Neuronal expression of a thyroid hormone receptor α mutation alters mouse behaviour.

In humans, alterations in thyroid hormone signalling are associated with mood and anxiety disorders, but the neural mechanisms underlying such association are poorly understood. The present study investigates the involvement of neuronal thyroid hormone receptor α (TRα) in anxiety, using mouse genetics and Cre/loxP technology to specifically alter TRα signalling in neurons. We evaluated the behaviour of mice expressing a dominant negative, neuron-specific mutation of TRα (TRαAMI/Cre3 mice), using the elevated-plus maze, light-dark box and open-field tests. In a first experiment, mice were housed individually, and the behaviour of TRαAMI/Cre3 mice differed significantly from that of control littermates in these 3 tests, suggesting heightened anxiety. In a second experiment, designed to evaluate the robustness of the results with the same 3 tests, mice were housed in groups. In these conditions, the behaviour of TRαAMI/Cre3 mice differed from that of control littermates only in the light-dark box. Thus, TRαAMI/Cre3 mice appear to be more likely to develop anxiety under stressful housing conditions than control mice. These results suggest that in adult mice, thyroid hormone signalling in neurons, via TRα, is involved in the control of anxiety behaviour.

A temporary compendium of thyroid hormone target genes in brain.

BACKGROUND: Thyroid hormone controls a number of developmental and physiological processes in the brain by directly acting on gene expression. Transcriptome analyses in rodent identified a number of thyroid hormone regulated genes in several brain areas at different stages. Genome wide analysis of chromatin occupancy in a neural cell line also identified a subset of genes which transcription is likely to be directly regulated by thyroid hormone receptors in neurons. However, the abundance of these data and apparent discrepancies between studies brought some confusion. RESULTS: We present here a meta-analysis of available data to identify recurrent themes in thyroid hormone action in brain cells. This provides a curated list of 734 regulated genes in rodent brain, and highlights a small number of likely direct target genes. Some of these genes are also regulated in amphibians during metamorphosis. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

[Thyroid hormone and cerebellum development: direct and indirect effects?]. / Hormone thyroïdienne et développement du cervelet : effets directs ou indirects ?

Thyroid hormone (T3) exerts an important influence on neurodevelopment, which can be analysed by using the postnatal development of rodent cerebellum as a model. T3 acts on all types of neuronal and glial cells, which express at least the TRα1 nuclear receptor, and, for some of them, the TRß1 isoform. However, as T3 also activates the secretion of neurotrophins, it can also affect cellular differentiation in an indirect manner. Ongoing experiments, based on mouse genetics and genome wide analysis of gene expression, provide a promising way to study the basic mechanisms of neurodevelopment. This review describes new mouse genetics models and recent advance in this field.

Thyroid hormone signaling is highly heterogeneous during pre- and postnatal brain development.

We have generated transgenic reporter mice to analyze the spatio-temporal distribution of thyroid hormone signaling during mouse brain development. The reporter system, utilizing a chimeric yeast Gal4 DNA-binding domain-thyroid hormone alpha ligand-binding domain fusion protein to drive lacZ expression, revealed that thyroid hormone signaling starts in the midbrain roof several days before the onset of thyroid gland function, and that it remains highly heterogeneous in the central nervous system throughout pre- and postnatal development. We speculate that this heterogeneity might provide neural cells with positional information during development.

The transcription factor RFX3 directs nodal cilium development and left-right asymmetry specification.

There are five members of the RFX family of transcription factors in mammals. While RFX5 plays a well-defined role in the immune system, the functions of RFX1 to RFX4 remain largely unknown. We have generated mice with a deletion of the Rfx3 gene. RFX3-deficient mice exhibit frequent left-right (LR) asymmetry defects leading to a high rate of embryonic lethality and situs inversus in surviving adults. In vertebrates, specification of the LR body axis is controlled by monocilia in the embryonic node, and defects in nodal cilia consequently result in abnormal LR patterning. Consistent with this, Rfx3 is expressed in ciliated cells of the node and RFX3-deficient mice exhibit a pronounced defect in nodal cilia. In contrast to the case for wild-type embryos, for which we document for the first time a twofold increase in the length of nodal cilia during development, the cilia are present but remain markedly stunted in mutant embryos. Finally, we show that RFX3 regulates the expression of D2lic, the mouse orthologue of a Caenorhabditis elegans gene that is implicated in intraflagellar transport, a process required for the assembly and maintenance of cilia. In conclusion, RFX3 is essential for the differentiation of nodal monocilia and hence for LR body axis determination.

