Biology subjects

Zartman, J. J.

Publications and source records attributed to Zartman, J. J..

18 recordsLinked to original sources

bioRxiv2026

Harnessing NCX-IP3R-dependent Calcium Oscillations to Regulate Angiogenic Signaling in Endothelial Cells

The blood vasculature has a high capacity for structural regeneration, driven by the blood endothelial cells (BECs) that comprise it. This regenerative process, which involves BEC migration and proliferation to form these complex tissues, is linked to low frequency (< 0.1 Hz) calcium spiking that precedes these activities. However, we need new approaches to stimulating angiogenic responses in tissue engineering applications. By conducting experiments that manipulate local ionic concentrations and developing a simple, yet powerful, computational analysis, we demonstrate that sodium-calcium cross-talk is a crucial component that regulates the calcium signaling and downstream angiogenic responses. Activation and deactivation of the inositol triphosphate 3 receptors (IP3Rs) on the endoplasmic reticulum (ER) and the switch between forward and reverse modes of the sodium-calcium exchanger (NCX) are proposed to be the key mechanisms underlying calcium oscillations when cells are exposed to temporary cationic depletion. The spiking is suggested to be a release of intracellular calcium mediated by IP3R activity, and transport in or out of the cell is driven by NCX for the calcium oscillatory signaling pattern. The NCX and IP3R both contribute to manage intracellular calcium and ionic concentration as initially there is a long ER deactivation period while intracellular sodium slowly increases until a sudden onset of calcium is released by the ER. Other calcium and sodium ion channels can change this resonant coupling of ER and NCX to alter the inter-spike duration. Synchronization of the spiking intervals between cells is triggered by stimulating with vascular endothelial growth factor (VEGF), which induces a propagating wave of intracellular calcium across the 2D tissue culture, prior to coordinated cell migration and proliferation towards the VEGF source. This wave, which can be artificially induced and studied using electrical stimulation, suggests that the underlying sodium-calcium crosstalk mechanism introduces intracellular calcium polarization, whose orientation is transferred across cells through spike synchronization. Thus, control of calcium signaling dynamics through regulation of ionic depletion can serve as useful method for generating angiogenic responses in engineered tissue constructs.

cell biology↗

bioRxiv2025

CalciumInsights: An Open-Source, Tissue-Agnostic Graphical Interface for High-Quality Analysis of Calcium Signals

Fluctuations and propagation of cytosolic calcium levels at both the cellular and tissue levels show complex patterns, referred to as calcium signatures, that regulate growth, organ development, damage responses, and survival. The quantitative analysis of calcium signatures at the cellular level is essential for identifying unique patterns that coordinate biological processes. However, a versatile framework applicable to multiple tissue types, allowing researchers to compare, measure, and validate diverse responses and recognize conserved patterns across model organisms, is missing. Here, we present a post-processing tool, CalciumInsights, which leverages the R packages Shiny and Golem. This tool has a graphical user interface and does not require software programming experience to perform calcium signal analysis. The open-source software has a modular framework with standardized functionalities that can be tailored for various research approaches. CalciumInsights provides descriptive statistical analysis through various metrics extracted from dynamic calcium transients and oscillations, such as peak amplitude, area under the curve, frequency, among others. The tool was evaluated with fluorescence imaging data from three model organisms: Danio rerio, Arabidopsis thaliana, and Drosophila melanogaster, demonstrating its ability to analyze diverse biological responses and models. Finally, the open-source nature of CalciumInsights enables community-driven improvements and developments for enabling new applications. Author SummaryThis manuscript introduces CalciumInsights, an open-source tool for calcium signature analysis. Designed to be a versatile tool that works with various tissue types and biological systems, CalciumInsights has an easy-to-use graphical user interface. Our program simplifies metrics extraction while maintaining the quality of the analysis by integrating several algorithms. CalciumInsights stands out for its user-friendliness, ease of use, and robust data exploration features, such as tunable filters for improved accuracy. These features promote inclusivity and lower barriers to scientific research by making calcium signature analysis accessible to users of all programming skill levels.

