Mice deficient for delta- and mu-opioid receptors exhibit opposing alterations of emotional responses.

The role of the opioid system in controlling pain, reward and addiction is well established, but its role in regulating other emotional responses is poorly documented in pharmacology. The mu-, delta- and kappa- opioid receptors (encoded by Oprm, Oprd1 and Oprk1, respectively) mediate the biological activity of opioids. We have generated Oprd1-deficient mice and compared the behavioural responses of mice lacking Oprd1, Oprm (ref. 6) and Oprk1 (ref. 7) in several models of anxiety and depression. Our data show no detectable phenotype in Oprk1-/- mutants, suggesting that kappa-receptors do not have a role in this aspect of opioid function; opposing phenotypes in Oprm-/- and Oprd1-/- mutants which contrasts with the classical notion of similar activities of mu- and delta-receptors; and consistent anxiogenic- and depressive-like responses in Oprd1-/- mice, indicating that delta-receptor activity contributes to improvement of mood states. We conclude that the Oprd1-encoded receptor, which has been proposed to be a promising target for the clinical management of pain, should also be considered in the treatment of drug addiction and other mood-related disorders.

The breast cancer-associated stromelysin-3 gene is expressed during mouse mammary gland apoptosis.

We have cloned from a mouse placenta cDNA library a mouse homologue of the human stromelysin-3 (ST3) cDNA, which codes for a putative matrix metalloproteinase expressed in breast carcinomas. The ST3 protein is well conserved between humans and mice, and the pattern of ST3 gene expression is similar in both species, and shows expression in the placenta, in the uterus, and during limb bud morphogenesis. We show that the ST3 gene can also be expressed in the normal mouse mammary gland. ST3 gene expression was not detected during mammary growth, neither in virgin nor in pregnant mice, but was specifically observed during postlactating involution of the gland, an apoptotic process associated with intense extracellular matrix remodeling. ST3 transcripts were found in fibroblasts immediately surrounding degenerative ducts, suggesting that ST3 gene expression may be associated with the basement membrane dissolution, which occurs during mammary gland involution. Since the ST3 gene is also specifically expressed in fibroblastic cells surrounding invasive neoplastic cells of breast carcinomas, we suggest that ST3 is implicated in extracellular matrix remodeling processes common to mammary apoptosis and breast cancer progression.

In vivo evidence that the stromelysin-3 metalloproteinase contributes in a paracrine manner to epithelial cell malignancy.

Stromelysin-3 (ST3; Basset, P., J.P. Bellocq, C. Wolf, I. Stoll, P. Hutin, J.M. Limacher, O.L. Podhajcer, M.P. Chenard, M.C. Rio, P. Chambon. 1990. Nature. 348:699-704) is a matrix metalloproteinase (MMP) expressed in mesenchymal cells located close to epithelial cells, during physiological and pathological tissue remodeling processes. In human carcinomas, high ST3 levels are associated with a poor clinical outcome, suggesting that ST3 plays a role during malignant processes. In this study we report the ST3 gene inactivation by homologous recombination. Although ST3 null mice (ST3-/-) were fertile and did not exhibit obvious alterations in appearance and behavior, the lack of ST3 altered malignant processes. Thus, the suppression of ST3 results in a decreased 7, 12-dimethylbenzanthracene-induced tumorigenesis in ST3-/- mice. Moreover, ST3-/- fibroblasts have lost the capacity to promote implantation of MCF7 human malignant epithelial cells in nude mice (P < 0.008). Finally, we show that this ST3 paracrine function requires extracellular matrix (ECM)-associated growth factors. Altogether, these findings give evidence that ST3 promotes, in a paracrine manner, homing of malignant epithelial cells, a key process for both primary tumors and metastases. Therefore, ST3 represents an appropriate target for specific MMP inhibitor(s) in future therapeutical approaches directed against the stromal compartment of human carcinomas.

Mice lacking TdT: mature animals with an immature lymphocyte repertoire.