Florid plaques in ovine PrP transgenic mice infected with an experimental ovine BSE.

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.

Efficient transmission of two different sheep scrapie isolates in transgenic mice expressing the ovine PrP gene.

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.

Conservative treatment for girls with nonmetastatic rhabdomyosarcoma of the genital tract: A report from the Study Committee of the International Society of Pediatric Oncology.

PURPOSE: To report the results of a conservative multimodal approach in girls with nonmetastatic rhabdomyosarcoma (RMS) of the genital tract, treated in International Society of Pediatric Oncology (SIOP) Malignant Mesenchymal Tumors 84 and 89 protocols. PATIENTS AND METHODS: From 1984 to 1994, 38 girls with RMS of the genital tract (vulva, vagina, uterus) were treated in SIOP protocols. With the exception of patients with rare small tumors, which were resected at the start of the studies, all patients received initial chemotherapy (CHT) (ifosfamide, vincristine, and actinomycin D). Local treatment including surgery, brachytherapy (BT), and external-beam radiotherapy (ERT) was given only to girls who did not achieve complete remission (CR) with CHT or who subsequently relapsed. RESULTS: The primary tumor originated in the vulva or vagina in 27 girls and in the uterus in 11. The overall survival rate (+/- SE) was 91% +/- 6% at 5 years, and the event-free survival rate was 78% +/- 7%. At a median follow-up of 5 years, 30 girls were alive and in first CR and five were alive and in second CR. Four patients treated with complete resection of the tumor at diagnosis received less CHT. Thirteen patients were treated with CHT alone. In 17 patients, local treatment was necessary to achieve complete local control, for a residual mass after initial CHT (10 patients), for viable tumor on biopsy (three patients), or for local relapse (four patients). The local treatment used was radiotherapy (RT) (ERT in three patients, BT in seven), radical surgery with uterine ablation (three patients), RT and radical surgery (three patients), and conservative surgery with RT (one patient). CONCLUSION: Girls with nonmetastatic RMS of the genital tract have an excellent prognosis. We found no difference in outcome between uterine and vulvovaginal RMS. Local treatment does not seem necessary in patients who have a complete response to CHT. When a local treatment is needed, BT may be an alternative to radical surgery or ERT.

Eukaryotic conditional expression system.

Existing conditional expression systems can be classified in two major categories that are based either on the induction or on the de-repression of transcription. The system described here combines both mechanisms, since a unique transcription factor can be shifted from a repression to a stimulation activity by simply changing its ligand. The resulting advantage of this system is the complete absence of basal expression before active induction. The principle of this method is based on the unexpected ability of the chimeric protein containing the DNA-binding domain of the yeast Gal4 transcription factor fused to the COOH half of the estradiol receptor (GalER), to act as a repressor when bound to the drug 4OH-tamoxifen, in the context of a previously described optimized Gal4-responsive promoter. The efficacy of this system has been assessed in transient expression assays using the chloramphenicol acetyl transferase (CAT), and in situ, through the activity of a Gal4 responsive beta-galactosidase gene.

[Malignant non-seminomatous germinal tumors of the ovary. Experience of the French Society of Pediatric Oncology. 64 cases]. / Les tumeurs germinales malignes non séminomateuses de l'ovaire. Expérience de la Société Française d'Oncologie Pédiatrique. A propos de 64 cas.

Ovary seminomatous malignant germ cell tumours are a particular histopathologica entity. The presence of yolk salk tumor or choriocarcinoma is respectively correlated with elevation of alpha FP or beta hCG. This markers elevation permits to assess diagnosis, appreciate response to treatment, and detect relapses. The study of 64 patients registered in two successive S.F.O.P. protocols (TGM 85-TGM 90) precise indications of surgery, platin-based chemotherapy and results. Malignant non seminomatous germ cell tumors are separated in not secreting pure immature teratoma and secreting malignant germ cell tumours. Surgery is essential for treatment of not secreting pure immature teratoma. Secreting germ cell tumors are very chemosensitive and surgery must be as conservative as possible in all cases even metastatic tumour or relapse. If markers are normalized second-look surgery of secreting malignant germ cell tumours is only necessary in case of evident residual tumour. However in case of initial chemotherapy, resection of a pathological ovary is always performed at the end of treatment. These tumours have a good prognosis: 5-years overall survival and disease-free survival are 85%.

