophila wing disc. However, the functional in vivo significance of ICTs remains largely unknown. Here we demonstrate the in vivo relevance of intercellular Ca2+ signaling and its impact on wing development. We report that Ca2+ signaling in vivo decreases as wing discs mature. Ca2+ signaling ex vivo responds to fly extract in a dose-dependent manner. This suggests ICTs occur in vivo due to chemical stimulus that varies in concentration during development. RNAi mediated inhibition of genes required for ICTs results in defects in the size, shape, and vein patterning of adult wings. It also leads to reduction or elimination of in vivo Ca2+ transients. Further, perturbations to the extracellular matrix along the basal side of the wing disc stimulates intercellular Ca2+ waves. This is the first identified chemically defined, non-wounding stimulus of ICTs. Together, these results point toward specific in vivo functions of intercellular Ca2+ signaling to mediate mechanical stress dissipation and ensure robust patterning during development.

Intercellular calcium waves are controlled by morphogen signaling during organ development

Organ development is driven by a set of patterned inductive signals. However, how these signals are integrated to coordinate tissue patterning is still poorly understood. Calcium ions (Ca 2+ ) are critical signaling components involved in signal integration and are regulated by a core Ca 2+ signaling toolkit. Ca 2+ signaling encodes a significant fraction of information in cells through both amplitude and frequency-dependent regulation of transcription factors and key regulatory enzymes. A range of intercellular Ca 2+ transients, including coordinated oscillations, recently have been reported in Drosophila wing discs. In an accompanying paper, we show that impaired Ca 2+ signaling impacts the final size and shape of the wing. Here, we discover specific spatiotemporal signatures of Ca 2+ transients during wing disc development. To do so, we developed a new neural-network-based approach for registration of oscillatory signals in organs that frequently move during imaging, and a pipeline for spatiotemporal analysis of intercellular Ca 2+ oscillations. As a specific test case, we further demonstrated that the morphogen pathway, Hedgehog, controls frequencies of Ca 2+ oscillations uniformly in the tissue and is required for spatial patterning of oscillation amplitudes. Thus, the time-averaged dynamics of spontaneous intercellular Ca 2+ transients reflect the morphogenetic signaling state of the tissue during development. This suggests a general mechanism of physiological signaling that provides a memory of morphogenetic patterns. Additionally, our study provides a powerful approach for registering and quantifying oscillatory dynamics in developing organs.Spontaneous and dramatic intercellular calcium waves are frequently observed during organ development, but are poorly understood. Calcium ions are ubiquitous second messengers that carry out a wide-range of functions, including the regulation of cell proliferation, metabolism and death. Consequently, regulation of calcium signaling encodes a significant portion of the cellular decision making state of cells through both amplitude and frequency-dependent regulation of transcription factors and key regulatory enzymes. Here we report that intercellular calcium waves exhibit spatiotemporal patterns at the organ-level using a quantitative image analysis pipeline. Intercellular calcium waves in the Drosophila wing disc require a specific phospholipase C, Plc21C. Further, we demonstrate that the morphogen signaling pathway, Hedgehog, controls frequencies of calcium oscillations uniformly in the tissue and is required for non-uniform spatial patterning of oscillation amplitudes. Thus, the dynamics of spontaneous intercellular calcium waves are regulated by morphogenetic signaling. Intercellular calcium waves propagate information at the organ-scale that reflects the differentiation state of the developing wing disc.

Intercellular calcium signaling is regulated by morphogens during Drosophila wing development

Organ development is driven by a set of patterned inductive signals. However, how these signals are integrated to coordinate tissue patterning is still poorly understood. Calcium ions (Ca 2+ ) are critical signaling components involved in signal integration and are regulated by a core Ca 2+ signaling toolkit. Ca 2+ signaling encodes a significant fraction of information in cells through both amplitude and frequency-dependent regulation of transcription factors and key regulatory enzymes. A range of intercellular Ca 2+ transients, including coordinated oscillations, recently have been reported in Drosophila wing discs. In an accompanying paper, we show that impaired Ca 2+ signaling impacts the final size and shape of the wing. Here, we discover specific spatiotemporal signatures of Ca 2+ transients during wing disc development. To do so, we developed a new neural-network-based approach for registration of oscillatory signals in organs that frequently move during imaging, and a pipeline for spatiotemporal analysis of intercellular Ca 2+ oscillations. As a specific test case, we further demonstrated that the morphogen pathway, Hedgehog, controls frequencies of Ca 2+ oscillations uniformly in the tissue and is required for spatial patterning of oscillation amplitudes. Thus, the time-averaged dynamics of spontaneous intercellular Ca 2+ transients reflect the morphogenetic signaling state of the tissue during development. This suggests a general mechanism of physiological signaling that provides a memory of morphogenetic patterns. Additionally, our study provides a powerful approach for registering and quantifying oscillatory dynamics in developing organs.Spontaneous and dramatic intercellular calcium waves are frequently observed during organ development, but are poorly understood. Calcium ions are ubiquitous second messengers that carry out a wide-range of functions, including the regulation of cell proliferation, metabolism and death. Consequently, regulation of calcium signaling encodes a significant portion of the cellular decision making state of cells through both amplitude and frequency-dependent regulation of transcription factors and key regulatory enzymes. Here we report that intercellular calcium waves exhibit spatiotemporal patterns at the organ-level using a quantitative image analysis pipeline. Intercellular calcium waves in the Drosophila wing disc require a specific phospholipase C, Plc21C. Further, we demonstrate that the morphogen signaling pathway, Hedgehog, controls frequencies of calcium oscillations uniformly in the tissue and is required for non-uniform spatial patterning of oscillation amplitudes. Thus, the dynamics of spontaneous intercellular calcium waves are regulated by morphogenetic signaling. Intercellular calcium waves propagate information at the organ-scale that reflects the differentiation state of the developing wing disc.