In adult animals, template-independent (or N) nucleotides are frequently added during the rearrangement of variable (V), diversity (D), and joining (J) segments of lymphocyte receptor genes, greatly enhancing junctional diversity. Receptor genes from adult mice carrying a mutation in the terminal deoxynucleotidyl transferase (TdT) gene have few N nucleotides, providing proof that this enzyme is essential for creating diversity. Unlike those from normal adults, receptor genes from adult mutant mice show extensive evidence of homology-directed recombination, suggesting that TdT blocks this process. Thus, switch-on of the TdT gene during the first week after birth provokes an even greater expansion of lymphocyte receptor diversity than had previously been thought.

Enhanced aggressive behavior in mice lacking 5-HT1B receptor.

The neuromodulator serotonin (5-hydroxytryptamine, 5-HT) has been associated with mood disorders such as depression, anxiety, and impulsive violence. To define the contribution of 5-HT receptor subtypes to behavior, mutant mice lacking the 5-HT1B receptor were generated by homologous recombination. These mice did not exhibit any obvious developmental or behavioral defects. However, the hyperlocomotor effect of the 5-HT1A/1B agonist RU24969 was absent in mutant mice, indicating that this effect is mediated by 5-HT1B receptors. Moreover, when confronted with an intruder, mutant mice attacked the intruder faster and more intensely than did wild-type mice, suggesting the participation of 5-HT1B receptors in aggressive behavior.

Transgenic mice with I-A on islet cells are normoglycemic but immunologically intolerant.

Insulin-dependent diabetes mellitus (IDDM) is caused by a specific loss of the insulin-producing beta cells from pancreatic Langerhans islets. It has been proposed that aberrant expression of major histocompatibility complex (MHC) class II molecules on these cells could be a triggering factor for their autoimmune destruction. This proposal was tested in transgenic mice that express allogeneic or syngeneic class II molecules on the surface of islet cells at a level comparable with that normally found on resting B lymphocytes. These animals do not develop diabetes, nor is lymphocyte infiltration of the islets observed. This immunological inactivity does not result from tolerance to the "foreign" class II molecules.

Gastric mucosa abnormalities and tumorigenesis in mice lacking the pS2 trefoil protein.

To determine the function of the pS2 trefoil protein, which is normally expressed in the gastric mucosa, the mouse pS2 (mpS2) gene was inactivated. The antral and pyloric gastric mucosa of mpS2-null mice was dysfunctional and exhibited severe hyperplasia and dysplasia. All homozygous mutant mice developed antropyloric adenoma, and 30 percent developed multifocal intraepithelial or intramucosal carcinomas. The small intestine was characterized by enlarged villi and an abnormal infiltrate of lymphoid cells. These results indicate that mpS2 is essential for normal differentiation of the antral and pyloric gastric mucosa and may function as a gastric-specific tumor suppressor gene.

The bHLH protein NEUROGENIN 2 is a determination factor for epibranchial placode-derived sensory neurons.

neurogenin2 encodes a neural-specific basic helix-loop-helix (bHLH) transcription factor related to the Drosophila proneural factor atonal. We show here that the murine ngn2 gene is essential for development of the epibranchial placode-derived cranial sensory ganglia. An ngn2 null mutation blocks the delamination of neuronal precursors from the placodes, the first morphological sign of differentiation in these lineages. Mutant placodal cells fail to express downstream bHLH differentiation factors and the Notch ligand Delta-like 1. These data suggest that ngn2 functions like the Drosophila proneural genes in the determination of neuronal fate in distal cranial ganglia. Interestingly, the homeobox gene Phox2a is activated independently of ngn2 in epibranchial placodes, suggesting that neuronal fate and neuronal subtype identity may be specified independently in cranial sensory ganglia.

Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice.

ADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y(1) receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y(1)-null mice were generated to define the role of the P2Y(1) receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y(1). These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y(1)-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y(1). P2Y(1)-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y(1) receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs.