A novel growth-factor-dependent myeloid cell line derived from mouse bone marrow cells contains progenitors endowed with high proliferative potential.

Constitutive expression of human colony-stimulating factor-1 receptor (CSF-1R) confers long-lasting CSF-1-dependent proliferation to mouse myeloid cell lines. We developed mice transgenic for human CSF-1R because mouse CSF-1 cannot activate human CSF-1R. Then bone marrow cells from transgenic mice were plated onto MS-5 stromal cells expressing the membrane form of human CSF-1 (2M-1 cells) in order to combine the hematopoietic supporting properties of stromal cells and the proliferative effects of CSF-1. Thus, we were able to derive a hematopoietic cell line, called 47.10, that grew indefinitely under these conditions, whereas no cell line could be developed from nontransgenic mice. Proliferation of 47.10 cells is severely affected by neutralizing anti-CSF-1R monoclonal antibodies. Morphologic and cytofluorometry analysis established that most 47.10 cells are immature myelomonocytic cells. Consistent with this phenotype, the myeloid transcription factor PU.1, but not the erythroid transcription factor GATA-1, is expressed in 47.10 cells. A few 47.10 cells (3-5%) do not express lineage specific markers; they differentiate spontaneously to lineage-positive cells after replating on 2M-1 cells. In agar cultures, 47.10 cells form 7- and 14-day colonies in response to a cocktail of granulocyte/macrophage colony-stimulating factor (2.5 ng/mL), interleukin-3 (1 ng/mL), and mouse CSF-1 (10 ng/mL). Under these conditions, about 0.5% of 47.10 cells formed large 14-day colonies (>1 mm) composed of mature monocytes and granulocytes, reflecting the presence of progenitors endowed with high proliferative potential (HPP-47.10 cells). In conclusion, we have characterized a novel continuous myeloid cell line presenting a hierarchical structure similar to that of the bone marrow progenitor cell compartment.

Treatment of non-metastatic rhabdomyosarcomas in childhood and adolescence. Results of the second study of the International Society of Paediatric Oncology: MMT84.

The second International Society of Paediatric Oncology (SIOP) study for rhabdomyosarcoma (MMT84) had several goals. The two principal aims were: (1) to improve the survival of children with rhabdomyosarcoma; and (2) to reduce the late effects from therapy by restricting the indications for surgery and/or radiotherapy after good response to initial chemotherapy. A further aim was to investigate the role of high-dose chemotherapy in young patients with parameningeal primary tumours. 186 previously untreated eligible patients entered the study. Patients with completely resected primary tumour received three courses of IVA (ifosfamide, vincristine and actinomycin D). Patients with incompletely resected tumour received six to 10 courses of IVA according to stage. Patients achieving complete remission with chemotherapy alone did not usually receive radiotherapy or undergo extensive surgery, but patients remaining in partial remission received local therapy with surgery and/or radiotherapy. Only patients over 5 years of age with parameningeal disease and patients over 12 years with tumours at any site were given systematic irradiation. Complete remission was achieved in 91% (170/186) of all patients. With a median follow-up of 8 years, the 5-year overall survival was 68% (+/- 3% standard error of the mean (SEM) and the 5-year event-free survival 53% (+/- 4% SEM). These results show an improvement over previous SIOP study (RMS75) in which survival was 52% and event-free survival was 47%. Among the 54 patients who exhibited isolated local relapse, 35% (19/54) survived in further remission longer than 2 years after retreatment, including local therapy (surgery +/- radiotherapy). Analysis of the overall burden of therapy received by all surviving children (including primary treatment and treatment for relapse if required) showed that 24% (28/116) were treated by limited surgery followed by three courses of IVA, 29% (34/116) were treated by chemotherapy alone (after initial biopsy) and 13% (15/116) received chemotherapy plus conservative local treatment (limited surgery or radiotherapy for residual disease). Only 34% (39/116) received intensive local therapy defined as radical wide field radiotherapy or radical surgery or both. Compared with the results obtained in the previous SIOP study, treatment in MMT84 was based on response to initial chemotherapy and, despite an overall reduction of the use of local therapy, significantly improved survival for patients with non-metastatic disease. This trial, also for the first time, provides evidence that retreatment after local relapse can achieve long-term second remissions.

Involvement of thyroid hormone and its alpha receptor in avian neurulation.

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.

Pheochromocytoma and paraganglioma in children: a report of 24 cases of the French Society of Pediatric Oncology.

