Rachel A. Heimeier

The Xenoestrogen Bisphenol A Inhibits Postembryonic Vertebrate Development by Antagonizing Gene Regulation by Thyroid Hormone

Bisphenol A (BPA), a chemical widely used to manufacture plastics, is estrogenic and capable of disrupting sex differentiation. However, recent in vitro studies have shown that BPA can also antagonize T(3) activation of the T(3) receptor. The difficulty in studying uterus-enclosed mammalian embryos has hampered the analysis on the direct effects of BPA during vertebrate development. This study proposed to identify critical T(3) pathways that may be disrupted by BPA based on molecular analysis in vivo. Because amphibian metamorphosis requires T(3) and encompasses the postembryonic period in mammals when T(3) action is most critical, we used this unique model for studying the effect of BPA on T(3)-dependent vertebrate development at both the morphological and molecular levels. After 4 d of exposure, BPA inhibited T(3)-induced intestinal remodeling in premetamorphic Xenopus laevis tadpoles. Importantly, microarray analysis revealed that BPA antagonized the regulation of most T(3)-response genes, thereby explaining the inhibitory effect of BPA on metamorphosis. Surprisingly, most of the genes affected by BPA in the presence of T(3) were T(3)-response genes, suggesting that BPA predominantly affected T(3)-signaling pathways during metamorphosis. Our finding that this endocrine disruptor, well known for its estrogenic activity in vitro, functions to inhibit T(3) pathways to affect vertebrate development in vivo and thus not only provides a mechanism for the likely deleterious effects of BPA on human development but also demonstrates the importance of studying endocrine disruption in a developmental context in vivo.

Identification of direct thyroid hormone response genes reveals the earliest gene regulation programs during frog metamorphosis

Thyroid hormone (T3) is essential for normal development and organ function throughout vertebrates. Its effects are mainly mediated through transcriptional regulation by T3 receptor (TR). The identification and characterization of the immediate early, direct target genes are thus of critical importance in understanding the molecular pathways induced by T3. Unfortunately, this has been hampered by the difficulty to study gene regulation by T3 in uterus-enclosed mammalian embryos. Here we used Xenopus metamorphosis as a model for vertebrate postembryonic development to identify direct T3 response genes in vivo. We took advantage of the ability to easily induce metamorphosis with physiological levels of T3 and to carry out microarray analysis in Xenopus laevis and genome-wide sequence analysis in Xenopus tropicalis. This allowed us to identify 188 up-regulated and 249 down-regulated genes by T3 in the absence of new protein synthesis in whole animals. We further provide evidence to show that these genes contain functional TREs that are bound by TR in tadpoles and that their promoters are regulated by TR in vivo. More importantly, gene ontology analysis showed that the direct up-regulated genes are enriched in categories important for transcriptional regulation and protein degradation-dependent signaling processes but not DNA replication. Our findings thus revealed the existence of interesting pathways induced by T3 at the earliest step of metamorphosis.

Studies on Xenopus laevis intestine reveal biological pathways underlying vertebrate gut adaptation from embryo to adult

BackgroundTo adapt to its changing dietary environment, the digestive tract is extensively remodeled from the embryo to the adult during vertebrate development. Xenopus laevis metamorphosis is an excellent model system for studying mammalian gastrointestinal development and is used to determine the genes and signaling programs essential for intestinal development and maturation.ResultsThe metamorphosing intestine can be divided into four distinct developmental time points and these were analyzed with X. laevis microarrays. Due to the high level of conservation in developmental signaling programs and homology to mammalian genes, annotations and bioinformatics analysis were based on human orthologs. Clustering of the expression patterns revealed co-expressed genes involved in essential cell processes such as apoptosis and proliferation. The two largest clusters of genes have expression peaks and troughs at the climax of metamorphosis, respectively. Novel conserved gene ontology categories regulated during this period include transcriptional activity, signal transduction, and metabolic processes. Additionally, we identified larval/embryo- and adult-specific genes. Detailed analysis revealed 17 larval specific genes that may represent molecular markers for human colonic cancers, while many adult specific genes are associated with dietary enzymes.ConclusionsThis global developmental expression study provides the first detailed molecular description of intestinal remodeling and maturation during postembryonic development, which should help improve our understanding of intestinal organogenesis and human diseases. This study significantly contributes towards our understanding of the dynamics of molecular regulation during development and tissue renewal, which is important for future basic and clinical research and for medicinal applications.

