In this study, we defined the EEC lineage trajectory as the following: (1) Lgr5+ aISCs, (2) Sox9-Low cells and Hopx+ cells that exhibit features of the EEC lineage, (3) Prox1+ EEC progenitors, (4) Sox9-High and lower side population (LSP) cells that represent a mixed population of rISCs and mature EECs, and (5) Pyy+ cells that represent mature EECs. To define the miRNA landscape across the EEC lineage trajectory, we first investigated Sox9-EGFP reporter mice (Figure 1Figure 1A). From the jejunal crypts of the Sox9-EGFP mice, we sorted and performed small RNA sequencing (RNA-seq) analysis on 4 different epithelial cell populations enriched in enterocytes (Sox9-Negative), stem cells or EEC progenitors (Sox9-Low) (hereafter referred to as EEC progenitors), transit amplifying cells (Sox9-Sublow), and mature EECs (Sox9-High), and demonstrated that each fraction is enriched for the expected markers (Figure 1Figure 1B). We then focused our analysis on the cell populations in the EEC lineage trajectory, Sox9-Low and Sox9-High. The small RNA-seq analysis identified a total of 187 miRNAs in these 2 populations. Of these, we found that only 8 miRNAs are enriched (>5-fold) in mature EECs (class A), 2 in stem or EEC progenitors (class B), and 14 in both (class C) relative to unsorted intestinal epithelial cells (Table 1Table 1). Class A miRNAs represent candidate regulators of mature EEC function, class B miRNAs represent candidate regulators of EEC progenitor cell behavior, and class C miRNAs represent candidate regulators of both mature EEC function and EEC progenitor cell behavior. Notably, class C miRNAs include miR-7b, which has been previously extensively studied in the context of endocrine pancreatic development and function.8x8Latreille, M., Hausser, J., Stutzer, I., Zhang, Q., Hastoy, B., Gargani, S., Kerr-Conte, J., Pattou, F., Zavolan, M., Esguerra, J.L., Eliasson, L., and Stoffel, M. MicroRNA-7a regulates pancreatic beta cell function. J Clin Invest. 2014;
Crossref | PubMed | Scopus (131) | Google ScholarSee all References,9x9Poy, M.N. MicroRNAs: An adaptive mechanism in the pancreatic beta-cell…and beyond?. Best Pract Res Clin Endocrinol Metab. 2016;
Crossref | PubMed | Scopus (3) | Google ScholarSee all References,10x10Lopez-Beas, J., Capilla-Gonzalez, V., Aguilera, Y., Mellado, N., Lachaud, C.C., Martin, F., Smani, T., Soria, B., and Hmadcha, A. miR-7 modulates hESC differentiation into insulin-producing beta-like cells and contributes to cell maturation. Mol Ther Nucleic Acids. 2018;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (5) | Google ScholarSee all References,11x11Xu, H., Guo, S., Li, W., and Yu, P. The circular RNA Cdr1as, via miR-7 and its targets, regulates insulin transcription and secretion in islet cells. Sci Rep. 2015;
Crossref | PubMed | Scopus (230) | Google ScholarSee all References,12x12Wang, Y., Liu, J., Liu, C., Naji, A., and Stoffers, D.A. MicroRNA-7 regulates the mTOR pathway and proliferation in adult pancreatic beta-cells. Diabetes. 2013;
Crossref | PubMed | Scopus (116) | Google ScholarSee all References,13x13Bravo-Egana, V., Rosero, S., Molano, R.D., Pileggi, A., Ricordi, C., Dominguez-Bendala, J., and Pastori, R.L. Quantitative differential expression analysis reveals miR-7 as major islet microRNA. Biochem Biophys Res Commun. 2008;
Crossref | PubMed | Scopus (103) | Google ScholarSee all References,14x14Kredo-Russo, S., Mandelbaum, A.D., Ness, A., Alon, I., Lennox, K.A., Behlke, M.A., and Hornstein, E. Pancreas-enriched miRNA refines endocrine cell differentiation. Development. 2012;
Crossref | PubMed | Scopus (55) | Google ScholarSee all References,15x15Downing, S., Zhang, F., Chen, Z., and Tzanakakis, E.S. MicroRNA-7 directly targets Reg1 in pancreatic cells. Am J Physiol Cell Physiol. 2019;
