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Thyroid hormones are involved in the regulation of many physiological processes and regulate gene transcription by binding to their nuclear receptors TRalpha and TRbeta. In the absence of triiodothyronine (T3), the unliganded receptors (aporeceptors) do bind DNA and repress the transcription of target genes. The role of thyroid hormone aporeceptors as repressors was observed in hypothyroid adult mice, but its physiological relevance in nonpathological hypothyroid conditions remained to be determined. Here we show that, in the normal mouse fetus, TRalpha aporeceptors repress heart rate as well as the expression of TRbeta and several genes encoding ion channels involved in cardiac contractile activity. Right after birth, when T3 concentration sharply increases, liganded TRalpha (holoreceptors) turn on the expression of some of these same genes concomitantly with heart rate increase. These data describe a physiological situation under which conversion of TRalpha from apo-receptors into holo-receptors, upon changes in T3 availability, plays a determinant role in a developmental process.

The existence of different strains of infectious agents involved in scrapie, a transmissible spongiform encephalopathy (TSE) of sheep and goats, remains poorly explained. These strains can, however, be differentiated by characteristics of the disease in mice and also by the molecular features of the protease-resistant prion protein (PrP(res)) that accumulates into the infected tissues. For further analysis, we first transmitted the disease from brain samples of TSE-infected sheep to ovine transgenic [Tg(OvPrP4)] and to wild-type (C57BL/6) mice. We show that, as in sheep, molecular differences of PrP(res) detected by Western blotting can differentiate, in both ovine transgenic and wild-type mice, infection by the bovine spongiform encephalopathy (BSE) agent from most scrapie sources. Similarities of an experimental scrapie isolate (CH1641) with BSE were also likewise found following transmission in ovine transgenic mice. Secondly, we transmitted the disease to ovine transgenic mice by inoculation of brain samples of wild-type mice infected with different experimental scrapie strains (C506M3, 87V, 79A, and Chandler) or with BSE. Features of these strains in ovine transgenic mice were reminiscent of those previously described for wild-type mice, by both ratios and by molecular masses of the different PrP(res) glycoforms. Moreover, these studies revealed the diversity of scrapie strains and their differences with BSE according to labeling by a monoclonal antibody (P4). These data, in an experimental model expressing the prion protein of the host of natural scrapie, further suggest a genuine diversity of TSE infectious agents and emphasize its linkage to the molecular features of the abnormal prion protein.

Thyroid hormones (TH) are essential regulators of vertebrate development and metabolism. Central mechanisms governing their production have evolved, with the beta-TH receptor (TRbeta) playing a key regulatory role in the negative feedback effects of circulating TH levels on production of hypothalamic TRH and hypophyseal TSH. Both TRbeta-isoforms (TRbeta1 and TRbeta2) are expressed in the hypothalamus and pituitary. However, their respective roles in TH-dependent transcriptional regulation of TRH are undefined. We confirmed the preferential role of TRbeta vs. TRalpha isoforms in TRH regulation in wild-type mice in vivo by using the TRbeta preferential agonist GC-1. We next determined the effects of tissue-specific rescue of TRbeta1 and TRbeta2 isoforms by somatic gene transfer in hypothalami of TRbeta null (TRbeta(-/-)) mice. TH-dependent TRH transcriptional repression was impaired in TRbeta(-/-) mice, but was restored by cotransfection of either TRbeta1 or TRbeta2 into the hypothalamus. TRbeta1, but not TRbeta2, displayed a role in ligand-independent activation. In situ hybridization was used to examine endogenous TRH expression in the paraventricular nucleus of the hypothalamus of TRbeta(-/-) or TRalpha null (TRalpha(o/o)) mice under different thyroid states. In contrast to published data on TRbeta2(-/-) mice, we found that both ligand-independent TRH activation and ligand-dependent TRH repression were severely impaired in TRbeta(-/-) mice. This study thus provides functional in vivo data showing that both TRbeta1 and TRbeta2 isoforms have specific roles in regulating TRH transcription.

cENS-1/cERNI genes have been shown to be expressed very early during chicken embryonic development and as well as in pluripotent chicken embryonic stem (CES) cells. We have previously identified a promoter region, which is specifically active in CES cells compared to differentiated cells. In order to understand the molecular mechanisms which regulate the cENS-1/cERNI promoter, we analyzed the cis-acting elements of this promoter in CES and differentiated cells. We identified a short sequence, named the B region, 5'-CAAG TCCAGG CAAG-3', that exhibits a strong enhancer activity in CES and differentiated cells. Mutation of the B region in the whole cENS-1 promoter strongly decreases the promoter activity in CES cells, suggesting that this region is essential for activating the promoter. The B region is similar to the previously described response element for the transcription factor CP2 and we show by supershift experiments that a protein complex containing CP2 is bound to this B response element. All these results identify a nuclear factor belonging to the CP2 transcription factor family that is crucial for the activation of the cENS-1/cERNI promoter. The pattern of expression of cCP2 in early chicken embryo before gastrulation is very similar to that of cENS-1/cERNI which strongly suggests that cCP2 also plays an essential role in gene expression early in embryonic development.

