1. Volume 137, Issue 1, Pages 136-144.e3 (July 2009)
Colony Stimulating Factor-1 Dependence of Paneth Cell Development in the Mouse Small Intestine 
Duy Huynh, Xu–Ming Dai, Sayan Nandi, Sally Lightowler, Melanie Trivett, Chee–Kai Chan, Ivan Bertoncello, Robert G. Ramsay, E. Richard Stanley 
Gastroenterology 
Volume 137, Issue 1, Pages e3 (July 2009)
DOI: /j.gastro
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2. Figure 1
Abnormal SI crypts and villi in 2-week-old Csf1r−/− and Csf1op/op mice. (A) Longitudinal sections stained for neutral mucins (PAS stain) show intense staining of goblet cells in the SIs of the mutant mice. (B) Higher power images of the regions boxed in panel A show the enlarged mucin-containing deposits in goblet cells (arrows). In addition, the less-ordered organization of nuclei in the mutant villi is also evident. Bars = 50 μm. (C) Numbers of cells/crypt length and (D) number of cells/villus length (mean ± SEM; n = 3–5/genotype; ANOVA P values shown).
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3. Figure 2
Lysozyme staining shows a dramatic reduction of PCs in the SI crypts of 2-week-old Csf1r−/− and Csf1op/op mice. Low-power images of lysozyme-positive PCs in the SIs of (A) WT, (B) Csf1r−/−, and (C) Csf1op/op mice. (D) Quantitation of the number of lysozyme-positive cells/crypt section (mean ± SEM; n = 3; ANOVA P values shown) showed significant differences the number of PCs at the crypt base (gray arrow in E) between both mutant and WT, as well as between mutants. (E) Higher power images of WT crypts showing the presence of lysozyme-positive cells within the lamina propria indicative of macrophages (black arrow). (F) Only background lysozyme staining is evident in the Csf1r−/− SI. White arrow indicates goblet cell with enlarged mucin body. Bar = 50 μm.
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4. Figure 3
Treatment of WT mice with anti–CSF-1R antibody inhibits the development of PCs and villus macrophages. Newborn mice were subcutaneously injected with (A) control IgG or (B) anti–CSF-1R antibody for 14 days before fixation and sectioning of the SIs and staining with antilysozyme antibody and 4′,6-diamidino-2-phenylindole (DAPI). Bar = 60 μm.
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5. Figure 4
SI crypts of 2-week-old Csf1r−/− and Csf1op/op mice display hypoproliferative defects. PCNA staining of (A) WT, (B) Csf1r−/−, and (C) Csf1op/op SI shows proliferation deficits in the mutant crypts. Bar = 50 μm. (D) Reduced numbers of PCNA+ nuclei/Csf1r−/− or Csf1op/op crypt compared with WT littermate crypts (mean ± SEM; n = 3/genotype, 25 crypt/villi per mouse counted; ANOVA P values shown). (E) Quantitative RT-PCR analysis of Csf1r, cyclin D1, and Lgr5 in crypts of Csf1r−/− and WT littermates (mean ± SEM; ND = no data, n = 3; ANOVA P values shown).
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6. Figure 5
PCs and SI macrophages express the CSF-1R. Immunofluorescence micrography showing (A) CSF-1R staining (red) of cells within the crypt (PC), below the crypt, and within interstitial regions of the villi (macrophages); (B) staining for lysozyme (green); and (C) merged images, showing colocalization of the CSF-1R and lysozyme staining. P indicates PCs; M, macrophages. Nuclei were counterstained with DAPI (blue). Bar = 30 μm.
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7. Figure 6
CSF-1 is expressed in close proximity to PCs. Histochemical staining for β-galactosidase in the SIs of 2-week-old (A) control WT and (B–D) TgZ9/TgZ9 mice. (B) Low-power and (C and D) high-power fields, showing the close proximity of β-gal staining (blue; nuclear, arrows) to the PCs (P). Note: There is spillover of nuclear β-galactosidase expression into the cytoplasm in some cells.
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8. Figure 7
Expression of the membrane-spanning, csCSF-1 is sufficient to rescue the SI defects of 2-week-old Csf1op/op mice. (A) Rescue of proliferating (PCNA), crypt, and villus cells. (B) Rescue of PCs. Bar = 50 μm.