bioengineering↗

bioRxiv2025

Forward Engineering Organ Development and Cancer Therapeutics with Optogenetics

Robust growth control is an essential requirement for the survival of living organisms, while its dysregulation results in diseases such as cancer. However, a significant knowledge gap exists in understanding how precise organ growth control is achieved. The growing arsenal of optogenetic toolkits allows precise, noninvasive control of cellular signaling in vivo, enabling research into how bioelectrical and chemical cues regulate organ growth. Here, we used the red-light-activated channelrhodopsin, CsChrimson, to stimulate intracellular calcium signaling dynamics in the wing epithelium of Drosophila melanogaster, an established model system for investigating organ size control. By varying light intensity and activation dynamics systematically, we identified a biphasic regulation of final organ size. Illumination of CsChrimson depolarizes cells and stimulates spikes of cytosolic calcium concentrations, a phenomenon explained by a computational model that incorporates the inclusion of both gap junction closure and voltage-gated calcium channel activation. This calcium regulation tunes downstream effectors involved in growth regulation and apoptosis. In particular, we found that prolonged bright red light exposure (100 lux/12 hours) increased cell death in wing imaginal discs and caused severe morphological abnormalities in adult wings, with phenotypic severity dependent on stimulation parameters defined by illumination intensity and period of activation. Strikingly, an optimum level of dim, pulsatile light (5 lux, 1 minute on/off pulse train) resulted in overgrown organs and significantly upregulated cell proliferation. We also co-expressed an oncogene, RasV12, with CsChrimson and showed that experimental optical simulation parameters can be exploited to control the morphology of tumorous tissues and initiate targeted remission of tumorous growth. Our findings and approach provide a powerful framework to dissect the role of dynamic physiological signaling events in organogenesis and offer translational insights into new therapeutic strategies with applications in cancer and regenerative medicine.

bioengineering↗

bioRxiv2024

Optimal Performance Objectives in the Highly Conserved Bone Morphogenetic Protein Signaling Pathway

Throughout development, complex networks of cell signaling pathways drive cellular decision-making across different tissues and contexts. The transforming growth factor {beta} (TGF-{beta}) pathways, including the BMP/Smad pathway, play crucial roles in these cellular responses. However, as the Smad pathway is used reiteratively throughout the life cycle of all animals, its systems-level behavior varies from one context to another, despite the pathway connectivity remaining nearly constant. For instance, some cellular systems require a rapid response, while others require high noise filtering. In this paper, we examine how the BMP- Smad pathway balances trade-offs among three such systems-level behaviors, or "Performance Objectives (POs)": response speed, noise amplification, and the sensitivity of pathway output to receptor input. Using a Smad pathway model fit to human cell data, we show that varying non-conserved parameters (NCPs) such as protein concentrations, the Smad pathway can be tuned to emphasize any of the three POs and that the concentration of nuclear phosphatase has the greatest effect on tuning the POs. However, due to competition among the POs, the pathway cannot simultaneously optimize all three, but at best must balance trade-offs among the POs. We applied the multi-objective optimization concept of the Pareto Front, a widely used concept in economics to identify optimal trade-offs among various requirements. We show that the BMP pathway efficiently balances competing POs across species and is largely Pareto optimal. Our findings reveal that varying the concentration of NCPs allows the Smad signaling pathway to generate a diverse range of POs. This insight identifies how signaling pathways can be optimally tuned for each context.

systems biology↗

bioRxiv2023

Reverse engineering morphogenesis through Bayesian optimization of physics-based models

