Combining BrdU-Labeling to Detection of Neuronal Markers to Monitor Adult Neurogenesis in Hydra

  • Wanda Buzgariu
  • Marie-Laure Curchod
  • Chrystelle Perruchoud
  • Brigitte GalliotEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)


The nervous system is produced and maintained in adult Hydra through the continuous production of nerve cells and mechanosensory cells (nematocytes or cnidocytes). De novo neurogenesis occurs slowly in intact animals that replace their dying nerve cells, at a faster rate in animals regenerating their head as a complete apical nervous system is built in few days. To dissect the molecular mechanisms that underlie these properties, a precise monitoring of the markers of neurogenesis and nematogenesis is required. Here we describe the conditions for an efficient BrdU-labeling coupled to an immunodetection of neuronal markers, either regulators of neurogenesis, here the homeoprotein prdl-a, or neuropeptides such as RFamide or Hym-355. This method can be performed on whole-mount animals as well as on macerated tissues when cells retain their morphology. Moreover, when antibodies are not available, BrdU-labeling can be combined with the analysis of gene expression by whole-mount in situ hybridization. This co-immunodetection procedure is well adapted to visualize and quantify the dynamics of de novo neurogenesis. Upon continuous BrdU labeling, the repeated measurements of BrdU-labeling indexes in specific cellular populations provide a precise monitoring of nematogenesis as well as neurogenesis, in homeostatic or developmental conditions.


Hydra nervous system Interstitial stem cells Neurogenesis Nematogenesis In situ hybridization Immunofluorescence Hydroxyurea BrdU prdl-a Hym-355 RFamide 



This work was supported by the Swiss National Science Foundation (SNF grants 31003A_149630, 31003_169930), the Claraz donation, and the Canton of Geneva.


  1. 1.
    Koizumi O (2016) Origin and evolution of the nervous system considered from the diffuse nervous system of cnidarians. In: Goffredo S, Dubinsky Z (eds) The cnidaria, past, present and future. Springer International Publishing, Cham, pp 73–91CrossRefGoogle Scholar
  2. 2.
    Galliot B, Quiquand M, Ghila L, de Rosa R, Miljkovic-Licina M, Chera S (2009) Origins of neurogenesis, a cnidarian view. Dev Biol 332:2–24CrossRefGoogle Scholar
  3. 3.
    Takahashi T, Koizumi O, Ariura Y, Romanovitch A, Bosch TC, Kobayakawa Y, Mohri S, Bode HR, Yum S, Hatta M et al (2000) A novel neuropeptide, Hym-355, positively regulates neuron differentiation in Hydra. Development 127:997–1005PubMedGoogle Scholar
  4. 4.
    Chera S, Ghila L, Dobretz K, Wenger Y, Bauer C, Buzgariu W, Martinou JC, Galliot B (2009) Apoptotic cells provide an unexpected source of Wnt3 signaling to drive hydra head regeneration. Dev Cell 17:279–289CrossRefGoogle Scholar
  5. 5.
    Richards GS, Simionato E, Perron M, Adamska M, Vervoort M, Degnan BM (2008) Sponge genes provide new insight into the evolutionary origin of the neurogenic circuit. Curr Biol 18:1156–1161CrossRefGoogle Scholar
  6. 6.
    Teragawa CK, Bode HR (1995) Migrating interstitial cells differentiate into neurons in hydra. Dev Biol 171:286–293CrossRefGoogle Scholar
  7. 7.
    Fujisawa T, Nishimiya C, Sugiyama T (1986) Nematocyte differentiation in hydra. Curr Top Dev Biol 20:281–290CrossRefGoogle Scholar
  8. 8.
    Tardent P (1995) The cnidarian cnidocyte, a high-tech cellular weaponry. BioEssays 17:351–362CrossRefGoogle Scholar
  9. 9.
    Galliot B, Quiquand M (2011) A two-step process in the emergence of neurogenesis. Eur J Neurosci 34:847–862CrossRefGoogle Scholar
  10. 10.
    Bode HR (1992) Continuous conversion of neuron phenotype in hydra. Trends Genet 8:279–284CrossRefGoogle Scholar
  11. 11.
    Koizumi O (2007) Nerve ring of the hypostome in hydra: is it an origin of the central nervous system of bilaterian animals? Brain Behav Evol 69:151–159CrossRefGoogle Scholar
  12. 12.
