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The System L1-type amino acid transporter mediates transport of large neutral amino acids (LNAA) in many mammalian cell-types. LNAA such as leucine are required for full activation of the mTOR-S6K signalling pathway promoting protein synthesis and cell growth. The SLC7A5 (LAT1) catalytic subunit of high-affinity System L1 functions as a glycoprotein-associated heterodimer with the multifunctional protein SLC3A2 (CD98). We generated a floxed Slc7a5 mouse strain which, when crossed with mice expressing Cre driven by a global promoter, produced Slc7a5 heterozygous knockout (Slc7a5+/-) animals with no overt phenotype, although homozygous global knockout of Slc7a5 was embryonically lethal. Muscle-specific (MCK Cre-mediated) Slc7a5 knockout (MS-Slc7a5-KO) mice were used to study the role of intracellular LNAA delivery by the SLC7A5 transporter for mTOR-S6K pathway activation in skeletal muscle. Activation of muscle mTOR-S6K (Thr389 phosphorylation) in vivo by intraperitoneal leucine injection was blunted in homozygous MS-Slc7a5-KO mice relative to wild-type animals. Dietary intake and growth rate were similar for MS-Slc7a5-KO mice and wild-type littermates fed for 10 weeks (to age 120 days) with diets containing 10%, 20% or 30% of protein. In MS-Slc7a5-KO mice, Leu and Ile concentrations in gastrocnemius muscle were reduced by ∼40% as dietary protein content was reduced from 30 to 10%. These changes were associated with >50% decrease in S6K Thr389 phosphorylation in muscles from MS-Slc7a5-KO mice, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet. MS-Slc7a5-KO mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals. Thus, SLC7A5 modulates LNAA-dependent muscle mTOR-S6K signalling in mice, although it appears non-essential (or is sufficiently compensated by e.g. SLC7A8 (LAT2)) for maintenance of normal muscle mass.

Intestinal remodeling during amphibian metamorphosis resembles the maturation of the adult intestine during mammalian postembryonic development when the adult epithelial self-renewing system is established under the influence of high concentrations of plasma thyroid hormone (T3). This process involves de novo formation and subsequent proliferation and differentiation of the adult stem cells.The T3-dependence of the formation of adult intestinal stem cell during Xenopus laevis metamorphosis offers a unique opportunity to identify genes likely important for adult organ-specific stem cell development. We have cloned and characterized the ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI generated via transcription from the upstream MDS promoter and alternative splicing. EVI and MDS/EVI have been implicated in a number of cancers including breast, leukemia, ovarian, and intestinal cancers. We show that EVI and MDS/EVI transcripts are upregulated by T3 in the epithelium but not the rest of the intestine in Xenopus laevis when adult stem cells are forming in the epithelium.Our results suggest that EVI and MDS/EVI are likely involved in the development and/or proliferation of newly forming adult intestinal epithelial cells.

Frog metamorphosis is totally dependent on thyroid hormone (T3) and mimics the postembryonic period around birth in mammals. It is an excellent model to study the molecular basis of postembryonic development in vertebrate. We and others have shown that many, if not all, matrix metalloproteinases (MMPs), which cleave proteins of the extracellular matrix as well as other substrates, are induced by T3 and important for metamorphosis. MMP activity can be inhibited by tissue inhibitors of metalloproteinase (TIMPs). There are 4 TIMPs in vertebrates and their roles in postembryonic development are poorly studied.We analyzed the TIMP2 genes in Xenopus laevis and the highly related species Xenopus tropicalis and discovered that TIMP2 is a single copy gene in Xenopus tropicalis as in mammals but is duplicated in Xenopus laevis. Furthermore, the TIMP2 locus in Xenopus tropicalis genome is different from that in human, suggesting an evolutionary reorganization of the locus. More importantly, we found that the duplicated TIMP2 genes were similarly regulated in the developing limb, remodeling intestine, resorbing tail during metamorphosis. Unexpectedly, like its MMP target genes, the TIMP2 genes were upregulated by T3 during both natural and T3-induced metamorphosis.Our results indicate that TIMP2 is highly conserved among vertebrates and that the TIMP2 locus underwent a chromosomal reorganization during evolution. Furthermore, the unexpected upregulation of TIMP2 genes during metamorphosis suggests that proper balance of MMP activity is important for metamorphosis.