Expression Profiling of Intestinal Tissues Implicates Tissue-Specific Genes and Pathways Essential for Thyroid Hormone-Induced Adult Stem Cell Development

The study of the epithelium during development in the vertebrate intestine touches upon many contemporary aspects of biology: to name a few, the formation of the adult stem cells (ASCs) essential for the life-long self-renewal and the balance of stem cell activity for renewal vs cancer development. Although extensive analyses have been carried out on the property and functions of the adult intestinal stem cells in mammals, little is known about their formation during development due to the difficulty of manipulating late-stage, uterus-enclosed embryos. The gastrointestinal tract of the amphibian Xenopus laevis is an excellent model system for the study of mammalian ASC formation, cell proliferation, and differentiation. During T3-dependent amphibian metamorphosis, the digestive tract is extensively remodeled from the larval to the adult form for the adaptation of the amphibian from its aquatic herbivorous lifestyle to that of a terrestrial carnivorous frog. This involves de novo formation of ASCs that requires T3 signaling in both the larval epithelium and nonepithelial tissues. To understand the underlying molecular mechanisms, we have characterized the gene expression profiles in the epithelium and nonepithelial tissues by using cDNA microarrays. Our results revealed that T3 induces distinct tissue-specific gene regulation programs associated with the remodeling of the intestine, particularly the formation of the ASCs, and further suggested the existence of potentially many novel stem cell-associated genes, at least in the intestine during development.

Participation of Brahma-Related Gene 1 (BRG1)-Associated Factor 57 and BRG1-Containing Chromatin Remodeling Complexes in Thyroid Hormone-Dependent Gene Activation during Vertebrate Development

Multiple cofactors and chromatin remodeling complexes have been identified to contribute to the transcriptional activation regulated by thyroid hormone receptors (TRs) in vitro. However, their role and function during development in vivo remains to be elucidated. The total dependence of amphibian metamorphosis on thyroid hormone T3 provides a unique vertebrate model for studying the molecular mechanism of TR function in vivo. In this study, we show that the expression of Brahma-related gene 1 (BRG1), a chromatin-remodeling enzyme, is up-regulated at the climax of Xenopus laevis metamorphosis, whereas BRG1-associated factor 57 (BAF57), a BRG1-binding protein in BRG1-containing chromatin remodeling complexes, is constitutively expressed during development. Consistently, T3 treatment of premetamorphic tadpoles led to up-regulation of the expression of BRG1 but not BAF57. Studies using a reconstituted T3-dependent Xenopus oocyte transcription system, where we could study TR function in the context of chromatin, revealed that BRG1 enhances the transcriptional activation by ligand-bound TRs in a dose-dependent manner, whereas a remodeling-defective BRG1 mutant inhibited the activation, suggesting that this process relies on chromatin remodeling. Additional studies showed that BAF57 interacted with BRG1 in oocytes and enhanced gene activation by TR cooperatively with BRG1 in vivo. Chromatin immunoprecipitation revealed that BAF57 was recruited to the TR-regulated promoter in the presence of TR and T3. Together, these findings suggest a role of BRG1/BAF57-containing chromatin remodeling complexes in TR-regulated gene expression during postembryonic development.

Identification and Developmental Expression of Xenopus laevis SUMO Proteases

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-β) pathway. These findings collectively indicate that SUMO proteases are both highly regulated and essential for normal development.