The capacity of embryonic stem cells (ES cells) to differentiate into neuronal cells represents a potential source for neuronal replacement and a model for studying factors controlling early stages of neuronal differentiation. Various molecules have been used to induce such differentiation but so far neuropeptides acting via functional G-protein-coupled receptors (GPCRs) have not been investigated. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides expressed in early development which affect neuronal precursor proliferation and neuronal differentiation. VIP and PACAP share two common receptors (VPAC1 and VPAC2 receptors) while only PACAP binds with high affinity to PAC1 receptors. The aim of the study was to determine whether VIP and PACAP could produce functional neuronal differentiation of ES cells. Mouse ES cells were allowed to aggregate in embryoid bodies (EBs) in the presence or not of VIP and PACAP for 1 week. VIP and PACAP potently increased the proportion of EB-derived cells expressing specifically a neuronal phenotype shown by immunocytochemistry and neurite outgrowth without altering glial cell number. Binding and RT-PCR analyses demonstrated the presence of VPAC2 and PAC1 receptors on ES cells. Accordingly, both peptides increased cyclic AMP and intracellular calcium. In contrast, EB-derived cells only expressed a functional PAC1 receptor, suggesting a switch in GPCR phenotype during ES cell differentiation. These original data demonstrate that functional GPCRs for VIP and PACAP are present on ES cells and that these neuropeptides may induce their differentiation into a neuronal phenotype. It opens an exciting new field for neuropeptide regulation of tissue ontogenesis.

Thyroid hormone (T3) and the T3 receptor (TR) alpha gene are essential for bone development whereas adult hyperthyroidism increases the risk of osteoporotic fracture. We isolated fibroblast growth factor receptor-1 (FGFR1) as a T3-target gene in osteoblasts by subtraction hybridization. FGFR1 mRNA was induced 2- to 3-fold in osteoblasts treated with T3 for 6-48 h, and FGFR1 protein was stimulated 2- to 4-fold. Induction of FGFR1 was independent of mRNA half-life and abolished by actinomycin D and cycloheximide, indicating the involvement of an intermediary protein. Fibroblast growth factor 2 (FGF2) stimulated MAPK in osteoblasts, and pretreatment with T3 for 6 h induced a more rapid response to FGF that was increased in magnitude by 2- to 3-fold. Similarly, T3 enhanced FGF2-activated autophosphorylation of FGFR1, but did not modify FGF2-induced phosphorylation of the docking protein FRS2. These effects were abolished by the FGFR-selective inhibitors PD166866 and PD161570. In situ hybridization analyses of TRalpha-knockout mice, which have impaired ossification and skeletal mineralization, revealed reduced FGFR1 mRNA expression in osteoblasts and osteocytes, whereas T3 failed to stimulate FGFR1 mRNA or enhance FGF2-activated MAPK signaling in TRalpha-null osteoblasts. These findings implicate FGFR1 signaling in T3-dependent bone development and the pathogenesis of skeletal disorders resulting from thyroid disease.

Little is known about the overall patterns of thyroid hormone (Th)-mediated gene regulation by the main Th receptor (Tr) isoforms, Tr-alpha and Tr-beta, in vivo. We used 48 complementary DNA microarrays to examine hepatic gene expression profiles of wild-type and Thra and Thrb knockout mice under different Th conditions: no treatment, treatment with 3,3',5-triiodothyronine (T(3)), Th-deprivation using propylthiouracil (PTU), and treatment with a combination of PTU and T(3). Hierarchical clustering analyses showed that positively regulated genes fit into three main expression patterns. In addition, only a subpopulation of target genes repressed basal transcription in the absence of ligand. Interestingly, Thra and Thrb knockout mice showed similar gene expression patterns to wild-type mice, suggesting that these isoforms co-regulate most hepatic target genes. Differences in the gene expression patterns of Thra/Thrb double-knockout mice and Th-deprived wild-type mice show that absence of receptor and of hormone can have different effects. This large-scale study of hormonal regulation reveals the functions of Th and of Tr isoforms in the regulation of gene expression patterns.

Autoregulation of thyroid hormone (TH) receptors (TRs) is a mechanism whereby a cell can regulate its responsiveness to TH. Nuclear coactivators (NCoAs) modulate TH action and may also be important for regulation of TR expression. We have determined the effect of TH withdrawal and treatment on the expression of different isoforms of TR as well as expression of the NCoAs SRC-1, TIF-2 and SRC-3 using quantitative real time polymerase chain reaction. In order to identify the effect that each TR isoform exerts over the expression of the other, NCoA and TR transcripts were measured in liver and heart tissue from wild type mice or mice with deletion of either TR isoform or SRC-1 genes. In liver, regulation of TR beta1 and TR alpha2 subtype expression is inversely related to TH levels and the regulation of TR beta expression is, in part, controlled by TR alpha. In the heart, the opposite is the case, regulation of TR alpha2 and TR beta1 isoform expression is directly related to TH levels and this regulation is primarily controlled by TR alpha. Although NCoAs are, in general, increased in response to hypothyroidism or in states of TH resistance, SRC-1 specifically does not regulate TR isoform expression. We have demonstrated that TR isoforms and NCoAs are autoregulated transcription factors with tissue specificity.

Steroid receptor coactivator-1 (SRC-1) is a transcription cofactor that enhances the hormone-dependent action mediated by the thyroid hormone (TH) receptor (TR) as well as other nuclear receptors. However, it is not known whether the SRC-1-mediated activation of TH-regulated gene transcription is TR isoform specific in the pituitary. We generated mice that were deficient in TRalpha and SRC-1 (TRalpha(0/0)SRC-1(-/-)), as well in TRbeta and SRC-1 (TRbeta(-/-)SRC-1(-/-)), and thyroid function tests and effects of TH deprivation and TH treatment were compared with wild-type mice or mice with deletion of either TRs or SRC-1 alone. We have shown that 1) TRbeta(-/-)SRC-1(-/-) mice demonstrate more severe TH resistance than either the SRC-1(-/-) or TRbeta(-/-) mice; the additive effect indicates that SRC-1 has an independent role in TH action over that of TRbeta; 2) SRC-1 facilitates TRbeta and TRalpha-mediated down-regulation of TSH, as TRalpha(0/0)SRC-1(-/-) mice demonstrate TH resistance rather than hypersensitivity as seen in TRalpha(0/0)mice; and 3) a compensatory increase in SRC-1 expression is associated with the TH hypersensitivity seen in TRalpha-deficient animals. We conclude that SRC-1 action in the pituitary mediates TH action via specific TR subtypes.

