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Neural Crest Cells pp 97–105Cite as

Technologies for Live Imaging of Enteric Neural Crest-Derived Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1976))

Abstract

Time-lapse imaging of gut explants from embryonic mice in which neural crest-derived cells express fluorescent proteins allows the behavior of enteric neural crest cells to be observed and analyzed. Explants of embryonic gut are dissected, mounted on filter paper supports so the gut retains its tubular three-dimensional structure, and then placed in coverglass bottom culture dishes in tissue culture medium. A stainless steel ring is placed on top of the filter support to prevent movement. Imaging is performed using a confocal microscope in an environmental chamber. A z series of images through the network of fluorescent cells is collected every 3, 5, or 10 min. At the end of imaging, the z series are projected.

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References

  1. Burns AJ, Le Douarin NM (1998) The sacral neural crest contributes neurons and glia to the post- umbilical gut: spatiotemporal analysis of the development of the enteric nervous system. Development 125:4335–4347

    CAS  PubMed  Google Scholar 

  2. Le Douarin NM, Teillet MA (1973) The migration of neural crest cells to the wall of the digestive tract in avian embryo. J Embryol Exp Morphol 30:31–48

    PubMed  Google Scholar 

  3. Yntema CL, Hammond WS (1954) The origin of intrinsic ganglia of trunk viscera from vagal neural crest in the chick embryo. J Comp Neurol 101:515–541

    Article  CAS  Google Scholar 

  4. Young HM, Bergner AJ, Anderson RB et al (2004) Dynamics of neural crest-derived cell migration in the embryonic mouse gut. Dev Biol 270:455–473

    Article  CAS  Google Scholar 

  5. Heanue TA, Pachnis V (2007) Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies. Nat Rev Neurosci 8:466–479

    Article  CAS  Google Scholar 

  6. Mckeown SJ, Stamp L, Hao MM et al (2013) Hirschsprung disease: a developmental disorder of the enteric nervous system. WIRES Dev Biol 2:113–129

    Article  CAS  Google Scholar 

  7. Lake JI, Heuckeroth RO (2013) Enteric nervous system development: migration, differentiation, and disease. Am J Physiol 305:G1–G24

    Article  CAS  Google Scholar 

  8. Goldstein AM, Thapar N, Karunaratne TB et al (2016) Clinical aspects of neurointestinal disease: pathophysiology, diagnosis, and treatment. Dev Biol

    Google Scholar 

  9. Lichtman JW, Fraser SE (2001) The neuronal naturalist: watching neurons in their native habitat. Nat Neurosci 4(Suppl):1215–1220

    Article  CAS  Google Scholar 

  10. Kulesa PM, Mckinney MC, Mclennan R (2013) Developmental imaging: the avian embryo hatches to the challenge. Birth Defects Res C Embryo Today 99:121–133

    Article  CAS  Google Scholar 

  11. Theveneau E, Mayor R (2012) Neural crest migration: interplay between chemorepellents, chemoattractants, contact inhibition, epithelial-mesenchymal transition, and collective cell migration. Wiley Interdiscip Rev Dev Biol 1:435–445

    Article  CAS  Google Scholar 

  12. Richardson J, Gauert A, Briones Montecinos L et al (2016) Leader cells define directionality of trunk, but not cranial, neural crest cell migration. Cell Rep 15:2076–2088

    Article  CAS  Google Scholar 

  13. Young HM, Bergner AJ, Simpson MJ et al (2014) Colonizing while migrating: how do individual enteric neural crest cells behave? BMC Biol 12:23

    Article  Google Scholar 

  14. Theveneau E, Mayor R (2011) Can mesenchymal cells undergo collective cell migration? The case of the neural crest. Cell Adhes Migr 5:490–498

    Article  Google Scholar 

  15. Hearn CJ, Young HM, Ciampoli D et al (1999) Catenary cultures of embryonic gastrointestinal tract support organ morphogenesis, motility, neural crest cell migration, and cell differentiation. Dev Dyn 214:239–247

    Article  CAS  Google Scholar 

  16. Druckenbrod NR, Epstein ML (2005) The pattern of neural crest advance in the cecum and colon. Dev Biol 287:125–133

    Article  CAS  Google Scholar 

  17. Druckenbrod NR, Epstein ML (2007) Behavior of enteric neural crest-derived cells varies with respect to the migratory wavefront. Dev Dyn 236:84–92

    Article  Google Scholar 

  18. Druckenbrod NR, Epstein ML (2009) Age-dependent changes in the gut environment restrict the invasion of the hindgut by enteric neural progenitors. Development 136:3195–3203

    Article  CAS  Google Scholar 

  19. Breau MA, Dahmani A, Broders-Bondon F et al (2009) Beta1 integrins are required for the invasion of the caecum and proximal hindgut by enteric neural crest cells. Development 136:2791–2801

    Article  CAS  Google Scholar 

  20. Breau MA, Pietri T, Eder O et al (2006) Lack of beta1 integrins in enteric neural crest cells leads to a Hirschsprung-like phenotype. Development 133:1725–1734

