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. 2017 Sep 19;32(1):431–439. doi: 10.1096/fj.201700424R

EVI and MDS/EVI are required for adult intestinal stem cell formation during postembryonic vertebrate development

Morihiro Okada 1, Yun-Bo Shi 1,1
PMCID: PMC6207364  PMID: 28928245

Abstract

The gene ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI encode zinc-finger proteins that have been recognized as important oncogenes in various types of cancer. In contrast to the established role of EVI and MDS/EVI in cancer development, their potential function during vertebrate postembryonic development, especially in organ-specific adult stem cells, is unclear. Amphibian metamorphosis is strikingly similar to postembryonic development around birth in mammals, with both processes taking place when plasma thyroid hormone (T3) levels are high. Using the T3-dependent metamorphosis in Xenopus tropicalis as a model, we show here that high levels of EVI and MDS/EVI are expressed in the intestine at the climax of metamorphosis and are induced by T3. By using the transcription activator–like effector nuclease gene editing technology, we have knocked out both EVI and MDS/EVI and have shown that EVI and MDS/EVI are not essential for embryogenesis and premetamorphosis in X. tropicalis. On the other hand, knocking out EVI and MDS/EVI causes severe retardation in the growth and development of the tadpoles during metamorphosis and leads to tadpole lethality at the climax of metamorphosis. Furthermore, the homozygous-knockout animals have reduced adult intestinal epithelial stem cell proliferation at the end of metamorphosis (for the few that survive through metamorphosis) or during T3-induced metamorphosis. These findings reveal a novel role of EVI and/or MDS/EVI in regulating the formation and/or proliferation of adult intestinal adult stem cells during postembryonic development in vertebrates.—Okada, M., Shi, Y.-B. EVI and MDS/EVI are required for adult intestinal stem cell formation during postembryonic vertebrate development.

Keywords: stem cells, intestine, amphibian metamorphosis, organogenesis, Xenopus tropicalis

Continuous cellular turnover, which is driven by small populations of adult stem cells, is important for proper organ development and organ homeostasis in adult tissues, such as the intestinal epithelium. The epithelium of the small intestine is one of the most architecturally and functionally complex tissues and is an excellent in vivo model system to study stem cells. This is because it has a higher self-renewal rate than other tissues in vertebrates, with a turnover time of <5 d in humans (1–4). This self-renewal system is established late during development (i.e., around birth) in mammals when endogenous thyroid hormone (T3) levels are high (5). Several signaling pathways, such as the Wnt and Notch pathways, are known to regulate the continuous cell renewal and the homeostasis of the intestinal epithelium in adult vertebrates (4, 6, 7). However, much less is known about how the adult stem cells are formed during development, in part due to the difficulties in studying the uterus-enclosed mammalian embryos.

Amphibian metamorphosis is controlled by T3 and resembles postembryonic development in mammals, a period around birth when many organs mature into the adult form (8, 9). Many unique features of amphibian metamorphosis make it an attractive model for studying the mechanisms of T3-dependent adult stem cell development. For example, amphibian embryos develop outside the mother in the absence of maternal interference, and their metamorphosis is dependent on T3, allowing the process to be easily manipulated by controlling the availability of T3 to the tadpoles.

Using Xenopus metamorphosis as a model, we have been studying the genes and signaling pathways that are required for adult intestinal stem cell formation. During Xenopus metamorphosis, the remodeling of the intestine occurs via extensive apoptosis of larval epithelial cells, followed by de novo formation and proliferation and differentiation of adult stem cells to form the adult intestine (10, 11). A genome-wide microarray analysis of intestinal gene expression during metamorphosis (12) identified a number of novel candidate adult stem cell–related genes (13–16), among which are the ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI, generated from the alternative splicing of the transcript of the MDS gene that linked exon 2 of MDS to exon 2 of EVI (16). Both EVI and MDS/EVI have been implicated in a number of epithelial cancers and have been directly linked to the severity of breast, leukemia, ovarian, and intestinal cancers (17–23). Although they are known to be important for hematopoietic stem cell maintenance in adult mammals (22, 24–27), their roles in vertebrate development are largely unexplored. We have shown previously that EVI and MDS/EVI transcripts are strongly up-regulated by T3 in the epithelium but not in the rest of the intestine at the climax of metamorphosis in Xenopus laevis when adult stem cells are forming in the epithelium (16). This temporal expression profile suggests that the expression of EVI and MDS/EVI correlates with adult epithelial stem cell formation. To investigate the possibility that they are involved in adult intestinal stem cell formation, we have investigated here the physiologic roles of endogenous EVI and MDS/EVI during development in Xenopus tropicalis, a diploid species highly related to X. laevis (28, 29). By using transcriptional activator like effector nuclease (TALEN)-mediated in vivo gene mutation technology, we show that knocking out both EVI and MDS/EVI leads to growth retardation and lethality in tadpoles at the climax of metamorphosis, suggesting an important role of EVI and MDS/EVI for the transformation of a tadpole to a frog. We further show that knockout of EVI and MDS/EVI decreases the level of proliferating cells in the intestine at the end of metamorphosis or after T3 treatment of premetamorphic tadpoles. These data thus provide the first evidence for a role of EVI and/or MDS/EVI in adult organ-specific stem cell development.

