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Technologies for Live Imaging of Enteric Neural Crest-Derived Cells

  • Marlene M. Hao
  • Annette J. Bergner
  • Donald F. Newgreen
  • Hideki Enomoto
  • Heather M. Young
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Time-lapse imaging Live imaging Enteric nervous system Cell migration Vagal neural crest 

Notes

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.

References

  1. 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 125:4335–4347PubMedGoogle Scholar
  2. 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 30:31–48PubMedGoogle Scholar
  3. 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 101:515–541CrossRefGoogle Scholar
  4. 4.
    Young HM, Bergner AJ, Anderson RB et al (2004) Dynamics of neural crest-derived cell migration in the embryonic mouse gut. Dev Biol 270:455–473CrossRefGoogle Scholar
  5. 5.
    Heanue TA, Pachnis V (2007) Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies. Nat Rev Neurosci 8:466–479CrossRefGoogle Scholar
  6. 6.
    Mckeown SJ, Stamp L, Hao MM et al (2013) Hirschsprung disease: a developmental disorder of the enteric nervous system. WIRES Dev Biol 2:113–129CrossRefGoogle Scholar
  7. 7.
    Lake JI, Heuckeroth RO (2013) Enteric nervous system development: migration, differentiation, and disease. Am J Physiol 305:G1–G24CrossRefGoogle Scholar
  8. 8.
    Goldstein AM, Thapar N, Karunaratne TB et al (2016) Clinical aspects of neurointestinal disease: pathophysiology, diagnosis, and treatment. Dev BiolGoogle Scholar
  9. 9.
    Lichtman JW, Fraser SE (2001) The neuronal naturalist: watching neurons in their native habitat. Nat Neurosci 4(Suppl):1215–1220CrossRefGoogle Scholar
  10. 10.
    Kulesa PM, Mckinney MC, Mclennan R (2013) Developmental imaging: the avian embryo hatches to the challenge. Birth Defects Res C Embryo Today 99:121–133CrossRefGoogle Scholar
  11. 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 1:435–445CrossRefGoogle Scholar
  12. 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 15:2076–2088CrossRefGoogle Scholar
  13. 13.
    Young HM, Bergner AJ, Simpson MJ et al (2014) Colonizing while migrating: how do individual enteric neural crest cells behave? BMC Biol 12:23CrossRefGoogle Scholar
  14. 14.
    Theveneau E, Mayor R (2011) Can mesenchymal cells undergo collective cell migration? The case of the neural crest. Cell Adhes Migr 5:490–498CrossRefGoogle Scholar
  15. 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 214:239–247CrossRefGoogle Scholar
  16. 16.
    Druckenbrod NR, Epstein ML (2005) The pattern of neural crest advance in the cecum and colon. Dev Biol 287:125–133CrossRefGoogle Scholar
  17. 17.
    Druckenbrod NR, Epstein ML (2007) Behavior of enteric neural crest-derived cells varies with respect to the migratory wavefront. Dev Dyn 236:84–92CrossRefGoogle Scholar
  18. 18.
    Druckenbrod NR, Epstein ML (2009) Age-dependent changes in the gut environment restrict the invasion of the hindgut by enteric neural progenitors. Development 136:3195–3203CrossRefGoogle Scholar
  19. 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 136:2791–2801CrossRefGoogle Scholar
  20. 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 133:1725–1734CrossRefGoogle Scholar
  21. 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 237:1119–1132CrossRefGoogle Scholar
  22. 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 26:69–81CrossRefGoogle Scholar
  23. 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.e1-6CrossRefGoogle Scholar
  24. 24.
    Uesaka T, Nagashimada M, Enomoto H (2013) GDNF signaling levels control migration and neuronal differentiation of enteric ganglion precursors. J Neurosci 33:16372–16382CrossRefGoogle Scholar
  25. 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 32:3–7CrossRefGoogle Scholar
  26. 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:e1005093CrossRefGoogle Scholar
  27. 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):1283–1293CrossRefGoogle Scholar
  28. 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 128:3963–3974PubMedGoogle Scholar
  29. 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 399:366–370CrossRefGoogle Scholar
  30. 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 216:137–152CrossRefGoogle Scholar
  31. 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 15:1211–1218CrossRefGoogle Scholar
  32. 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 116:167–175PubMedGoogle Scholar
  33. 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 202:67–84CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Marlene M. Hao
    • 1
    • 2
  • Annette J. Bergner
    • 1
  • Donald F. Newgreen
    • 3
  • Hideki Enomoto
    • 4
    • 5
  • Heather M. Young
    • 1
  1. 1.Department of Anatomy and NeuroscienceUniversity of MelbourneMelbourneAustralia
  2. 2.Laboratory for Enteric Neuroscience, TARGIDUniversity of LeuvenLeuvenBelgium
  3. 3.Murdoch Children’s Research InstituteRoyal Children’s HospitalParkvilleAustralia
  4. 4.Division of Neural Differentiation and Regeneration, Department of Physiology and Cell Biology, Graduate School of MedicineKobe UniversityKobeJapan
  5. 5.Core Research for Evolutional Science and TechnologyJapan Science and Technology AgencySaitamaJapan

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