The genes encoding thyroid hormone receptor alpha and beta (TRalpha and TRbeta) encode four thyroid hormone receptors and four variant isoforms with antagonistic properties. Because of this complexity, numerous models of TR mutation have been developed to understand the functions of specific receptors. In total, 13 mutant strains are now available. Phenotype analysis has shown that the two genes serve distinct functions: TRalpha is crucial for postnatal development and cardiac function, whereas TRbeta mainly controls inner ear and retina development, liver metabolism and thyroid hormone levels. These mouse mutant strains also provide us with the unique opportunity to address the respective contribution of each receptor isoform and isotype in vivo and highlight the in vivo importance of the ligand-independent function of the TR gene products.

Thyroid hormone is a major regulator of postnatal brain development, but the precise molecular mechanisms underlying its action in this organ remain poorly understood. We used microarray analysis to identify new target genes in brain. Thyroid hormone treatment of hypothyroid Pax8(-/-) knockout mice, which lack thyroid follicular cells, had a very limited global effect on brain transcripts. This analysis mainly identified cyclin D2 as a new thyroid hormone target gene in the cerebellum of hypothyroid mice. Thyroid hormone receptor (TRalpha and/or TRbeta) knockout mice studies provided further genetic evidence that cyclin D2 is likely to mediate the antiapoptotic effect exerted by thyroid hormone on the cerebellum external granular layer neuroblasts but that this transcriptional activation is not directly exerted by the thyroid hormone receptors., (Copyright 2003 Elsevier Science (USA))

Thyroid hormone modulates the expression of numerous genes that in turn regulate lipoprotein metabolism in vivo. We have examined the thyroid hormone-dependent regulation of apolipoprotein B (apoB) RNA editing in a strain of congenitally hypothyroid mice (Pax8(-/-)) that lacks thyroid follicular cells. Neonatal Pax8(-/-) mice demonstrate an approximately 10-fold increase in hepatic triglyceride content associated with a decrease in hepatic apoB RNA editing. Thyroid hormone administration resulted in hepatic triglyceride mobilization in conjunction with an increase in hepatic, but not intestinal, apoB RNA editing and without changing total apoB RNA abundance. ApoB RNA editing is mediated by a multicomponent enzyme complex whose catalytic core contains two proteins, apobec-1 and apobec-1 complementation factor (ACF). Hepatic ACF mRNA and protein abundance decreased in Pax8(-/-) mice, with restoration after thyroid hormone administration, whereas apobec-1 mRNA and protein abundance were unchanged. Immunohistochemical analysis revealed increased staining intensity of ACF within hepatocyte nuclei of treated mice, findings confirmed by Western analysis of isolated nuclei. In vitro RNA editing assays demonstrated that supplementation with recombinant ACF alone restored enzymatic activity of S100 extracts from hypothyroid, Pax8(-/-) mice. These data demonstrate that thyroid hormone modulates murine hepatic lipoprotein metabolism in association with tissue-specific effects on apoB RNA editing mediated through alterations in ACF gene expression.

Isoforms of the thyroid hormone receptor (TR)alpha and TRbeta genes mediate thyroid hormone action. How TR isoforms modulate tissue-specific thyroid hormone (TH) action remains largely unknown. The steroid receptor coactivator-1 (SRC-1) is among a group of transcriptional coactivator proteins that bind to TRs, along with other members of the nuclear receptor superfamily, and modulate the activity of genes regulated by TH. Mice deficient in SRC-1 possess decreased tissue responsiveness to TH and many steroid hormones; however, it is not known whether or not SRC-1-mediated activation of TH-regulated gene transcription in peripheral tissues, such as heart and liver, is TR isoform specific. We have generated mice deficient in TRalpha and SRC-1, as well as in TRbeta and SRC-1, and investigated thyroid function tests and effects of TH deprivation and TH treatment compared with wild-type (WT) mice or those deficient in either TR or SRC-1 alone. The data show that 1) in the absence of TRalpha or TRbeta, SRC-1 is important for normal growth; 2) SRC-1 modulates TRalpha and TRbeta effects on heart rate; 3) two new TRbeta-dependent markers of TH action in the liver have been identified, osteopontin (upregulated) and glutathione S-transferase (downregulated); and 4) SRC-1 may mediate the hypersensitivity to TH seen in liver of TRalpha-deficient mice.