MATERIALS AND METHODS

Experimental animals

Wild-type X. tropicalis adults were purchased from Nasco (Fort Atkinson, WI, USA) or from Xenopus-1, Inc. (Dexter, MI, USA). The developmental stages were based on Nieuwkoop and Faber (30). Premetamorphic X. tropicalis tadpoles at stage 54 were treated with 5 or 10 nM T3 at 25°C. At least 3 tadpoles were analyzed for each stage or day of T3 treatment. All experiments involving animals were carried out as approved by the National Institute of Child Health and Human Development Animal Use and Care Committee.

Gene expression analysis

Total RNA was isolated from tadpole tissues by using the Small Viral (SV) Total RNA Isolation System kit (Promega, Madison, WI, USA). A reverse transcription reaction was carried out by using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative RT-PCR was done as described in Okada et al. (31). Briefly, 1 µg total RNA was reverse transcribed into cDNA by using the RT2 Easy First Strand Kit (Qiagen, Valencia, CA, USA). The resulting cDNA was diluted 1:10, and the diluted products (2 µl) were subjected to PCR by using a SYBR Green PCR Master Mix (Applied Biosystems) in 20 µl of reaction solution and the StepOnePlus Real-Time PCR System (Applied Biosystems) according to the manufacturer’s protocol. The primers used were forward: 5′-CGCACCATACCTGCCAGC-3′ and reverse: 5′-ATAACCTGCCAACTGTCCCCAG-3′ for X. tropicalis EVI and MDS/EVI, and forward: 5′-AAGAGGGATCTGGCAGCGG-3′ and reverse: 5′-AAGGACACCAGTCTCCACAC-3′ for X. tropicalis elongation factor (EF) 1-α. The level of EVI and MDS/EVI mRNA was normalized against the level of EF1-α mRNA for each sample.

Construction of TALENs

A TALEN targeting X. tropicalis EVI and MDS/EVI was assembled as previously described (32, 33). Briefly, the left and right arms of the TALEN were designed by using TAL Effector-Nucleotide Targeter 2.0 (https://tale-nt.cac.cornell.edu), and the assemblies were performed as previously described (34). The target sequences of the TALEN left arm (-ELD) and right arm (-KKR) were 5′-GTTCATGAAGAGTGAAGATT-3′ and 5′-TGGCTCCAGATATCC-3′, respectively.

TALEN mRNA microinjection

TALEN left and right arm mRNAs were transcribed in vitro by using the mMessage mMachine SP6 Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). TALEN mRNA (400 pg) was injected into X. tropicalis embryos at the 1-cell stage, along with 200 pg of DsRed mRNA (Clontech Laboratories, Palo Alto, CA, USA). The fluorescent product of the latter was used to identify successfully injected embryos and to confirm that the injected mRNA had been translated.

