Murine intestinal stem cells are highly sensitive to modulation of the T3/TRα1-dependent pathway.

The thyroid hormone T3 and its nuclear receptor TRα1 control gut development and homeostasis through the modulation of intestinal crypt cell proliferation. Despite increasing data, in-depth analysis on their specific action on intestinal stem cells is lacking. By using ex vivo 3D organoid cultures and molecular approaches, we observed early responses to T3 involving the T3-metabolizing enzyme Dio1 and the transporter Mct10, accompanied by a complex response of stem cell- and progenitor-enriched genes. Interestingly, specific TRα1 loss-of-function (inducible or constitutive) was responsible for low ex vivo organoid development and impaired stem cell activity. T3 treatment of animals in vivo not only confirmed the positive action of this hormone on crypt cell proliferation but also demonstrated its key action in modulating the number of stem cells, the expression of their specific markers and the commitment of progenitors into lineage-specific differentiation. In conclusion, T3 treatment or TRα1 modulation has a rapid and strong effect on intestinal stem cells, broadening our perspectives in the study of T3/TRα1-dependent signaling in these cells.

Comparative analysis of novel and common reference genes in adult tissues of the mussel Mytilus galloprovincialis.

BACKGROUND: Real-time quantitative PCR is a widely used method for gene expression analyses in various organisms. Its accuracy mainly relies on the correct selection of reference genes. Any experimental plan involving real-time PCR needs to evaluate the characteristics of the samples to be examined and the relative stability of reference genes. Most studies in mollusks rely on reference genes commonly used in vertebrates. RESULTS: In this study, we focused on the transcriptome of the bivalve mollusk Mytilus galloprovincialis in physiological state to identify suitable reference genes in several adult tissues. Candidate genes with highly stable expression across 51 RNA-seq datasets from multiple tissues were selected through genome-wide bioinformatics analysis. This approach led to the identification of three genes (Rpl14, Rpl32 and Rpl34), whose suitability was evaluated together with 7 other reference genes commonly reported in literature (Act, Cyp-A, Ef1α, Gapdh, 18S, 28S and Rps4). The stability analyses performed with geNorm, NormFinder and Bestkeeper identified specific either single or pairs of genes suitable as references for gene expression analyses in specific tissues and revealed the Act/Cyp-A pair as the most appropriate to analyze gene expression across different tissues. CONCLUSION: Mytilus galloprovincialis is a model system increasingly used in ecotoxicology and molecular studies. Our transcriptome-wide approach represents the first comprehensive investigation aimed at the identification of suitable reference genes for expression studies in this species.

The thyroid hormone nuclear receptors and the Wnt/ß-catenin pathway: An intriguing liaison.

The thyroid hormones, T3 and T4, control several developmental and homeostatic processes. From a molecular point of view, most of their actions depend on the activity of the thyroid hormone nuclear receptors (TRs), which are T3-modulated transcription factors. Recent studies have not only highlighted that the physiological response induced by T3 within a cell depends on the expression of specific TRs, but also that the functions of TRs are coordinated by and integrated in other signalling pathways. This is particularly the case for the multilevel interactions between TRs and the Wnt signalling pathway. Interestingly both signals are involved in development and homeostasis, and their alterations are responsible for the development of pathologies, such as cancer. Here, we present findings on the complex crosstalk between TRs and Wnt in several organisms and in different tissue contexts, and speculate on the biological relevance of modulating TR-Wnt functionality in therapeutic approaches aimed to target cancer cells or applications for regenerative medicine.

The thyroid hormone nuclear receptor TRα1 controls the Notch signaling pathway and cell fate in murine intestine.

Thyroid hormones control various aspects of gut development and homeostasis. The best-known example is in gastrointestinal tract remodeling during amphibian metamorphosis. It is well documented that these hormones act via the TR nuclear receptors, which are hormone-modulated transcription factors. Several studies have shown that thyroid hormones regulate the expression of several genes in the Notch signaling pathway, indicating a possible means by which they participate in the control of gut physiology. However, the mechanisms and biological significance of this control have remained unexplored. Using multiple in vivo and in vitro approaches, we show that thyroid hormones positively regulate Notch activity through the TRα1 receptor. From a molecular point of view, TRα1 indirectly controls Notch1, Dll1, Dll4 and Hes1 expression but acts as a direct transcriptional regulator of the Jag1 gene by binding to a responsive element in the Jag1 promoter. Our findings show that the TRα1 nuclear receptor plays a key role in intestinal crypt progenitor/stem cell biology by controlling the Notch pathway and hence the balance between cell proliferation and cell differentiation.

