
Intestinal epithelium is a rapidly self-renewing tissue, which consists of a crypt/villus unit. The renewal process is driven by leucine-rich G-protein-coupled receptor 5 (LGR5)+ intestinal stem cells (ISCs) at the crypt bottom (1). ISC has another population of the cells for tissue maintenance, named reserve ISC (rISC) located around +4 position from the bottom of the crypt (2). The rISCs are slowly cycling, injury-resistant and are capable of giving rise to multiple cell types upon injury or LGR5+ ISC loss (2, 3). B-cell-specific Moloney murine leukemia virus integrin site 1 (BMI1) is known as one of the rISC marker (4). Previous studies demonstrated that BMI1+ ISCs contribute to the regenerative response after injury to the small intestinal epithelium while LGR5+ ISCs are ablated (5, 6).
During homeostasis, ISCs produce the progenitors in the transit-amplifying cell zone that are committed to becoming differentiated cells (1). Differentiated cells in the small intestine are classified into two groups, absorptive and secretory cells (7). The absorptive progenitors differentiate into enterocyte. Secretory cells include goblet, Paneth, enteroendocrine and tuft cells. Previous studies suggest that during homeostasis secretory cell lineage originates from BMI1+ ISC, however it remains unclear how secretory cell differentiation from BMI1+ ISC is regulated (6, 8).
The zinc finger transcription factor, Krüppel-like factor 4 (KLF4) is normally expressed in the differentiated epithelial cells of the intestine, and contributes to epithelial homeostasis (9). Previously, we reported that KLF4 modulates the fate of BMI1+ ISCs and contributes to crypt regeneration from the BMI1+ cell-derived lineage by promoting its clonal expansion after radiation-induced injury (10). Furthermore, several studies suggested that goblet cell differentiation is regulated by KLF4 in the small intestine (11-13). However, it has not been elucidated how KLF4 regulates intestinal BMI1+ ISC during homeostasis.
These findings provide definitive evidence that KLF4 is essential in regulating goblet cell differentiation in the BMI1-expressing intestinal stem cells both
Proximal parts of small intestines were swiss-rolled as described previously (10), and paraffin embedded blocks were cut into 5
Proximal small intestine was harvested from mice at 48 h after tamoxifen injection. Intestinal epithelial cells were dissociated as previously described (14). EYFP+ cells were sorted by flow cytometry (BD FACSARIA III; BD Biosciences, San Jose, CA) from
Cultured organoids were washed with PBS. Matrigel was dissolved with Cell Recovery Solution (Corning) and collected organoids were resuspended in iPGell (GenoStaff, Tokyo, Japan). The iPGell-embedded organoids were fixed in 4% paraformaldehyde and embedded in paraffin.
Statistical analysis was carried out using GraphPad Prism version 8.3.0 for Windows (GraphPad). Student t test, Dunn’s multiple comparisons test with Kruskal-Wallis test, Spearman correlation were used. Values of p<0.05 were considered significant. All data are shown as Mean± SEM.
As previously reported, KLF4 is expressed mainly in the terminally differentiated epithelial cells lining the intestinal villi with the highest levels of expression within the proximal intestinal epithelium and also in a small population of the cells within the intestinal crypts of
Next, we investigated the effect of
To confirm relationship between KLF4 expression and goblet cell differentiation, we counted BMI1-eYFP positive cells from proximal small intestinal crypts of
To determine the effect of
In this study, we have shown that goblet cell differentiation is regulated by KLF4 in BMI1+ ISC lineage during homeostasis. Initially, BMI1+ ISC was identified as quiescent and radiation-resistant cell located around +4 position in the proximal small intestinal crypt (4, 6). Afterward, several studies observed controversial results that
Goblet cells are the most abundant intestinal secretory lineage, comprising ∼15% of the small intestinal epithelial cells (2, 19). Although turnover time of MUC2+ goblet cells is slower in the crypt compared to goblet cells along the villi, newly produced MUC2 expressing goblet cell will disappear within 24 h (2, 19). In this study, MUC2 positive cells were detected in BMI1+ cell lineage at 7 days after tamoxifen injection. Thus, BMI1+ cells act as not only rISC but also active intestinal stem cells (aISC) with self-renewal and multipotency during homeostasis. Previous studies indicated that KLF4 had a critical role in the development of goblet cells and thefunction was regulated by the Notch pathway (11, 12, 20, 21). Another study suggested that KLF4 cooperated with Hippo signaling effectors, YAP and TAZ in promoting differentiation into goblet cells (13). In these studies, the effect of
In conclusion, we have shown that KLF4 regulates goblet cell differentiation in BMI1+ intestinal stem cell lineage during homeostasis.
Authors thank Dr. Jie Yang and Lizhou Nie for support with statistical analysis, and Rebecca C. Connor and Todd P. Rueb (Research Flow Cytometry Core, Department of Pathology, Stony Brook University) for assistance with flow cytometry. This study was supported by National Institutes of Health grants awarded to Vincent W. Yang (CA084197 and DK052230).
The authors have no conflicting financial interest.
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