Mutation analysis and generation of EVI- and MDS/EVI-knockout tadpoles

Genomic DNA was extracted from whole embryos at stage 35/36 by using DNeasy Blood and Tissue Kit (Qiagen) to check whether tadpoles had mutations in the TALEN-targeted region in the EVI and MDS/EVI gene. DNA fragments containing the target site were amplified by using the PrimeStar Max DNA Polymerase (Takara, Shiga, Japan) and the primers F1 (5′-TCCGTTTGCAGTGAAGTTTGTTGATATGA-3′) and R1 (5′-ACTCTTCACAGCGATACTGGCGT-3′) for 35 cycles (98°C, 10 s; 60°C, 5 s; 72°C, 5 s). The amplified fragment was inserted into the TOPO vector (Invitrogen, Grand Island, NY, USA), and the nucleotide sequences were subsequently determined. To obtain knockout animals, the F0 TALEN mRNA–injected animals were raised to maturity and crossed with wild-type animals. The resulting F1 generation tadpoles were genotyped by tail clipping to identify tadpoles carrying mutations. Tadpoles carrying frame-shift mutations were raised to adulthood for mating. One heterozygous F1 male and one female tadpole, each with a 20-nt deletion in the targeted region affecting both EVI and MDS/EVI, were mated to obtain F2 offspring with disrupted EVI and MDS/EVI.

5-Ethynyl-2′-deoxyuridine labeling

Staining by 5-ethynyl-2′-deoxyuridine (EdU) was performed as previously described (15, 35). Briefly, 1.25 µl of 10 mg/ml EdU were injected into stage 54 tadpoles with or without T3 treatment. Thirty minutes after injection, the tadpoles were killed, and the anterior part of the intestine was fixed in 4% magnesium sulfate/formaldehyde buffer and processed for paraffin sectioning. Tissue sections (7 μm) were subjected to double staining for EdU (cell proliferation) and Hoechst (nuclear DNA) by using the Click-iT Plus EdU Alexa Fluor 594 Imaging Kit (Thermo Fisher Scientific) according to the supplier’s instructions. EdU-positive areas in epithelium were measured and normalized against the total cellular area in epithelium determined by Hoechst staining using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Methyl green-pyronin Y staining

Sections were stained with methyl green-pyronin Y (MGPY) (Muto, Tokyo, Japan), a mixture of methyl green, which binds strongly to DNA, and pyronin Y, which binds strongly to RNA, for 5 min at room temperature. Adult epithelial stem cells stained red intensely because of their RNA-rich cytoplasm (35, 36).

Statistical analysis

Data are presented as means ± sd. The significance of differences between groups was evaluated by a Student’s t or log-rank (Mantel-Cox) test with Prism 5 (GraphPad Software, La Jolla, CA, USA).

RESULTS

High levels of EVI and/or MDS/EVI are expressed in the intestine at the climax of metamorphosis and induced by T3 in X. tropicalis

Our earlier study showed that EVI and MDS/EVI mRNAs are strongly up-regulated in the intestine at the climax of metamorphosis in X. laevis (16). To analyze the function of the endogenous EVI and MDS/EVI, we turned to X. tropicalis because X. laevis is pseudo tetraploid, making it difficult to carry out knockout studies. We first investigated whether the expression of EVI and MDS/EVI in the intestine was conserved between the two species by using RT-PCR with a primer set to amplify both EVI and MDS/EVI. Treating premetamorphic X. tropicalis tadpoles with T3 led to induction of EVI and MDS/EVI in the intestine but not in the tail or liver (Fig. 1A). In addition, the mRNAs for EVI and MDS/EVI were highly up-regulated in X. tropicalis intestine at the climax of metamorphosis (Fig. 1B). These findings indicate that their expression is conserved between X. tropicalis and X. laevis.

Figure 1.

Figure 1.

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The expression of EVI and MDS/EVI is up-regulated during T3-induced or natural metamorphosis. A) EVI and MDS/EVI mRNAs are increased in the intestine but not in the liver or tail of premetamorphic X. tropicalis tadpoles treated with T3. Stage 54 X. tropicalis tadpoles were treated with or without 10 nM T3 for 2 d. Total RNA was isolated from the intestine, tail, and liver and analyzed by RT-PCR with a primer set for both EVI and MDS/EVI. B) EVI and MDS/EVI are up-regulated during intestinal metamorphosis. Total RNA was isolated from the whole intestine of X. tropicalis tadpoles at the indicated stages and analyzed by RT-PCR. The mRNA level was normalized against that of EF1-α mRNA. Data are shown as the mean ± sd (n = 3).

