A Mouse Model of Targeted Musashi1 Expression in Whole Intestinal Epithelium Suggests Regulatory Roles in Cell Cycle and Stemness.

The intestinal epithelium is very peculiar for its continuous cell renewal, fuelled by multipotent stem cells localized within the crypts of Lieberkühn. Several lines of evidence have established the evolutionary conserved RNA-binding protein Musashi1 as a marker of adult stem cells, including those of the intestinal epithelium, and revealed its roles in stem cell self-renewal and cell fate determination. Previous studies from our laboratories have shown that Musashi1 controls stem cell-like features in medulloblastoma, glioblastoma, and breast cancer cells, and has pro-proliferative and pro-tumorigenic properties in intestinal epithelial progenitor cells in vitro. To undertake a detailed study of Musashi1's function in the intestinal epithelium in vivo, we have generated a mouse model, referred to as v-Msi, overexpressing Musashi1 specifically in the entire intestinal epithelium. Compared with wild type litters, v-Msi1 mice exhibited increased intestinal crypt size accompanied by enhanced proliferation. Comparative transcriptomics by RNA-seq revealed Musashi1's association with gut stem cell signature, cell cycle, DNA replication, and drug metabolism. Finally, we identified and validated three novel mRNA targets that are stabilized by Musashi1, Ccnd1 (Cyclin D1), Cdk6, and Sox4. In conclusion, the targeted expression of Musashi1 in the intestinal epithelium in vivo increases the cell proliferation rate and strongly suggests its action on stem cells activity. This is due to the modulation of a complex network of gene functions and pathways including drug metabolism, cell cycle, and DNA synthesis and repair.

Genetic analysis reveals different functions for the products of the thyroid hormone receptor alpha locus.

Thyroid hormone receptors are encoded by the TRalpha (NR1A1) and TRbeta (NR1A2) loci. These genes are transcribed into multiple variants whose functions are unclear. Analysis by gene inactivation in mice has provided new insights into the functional complexity of these products. Different strategies designed to modify the TRalpha locus have led to strikingly different phenotypes. In order to analyze the molecular basis for these alterations, we generated mice devoid of all known isoforms produced from the TRalpha locus (TRalpha(0/0)). These mice are viable and exhibit reduced linear growth, bone maturation delay, moderate hypothermia, and reduced thickness of the intestinal mucosa. Compounding TRalpha(0) and TRbeta(-) mutations produces viable TRalpha(0/0)beta(-/-) mice, which display a more severe linear growth reduction and a more profound hypothermia as well as impaired hearing. A striking phenotypic difference is observed between TRalpha(0/0) and the previously described TRalpha(-/-) mice, which retain truncated TRDeltaalpha isoforms arising from a newly described promoter in intron 7. The lethality and severe impairment of the intestinal maturation in TRalpha(-/-) mice are rescued in TRalpha(0/0) animals. We demonstrate that the TRDeltaalpha protein isoforms, which are natural products of the TRalpha locus, are the key determinants of these phenotypical differences. These data reveal the functional importance of the non-T3-binding variants encoded by the TRalpha locus in vertebrate postnatal development and homeostasis.

Functional interference between thyroid hormone receptor alpha (TRalpha) and natural truncated TRDeltaalpha isoforms in the control of intestine development.