Crossref | PubMed | Scopus (2) | Google ScholarSee all References MiR-7 was also shown to be enriched in a specific subtype of mature EECs and cholecystokinin-producing EECs,16x16Knudsen, L.A., Petersen, N., Schwartz, T.W., and Egerod, K.L. The MicroRNA Repertoire in enteroendocrine cells: identification of miR-375 as a potential regulator of the enteroendocrine lineage. Endocrinology. 2015;
Crossref | PubMed | Scopus (15) | Google ScholarSee all References and also in enterochromaffin cell-derived tumors17x17Heverhagen, A.E., Legrand, N., Wagner, V., Fendrich, V., Bartsch, D.K., and Slater, E.P. Overexpression of MicroRNA miR-7-5p is a potential biomarker in neuroendocrine neoplasms of the small intestine. Neuroendocrinology. 2018;
Crossref | PubMed | Scopus (7) | Google ScholarSee all References; however, importantly, the expression pattern of miR-7 (or any other miRNA) across the entire EEC lineage trajectory has never before been reported.
miRNAs enriched in mature EEC (Sox9-High)
miRNAs enriched in progenitor EEC (Sox9-Low)
miRNAs enriched in both mature and progenitor EECs
Next, from the jejunal crypts of Lgr5-EGFP, Prox1-EGFP, and Hopx-CreERT2;Rosa26-tdTomato reporter mice (Figure 1Figure 1A), we sorted Lgr5+, Prox1+, and Hopx+ cells, respectively, and performed small RNA-seq to define miRNA profiles in each population (Figure 1Figure 1C). We found that the level of expression of miR-7a and miR-7b increases steadily along the EEC trajectory from Lgr5+ aISCs to Sox9-Low EEC progenitors to Sox9-High mature EECs, in contrast to other miRNAs such as miR-194 and miR-215, which are depleted in the EEC lineage and enriched in the non-EEC, absorptive lineage (Sox9-Sublow and Sox9-Negative) (Figure 1Figure 1D). We also found by quantitative polymerase chain reaction (qPCR) that miR-7 is significantly enriched in Hopx+ cells (Figure 1Figure 1E), which have been shown previously to exhibit molecular features of EEC progenitors.18x18von Furstenberg, R.J., Buczacki, S.J., Smith, B.J., Seiler, K.M., Winton, D.J., and Henning, S.J. Side population sorting separates subfractions of cycling and non-cycling intestinal stem cells. Stem Cell Res. 2014;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (15) | Google ScholarSee all References Moreover, through small RNA-seq analysis, we found that miR-7b is one of the top 3 miRNAs to be significantly enriched (>5-fold enrichment based on expression as measured by reads per million mapped to miRNAs and P < .05 by 2-tailed Student t test) in Hopx+ cells relative to Hopx– cells, further underscoring the potential importance of miR-7b in EEC progenitors.
To validate that the miR-7 family is enriched in EEC progenitors, we next performed side population sorting of the intestinal epithelium and isolated the LSP and upper side population (USP) of cells, which correspond to rISCs and aISCs, respectively (Figure 1Figure 1A). Consistent with the notion of overlapping identity between rISCs and cell populations in the EEC lineage,18x18von Furstenberg, R.J., Buczacki, S.J., Smith, B.J., Seiler, K.M., Winton, D.J., and Henning, S.J. Side population sorting separates subfractions of cycling and non-cycling intestinal stem cells. Stem Cell Res. 2014;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (15) | Google ScholarSee all References we found that LSP cells exhibit molecular features of mature EECs and EEC progenitors, including expression of Chga (Figure 1Figure 1F), and are depleted for markers of aISCs, including Lgr5 (Figure 1Figure 1F). RT-qPCR analysis showed that miR-7 is significantly enriched in LSP relative to both USP and Lgr5+ cells (Figure 1Figure 1F), confirming miR-7 enrichment in cells with EEC progenitor features.