Morphogenetic programs direct the cell signaling and nonlinear mechanical interactions between multiple cell types and tissue layers to define organ shape and size. A key challenge for systems and synthetic biology is determining optimal combinations of intra- and inter-cellular interactions to predict an organs shape, size, and function. Physics-based mechanistic models that define the subcellular force distribution facilitate this, but it is extremely challenging to calibrate parameters in these models from data. To solve this inverse problem, we created a Bayesian optimization framework to determine the optimal cellular force distribution such that the predicted organ shapes match the desired organ shapes observed within the experimental imaging data. This integrative framework employs Gaussian Process Regression (GPR), a non-parametric kernel-based probabilistic machine learning modeling paradigm, to learn the mapping functions relating to the morphogenetic programs that generate and maintain the final organ shape. We calibrated and tested the method on cross-sections of Drosophila wing imaginal discs, a highly informative model organ system, to study mechanisms that regulate epithelial processes that range from development to cancer. As a specific test case, the parameter estimation framework successfully infers the underlying changes in core parameters needed to match simulation data with time series imaging data of wing discs perturbed with collagenase. Unexpectedly, the framework also identifies multiple distinct parameter sets that generate shapes similar to wild-type organ shapes. This platform enables an efficient, global sensitivity analysis to support the necessity of both actomyosin contractility and basal ECM stiffness to generate and maintain the curved shape of the wing imaginal disc. The optimization framework, combined with fixed tissue imaging, identified that Piezo, a mechanosensitive ion channel, impacts fold formation by regulating the apical-basal balance of actomyosin contractility and elasticity of ECM. This framework is extensible toward reverse-engineering the morphogenesis of any organ system and can be utilized in real-time control of complex multicellular systems.

systems biology↗

bioRxiv2023

Piezo regulates epithelial topology and promotes precision in organ size control

Mechanosensitive Piezo channels regulate cell division through calcium-mediated activation of ERK signaling or activate Rho signaling to mediate cell extrusion and cell death. However, systems-level functions of Piezo in regulating organogenesis remain poorly understood. Here, we demonstrate that Piezo controls epithelial cell topology to ensure precise organ growth through the integration of live imaging experiments with pharmacological and genetic perturbations and computational modeling. Notably, knockout or knockdown of Piezo led to bilateral asymmetry in wing phenotypes. While pharmacological activation of Piezo stimulated an increase in the frequency of spikes in cytosolic Ca2+, we discovered that Piezo overexpression counterintuitively reduces Ca2+ signaling dynamics. Knockdown of Piezo inhibited proliferation and decreased apoptosis, resulting in an overall increase in epithelial overcrowding. In contrast, either genetic overexpression or pharmacological activation of Piezo increased cell proliferation and cell removal through basal extrusion. Surprisingly, Piezo overexpression increased the hexagonality of cellular topology. To test whether Piezo regulates cell topology, we formulated computational simulations to investigate how expression levels of Piezo protein regulate cell proliferation and apoptosis through modulation of the cut-off tension required for Piezo channel activation. Quantitative analysis validated computational simulation predictions of how perturbations to Piezo impacted epithelial topology. Overall, our findings demonstrate that Piezo promotes robustness in regulating epithelial topology and is necessary for precise organ size control.

developmental biology↗

bioRxiv2023

The multimodal action of G alpha q in coordinating growth and homeostasis in the Drosophila wing imaginal disc

