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Mapping the calcium signalsome during Drosophila wing development
Proceedings, 7th IFAC Conference on Proceedings, 7th IFAC Conference Foundations of Systems Biology inon Engineering Proceedings, 7th IFAC Conference on Available online at www.sciencedirect.com Foundations of Systems Biology in2018 Engineering Chicago, Illinois, USA, August 5-8, Proceedings, 7th IFAC Conference on Proceedings,of 7th IFAC Conference on Foundations Systems Biology Engineering Chicago, Illinois, USA, August 5-8,in 2018 Foundations of Systems Biology in Engineering Foundations of Systems Biology Engineering Chicago, Illinois, USA, August 5-8,in2018 Chicago, Illinois, Illinois, USA, USA, August August 5-8, 5-8, 2018 2018 Chicago,
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IFAC PapersOnLine 51-19 (2018) 108–109
Mapping the calcium signalsome during Drosophila wing development Mapping the calcium signalsome during Drosophila wing development Mapping the calcium signalsome during Drosophila wing development *,† * Drosophila wing development Mapping the calcium signalsome during Pavel A. Brodskiy , Qinfeng Wu*,† , Nilay Kumar Kumar , Vijay Kumar Naidu Velagala**, *,† *,† *
*,†, Qinfeng Wu*,† *, Vijay Kumar *, * * , Nilay Kumar Kumar * * * Pavel A. Brodskiy Naidu Velagala *,† * * Kara L. Snyder , Francisco J. Huizar , Seth J.Kumar TautgesKumar , Meaghan Snyder , Jeremiah J. Zartman Pavel A. Brodskiy Wu*,† Nilay Kumar Naidu Velagala * * * * * *,†, Qinfeng *,† *, Vijay *, *, Francisco *, ,Seth *, Meaghan *, Jeremiah *,† *,† * * Kara L. Snyder J. Huizar J. Tautges Snyder J. Zartman Pavel A. A. Brodskiy Brodskiy Qinfeng Wu Wu * ,, Nilay Nilay Kumar Kumar Kumar Kumar Vijay Kumar Kumar Naidu Velagala Velagala ,, ** * * * Pavel ,, Qinfeng ,, Vijay Naidu , Francisco J. Huizar , Seth J. Tautges , Meaghan Snyder , Jeremiah J. Zartman Kara L. Snyder * * * * * * *, Francisco *, Seth *, Meaghan University Kara J. J. J. Department of Chemical and Biomolecular Engineering, of Notre Dame, Kara L. L. Snyder Snyder , Francisco J. Huizar Huizar , Seth J. Tautges Tautges , Meaghan Snyder Snyder*,, Jeremiah Jeremiah J. Zartman Zartman* ** of Notre Chemical andINBiomolecular Engineering, University of Notre Dame, *Department 205 McCourtney Hall, Dame, 46556 USA (Tel: 574-631-0455; e-mail: [email protected]). and Biomolecular Engineering, University of Notre Dame, **Department of Chemical †Dame, Department of Notre Chemical and Biomolecular Engineering, University of Notre Dame, 205 McCourtney Hall, IN 46556 USA (Tel: 574-631-0455; e-mail: [email protected]). Department of Chemical and Biomolecular Engineering, University of Notre Dame, Authors contributed equally to this work e-mail: [email protected]). 205 McCourtney Hall, Notre ††Dame, IN 46556 USA (Tel: 574-631-0455; Authors to this work e-mail: 205 McCourtney McCourtney Hall, Hall, Notre Notre †Dame, Dame, INcontributed 46556 USA USAequally (Tel: 574-631-0455; 574-631-0455; e-mail: [email protected]). [email protected]). 205 IN 46556 (Tel: †Authors contributed equally to this work † Authors contributed contributed equally equally to to this this work work Authors Abstract: Second messengers such as calcium (Ca2+ ) play critical roles in signal integration for complex, 2+ 2+) play critical Abstract: Second messengers such as calcium (Ca roles inregulates signal integration for complex, 2+ multicellular processes. However, how components of Ca signaling tissue development is Abstract: Second messengers such as calcium (Ca2+ critical roles inregulates signal integration for complex, 2+ 2+) play 2+ signaling 2+ multicellular processes. However, how components of Ca tissue development is 2+ Abstract: SecondRecently, messengers such asdiscovered calcium (Ca (Ca ) play play critical rolessignaling in signal signal differentially integration for foracross complex, Abstract: Second messengers such as calcium ) critical roles in integration complex, 2+ the poorly studied. we have that perturbing Ca multicellular processes. However, components of Ca2+ regulatesdifferentially tissue development is 2+ signaling 2+ signaling poorly studied. Recently, weresults havehow discovered that perturbing Ca2+ acrossThis the multicellular processes. However, how components of Ca Ca signaling regulates tissuearchitecture. development is multicellular processes. However, how components of signaling regulates tissue development is 2+ to the developing Drosophila wing in three-dimensional distortions finaldifferentially wing poorly studied. Recently, we have discovered that perturbing Ca signaling across the 2+ 2+ to developing Drosophila wing results in three-dimensional distortions the finaldifferentially wing architecture. This poorly studied. Recently, we have discovered that perturbing Ca signaling across the poorly studied. Recently, we have discovered that perturbing Ca signaling differentially across the finding prompted us to pursue a comprehensive high-content screen for phenotypes generated by perturbing developing Drosophila wing results in three-dimensional the final generated wing architecture. This finding prompted us to pursue a comprehensive high-contentdistortions screen for to phenotypes by perturbing developing Drosophila wing results in distortions to the wing This developing Drosophila wing results in three-dimensional three-dimensional distortions to the final final generated wing architecture. This known calcium-related genes. The rapidly-expanding dataset includes deep architecture. coverage (~30 finding prompted us to pursue a comprehensive high-content screen for phenotypes by perturbing known calcium-related genes. The rapidly-expanding dataset includes deep coverage (~30 finding prompted us to pursue a comprehensive high-content screen for phenotypes generated by perturbing finding prompted us to pursue a comprehensive high-content screen for phenotypes generated by perturbing wings/perturbation) and a high-resolution dataset of over 7,000 fly wing images. Preliminary findings known calcium-related genes. The rapidly-expanding dataset includes deep coveragefindings (~30 wings/perturbation) and a of high-resolution dataset of several over 7,000 fly of wing images. Preliminary known calcium-related genes. The dataset includes deep coverage (~30 known calcium-related genes. The rapidly-expanding rapidly-expanding