The novel enterochromaffin marker Lmx1a regulates serotonin biosynthesis in enteroendocrine cell lineages downstream of Nkx2.2

Abstract

Intestinal hormone-producing cells represent the largest endocrine system in the body, but remarkably little is known about enteroendocrine cell type specification in the embryo and adult. We analyzed stage- and cell type-specific deletions of Nkx2.2 and its functional domains in order to characterize its role in the development and maintenance of enteroendocrine cell lineages in the mouse duodenum and colon. Although Nkx2.2 regulates enteroendocrine cell specification in the duodenum at all stages examined, it controls the differentiation of progressively fewer enteroendocrine cell populations when deleted from Ngn3(+) progenitor cells or in the adult duodenum. During embryonic development Nkx2.2 regulates all enteroendocrine cell types, except gastrin and preproglucagon. In developing Ngn3(+) enteroendocrine progenitor cells, Nkx2.2 is not required for the specification of neuropeptide Y and vasoactive intestinal polypeptide, indicating that a subset of these cell populations derive from an Nkx2.2-independent lineage. In adult duodenum, Nkx2.2 becomes dispensable for cholecystokinin and secretin production. In all stages and Nkx2.2 mutant conditions, serotonin-producing enterochromaffin cells were the most severely reduced enteroendocrine lineage in the duodenum and colon. We determined that the transcription factor Lmx1a is expressed in enterochromaffin cells and functions downstream of Nkx2.2. Lmx1a-deficient mice have reduced expression of Tph1, the rate-limiting enzyme for serotonin biosynthesis. These data clarify the function of Nkx2.2 in the specification and homeostatic maintenance of enteroendocrine populations, and identify Lmx1a as a novel enterochromaffin cell marker that is also essential for the production of the serotonin biosynthetic enzyme Tph1.

Keywords: Enterochromaffin; Enteroendocrine cells; Intestine; Lmx1a; Nkx2.2; Serotonin.

Fig. 1.

Expression analysis of the duodenum and colon of Nkx2.2^Δint mice. (A) RNA-Seq analysis of the duodenum and colon of 6-week-old Nkx2.2^Δint mice revealed 211 significantly upregulated and 184 significantly downregulated genes in the duodenum and 36 significantly upregulated and 53 significantly downregulated genes in the colon (P<0.05). Among the significantly downregulated genes in the duodenum are most of the enteroendocrine cell hormones, except Ghrl, which is significantly upregulated (see Table 1). In the colon, few enteroendocrine hormones are changed. (B-I) Immunofluorescence of the duodenum (B,D,F,H) and qPCR analysis of the duodenum and colon (C,E,G,I; n=5) of 6-week-old Nkx2.2^Δint mice, showing significant reduction in expression of the enteroendocrine marker Chga (B,C) and the rate-limiting enzyme for 5-HT biosynthesis Tph1 (D,E). The expression of the hormone Ghrl is significantly higher in the duodenum, as well as in the colon, than in controls (F,G). Expression of the hormone Sst is significantly reduced in the duodenum of Nkx2.2^Δint mice, but is unchanged in the colon (H,I). Arrows indicate hormone-positive cells. **P<0.01, ***P<0.001.

Fig. 2.

qPCR expression analysis of the duodenum or colon of Nkx2.2^Δint, Nkx2.2^Δprogenitor and Nkx2.2^Δadult mice. (A) Analysis of the duodenum and colon of 6-week-old Nkx2.2^Δint mice (n=5) shows no changes in Atoh1, Foxa2, Hes1, Klf4, Klf5 and Ngn3. (B) The P0 Nkx2.2^Δprogenitor duodenum (n=5) revealed significantly decreased expression of most hormones analyzed, except for higher expression of Ghrl and no change in expression for ppGcg, Npy and Vip. (C) Nkx2.2^Δadult mice were tamoxifen injected at 7 weeks of age for 5 consecutive days (days 1-5) and the duodenum harvested 14 days after the last injection (day 19; n=4). The duodenum of 7-week-old control mice was harvested at day 1 (n=3). qPCR analysis showed a significant reduction in expression of Chga, ppGcg, Gip, Nts, Pyy, Sst, Tac1 and Tph1, but an increase in Ghrl, in the duodenum of the 7- to 10-week-old Nkx2.2^Δadult mice. Expression of the hormones Cck, Gast, Npy, Sct and Vip was unchanged. *P<0.05, **P<0.01, ***P<0.001.

Fig. 3.

Comparison of gene expression changes in the duodenum of Nkx2.2^Δint, SD^int and TN^int mice. (A) Venn diagram summarizing gene expression changes in the duodenum of Nkx2.2^Δint, SD^int and TN^int mice identified by RNA-Seq. (B) List of the eight genes with significantly changed expression in the duodenum of Nkx2.2^Δint, SD^int and TN^int mice; the 11 genes significantly changed in Nkx2.2^Δint and SD^int mice; the 26 genes that are changed in both Nkx2.2^Δint and TN^int mice; and the nine genes significantly differentially expressed in SD^int and TN^int.

Fig. 4.

Lmx1a is expressed in 5-HT-expressing cells. (A) qPCR analysis of the duodenum of Nkx2.2^Δprogenitor (n=5) and Nkx2.2^Δadult (control, n=3; mutant, n=4) mice reveals a significant reduction in Lmx1a in Nkx2.2^Δprogenitor mice and absence of Lmx1a expression in Nkx2.2^Δadult mice compared with controls. (B-E) Immunofluorescence analysis of the duodenum of 6-week-old Lmx1a^eGFP/+ mice shows that Lmx1a is expressed in Chga⁺ enteroendocrine cells (B,D) and in 5-HT⁺ cells (C,E) in the small (B,C) and large (D,E) intestine. Arrows indicate co-expressing cells that are shown at higher magnification in the insets. (F) qPCR analysis of the small intestine of P0 Lmx1a^eGFP/EGFP mice shows that Chga and Tph1 expression is significantly reduced, whereas Gip and Sst are upregulated (n=4). (G) Luciferase reporter assays in MIN6 cells. The pGL4.27:Lmx1a enhancer element (Lmx1a-Enh) and pcDNA3:myc-Nkx2.2 expression plasmid were co-transfected into MIN6 cells. Luciferase values were normalized to Renilla activity to account for transfection efficiencies (n=3). *P<0.05, **P<0.01, ***P<0.001.

Fig. 5.

Regulation of 5-HT biosynthesis in the CNS and intestine by Nkx2.2. In the CNS, Lmx1b is downstream of Nkx2.2 and is required for 5-HT biosynthesis by regulating Tph2 expression. Ngn3 represses the serotonergic fate. In the intestine, Nkx2.2 is downstream of the enteroendocrine progenitor marker Ngn3. We identified Lmx1a, a paralog of Lmx1b, downstream of Nkx2.2 as a regulator of Tph1 expression and thereby controls 5-HT biosynthesis in enterochromaffin cells.