Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice
Ryotaro Sakamori, Soumyashree Das, Shiyan Yu, Shanshan Feng, Ewa Stypulkowski, Yinzheng Guan, Veronique Douard, Waixing Tang, Ronaldo P. Ferraris, Akihiro Harada, Cord Brakebusch, Wei Guo, Nan Gao
Ryotaro Sakamori, Soumyashree Das, Shiyan Yu, Shanshan Feng, Ewa Stypulkowski, Yinzheng Guan, Veronique Douard, Waixing Tang, Ronaldo P. Ferraris, Akihiro Harada, Cord Brakebusch, Wei Guo, Nan Gao
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Research Article Gastroenterology

Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice

Abstract

The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases Cdc42 and Rab8a are critical regulators of these processes in mice. Conditional ablation of Cdc42 in the mouse intestinal epithelium resulted in the formation of large intracellular vacuolar structures containing microvilli (microvillus inclusion bodies) in epithelial enterocytes, a phenotype reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells, and increased apoptosis. Cdc42 deficiency impaired Rab8a activation and its association with multiple effectors, and prevented trafficking of Rab8a vesicles to the midbody. This impeded cytokinesis, triggering crypt apoptosis and disrupting epithelial morphogenesis. Rab8a was also required for Cdc42-GTP activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell–based approaches could be beneficial to infants with this often lethal condition.

Authors

Ryotaro Sakamori, Soumyashree Das, Shiyan Yu, Shanshan Feng, Ewa Stypulkowski, Yinzheng Guan, Veronique Douard, Waixing Tang, Ronaldo P. Ferraris, Akihiro Harada, Cord Brakebusch, Wei Guo, Nan Gao

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Figure 4

Cdc42 deficiency reduces the clonal expansion capacity of Lgr5 stem cells.

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Cdc42 deficiency reduces the clonal expansion capacity of Lgr5 stem cel...
(A) Experimental scheme for the genetic tracing experiments. (B and C) GFP and lysozyme staining for control and Cdc42-deficient crypts 2 weeks after tamoxifen treatment. Arrow in B points to cells (in yellow) positive for both GFP and lysozyme. (D and E) GFP and basement membrane staining for control and Cdc42-deficient crypts 3 weeks after tamoxifen treatment. Arrows in E point to small clusters of cells derived from Cdc42-deficient stem cells. Schematic diagrams at the right of each panel summarize the results shown at left. (F) Quantification of RosaYFP-labeled villus epithelial cells derived from control and Cdc42-deficient Lgr5 stem cells 3 weeks after tamoxifen administration. ***P < 0.001. (G–L) GFP and basement membrane staining illustrate columnar shapes of labeled control intestinal epithelial cells (arrows in G) but abnormal morphology of labeled Cdc42-deficient cells (arrows in J). Arrowheads in H and K indicate positively labeled villus epithelial cells. White arrowheads in I and L indicate normal nuclear alignment. Yellow arrowheads in L indicate disrupted nuclear organization and cell polarity in villus cells derived from Cdc42-deficient stem cells. (M and N) Costaining of pHH3 and GFP. Dotted line in M indicates an unlabeled crypt. Dotted lines in N encircle the wild-type crypts that escaped Cre recombination. Arrows in M and N indicate GFP+/pHH3+ cells. Scale bars: 5 μm.