dataset includes deep coverage (~30 signaling leads to classes wing developmental phenotypes indicate that perturbation Ca2+ wings/perturbation) and a high-resolution dataset of over 7,000 fly wing images. Preliminary findings 2+ 2+ indicate that perturbation of Ca signaling leads to several classes of wing developmental phenotypes wings/perturbation) and a high-resolution dataset of over 7,000 fly wing images. Preliminary findings wings/perturbation) and a high-resolution dataset of over 7,000 fly wing images. Preliminary findings 2+ including thickening of veins, bifurcations, and wing crumpling. High-dimensional fingerprinting of these indicate that perturbation of Ca signaling leads to several classes of wing developmental phenotypes 2+ 2+ including thickening of veins, bifurcations, and wing High-dimensional of these indicate that that perturbation of clusters Ca signaling leads to crumpling. several classes of wing wing developmental developmental phenotypes indicate perturbation of Ca signaling leads to several classes of phenotypes phenotypes revealed several of genes, implying mechanistic interactions as fingerprinting well as novel roles for including thickening of veins, bifurcations, and wing crumpling. High-dimensional fingerprinting of these phenotypes revealed several clusters of genes, implying mechanistic interactions as well as novel roles for including thickening thickening ofinveins, veins, bifurcations, and wing wing crumpling. High-dimensional fingerprinting of these these including of bifurcations, and crumpling. High-dimensional fingerprinting of calcium-related genes morphogenesis. Future work will focus on validation of interactions within gene phenotypes revealed clusters of genes, implying mechanistic interactions as well as novel rolesgene for calcium-related genesseveral innovel morphogenesis. Future work will focus interactions within 2+ validation phenotypes revealed several clusters of genes, genes, implying mechanistic interactions as well as as novel novel roles for for phenotypes clusters of mechanistic interactions as well roles signaling onof development. clusters andrevealed identifying consequences ofimplying perturbation of Caon calcium-related genesseveral in morphogenesis. Future work will focus on validation of interactions within gene 2+ 2+ calcium-related genes in morphogenesis. Future work will focus validation interactions within gene clusters and identifying novel consequences of perturbation of Caon onof development. calcium-related genes in morphogenesis. Future work will focus on validation of interactions within gene 2+ signaling clusters identifying novel consequences of perturbation Ca2+ signaling onLtd. development. Keywords: Organogenesis, high-content machine learning, analysis. © 2018, and IFAC (International Federation ofscreening, Automatic Control)of Hosting byimage Elsevier All rights reserved. clusters and identifying novel consequences of of Ca on clusters and identifying novel consequences of perturbation perturbation of Ca2+ signaling signaling on development. development. Keywords: Organogenesis, high-content screening, machine learning, image analysis. Keywords: Organogenesis, high-content screening, machine learning, image analysis. Keywords: Organogenesis, high-content high-content screening, screening, machine machine learning, learning, image image analysis. analysis. Keywords: Organogenesis, Drosophila wing phenotypes (Fig 1), and to characterize 1. INTRODUCTION Drosophila wing phenotypes (Figof 1), and to characterize phenotypes caused by perturbation Ca2+ signaling. 1. INTRODUCTION Drosophila wing phenotypes (Figof 1), and to characterize 2+ 2+ 2+ phenotypes caused by perturbation Ca signaling. Calcium (Ca ) integrates a diverse range of chemical, genetic, Drosophila wing wing phenotypes phenotypes (Fig (Fig 1), 1), 2+and and to to characterize characterize 1. INTRODUCTION Drosophila 2+ 2+) integrates a diverse range of chemical, genetic, phenotypes caused by perturbation of Ca signaling. 1. INTRODUCTION Calcium (Ca 2+ 1. INTRODUCTION 2+ 2+ mechanical inputs to regulate many cellular electrical, and phenotypes caused by perturbation of Ca signaling. 2. METHODS phenotypes caused by perturbation of Ca signaling. Calcium (Ca ) integrates a diverse range of chemical, genetic, 2+ mechanical inputs to regulate many cellular 2+ electrical, and Calcium (Ca ) integrates diverse range of genetic, Calcium integrates diverse range of chemical, chemical, genetic, METHODS processes (Berridge et al.,aa2000). include proliferation, electrical,(Ca and) mechanical inputsThese to regulate many cellular 2. 2. METHODS (Berridge et al., 2000). These include proliferation, electrical, and mechanical inputs to regulate many cellular processes electrical, and mechanical inputs to regulate many cellular 2. METHODS death, motility, properties, and differentiation. As 2.1 Gene selection 2. METHODS processes (Berridge et al., 2000). These include proliferation, 2+ 2000). processes (Berridge et al., These include proliferation, death, motility, mechanical properties, and differentiation. As 2.1 Gene selection processes (Berridge et al., 2000). These include proliferation, adeath, result, intercellular Ca signals serve as a key module for motility, mechanical 2.1 Gene selection 2+ properties, and differentiation. As motility, mechanical properties, As serveand as differentiation. a key module for adeath, result, intercellular Ca2+ 2.1 selection screen currently consists of 132 RNAi mediated death, motility, mechanical properties, and differentiation. As The 2+ signals coordinating large-scale collective behaviors including 2.1 Gene Gene selection acoordinating result, intercellular Ca signals serve as a key module for 2+ The screen of 132 binary RNAi expression mediated 2+ signals large-scale collective behaviors including aamorphogenesis result, intercellular Ca serve as a key module for knockdowns driven by consists the GAL4/UAS result, intercellular signals servebehaviors as a key module for The screen currently and Ca wound healing. Second messenger currently consists of 132 binary RNAi expression mediated coordinating large-scale collective including knockdowns driven by the GAL4/UAS morphogenesis and such wound healing. Secondaremessenger 2+ The screen screen currently consists of 67132 132 RNAi mediated coordinating large-scale collective behaviors including The currently consists of RNAi mediated system (Duffy,driven 2002, by p. 4). OfGAL4/UAS these, genes are involved in coordinating large-scale collective behaviors including signaling pathways as Ca signaling therefore knockdowns the binary expression morphogenesis and wound healing. Second messenger 2+ 2+ signaling are therefore system (Duffy, 2002, p. 4). OfGAL4/UAS these, 67 genes are involved in 2+ by 2+ signaling pathways such as Ca knockdowns driven by the GAL4/UAS binary expression morphogenesis and wound healing. Second messenger knockdowns driven the binary expression transport of Ca or expressed Ca -binding domains. morphogenesis and wound healing. Second messenger 2+ composed of both encoding and decoding modules, forming a (Duffy, 2002, Of these, 67 2+ genes are involved in 2+ signaling pathways such as Ca are forming thereforea system 2+ p. 4). 