    Article  CAS  Google Scholar 

  21. Corpening JC, Cantrell VA, Deal KK et al (2008) A Histone2BCerulean BAC transgene identifies differential expression of Phox2b in migrating enteric neural crest derivatives and enteric glia. Dev Dyn 237:1119–1132

    Article  CAS  Google Scholar 

  22. Zhang Y, Kim TH, Niswander L (2012) Phactr4 regulates directional migration of enteric neural crest through PP1, integrin signaling, and cofilin activity. Genes Dev 26:69–81

    Article  Google Scholar 

  23. Wang X, Chan AK, Sham MH et al (2011) Analysis of the sacral neural crest cell contribution to the hindgut enteric nervous system in the mouse embryo. Gastroenterology 141:992–1002.e1-6

    Article  Google Scholar 

  24. Uesaka T, Nagashimada M, Enomoto H (2013) GDNF signaling levels control migration and neuronal differentiation of enteric ganglion precursors. J Neurosci 33:16372–16382

    Article  CAS  Google Scholar 

  25. Nakazawa-Tanaka N, Miyahara K, Fujiwara N et al (2016) Three- and four-dimensional analysis of altered behavior of enteric neural crest derived cells in the Hirschsprung's disease mouse model. Pediatr Surg Int 32:3–7

    Article  Google Scholar 

  26. Bergeron KF, Cardinal T, Toure AM et al (2015) Male-biased aganglionic megacolon in the TashT mouse line due to perturbation of silencer elements in a large gene desert of chromosome 10. PLoS Genet 11:e1005093

    Article  Google Scholar 

  27. Bergeron KF, Nguyen CM, Cardinal T et al (2016) Upregulation of Nr2f1-A830082K12Rik gene pair in murine neural crest cells results in a complex phenotype reminiscent of waardenburg syndrome type 4. Dis Model Mech 9(11):1283–1293

    Article  CAS  Google Scholar 

  28. Enomoto H, Crawford PA, Gorodinsky A et al (2001) RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons. Development 128:3963–3974

    CAS  PubMed  Google Scholar 

  29. Pattyn A, Morin X, Cremer H et al (1999) The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature 399:366–370

    Article  CAS  Google Scholar 

  30. Young HM, Ciampoli D, Hsuan J et al (1999) Expression of ret-, p75(NTR)-, Phox2a-, Phox2b-, and tyrosine hydroxylase-immunoreactivity by undifferentiated neural crest-derived cells and different classes of enteric neurons in the embryonic mouse gut. Dev Dyn 216:137–152

    Article  CAS  Google Scholar 

  31. Nishiyama C, Uesaka T, Manabe T et al (2012) Trans-mesenteric neural crest cells are the principal source of the colonic enteric nervous system. Nat Neurosci 15:1211–1218

    Article  CAS  Google Scholar 

  32. Kapur RP, Yost C, Palmiter RD (1992) A transgenic model for studying development of the enteric nervous system in normal and aganglionic mice. Development 116:167–175

    CAS  PubMed  Google Scholar 

  33. Young HM, Hearn CJ, Ciampoli D et al (1998) A single rostrocaudal colonization of the rodent intestine by enteric neuron precursors is revealed by the expression of Phox2b, ret, and p75 and by explants grown under the kidney capsule or in organ culture. Dev Biol 202:67–84

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Australian Research Council Discovery Grant DP150103709 to H.M.Y., National Health and Medical Research Council (NHMRC) Training Fellowship APP1071153 to M.M.H., and NHMRC Senior Research Fellowship APP1103297 to H.M.Y.

Author information

Authors and Affiliations

  1. Department of Anatomy and Neuroscience, University of Melbourne, Melbourne, VIC, Australia

    Marlene M. Hao, Annette J. Bergner & Heather M. Young

  2. Laboratory for Enteric Neuroscience, TARGID, University of Leuven, Leuven, Belgium

    Marlene M. Hao

  3. Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, VIC, Australia

    Donald F. Newgreen

  4. Division of Neural Differentiation and Regeneration, Department of Physiology and Cell Biology, Graduate School of Medicine, Kobe University, Kobe, Japan

    Hideki Enomoto

  5. Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama, Japan

    Hideki Enomoto

Authors
  1. Marlene M. Hao
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  2. Annette J. Bergner
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  3. Donald F. Newgreen
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  4. Hideki Enomoto
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  5. Heather M. Young
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Editor information

Editors and Affiliations

  1. Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA, Australia

    Quenten Schwarz

  2. Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA, Australia

    Sophie Wiszniak

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Hao, M.M., Bergner, A.J., Newgreen, D.F., Enomoto, H., Young, H.M. (2019). Technologies for Live Imaging of Enteric Neural Crest-Derived Cells. In: Schwarz, Q., Wiszniak, S. (eds) Neural Crest Cells. Methods in Molecular Biology, vol 1976. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9412-0_8

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  • DOI: https://doi.org/10.1007/978-1-4939-9412-0_8

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9411-3

  • Online ISBN: 978-1-4939-9412-0

  • eBook Packages: Springer Protocols

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