Thyroid hormone is known to participate in the control of intestine maturation at weaning. Its action is mediated by the thyroid hormone nuclear receptors, encoded by the TRalpha and TRbeta genes. Since previous studies have shown that TRbeta plays a minor role in the gut, we focused here our analysis on the TRalpha gene. The TRalpha locus generates the TRalpha1 receptor together with the splicing variant TRalpha2 and the truncated products TRDeltaalpha1 and TRDeltaalpha2, which all lack an intact ligand binding domain. The TRDeltaalpha isoforms are transcribed from an internal promoter located in intron 7, and their distribution is restricted to a few tissues including those of the intestine. In order to define the functions of the different isoforms encoded by the TRalpha locus in the intestinal mucosa, we produced mice either lacking all known TRalpha products or harboring a mutation which inactivates the intronic promoter. We performed a detailed analysis of the intestinal phenotypes in these mice and compared it to that of the previously described TRalpha(-/-) mice, in which TRalpha isoforms are abolished but the TRDeltaalpha isoforms remain. This comparative analysis leads us to the following conclusions: (i) the TRalpha1 receptor mediates the T3-dependent functions in the intestine at weaning time and (ii) the TRDeltaalpha products negatively control the responsiveness of the epithelial cells to T3. Moreover, we show that TRDeltaalpha proteins can interfere with the transcription of the intestine-specific homeobox genes cdx1 and cdx2 and that their activity is regulated by TRalpha1. Altogether these data demonstrate that cooperation of TRalpha and TRDeltaalpha products is essential to ensure the normal postnatal development of the intestine and that mutations in the TRalpha locus can generate different phenotypes caused by the disruption of the equilibrium between these products.

Cytokine gene expression during postnatal small intestinal development: regulation by glucocorticoids.

BACKGROUND: In the intestinal mucosa, numerous cytokines produced by the epithelium, fibroblasts, and immune cells were shown to affect epithelial differentiation and proliferation through epithelial-mesenchymal and epithelial-immune cell interactions. To date, the importance of cytokines in postnatal development of the rat small intestine has not been established. AIM: To investigate the developmental changes in expression of mucosal cytokines in the postnatal maturation of the rat small intestinal epithelium and their regulation by glucocorticoids (GC). METHODS: Mucosal maturation was assessed by the onset of sucrase-isomaltase (SI) mRNA, analysed by in situ hybridisation. The amount of transforming growth factor beta1 (TGF-beta1), beta2 (TGF-beta2), tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and TGF-alpha was analysed by reverse transcription-polymerase chain reaction (RT-PCR) in mucosal extracts from weaning (14-21 days old) and adult rats, or one day after an injection of hydrocortisone (HC) in 11 day old rats. Similarly, expression of cytokines and the regulatory effect of GC were studied on cultured subepithelial myofibroblasts cloned from postnatal jejunum and ileum cultured in the absence or presence of dexamethasone (DX). RESULTS: TGF-beta1, TGF-beta2, and IL-1beta decreased during the third week of life while levels of TNF-alpha increased and TGF-alpha remained constant. In parallel, SI transcripts increased and showed a progressive accumulation in the apical part of the enterocytes first localised at the base of the villi from 18 days onwards. Interestingly, precocious induction of SI mRNA by HC paralleled the decrease in expression of TGF-beta isoforms and of IL-1beta. All cytokines were expressed in the myofibroblast cell lines. In addition, the results showed that TNF-alpha was differentially expressed in basal culture conditions and after DX stimulation in jejunal and ileal myofibroblasts. DX decreased IL-1beta but not the TGF-beta isoforms, similar to that in vivo. CONCLUSIONS: This study shows that mucosal cytokines are developmentally regulated and that GC are potentially involved in this regulation in parallel with maturation of the gut mucosa at weaning.

Involvement of T3Ralpha- and beta-receptor subtypes in mediation of T3 functions during postnatal murine intestinal development.

BACKGROUND & AIMS: Thyroid hormones are implicated in intestinal development. Their effects are mediated by nuclear receptors, which are transcriptional regulators activated upon binding of triiodothyronine. The aim of this study was to define the involvement of the receptor subtypes during intestinal development. METHODS: We used strains of knockout mice lacking T3Ralpha, T3Rbeta, or both receptors, encoded by T3Ralpha and T3Rbeta genes. RESULTS: Morphological features and expression of digestive enzymes and of two intestinal regulators, Cdx-1 and Cdx-2, were compared in wild-type and T3Ralpha, T3Rbeta, and T3Ralphabeta knockout animals. T3Ralpha-/- mice had abnormal intestinal morphology, assessed by a decrease in the number of epithelial cells along the crypt-villus axis and a decrease in proliferating crypt cells. Expression of Cdx-1 and Cdx-2, and of the digestive enzymes, was down-regulated. These parameters can be partially reversed by T3 injection. A similar (jejunum) or more severe (ileum) phenotype was found in T3Ralphabeta double mutants. In contrast, no changes occurred in T3Rbeta mice. CONCLUSIONS: These data describe for the first time a direct effect of TH through the T3Ralpha-receptor subtypes on postnatal intestinal mucosa maturation. They also suggest that T3Rbeta receptors are dispensable but can partially substitute for T3Ralpha.