    Grimmelikhuijzen CJP, Westfall JA (1995) The nervous systems of Cnidarians. In: Breidbach O, Kutsch W (eds) The nervous systems of invertebrates: an evolutionary and comparative approach. Birkhaüser Verlag, Basel, pp 7–24CrossRefGoogle Scholar
  13. 13.
    Grunder S, Assmann M (2015) Peptide-gated ion channels and the simple nervous system of Hydra. J Exp Biol 218:551–561CrossRefGoogle Scholar
  14. 14.
    Anderson PA, Spencer AN (1989) The importance of cnidarian synapses for neurobiology. J Neurobiol 20:435–457CrossRefGoogle Scholar
  15. 15.
    Kass-Simon G, Pierobon P (2007) Cnidarian chemical neurotransmission, an updated overview. Comp Biochem Physiol A Mol Integr Physiol 146:9–25CrossRefGoogle Scholar
  16. 16.
    Steinmetz PRH, Kraus JEM, Larroux C, Hammel JU, Amon-Hassenzahl A, Houliston E, Wörheide G, Nickel M, Degnan BM, Technau U (2012) Independent evolution of striated muscles in cnidarians and bilaterians. Nature 487:231–234CrossRefGoogle Scholar
  17. 17.
    Grens A, Mason E, Marsh JL, Bode HR (1995) Evolutionary conservation of a cell fate specification gene: the Hydra achaete-scute homolog has proneural activity in Drosophila. Development 121:4027–4035PubMedGoogle Scholar
  18. 18.
    Gauchat D, Kreger S, Holstein T, Galliot B (1998) prdl-a, a gene marker for hydra apical differentiation related to triploblastic paired-like head-specific genes. Development 125:1637–1645PubMedGoogle Scholar
  19. 19.
    Gauchat D, Escriva H, Miljkovic-Licina M, Chera S, Langlois MC, Begue A, Laudet V, Galliot B (2004) The orphan COUP-TF nuclear receptors are markers for neurogenesis from cnidarians to vertebrates. Dev Biol 275:104–123CrossRefGoogle Scholar
  20. 20.
    Miljkovic-Licina M, Gauchat D, Galliot B (2004) Neuronal evolution: analysis of regulatory genes in a first-evolved nervous system, the hydra nervous system. Biosystems 76:75–87CrossRefGoogle Scholar
  21. 21.
    Miljkovic-Licina M, Chera S, Ghila L, Galliot B (2007) Head regeneration in wild-type hydra requires de novo neurogenesis. Development 134:1191–1201CrossRefGoogle Scholar
  22. 22.
    Wenger Y, Buzgariu W, Galliot B (2016) Loss of neurogenesis in Hydra leads to compensatory regulation of neurogenic and neurotransmission genes in epithelial cells. Philos Trans R Soc Lond Ser B Biol Sci 371:20150040CrossRefGoogle Scholar
  23. 23.
    Wenger Y, Buzgariu W, Perruchoud C, Loichot G, Galliot B (2019) Generic and context-dependent gene modulations during Hydra whole body regeneration. BioRXiv 587147.
  24. 24.
    Darmer D, Hauser F, Nothacker HP, Bosch TC, Williamson M, Grimmelikhuijzen CJ (1998) Three different prohormones yield a variety of Hydra-RFamide (Arg-Phe- NH2) neuropeptides in Hydra magnipapillata. Biochem J 332:403–412CrossRefGoogle Scholar
  25. 25.
    Hansen GN, Williamson M, Grimmelikhuijzen CJ (2000) Two-color double-labeling in situ hybridization of whole-mount Hydra using RNA probes for five different Hydra neuropeptide preprohormones: evidence for colocalization. Cell Tissue Res 301:245–253CrossRefGoogle Scholar
  26. 26.
    Hansen GN, Williamson M, Grimmelikhuijzen CJ (2002) A new case of neuropeptide coexpression (RGamide and LWamides) in Hydra, found by whole-mount, two-color double-labeling in situ hybridization. Cell Tissue Res 308:157–165CrossRefGoogle Scholar
  27. 27.
    Hwang JS, Ohyanagi H, Hayakawa S, Osato N, Nishimiya-Fujisawa C, Ikeo K, David CN, Fujisawa T, Gojobori T (2007) The evolutionary emergence of cell type-specific genes inferred from the gene expression analysis of Hydra. Proc Natl Acad Sci U S A 104:14735–14740CrossRefGoogle Scholar
  28. 28.