2+ 2+ signaling transport of Ca expressed -binding 2+ composed of both encoding and decoding modules, 2+ system (Duffy, 2002, p. 4). Of these, 67 genes are involved in or Ca domains. signaling pathways pathways such such asCaCa Ca is signaling signaling are for therefore (Duffy, 2002, p. 4). Of selected these, 67 genes are involved in 2+ 2+ Additionally, genes were signaling genes signaling are therefore bow-tie-shaped important winga system transport of 65 Ca or expressed Cafrom -binding domains. composed of bothnetwork. encodingas and modules, forming 2+ 2+ 2+ 2+decoding 2+ 2+ bow-tie-shaped network. Ca is important for wing transport of Ca or expressed Ca -binding domains. Additionally, 65 genes were selected from signaling genes composed of of both both encoding and2+the decoding modules, forming a transport of Ca or expressed Ca -binding domains. with known phenotypes and human disease genes highly composed encoding and decoding modules, forming a development (Fig 1). However, roles of different modules Additionally, 65 genes were selected from signaling genes bow-tie-shaped Ca2+ is important wing with development (Fignetwork. 1).the However, roles of differentfor known phenotypes and human disease genescontrols highly 65 genes selected from signaling genes bow-tie-shaped network. Ca is for wing Additionally, 65developing genes were were selected from signaling genes expressed in the wing to serve as positive bow-tie-shaped network. Ca2+the is important formodules wing Additionally, of the Ca2+ toolkit in development ofimportant multicellular systems with known phenotypes and human disease genes highly development (Fig 1). However, the roles of different modules 2+ 2+ of the Ca with known phenotypes and human disease genes highly expressed in the developing wing to serve as positive controls toolkit in the development of multicellular systems development (Fig 1). However, the roles of different modules with known phenotypes and human disease genes highly for image processing. 2+ scale development (Fig 1). However, the roles of different modules at a systems are poorly understood. expressed in the developing wing to serve as positive controls of Ca2+ toolkit in the development of multicellular systems for at athe systems scale are poorly understood. expressed in the the developing developing wing wing to to serve serve as as positive positive controls controls image processing. of the Ca in development of in of Ca2+ toolkit toolkit in the the development of multicellular multicellular systems systems expressed for image processing. at athe systems scale are poorly understood. for image processing. at a systems scale are poorly understood. 2.2 Fly culture and wing collection for image processing. at a systems scale are poorly understood. 2.2 Fly culture and wing collection 2.2 Fly culture and wing collection 2.2 wing collection MS1096-GAL4 (BL# 8860) virgins were crossed to TRiP line 2.2 Fly Fly culture culture and and wing collection MS1096-GAL4 (BL# 8860) virgins were crossed TRiP line males under fly culture conditions described in to (Greenspan, MS1096-GAL4 (BL# 8860) virgins were crossed to TRiP line males under fly culture conditions described in (Greenspan, MS1096-GAL4 (BL# 8860) virgins were crossed to MS1096-GAL4 (BL# 8860) virgins were crossed to TRiP line 2004). Crosses were passaged every 4-5 days at TRiP 25ooC.line A males under fly culture conditions described in (Greenspan, o 2004). Crosses were A males under fly culture conditions described in (Greenspan, passaged every 4-5 days at 25oC. to males under fly culture conditions described in (Greenspan, custom laboratory data management system was developed 2004). Crosses were passaged every 4-5 days at 25ooC. A 2004). Crosses were passaged every 4-5 days at 25 C. A custom laboratory data management system was developed to 2004). were every 4-5 placed days atin25 C. to A streamline the pipeline (Fig. 2). Wings were ethanol, custom Crosses laboratory datapassaged management system was developed pipeline custom laboratory data management system was developed to streamline the (Fig. 2). Wings were placed in ethanol, custom laboratory data management system was developed to and approximately 15 wings were mounted on each slide in streamline the pipeline (Fig. 2). Wings were placed in ethanol, streamline the pipeline (Fig. Wings were placed in and approximately 15 wings2). were mounted on each slide in streamline the pipeline (Fig. Wings were placed in ethanol, ethanol, Permount medium (Fisher Scientific, SP15). Slides were and approximately 15 wings2). were mounted on each slide in and approximately approximately 15 wings were mounted on each slide in Permount medium (Fisher Scientific, SP15). Slidesslide were and 15 wings were mounted on each in imaged on a Leica Aperio slide scanner at 5x magnification. Permount medium (Fisher Scientific, SP15). Slides were imaged on amedium Leica Aperio slide scanner atSP15). 5x magnification. Permount medium (Fisher Scientific, SP15). Slides were were Permount (Fisher Scientific, Slides imaged on a Leica Aperio slide scanner at 5x magnification. imaged on analysis Leica Aperio Aperio slide slide scanner scanner at at 5x 5x magnification. magnification. 2.3 Image imaged on aa Leica 2.3 Image analysis Fig 1. Perturbations to core regulators of Ca2+ signaling lead to 2.3 Image analysis 2+ Fig 1. Perturbations to adult core regulators 2.3 analysis signaling lead in to Wing of Ca2+ images were isolated from slide scans and rotated using 2.3 Image Image analysis several characteristic wing phenotypes, as reported Fig 1. Perturbations to core regulators of Ca2+ 2+ signaling lead to images MATLAB were isolated from slide scans and using 2+ Fig 1. 