We investigated the effect of thyroid hormone (TH) receptor (TR)alpha and -beta isoforms in TH action in the heart. Noninvasive echocardiographic measurements were made in mice homozygous for disruption of TRalpha (TRalpha(0/0)) or TRbeta (TRbeta(-/-)). Mice were studied at baseline, 4 wk after TH deprivation (using a low-iodine diet containing propylthiouracil), and after 4-wk treatment with TH. Baseline heart rates (HR) were similar in wild-type (WT) and TRalpha(0/0) mice but were greater in TRbeta(-/-) mice. With TH deprivation, HR decreased 49% in WT and 37% in TRbeta(-/-) mice and decreased only 5% in TRalpha(0/0) mice from baseline, whereas HR increased in all genotypes with TH treatment. Cardiac output (CO) and cardiac index (CI) in WT mice decreased (-31 and -32%, respectively) with TH deprivation and increased (+69 and +35%, respectively) with TH treatment. The effects of CO and CI were blunted with TH withdrawal in both TRalpha(0/0) (+8 and -2%, respectively) and TRbeta(-/-) mice (-17 and -18%, respectively). Treatment with TH resulted in a 64% increase in LV mass in WT and a 44% increase in TRalpha(0/0) mice but only a 6% increase in TRbeta(-/-) mice (ANOVA P < 0.05). Taken together, these data suggest that TRalpha and TRbeta play different roles in the physiology of TH action on the heart.

High-affinity agonists for the retinoic acid X receptors (RXR) have pleotropic effects when administered to humans. These include induction of hypertriglyceridemia and hypothyroidism. We determined the effect of a novel high-affinity RXR agonist with potent antihyperglycemic effects on thyroid function of female Zucker diabetic rats and nondiabetic littermates and in db/db mice. In both nondiabetic and ZFF rats, AGN194204 causes a 70-80% decrease in thyrotropin (TSH), 3,3',5-triiodothyronine, and thyroxine (T(4)) concentrations. In the db/db mouse, AGN194204 causes a time-dependent decrease in thyroid hormone levels with the fall in TSH that was significant after 1 day of treatment preceding the fall in T(4) levels that was significant at 3 days of treatment. Treatment with AGN194204 caused an initial increase in hepatic 5'-deiodinase mRNA levels which then fell to undetectable levels by 3 days of treatment and continued to be low at 7 days of treatment. After treatment for 5 days with AGN194204, both wild-type and thyroid hormone receptor beta (TR beta(-/-))-deficient mice demonstrated a nearly 50% decrease in serum TSH and T(4) concentrations. The results suggest that a high-affinity RXR agonist with antihyperglycemic activity can cause central hypothyroidism independently of TR beta, the main mediator of hormone-induced TSH suppression.

T2EC are chicken erythrocytic progenitors that balance between self-renewal and differentiation as a function of response to specific growth factors. Their transformation by the v-erbA oncogene locks them into the self-renewal program. We show here that the expression of the VLA-2 integrin alpha2 subunit mRNA is downregulated by v-erbA and that VLA-2 engagement and clustering, brought about by treatment with an alpha2-specific antibody or by culture on the VLA-2 ligand collagen I, inhibits T2EC proliferation. From competition studies using antibodies, VLA-2 was shown to be involved in the collagen-induced response. While engagement of VLA-2 inhibited proliferation, it was not sufficient to induce differentiation. The transformation of T2EC by v-erbA decreased their interaction with collagen I and the VLA-2 brake on cell proliferation, which may account for the increased proliferation potential of transformed erythrocytic progenitors and for their shedding into the blood of infected chickens. Our data suggest that the interaction between erythroid progenitors and collagen, mediated by VLA-2, play a major role in the control of erythropoiesis in vitro and that this pathway is a target of the v-erbA oncogene.

Thyroid hormone (3,5,3'-triiodo-l-thyronine or T3) exerts a pleiotropic activity during central nervous system development. Hypothyroidism during the fetal and postnatal life results in an irreversible mental retardation syndrome. At the cellular level, T3 is known to act on neuronal and glial lineages and to control cell proliferation, apoptosis, migration, and differentiation. Oligodendrocyte precursor cells (OPC) found at birth in the optic nerves are self-renewing cells that normally differentiate during the first 3 weeks of rodent postnatal life into postmitotic myelinating oligodendrocytes. In vitro, the addition of T3 to OPC is sufficient to trigger their terminal differentiation. The present analysis of T3 receptor knockout mice reveals that the absence of all T3 receptor results in the persistence of OPC proliferation in adult optic nerves, in a default in myelination, and sometimes in the degeneration of the retinal ganglion neurons. Thus, T3 signaling is necessary in vivo to promote the complete differentiation of OPC.

Mice devoid of all TRs are viable, whereas Pax8(-/-) mice, which lack the follicular cells producing T4 and T3 in the thyroid gland, die during the first weeks of postnatal life. A precise comparison between the two types of mutants reveals that their phenotypes are similar, but the defects in spleen, bone, and small intestine are more pronounced in Pax8(-/-) mice. This is interpreted as the result of a negative effect of the unliganded TR on thyroid hormone target genes expression in the Pax8(-/-) mutants. Pax8/TRalpha compound mutants can survive to adulthood, and the expression of target genes is partially restored. This demonstrates the importance of TRalpha aporeceptor activity in several aspects of postnatal development.

The occurrence of the variant Creutzfeldt-Jakob disease (vCJD), related to bovine spongiform encephalopathy (BSE), raises the important question of the sources of human contamination. The possibility that sheep may have been fed with BSE-contaminated foodstuff raises the serious concern that BSE may now be present in sheep without being distinguishable from scrapie. Sensitive models are urgently needed given the dramatic consequences of such a possible contamination on animal and human health. We inoculated transgenic mice expressing the ovine PrP gene with a brain homogenate from sheep experimentally infected with BSE. We found numerous typical florid plaques in their brains. Such florid plaques are a feature of vCJD in humans and experimental BSE infection in macaques. Our observation represents the first description, after a primary infection, of this hallmark in a transgenic mouse model. Moreover, these mice appear to be a promising tool in the search for BSE in sheep.

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.

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.