The adenoviral E1A protein displaces corepressors and relieves gene repression by unliganded thyroid hormone receptors in vivo

The human adenovirus type 5 early region 1A (E1A) is one of two oncogenes present in the adenovirus genome and functions by interfering with the activities of cellular regulatory proteins. The E1A gene is alternatively spliced to yield five products. Earlier studies have revealed that E1A can regulate the function of thyroid hormone (T3) receptors (TRs). However, analysis in yeast compared with transfection studies in mammalian cell cultures yields surprisingly different effects. Here, we have examined the effect of E1A on TR function by using the frog oocyte in vivo system, where the effects of E1A can be studied in the context of chromatin. We demonstrate that different isoforms of E1A have distinct effects on TR function. The two longest forms inhibit both the repression by unliganded TR and activation by T3-bound TR. We further show that E1A binds to unliganded TR to displace the endogenous corepressor nuclear receptor corepressor, thus relieving the repression by unliganded TR. On the other hand, in the presence of T3, E1A inhibits gene activation by T3-bound TR indirectly, through a mechanism that requires its binding domain for the general coactivator p300. Taken together, our results thus indicate that E1A affects TR function through distinct mechanisms that are dependent upon the presence or absence of T3.

Extracellular domain of CD98hc is required for early murine development

BackgroundThe multifunctional protein CD98 heavy chain (CD98hc, Slc3a2) associates with integrin β1 through its cytoplasmic and transmembrane domains and the CD98hc-mediated integrin signaling is required for maintenance of ES cell proliferation. CD98hc-null mice exhibit early post-implantation lethality similar to integrin β1-null mice, supporting the importance of its interaction with integrin β1. On the other hand, the extracellular domain of CD98hc interacts with L-type amino acid transporters (LATs) and is essential for appropriate cell surface distribution of LATs. LATs mediate the transport of amino acids and other molecules such as thyroid hormone. In this respect, CD98hc may also affect development via these transporters.ResultsIn this study, mice were generated from embryonic stem (ES) cell line (PST080) harboring a mutant CD98hc allele (CD98hcΔ/+). Expression of the CD98hc mutant allele results in ΔCD98hc-β geo fusion protein where extracellular C-terminal 102 amino acids of CD98hc are replaced with β geo. Analyses of PST080 ES cells as well as reconstituted frog oocytes demonstrated that ΔCD98hc-β geo fusion protein preserved its ability to interact with integrin β1 although this mutant protein was hardly localized on the cell surface. These findings suggest that ΔCD98hc-β geo protein can mediate integrin signaling but cannot support amino acid transport through LATs. CD98hcΔ/+ mice were normal. Although some of the implantation sites lacked embryonic component at E9.5, all the implantation sites contained embryonic component at E7.5. Thus, CD98hcΔ/Δ embryos are likely to die between E7.5 and E9.5.ConclusionsConsidering that CD98hc complete knockout (CD98hc-/-) embryos are reported to die shortly after implantation, our findings suggest potential stage-specific roles of CD98hc in murine embryonic development. CD98hc may be essential for early post-implantation development by regulating integrin-dependent signaling, while the other function of CD98hc as a component of amino acid transporters may be required for embryonic development at later stages.

Tissue-Specific Upregulation of MDS/EVI Gene Transcripts in the Intestine by Thyroid Hormone during Xenopus Metamorphosis

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.

Modulation of Thyroid Hormone-Dependent Gene Expression in Xenopus laevis by INhibitor of Growth (ING) Proteins

Background 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. Methodology/Principal Findings Tadpoles expressing a Xenopus ING2 transgene (TransING2) were significantly smaller than tadpoles not expressing the transgene (TransGFP). When exposed to 10 nM 3,5,3′-triiodothyronine (T3), premetamorphic TransING2 tadpoles exhibited a greater reduction in tail, head, and brain areas, and a protrusion of the lower jaw than T3-treated TransGFP tadpoles. Quantitative real time polymerase chain reaction (QPCR) demonstrated elevated TH receptor β (TRβ) and TH/bZIP transcript levels in TransING2 tadpole tails compared to TransGFP 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 TransING2 and TransGFP 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, T3-induced TRβ gene promoter activity. Examination of endogenous T3-responsive promoters (TRβ and TH/bZIP) in the tail by chromatin immunoprecipitation assays showed that ING proteins were recruited to TRE-containing regions in T3-dependent and independent ways, respectively. Moreover, ING and TR proteins coimmunoprecipitated from tail protein homogenates derived from metamorphic climax animals. Conclusions/Significance 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.