A requirement for hedgehog signaling in thyroid hormone-induced postembryonic intestinal remodeling

Abstract

Background: Intestinal remodeling during amphibian metamorphosis has long been studied as a model for the formation of the adult organs in vertebrates, especially the formation of adult organ-specific stem cells. Like all other processes during metamorphosis, this process is controlled by thyroid hormone (T3), which affects cell fate and behavior through transcriptional regulation of target genes by binding to T3 receptors (TRs). Earlier studies have shown that Sonic hedgehog (Shh) is induced by T3 in the developing adult stem cells and that the Shh receptor and other downstream components are present in the connective tissue and at lower levels in the muscles at the climax of intestinal remodeling. However, no in vivo studies have carried out to investigate whether Shh produced in the adult cells can regulate the connective tissue to promote intestinal maturation.

Results: We have addressed this issue by treating tadpoles with Shh inhibitor cyclopamine. We showed that cyclopamine but not the structurally related chemical tomatidine inhibited the expression of Shh response genes BMP4, Snai2, and Twist1. More importantly, we showed that cyclopamine reduced the cell proliferation of both the developing adult stem cells as well as cells in the other intestinal tissues at the climax of metamorphosis, leading to delayed/incomplete remodeling of the intestine at the end of metamorphosis. We further revealed that both Snai2 and Twist1 were strongly upregulated during metamorphosis in the intestine and their expression was restricted to the connective tissue.

Conclusions: Our results suggest that Shh indeed signals the connective tissue whereby it can increase adult stem cell proliferation and promote formation of the adult intestine.

Keywords: Adult stem cells; Amphibian metamorphosis; Postembryonic development; Thyroid hormone receptor; Xenopus laevis.

Figure 1

Inhibition of hedgehog (Shh) signaling by cyclopamine suppresses intestinal remodeling during Xenopus laevis metamorphosis . A: Cyclopamine-treated animals have longer intestine at the end of metamorphosis. Tadpoles at stage 58 were treated with vehicle (100% ethanol, final concentration in rearing water: 0.1%) (3 tadpoles) or tomatidine (7 tadpoles), or cyclopamine (8 tadpoles) till they reached stage 66. Intestinal length was measured from bile duct junction to colon for all animals. * indicated significant difference between the cyclopamine group and the other two groups (p < 0.05). Note that the cyclopamine-treated tadpoles completed metamorphosis, as judged based on external morphology, e.g., resorption of the tail, similarly as the animals in the tomatidine and vehicle groups. However, they had significant longer intestine. B: Retarded intestinal maturation in the cyclopamine-treated animals. Transverse sections of the intestine from the animals above were stained with methyl green pyronin Y (MGPY), which stained DNA in blue (methyl green) and RNA in red (pyronin Y). Note that the cyclopamine-treated animals had intestines with fewer and less structured epithelial folds as well as reduced connective tissue and muscles (outer layers). C: Immunofluorescence staining showing reduced intestinal muscle development in the cyclopamine-treated animals. The intestinal sections above were stained with anti-smooth muscle actin (SMA) antibody as well as DAPI for cell nuclei.

Figure 2

Inhibition of hedgehog signaling suppresses intestine shortening and remodeling at metamorphic climax. A: Cyclopamine-treated tadpoles have longer intestine at the climax. Tadpoles at stage 58 were treated with vehicle (100% ethanol, final concentration in rearing water: 0.1%) (9 tadpoles) or tomatidine (10 tadpoles), or cyclopamine (11 tadpoles) till they reached climax (stage 62). Intestine was isolated and measured from bile duct junction to colon. The animals in all three groups reached developed to stage 62 similarly as judged based on external morphology. ** indicate significant difference between the cyclopamine group and the two control groups (p < 0.001). B: Cyclopamine-treated tadpoles have fewer islets of adult epithelial stem cells. Transverse sections of the intestine from above were stained with methyl green pyronin Y (MGPY). The boxed area in the left panel is enlarged in the right panel. Note that at the climax of metamorphosis, the clusters (islets) of adult epithelial stem cells are stained strongly by MGPY (arrows) while the dying larval epithelial cells are only weakly stained [15,49,50]. The cyclopamine-treated tadpoles had smaller intestinal cross-section and fewer islets of adult cells. C: Cyclopamine-treated tadpoles have reduced cell proliferation. The intestinal sections above were stained with DAPI for nuclear DNA and anti-PCNA antibody for mitotic cells. The boxed area in the middle panel is enlarged in the right panel. Cyclopamine treatment significantly reduced cell proliferation in the intestine, especially in the epithelium. Note that the labeling in the epithelium was limited to the islet of the adult cells (arrows) but not in the dying larval epithelial cells, as expected.

Figure 3

Cyclopamine inhibits Shh target gene expression. Tadpoles at stage 58 were treated as in Figure 2 until they reached stage 62. Total intestinal RNA was isolated for qRT-PCR analysis of the expression of Shh target genes BMP4 [51] (A), Snai2 [52] (B) and Twist1 [52] (C). ** indicate significant difference between the cyclopamine group and the other groups (p < 0.001).

Figure 4

Snai2 expression is restricted to the connective tissue and peaks at the climax of intestinal metamorphosis. A: Snai2 mRNA level peaks at the climax (Stage 62). Quantitative RT-PCR analysis of Snai2 mRNA level in intestine from tadpoles at indicated stages, normalized against the control gene EF1a. B-G: In situ hybridization reveals connective tissue-specific expression of Snai2 in intestine. Note that high levels of Snai2 mRNA were detected fairly uniformly in the connective tissue at the climax (St61-62). H-I: Snai2 is not expressed in the muscle layers. In situ hybridization sections of Snai2 at the stage 61 were double-stained with smooth muscle actin antibody (red) to label the muscle layers (Ms). Ty: typhlosole, the single epithelial fold in in premetamorphic tadpole intestine where connective tissue is abundant; Lu: lumen; Ep: epithelium. Note that Snai2 labeled cells are localized between epithelium and muscle layers.

Figure 5

Twist1 has similar spatiotemporal expression profiles as Snai2 in the intestine. A: Twist1 mRNA level peaks at the climax (Stage 62). Quantitative RT-PCR analysis of Twist1 mRNA level in intestine from tadpoles at indicated stages, normalized against the control gene EF1a. B-G: In situ hybridization reveals connective tissue-specific expression of Twist1 in intestine. Note that Twist1 mRNA level peaked in the connective tissue at the climax (St61-62). H-I: Twist1 is not expressed in the muscle layers. In situ hybridization sections of Twist1 at the stage 61 were double-stained with smooth muscle actin antibody (red) to labeled the muscle layers (Ms). Ty: typhlosole, the single epithelial fold in in premetamorphic tadpole intestine where connective tissue is abundant; Lu: lumen; Ep: epithelium. Note that Twist1 labeled cells are localized between epithelium and muscle layers.

Figure 6

A schematic diagram showing the effects of Shh signaling during intestinal remodeling. Shh appears to enhance the proliferation of cells in all three tissue layers during metamorphosis. Shh receptors and downstream transcription factors are highly expressed in the connective tissue, which are likely responsible for the activation of the expression of BMP-4, Snai2, and Twist1. The connective tissue may in turn help to mediate the cell proliferation effects of Shh on the adult epithelial stem cells and the circular muscle cells. Cyclopamine directly inhibits Shh signaling in the epithelium and thus blocks all the effects of Shh.