H-ras induced transformation of mammary epithelium is favoured by increased oncogene expression or by inhibition of mammary regression.

The promoter of the mammary specific murine whey acidic protein gene was used to direct Ha-ras expression in different lines of transgenic mice. We found that this promoter contains a tissue specific enhancer which directed expression in both orientations albeit to different levels. We used this feature to generate low and high ras expressing transgenic lines. The reversed orientation led to a weak expression in lines 3 and 58 and to a tumor frequency of 2%. In contrast, 72% of mice from line 25 showing high ras expression developed mammary tumors. Nulliparity is one risk factor for human breast cancer, suggesting a protective effect of post-lactational mammary regression. In order to investigate the effect of post-lactational regression, the low tumor frequency lines were crossed with mice expressing ubiquitously the human growth hormone gene, which induces permanent development of the mammary epithelium. Indeed, mammary tumors were observed in 76% of double transgenic females. Thus, the tumorigenic potential of the ras oncogene in mammary cells in vivo correlates with the level of its expression and with the developmental history of the mammary gland. Transformation coincides with the escape of oncogene expression from the regulation of the Wap promoter and the extinction of endogenous Wap gene expression.

A transgenic mouse model for inducible and reversible dysmyelination.

Oligodendrocytes are glial cells devoted to the production of myelin sheaths. Myelination of the CNS occurs essentially after birth. To delineate both the times of oligodendrocyte proliferation and myelination, as well as to study the consequence of dysmyelination in vivo, a model of inducible dysmyelination was developed. To achieve oligodendrocyte ablation, transgenic animals were generated that express the herpes virus 1 thymidine kinase (HSV1-TK) gene under the control of the myelin basic protein (MBP) gene promoter. The expression of the MBP-TK transgene in oligodendrocytes is not toxic on its own; however, toxicity can be selectively induced by the systemic injection of animals with nucleoside analogs, such as FIAU [1-(2-deoxy-2-fluoro-beta-delta-arabinofuranosyl)-5-iodouracil]. This system allows us to control the precise duration of the toxic insult and the degree of ablation of oligodendrocytes in vivo. We show that chronic treatment of MBP-TK mice with FIAU during the first 3 postnatal weeks triggers almost a total depletion of oligodendrocytes in the CNS. These effects are accompanied by a behavioral phenotype characterized by tremors, seizures, retarded growth, and premature animal death. We identify the period of highest oligodendrocytes division in the first 9 postnatal days. Delaying the beginning of FIAU treatments results in different degrees of dysmyelination. Dysmyelination in MBP-TK mice is always accompanied by astrocytosis. Thus, this transgenic line provides a model to study the events occurring during dysmyelination of various intensities. It also represents an invaluable tool to investigate remyelination in vivo.

RAR beta isoforms: distinct transcriptional control by retinoic acid and specific spatial patterns of promoter activity during mouse embryonic development.

That both deficiency and excess of vitamin A lead to a wide spectrum of congenital abnormalities has strongly implicated the active metabolite, retinoic acid (RA), in normal embryonic development. There are 3 families of RA receptors (RARs), RAR alpha, RAR beta and RAR gamma, each having at least two isoforms derived from primary transcripts initiated at two promoters P1 and P2 (reviewed in Leid et al., 1992) Transcripts encoding all 4 isoforms of RAR beta (RAR beta 1 to RAR beta 4) accumulate in embryonal carcinoma (EC) cells in the presence of RA. It has been previously shown that the RA modulation of RAR beta 2/beta 4 transcripts is achieved at the level of transcriptional initiation via a RA response element (RARE) present in the P2 RAR beta 2/beta 4 promoter. In contrast, the mechanism by which RA up-regulates RAR beta 1/beta 3 transcripts has not yet been elucidated. We describe here the isolation of the P1 RAR beta 1/beta 3 promoter and characterization of its activity in transgenic animals. We find that RAR beta 1/beta 3 promoter activity, which is apparently confined to the embryonic CNS, is not modified by RA treatment, unlike that of the RAR beta 2/beta 4 promoter (Mendelsohn et al., 1991). Nuclear run-on transcription analysis in EC cells supports the conclusion that RAR beta 1/beta 3 transcript initiation is not modulated by RA, and that the RA-induced accumulation of RAR beta 1/beta 3 transcripts occur via a RA-dependent release of a block in RNA chain elongation.