The secreted Frizzled-Related Protein 2 modulates cell fate and the Wnt pathway in the murine intestinal epithelium.

The secreted Frizzled-Related Proteins (sFRPs) are generally considered antagonistic to Wnt signaling. However, several studies have described their synergy and/or activation of this pathway. Our own data indicated that in the intestinal epithelium, thyroid hormone induced-expression of sFRP2 stabilizes ß-catenin, leading to induction of Wnt. The aim of this work was to investigate the role of sFRP2 in the intestinal epithelium homeostasis and its specific effect on canonical Wnt pathway. In wild type animals we observed a restricted pattern of sFRP2 protein expression at the level of the intestinal crypts. Interestingly, sFRP2(-/-) mice displayed increased apoptosis within the crypts together with a defect in cell migration. Because of altered proportion of lineage-specific committed progenitors, the sFRP2(-/-) animals also showed a decrease of absorptive differentiation counterbalanced by an increase of secretory differentiation. Regarding the action of sFRP2 on canonical Wnt pathway, the lack of sFRP2 expression in sFRP2(-/-)/TopGal animals in vivo reduced the Wnt activity. This positive action of sFRP2 on Wnt was further confirmed by in vitro studies. In conclusion, in accordance with its restricted expression profile, sFRP2 contributes to the physiology of the intestinal epithelial crypt progenitors by controlling apoptosis, cell fate decisions and the Wnt pathway.

Telomere protection and TRF2 expression are enhanced by the canonical Wnt signalling pathway.

The DNA-binding protein TRF2 is essential for telomere protection and chromosome stability in mammals. We show here that TRF2 expression is activated by the Wnt/ß-catenin signalling pathway in human cancer and normal cells as well as in mouse intestinal tissues. Furthermore, ß-catenin binds to TRF2 gene regulatory regions that are functional in a luciferase transactivating assay. Reduced ß-catenin expression in cancer cells triggers a marked increase in telomere dysfunction, which can be reversed by TRF2 overexpression. We conclude that the Wnt/ß-catenin signalling pathway maintains a level of TRF2 critical for telomere protection. This is expected to have an important role during development, adult stem cell function and oncogenesis.

Thyroid hormones and their nuclear receptors: new players in intestinal epithelium stem cell biology?

Thyroid hormones participate in the development and homeostasis of several organs and tissues. It is well documented that they act via nuclear receptors, the TRs, which are transcription factors whose function is modulated by the hormone T3. Importantly, T3-induced physiological response within a cell depends on the specific TR expression and on the T3 bioavailability. However, in addition to this T3-dependent control of TR functionality, increasing data show that the action of TRs is coordinated and integrated with other signaling pathways, specifically at the level of stem/progenitor cell populations. By focusing on the intestinal epithelium of both amphibians and mammals we summarize here new data in support of a role for thyroid hormones and the TR nuclear receptors in stem cell biology. This new concept may be extended to other organs and have biological relevance in therapeutic approaches aimed to target stem cells such as tissue engineering and cancer.

The Xenopus doublesex-related gene Dmrt5 is required for olfactory placode neurogenesis.