We produced transgenic mice expressing the sheep prion protein to obtain a sensitive model for sheep spongiform encephalopathies (scrapie). The complete open reading frame, with alanine, arginine, and glutamine at susceptibility codons 136, 154, and 171, respectively, was inserted downstream from the neuron-specific enolase promoter. A mouse line, Tg(OvPrP4), devoid of the murine PrP gene, was obtained by crossing with PrP knockout mice. Tg(OvPrP4) mice were shown to selectively express sheep PrP in their brains, as demonstrated in mRNA and protein analysis. We showed that these mice were susceptible to infection by sheep scrapie following intracerebral inoculation with two natural sheep scrapie isolates, as demonstrated not only by the occurrence of neurological signs but also by the presence of the spongiform changes and abnormal prion protein accumulation in their brains. Mean times to death of 238 and 290 days were observed with these isolates, but the clinical course of the disease was strikingly different in the two cases. One isolate led to a very early onset of neurological signs which could last for prolonged periods before death. Independently of the incubation periods, some of the mice inoculated with this isolate showed low or undetectable levels of PrPsc, as detected by both Western blotting and immunohistochemistry. The development of experimental scrapie in these mice following inoculation of the scrapie infectious agent further confirms that neuronal expression of the PrP open reading frame alone is sufficient to mediate susceptibility to spongiform encephalopathies. More importantly, these mice provide a new and promising tool for studying the infectious agents in sheep spongiform encephalopathies.

We have developed a universally applicable system for conditional gene expression in embryonic stem (ES) cells that relies on tamoxifen-dependent Cre recombinase-loxP site-mediated recombination and bicistronic gene-trap expression vectors that allow transgene expression from endogenous cellular promoters. Two vectors were introduced into the genome of recipient ES cells, successively: (i) a bicistronic gene-trap vector encoding the beta-galactosidase/neo(R) fusion protein and the Cre-ER(T2) (Cre recombinase fused to a mutated ligand-binding domain of the human estrogen receptor) and (ii) a bicistronic gene-trap vector encoding the hygro(R) protein and the human alkaline phosphatase (hAP), the expression of which is prevented by tandemly repeated stop-of-transcription sequences flanked by loxP sites. In selected clones, hAP expression was shown to be regulated accurately by 4'hydroxy-tamoxifen. Strict hormone-dependent expression of hAP was achieved (i) in vitro in undifferentiated ES cells and embryoid bodies, (ii) in vivo in virtually all the tissues of the 10-day-old chimeric fetus (after injection of 4'hydroxy-tamoxifen to foster mothers), and (iii) ex vivo in primary embryonic fibroblasts isolated from chimeric fetuses. Therefore, this approach can be applied to drive conditional expression of virtually any transgene in a large variety of cell types, both in vitro and in vivo.

Cardiac myocytes express the two thyroid hormone receptors (T(3)Rs), T(3)Ralpha and T(3)Rbeta. However, which isoform contributes to specific, T(3)-induced alterations of cardiac function remains unclear. Here, we used individual T(3)R isoform knockout (KO) mice to study the effects of T(3)Ralpha and T(3)Rbeta in the heart. Our findings indicate that potassium channel genes that code for K(+) channels involved in action potential repolarization, like KV 4.2 and minK, are T(3)Ralpha targets. Both are markedly regulated by thyroid status. The recently identified cyclic nucleotide-gated channels, HCN2 and HCN4, are targets of T(3)Ralpha and are unchanged in a euthyroid T(3)Rbeta KO. However, these transcripts respond markedly to altered T(3) signaling concomitant with bradycardia in T(3)Ralpha KO and hypothyroid animals, as well as tachycardia in hyperthyroid T(3)Rss KO mice. SERCA2a and myosins are T(3) regulated and were also targets of T(3)Ralpha, and the papillary muscles of alphaKO animals showed a slowed rate of force development. Because of the absence of significant cardiac effects in euthyroid T(3)Rss KO mice, we determined messenger RNA levels for both T(3)Ralpha and T(3)Rss in the heart. We found that T(3)Rss is present at a 1:3 ratio to T(3)Ralpha1. We conclude that the cardiac phenotype regulated by T(3) is predominantly mediated by T(3)Ralpha and that the lack of T(3)Ralpha cannot be compensated by T(3)Rss in the heart.

Only three of the four thyroid hormone receptor (TR) isoforms, alpha1, beta1, and beta2, bind thyroid hormone (TH) and are considered to be true TRs. TRalpha2 binds to TH response elements on DNA, but its role in vivo is still unknown. We produced mice completely deficient in TRalpha (TRalpha(o/o)) that maintain normal serum thyroid-stimulating hormone (TSH) concentration despite low serum thyroxine (T(4)), suggesting increased sensitivity to TH. We therefore examined the effects of TH (L-3,3',5-triiodothyronine, L-T3) given to TH-deprived and to intact TRalpha(o/o) mice. Controls were wild-type (WT) mice of the same strain and mice resistant to TH due to deficiency in TRbeta (TRbeta(-/-)). In liver, T3 produced significantly greater responses in TRalpha(o/o) and smaller responses in TRbeta(-/-) as compared with WT mice. In contrast, cardiac responses to L-T3 were absent or reduced in TRalpha(o/o), whereas they were similar in WT and TRbeta(-/-) mice, supporting the notion that TRalpha1 is the dominant TH-dependent TR isoform in heart. 5-Triiodothyronine (L-T3) given to intact mice produced a greater suppression of serum T(4) in TRalpha(o/o) than it did in WT mice and reduced by a greater amount the TSH response to TSH-releasing hormone. This is an in vivo demonstration that a TR deficiency can enhance sensitivity to TH. This effect is likely due to the abrogation of the constitutive "silencing" effect of TRalpha2 in tissues expressing the TRbeta isoforms.

