Automated high-throughput measurement of body movements and cardiac activity of Xenopus tropicalis tadpoles

Authors

  • Kay Eckelt Institute for Bioengineering of Catalonia (IBEC)
  • Helena Masanas Institute for Bioengineering of Catalonia (IBEC)
  • Artur Llobet Bellvitge Biomedical Research Institute (IDIBELL)
  • Pau Gorostiza Institute for Bioengineering of Catalonia (IBEC)

DOI:

https://doi.org/10.14440/jbm.2014.29

Keywords:

Xenopus tropicalis, animal behavior, cardiac imaging, motion analysis, animal tracking, high-throughput in vivo assay

Abstract

Xenopus tadpoles are an emerging model for developmental, genetic and behavioral studies. A small size, optical accessibility of most of their organs, together with a close genetic and structural relationship to humans make them a convenient experimental model. However, there is only a limited toolset available to measure behavior and organ function of these animals at medium or high-throughput. Herein, we describe an imaging-based platform to quantify body and autonomic movements of Xenopus tropicalis tadpoles of advanced developmental stages. Animals alternate periods of quiescence and locomotor movements and display buccal pumping for oxygen uptake from water and rhythmic cardiac movements. We imaged up to 24 animals in parallel and automatically tracked and quantified their movements by using image analysis software. Animal trajectories, moved distances, activity time, buccal pumping rates and heart beat rates were calculated and used to characterize the effects of test compounds. We evaluated the effects of propranolol and atropine, observing a dose-dependent bradycardia and tachycardia, respectively. This imaging and analysis platform is a simple, cost-effective high-throughput in vivo assay system for genetic, toxicological or pharmacological characterizations.


References

Schmitt SM, Gull M, Brändli AW (2014) Engineering Xenopus embryos for phenotypic drug discovery screening. Adv Drug Deliv Rev 69-70: 225–246. doi:10.1016/j.addr.2014.02.004.

Hellsten U, Harland RM, Gilchrist MJ, Hendrix D, Jurka J, et al. (2010) The Genome of the Western Clawed Frog Xenopus tropicalis. Science 328: 633–636. doi:10.1126/science.1183670.

Khokha MK, Chung C, Bustamante EL, Gaw LWK, Trott KA, et al. (2002) Techniques and probes for the study of Xenopus tropicalis development. Dev Dyn Off Publ Am Assoc Anat 225: 499–510. doi:10.1002/dvdy.10184.

Ogino H, McConnell WB, Grainger RM (2006) High-throughput transgenesis in Xenopus using I-SceI meganuclease. Nat Protoc 1: 1703–1710. doi:10.1038/nprot.2006.208.

Love NR, Thuret R, Chen Y, Ishibashi S, Sabherwal N, et al. (2011) pTransgenesis: a cross-species, modular transgenesis resource. Development 138: 5451–5458. doi:10.1242/dev.066498.

Ishibashi S, Cliffe R, Amaya E (2012) Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis. Biol Open 1: 1273–1276. doi:10.1242/bio.20123228.

Wheeler GN, Brändli AW (2009) Simple vertebrate models for chemical genetics and drug discovery screens: Lessons from zebrafish and Xenopus. Dev Dyn 238: 1287–1308. doi:10.1002/dvdy.21967.

Mohun TJ, Leong LM, Weninger WJ, Sparrow DB (2000) The morphology of heart development in Xenopus laevis. Dev Biol 218: 74–88. doi:10.1006/dbio.1999.9559.

Warkman AS, Krieg PA (2007) Xenopus as a model system for vertebrate heart development. Semin Cell Dev Biol 18: 46–53. doi:10.1016/j.semcdb.2006.11.010.

Tomlinson ML, Rejzek M, Fidock M, Field RA, Wheeler GN (2009) Chemical genomics identifies compounds affecting Xenopus laevis pigment cell development. Mol Biosyst 5: 376. doi:10.1039/b818695b.

Kälin RE, Bänziger-Tobler NE, Detmar M, Brändli AW (2009) An in vivo chemical library screen in Xenopus tadpoles reveals novel pathways involved in angiogenesis and lymphangiogenesis. Blood 114: 1110–1122. doi:10.1182/blood-2009-03-211771.