To cement the finding of miR-7 enrichment in EEC progenitors, we next turned our attention to the Prox1+ cells sorted from the intestinal epithelium of Prox1-EGFP reporter mice (Figure 1Figure 1A, C). Prox1 was recently shown to mark intestinal secretory progenitors with the capacity to either differentiate to mature EECs or exhibit proliferative stem cell–like activity,4x4Yan, K.S., Gevaert, O., Zheng, G.X.Y., Anchang, B., Probert, C.S., Larkin, K.A., Davies, P.S., Cheng, Z.F., Kaddis, J.S., Han, A., Roelf, K., Calderon, R.I., Cynn, E., Hu, X., Mandleywala, K., Wilhelmy, J., Grimes, S.M., Corney, D.C., Boutet, S.C., Terry, J.M., Belgader, P., Ziraldo, S.B., Mikelsen, T.S., Wang, F., von Furstenberg, R.J., Smith, N.R., CHandrakesan, P., May, R., Chrissy, M.A.S., Jain, R., Cartwright, C.A., Niland, J.C., Hong, Y.K., Carrington, J., Breault, D.T., Epstein, J., Houchen, C.W., Lynch, J.P., Martin, M.G., Plevritis, S.K., Curtis, C., Ji, H.P., Li, L., Henning, S.J., Wong, M.H., and Kuo, C.J. Intestinal enteroendocrine lineage cells possess homeostatic and injury-inducible stem cell activity. Cell Stem Cell. 2017;
Abstract | Full Text | Full Text PDF | PubMed | Scopus (99) | Google ScholarSee all References and our small RNA-seq analysis showed that the miRNA profile of intestinal epithelial Prox1+ cells most closely resembles that of Hopx+ cells (rISCs/EEC progenitors) and Sox9-Low cells (EEC progenitors) (Figure 1Figure 1C). We first demonstrated by qPCR that the traditional EEC lineage marker, Chga, and miR-7 are significantly enriched in Prox1+ cells (Figure 1Figure 1G). Then, we analyzed the small RNA-seq data and found that only 10 miRNAs are >5-fold enriched in Prox1+ cells relative to Prox1– cells (Figure 1Figure 1H). Several of these, including miR-7b, overlap with the class C miRNAs defined in Table 1Table 1. Notably, among these 10 miRNAs, we found that miR-7b is by far the most dramatically enriched (∼41-fold) in Prox1+ EEC progenitors relative to Lgr5+ aISCs (Figure 1Figure 1I).
Prox1+ progenitor cells are thought to give rise not only to mature EECs but also to differentiated tuft cells. To determine whether miR-7 is truly enriched along the EEC lineage trajectory, or also highly abundant in tuft cells, we next measured miR-7 in mouse jejunal tuft cells (Epcam+/Siglecf+/Cd45– cells sorted from wild-type C57BL/6J mice), which are highly enriched as expected for the tuft cell marker Dclk1 (Figure 1Figure 1J). This analysis revealed that miR-7 is >350-fold enriched in Sox-9 High EECs relative to Dclk1+ tuft cells (Figure 1Figure 1J). As a control, we also included Lyz1+ Paneth cells sorted from the Defa6-Cre;tdTomato line (Figure 1Figure 1J), and demonstrated that miR-7 is indeed significantly depleted in these cells relative to Sox9-High EECs (Figure 1Figure 1J). As additional validation, we sorted Pyy+ EECs from the jejunum of Pyy-EGFP reporter mice and found that miR-7 is >600-fold more highly expressed in Pyy+ cells than in tuft cells (data not shown). These findings provide strong support for the enrichment of miR-7 along the entire EEC lineage trajectory.
Taken together, these data define a clear EEC lineage trajectory (from Lgr5+ aISCs to mature EECs) miRNA signature for the first time and reveal that miR-7 is the most enriched miRNA in EEC progenitors compared with Lgr5+ aISCs.