Distinct SOX9 levels differentially mark stem/progenitor populations and enteroendocrine cells of the small intestine epithelium

Abstract

SOX transcription factors have the capacity to modulate stem/progenitor cell proliferation and differentiation in a dose-dependent manner. SOX9 is expressed in the small intestine epithelial stem cell zone. Therefore, we hypothesized that differential levels of SOX9 may exist, influencing proliferation and/or differentiation of the small intestine epithelium. Sox9 expression levels in the small intestine were investigated using a Sox9 enhanced green fluorescent protein (Sox9(EGFP)) transgenic mouse. Sox9(EGFP) levels correlate with endogenous SOX9 levels, which are expressed at two steady-state levels, termed Sox9(EGFPLO) and Sox9(EGFPHI). Crypt-based columnar cells are Sox9(EGFPLO) and demonstrate enriched expression of the stem cell marker, Lgr5. Sox9(EGFPHI) cells express chromogranin A and substance P but do not express Ki67 and neurogenin3, indicating that Sox9(EGFPHI) cells are postmitotic enteroendocrine cells. Overexpression of SOX9 in a crypt cell line stopped proliferation and induced morphological changes. These data support a bimodal role for SOX9 in the intestinal epithelium, where low SOX9 expression supports proliferative capacity, and high SOX9 expression suppresses proliferation.

Fig. 1.

Expression analysis of Sox9^EGFP in the small intestine. A: Sox9^EGFP expression in jejunal crypts is localized to the base in intercalating crypt-based columnar cells (CBCs) and supra-Paneth cell locations. There are two distinct levels termed “HI” and “LO”. EGFP, enhanced green fluorescent protein. B: sporadic Sox9^EGFPHI-expressing cells are found throughout the villus epithelium. White arrows mark representative Sox9^EGFPHI cells. Image at far right is a high magnification to demonstrate morphology. C: Sox9^EGFP expression does not colocalize with granulated Paneth cells. D: molecular confirmation that Sox9^EGFP is not expressed in Paneth cells as marked by lysozyme.

Fig. 2.

Validation of the Sox9^EGFP transgene and whole crypt analysis of SOX9 expression in Paneth cells. A: SOX9 protein levels directly correlate with Sox9^EGFP transgene expression levels. The nucleus of the Sox9^EGFPHI cell located higher up the crypt is not in the focal plane of the confocal image, and thus endogenous SOX9 is not observable. B: numbering scheme used to quantify the location of Sox9^EGFPHI cells. Cell position 1 is located at the very base of the crypt. C: distribution of Sox9^EGFPHI cells in the small intestine crypt. Variation is depicted as the standard error of the mean of 3 individuals counting one cell position difference either up or down the average cell position along crypt-villus axis. D: whole crypts were immunostained for lysozyme and SOX9 and digitally reconstructed from 1-μm confocal slices. By assessing every Paneth cell in an intact jejunal crypt, we can define two populations of Paneth cells by either the presence or absence of SOX9. The ratio of SOX9⁺ to SOX9⁻ Paneth cells was on average 1:1 (n = 5 intact crypts, SOX9⁺ mean = 2.6/crypt, SE = 1.5; SOX9⁻ mean = 2.6/crypt, SE = 1.1). The images represent 2 different representative crypts (designated A and B). Each image represents a different optical slice on the z-plane. Blue = nuclei, red = SOX9, green = lysozyme. Red arrows point to SOX9-positive Paneth cells. White arrows point to SOX9-negative Paneth cells.

Fig. 3.

Gene expression analysis in isolated populations of Sox9^EGFP-expressing cells. A: FACS histogram demonstrates the 3 distinct populations of Sox9^EGFP-expressing cells. Sox9^EGFP cells were sorted on the basis of EGFP intensities [either negative (NEG), HI, or LO]. B: FACS on the basis of EGFP levels (and thus Sox9 mRNA levels) was validated because Sox9^EGFPHI cells express 5-fold higher (SE = 1.2-fold) Sox9 mRNA than Sox9^EGFPLO cells. Sox9^EGFPLO cells have 10.2-fold more Notch1 (SE = 0.65-fold) and 11.4-fold more Hes1 (SE = 1.3-fold) mRNA compared with Sox9^EGFPHI cells. Images to the right of the graph represent the cells postsort. C: Sox9^EGFPLO cells are enriched in stem/progenitor cell marker genes. Sox9 (mean = 6.7-fold higher, SE = 2.3-fold), Lgr5 (mean = 8.9-fold, SE = 3.8-fold), Msi1 (mean = 13.3, SE = 3.6-fold); all P values <0.001.

Fig. 4.

Sox9^EGFPHI cells are postmitotic enteroendocrine cells. Sox9^EGFPHI cell lineage was identified by colocalization of EGFP with enteroendocrine-specific markers, substance P (A) and chromogranin A (B). Postmitotic status of Sox9^EGFPHI cells was assessed by colocalization of EGFP with Ki67 (C) and Neurogenin3 (Ngn3) (D). White arrows depict representative cells. All images are 1-μm confocal optical slices. Note: A is EGFP fluorescence, and is immunofluorescence staining for EGFP (B–C). Immunodetection of EGFP is required because of destruction of EGFP fluorescence by the antigen retrieval methods. SUB P, substance P; CHG A, chromogranin A.

Fig. 5.

Phenotypic analysis of lentiviral-mediated increases in SOX9 levels. A: schematic of lentiviral construct generated. The control lentiviral vector contains all the elements as the SOX9 lentiviral vector except the Sox9 cDNA. EGFP is translated from an internal ribosomal entry site (IRES) as a reporter gene to assess infection. The puromycin resistance (PURO) gene is included to allow positive selection for viral integration into the genome. cDNA expression is driven by strong constitutive promoters [either cytomegalovirus (CMV) or phosphoglycerate kinase (PGK)]. LTR, long terminal repeats. B: validation of the SOX9 lentivirus. 48 h postinfection, intestinal epithelial cell (IEC)-18 cells were assessed for SOX9 by immunostaining (red), and EGFP autofluorescence (green). C: IEC-18 cells were infected with equivalent titers of either control lentivirus or SOX9 lentivirus and selected with puromycin to deplete the cultures of untransduced cells. Equivalent numbers of cells were plated and allowed to grow for an additional 5 days. Images (left and middle left) depict monolayers of IEC-18 cells that emerged in the control infected cells that express low endogenous levels of SOX9 (red). Other images (right and middle right) depict cells infected with the SOX9 lentivirus. No proliferation (as detected by clonal populations) was observed in SOX9 lentivirus-infected cells. Immunostaining for SOX9 (red) validates high expression of SOX9 in these cells. D: immunoblotting analysis of whole cell extracts made from control-virus (left lane) or SOX9-virus infected (right lane) IEC-18 cells. Blots were probed for c-MYC and proliferating cell nucleus antigen (PCNA) using β-actin expression as an internal control. E: morphological phenotypes of SOX9 lentivirus-infected IEC-18 cells. Note neuroendocrine-like morphologies (axonal/dendritic processes and dense vesicle formations) were evident 6–8 days postinfection.

Fig. 6.

Model describing the relationship between Wnt and Sox9 in the adult intestinal epithelium. Multipotent CBCs universally express low levels of Sox9 in a Wnt-dependent manner. Post-Ngn3 lineage specification, instructive intrinsic and/or extrinsic signaling increases Sox9 expression to high levels in a Wnt-independent manner decreasing proliferative capacity and promoting terminal differentiation/maturation of enteroendocrine cells.