The formation and/or maturation of adult organs in vertebrates often takes place during postembryonic development, a period around birth in mammals when thyroid hormone (T3) levels are high. The T3-dependent anuran metamorphosis serves as a model to study postembryonic development. Studies on the remodeling of the intestine during Xenopus (X.) laevis metamorphosis have shown that the development of the adult intestine involves de novo formation of adult stem cells in a process controlled by T3. On the other hand, X. tropicalis, highly related to X. laevis, offers a number of advantages for studying developmental mechanisms, especially at genome-wide level, over X. laevis, largely due to its shorter life cycle and sequenced genome. To establish X. tropicalis intestinal metamorphosis as a model for adult organogenesis, we analyzed the morphological and cytological changes in X. tropicalis intestine during metamorphosis.We observed that in X. tropicalis, the premetamorphic intestine was made of mainly a monolayer of larval epithelial cells surrounded by little connective tissue except in the single epithelial fold, the typhlosole. During metamorphosis, the larval epithelium degenerates and adult epithelium develops to form a multi-folded structure with elaborate connective tissue and muscles. Interestingly, typhlosole, which is likely critical for adult epithelial development, is present along the entire length of the small intestine in premetamorphic tadpoles, in contrast to X. laevis, where it is present only in the anterior 1/3. T3-treatment induces intestinal remodeling, including the shortening of the intestine and the typhlosole, just like in X. laevis.Our observations indicate that the intestine undergoes similar metamorphic changes in X. laevis and X. tropicalis, making it possible to use the large amount of information available on X. laevis intestinal metamorphosis and the genome sequence information and genetic advantages of X. tropicalis to dissect the pathways governing adult intestinal development.

INhibitor of Growth (ING) proteins belong to a large family of plant homeodomain finger-containing proteins important in epigenetic regulation and carcinogenesis. We have previously shown that ING1 and ING2 expression is regulated by thyroid hormone (TH) during metamorphosis of the Xenopus laevis tadpole. The present study investigates the possibility that ING proteins modulate TH action.Tadpoles expressing a Xenopus ING2 transgene (Trans(ING2)) were significantly smaller than tadpoles not expressing the transgene (Trans(GFP)). When exposed to 10 nM 3,5,3'-triiodothyronine (T(3)), premetamorphic Trans(ING2) tadpoles exhibited a greater reduction in tail, head, and brain areas, and a protrusion of the lower jaw than T(3)-treated Trans(GFP) tadpoles. Quantitative real time polymerase chain reaction (QPCR) demonstrated elevated TH receptor β (TRβ) and TH/bZIP transcript levels in Trans(ING2) tadpole tails compared to Trans(GFP) tadpoles while TRα mRNAs were unaffected. In contrast, no difference in TRα, TRβ or insulin-like growth factor (IGF2) mRNA abundance was observed in the brain between Trans(ING2) and Trans(GFP) tadpoles. All of these transcripts, except for TRα mRNA in the brain, were inducible by the hormone in both tissues. Oocyte transcription assays indicated that ING proteins enhanced TR-dependent, T(3)-induced TRβ gene promoter activity. Examination of endogenous T(3)-responsive promoters (TRβ and TH/bZIP) in the tail by chromatin immunoprecipitation assays showed that ING proteins were recruited to TRE-containing regions in T(3)-dependent and independent ways, respectively. Moreover, ING and TR proteins coimmunoprecipitated from tail protein homogenates derived from metamorphic climax animals.We show for the first time that ING proteins modulate TH-dependent responses, thus revealing a novel role for ING proteins in hormone signaling. This has important implications for understanding hormone influenced disease states and suggests that the induction of ING proteins may facilitate TR function during metamorphosis in a tissue-specific manner.

The intestinal epithelium undergoes constant self-renewal throughout adult life across vertebrates. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells. This self-renewal system is established in the so-called postembryonic developmental period in mammals when endogenous thyroid hormone (T3) levels are high.The T3-dependent metamorphosis in anurans like Xenopus laevis resembles the mammalian postembryonic development and offers a unique opportunity to study how the adult stem cells are developed. The tadpole intestine is predominantly a monolayer of larval epithelial cells. During metamorphosis, the larval epithelial cells undergo apoptosis and, concurrently, adult epithelial stem/progenitor cells develop de novo, rapidly proliferate, and then differentiate to establish a trough-crest axis of the epithelial fold, resembling the crypt-villus axis in the adult mammalian intestine. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a well-established stem cell marker in the adult mouse intestinal crypt. Here we have cloned and analyzed the spatiotemporal expression profile of LGR5 gene during frog metamorphosis. We show that the two duplicated LGR5 genes in Xenopus laevis and the LGR5 gene in Xenopus tropicalis are highly homologous to the LGR5 in other vertebrates. The expression of LGR5 is induced in the limb, tail, and intestine by T3 during metamorphosis. More importantly, LGR5 mRNA is localized to the developing adult epithelial stem cells of the intestine.These results suggest that LGR5-expressing cells are the stem/progenitor cells of the adult intestine and that LGR5 plays a role in the development and/or maintenance of the adult intestinal stem cells during postembryonic development in vertebrates.