The Dmrt (doublesex and mab-3 related transcription factor) genes encode a large family of evolutionarily conserved transcription factors whose function in sex specific differentiation has been well studied in all animal lineages. In vertebrates, their function is not restricted to the developing gonads. For example, Xenopus Dmrt4 is essential for neurogenesis in the olfactory system. Here we have isolated and characterized Xenopus Dmrt5 and found that it is coexpressed with Dmrt4 in the developing olfactory placodes. As Dmrt4, Dmrt5 is positively regulated in the ectoderm by neural inducers and negatively by proneural factors. Both Dmrt5 and Dmrt4 genes are also activated by the combined action of the transcription factor Otx2, broadly transcribed in the head ectoderm and of Notch signaling, activated in the anterior neural ridge. As for Dmrt4, knockdown of Dmrt5 impairs neurogenesis in the embryonic olfactory system and in neuralized animal caps. Conversely, its overexpression promotes neuronal differentiation in animal caps, a property that requires the conserved C-terminal DMA and DMB domains. We also found that the sea anenome Dmrt4/5 related gene NvDmrtb also induces neurogenesis in Xenopus animal caps and that conversely, its knockdown in Nematostella reduces elav-1 positive neurons. Together, our data identify Dmrt5 as a novel important regulator of neurogenesis whose function overlaps with that of Dmrt4 during Xenopus olfactory system development. They also suggest that Dmrt may have had a role in neurogenesis in the last common ancestor of cnidarians and bilaterians.

Thyroid hormone's action on progenitor/stem cell biology: new challenge for a classic hormone?

BACKGROUND: Thyroid hormones are involved in developmental and homeostatic processes in several tissues. Their action results in different outcomes depending on the developmental stage, tissue and/or cellular context. Interestingly, their pleiotropic roles are conserved across vertebrates. It is largely documented that thyroid hormones act via nuclear receptors, the TRs, which are transcription factors and whose activity can be modulated by the local availability of the hormone T3. In the "classical view", the T3-induced physiological response depends on the expression of specific TR isoforms and the iodothyronine deiodinase selenoenzymes that control the local level of T3, thus TR activity. SCOPE OF THE REVIEW: Recent data have clearly established that the functionality of TRs is coordinated and integrated with other signaling pathways, specifically at the level of stem/progenitor cell populations. Here, we summarize these data and propose a new and intriguing role for thyroid hormones in two selected examples. MAJOR CONCLUSIONS: In the intestinal epithelium and the retina, TRα1 and TRß2 are expressed at the level of the precursors where they induce cell proliferation and differentiation, respectively. Moreover, these different functions result from the integration of the hormone signal with other intrinsic pathways, which play a fundamental role in progenitor/stem cell physiology. GENERAL SIGNIFICANCE: Taken together, the interaction of TRs with other signaling pathways, specifically in stem/progenitor cells, is a new concept that may have biological relevance in therapeutic approaches aimed to target stem cells such as tissue engineering and cancer. This article is part of a Special Issue entitled Thyroid hormone signalling.

Expanding roles for the evolutionarily conserved Dmrt sex transcriptional regulators during embryogenesis.

Dmrt genes encode a large family of transcription factors characterized by the presence of a DM domain, an unusual zinc finger DNA binding domain. While Dmrt genes are well known for their important role in sexual development in arthropodes, nematodes and vertebrates, several new findings indicate emerging functions of this gene family in other developmental processes. Here, we provide an overview of the evolution, structure and mechanisms of action of Dmrt genes. We summarize recent findings on their function in sexual regulation and discuss more extensively the role played by these proteins in somitogenesis and neural development.

The thyroid hormones and their nuclear receptors in the gut: from developmental biology to cancer.

The thyroid hormones control the development and the homeostasis of several organs in vertebrates. Their actions depend, for the most part, on nuclear receptors, the TRs, which are transcription factors whose activity is modulated by the hormone T3. The gastrointestinal tract is a well characterized target of thyroid hormones and TRs, as extensively described in the literature. In fact, its remodeling in amphibians during thyroid hormone-dependent metamorphosis is well characterized at the cellular and the molecular levels. However, whereas a great attention has been paid to the nervous system and to cardiac development and physiology, the function of thyroid hormones and TRs in the mammalian gastrointestinal tract has been, until recently, underestimated. Several studies have described an important conservation of this hormonal signal during intestinal development and have suggested that it may play a role in stem cell physiology in both amphibians and mammals. These findings show the importance of the thyroid hormones and TRs, whose homologous actions are maintained across species. In the present review, we summarize the most recent data on this issue, starting from work that has been conducted on amphibian metamorphosis to results on postnatal development, homeostasis, and tumorigenesis in mammals. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

Thyroid Hormone Signaling and Function: News from Classical and Emerging Models.