    Hayakawa E, Fujisawa C, Fujisawa T (2004) Involvement of Hydra achaete-scute gene CnASH in the differentiation pathway of sensory neurons in the tentacles. Dev Genes Evol 214:486–492PubMedGoogle Scholar
  29. 29.
    Chera S, Kaloulis K, Galliot B (2007) The cAMP response element binding protein (CREB) as an integrative HUB selector in metazoans: clues from the hydra model system. Biosystems 87:191–203CrossRefGoogle Scholar
  30. 30.
    Lindgens D, Holstein TW, Technau U (2004) Hyzic, the Hydra homolog of the zic/odd-paired gene, is involved in the early specification of the sensory nematocytes. Development 131:191–201CrossRefGoogle Scholar
  31. 31.
    Hartl M, Mitterstiller AM, Valovka T, Breuker K, Hobmayer B, Bister K (2010) Stem cell-specific activation of an ancestral myc protooncogene with conserved basic functions in the early metazoan Hydra. Proc Natl Acad Sci U S A 107:4051–4056CrossRefGoogle Scholar
  32. 32.
    Ambrosone A, Marchesano V, Tino A, Hobmayer B, Tortiglione C (2012) Hymyc1 downregulation promotes stem cell proliferation in Hydra vulgaris. PLoS One 7:e30660CrossRefGoogle Scholar
  33. 33.
    Juliano CE, Reich A, Liu N, Götzfried J, Zhong M, Uman S, Reenan RA, Wessel GM, Steele RE, Lin H (2014) PIWI proteins and PIWI-interacting RNAs function in Hydra somatic stem cells. Proc Natl Acad Sci U S A 111:337–342CrossRefGoogle Scholar
  34. 34.
    Takaku Y, Hwang JS, Wolf A, Bottger A, Shimizu H, David CN, Gojobori T (2014) Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps. Sci Rep 4:3573CrossRefGoogle Scholar
  35. 35.
    Engel U (2001) A switch in disulfide linkage during minicollagen assembly in Hydra nematocysts. EMBO J 20:3063–3073CrossRefGoogle Scholar
  36. 36.
    Engel U, Ozbek S, Streitwolf-Engel R, Petri B, Lottspeich F, Holstein TW (2002) Nowa, a novel protein with minicollagen Cys-rich domains, is involved in nematocyst formation in Hydra. J Cell Sci 115:3923–3934CrossRefGoogle Scholar
  37. 37.
    Koch AW, Holstein TW, Mala C, Kurz E, Engel J, David CN (1998) Spinalin, a new glycine- and histidine-rich protein in spines of Hydra nematocysts. J Cell Sci 111:1545–1554PubMedGoogle Scholar
  38. 38.
    Adamczyk P, Meier S, Gross T, Hobmayer B, Grzesiek S, Bachinger HP, Holstein TW, Ozbek S (2008) Minicollagen-15, a novel minicollagen isolated from Hydra, forms tubule structures in nematocysts. J Mol Biol 376:1008–1020CrossRefGoogle Scholar
  39. 39.
    Hwang JS, Takaku Y, Momose T, Adamczyk P, Ozbek S, Ikeo K, Khalturin K, Hemmrich G, Bosch TC, Holstein TW et al (2010) Nematogalectin, a nematocyst protein with GlyXY and galectin domains, demonstrates nematocyte-specific alternative splicing in Hydra. Proc Natl Acad Sci U S A 107:18539–18544CrossRefGoogle Scholar
  40. 40.
    Balasubramanian PG, Beckmann A, Warnken U, Schnolzer M, Schuler A, Bornberg-Bauer E, Holstein TW, Ozbek S (2012) Proteome of Hydra nematocyst. J Biol Chem 287:9672–9681CrossRefGoogle Scholar
  41. 41.
    David CN (1973) A quantitative method for maceration of hydra tissue. Wilhelm Roux Arch Dev Biol 171:259–268CrossRefGoogle Scholar
  42. 42.
    Bode HR, Berking S, David C, Gierer A, Schaller H, Trenker E (1973) Quantitative analysis of cell types during growth and regeneration in hydra. Wilhelm Roux Arch Entw Mech Org 171:269–285CrossRefGoogle Scholar
  43. 43.
    Fujisawa T (1989) Role of interstitial cell migration in generating position-dependent patterns of nerve cell differentiation in Hydra. Dev Biol 133:77–82CrossRefGoogle Scholar
  44. 44.
