Abstract
Despite the continuous renewal and turnover of the small intestinal epithelium, the intestinal crypt maintains a ‘soccer ball-like’, alternating pattern of stem and Paneth cells at the base of the crypt. To study the robustness of the alternating pattern, we used intravital two-photon microscopy in mice with fluorescently-labeled Lgr5+ intestinal stem cells and precisely perturbed the mosaic pattern with femtosecond laser ablation. Ablation of one to three cells initiated rapid motion of crypt cells that restored the alternation in the pattern within about two hours with only the rearrangement of pre-existing cells, without any cell division. Crypt cells then performed a coordinated dilation of the crypt lumen, which resulted in peristalsis-like motion that forced damaged cells out of the crypt. Crypt cell motion was reduced with inhibition of the ROCK pathway and attenuated with old age, and both resulted in incomplete pattern recovery. This suggests that in addition to proliferation and self-renewal, motility of stem cells is critical for maintaining homeostasis. Reduction of this newly-identified behavior of stem cells could contribute to disease and age-related changes.
Highlights
Despite the continuous renewal and turnover of the small intestinal epithelium, the intestinal crypt maintains a ‘soccer ball-like’, alternating pattern of stem and Paneth cells at the base of the crypt
Lgr5+ crypt cells were visualized in mice expressing green fluorescent protein driven by the Lgr[5] promoter (Lgr5-GFP)[3], while Paneth cells appeared dark (Fig. 1d)
Time-lapse images show that the pattern of GFP and the number of Hoechst-labeled nuclei within a crypt was stable over the course of several hours
Summary
Despite the continuous renewal and turnover of the small intestinal epithelium, the intestinal crypt maintains a ‘soccer ball-like’, alternating pattern of stem and Paneth cells at the base of the crypt. Crypt cell motion was reduced with inhibition of the ROCK pathway and attenuated with old age, and both resulted in incomplete pattern recovery This suggests that in addition to proliferation and self-renewal, motility of stem cells is critical for maintaining homeostasis. To investigate the robustness of the patterning and its maintenance in vivo, we ablated individual cells in the crypt with high-pulse-energy femtosecond (fs) laser ablation and imaged the real-time dynamics of recovery with multiphoton microscopy[7,8]. The repair movements were impaired by both inhibition of cellular movement and aging, highlighting the importance of this dynamic response for the integrity of the niche
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