Dush MK, McIver AL, Parr MA, Young DD, Fisher J, et al. (2011) Heterotaxin: a TGF-β signaling inhibitor identified in a multi-phenotype profiling screen in Xenopus embryos. Chem Biol 18: 252–263. doi:10.1016/j.chembiol.2010.12.008.

Tomlinson ML, Hendry AE, Wheeler GN (2012) Chemical Genetics and Drug Discovery in Xenopus. In: Hoppler S, Vize PD, editors. Xenopus Protocols. Totowa, NJ: Humana Press, Vol. 917. pp. 155–166. Available: http://link.springer.com/10.1007/978-1-61779-992-1_9. Accessed 24 March 2014.

Fini JB, Le Mével S, Palmier K, Darras VM, Punzon I, et al. (2012) Thyroid hormone signaling in the Xenopus laevis embryo is functional and susceptible to endocrine disruption. Endocrinology 153: 5068–5081. doi:10.1210/en.2012-1463.

Kokel D, Bryan J, Laggner C, White R, Cheung CYJ, et al. (2010) Rapid behavior-based identification of neuroactive small molecules in the zebrafish. Nat Chem Biol 6: 231–237. doi:10.1038/nchembio.307.

Kokel D, Cheung CYJ, Mills R, Coutinho-Budd J, Huang L, et al. (2013) Photochemical activation of TRPA1 channels in neurons and animals. Nat Chem Biol 9: 257–263. doi:10.1038/nchembio.1183.

Rihel J, Prober DA, Arvanites A, Lam K, Zimmerman S, et al. (2010) Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake Regulation. Science 327: 348–351. doi:10.1126/science.1183090.

Burns CG, Milan DJ, Grande EJ, Rottbauer W, MacRae CA, et al. (2005) High-throughput assay for small molecules that modulate zebrafish embryonic heart rate. Nat Chem Biol 1: 263–264. doi:10.1038/nchembio732.

Letamendia A, Quevedo C, Ibarbia I, Virto JM, Holgado O, et al. (2012) Development and validation of an automated high-throughput system for zebrafish in vivo screenings. PloS One 7: e36690. doi:10.1371/journal.pone.0036690.

Blackiston D, Shomrat T, Nicolas CL, Granata C, Levin M (2010) A Second-Generation Device for Automated Training and Quantitative Behavior Analyses of Molecularly-Tractable Model Organisms. PLoS ONE 5: e14370. doi:10.1371/journal.pone.0014370.

Blackiston DJ, Levin M (2012) Aversive Training Methods in Xenopus laevis: General Principles. Cold Spring Harb Protoc 2012: pdb.top068338–pdb.top068338. doi:10.1101/pdb.top068338.

Blackiston DJ, Levin M (2013) Ectopic eyes outside the head in Xenopus tadpoles provide sensory data for light-mediated learning. J Exp Biol 216: 1031–1040. doi:10.1242/jeb.074963.

Schriks M, Vanhoorn M, Faassen E, Vandam J, Murk A (2006) Real-time automated measurement of Xenopus leavis tadpole behavior and behavioral responses following triphenyltin exposure using the multispecies freshwater biomonitor (MFB). Aquat Toxicol 77: 298–305. doi:10.1016/j.aquatox.2005.12.011.

Lavorato M, Bernabò I, Crescente A, Denoël M, Tripepi S, et al. (2012) Endosulfan Effects on Rana dalmatina Tadpoles: Quantitative Developmental and Behavioural Analysis. Arch Environ Contam Toxicol 64: 253–262. doi:10.1007/s00244-012-9819-7.

Denoël M, Libon S, Kestemont P, Brasseur C, Focant J-F, et al. (2013) Effects of a sublethal pesticide exposure on locomotor behavior: a video-tracking analysis in larval amphibians. Chemosphere 90: 945–951. doi:10.1016/j.chemosphere.2012.06.037.

Villinger J, Waldman B (2008) Self-referent MHC type matching in frog tadpoles. Proc R Soc B Biol Sci 275: 1225–1230. doi:10.1098/rspb.2008.0022.

Villinger J, Waldman B (2012) Social discrimination by quantitative assessment of immunogenetic similarity. Proc R Soc B Biol Sci 279: 4368–4374. doi:10.1098/rspb.2012.1279.

