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. 2013 Mar;10(2):203-12.
doi: 10.1016/j.scr.2012.12.001. Epub 2012 Dec 9.

The CSF-1 receptor fashions the intestinal stem cell niche

Dilara Akcora  1 , Duy Huynh, Sally Lightowler, Markus Germann, Sylvie Robine, Jan R de May, Jeffrey W Pollard, E Richard Stanley, Jordane Malaterre, Robert G Ramsay
Affiliations

The CSF-1 receptor fashions the intestinal stem cell niche

Dilara Akcora et al. Stem Cell Res. 2013 Mar.

Abstract

Gastrointestinal (GI) homeostasis requires the action of multiple pathways. There is some controversy regarding whether small intestine (SI) Paneth cells (PCs) play a central role in orchestrating crypt architecture and their relationship with Lgr5+ve stem cells. Nevertheless, we previously showed that germline CSF-1 receptor (Csf1r) knock out (KO) or Csf1 mutation is associated with an absence of mature PC, reduced crypt proliferation and lowered stem cell gene, Lgr5 expression. Here we show the additional loss of CD24, Bmi1 and Olfm4 expression in the KO crypts and a high resolution 3D localization of CSF-1R mainly to PC. The induction of GI-specific Csf1r deletion in young adult mice also led to PC loss over a period of weeks, in accord with the anticipated long life span of PC, changed distribution of proliferating cells and this was with a commensurate loss of Lgr5 and other stem cell marker gene expression. By culturing SI organoids, we further show that the Csf1r(-/-) defect in PC production is intrinsic to epithelial cells as well as definitively affecting stem cell activity. These results show that CSF-1R directly supports PC maturation and that in turn PCs fashion the intestinal stem cell niche.

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Conflict of interest statement

The authors have no conflicts of interest.

Figures

Figure 1
Figure 1
Global CSF-1R loss affects on mature Paneth cells and intestinal stem cells (ISCs). (A) The Paneth cell (PC) and ISC marker, CD24, are expressed at the base of the SI crypt (brown arrow). (B) Reduced CD24 expression and loss of PC and ISC in Csf1r−/− mouse SI crypts. (C) When crypts are liberated from the SI villi are usually sheared away (broken lines) allowing the production of enriched crypts separated from the ISC niche. A cartoon depicting this is shown. (D) Confocal IF images reconstructed into a 3D format show coincident expression of CSF-1R (green) and lysozyme (red) at the crypt base. DAPI (blue) defines the crypt nuclei (see Movies in Supplementary data). (E) 2D confocal image of Csf1r−/− crypts shows no CSF-1R (green) and minimal lysozyme (red). Similarly prepared 2D confocal images of crypts show (F) DAPI-stained nuclei (blue) most (G) CSF-1R (green) associated with PC (green arrow). (H) Co-staining with β-catenin (red) as a merged image of (F) and (G) highlights the epithelial cells (red). Scale bar=50 µm. Magnification ×60.
Figure 2
Figure 2
Global CSF-1R loss affects on intestinal stem cell (ISC) function and gene expression signatures in vitro. (A) Organoid cultures generated by WT and Csf1r−/− ISC shown at days 1, 3 and 5 indicate delayed growth of the KO organoids (Bar=20 µM). (B) Quantitation of WT and Csf1r−/− organoid numbers after 7 days in culture (equivalent numbers of crypt nests were plated at day 0) (*p=0.05; ***=0.001; student t-test). Organoid cultures initiated from WT and Csf1r−/− mice were harvested at day 7, RNA prepared and subjected to qRT-PCR using primers for ISC genes (C) Bmi1, (D) Olfm4 and (E) Lgr5 (*p<0.05; Mann Whitney test).
Figure 3
Figure 3
Conditional CSF-1R inactivation effects on Paneth cells and crypt proliferation. (A) CSF-1R (green) at the crypt base (green arrows) in association with lysozyme (red) as well as in the lamina propria and muscularis is shown. (B) PC loss in mice following the tamoxifen-induced inactivation (2 weeks) of the Csf1r gene and loss of Csf-1R expression (green arrow). (C) 3D image of a SI crypt from a Csf1rf//fl;VillinCreERT2 mouse after 2 weeks of tamoxifen exposure. (D) Lysozyme expression (brown arrow) determined by IHC in the ileum of Csf1rf//fl;VillinCreERT2 mice after 4 weeks of tamoxifen exposure indicates substantive loss of mature PC. (E) ISC Lgr5 expression is reduced in inducible, intestinal-specific Csf1r deleted mice. (F) Displacement of PC, or replacement by, PCNA+ve cells in the base of the SI crypts of Csf1rf//fl;VillinCreERT2 mice after 4 weeks of tamoxifen exposure leads to significantly more PCNA+ve cells at the crypt base. (*p=0.05, ***=0.001; Student t-test).
Figure 4
Figure 4
Reduced ISC gene expression is durable for several weeks following loss of Paneth cells. Significant gene expression changes for Csf1r, Lgr5 and Olfm4 in the SI crypts of Csf1rf//fl;VillinCreERT2 mice after 4 weeks of tamoxifen exposure, followed by a 4 week chase in its absence is shown. Cell cycle related genes c-Myb, CyclinD1 and c-Myc expressions were slightly but significantly reduced (*p=0.05; Student t-test).
Figure 5
Figure 5
Effect of in vitro deletion of Csf1r on organoid formation. (A) Organoid cultures were initiated from WT and Csf1rfl/fl;VillinCreERT2 mice and 4-hydroxytamoxifen (4OHT) added on day 1 and assessed on day 7. (B) The number of organoids formed from the same number of starting crypt nests Csf1rfl/fl:VillinCreERT2 and wild type (WT) cultures are shown (*p<0.05; Student t-test).
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