The Sox transcriptional factors: Functions during intestinal development in vertebrates

Abstract

The intestine has long been studied as a model for adult stem cells due to the life-long self-renewal of the intestinal epithelium through the proliferation of the adult intestinal stem cells. Recent evidence suggests that the formation of adult intestinal stem cells in mammals takes place during the thyroid hormone-dependent neonatal period, also known as postembryonic development, which resembles intestinal remodeling during frog metamorphosis. Studies on the metamorphosis in Xenopus laevis have revealed that many members of the Sox family, a large family of DNA binding transcription factors, are upregulated in the intestinal epithelium during the formation and/or proliferation of the intestinal stem cells. Similarly, a number of Sox genes have been implicated in intestinal development and pathogenesis in mammals. Futures studies are needed to determine the expression and potential involvement of this important gene family in the development of the adult intestinal stem cells. These include the analyses of the expression and regulation of these and other Sox genes during postembryonic development in mammals as well as functional investigations in both mammals and amphibians by using the recently developed gene knockout technologies.

Keywords: Intestine; Metamorphosis; Postembryonic development; Sox genes; Stem cells; Thyroid hormone; Thyroid hormone receptor; Xenopus.

Fig. 1A

Mouse intestinal maturation (upper panel) resembles Xenopus metamorphic intestinal remodeling (lower panel). In both species, the adult stem cells are formed from the preexisting epithelial cells when the plasma thyroid hormone (T3) levels become high. After birth, cells in the intervillus region of the mouse intestine develop into adult stem cells expressing protein arginine methyltransferase 1 (PRMT1) and hedgehog (hh) (green cells with irregular-shaped dark nuclei) and invaginate into the underlying connective tissue to form the crypts. During Xenopus metamorphosis, some larval epithelial cells undergo dedifferentiation to become the adult stem cells that express high levels of PRMT1 and sonic hedgehog (Shh) (green cells with irregular-shaped dark nuclei). Subsequently, the descendants of these adult stem cells in both mouse and Xenopus replace the suckling-type or larval-type epithelial cells via active proliferation and differentiation to generate the adult epithelium possessing a self-renewal system (green cells). Modified after [14].

Fig. 1B. Intestinal remodeling during Xenopus laevis metamorphosis

In premetamorphic tadpoles at stage 51, the intestine has a simple structure with only a single fold, the typhlosole. At the metamorphic climax around stage 61, the larval epithelial cells begin to undergo apoptosis, as indicated by the open circles. Concurrently, the proliferating adult stem cells are developed de novo from larval epithelial cells through dedifferentiation, as indicated by black dots. By the end of metamorphosis at stage 66, the newly differentiated adult epithelial cells form a multiply folded epithelium.

Fig. 2. MGPY stains strongly the proliferating adult intestinal stem cells

Premetamorphic stage 54 tadpoles treated with 10 nM T3 for 0, or 6 days were sacrificed one hour after injecting EdU. Cross-sections of the intestine from the resulting tadpoles were double-stained for EdU (5-ethynyl-2′-deoxyuridine) and with MGPY (methyl green pyronin Y, a mixture of methyl green, which stains DNA, and pyronin Y, which stains RNA [42, 145, 146]) (A) or for Edu and Lgr5 (in situ hybridization) (B). The approximate epithelium-mesenchyme boundary was drawn based on morphological differences between epithelial cells and mesenchyme cells in the pictures of the double-stained tissues (dotted lines). Note that the clusters (islets) of EdU labeled cells in the epithelium after 6 days of T3 treatment were strongly stained by MGPY and had high levels of Lgr5 mRNA, a well-established marker for adult intestinal stem cells in vertebrates. See [40] for more detail.

Fig. 3. A putative TRE in Xenopus Sox3 gene can mediate transcriptional activation by T3-bound TR/RXR in frog oocyte

(A) Putative TREs and promoter constructs. Sox3 gene sequence was obtained from Xenopus laevis genome sequences at http://xenopus.lab.nig.ac.jp/blast.php and searched for TREs by using NHR Scan at http://nhrscan.genereg.net/cgi-bin/nhr_scan.cgi?rm=advanced. Three putative TREs were found and are listed above the schematic diagram of the Sox3 gene with their positions relative to the first nucleotide of the start codon (designated as “+1”). Three promoter constructs, the full length Sox3 promoter including the putative TRE2 and TRE3 (Psox3), a truncated version of Sox3 promoter (Psox3Δ), and the truncated version of Sox3 promoter with the TRE2 (Psox3Δ+TRE) inserted immediately upstream of it, were generated to drive the firefly luciferase expression in pGL4 vector (Promega). TRE: thyroid hormone response element; F-luc: firefly luciferase gene. (B) TRE2 binds to TR/RXR in vitro. Double strand DNA oligos containing the putative TREs shown in (A) were used in competitive electrophoretic mobility shift assay (EMSA) against the infrared (IR) dye IR700 (LI-COR, Lincoln, NE)-labeled, well-characterized TRE of Xenopus laevis TRβ gene in the presence of in vitro translated TR/RXR proteins. Unlabeled TRE of the Xenopus laevis TRβ gene (TRE) and a mutant version of TRE of Xenopus laevis TRβ gene (mTRE) known to lack binding to TR/RXR were used as the positive and negative control, respectively [147, 148]. All unlabeled oligos were used in 100 times excess over the labeled IR700-TRE. Note that only TRE and TRE2 competed effectively, suggesting that TRE2, but not TRE1 or TRE3, binds to TR/RXR specifically. (C) Sox3 promoter can be activated by liganded TR/RXR in frog oocyte. Transcription assay was done in Xenopus laevis oocytes where the cytoplasm of stage VI oocytes were injected with 460 pg per oocyte of TR and RXR mRNA mixture or GFP mRNA. 2 hours later, the firefly luciferase reporter constructs shown in (A) (34.5 pg per oocyte) and the phRG-TK (Promega) expressing Renilla luciferase as an internal control (34.5 pg per oocyte) were coinjected into the nuclei of the oocytes. After incubation at 18°C overnight in the presence or absence of 100 nM T3, 5 oocytes were collected per sample and lysed in 75 μl of 1×Passive Lysis Buffer (Promega) for dual luciferase assay by following the manufacturer’s protocol. The relative expression of firefly luciferase to Renilla luciferase (F/R) was determined with the F/R value for oocytes injected with GFP mRNA instead of TR/RXR in the absence of T3 set as 1. Note that the full-length promoter was activated by TR/RXR in the presence of T3. This activation was drastically reduced when the TRE sequences were deleted and was restored when TRE2 was inserted into the truncated promoter, suggesting that TRE2 is capable of mediating T3 induction. Each data point shows the average of 5 samples with the standard error. Statistical analysis was done through ANOVA with Tukey’s Multiple Component Test. *: p<0.05. (L. Fu and Y.-B. Shi, unpublished observations).

Fig. 4. Tissue-specific developmental regulation of Sox3 in the intestine during Xenopus laevis metamorphosis

(A) Sox3 is highly expressed only during metamorphosis. Total intestinal RNA at different stages was analyzed by qRT-PCR. Note that little Sox3 expression was found either before (stage 54–56) or at the end of metamorphosis (stage 66). (B) Sox3 is expressed only in the intestinal epithelium. Total RNA was isolated from intestinal epithelium (Ep) and the rest of the intestine (non-Ep) at different stages of development, 56 (premetamorphosis), 61 (climax), and 66 (end of metamorphosis), and analyzed by qRT-PCR. Note that Sox3 was highly expressed only in the Ep at the climax of metamorphosis when stem cells were forming or proliferating. See [105] for details.