SUMO proteins are small ubiquitin-related modifiers. All SUMOs are synthesized as propeptides that are post-translationally cleaved prior to conjugation. After processing, SUMOs become covalently conjugated to cellular targets through a pathway that is similar to ubiquitination. Ubiquitin like protein proteases/Sentrin specific proteases (Ulp/SENPs) mediate both processing and deconjugation of SUMOs. The action of Ulp/SENPs makes SUMOylation a highly dynamic post-translational modification. To investigate how Ulp/SENPs are regulated in a developmental context, we isolated and characterized all Ulp/SENPs in Xenopus laevis. Xenopus possess homologues of mammalian SENP3, 5, 6 and 7. All of these enzymes reacted with HA-tagged vinyl sulfone derivatives of SUMO-2 (HA-SU2-VS) but not SUMO-1 (HA-SU1-VS), suggesting that they act primarily on SUMO-2 and -3. In contrast, Xenopus possess a single member of the SENP1/SENP2 subfamily of Ulp/SENPs, most closely related to mammalian SENP1. Xenopus SENP1 reacted with HA-SU1-VS and HA-SU2-VS, suggesting that it acts on all SUMO paralogues. We analyzed the mRNA and protein levels for each of the Ulp/SENPs through development; we found that they show distinct patterns of expression that may involve both transcriptional and post-transcriptional regulation. Finally, we have characterized the developmental function of the most abundant Ulp/SENP found within Xenopus eggs, SENP3. Depletion of SENP3 using morpholino antisense oligonucleotides (morpholinos) caused accumulation of high molecular weight SUMO-2/3 conjugated species, defects in developing embryos and changes in the expression of some genes regulated by the transforming growth factor beta (TGF-beta) pathway. These findings collectively indicate that SUMO proteases are both highly regulated and essential for normal development.

Background: Intestinal remodeling during amphibian metamorphosis resembles the maturation of the adult intestine during mammalian postembryonic development when the adult epithelial self-renewing system is established under the influence of high concentrations of plasma thyroid hormone (T3). This process involves de novo formation and subsequent proliferation and differentiation of the adult stem cells. Methodology/Principal Findings: The T3-dependence of the formation of adult intestinal stem cell during Xenopus laevis metamorphosis offers a unique opportunity to identify genes likely important for adult organ-specific stem cell development. We have cloned and characterized the ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI generated via transcription from the upstream MDS promoter and alternative splicing. EVI and MDS/ EVI have been implicated in a number of cancers including breast, leukemia, ovarian, and intestinal cancers. We show that EVI and MDS/EVI transcripts are upregulated by T3 in the epithelium but not the rest of the intestine in Xenopus laevis when adult stem cells are forming in the epithelium. Conclusions/Significance: Our results suggest that EVI and MDS/EVI are likely involved in the development and/or proliferation of newly forming adult intestinal epithelial cells. [ABSTRACT FROM AUTHOR]

Background: The formation and/or maturation of adult organs in vertebrates often takes place during postembryonic development, a period around birth in mammals when thyroid hormone (T3) levels are high. The T3-dependent anuran metamorphosis serves as a model to study postembryonic development. Studies on the remodeling of the intestine during Xenopus (X.) laevis metamorphosis have shown that the development of the adult intestine involves de novo formation of adult stem cells in a process controlled by T3. On the other hand, X. tropicalis, highly related to X. laevis, offers a number of advantages for studying developmental mechanisms, especially at genome-wide level, over X. laevis, largely due to its shorter life cycle and sequenced genome. To establish X. tropicalis intestinal metamorphosis as a model for adult organogenesis, we analyzed the morphological and cytological changes in X. tropicalis intestine during metamorphosis. Methodology/Principal Findings: We observed that in X. tropicalis, the premetamorphic intestine was made of mainly a monolayer of larval epithelial cells surrounded by little connective tissue except in the single epithelial fold, the typhlosole. During metamorphosis, the larval epithelium degenerates and adult epithelium develops to form a multi-folded structure with elaborate connective tissue and muscles. Interestingly, typhlosole, which is likely critical for adult epithelial development, is present along the entire length of the small intestine in premetamorphic tadpoles, in contrast to X. laevis, where it is present only in the anterior 1/3. T3-treatment induces intestinal remodeling, including the shortening of the intestine and the typhlosole, just like in X. laevis. Conclusions/Significance: Our observations indicate that the intestine undergoes similar metamorphic changes in X. laevis and X. tropicalis, making it possible to use the large amount of information available on X. laevis intestinal metamorphosis and the genome sequence information and genetic advantages of X. tropicalis to dissect the pathways governing adult intestinal development. [ABSTRACT FROM AUTHOR]