    Technau U, Holstein TW (1996) Phenotypic maturation of neurons and continuous precursor migration in the formation of the peduncle nerve net in Hydra. Dev Biol 177:599–615CrossRefGoogle Scholar
  45. 45.
    Campbell RD (1976) Elimination by Hydra interstitial and nerve cells by means of colchicine. J Cell Sci 21:1–13PubMedGoogle Scholar
  46. 46.
    Marcum BA, Campbell RD (1978) Development of Hydra lacking nerve and interstitial cells. J Cell Sci 29:17–33PubMedGoogle Scholar
  47. 47.
    Marcum BA, Fujisawa T, Sugiyama T (1980) A mutant hydra strain (sf-1) containing temperature-sensitive interstitial cells. In: Tardent P, Tardent R (eds) Developmental and cellular biology of coelenterates. Elsevier, Amsterdam, pp 429–434Google Scholar
  48. 48.
    Takahashi T, Fujisawa T (2009) Important roles for epithelial cell peptides in hydra development. BioEssaysGoogle Scholar
  49. 49.
    Takahashi T, Takeda N (2015) Insight into the molecular and functional diversity of cnidarian neuropeptides. Int J Mol Sci 16:2610–2625CrossRefGoogle Scholar
  50. 50.
    Plickert G, Kroiher M (1988) Proliferation kinetics and cell lineages can be studied in whole mounts and macerates by means of BrdU/anti-BrdU technique. Development 103:791–794PubMedGoogle Scholar
  51. 51.
    Bode H, Lengfeld T, Hobmayer B, Holstein TW (2008) Detection of expression patterns in Hydra pattern formation. Methods Mol Biol 469:69–84CrossRefGoogle Scholar
  52. 52.
    Kaloulis K, Chera S, Hassel M, Gauchat D, Galliot B (2004) Reactivation of developmental programs: the cAMP-response element-binding protein pathway is involved in hydra head regeneration. Proc Natl Acad Sci U S A 101:2363–2368CrossRefGoogle Scholar
  53. 53.
    Grimmelikhuijzen CJ (1985) Antisera to the sequence Arg-Phe-amide visualize neuronal centralization in hydroid polyps. Cell Tissue Res 241:171–182CrossRefGoogle Scholar
  54. 54.
    Koizumi O, Bode HR (1991) Plasticity in the nervous system of adult hydra. III. Conversion of neurons to expression of a vasopressin-like immunoreactivity depends on axial location. J Neurosci 11:2011–2020CrossRefGoogle Scholar
  55. 55.
    Dunne JF, Javois LC, Huang LW, Bode HR (1985) A subset of cells in the nerve net of Hydra oligactis defined by a monoclonal antibody: its arrangement and development. Dev Biol 109:41–53CrossRefGoogle Scholar
  56. 56.
    Galliot B, Welschof M, Schuckert O, Hoffmeister S, Schaller HC (1995) The cAMP response element binding protein is involved in hydra regeneration. Development 121:1205–1216PubMedGoogle Scholar
  57. 57.
    Chera S, Ghila L, Wenger Y, Galliot B (2011) Injury-induced activation of the MAPK/CREB pathway triggers apoptosis-induced compensatory proliferation in hydra head regeneration. Develop Growth Differ 53:186–201CrossRefGoogle Scholar
  58. 58.
    Ott SR (2008) Confocal microscopy in large insect brains: zinc-formaldehyde fixation improves synapsin immunostaining and preservation of morphology in whole-mounts. J Neurosci Methods 172:220–230CrossRefGoogle Scholar
  59. 59.
    Loomis WF (1956) Growth and sexual differentiation of hydra in mass culture. J Exp Zool 132CrossRefGoogle Scholar
  60. 60.
    Gierer A, Berking S, Bode H, David CN, Flick K, Hansmann G, Schaller H, Trenkner E (1972) Regeneration of hydra from reaggregated cells. Nat New Biol 239:98–101CrossRefGoogle Scholar
  61. 61.
    Macklin M (1976) The effect of urethan on hydra. Biol Bull 150:442–452CrossRefGoogle Scholar
  62. 62.
    Latt SA (1973) Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc Natl Acad Sci U S A 70:3395–3399CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Wanda Buzgariu
    • 1
  • Marie-Laure Curchod
    • 1
  • Chrystelle Perruchoud
    • 1
  • Brigitte Galliot
    • 1
    Email author
  1. 1.Department of Genetics and Evolution, iGE3, Faculty of SciencesUniversity of GenevaGenevaSwitzerland

Personalised recommendations