Lymphoproliferative disorders in an IL-7 transgenic mouse line.

A high incidence of severe lymphoproliferative disease was observed in a newly generated strain of mice carrying murine IL-7 as a transgene under the control of the E alpha (MHC class II) promoter. An analysis of the cells from lesions in these mice shows the selective expansion of cells at an early stage of B cell development and, more interestingly, expansion of cells phenotypically identical to the recently reported bipotent (B/macrophage) stem cell populations described in midgestation embryonic liver. Such cells can be propagated (and remain dependent upon) bone marrow feeder cell lines obtained from IL-7 transgenic mice. A molecular analysis of fresh and cultured cells reveals that the lesions are oligoclonal, or in rare cases monoclonal, and include clones of cells with unrearranged Ig heavy chain loci. These data suggest that IL-7 acts at multiple stages of B cell development. Furthermore cell lines derived from IL-7 transgenic mice may provide a novel source of rare factor-dependent bipotent stem cells.

Breast cancer protein PS2 synthesis in mammary gland of transgenic mice and secretion into milk.

PS2, a small estrogen-inducible secretory polypeptide with structural analogies to a growth factor, is produced by approximately 50% of human breast tumors. The function of PS2 is, however, unknown. To determine whether PS2 may play an autocrine role in the development of mammary tumors we constructed transgenic mice bearing fusion constructs designed to direct the expression of human PS2 in the lactating mammary gland under the control of the whey acidic protein (WAP) promoter. Mouse lines bearing the genomic PS2 gene under the control of the WAP promoter region (WAP-PS2-2) failed to express the transgene. However, mice harboring the fusion construct WAP-PS2-1, in which the PS2 coding sequence is inserted into the 5' untranslated region of the complete WAP gene, were observed to express the transgene. Expression was restricted to the secretory epithelium of the mammary gland during lactation, and PS2 protein was secreted into the milk. Nevertheless, no mammary gland dysplasia was observed, and PS2 expression had no discernable effect upon the physiology and/or development of the suckling young or the transgenic mother.

In vivo functional analysis of the mouse estrogen receptor gene promoter: a transgenic mouse model to study tissue-specific and developmental regulation of estrogen receptor gene transcription.

Understanding the molecular and morphological basis of estrogen responsiveness in the various tissues and organs that make up an adult organism and its onset during ontogenesis requires identification of the genetic controls that determine timed expression of the estrogen receptor (ER) gene in multiple cell types. With this goal in mind, we describe here the results of the functional analysis of the mouse (m) ER gene promoter, carried out in vivo in transgenic mice. The mER gene promoter was cloned and spliced to the coding sequence of the bacterial lacZ gene (fused to the nuclear localization signal of SV40 large T: nls-beta-GAL) and then stably reintegrated into the genome of mice. Analysis of beta-GAL mRNA and protein expression in multiple organs of both female and male transgenic animals was then performed. Results show that the transgenic mER promoter, much like the endogenous one, is active in several organs and tissues of adult female and male mice. The first 0.4 kilobases of 5'-flanking DNA (up to -364) are sufficient to direct widespread expression of the transgene in mouse organs. This indicates that genetic elements functional in various cell types are included in this segment. Furthermore, the first exon and intron of the mER gene are necessary to achieve sexually dimorphic expression of the transgene in neurons located at specific sites within the central nervous system. These mER promoter transgenic mice will be useful in mapping estrogen- responsive cell types under different physiological and pathological conditions in vivo, in defining ontogenesis of estrogen action in the mouse, and in studying the mechanisms that regulate ER gene transcription.