EVI and MDS/EVI are not essential for embryogenesis and premetamorphosis

To investigate the role of EVI and MDS/EVI during metamorphosis, especially for intestinal adult stem cell formation, we knocked out the endogenous EVI and MDS/EVI by using TALEN. We designed a TALEN nuclease made of a pair of left and right arms targeting the second ATG codon in exon 3 of X. tropicalis EVI that is present in both EVI and MDS/EVI in order to knock out both of them (16) (Fig. 2A). We microinjected mRNAs encoding the 2 TALEN arms into fertilized eggs and analyzed the mutation in the targeted region by using a genomic DNA–derived pooled, TALEN mRNA-injected F0 tadpoles. The results showed that the TALEN could specifically mutate the endogenous target with 70–95% efficiency (data not shown). We allowed F0 animals to develop naturally. The F0 tadpoles underwent normal metamorphosis and developed into sexually mature adult frogs. The F0 frogs were mated with wild-type frogs to obtain F1 animals that might contain heterozygous mutations in the TALEN-targeted region. We genotyped the resulting F1 tadpoles and selected the tadpoles with an out-of-frame, 20-nt deletion in the targeted region. There are multiple functional domains in EVI and MDS/EVI that are conserved among vertebrates, and the first zinc finger domain and the CtBP binding domain have been shown to play a critical role in the early formation of X. laevis kidney (37). The 20-nt deletion at the TALEN target site caused a frame shift that would abolish the zinc finger domain and CtBP binding domain by creating stop codons (Fig. 2B, C). Thus, the mutation would lead to the loss of both EVI and MDS/EVI proteins.

Figure 2.

Figure 2.

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EVI and MDS/EVI are not required for embryogenesis and premetamorphic development. A) Genomic structure and target sequences of a TALEN against X. tropicalis EVI locus. There are 3 transcripts around the EVI locus: MDS, MDS/EVI, and EVI, with 3, 17, and 16 exons, respectively (16). MDS/EVI shares the first 2 exons with MDS and the last 15 exons with EVI. A TALEN targeting both EVI and MDS/EVI was designed to have the left (L) and right (R) arms targeting exon 3 of EVI. The left and right TALEN arm binding sequences are shown in red and blue, respectively, and the spacer region is in black. B) A mutant line containing an out-of-frame 20-bp deletion (Δ20) in the TALEN-targeted region. The wild-type and mutant sequences are aligned to show the Δ20 deletion. Deletions are indicated by dashes. The deletion results in the inactivation of EVI and MDS/EVI (A). C) Representative sequencing profiles for genotyping. Genomic DNA from wild-type, heterozygous (Evi+/−), or homozygous (Evi−/−) mutant animals was sequenced, revealing a Δ20 deletion in 1 or both copies of the EVI and MDS/EVI gene in the heterozygous or homozygous animals, respectively.

Adult heterozygous mutant F1 male and F1 female frogs were mated to obtain F2 offspring. When the F2 animals were genotyped at different stages by sequencing (Fig. 2C), we observed that that homozygous knockout, heterozygous (with 1 wild-type copy of EVI and MDS/EVI), and wild-type animals were present in the expected mendelian ratio at premetamorphic stage 45 (4 d after fertilization), premetamorphic stage 49 (11 d after fertilization), and the early metamorphic climax (stage 58) (Table 1). Furthermore, as late as stage 58, there were no detectable external morphologic differences among the 3 genotypes, including body weight (Fig. 3A) and body length (Fig. 3B). Thus, neither EVI nor MDS/EVI is essential for embryogenesis, premetamorphic development, and metamorphosis up to early climax of metamorphosis (stage 58).

TABLE 1.

Genotypes of tadpoles at stages 45, 49, and 58

Developmental stage Wild-type Evi+/− Evi−/− Total
45 7 14 9 30
49 10 13 7 30
58 14 24 11 49
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Animals were obtained by intercrossing heterozygous frogs and were genotyped by genomic DNA sequencing at the indicated stages. Wild-type, heterozygous, and homozygous tadpoles were approximately in the expected mendelian ratio at these premetamorphic and metamorphic stages.

Figure 3.

Figure 3.

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EVI and MDS/EVI are not required for embryogenesis and premetamorphic development. Tadpoles were reared identically to stage 58 and then genotyped as wild-type, heterozygous (Evi+/−), and homozygous knockout of both EVI and MDS/EVI (Evi−/−). Body weight (A) and length (B) at stage 58 for each animal were determined and plotted with the mean (marked as a line) and se. No significant difference was observed for both parameters for the 3 genotypes, indicating no gross defect in the animals up to early metamorphic climax (stage 58) caused by EVI and MDS/EVI knockout (Evi−/−).