1.etPerturbations Perturbations to adult core regulators regulators of Ca Ca signaling signaling lead in to Wing several characteristic wing phenotypes, as reported a custom Images wererotated manually Fig to core of lead to (Wu al., 2017). images were isolatedscript. from slide scans and rotated using several characteristic adult wing phenotypes, as reported in aWing Wing images were isolated from slide scans and rotated using custom MATLAB script. Images were manually (Wu et al., 2017). several characteristic characteristic adult adult wing wing phenotypes, phenotypes, as as reported reported in in Wing images MATLAB were isolated from slide and using phenotypically scored withscript. 61 classes ofscans controlled vocabulary several a custom Images wererotated manually (Wu et al., 2017). a custom MATLAB script. Images were manually phenotypically scored with 61 classes of controlled vocabulary (Wu primary et al., al., 2017). 2017). custom MATLAB script. Images were manually The goal of this study is to identify additional genes a(Fig 3). To extract quantitative features, pixels were classified (Wu et phenotypically scored with 61 classes of controlled vocabulary phenotypically scored with of controlled vocabulary The primary goal of thisthat study to identify 3). To extract quantitative features, pixelsand were classified phenotypically scored with 61 61 classes classes ofhairs, controlled vocabulary in Ca2+ signalsome areis involved in additional regulating genes these (Fig as background, veins, intervein regions, debris using Thethe primary goal of thisthat study is to identify additional genes (Fig 3). To extract quantitative features, pixels were classified 2+ 2+ signalsome are involved in regulating these The primary goal of this study is to identify additional genes (Fig 3). To extract quantitative features, pixels were classified in the Ca as background, veins, intervein regions, hairs, and debris using The primary goal of thisthat study isinvolved to identify additional genes (Fig 3). To extract quantitative features,hairs, pixelsand were classified in the Ca2+ signalsome are in regulating these as background, veins, intervein regions, debris using 2+ signalsome that are involved in regulating these in the the Ca Ca2+ as background, background, veins, veins, intervein intervein regions, regions, hairs, hairs, and and debris debris using using in signalsome that are involved in regulating these as
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the pixel classifier Ilastik (Sommer et al., 2011). Intervein regions were extracted using a custom MATLAB pipeline. A bagged tree classifier was trained with 500-1,000 images per intervein region class to predict the identity of intervein regions from their morphometric features.
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organogenesis. We expect to generate a dataset of >10,000 images encompassing approximately 500 Ca2+-related genes. This dataset will provide materials to the community to advance the study of developmental biology.
Fig 4. t-distributed stochastic neighbor embedding representation of fingerprints to map the consequences of perturbing Ca2+ signaling (Maaten and Hinton, 2008).
Fig. 2. We constructed a custom MySQL database and web front to manage experiments and record results. Operational data generated from the database is used to optimize the screening process.
ACKNOWLEDGEMENTS We would like to thank the South Bend Medical Foundation for generous access to their Apero Slide Scanner, and CzierAnne Gone, Isaac Nordan, Ryan Govi, Heather Flynn, Adriana Szpynda, Quincey Hogue, and Maria Unger for technical assistance. The work in this manuscript was supported in part by NIH Grant R35GM124935 and CBET-1553826 and the Walther Cancer Foundation Interdisciplinary Interface Training Project (PB). REFERENCES Berridge, M.J., Lipp, P., Bootman, M.D., 2000. The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1, 11–21. https://doi.org/10.1038/35036035 Duffy, J.B., 2002. GAL4 system in drosophila: A fly geneticist’s swiss army knife. genesis 34, 1–15. https://doi.org/10.1002/gene.10150 Eid, J.-P., Arias, A.M., Robertson, H., Hime, G.R., Dziadek, M., 2008. The DrosophilaSTIM1 orthologue, dSTIM, has roles in cell fate specification and tissue patterning. BMC Dev. Biol. 8, 104. https://doi.org/10.1186/1471-213X-8-104 Greenspan, R., 2004. Fly Pushing: The Theory and Practice of Drosophila Genetics. Cold Spring Harbor Laboratory Press. Maaten, L. van der, Hinton, G., 2008. Visualizing Data using t-SNE. J. Mach. Learn. Res. 9, 2579–2605. Sommer, C., Straehle, C., Köthe, U., Hamprecht, F.A., 2011. Ilastik: Interactive learning and segmentation toolkit, in: 2011 IEEE International Symposium on Biomedical Imaging: From Nano to Macro. Presented at the 2011 IEEE International Symposium on Biomedical Imaging: From Nano to Macro, pp. 230–233. https://doi.org/10.1109/ISBI.2011.5872394 Wu, Q., Brodskiy, P.A., Huizar, F.J., Jangula, J.J., Narciso, C., Levis, M.K., Brito-Robinson, T., Zartman, J.J., 2017. In vivo relevance of intercellular calcium signaling in Drosophila wing development. bioRxiv 187401. https://doi.org/10.1101/187401
Fig. 3. The pipeline on the left was used to qualitatively score controlled vocabulary. The pipeline on the right was used to extract quantitative features in an automated fashion. 3. RESULTS 3.1 Genetic perturbations cluster into phenotypic categories Clusters of phenotypes include bent, blistered, and crumpled wings (Eid et al., 2008). Additionally, we discovered unexpected phenotypic clusters including genes that caused thickening and bifurcation of veins, implying previously unknown patterning functions for several Ca2+-related genes (orange arrows, Fig 4). Overall, these findings indicate several mechanisms by which the “calcium signaling toolkit” can be manipulated to alter the three-dimensional shape of developing tissues (Berridge et al., 2000). Due to high conservation, these findings can inform studies in other multicellular systems, and this method can be expanded to quantify the contributions of other second messenger pathways including cyclic nucleotides, phospholipids, and reactive oxygen species to organogenesis. 4. CONCLUSIONS Large-scale, high-throughput screening using Drosophila wings enables systematical investigations into functional roles of Ca2+ signalsome during tissue development. The highdimensional dataset reveals perturbations with similar phenotypes and provides data for inferring the role of Ca2+ in 109