Different functions for the thyroid hormone receptors TRalpha and TRbeta in the control of thyroid hormone production and post-natal development.

The biological activities of thyroid hormones are thought to be mediated by receptors generated by the TRalpha and TRbeta loci. The existence of several receptor isoforms suggests that different functions are mediated by specific isoforms and raises the possibility of functional redundancies. We have inactivated both TRalpha and TRbeta genes by homologous recombination in the mouse and compared the phenotypes of wild-type, and single and double mutant mice. We show by this method that the TRbeta receptors are the most potent regulators of the production of thyroid stimulating hormone (TSH). However, in the absence of TRbeta, the products of the TRalpha gene can fulfill this function as, in the absence of any receptors, TSH and thyroid hormone concentrations reach very high levels. We also show that TRbeta, in contrast to TRalpha, is dispensable for the normal development of bone and intestine. In bone, the disruption of both TRalpha and TRbeta genes does not modify the maturation delay observed in TRalpha -/- mice. In the ileum, the absence of any receptor results in a much more severe impairment than that observed in TRalpha -/- animals. We conclude that each of the two families of proteins mediate specific functions of triiodothyronin (T3), and that redundancy is only partial and concerns a limited number of functions.

Subepithelial fibroblast cell lines from different levels of gut axis display regional characteristics.

The intestine is characterized by morphofunctional differences along the proximodistal axis. The aim of this study was to derive mesenchymal cell lines representative of the gut axis. We isolated and cloned rat intestinal subepithelial myofibroblasts raised from 8-day proximal jejunum, distal ileum, and proximal colon lamina propria. Two clonal cell lines from each level of the gut were characterized. They 1) express the specific markers vimentin, smooth muscle alpha-actin, and smooth muscle myosin heavy chain, revealed by immunofluorescence microscopy and 2) distinctly support endodermal cell growth in a coculture model, depending on their regional origin, and 3) the clones raised from the various proximodistal regions maintain the same pattern of morphogenetic and growth and/or differentiation factor gene expression as in vivo: hepatocyte growth and/or scatter factor and transforming growth factor-beta 1 mRNAs analyzed by RT-PCR were more abundant, in the colon and ileal clones and mucosal connective tissue, respectively. In addition, epimorphin mRNA studied by Northern blot was also the highest in one ileal clone, in which it was selectively upregulated by all-trans retinoic acid (RA) treatment. Epimorphin expression in isolated 8-day intestinal lamina propria was higher in the distal small intestine and proximal colon than in the proximal small intestine. In conclusion, we isolated and characterized homogeneous cell subtypes that can now be used to approach the molecular regulation of the epithelium-mesenchyme-dependent regional specificity along the gut.

Effects of retinoids on gene expression in different epithelial models in vivo and in vitro.

We have previously reported that the induction of Vitamin A deficiency results in a threefold decrease in the hepatic expression of cellular retinol binding protein I (CRBP I) mRNA in vivo and that the treatment of intestinal cell lines in vitro with retinoids leads to the induction of CRBP I transcription. In the present paper we extend the analysis to retinoid-dependent gene expression in the testicular epithelium in vivo and in the intestinal cell line FRIC B. In rat testis excess Vitamin A results in the reduced production of mature spermatozoa and in the premature release of immature germ cells in the lumen, while Vitamin A deficiency leads to almost complete degeneration of the germinal epithelium. We show reduced level of expression of CRBP I mRNA in vitamin A deficient testis. Retinoid treatment of cultured intestinal cells, which induces a reorganization of the actin cytoskeleton, has no effect on the expression of the differentiation induced gene Dri 42. The results show that even though unable to trigger by themselves the differentiation process, retinoids exert a direct effect on the expression of specific genes.