According to Brown and Cai, Thyroid hormones (THs) have been considered "the first developmental morphogen ever discovered" [...].

Cooperation between the thyroid hormone receptor TRalpha1 and the WNT pathway in the induction of intestinal tumorigenesis.

BACKGROUND & AIMS: Colorectal tumorigenesis is a multistep process involving the alteration of oncogenes and tumor suppressor genes, leading to the deregulation of molecular pathways that govern intestinal homeostasis. We have previously shown that the thyroid hormone receptor alpha1 (TRalpha1) controls intestinal development and homeostasis through the WNT pathway. More precisely, TRalpha1 directly enhances the transcription of several components of this pathway, allowing increased expression of beta-catenin/Tcf4 target genes and stimulation of cell proliferation. Because the WNT pathway is a major player in controlling intestinal homeostasis, we addressed whether the TRalpha1 receptor has tumor-inducing potential. METHODS: We generated mice overexpressing TRalpha1 specifically in the intestinal epithelium in a wild-type (vil-TRalpha1) or a WNT-activated (vil-TRalpha1/Apc(+/1638N)) genetic background. RESULTS: The intestine of vil-TRalpha1 mice presents aberrant intestinal mucosal architecture and increased cell proliferation and develops adenoma at a low rate. However, TRalpha1 overexpression is unable to induce cancer development. On the contrary, we observed accelerated tumorigenesis in vil-TRalpha1/Apc(+/1638N) mice compared with the Apc(+/1638N) mutants. CONCLUSION: Our results suggest that this phenotype is due to cooperation between the activated TRalpha1 and WNT pathways. This is the first report describing the tumor-inducing function of TRalpha1 in the intestine.

Thyroid Hormone Nuclear Receptor TRα1 and Canonical WNT Pathway Cross-Regulation in Normal Intestine and Cancer.

A pivotal role of thyroid hormones and their nuclear receptors in intestinal development and homeostasis have been described, whereas their involvement in intestinal carcinogenesis is still controversial. In this perspective article we briefly summarize the recent advances in this field and present new data regarding their functional interaction with one of the most important signaling pathway, such as WNT, regulating intestinal development and carcinogenesis. These complex interactions unveil new concepts and will surely be of importance for translational research.

Cyanidiophyceae (Rhodophyta) Tolerance to Precious Metals: Metabolic Response to Palladium and Gold.

Polyextremophilic red algae, which belong to the class Cyanidiophyceae, are adapted to live in geothermal and volcanic sites. These sites often have very high concentrations of heavy and precious metals. In this study, we assessed the capacity of three strains of Galdieria (G. maxima, G. sulphuraria, and G. phlegrea) and one strain of Cyanidiumcaldarium to tolerate different concentrations of precious metals, such as palladium (Cl4K2Pd) and gold (AuCl4K) by monitoring algal growths in cultures exposed to metals, and we investigated the algae potential oxidative stress induced by the metals. This work provides further understanding of metals responses in the Cyanidiophyceae, as this taxonomic class is developed as a biological refinement tool.

Selection and validation of a set of reliable reference genes for quantitative RT-PCR studies in the brain of the Cephalopod Mollusc Octopus vulgaris.

BACKGROUND: Quantitative real-time polymerase chain reaction (RT-qPCR) is valuable for studying the molecular events underlying physiological and behavioral phenomena. Normalization of real-time PCR data is critical for a reliable mRNA quantification. Here we identify reference genes to be utilized in RT-qPCR experiments to normalize and monitor the expression of target genes in the brain of the cephalopod mollusc Octopus vulgaris, an invertebrate. Such an approach is novel for this taxon and of advantage in future experiments given the complexity of the behavioral repertoire of this species when compared with its relatively simple neural organization. RESULTS: We chose 16S, and 18S rRNA, actB, EEF1A, tubA and ubi as candidate reference genes (housekeeping genes, HKG). The expression of 16S and 18S was highly variable and did not meet the requirements of candidate HKG. The expression of the other genes was almost stable and uniform among samples. We analyzed the expression of HKG into two different set of animals using tissues taken from the central nervous system (brain parts) and mantle (here considered as control tissue) by BestKeeper, geNorm and NormFinder. We found that HKG expressions differed considerably with respect to brain area and octopus samples in an HKG-specific manner. However, when the mantle is treated as control tissue and the entire central nervous system is considered, NormFinder revealed tubA and ubi as the most suitable HKG pair. These two genes were utilized to evaluate the relative expression of the genes FoxP, creb, dat and TH in O. vulgaris. CONCLUSION: We analyzed the expression profiles of some genes here identified for O. vulgaris by applying RT-qPCR analysis for the first time in cephalopods. We validated candidate reference genes and found the expression of ubi and tubA to be the most appropriate to evaluate the expression of target genes in the brain of different octopuses. Our results also underline the importance of choosing a proper normalization strategy when analyzing gene expression by qPCR taking into appropriate account the experimental setting and variability of the sample of animals (and tissues), thus providing a set of HGK which expression appears to be unaffected by the experimental factor(s).