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IFAC PapersOnLine 51-19 (2018) 108–109
Mapping the calcium signalsome during Drosophila wing development Mapping the calcium signalsome during Drosophila wing development Mapping the calcium signalsome during Drosophila wing development *,† * Drosophila wing development Mapping the calcium signalsome during Pavel A. Brodskiy , Qinfeng Wu*,† , Nilay Kumar Kumar , Vijay Kumar Naidu Velagala**, *,† *,† *
*,†, Qinfeng Wu*,† *, Vijay Kumar *, * * , Nilay Kumar Kumar * * * Pavel A. Brodskiy Naidu Velagala *,† * * Kara L. Snyder , Francisco J. Huizar , Seth J.Kumar TautgesKumar , Meaghan Snyder , Jeremiah J. Zartman Pavel A. Brodskiy Wu*,† Nilay Kumar Naidu Velagala * * * * * *,†, Qinfeng *,† *, Vijay *, *, Francisco *, ,Seth *, Meaghan *, Jeremiah *,† *,† * * Kara L. Snyder J. Huizar J. Tautges Snyder J. Zartman Pavel A. A. Brodskiy Brodskiy Qinfeng Wu Wu * ,, Nilay Nilay Kumar Kumar Kumar Kumar Vijay Kumar Kumar Naidu Velagala Velagala ,, ** * * * Pavel ,, Qinfeng ,, Vijay Naidu , Francisco J. Huizar , Seth J. Tautges , Meaghan Snyder , Jeremiah J. Zartman Kara L. Snyder * * * * * * *, Francisco *, Seth *, Meaghan University Kara J. J. J. Department of Chemical and Biomolecular Engineering, of Notre Dame, Kara L. L. Snyder Snyder , Francisco J. Huizar Huizar , Seth J. Tautges Tautges , Meaghan Snyder Snyder*,, Jeremiah Jeremiah J. Zartman Zartman* ** of Notre Chemical andINBiomolecular Engineering, University of Notre Dame, *Department 205 McCourtney Hall, Dame, 46556 USA (Tel: 574-631-0455; e-mail: [email protected]). and Biomolecular Engineering, University of Notre Dame, **Department of Chemical †Dame, Department of Notre Chemical and Biomolecular Engineering, University of Notre Dame, 205 McCourtney Hall, IN 46556 USA (Tel: 574-631-0455; e-mail: [email protected]). Department of Chemical and Biomolecular Engineering, University of Notre Dame, Authors contributed equally to this work e-mail: [email protected]). 205 McCourtney Hall, Notre ††Dame, IN 46556 USA (Tel: 574-631-0455; Authors to this work e-mail: 205 McCourtney McCourtney Hall, Hall, Notre Notre †Dame, Dame, INcontributed 46556 USA USAequally (Tel: 574-631-0455; 574-631-0455; e-mail: [email protected]). [email protected]). 205 IN 46556 (Tel: †Authors contributed equally to this work † Authors contributed contributed equally equally to to this this work work Authors Abstract: Second messengers such as calcium (Ca2+ ) play critical roles in signal integration for complex, 2+ 2+) play critical Abstract: Second messengers such as calcium (Ca roles inregulates signal integration for complex, 2+ multicellular processes. However, how components of Ca signaling tissue development is Abstract: Second messengers such as calcium (Ca2+ critical roles inregulates signal integration for complex, 2+ 2+) play 2+ signaling 2+ multicellular processes. However, how components of Ca tissue development is 2+ Abstract: SecondRecently, messengers such asdiscovered calcium (Ca (Ca ) play play critical rolessignaling in signal signal differentially integration for foracross complex, Abstract: Second messengers such as calcium ) critical roles in integration complex, 2+ the poorly studied. we have that perturbing Ca multicellular processes. However, components of Ca2+ regulatesdifferentially tissue development is 2+ signaling 2+ signaling poorly studied. Recently, weresults havehow discovered that perturbing Ca2+ acrossThis the multicellular processes. However, how components of Ca Ca signaling regulates tissuearchitecture. development is multicellular processes. However, how components of signaling regulates tissue development is 2+ to the developing Drosophila wing in three-dimensional distortions finaldifferentially wing poorly studied. Recently, we have discovered that perturbing Ca signaling across the 2+ 2+ to developing Drosophila wing results in three-dimensional distortions the finaldifferentially wing architecture. This poorly studied. Recently, we have discovered that perturbing Ca signaling across the poorly studied. Recently, we have discovered that perturbing Ca signaling differentially across the finding prompted us to pursue a comprehensive high-content screen for phenotypes generated by perturbing developing Drosophila wing results in three-dimensional the final generated wing architecture. This finding prompted us to pursue a comprehensive high-contentdistortions screen for to phenotypes by perturbing developing Drosophila wing results in distortions to the wing This developing Drosophila wing results in three-dimensional three-dimensional distortions to the final final generated wing architecture. This known calcium-related genes. The rapidly-expanding dataset includes deep architecture. coverage (~30 finding prompted us to pursue a comprehensive high-content screen for phenotypes by perturbing known calcium-related genes. The rapidly-expanding dataset includes deep coverage (~30 finding prompted us to pursue a comprehensive high-content screen for phenotypes generated by perturbing finding prompted us to pursue a comprehensive high-content screen for phenotypes generated by perturbing wings/perturbation) and a high-resolution dataset of over 7,000 fly wing images. Preliminary findings known calcium-related genes. The rapidly-expanding dataset includes deep coveragefindings (~30 wings/perturbation) and a of high-resolution dataset of several over 7,000 fly of wing images. Preliminary known calcium-related genes. The dataset includes deep coverage (~30 known calcium-related genes. The rapidly-expanding rapidly-expanding dataset includes deep coverage (~30 signaling leads to classes wing developmental phenotypes indicate that perturbation Ca2+ wings/perturbation) and a high-resolution dataset of over 7,000 fly wing images. Preliminary findings 2+ 2+ indicate that perturbation of Ca signaling leads to several classes of wing developmental phenotypes wings/perturbation) and a high-resolution dataset of