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Methods Mol Biol. 2020;2047:3-24. doi: 10.1007/978-1-4939-9732-9_1.

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

Author information

1
Department of Genetics and Evolution, iGE3, Faculty of Sciences, University of Geneva, Geneva, Switzerland.
2
Department of Genetics and Evolution, iGE3, Faculty of Sciences, University of Geneva, Geneva, Switzerland. Brigitte.Galliot@unige.ch.

Abstract

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.

KEYWORDS:

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

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