The CSF-1 receptor fashions the intestinal stem cell niche

AbstractGastrointestinal (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

Tripartite interactions between Wnt signaling, Notch and Myb for stem/progenitor cell functions during intestinal tumorigenesis

Deletion studies confirm Wnt, Notch and Myb transcriptional pathway engagement in intestinal tumorigenesis. Nevertheless, their contrasting and combined roles when activated have not been elucidated. This is important as these pathways are not ablated but rather are aberrantly activated during carcinogenesis. Using ApcMin/+ mice as a source of organoids we documented their transition, on a clone-by-clone basis, to cyst-like spheres with constitutively activated Wnt pathway, increased self-renewal and growth and reduced differentiation. We then looked at this transition when Myb and/or Notch1 are activated. Activated Notch promoted cyst-like organoids. Conversely growth and propagation of cyst-like, but not normal organoids were Notch-independent. Activated Myb promoted normal, but not cyst-like organoids. Interestingly the Wnt, Notch and Myb pathways were all involved in regulating the expression of the intestinal stem cell (ISC) gene Lgr5 in organoids, while ISC gene and Notch target Olfm4 was dominantly repressed by Wnt. These findings parallel mouse intestinal adenoma formation where Notch promoted the initiation, but not growth, of Wnt-driven Olfm4-repressed colon tumors. Also Myb was essential for colon tumor initiation and collateral mouse pathologies. These data reveal the complex interplay and hierarchy of transcriptional networks that operate in ISCs and uncover a shift in pathway-dependencies during tumor initiation

CSF-1 Receptor-Dependent Colon Development, Homeostasis and Inflammatory Stress Response

<div><p>The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) directly regulates the development of Paneth cells (PC) and influences proliferation and cell fate in the small intestine (SI). In the present study, we have examined the role of CSF-1 and the CSF-1R in the large intestine, which lacks PC, in the steady state and in response to acute inflammation induced by dextran sulfate sodium (DSS). As previously shown in mouse, immunohistochemical (IHC) analysis of CSF-1R expression showed that the receptor is baso-laterally expressed on epithelial cells of human colonic crypts, indicating that this expression pattern is shared between species. Colons from <i>Csf1r</i> null and <i>Csf1^op/op</i> mice were isolated and sectioned for IHC identification of enterocytes, enteroendocrine cells, goblet cells and proliferating cells. Both <i>Csf1r^−/−</i> and <i>Csf1^op/op</i> mice were found to have colon defects in enterocytes and enteroendocrine cell fate, with excessive goblet cell staining and reduced cell proliferation. In addition, the gene expression profiles of the cell cycle genes, <i>cyclinD1</i>, <i>c-myc</i>, <i>c-fos</i>, and <i>c-myb</i> were suppressed in <i>Csf1r^−/−</i> colonic crypt, compared with those of WT mice and the expression of the stem cell marker gene <i>Lgr5</i> was markedly reduced. However, analysis of the proliferative responses of immortalized mouse colon epithelial cells (lines; Immorto-5 and YAMC) indicated that CSF-1R is not a major regulator of colonocyte proliferation and that its effects on proliferation are indirect. In an examination of the acute inflammatory response, <i>Csf1r</i>^+/− male mice were protected from the adverse affects of DSS-induced colitis compared with WT mice, while <i>Csf1r</i>^+/− female mice were significantly less protected. These data indicate that CSF-1R signaling plays an important role in colon homeostasis and stem cell gene expression but that the receptor exacerbates the response to inflammatory challenge in male mice.</p> </div

Enhanced lithium-induced brain recovery following cranial irradiation is not impeded by inflammation

Radiation-induced brain injury occurs in many patients receiving cranial radiation therapy, and these deleterious effects are most profound in younger patients. Impaired neurocognitive functions in both humans and rodents are associated with inflammation, demyelination, and neural stem cell dysfunction. Here we evaluated the utility of lithium and a synthetic retinoid receptor agonist in reducing damage in a model of brain-focused irradiation in juvenile mice. We found that lithium stimulated brain progenitor cell proliferation and differentiation following cranial irradiation while also preventing oligodendrocyte loss in the dentate gyrus of juvenile mice. In response to inflammation induced by radiation, which may have encumbered the optimal reparative action of lithium, we used the anti-inflammatory synthetic retinoid Am80 that is in clinical use in the treatment of acute promyelocytic leukemia. Although Am80 reduced the number of cyclooxygenase-2-positive microglial cells following radiation treatment, it did not enhance lithium-induced neurogenesis recovery, and this alone was not significantly different from the effect of lithium on this proinflammatory response. Similarly, lithium was superior to Am80 in supporting the restoration of new doublecortin-positive neurons following irradiation. These data suggest that lithium is superior in its restorative effects to blocking inflammation alone, at least in the case of Am80. Because lithium has been in routine clinical practice for 60 years, these preclinical studies indicate that this drug might be beneficial in reducing post-therapy late effects in patients receiving cranial radiotherapy and that blocking inflammation in this context may not be as advantageous as previously suggested

Two-week old <i>Csf1r^−/−</i> and <i>Csf1^op/op</i> mutant mice have shorter colons, fewer cells per crypt and fewer enteroendocrine cells.

<p>(A) Colon lengths for each genotype are represented (n = 3). (<b>B,C</b>) Colon sections from proximal (<b>B</b>) and distal (<b>C</b>) regions were stained with PAS to highlight neutral mucin production. The crypt length bars defined in WT colons (<b>B</b>) were duplicated and superimposed on the images in (<b>C</b>) to illustrate the shortened crypts of the mutant mice. Bars = 50 µm. Lower panels show the number of cells per crypt cross section. (<b>D</b>) Average numbers of enteroendocrine cells as visualized by Chromogranin A immunohistochemistry are plotted. Means are depicted by horizontal bars, n = 3 per group, (**P<0.01 using unpaired two-tailed t-tests).</p

Decreased proliferation in <i>Csf1r^−/−</i> and <i>Csf1^op/op</i> mouse colonic epithelium.

<p>(<b>A</b>) Representative PCNA immunostaining in proximal and distal colon sections are shown. (<b>B</b>) Enumeration of the total number of positively stained nuclei reveals that average number of proliferating cells/crypt is significantly reduced in proximal (left panel) and distal (right panel) colons of <i>Csf1^op/op</i> and <i>Csf1r^−/−,</i> compared to WT, mice. Bar = 50 µm (n = 3). (<b>C</b>) Staining with the G₂/M phase marker, phosphorylated histone H3 (PH-3, black arrows) also shows decreased numbers of cells in cycle per crypt in both mutants. Bar = 50 µm. Means are depicted by horizontal bars, n = 3 per group, *<b>*</b>P<0.01, *P<0.05, using unpaired two-tailed t-test. (<b>D</b>) Quantitation the PH-3⁺ staining where means are depicted by horizontal bars, (n = 3 per group). Having predicted a reduced level of PH3 in <i>Csf1r^−/−</i> crypts data was analyzed using one-tailed t-tests, (*<b>*</b>P<0.01, *P<0.05).</p