over 7,000 fly wing images. Preliminary findings wings/perturbation) and a high-resolution dataset of over 7,000 fly wing images. Preliminary findings 2+ including thickening of veins, bifurcations, and wing crumpling. High-dimensional fingerprinting of these indicate that perturbation of Ca signaling leads to several classes of wing developmental phenotypes 2+ 2+ including thickening of veins, bifurcations, and wing High-dimensional of these indicate that that perturbation of clusters Ca signaling leads to crumpling. several classes of wing wing developmental developmental phenotypes indicate perturbation of Ca signaling leads to several classes of phenotypes phenotypes revealed several of genes, implying mechanistic interactions as fingerprinting well as novel roles for including thickening of veins, bifurcations, and wing crumpling. High-dimensional fingerprinting of these phenotypes revealed several clusters of genes, implying mechanistic interactions as well as novel roles for including thickening thickening ofinveins, veins, bifurcations, and wing wing crumpling. High-dimensional fingerprinting of these these including of bifurcations, and crumpling. High-dimensional fingerprinting of calcium-related genes morphogenesis. Future work will focus on validation of interactions within gene phenotypes revealed clusters of genes, implying mechanistic interactions as well as novel rolesgene for calcium-related genesseveral innovel morphogenesis. Future work will focus interactions within 2+ validation phenotypes revealed several clusters of genes, genes, implying mechanistic interactions as well as as novel novel roles for for phenotypes clusters of mechanistic interactions as well roles signaling onof development. clusters andrevealed identifying consequences ofimplying perturbation of Caon calcium-related genesseveral in morphogenesis. Future work will focus on validation of interactions within gene 2+ 2+ calcium-related genes in morphogenesis. Future work will focus validation interactions within gene clusters and identifying novel consequences of perturbation of Caon onof development. calcium-related genes in morphogenesis. Future work will focus on validation of interactions within gene 2+ signaling clusters identifying novel consequences of perturbation Ca2+ signaling onLtd. development. Keywords: Organogenesis, high-content machine learning, analysis. © 2018, and IFAC (International Federation ofscreening, Automatic Control)of Hosting byimage Elsevier All rights reserved. clusters and identifying novel consequences of of Ca on clusters and identifying novel consequences of perturbation perturbation of Ca2+ signaling signaling on development. development. Keywords: Organogenesis, high-content screening, machine learning, image analysis. Keywords: Organogenesis, high-content screening, machine learning, image analysis. Keywords: Organogenesis, high-content high-content screening, screening, machine machine learning, learning, image image analysis. analysis. Keywords: Organogenesis, Drosophila wing phenotypes (Fig 1), and to characterize 1. INTRODUCTION Drosophila wing phenotypes (Figof 1), and to characterize phenotypes caused by perturbation Ca2+ signaling. 1. INTRODUCTION Drosophila wing phenotypes (Figof 1), and to characterize 2+ 2+ 2+ phenotypes caused by perturbation Ca signaling. Calcium (Ca ) integrates a diverse range of chemical, genetic, Drosophila wing wing phenotypes phenotypes (Fig (Fig 1), 1), 2+and and to to characterize characterize 1. INTRODUCTION Drosophila 2+ 2+) integrates a diverse range of chemical, genetic, phenotypes caused by perturbation of Ca signaling. 1. INTRODUCTION Calcium (Ca 2+ 1. INTRODUCTION 2+ 2+ mechanical inputs to regulate many cellular electrical, and phenotypes caused by perturbation of Ca signaling. 2. METHODS phenotypes caused by perturbation of Ca signaling. Calcium (Ca ) integrates a diverse range of chemical, genetic, 2+ mechanical inputs to regulate many cellular 2+ electrical, and Calcium (Ca ) integrates diverse range of genetic, Calcium integrates diverse range of chemical, chemical, genetic, METHODS processes (Berridge et al.,aa2000). include proliferation, electrical,(Ca and) mechanical inputsThese to regulate many cellular 2. 2. METHODS (Berridge et al., 2000). These include proliferation, electrical, and mechanical inputs to regulate many cellular processes electrical, and mechanical inputs to regulate many cellular 2. METHODS death, motility, properties, and differentiation. As 2.1 Gene selection 2. METHODS processes (Berridge et al., 2000). These include proliferation, 2+ 2000). processes (Berridge et al., These include proliferation, death, motility, mechanical properties, and differentiation. As 2.1 Gene selection processes (Berridge et al., 2000). These include proliferation, adeath, result, intercellular Ca signals serve as a key module for motility, mechanical 2.1 Gene selection 2+ properties, and differentiation. As motility, mechanical properties, As serveand as differentiation. a key module for adeath, result, intercellular Ca2+ 2.1 selection screen currently consists of 132 RNAi mediated death, motility, mechanical properties, and differentiation. As The 2+ signals coordinating large-scale collective behaviors including 2.1 Gene Gene selection acoordinating result, intercellular Ca signals serve as a key module for 2+ The screen of 132 binary RNAi expression mediated 2+ signals large-scale collective behaviors including aamorphogenesis result, intercellular Ca serve as a key module for knockdowns driven by consists the GAL4/UAS result, intercellular signals servebehaviors as a key module for The screen currently and Ca wound healing. Second messenger currently consists of 132 binary RNAi expression mediated coordinating large-scale collective including knockdowns driven by the GAL4/UAS morphogenesis and such wound healing. Secondaremessenger 2+ The screen screen currently consists of 67132 132 RNAi mediated coordinating large-scale collective behaviors including The currently consists of RNAi mediated system (Duffy,driven 2002, by p. 4). OfGAL4/UAS these, genes are involved in coordinating large-scale collective behaviors including signaling pathways as Ca signaling therefore knockdowns the binary expression morphogenesis and wound healing. Second messenger 2+ 2+ signaling are therefore system (Duffy, 2002, p. 4). OfGAL4/UAS these, 67 genes are involved in 2+ by 2+ signaling pathways such as Ca knockdowns driven by the GAL4/UAS binary expression morphogenesis and wound healing. Second messenger knockdowns driven the binary expression transport of Ca or expressed Ca -binding domains. morphogenesis and wound healing. Second messenger 2+ composed of both encoding and decoding modules, forming a (Duffy, 2002, Of these, 67 2+ genes are involved in 2+ signaling pathways such as Ca are forming thereforea system 2+ p. 4). 