Intestinal epithelial-mesenchymal cell interactions.

Intestinal morphogenesis, as well as maintenance of the stem cell population and of the steady state between cell proliferation and differentiation, results from controlled cell interactions. There is growing evidence that the mesenchymal cells control epithelial cell behavior via their own expression and induction in the epithelial cells of key regulatory genes. This heterologous cross talk involves basement membrane molecules and paracrine factors. New in vitro/in vivo cellular models allowed us to analyze various mesenchymal cell phenotypes and to show that they exhibit different inductive properties on epithelial cells and that their proliferation and metabolic properties are differentially modulated by cytokines. Finally the epithelial-mesenchymal unit is controlled by hormonal and exogenous factors.

The T3R alpha gene encoding a thyroid hormone receptor is essential for post-natal development and thyroid hormone production.

The diverse functions of thyroid hormones are thought to be mediated by two nuclear receptors, T3R alpha1 and T3R beta, encoded by the genes T3R alpha and T3R beta respectively. The T3R alpha gene also produces a non-ligand-binding protein T3R alpha2. The in vivo functions of these receptors are still unclear. We describe here the homozygous inactivation of the T3R alpha gene which abrogates the production of both T3R alpha1 and T3R alpha2 isoforms and that leads to death in mice within 5 weeks after birth. After 2 weeks of life, the homozygous mice become progressively hypothyroidic and exhibit a growth arrest. Small intestine and bones showed a strongly delayed maturation. In contrast to the negative regulatory function of the T3R beta gene on thyroid hormone production, our data show that the T3R alpha gene products are involved in up-regulation of thyroid hormone production at weaning time. Thus, thyroid hormone production might be balanced through a positive T3R alpha and a negative T3R beta pathway. The abnormal phenotypes observed on the homozygous mutant mice strongly suggest that the T3R alpha gene is essential for the transformation of a mother-dependent pup to an 'adult' mouse. These data define crucial in vivo functions for thyroid hormones through a T3R alpha pathway during post-natal development.

Mesenchyme-mediated effects of retinoic acid during rat intestinal development.

In previous experiments we showed that intestinal development was dependent upon epithelial-mesenchymal cell interactions. The aim of this study was to investigate the possible role of retinoic acid (RA), a morphogenetic and differentiating agent, on the gut epithelial-mesenchymal unit. For this purpose we first analyzed the effects of a physiological dose of RA on 14-day fetal rat intestine using short-term organ culture experiments, or long-term grafts under the skin of nude mice. In these conditions, RA accelerated villus outgrowth and epithelial cell differentiation as assessed by the onset of lactase expression, and it also stimulated muscle and crypt formation. In order to analyze potential effects of RA mediated by mesenchymal cells, we isolated and characterized gut mucosa mesenchyme-derived cell cultures (mesenchyme-derived intestinal cell lines, MIC). These cells were shown to express mRNAs for retinoid binding proteins similar to those expressed in situ in the intestinal mesenchyme. MIC cells co-cultured with 14-day intestinal endoderms promoted endodermal cell adhesion and growth, and the addition of exogeneous RA enhanced epithelial cell polarization and differentiation assessed by cytokeratin and lactase immunostaining. Such a differentiating effect of RA was not observed on endodermal cells when cultured without a mesenchymal feeder layer or maintained in conditioned medium from RA-treated MIC cells. In the co-cultures, immunostaining of laminin and collagen IV with polyclonal antibodies, as well as alpha1 and beta1 laminin chains mRNAs (analyzed by RT-PCR) increased concurrently with the RA-enhanced differentiation of epithelial cells. It is worth noting that this stimulation by RA was also obvious on the mesenchymal cells cultured alone. These results show that RA plays a role in intestinal morphogenesis and differentiation. In addition, they indicate that RA acts on the mesenchymal cell phenotype and suggest that RA may modify the mesenchymal-epithelial cell interactions during intestinal development.

