In this study, the second type of granular goblet cells with secretory granules enclosed a larger and irregular dense core appeared in the ileal epithelium, later than the first granular goblet cells, and earlier than Paneth cells. The cells were located in the ileal crypt, deeper than the first granular goblet cells, and shallower than Paneth cells.
Granular goblet cells in the upper part of the crypts, which we call the first type, differ from other goblet cells because of the presence of small dense granules embedded within the mucus globules (Calvert, et al., 1988, Cheng, 1974a). In addition, the presence of intermediate cells between Paneth and goblet cells has been reported in normal mice (Cheng, 1974b, Troughton and Trier, 1969) and humans (Subbuswamy, 1973). Furthermore, the appearance of intermediate cells has recently been reported in various knockout mice (Jackson, et al., 2020, Khaloian, et al., 2020, Watanabe, et al., 2016), ileitis (Jackson, et al., 2020) and inflammatory bowel disease (IBD) (Khaloian, et al., 2020).
Previously, intermediate cells were considered immature Paneth cells or young goblet cells in the small intestine (Troughton and Trier, 1969) or independent of the presence of the Paneth cells (Calvert, et al., 1988). However, the cellular differentiation between Paneth and goblet cells is considered to probably occur via intermediate cells (Mantani, et al., 2014). In our study, the first granular goblet cells appeared in the epithelium of the villi and intervillous region. Second granular goblet cells that were similar to intermediate cells appeared below the first granular goblet cells. Moreover, Paneth cells appeared at the bottom of the crypt after P14. The appearance of these three cell types differs temporally and spatially. In addition, we observed another cell type that had a few coreless granules (data not shown). Recently, a study revealed that the phenotype of intermediate cells is consistent with an immature Paneth cell (Dekaney, et al., 2019). Our findings indicate that two types of granular goblet cells, especially in the second type, are precursors of Paneth cells; however, further research is needed on the expression of specific marker genes and proteins.
In the present study, Paneth cells located at the bottom of the crypt in P14 mice ilea had trimethylated histone H3 lysine 27. Inhibition of EZH2 in organoid culture suppressed stage-specific crypt budding. Post-transcriptional modification of histones plays an essential role in the regulation of chromatin structure and gene transcription. Histone methylation occurs in histone H3 at lysine 4, 9, 14, 27, 36 and 79 residues, and in histone H4 at lysine 20 and 59 residues. Generally, H3 methylation at lysine 4, 36, and 79 is correlated with euchromatin and transcriptional activation, whereas H3 methylation at lysine 9 and 27, and H4 at lysine 20 is associated with heterochromatin and transcriptional repression. However, post-transcriptional modification of histones fluctuates with the developmental stage (Lee, et al., 2005). The reduction or suppression of histone H3K27 trimethylation is also related to intestinal tumorigenesis and cancer (McCleland, et al., 2015).
Enhancer of zeste homolog 2 (EZH2), as part of the polycomb repressive complex 2 (PRC2), selectively catalyzes H3 lysine 27 trimethylation. In the murine jejunum and ileum, EZH2 and suppressor of Zeste-12 (SUZ12) were expressed in non-differentiated proliferative crypt IECs, and the PRC2 complex ensures the proper response of IECs to cell density (Turgeon, et al., 2013). PRC2 regulates intestinal homeostasis, maintaining progenitor cell proliferation and an optimal balance between secretory and absorptive lineage differentiation programs (Chiacchiera, et al., 2016, Koppens, et al., 2016). PRC2 activity is required to maintain cell plasticity at the bottom of the intestinal crypt and the repression of Atoh1 and Gfi1, which are master regulators of goblet cells (Chiacchiera, et al, 2016). However, cell-specific trimethylation at H3K27 has not been reported in the developing small intestine.
In this study, H3K27 trimethylation was detected in Paneth cells at the bottom of the crypts during the second week of postnatal mice. Organoid culture from early in the second postnatal week mouse was suppressed crypt budding and Paneth cell maturation by the EZH2 inhibitor, unlike late in the second week. These results suggest that H3K27 trimethylation in Paneth cells at the bottom of the crypts began early in the second postnatal week and was almost complete during the second week, and it was related to its functional and morphological maturation. In our observation, the transition of the absorptive cell from sucking to weaning was assumed to be completed after Paneth cell maturation. Paneth cell maturation, which is involved not only in the secretion of antimicrobial peptides but also in the construction of the ISC niche, is related to the formation of the crypt and its localization. However, crypts have been reported to form independently of Paneth cells in mice lacking lysine-specific demethylase 1A (Zwiggelaar, et al., 2020). Consistent with our observations, organoids derived from fetal IECs undergo suckling–weaning transition, and organoids with crypts gradually increase, contrary to decreasing spheroids (Navis, et al., 2019). However, the authors of that paper concluded that spheroids transition to organoids and do not reflect the maturation stages. Further research is needed to determine whether all cell types mature or not. The intestinal transcription factor Blimp-1, which is selectively expressed in mouse IECs during embryonic and postnatal development, lost its expression during suckling–weaning transition (Muncan et al., 2011). Although it was assumed that H3 trimethylation at lysine 27 participates in suppression of Blimp-1, the gene expression that is suppressed by H3K27 trimethylation remains unknown. To address this, understanding the mechanism by which intermediate cells that are regarded as premature Paneth cells, appearing aberrantly in intestinal diseases such as IBD, is required.
In conclusion, our data show that post-transcriptional modification of histones, particularly H3 at lysine 27 trimethylation, exerted structural and functional maturation of Paneth cells during postnatal development in mice.