Elsevier

Differentiation

Volume 87, Issues 1–2, January–February 2014, Pages 83-99
Differentiation

Robust G2 pausing of adult stem cells in Hydra

https://doi.org/10.1016/j.diff.2014.03.001Get rights and content
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Highlights

  • We established an improved FACS procedure to monitor the DNA profiles in Hydra.

  • DNA profiles along Hydra axis distribute as differentiated and cycling stem cells.

  • Terminal differentiation is pre-mitotic for epithelial cells in Hydra.

  • Adult stem cells in Hydra cycle by alternating between G2 and S with a minimal G1.

  • Interstitial progenitors have a distinct cycling behavior with a shortened G2.

Abstract

Hydra is a freshwater hydrozoan polyp that constantly renews its two tissue layers thanks to three distinct stem cell populations that cannot replace each other, epithelial ectodermal, epithelial endodermal, and multipotent interstitial. These adult stem cells, located in the central body column, exhibit different cycling paces, slow for the epithelial, fast for the interstitial. To monitor the changes in cell cycling in Hydra, we established a fast and efficient flow cytometry procedure, which we validated by confirming previous findings, as the Nocodazole-induced reversible arrest of cell cycling in G2/M, and the mitogenic signal provided by feeding. Then to dissect the cycling and differentiation behaviors of the interstitial stem cells, we used the AEP_cnnos1 and AEP_Icy1 transgenic lines that constitutively express GFP in this lineage. For the epithelial lineages we used the sf-1 strain that rapidly eliminates the fast cycling cells upon heat-shock and progressively becomes epithelial. This study evidences similar cycling patterns for the interstitial and epithelial stem cells, which all alternate between the G2 and S-phases traversing a minimal G1-phase. We also found interstitial progenitors with a shorter G2 that pause in G1/G0. At the animal extremities, most cells no longer cycle, the epithelial cells terminally differentiate in G2 and the interstitial progenitors in G1/G0. At the apical pole ~80% cells are post-mitotic differentiated cells, reflecting the higher density of neurons and nematocytes in this region. We discuss how the robust G2 pausing of stem cells, maintained over weeks of starvation, may contribute to regeneration.

Keywords

Flow cytometry method
Epithelial stem cells
Interstitial stem cells
G2 pausing
Cell cycling
AEP_cnnos1 and AEP_Icy1 transgenic Hydra

Abbreviations

ASCs
adult stem cells
BLI
BrdU-labeling index
BrdU
5-bromo-2׳-deoxyuridine
CV
coefficient of variation
DM
dissociation medium
eGFP
enhanced green fluorescent protein
ESCs
embryonic stem cells
FC
flow cytometry
FSC
forward scatter
HS
heat-shock
IPs
interstitial progenitors
ISCs
interstitial stem cells
MECs
myoepithelial cells
NDZ
nocodazole
PI
propidium iodide
SCs
stem cells
SSC
side scatter

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