In Vitro Approaches to Identify Thyroid Hormone Receptor-Dependent Transcriptional Response.

The thyroid hormones act through their nuclear receptor TRs that are T3-modulated transcription factors. To elucidate the molecular mechanisms at the basis of specific physiological responses to TH-TR, in vitro approaches are commonly used to demonstrate the activation or repression of target genes. These approaches allow identifying direct transcriptional targets of TRs which can eventually be confirmed by using in vivo chromatin binding assays.Here, we describe two classical approaches in vitro and in cell lines such as Electro-Mobility Shift Assay and thyroid-hormone-responsive Luciferase-reporter assay (EMSA and Luc, respectively) we have been largely using to investigate TRα1-driven TH-TRs intestinal epithelium cell autonomous action.

Defining suitable reference genes for RT-qPCR analysis on intestinal epithelial cells.

The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopathological conditions. It is worth mentioning that real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) approaches are commonly used, but only a few studies are available regarding suitable reference genes to normalize gene expression data. The present study was designed to validate potential reference genes in freshly isolated proliferating or differentiated epithelial cells from the mouse intestine. We also extended our analysis to the IEC6 intestinal epithelial cells, as a promising model to study intestinal physiopathology in vitro. The stability of six potential reference genes (Hprt1, Ppia, Gapdh, Rplp0, Ppib, and Vil1) has been tested both in epithelial cells isolated from the mouse intestine and in the IEC6 cell line. The software programs-geNorm and Normfinder-were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for RT-qPCR data normalization. These multiple approaches allowed us to select different suitable reference genes for the correct quantification of mRNAs depending on the differentiated or proliferative nature of the cells.

Multi-level interactions between the nuclear receptor TRα1 and the WNT effectors ß-catenin/Tcf4 in the intestinal epithelium.

Intestinal homeostasis results from complex cross-regulation of signaling pathways; their alteration induces intestinal tumorigenesis. Previously, we found that the thyroid hormone nuclear receptor TRα1 activates and synergizes with the WNT pathway, inducing crypt cell proliferation and promoting tumorigenesis. Here, we investigated the mechanisms and implications of the cross-regulation between these two pathways in gut tumorigenesis in vivo and in vitro. We analyzed TRα1 and WNT target gene expression in healthy mucosae and tumors from mice overexpressing TRα1 in the intestinal epithelium in a WNT-activated genetic background (vil-TRα1/Apc mice). Interestingly, increased levels of ß-catenin/Tcf4 complex in tumors from vil-TRα1/Apc mice blocked TRα1 transcriptional activity. This observation was confirmed in Caco2 cells, in which TRα1 functionality on a luciferase reporter-assay was reduced by the overexpression of ß-catenin/Tcf4. Moreover, TRα1 physically interacted with ß-catenin/Tcf4 in the nuclei of these cells. Using molecular approaches, we demonstrated that the binding of TRα1 to its DNA target sequences within the tumors was impaired, while it was newly recruited to WNT target genes. In conclusion, our observations strongly suggest that increased ß-catenin/Tcf4 levels i) correlated with reduced TRα1 transcriptional activity on its target genes and, ii) were likely responsible for the shift of TRα1 binding on WNT targets. Together, these data suggest a novel mechanism for the tumor-promoting activity of the TRα1 nuclear receptor.