Thyroid hormones bind to several nuclear receptors encoded by T3R alpha and T3R beta genes. There is now accumulating evidence that thyroid hormones act on the immune system. Indeed, mice deficient for thyroid hormones show a reduction in lymphocyte production. However, the mechanisms involved and, in particular, the role of the different thyroid hormone receptors in lymphocyte development have not been investigated. To address that question, we have studied lymphocyte development in mice deficient for the T3R alpha 1 and T3R alpha 2 gene products. A strong decrease in spleen cell numbers was found compared with wild-type littermates, B lymphocytes being more severely affected than T lymphocytes. A significant decrease in splenic macrophage and granulocyte numbers was also found. In bone marrow, a reduction in CD45+/IgM- pro/pre-B cell numbers was found in these mice compared with wild-type littermates. This decrease seems to result from a proliferation defect, as CD45+/IgM- cells incorporate less 5-bromo-2'-deoxyuridine in vivo. To define the origin of the bone marrow development defect, chimeric animals between T3R alpha-/- and Rag1-/- mice were generated. Results indicate that for B cells the control of the population size by T3R alpha 1 and T3R alpha 2 is intrinsic. Altogether, these results show that T3R alpha 1 or T3R alpha 2 gene products are implicated in the control of the B cell pool size.

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.

Simultaneous addition of both TGF-alpha and TGF-beta induces the sustained, long-term outgrowth of chicken erythrocytic progenitor cells, referred to as T2ECs from both chick bone marrow and 2-day-old chicken embryos. By analysis for differentiation antigens and gene expression, these cells were shown to represent very immature haematopoietic progenitors committed to the erythrocytic lineage. T2ECs differentiate into almost pure populations of fully mature erythrocytes within 6 days, when TGF-alpha and TGF-beta are withdrawn and the cells exposed to anaemic chicken serum plus insulin. Outgrowth of these cells from various sources invariably required both TGF-alpha and TGF-beta, as well as glucocorticoids. Proliferating, established T2ECs still require TGF-alpha, but are independent of exogenous TGF-beta. Using a TGF-beta-neutralizing antibody or expressing a dominant-negative TGF-beta receptor II, we demonstrate that T2ECs generate an autocrine loop involving TGF-beta during their establishment, which is required for sustained proliferation. Using specific inhibitors, we also show that signalling via Mek-1 is specifically required for induction and maintenance of cell proliferation driven by cooperation between the TGF-alpha and -beta receptors. These results establish a novel mechanism by which self-renewal of erythrocytic progenitors is induced and establish avian T2ECs as a new, quasi-optimal model system to study erythrocytic progenitors.

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.

We have analyzed the expression pattern of c-erb A alpha and c-erb A beta which encode the thyroid hormone receptors (T3R alpha and T3R beta) during early chicken embryogenesis. Only c-erb A alpha expression was detected by RT-PCR and whole-mount in situ hybridization. c-erb A alpha transcripts were found to be already present at low level in embryos before egg incubation. During neurulation a marked increase was observed in neurectoderm. A reporter cell line was then constructed and used to demonstrate the release of significant amount of thyroid hormone (T3) from egg yolk by area opaca cells before gastrulation. During gastrulation T3 was found to be enriched in the primitive streak and Hensen's node. Introduction of excess T3 frequently resulted in abnormal development of anterior structures, mainly neural tube defects and anencephalia. These observations suggest that T3R alpha, like the closely related retinoic acid receptors, fulfills functions which are important for embryonic development well before the onset of thyroid gland function., (Copyright 1998 Academic Press.)

Little is known as to how the nuclear oncogenes v-erbA and p135gag-myb-ets do transform cells. The elucidation of their molecular mechanisms of action requires the identification of relevant target genes. We analysed the possibility for the RARbeta gene to represent such a target gene. We first show that the RARbeta gene induction is a specific and direct process, requiring the continuous presence of retinoids and under the control of the RARalpha isoform exclusively. We then show that the expression of either the v-erbA or the p135gag-myb-ets oncogene is not sufficient to block the RARbeta gene induction. We confirmed the loss of RARbeta gene response in certain cell lines but we discarded the possibility that this loss might represent a necessary step for cell lines immortalization. We further show that the RARalpha isoform activation is necessary and sufficient to induce the growth inhibition and the differentiation stimulation characteristic for the commitment-inducing ability of retinoids in chicken erythrocytic progenitor cells. We therefore propose a model showing that RARalpha but not RARbeta is the key mediator for commitment to differentiation and that it should control two different set of genes whose expression is differentially affected by the v-erbA and the p135gag-myb-ets oncogenes.

We have previously shown that v-erb A stimulates quail myoblast differentiation in a T3 independent, cell-specific manner. In this work, we have studied the influence of v-erb B (the second oncogene carried in the AEV genome) upon quail myoblast proliferation and differentiation. v-erb B expression,moderately stimulates myoblast proliferation, and inhibits differentiation. Moreover, this oncoprotein fully inhibits the v-erb A myogenic influence. These data provide evidence that these two oncogenes do not cooperate in avian myoblasts. Consequently, in contrast to results obtained in other cell-types, coexpression of both oncogenes does not transform quail myoblasts.