BackgroundG proteins mediate cell responses to various ligands and play key roles in organ development. Dysregulation of G-proteins or Ca2+ signaling impacts many human diseases and results in birth defects. However, the downstream effectors of specific G proteins in developmental regulatory networks are still poorly understood. MethodsWe employed the Gal4/UAS binary system to inhibit or overexpress Gq in the wing disc, followed by phenotypic analysis. Immunohistochemistry and next-gen RNA sequencing identified the downstream effectors and the signaling cascades affected by the disruption of Gq homeostasis. ResultsHere, we characterized how the G protein subunit Gq tunes the size and shape of the wing in the larval and adult stages of development. Downregulation of Gq in the wing disc reduced wing growth and delayed larval development. Gq overexpression is sufficient to promote global Ca2+ waves in the wing disc with a concomitant reduction in the Drosophila final wing size and a delay in pupariation. The reduced wing size phenotype is further enhanced when downregulating downstream components of the core Ca2+ signaling toolkit, suggesting that downstream Ca2+ signaling partially ameliorates the reduction in wing size. In contrast, Gq-mediated pupariation delay is rescued by inhibition of IP3R, a key regulator of Ca2+ signaling. This suggests that Gq regulates developmental phenotypes through both Ca2+-dependent and Ca2+-independent mechanisms. RNA seq analysis shows that disruption of Gq homeostasis affects nuclear hormone receptors, JAK/STAT pathway, and immune response genes. Notably, disruption of Gq homeostasis increases expression levels of Dilp8, a key regulator of growth and pupariation timing. ConclusionGq activity contributes to cell size regulation and wing metamorphosis. Disruption to Gq homeostasis in the peripheral wing disc organ delays larval development through ecdysone signaling inhibition. Overall, Gq signaling mediates key modules of organ size regulation and epithelial homeostasis through the dual action of Ca2+-dependent and independent mechanisms.

developmental biology↗

bioRxiv2022

Balancing competing effects of tissue growth and cytoskeletal regulation during Drosophila wing disc development

Cytoskeletal structure and force generation within cells must be carefully regulated as the developing organ grows to reach a final size and shape. However, how the complex regulation of multiple features of tissue architecture is simultaneously coordinated remains poorly understood. Through iterations between experiments and novel computational multi-scale model simulations, we investigate the combined regulation of cytoskeletal regulation and proliferation in the growing wing imaginal disc. First, we found through experiments and calibrated model simulations that the local curvature and nuclear positioning of cells in the growing wing disc are defined by patterning of nested spatial domains of peaks in apical and basal contractility. Additionally, predictions from model simulations that incorporate a mechanistic description of interkinetic nuclear migration demonstrate that cell proliferation increases the local basal curvature of the wing disc. This is confirmed experimentally as basal curvature increases when growth and proliferation are increased through insulin signaling. In surprising contrast, we experimentally found that Decapentaplegic (Dpp), the key morphogen involved in both growth control and patterning of the anterior-posterior axis, counteracts increases in tissue bending due to cell proliferation via a combined mechanism that balances the competing impacts of both proliferation and patterning of cell contractility. Overall, the high conservation of these regulatory interactions suggests an important balancing mechanism through dual regulation of proliferation and cytoskeleton to meet the multiple criteria defining tissue morphogenesis.

developmental biology↗

bioRxiv2022

Vestigial-dependent recruitment contributes to robust patterning but is not required for wing-fate induction in Drosophila

Cell recruitment is a process by which a differentiated cell induces neighboring cells to adopt its same cell fate. In Drosophila, cells expressing the wing selector gene, vestigial (vg), drive a feed-forward recruitment signal that propagates Vg expression suggesting that the Vg pattern is established as a wavefront throughout the wing pouch. However, the dynamics of Vg pattern formation does not meet this expectation. Here we show that an induction signal may drive Vg expression without Vg feedforward recruitment several cells away from the dorsal-ventral (DV) boundary. Particularly, when Vg expression is strongly inhibited either at the DV boundary or away from it, the activation of the vg Quadrant Enhancer still occurs at a distance, although the levels and precision of the pattern are compromised. Using live imaging of a dual-fluorescent reporter system, we show that contact-dependent cell recruitment does occur in wild-type conditions suggesting that a combination of a long-range induction signal and a signal-relay recruitment establishes Vg patterning in a robust manner. Overall, our findings reveal a previously unidentified role of cell recruitment as a robustness-conferring patterning mechanism. Summary StatementRobust formation of the Vg pattern is established by two mechanisms: a fast, long-range, but noisy induction signal; and a more precise signal relay, contact-dependent, cell recruitment process.