EVI and MDS/EVI knockout leads to growth retardation and lethality of tadpoles at the climax of metamorphosis

Next, we analyzed the function of EVI and MDS/EVI at the climax of metamorphosis when intestinal adult stem cell development takes place. We investigated the rate of metamorphosis progress by determining the number of days to reach climax stage 62 as judged by external morphology. We noticed developmental retardation in the EVI- and MDS/EVI-knockout tadpoles because the homozygous knockout animals took longer to reach stage 62 compared with heterozygous and wild-type tadpoles (127 d on the average for the homozygous knockout tadpoles vs. 117 d for the heterozygous tadpoles and 116 d for the wild-type tadpoles) (Fig. 4A).

Figure 4.

Figure 4.

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EVI and MDS/EVI knockout causes developmental retardation and lethality at climax of metamorphosis. A) Tadpoles with homozygous knockout of both EVI and MDS/EVI (Evi−/−) take longer to develop to metamorphic climax stage 62. Tadpoles were reared identically to stage 62 and then genotyped as wild-type, heterozygous (Evi+/−), and Evi−/−. The time for individual animals to reach stage 62 was determined and plotted with the mean (marked as a line) and se. Mean time to reach stage 62 was 116, 117, and 127 d for wild-type, Evi+/−, and Evi−/−, respectively. The statistical significance of differences was determined by Student’s t test. *P < 0.05. B, C) EVI and MDS/EVI knockout leads to lethality at metamorphic climax. The survival rate of wild-type (n = 14), Evi+/− (n = 24), and Evi−/− (n = 11) tadpoles was determined from stage 58 (beginning of metamorphic climax) to stage 66 (end of metamorphosis) (B). The experiment was repeated independently to see whether the finding was reproducible by using wild-type (n = 11), Evi+/− (n = 29), and Evi−/− (n = 12) tadpoles (C). Note that 90% of the homozygous-knockout tadpoles died between stage 62 and stage 65, when most dramatic morphologic changes take place. Statistical significance was assessed by using log-rank (Mantel-Cox) test. *P < 0.05. No significant differences were observed between the wild-type and heterozygous animals.

The homozygous EVI- and MDS/EVI-knockout tadpoles began to die around stage 63–64, and only about 20% of the homozygous-knockout tadpoles were alive at the end of metamorphosis (stage 66) (Fig. 4B, C). In contrast, the wild-type and heterozygous animals completed metamorphosis normally (Fig. 4B, C). Although the mechanism underlying the metamorphic lethal phenotype remains to be investigated, this observation may be consistent with the finding in mouse where a total knockout of EVI and MDS/EVI leads to a lethal phenotype between embryonic d 13.5 and 16.5 (27), which temporally resembles the metamorphic period in frogs (8, 9).

EVI and MDS/EVI knockout reduces adult intestinal epithelial stem cell proliferation at the end of natural metamorphosis or during T3-induced metamorphosis

Because EVI and MDS/EVI are highly expressed in the intestine during metamorphosis, we next investigated whether there was any defect in adult stem cell development in the intestine during metamorphosis. We used EdU labeling to detect proliferating cells in the intestine at stages 60 and 62 during metamorphosis and at the end of metamorphosis (stage 66). We failed to detect any obvious difference among the wild-type and homozygous intestine at stages 60 and 62 (data not shown). On the other hand, at end of metamorphosis (stage 66), when EdU-labeled proliferating cells are localized mainly in the troughs of the newly formed epithelial folds (35), we observed much fewer proliferating cells in the epithelium of intestine of EVI- and MDS/EVI-knockout tadpoles compared with wild-type tadpoles (Fig. 5A), although no detectable external morphologic differences were observed between wild-type and homozygous-knockout tadpoles. Quantification of the EdU-labeled proliferating cells revealed that knocking out EVI and MDS/EVI led to a 10-fold reduction in epithelial cell proliferation at the end of metamorphosis (Fig. 5B).

Figure 5.

Figure 5.

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EVI and MDS/EVI knockout reduces adult intestinal stem cell proliferation at the end of metamorphosis. A) Cross sections of the intestine from wild-type and homozygous-knockout Evi−/− tadpoles at stage 66 were stained for EdU (cell proliferation). The number of epithelial cells labeled by EdU in the cross sections of the intestine from Evi−/− tadpoles was reduced compared with wild-type tadpoles. The dotted lines depict the epithelium–mesenchyme boundary. Scale bar, 50 μm. B) Quantitative analysis of proliferating cells by counting EdU-positive areas in the epithelium, normalized against the total area in epithelium as determined by Hoechst staining (nuclear DNA). The statistical significance of differences was determined by Student’s t test. *P < 0.05. Nine wild-type and 2 surviving Evi−/− tadpoles were used for counting EdU-positive areas in the epithelium.