Intestinal-type fibroblasts selectively influence proliferation rate and peptide synthesis in the murine entero-endocrine cell line STC-1.

The intestinal epithelium consists of enterocytes, endocrine cells, goblet cells and Paneth cells, which differentiate from pluripotent stem cells located at the crypt bases. The role of the epithelial-mesenchymal inter-actions has been well documented for the differentiation of enterocytes, but the mechanisms that control endocrine cell differentiation are poorly understood. We have cultured the intestinal endocrine cell line STC-1, which synthesizes most of the intestinal peptide hormones, in media conditioned by several subepithelial fibroblast cell lines from three distinct sites of intestine. The fibroblast Swiss 3T3 cell line was used as a non-intestinal control. Our results show that culture media from intestinal fibroblasts inhibit the proliferation rate of STC-1 cells, while those from Swiss 3T3 fibroblasts do not. As regards peptide hormone gene expression, Swiss 3T3-conditioned media have no effect, whereas media from intestinal fibroblasts variably affect cholecystokinin, glucagon, secretin and somatostatin mRNA levels. In particular, clonal subepithelial myofibroblasts do not exert the same effects as mixed subepithelial fibroblasts from homologous intestinal segment. Taken together, these results suggest that cultured fibroblasts of intestinal origin release soluble factors that inhibit STC-1 cell proliferation and modulate, in a region-specific manner, the expression of hormonal peptide genes in this nonspecialized endocrine cell line.

The Dri 42 gene, whose expression is up-regulated during epithelial differentiation, encodes a novel endoplasmic reticulum resident transmembrane protein.

A search for novel genes that are up-regulated during development and differentiation of the epithelial cells of the intestinal mucosa led us to the isolation of the Dri 42 cDNA clone (Dri, differentially expressed in rat intestine). The nucleotide sequence of the full-length cDNA has shown that it encodes a 35.5-kDa protein with one consensus sequence for N-linked glycosylation and alternating hydrophilic and hydrophobic domains. To determine the intracellular localization of Dri 42 we have raised polyclonal antibodies in hens against a bacterially produced Dri 42-glutathione S-transferase fusion protein. Immunofluorescence detection with these antibodies has shown specific staining of the endoplasmic reticulum (ER) in the relatively undifferentiated fetal rat intestinal cell line FRIC B and in sections of rat small intestine. ER membrane localization of Dri 42 was confirmed by laser confocal microscopy of polarized Madin-Darby canine kidney cells overexpressing a Dri 42-chloramphenicol acetyltransferase (CAT) fusion protein by transfection. Pulse labeling experiments on transiently transfected cells demonstrated that the protein does not acquire Golgi modifications up to 4 h after synthesis, thus indicating that Dri 42 is an ER resident protein. The transmembrane disposition of Dri 42 was studied using in vitro insertion of Dri 42-CAT fusion proteins into microsomal membranes. The fusion proteins consisted of several different lengths of truncated Dri 42 and a reporter protein, CAT, that was linked in-frame after each hydrophobic segment. We found that hydrophobic segments H1, H3, and H5 had a signal/anchor function, and that membrane insertion of Dri 42 was achieved co-translationally by the action of a series of alternating insertion signals and halt transfer signals, resulting in the exposure of both termini of the protein to the cytosolic side. The functional implications of the structure and localization of Dri 42, whose primary sequence does not share significant homology to any previously described protein, are discussed.

Synapsin I expression in spinal cord neurons during chick embryo development.