developmental biology↗

bioRxiv2022

G protein-coupled Receptor Contributions to Wing Growth and Morphogenesis in Drosophila melanogaster

The development of multicellular organisms relies on a symphony of spatiotemporally coordinated signals that regulate gene expression. G protein-coupled receptors (GPCRs) are the largest group of transmembrane receptors that play a pivotal role in transducing extracellular signals into physiological outcomes. Emerging research has implicated neurotransmitter GPCRs, classically associated with communication in neuronal tissues, as regulators of pattern formation and morphogenesis. However, how these receptors interact amongst themselves and signaling pathways to regulate organogenesis is still poorly understood. To address this gap, we performed a systematic RNA interference (RNAi)-based screening of 111 GPCRs along with 8 G, 3 G{beta}, and 2 G{gamma} protein subunits in Drosophila melanogaster. We performed a coupled, machine learning-based quantitative and qualitative analysis to identify both severe and more subtle phenotypes. Of the genes screened, 25 demonstrated at least 60% penetrance of severe phenotypes with several of the most severe phenotypes resulting from the knockdown of neuropeptide and neurotransmitter GPCRs that were not known previously to regulate epithelial morphogenesis. Phenotypes observed in positive hits mimic phenotypic manifestations of diseases caused by dysregulation of orthologous human genes. Quantitative reverse transcription polymerase chain reaction and meta-analysis of RNA expression validated positive hits. Overall, the combined qualitative and quantitative characterization of GPCRs and G proteins identifies an extensive set of GPCRs involved in regulating epithelial morphogenesis and relevant to the study of a broad range of human diseases.

developmental biology↗

bioRxiv2020

MAPPER: A new image analysis pipeline unmasks differential regulation of Drosophila wing features

Phenomics requires quantification of large volumes of image data, necessitating high throughput image processing approaches. Existing image processing pipelines for Drosophila wings, a powerful model for studying morphogenesis, are limited in speed, versatility, and precision. To overcome these limitations, we developed MAPPER, a fully-automated machine learning-based pipeline that quantifies high dimensional phenotypic signatures, with each dimension representing a unique morphological feature. MAPPER magnifies the power of Drosophila genetics by rapidly identifying subtle phenotypic differences in sample populations. To demonstrate its widespread utility, we used MAPPER to reveal new insights connecting patterning and growth across Drosophila genotypes and species. The morphological features extracted using MAPPER identified the presence of a uniform scaling of proximal-distal axis length across four different species of Drosophila. Observation of morphological features extracted by MAPPER from Drosophila wings by modulating insulin signaling pathway activity revealed the presence of a scaling gradient across the anterior-posterior axis. Additionally, batch processing of samples with MAPPER revealed a key function for the mechanosensitive calcium channel, Piezo, in regulating bilateral symmetry and robust organ growth. MAPPER is an open source tool for rapid analysis of large volumes of imaging data. Overall, MAPPER provides new capabilities to rigorously and systematically identify genotype-to-phenotype relationships in an automated, high throughput fashion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/422888v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@bdbecdorg.highwire.dtl.DTLVardef@139a7fdorg.highwire.dtl.DTLVardef@43d5b9org.highwire.dtl.DTLVardef@1846af9_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2020

Epithelial organ shape is generated by patterned actomyosin contractility and maintained by the extracellular matrix