Because natural metamorphosis takes a relatively long time, which may allow endogenous compensatory mechanisms to take place, and because there is considerable variation in the developmental rate among different sibling animals, it may be difficult to detect the effect of EVI and MDS/EVI knockout on adult intestinal stem cell development during metamorphosis. Thus, we investigated the effects of the knockout on T3-induced intestinal remodeling. We treated premetamorphic tadpoles at stage 54 with T3 for 3 d to induce metamorphosis. We have previously shown that Lgr5-positive (Lgr5 is a well-known adult intestinal stem cell marker of mammalian intestine) adult stem cell clusters are numerous in the epithelium after extended T3 treatment of premetamorphic tadpoles and that these epithelial cell clusters have more than 1 layer along the epithelium–connective tissue axis and are strongly stained with MGPY or labeled with EdU (35). Thus, we used MGPY and EdU labeling to analyze the animals treated with or without T3. The epithelium was uniformly stained with MGPY, and no stem cell clusters were present in premetamorphic intestine before T3 treatment in both wild-type and homozygous-knockout tadpoles (Fig. 6A). After 3 d of T3 treatment, MGPY-stained cell clusters were present in the epithelium, and there were much fewer such cells in homozygous-knockout tadpoles compared with wild-type tadpoles (Fig. 6A). Furthermore, EdU labeling showed that there were much fewer proliferating cells in the epithelium of the homozygous-knockout tadpoles compared with wild-type tadpoles after treatment with T3 for 3 d (Fig. 6B, C), indicating that EVI and/or MDS1/EVI play an important role in adult intestinal epithelial development during metamorphosis.

Figure 6.

Figure 6.

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EVI and MDS/EVI knockout reduces adult intestinal stem cell proliferation during T3-induced metamorphosis. A) MGPY staining reveals fewer adult stem cell clusters in the knockout animals compared with the wild-type tadpoles after 3 d of T3 treatment. Stage 54 premetamorphic tadpoles were treated with 5 nM T3 for 0 (−T3) or 3 (+T3) days and were killed 30 min after EdU injection. Cross sections of the intestine from the wild-type or homozygous animals were stained with MGPY, which stained the proliferating adult epithelial cells strongly (purple). Higher magnifications of boxed areas are also shown. After T3 treatment, many MGPY-stained cell clusters were present in the wild-type intestine. These epithelial cell clusters have more than 1 layer along the epithelium–connective tissue axis. MGPY-stained cells were also present in the epithelium prior to T3 treatment and were monolayer cells in the epithelium, although sometimes 2 or more MGPY-stained cells were present together. There were fewer epithelial cells/clusters strongly stained by MGPY in the cross sections of the intestine from Evi−/− tadpoles compared with wild-type tadpoles. Arrowheads indicate the clusters of epithelial cells. B) EdU labeling shows fewer proliferating cells in the knockout animals after T3 treatment. Cross-sections of the intestine from the tadpoles above were double stained for EdU (cell proliferation) and with Hoechst (nuclear DNA). The dotted lines depict the epithelium–mesenchyme boundary. Scale bar, 50 μm. C) Quantitative analysis reveals a drastic reduction in proliferating cells in the knockout animals. Cell proliferation was quantified for sections as in panel B by measuring red-colored, EdU-positive areas in epithelium and normalized against the total area in epithelium as determined by Hoechst staining. At least 3 tadpoles were analyzed for each stage or day of T3 treatment. The statistical significance of differences was determined by Student’s t test. *P < 0.05.

DISCUSSION

Research on stem cells has gained considerable recognition because of the promise of stem cell–based therapies for treating human diseases. On the other hand, to apply stem cell therapies to the treatment of organ-specific diseases, it is critical to understand how the organ-specific adult stem cells are formed during vertebrate development. T3-dependent frog metamorphosis resembles mammalian postembryonic development and offers a unique opportunity to study how adult stem cells are developed, largely due the ability to manipulate the externally developing frog embryos. Our earlier studies have shown that the formation of the adult intestine during Xenopus metamorphosis involves essentially complete degeneration of the larval epithelium and de novo formation of adult stem cells (38). Tissue-specific microarray analysis of intestinal gene expression during metamorphosis has identified a number of novel candidate stem cell genes. Among them are EVI and MDS/EVI (12, 16), which have been recognized as two of the important oncogenes associated with murine and human myeloid leukemia (17–23). Transcripts of EVI and MDS/EVI are up-regulated predominantly in the epithelium of the intestine by T3 during Xenopus metamorphosis (16). This suggests that EVI and MDS/EVI are likely involved in the development and/or proliferation of adult intestinal epithelial stem cells.