2+ 2+ signaling transport of Ca expressed -binding 2+ composed of both encoding and decoding modules, 2+ system (Duffy, 2002, p. 4). Of these, 67 genes are involved in or Ca domains. signaling pathways pathways such such asCaCa Ca is signaling signaling are for therefore (Duffy, 2002, p. 4). Of selected these, 67 genes are involved in 2+ 2+ Additionally, genes were signaling genes signaling are therefore bow-tie-shaped important winga system transport of 65 Ca or expressed Cafrom -binding domains. composed of bothnetwork. encodingas and modules, forming 2+ 2+ 2+ 2+decoding 2+ 2+ bow-tie-shaped network. Ca is important for wing transport of Ca or expressed Ca -binding domains. Additionally, 65 genes were selected from signaling genes composed of of both both encoding and2+the decoding modules, forming a transport of Ca or expressed Ca -binding domains. with known phenotypes and human disease genes highly composed encoding and decoding modules, forming a development (Fig 1). However, roles of different modules Additionally, 65 genes were selected from signaling genes bow-tie-shaped Ca2+ is important wing with development (Fignetwork. 1).the However, roles of differentfor known phenotypes and human disease genescontrols highly 65 genes selected from signaling genes bow-tie-shaped network. Ca is for wing Additionally, 65developing genes were were selected from signaling genes expressed in the wing to serve as positive bow-tie-shaped network. Ca2+the is important formodules wing Additionally, of the Ca2+ toolkit in development ofimportant multicellular systems with known phenotypes and human disease genes highly development (Fig 1). However, the roles of different modules 2+ 2+ of the Ca with known phenotypes and human disease genes highly expressed in the developing wing to serve as positive controls toolkit in the development of multicellular systems development (Fig 1). However, the roles of different modules with known phenotypes and human disease genes highly for image processing. 2+ scale development (Fig 1). However, the roles of different modules at a systems are poorly understood. expressed in the developing wing to serve as positive controls of Ca2+ toolkit in the development of multicellular systems for at athe systems scale are poorly understood. expressed in the the developing developing wing wing to to serve serve as as positive positive controls controls image processing. of the Ca in development of in of Ca2+ toolkit toolkit in the the development of multicellular multicellular systems systems expressed for image processing. at athe systems scale are poorly understood. for image processing. at a systems scale are poorly understood. 2.2 Fly culture and wing collection for image processing. at a systems scale are poorly understood. 2.2 Fly culture and wing collection 2.2 Fly culture and wing collection 2.2 wing collection MS1096-GAL4 (BL# 8860) virgins were crossed to TRiP line 2.2 Fly Fly culture culture and and wing collection MS1096-GAL4 (BL# 8860) virgins were crossed TRiP line males under fly culture conditions described in to (Greenspan, MS1096-GAL4 (BL# 8860) virgins were crossed to TRiP line males under fly culture conditions described in (Greenspan, MS1096-GAL4 (BL# 8860) virgins were crossed to MS1096-GAL4 (BL# 8860) virgins were crossed to TRiP line 2004). Crosses were passaged every 4-5 days at TRiP 25ooC.line A males under fly culture conditions described in (Greenspan, o 2004). Crosses were A males under fly culture conditions described in (Greenspan, passaged every 4-5 days at 25oC. to males under fly culture conditions described in (Greenspan, custom laboratory data management system was developed 2004). Crosses were passaged every 4-5 days at 25ooC. A 2004). Crosses were passaged every 4-5 days at 25 C. A custom laboratory data management system was developed to 2004). were every 4-5 placed days atin25 C. to A streamline the pipeline (Fig. 2). Wings were ethanol, custom Crosses laboratory datapassaged management system was developed pipeline custom laboratory data management system was developed to streamline the (Fig. 2). Wings were placed in ethanol, custom laboratory data management system was developed to and approximately 15 wings were mounted on each slide in streamline the pipeline (Fig. 2). Wings were placed in ethanol, streamline the pipeline (Fig. Wings were placed in and approximately 15 wings2). were mounted on each slide in streamline the pipeline (Fig. Wings were placed in ethanol, ethanol, Permount medium (Fisher Scientific, SP15). Slides were and approximately 15 wings2). were mounted on each slide in and approximately approximately 15 wings were mounted on each slide in Permount medium (Fisher Scientific, SP15). Slidesslide were and 15 wings were mounted on each in imaged on a Leica Aperio slide scanner at 5x magnification. Permount medium (Fisher Scientific, SP15). Slides were imaged on amedium Leica Aperio slide scanner atSP15). 5x magnification. Permount medium (Fisher Scientific, SP15). Slides were were Permount (Fisher Scientific, Slides imaged on a Leica Aperio slide scanner at 5x magnification. imaged on analysis Leica Aperio Aperio slide slide scanner scanner at at 5x 5x magnification. magnification. 2.3 Image imaged on aa Leica 2.3 Image analysis Fig 1. Perturbations to core regulators of Ca2+ signaling lead to 2.3 Image analysis 2+ Fig 1. Perturbations to adult core regulators 2.3 analysis signaling lead in to Wing of Ca2+ images were isolated from slide scans and rotated using 2.3 Image Image analysis several characteristic wing phenotypes, as reported Fig 1. Perturbations to core regulators of Ca2+ 2+ signaling lead to images MATLAB were isolated from slide scans and using 2+ Fig 1. 