The cellular distribution of synapsin I in chick spinal cord has been examined during embryo development and in cultured neurons from different developmental stages. Using immunocytochemical methods we have observed that synapsin I appears lightly detectable in spinal cord of embryonic day (E)5-E8 embryos when the motor neurons have already established functional contacts with muscle fibers, and increases at E9. Until E8 synapsin I immunoreactivity appeared mainly localized in the gray matter of spinal cord; immunostaining of white matter becomes clearly evident only at E9. These observations indicate that synapsin I expression and possibly its transport to the nerve terminals may be stimulated by sequential signals. The cellular distribution of synapsin I observed in vivo is maintained in E8 and E9 spinal cord neuron cell cultures. In fact, in E8 cultured neurons, synapsin I immunostaining is observed only in the cell body, while in E9 cultured neurons both cell body and fibers are stained. The addition of muscle extracts to E8 cultures induces synapsin I decoration of fibers similar to that observed in E9 cultured neurons. Indeed Western and Northern blot analysis and in situ hybridization demonstrate an increase of synapsin I and its mRNA in spinal cord neurons kept in the presence of muscle extracts. These data suggest that synapsin I expression, as previously reported for other neuronal markers, can be modulated by soluble factors present in target cells.

Cholinergic markers are expressed in developing and mature neurons of chick dorsal root ganglia.

The presence of acetylcholinesterase has been reported in chick dorsal root ganglia at early developmental stages although acetylcholine is not known to play a role in these ganglia. Recently, we reported that during development the level of acetylcholinesterase increases continuously and the enzyme becomes gradually expressed in all sensory neurons. These observations prompted the study of the developmental pattern of expression of other cholinergic markers, such as choline acetyltransferase (ChAT) and the high affinity transport mechanism for choline. ChAT activity is barely detectable at early developmental stages (E7) and increases markedly thereafter, with an activity profile similar to that described for acetylcholinesterase. A similar increase in enzyme activity is also observed when ChAT is measured in dorsal root ganglia explants and in dissociated cells in culture. The study of ChAT activity in cultured cells shows an increase over a period of 3 days, thus ruling out the hypothesis that motor fibers, still associated to the ganglia, may represent a possible source of the enzyme. Immunostaining of whole ganglia or cultured cells shows that ChAT immunoreactivity is not restricted to a specific neuronal sub-population but appears as a common marker of sensory neurons. High affinity choline uptake, blocked by hemicholinium, is present in sensory neurons cultured from E7 dorsal root ganglia. Observations on cultured neurons from later stages (E18) indicate that choline transport is not a transient property of sensory neurons. These observations show a similar pattern of expression of several cholinergic markers during development. Such a pattern is maintained at significant levels also in mature ganglia.

Expression of epithelial markers and retinoid-binding proteins in retinol- or retinoic acid-treated intestinal cells in vitro.

This study was undertaken with the aim of investigating the effects of retinoids on the expression of differentiated traits in intestinal cell models. The cell lines used included epithelial cells isolated from fetal rat intestines (FRIC), displaying a relatively undifferentiated phenotype, and the human colon adenocarcinoma cell lines Caco 2 and HT29, which express some of the traits of the mature enterocytes under defined culture conditions. The effects of retinoids were also studied in organ cultures of fetal rat intestine, where the epithelial-mesenchymal interactions are preserved. All-trans-retinol and all-trans-retinoic acid treatments were compared in their ability to regulate the expression of genes coding for proteins involved in retinoid metabolism and for cytoskeletal proteins. The results have shown that the effects of the two retinoids were qualitatively similar. A specific induction of the cellular retinol-binding protein CRBP I mRNA was observed following retinoid treatment in one of the two FRIC lines examined (FRIC B) and in organ culture. The expression of the retinoic acid receptors RAR alpha and gamma was not affected by treatment in any of the cultures examined, while RAR beta was expressed only by the organ cultures and was transcriptionally induced by retinoic acid treatment. The retinoids also induced a reorganization of the actin cytoskeleton in the FRIC B cell line, accompanied by a decrease in the expression of two components of the microvillar cytoskeleton, ezrin and villin. The results obtained in both cell and organ cultures suggest that retinoids alone are not able to trigger the differentiation program in the intestinal epithelial cell, irrespective of the level of differentiation already achieved at the time of treatment.