Epithelial sheets play important roles in defining organ architecture during development. Here, we employed an iterative experimental and multi-scale computational modeling approach to decouple direct and indirect effects of actomyosin-generated forces, nuclear positioning, extracellular matrix (ECM), and cell-cell adhesion in shaping Drosophila wing imaginal discs, a powerful system for elucidating general principles of epithelial morphogenesis. Basally generated actomyosin forces are found to regulate apically biased nuclear positioning and are required for generating epithelial bending and cell elongation of the wing disc pouch. Surprisingly, however, short-term pharmacological inhibition of ROCK-driven actomyosin contractility does not impact the maintenance of tissue height or curved shape. In comparison, the relative tautness of the extracellular basement membrane is also patterned between regions of the wing disc. However, computational simulations show that patterning of ECM tautness provides only a minor contribution to modulating tissue shape. Instead, the buildup of a passive ECM pre-strain serves a principle role in shape maintenance. Surprisingly, this is independent from the maintenance of actomyosin contractility. Furthermore, localized apical adhesion between the two cell layers within the wing disc requires ROCK-driven actomyosin activity in the absence of the basal extracellular matrix. This apical adhesion between the two cell layers provides additional mechanical support to help maintain tissue integrity. The combined experimental and computational approach provides general insight into how the subcellular forces are generated and maintained within individual cells to induce tissue curvature and suggests an important design principle of epithelial organogenesis whereby forces generated by actomyosin followed by maintenance as pre-strain within the ECM are interconnected, but functionally separable. Significance statementA major outstanding question in developmental biology is the elucidation of general principles of organ shape formation and maintenance. Here, an iterative experimental and multi-scale computational modeling approach reveals that actomyosin contractility generates the bent profile along the anterior-posterior axis while tension within the ECM is sufficient and necessary for preserving the bent shape even in the absence of continued actomyosin contractility once the shape is generated. The mechanisms tested in this study define the necessary factors for establishing the shape of the wing disc, which later everts to form the adult wing during pupal development. The method can be extended to test novel mechanisms of other epithelial systems that consist of several cellular and ECM layers.

biophysics↗

bioRxiv2019

Rab11b-mediated integrin recycling promotes brain metastatic adaptation and outgrowth

GRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC=\"FIGDIR/small/666750v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (68K):\norg.highwire.dtl.DTLVardef@16dc0c4org.highwire.dtl.DTLVardef@8ab7e2org.highwire.dtl.DTLVardef@12e5acforg.highwire.dtl.DTLVardef@4bf298_HPS_FORMAT_FIGEXP M_FIG C_FIG SUMMARYBreast cancer brain metastases (BCBM) have a 5-20 year latency and account for up to 30% of mortality. Developing new therapeutics requires a molecular understanding of adaptation to the brain microenvironment. Here, we combined RNA-sequencing of BCBM development with a reverse genetic screen in Drosophila melanogaster and identified Rab11b, an endosomal recycling protein, as a mediator of metastatic adaptation. We show that disseminated cells up-regulate Rab11b early after arrival in the brain, allowing control of the cell surface proteome through recycling of proteins required for successful interaction with the microenvironment, including integrin {beta}1. Rab11b-mediated control of integrin {beta}1 surface expression allows ligation to the brain ECM, activating mechanotransduction signaling to allow survival and proliferation. We propose a model in which up-regulation of Rab11b allows disseminated cells to recycle needed proteins during metastatic adaptation, without strictly requiring transcription and translation, to allow for metastatic outgrowth.\n\nManuscript SummaryRab11b up-regulation in the brain microenvironment promotes recycling of cargo proteins required for breast cancer brain metastasis, including increased surface expression of integrin {beta}1, which allows brain extracellular matrix attachment and mechanotransduction. Inhibition of the mevalonate pathway with statins prevents geranylgeranylation of Rab11b, decreasing cargo recycling, and inhibiting brain metastasis.

cancer biology↗

bioRxiv2019

From spikes to intercellular waves: tuning the strength of calcium stimulation modulates organ size control