The thyroid hormone receptor-coding locus, c-erbA alpha, generates several mRNAs originating from a single primary transcript that undergoes alternative splicing. We have identified for the first time two new transcripts, called TRdelta alpha1 and TRdelta alpha2 [mRNA for isoform alpha1 and alpha2 of the T3 receptor (TR), respectively], whose transcription is initiated from an internal promoter located within intron 7 of the c-erbA alpha gene. These two new transcripts exhibit tissue-specific patterns of expression in the mouse. These two patterns are in sharp contrast with the expression patterns of the full-length transcripts generated from the c-erbA alpha locus. TR alpha1 and TRdelta alpha2 mRNAs encode N-terminally truncated isoforms of T3R alpha1 and T3R alpha2, respectively. The protein product of TRdelta alpha1 antagonizes the transcriptional activation elicited by T3 and retinoic acid. This protein inhibits the ligand-induced activating functions of T3R alpha1 and 9-cis-retinoic acid receptor-alpha but does not affect the retinoic acid-dependent activating function of retinoic acid receptor-alpha. We predict that these truncated proteins may work as down-regulators of transcriptional activity of nuclear hormone receptors in vivo.

We have shown recently that a retrovirus vector expressing a natural mutant form of the PML-RAR alpha protein characteristic of human acute promyelocytic leukaemia can transform early chicken hematopoietic progenitors (Altabef et al., 1996). Neither truncated PML nor truncated RAR alpha alone could induce transformation which suggest that the two domains should cooperate for the oncogenicity of the fusion product. To further investigate the mechanisms of this co-operation, we have tested whether a truncated RAR alpha could cooperate with the v-erbB oncogene. This oncogene has previously been shown to co-operate with the rearranged thyroid hormone receptor, v-erbA, to transform erythrocytic progenitors. We show that v-erbB and a truncated RAR alpha co-operate when expressed simultaneously as independent products to transform very early chicken haematopoietic cells close to pluripotent stage. In addition, we show that v-erbB alters transcriptional abilities of RAR alpha by both enhancing its effects on RARE and reducing those on AP-1. Therefore, RAR alpha is able to co-operate with different kinds of proteins to induce transformation of early haematopoietic cells. This strongly suggests that RAR alpha are involved in the differentiation commitment of early haematopoietic progenitors during the normal process of haematopoietic differentiation. These data bring new insights in the mechanisms of oncogenic transformation by rearranged RAR alpha.

We have previously shown that v-erb A expression strongly stimulates quail myoblast proliferation and differentiation without alteration of the triiodothyronine (T3) influence in this cell type. In order to understand the molecular basis of v-erb A action in myoblasts, we have studied the influence of this oncoprotein on c-erb A alpha1 encoded T3 nuclear receptor (TR alpha) activity. In transfection experiments, v-erb A did not inhibit the T3-dependent c-erb A alpha1 transcriptional activity in QM7 myoblasts in contrast to its action in HeLa cells. However, it repressed the retinoic acid receptor RAR alpha activity in both cell-types, indicating that v-erb A interactions with T3 or RA mediated transcription significantly differs. In EMSA experiments using a TREpa1 probe, T3R alpha binds as three complexes in HeLa cells. We have previously identified the slow migrating complex, undetectable in QM7 myoblasts, as a T3R/RXR heterodimer. Interestingly, v-erb A inhibited binding of this complex in HeLa cells, but did not affect binding of the two other complexes in QM7 myoblasts. Expression of RXR (gamma isoform), the TR alpha dimerization partner absent in proliferating QM7 cells, restored inhibition of c-erb A alpha1 transcriptional activity in these cells and abrogated the v-erb A myogenic influence. Lastly, v-erb A induced a T3-independent c-erb A alpha1 activity in QM7 cells when cotransfected in equimolar ratio with the receptor, by inhibiting AP-1 activity and stimulating transcription of a reporter gene driven by a TRE sequence.

Cell cycle regulation is critical for maintenance of genome integrity. A prominent factor that guarantees genomic stability of cells is p53 (ref. 1). The P53 gene encodes a transcription factor that has a role as a tumour suppressor. Identification of p53-target genes should provide greater insight into the molecular mechanisms that mediate the tumour suppressor activities of p53. The rodent Pc3/Tis21 gene was initially described as an immediate early gene induced by tumour promoters and growth factors in PC12 and Swiss 3T3 cells. It is expressed in a variety of cell and tissue types and encodes a remarkably labile protein. Pc3/Tis21 has a strong sequence similarity to the human antiproliferative BTG1 gene cloned from a chromosomal translocation of a B-cell chronic lymphocytic leukaemia. This similarity led us to speculate that BTG1 and the putative human homologue of Pc3/Tis21 (named BTG2) were members of a new family of genes involved in growth control and/or differentiation. This hypothesis was recently strengthened by the identification of a new antiproliferative protein, named TOB, which shares sequence similarity with BTG1 and PC3/TIS21 (ref. 7). Here, we cloned and localized the human BTG2 gene. We show that BTG2 expression is induced through a p53-dependent mechanism and that BTG2 function may be relevant to cell cycle control and cellular response to DNA damage.

v-erbA is the oncogenic form of the c-erbA proto-oncogene, which encodes the receptor for thyroid hormones. The expression of the v-erbA oncogene in thyroid differentiated cells, PC Cl 3, inhibits iodide uptake and thyrotropin-dependent growth, whereas it has no effect on the expression of the other thyroid specific markers, i.e. thyroglobulin, thyroperoxidase and thyrotropin receptor. The activity of transcription factor AP-1, evaluated by a specific DNA binding assay and by transcription of AP-induced promoter (TRE) is enhanced in PC v-erbA cells. v-erbA mutants in the DNA binding domain do not affect the iodide uptake of thyroid cells nor AP-1 activity. We suggest that this transcriptional activation mediates the selective effects of v-erbA on the expression of thyroid specific markers.