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9. Supplementary Figure 1
Two-week old Csf1r−/− and Csf1op/op mutant mice are smaller in size and have shorter small intestines. (A) Body weight and (B) SI length (mean ± SEM; n = 3 for each genotype; ANOVA P value shown).
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10. Supplementary Figure 2
Two-week old Csf1r−/− and Csf1op/op mutant mice have multiple small intestinal defects. Csf1r−/− and Csf1op/op mutant mice have defects in villus morphology and apparent overproduction of acid mucins (black arrows) by goblet cells, that are particularly pronounced in the Csf1r−/− mice. Alcian blue staining, = 50 μm.
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11. Supplementary Figure 3
Enteroendocrine cell numbers are reduced in the SIs of two-week-old Csf1r−/− and Csf1op/op mutant mice compared to WT littermate controls. Enteroendocrine cells were visualized by staining for Chromogranin A (mean ± SEM; n = 3 for each genotype; ANOVA P value shown).
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12. Supplementary Figure 4
Reduced cryptdin 2+ cells in Csf1r−/− SI. Low power (×100) images of WT and Csf1r−/− SI, showing the absence of cryptdin 2+ cells in Csf1r−/− SI. High power (×400) images of the boxed region are also shown.
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13. Supplementary Figure 5
Reduced CRS4C+ cells in Csf1r−/− SI. Low power (×100) images of WT and Csf1r−/− SI, showing the absence of CRS4C+ cells in Csf1r−/− SI. High power (×400) images of the boxed region are also shown.
Gastroenterology  , e3DOI: ( /j.gastro )
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14. Supplementary Figure 6
Persistance of the reduction of PC and of macrophages in adult Csf1op/Csf1op SI. Immunofluorescence micrography showing images of lysozyme+ PCs in SI crypts of three-month-old (A) WT and (B) Csf1op/Csf1op mice. (E) Quantitation of number of lysozyme+ PCs/crypt length (mean ± SD, n = 2 for each genotype, 20 crypts/mouse). Immunofluorescence micrography showing images of F4/80+ macrophages in the SI villi of three-month-old (C) WT and (D) Csf1op/Csf1op mice. (F) Quantitation of number of F4/80+ macrophages/villus (mean ± SD, n = 2 for each genotype, 10 crypts/mouse).
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15. Supplementary Figure 7
Specificity of the anti CSF-1R antibody. (A) CSF-1R immunostaining of lamina propria macrophages (white arrows) in the SI of WT (a), but not of Csf1r−/− (b) mice at 2 weeks of age. (B) Failure of Csf1r−/− PC to to stain with anti-CSF-1R antibody. PC of 2 week-old Csf1r−/− mice immunostained with antibodies lysozyme failed to exhibit any CSF-1R immunostaining (a–c).
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16. Supplementary Figure 8
Colocalization of CSF-1R and lysozyme in PC by immunofluorescence micrography in WT mice. Three-month old SI sections showing (A) Low power merged images of anti-lysozyme (green), anti-CSF-1R (red) and DAPI (blue) staining of PCs. (B) Higher power image corresponding to the region boxed in A. (C–F) Higher power images corresponding to the region boxed in B, showing co-localization of lysozyme staining (C, green) with CSF-1R staining (D, red) in the merged image (F). The DAPI staining of nuclei (E) is outlined in E and F. Lysozyme staining in PC occurs in Golgi, lysosomes, and the mature secretory granules.45 Note: Major staining of the CSF-1R in cultured macrophages occurs in the Golgi, consistent with the large pool of mature intracellular CSF-1R (F.J. Pixley and E.R.S. unpublished observations).
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17. Supplementary Figure 9
Expression of the membrane-spanning, cell-surface isoform of CSF-1 is sufficient to rescue the SI defects of Csf1op/op mice. Rescue of macrophages by the csCSF1 transgene in two-week-old Csf1op/op mice as determined by lysozyme staining within the small intestinal lamina propria. Results for Csf1r−/− SI are shown for comparison.
Gastroenterology  , e3DOI: ( /j.gastro )
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