Information flow within and between cells depends in part on calcium (Ca2+) signaling dynamics. However, the biophysical mechanisms that govern emergent patterns of Ca2+ signaling dynamics at the organ level remain elusive. Recent experimental studies in developing Drosophila wing imaginal discs demonstrate the emergence of four distinct patterns of Ca2+ activity: Ca2+ spikes, intercellular Ca2+ transients, tissue-level Ca2+ waves, and a global "fluttering" state. Here, we used a combination of computational modeling and experimental approaches to identify two different populations of cells within tissues that are connected by gap junctional proteins. We term these two subpopulations "initiator cells" defined by elevated levels of Phospholipase C (PLC) activity and "standby cells," which exhibit baseline activity. We found that the strength of hormonal stimulation and extent of gap junctional communication jointly determine the predominate class of Ca2+ signaling activity. Further, single-cell Ca2+ spikes are stimulated by insulin, while intercellular Ca2+ waves depend on Gq activity. Our computational model successfully recapitulates how the dynamics of Ca2+ transients varies during organ growth. Phenotypic analysis of perturbations to Gq and insulin signaling support an integrated model of cytoplasmic Ca2+ as a dynamic reporter of overall tissue growth. Further, we show that perturbations to Ca2+signaling tune the final size of organs. This work provides a platform to further study how organ size regulation emerges from the crosstalk between biochemical growth signals and heterogeneous cell signaling states. Author SummaryCalcium (Ca2+) is a universal second messenger that regulates a myriad of cellular processes such as cell division, cell proliferation and apoptosis. Multiple patterns of Ca2+ signaling including single cell spikes, multicellular Ca2+ transients, large-scale Ca2+ waves, and global "fluttering" have been observed in epithelial systems during organ development. Key molecular players and biophysical mechanisms involved in formation of these patterns during organ development are not well understood. In this work, we developed a generalized multicellular model of Ca2+ that captures all the key categories of Ca2+ activity as a function of key hormonal signals. Integration of model predictions and experiments reveals two subclasses of cell populations and demonstrates that Ca2+ signaling activity at the organ scale is defined by a general decrease in gap junction communication as organ growth. Our experiments also reveal that a "goldilocks zone" of optimal Ca2+ activity is required to achieve optimal growth at the organ level.

systems biology↗

bioRxiv2017

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.

developmental biology↗

bioRxiv2017

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 (Ca2+) are critical signaling components involved in signal integration and are regulated by a core Ca2+ signaling toolkit. Ca2+ 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 Ca2+ transients, including coordinated oscillations, recently have been reported in Drosophila wing discs. In an accompanying paper, we show that impaired Ca2+ signaling impacts the final size and shape of the wing. Here, we discover specific spatiotemporal signatures of Ca2+ 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 Ca2+ oscillations. As a specific test case, we further demonstrated that the morphogen pathway, Hedgehog, controls frequencies of Ca2+ oscillations uniformly in the tissue and is required for spatial patterning of oscillation amplitudes. Thus, the time-averaged dynamics of spontaneous intercellular Ca2+ 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.

developmental biology↗

bioRxiv2016

QuickStitch for seamless stitching of confocal mosaics through high-pass filtering and recursive normalization

Fluorescence micrographs naturally exhibit darkening around their edges (vignetting), which makes seamless stitching challenging. If vignetting is not corrected for, a stitched image will have visible seams where the individual images (tiles) overlap, introducing a systematic error into any quantitative analysis of the image. Although multiple vignetting correction methods exist, there remains no open-source tool that robustly handles large 2D immunofluorescence-based mosaic images. Here, we develop and validate QuickStitch, a tool that applies a recursive normalization algorithm to stitch large-scale immunofluorescence-based mosaics without incurring vignetting seams. We demonstrate how the tool works successfully for tissues of differing size, morphology, and fluorescence intensity. QuickStitch requires no specific information about the imaging system. It is provided as an open-source tool that is both user friendly and extensible, allowing straightforward incorporation into existing image processing pipelines. This enables studies that require accurate segmentation and analysis of high-resolution datasets when parameters of interest include both cellular-level phenomena and larger tissue-level regions of interest.

bioinformatics↗

bioRxiv2016

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 organs 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.

developmental biology↗