We describe here the use of PCR-generated templates incorporating T3 polymerase sites in order to prepare digoxigenin (DIG)-labelled cRNA probes against any gene of known sequence. This method was applied to the preparation of probes specific for chicken glyceraldehyde-3-phosphate dehydrogenase messenger RNAs and we demonstrate that such probes can be used for in situ hybridization (ISH). This technique therefore represents a rapid and convenient means to prepare DIG-labelled cRNA probes for use in a non-radioactive ISH. It adds speed and convenience of probe preparation to the previously described advantages of non-radioactive detection techniques.

The expression of E and D-type cyclins, Cyclin-Dependent Kinase (CDK) 2 and 4, as well as CDK inhibitors p21Cip1 and p27Kip1 were examined during in vitro differentiation of mouse embryonic stem (ES) cells. ES cells cultured in presence of Differentiation Inhibitory Activity/Leukemia Inhibitory Factor (DIA/LIF) express very low levels of cyclin E/CDK2 complexes, p21Cip1 and p27Kip1 CDK inhibitors, while cyclin D/CDK4-associated kinase activity is undetectable. Withdrawal of DIA/LIF, which induces differentiation, results in the progressive up-regulation of all. Up-regulation of D cyclins occurs through an increase in the steady-state levels of mRNA, concomitantly with the activation of Brachyury and Goosecoid, two early markers of mesoderm differentiation. Similarly, cells from the epiblast of the early postimplantation mouse embryo do not express any cyclin D/CDK4 complexes. These are progressively upregulated at gastrulation and early organogenesis. DIA/LIF-stimulated ES cells are not growth-arrested by overexpression of p16Ink4a, a specific inhibitor of CDK4 and CDK6. We propose that the G1/S transition may be regulated by a minimal mechanism in mouse embryonic stem cells. Induction of differentiation triggers the establishment of a more sophisticated mechanism involving both cyclin D/CDK4- and CDK inhibitor-associated control of G1-phase progression.

Virofection is a simple new way to use replication-defective vectors. It consists of the cotransfection of two plasmids: one plasmid bearing the genome of the replication-defective retrovirus vector and a second "helper" plasmid expressing the gag, pol, and env retrovirus sequences. It provides stable integration into the chromosome and long-term expression of only vector-borne sequences. We present here several helper plasmids derived from avian leukosis viruses, which we have used for the virofection of the lacZ reporter gene in chicken cell cultures. Expression can be stabilized at a very high rate in both chicken fibroblasts and blastoderm cells, without giving rise to replication-competent virus.

In order to characterize Sterling's triiodothyronine (T3) mitochondrial receptor using photoaffinity labeling, we observed two specific T3-binding proteins in the inner membrane (28 kDa) and in the matrix (43 kDa) of rat liver mitochondria. Western blots and immunoprecipitation using antibodies raised against the T3-binding domain of the T3 nuclear receptor c-Erb A alpha 1 indicated that at least the 43-kDa protein was c-Erb A alpha 1-related. In addition, gel mobility shift assays demonstrated the occurrence of a c-Erb A alpha 1-related mitochondrial protein that specifically binds to a natural or a palindromic thyroid-responsive element. Moreover, this protein specifically binds to a direct repeat 2 sequence located in the D-loop of the mitochondrial genome. Furthermore, electron microscopy studies allowed the direct observation of a c-Erb A-related protein in mitochondria. Lastly, the relative amounts of the 43-kDa protein related to c-Erb A alpha 1 were in good correlation with the known mitochondrial mass in three typical tissues. Interestingly, expression of a truncated form of the c-Erb A alpha 1 nuclear receptor in CV1 cells was associated with a mitochondrial localization and a stimulation of mitochondrial activity. These results supply evidence of the localization of a member of the nuclear receptor superfamily in the mitochondrial matrix involved in the regulation of mitochondrial activity that could act as a mitochondrial T3-dependent transcription factor.

The v-erbA oncogene has been discovered as one of the two viral oncogenes carried by the avian leukemia retrovirus AEV. It is derived from the c-erbA protooncogene which encodes the alpha form of the nuclear receptor for the thyroid hormone triiodothyronine (T3R). This receptor belongs to a large family of nuclear hormone receptors that function as ligand-regulated transcription factors and the v-erbA oncoprotein has been shown to function as an antagonist of normal T3R and related receptors in the control of transcription. It is thus the first dominant negative transcription factor acting as an oncogene described to date. Functional and biochemical dissections of this oncogene have brought many informations on the mechanisms of action of normal receptors and on the ways through which altered receptors can contribute to oncogenic transformation. The v-erbA model is widely used as a reference to investigate the involvement of nuclear hormone receptors in the development of human cancers.

A new procedure, virofection, designed to stabilize the expression of transfected DNA has been developed. It exploits the capacity of retroviruses to integrate their genome into the chromosomes of host cells. The co-transfection of two plasmids, one carrying the genome of a defective retrovirus vector, the other one encoding all the retroviral proteins, results in a transient production of infectious virus particles. These particles can infect the neighboring cells and this leads to the stable integration of the vector genome. This procedure is time-saving and appears to be quite efficient. When applied to chicken embryonic fibroblasts cultured in vitro, it resulted in the stable expression of the lacZ gene in more than 30% of the cells, and did not induce chronic viremia. Stable lacZ expression was also achieved in chicken embryos in ovo. Virofection appears to be a promising and generally applicable method for implementing stable, safe and efficient gene transfer in vitro and in vivo.