Here, we have provided strong evidence to support an important role of EVI and/or MDS/EVI in the formation and/or proliferation of adult stem cells in Xenopus intestine during T3-induced metamorphosis and at the end of natural metamorphosis. We showed that EVI and MDS/EVI knockout leads to lethality in tadpoles at or around the climax of metamorphosis, when T3 levels peak and adult epithelial stem cells are forming and proliferating. In mouse, total knockout of EVI and MDS/EVI leads to a lethal phenotype between E13.5 and E16.5 (27), a period when plasma T3 levels start to increase and many organs develop and/or mature. Thus, EVI and MDS/EVI may have an evolutionarily conserved role during postembryonic development. Interestingly, we have reported a similar temporal conservation in the lethal phenotype caused by mutating Dot1L between X. tropicalis and mouse during postembryonic development (39). These findings further support the use of Xenopus intestinal metamorphosis as a model to study the development of adult organ-specific stem cells in vertebrates.

Our analyses indicate that EVI and MDS/EVI knockout reduces adult intestinal epithelial stem cell proliferation during T3-induced metamorphosis. This suggests that EVI and MDS/EVI are necessary for the formation and/or proliferation of the adult stem cells. Although we failed to observe significant differences between the intestine of wild-type and EVI- and MDS/EVI-knockout animals at the climax of metamorphosis during natural metamorphosis (data not shown), the knockout animals had reduced adult stem cell proliferation at the end of metamorphosis, indicating that EVI and/or MDS/EVI are important for the development and/or functioning of the adult intestinal stem cells. These findings further suggest that the slower and less synchronous development of tadpoles during natural metamorphosis may allow some endogenous compensatory mechanisms to take place, thus masking the effects of the knockout on adult stem cells at the climax of intestinal remodeling. In any case, our findings demonstrate a role of EVI and/or MDS/EVI in adult organ-specific stem cell development. Although such a role remains to be revealed in mammals, it has been shown that EVI and/or MDS/EVI are required for the maintenance of the hematopoietic stem cells in mammals (22, 24–27), suggesting that they may have a role in the development and/or function of adult stem cells in many different organs or tissues. Given that EVI and MDS/EVI can function as transcription factors, it would be interesting and important to determine their target genes that mediate their effect on stem cell development.

Although EVI and/or MDS/EVI are important for intestinal remodeling during metamorphosis, the lethal phenotype at the climax of metamorphosis is unlikely due to the defect in intestinal remodeling. The exact mechanism underlying the lethality in EVI- and MDS/EVI-knockout tadpoles or during late embryogenesis/postembryonic development in mouse remains unclear. This period in both frog and mouse involves the formation and/or maturation of adult organs. It is likely that EVI and/or MDS/EVI play an essential role in the development of an adult organ that is commonly critical for animal survival during this transition period in both frog and mouse. Identifying such an organ and the downstream targets and signaling pathways controlled by EVI and MDS/EVI is essential for understanding how EVI and MDS/EVI affect adult organ maturation and animal survival during postembryonic development.

ACKNOWLEDGMENTS

M.O. was supported, in part, by a Japan Society for the Promotion of Science Research Fellowship for Japanese Biomedical and Behavioral Researchers at the U.S. National Institutes of Health (NIH). This work was supported by the Intramural Research Program of the NIH National Institute of Child Health and Human Development. The authors declare no conflicts of interest.

Glossary

EdU

5-ethynyl-2′-deoxyuridine

EF

elongation factor

EVI

ectopic viral integration site 1

MDS

myelodysplastic syndrome 1

MGPY

methyl green-pyronin Y

T3

thyroid hormone

TALEN

transcription activator-like effector nuclease

AUTHOR CONTRIBUTIONS

M. Okada performed the research; and M. Okada and Y.-B. Shi designed research, analyzed the data, and wrote the paper.

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