1.etPerturbations Perturbations to adult core regulators regulators of Ca Ca signaling signaling lead in to Wing several characteristic wing phenotypes, as reported a custom Images wererotated manually Fig to core of lead to (Wu al., 2017). images were isolatedscript. from slide scans and rotated using several characteristic adult wing phenotypes, as reported in aWing Wing images were isolated from slide scans and rotated using custom MATLAB script. Images were manually (Wu et al., 2017). several characteristic characteristic adult adult wing wing phenotypes, phenotypes, as as reported reported in in Wing images MATLAB were isolated from slide and using phenotypically scored withscript. 61 classes ofscans controlled vocabulary several a custom Images wererotated manually (Wu et al., 2017). a custom MATLAB script. Images were manually phenotypically scored with 61 classes of controlled vocabulary (Wu primary et al., al., 2017). 2017). custom MATLAB script. Images were manually The goal of this study is to identify additional genes a(Fig 3). To extract quantitative features, pixels were classified (Wu et phenotypically scored with 61 classes of controlled vocabulary phenotypically scored with of controlled vocabulary The primary goal of thisthat study to identify 3). To extract quantitative features, pixelsand were classified phenotypically scored with 61 61 classes classes ofhairs, controlled vocabulary in Ca2+ signalsome areis involved in additional regulating genes these (Fig as background, veins, intervein regions, debris using Thethe primary goal of thisthat study is to identify additional genes (Fig 3). To extract quantitative features, pixels were classified 2+ 2+ signalsome are involved in regulating these The primary goal of this study is to identify additional genes (Fig 3). To extract quantitative features, pixels were classified in the Ca as background, veins, intervein regions, hairs, and debris using The primary goal of thisthat study isinvolved to identify additional genes (Fig 3). To extract quantitative features,hairs, pixelsand were classified in the Ca2+ signalsome are in regulating these as background, veins, intervein regions, debris using 2+ signalsome that are involved in regulating these in the the Ca Ca2+ as background, background, veins, veins, intervein intervein regions, regions, hairs, hairs, and and debris debris using using in signalsome that are involved in regulating these as
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the pixel classifier Ilastik (Sommer et al., 2011). Intervein regions were extracted using a custom MATLAB pipeline. A bagged tree classifier was trained with 500-1,000 images per intervein region class to predict the identity of intervein regions from their morphometric features.
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organogenesis. We expect to generate a dataset of >10,000 images encompassing approximately 500 Ca2+-related genes. This dataset will provide materials to the community to advance the study of developmental biology.
Fig 4. t-distributed stochastic neighbor embedding representation of fingerprints to map the consequences of perturbing Ca2+ signaling (Maaten and Hinton, 2008).
Fig. 2. We constructed a custom MySQL database and web front to manage experiments and record results. Operational data generated from the database is used to optimize the screening process.
ACKNOWLEDGEMENTS We would like to thank the South Bend Medical Foundation for generous access to their Apero Slide Scanner, and CzierAnne Gone, Isaac Nordan, Ryan Govi, Heather Flynn, Adriana Szpynda, Quincey Hogue, and Maria Unger for technical assistance. The work in this manuscript was supported in part by NIH Grant R35GM124935 and CBET-1553826 and the Walther Cancer Foundation Interdisciplinary Interface Training Project (PB). REFERENCES Berridge, M.J., Lipp, P., Bootman, M.D., 2000. The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1, 11–21. https://doi.org/10.1038/35036035 Duffy, J.B., 2002. GAL4 system in drosophila: A fly geneticist’s swiss army knife. genesis 34, 1–15. https://doi.org/10.1002/gene.10150 Eid, J.-P., Arias, A.M., Robertson, H., Hime, G.R., Dziadek, M., 2008. The DrosophilaSTIM1 orthologue, dSTIM, has roles in cell fate specification and tissue patterning. BMC Dev. Biol. 8, 104. https://doi.org/10.1186/1471-213X-8-104 Greenspan, R., 2004. Fly Pushing: The Theory and Practice of Drosophila Genetics. Cold Spring Harbor Laboratory Press. Maaten, L. van der, Hinton, G., 2008. Visualizing Data using t-SNE. J. Mach. Learn. Res. 9, 2579–2605. Sommer, C., Straehle, C., Köthe, U., Hamprecht, F.A., 2011. Ilastik: Interactive learning and segmentation toolkit, in: 2011 IEEE International Symposium on Biomedical Imaging: From Nano to Macro. Presented at the 2011 IEEE International Symposium on Biomedical Imaging: From Nano to Macro, pp. 230–233. https://doi.org/10.1109/ISBI.2011.5872394 Wu, Q., Brodskiy, P.A., Huizar, F.J., Jangula, J.J., Narciso, C., Levis, M.K., Brito-Robinson, T., Zartman, J.J., 2017. In vivo relevance of intercellular calcium signaling in Drosophila wing development. bioRxiv 187401. https://doi.org/10.1101/187401
Fig. 3. The pipeline on the left was used to qualitatively score controlled vocabulary. The pipeline on the right was used to extract quantitative features in an automated fashion. 3. RESULTS 3.1 Genetic perturbations cluster into phenotypic categories Clusters of phenotypes include bent, blistered, and crumpled wings (Eid et al., 2008). Additionally, we discovered unexpected phenotypic clusters including genes that caused thickening and bifurcation of veins, implying previously unknown patterning functions for several Ca2+-related genes (orange arrows, Fig 4). Overall, these findings indicate several mechanisms by which the “calcium signaling toolkit” can be manipulated to alter the three-dimensional shape of developing tissues (Berridge et al., 2000). Due to high conservation, these findings can inform studies in other multicellular systems, and this method can be expanded to quantify the contributions of other second messenger pathways including cyclic nucleotides, phospholipids, and reactive oxygen species to organogenesis. 4. CONCLUSIONS Large-scale, high-throughput screening using Drosophila wings enables systematical investigations into functional roles of Ca2+ signalsome during tissue development. The highdimensional dataset reveals perturbations with similar phenotypes and provides data for inferring the role of Ca2+ in 109