Pheochromocytoma and paraganglioma of childhood are rare neuroendocrine tumors. Urinary catecholamine measurements, metaiobenzylguanidine (MIBG) scanning, computed tomographic scanning, and magnetic resonance imaging have greatly facilitated diagnosis. Prognosis after surgical resection is excellent. In this retrospective series collected from French oncology centers, the risk of tumor progression was studied in order to assess prognostic factors and the optimal diagnostic and therapeutic management. Medical records of 24 children with paraganglioma were reviewed. This tumor occurred at a median age of 12.5 years and in most cases was revealed by arterial hypertension. The diagnosis was made by the demonstration of urinary excretion of catecholamines and their metabolites. Six patients had bilateral adrenal pheochromocytomas; two patients had extra-adrenal paragangliomas. In eight patients, the paraganglioma occurred as a familial disease. Surgical excision was the only therapeutic procedure. With a follow-up of 5.2 years, 14 of the patients are still in first complete remission and 6 have developed metastases or shown tumor progression. Despite a high long-term survival rate, the risk of malignancy and of multifocal involvement is of concern and is associated with a significant rate of late events. The outcome depends on adequacy of tumor resection and must be serially assessed.

Identification of transcripts initiated from an internal promoter in the c-erbA alpha locus that encode inhibitors of retinoic acid receptor-alpha and triiodothyronine receptor activities.

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.

Parameningeal rhabdomyosarcoma: results of an international workshop.

PURPOSE: A retrospective analysis was performed on children with nonmetastatic rhabdomyosarcomas (RMS) involving a parameningeal site treated by one of the four major cooperative groups: Intergroup Rhabdomyosarcoma Study (IRS), International Society of Pediatric Oncology (SIOP), German Cooperative Group (CWS), and Italian Cooperative Group (ICS) to analyse survival and prognostic factors. METHODS AND MATERIALS: Between 1979 and 1989, 230 children (median age 6 years) were treated in the IRS III, SIOP 84, CWS 81, and ICS 79 studies. All patients received chemotherapy, and 203 were irradiated. Radiotherapy doses were similar in the four studies, although treatment volumes were not similar. The SIOP patients had smaller volumes treated. In addition, the SIOP patients with a low risk of meningeal involvement and children under 5 years of age were not irradiated if they had a complete response (CR) to chemotherapy. Time to initiation of irradiation was earlier in the IRS and Italian studies. RESULTS: Median follow-up was 62 months (range 22-140). The 5-year survival and 5-year event-free survival were better for the IRS study (74% and 71%) than for the other study groups (55% and 36% for SIOP, 47% and 47% for CWS, and 39% and 39% for ICS). The low-risk (LR) patients in the IRS study had improved survival. However, patients with high risk of meningeal involvement had similar survival in all four studies. The most significant prognostic factor was the size of tumor (> 5 cm). CONCLUSION: The improved results from the IRS group, especially among the LR patients, could be related to the IRS treatment employed, particularly the systematic use of radiation, to the inclusion of patients with smaller tumors, and to the routine use of quality control of radiation.

The osteoclast generation: an in vitro and in vivo study with a genetically labelled avian monocytic cell line.

Osteoclasts are multinucleate giant cells responsible for bone resorption. Osteoclast precursors are hematopoietic mononucleate cells, which give rise to osteoclasts after fusion. Nevertheless, the precise stage of differentiation where osteoclast precursors diverge from other hematopoietic lineages is still debated. We describe here both in vitro and in vivo approaches to the study of the osteoclast differentiation pathway. We used cells of the BM2 avian monocytic cell line, which are able to differentiate into macrophages both in vitro and in vivo. In order to follow the progeny of BM2 monocytes, we have derived a BM2 cell clone expressing the nlslacZ gene (BM2nlslacZ) which has still retained the main features of the parental cell line. In vitro, when BM2nlslacZ cells were triggered toward macrophages, they participated in the formation of multinucleate osteoclast-like cells as seen by their blue nuclei. Furthermore, when BM2nlslacZ cells were injected into the blood stream of chicken embryos, they could give rise to blue nucleate macrophages in the bone marrow, as well as to osteoclasts with blue nuclei in bone. Finally, we have shown that fusion of tagged mononucleate precursor cells not only occurs with other mononucleate precursor cells but also with mature multinucleate osteoclasts. This work shows that cells already engaged in the late stages of the monocytic differentiation pathway are able to differentiate into osteoclasts and that osteoclast divergence takes place after the monocyte stage.

Virofection: a one-step procedure for using replication-defective retrovirus vectors.

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.