Feder ME, Wassersug, Richard J. (1984) Aerial Versus Aquatic Oxygen Consumption in Larvae of the Clawed Frog, Xenopus Laevis. J Exp Biol 108: 231–245.

Ryerson WG, Deban SM (2010) Buccal pumping mechanics of Xenopus laevis tadpoles: effects of biotic and abiotic factors. J Exp Biol 213: 2444–2452. doi:10.1242/jeb.038976.

Bartlett HL, Scholz TD, Lamb FS, Weeks DL (2004) Characterization of embryonic cardiac pacemaker and atrioventricular conduction physiology in Xenopus laevis using noninvasive imaging. Am J Physiol Heart Circ Physiol 286: H2035–2041. doi:10.1152/ajpheart.00807.2003.

Hou PC, Burggren WW (1995) Cardiac output and peripheral resistance during larval development in the anuran amphibian Xenopus laevis. Am J Physiol 269: R1126–1132.

Boppart SA, Tearney GJ, Bouma BE, Southern JF, Brezinski ME, et al. (1997) Noninvasive assessment of the developing Xenopus cardiovascular system using optical coherence tomography. Proc Natl Acad Sci U S A 94: 4256–4261.

Yang VXD, Gordon M, Seng-Yue E, Lo S, Qi B, et al. (2003) High speed, wide velocity dynamic range Doppler optical coherence tomography (Part II): Imaging in vivo cardiac dynamics of Xenopus laevis. Opt Express 11: 1650–1658.

Yelin R, Yelin D, Oh W-Y, Yun SH, Boudoux C, et al. (2007) Multimodality optical imaging of embryonic heart microstructure. J Biomed Opt 12: 064021. doi:10.1117/1.2822904.

Fritsche R, Burggren W (1996) Development of cardiovascular responses to hypoxia in larvae of the frog Xenopus laevis. Am J Physiol 271: R912–917.

Hou PC, Burggren WW (1995) Blood pressures and heart rate during larval development in the anuran amphibian Xenopus laevis. Am J Physiol 269: R1120–1125.

Nieuwkoop PD, Faber J (n.d.) Normal table of Xenopus laevis (Daudin), 1975. North-Holland Publishing, Amsterdam, The Netherlands.

Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9: 671–675. doi:10.1038/nmeth.2089.

Meijering E, Dzyubachyk O, Smal I (2012) Methods for cell and particle tracking. Methods Enzymol 504: 183–200. doi:10.1016/B978-0-12-391857-4.00009-4.

Zeileis A, Grothendieck G (2005) zoo: S3 Infrastructure for Regular and Irregular Time Series. J Stat Softw 14. Available: http://www.jstatsoft.org/v14/i06.

Benjamini Y, Hochberg J (1995) Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. J R Stat Soc 57. doi:10.2307/2346101.

Pittolo S, Gómez-Santacana X, Eckelt K, Rovira X, Dalton J, et al. (2014) An allosteric modulator to control endogenous G protein-coupled receptors with light; accepted manuscript. Nat Chem Biol. doi:DOI: 10.1038/NCHEMBIO.1612.

Blitz IL, Andelfinger G, Horb ME (2006) Germ layers to organs: using Xenopus to study “later” development. Semin Cell Dev Biol 17: 133–145. doi:10.1016/j.semcdb.2005.11.002.

Dong W, Lee RH, Xu H, Yang S, Pratt KG, et al. (2009) Visual avoidance in Xenopus tadpoles is correlated with the maturation of visual responses in the optic tectum. J Neurophysiol 101: 803–815. doi:10.1152/jn.90848.2008.

Tills O, Bitterli T, Culverhouse P, Spicer JI, Rundle S (2013) A novel application of motion analysis for detecting stress responses in embryos at different stages of development. BMC Bioinformatics 14: 37. doi:10.1186/1471-2105-14-37.

Landis R J, Koch G G (1977) The Measurement of Observer Agreement for Categorical Data. Biometrics 33: 159–174.

Downloads

Additional Files

Published

2014-11-05

How to Cite

1.
Eckelt K, Masanas H, Llobet A, Gorostiza P. Automated high-throughput measurement of body movements and cardiac activity of Xenopus tropicalis tadpoles. J Biol Methods [Internet]. 2014Nov.5 [cited 2021Jun.16];1(2):e9. Available from: https://jbmethods.org/jbm/article/view/29

Issue

Section

Articles