Prognostic factors for the recurrence of myxoid liposarcoma: 20 cases with up to 8 years follow-up.

INTRODUCTION: Myxoid liposarcomas (MLS) are the second most common type of liposarcoma. Although some MRI findings are distinctively characteristics of MLS, the diagnosis can be tricky in tumors with a large portion of round cells (RC). Known predictors of an unfavorable outcome include age, tumor size, high RC content and positive resection margins. The goal of this retrospective study was to define prognostic factors for recurrence, with special emphasis on the percentage of RCs and medical care provided in a non-specialized center. PATIENTS AND METHODS: Twenty patients (11 women, 9 men) with a mean age of 44.3 years (18-73) were reviewed after a mean of 55.9 months. Six of these patients had been operated at a non-specialized center. The diagnostic MRI was read by a specialized radiologist and the resection procedures performed by two specialized surgeons. Tumors were labeled as either "pure myxoid liposarcoma" or "myxoid/round-cell liposarcoma". The local recurrence-free survival rate and mortality rate were calculated. RESULTS: Fifteen patients had undergone an MRI during the initial assessment. The typical MRI findings of MLS were present in four of them. The MRI suggested a non-specific lesion in the other 11 patients. After correlation with pathology findings, these tumors contained more than 5% round cells. The fourteen patients treated at our facility had undergone a biopsy, while none of the ones treated outside did. Five patients had R0 resection margins and 15 had R1 margins. Prognostic factors for recurrence consisted of age, tumor size >10 cm, R1 resection margins, FNCLCC grade 2+R1 margins, medical care at a non-specialized center, and >5% round cells. There were eight local recurrences and three metastases (15%). Two patients died (90% overall survival rate). DISCUSSION: The risk of local recurrence was 3.86 times greater in this study when the tumor contained more than 5% RCs, which is consistent with published data. The MLS diagnosis was made only four times based on the initial MRI because misleading nature of high RC tumors. R1 resection margins are a risk factor for local recurrence. However, cases with R1 margins have a recurrence rate that is similar to R0 cases when the surgery is performed at a specialized cancer center. Treatment of MLS in a non-specialized center is a key negative prognostic factor. The reported rate of metastasis varies. Atypical extrapulmonary localizations are common, and often multifocal. MRI has been shown to be superior at detecting secondary lesions and some have suggested that a full-body MRI should be performed. CONCLUSION: Prognostic factors for the recurrence of myxoid liposarcomas have been identified. MRI analysis is not definitive and must be supplemented by a biopsy.

Precocious mammary gland development and milk protein synthesis in transgenic mice ubiquitously expressing human growth hormone.

A chimeric gene comprising the hydroxymethylglutaryl coenzyme-A reductase promoter and the human GH (hGH) genomic sequences was used to create transgenic mice expressing hGH in all tissues. In transgenic females, morphological development of the mammary gland and milk protein (WAP) expression commences at 3 weeks of age. At 8 weeks of age the mammary gland is morphologically and functionally comparable to that normally reached after 14-15 days of gestation. Precocious development correlated with local expression of hGH in mammary gland. Organ culture in the presence of different lactogenic hormones revealed that insulin and hydrocortisone are sufficient to maintain transcription of the WAP gene in transgenic mammary gland. In contrast, WAP transcription in normal gland required either hGH or PRL in addition to insulin and hydrocortisone. However, the effect of hGH on mammary differentiation does not appear to be solely mediated through an interaction with PRL receptors, since PRL, when added to cultured mammary tissues, did not elicit an equivalent response.

The methylation-free status of a housekeeping transgene is lost at high copy number.

Transgenic mouse lines were established bearing tandem arrays of a fusion construct comprising the promoter region of a housekeeping gene, HMGCR, encoding 3-hydroxy 3-methylglutaryl CoA reductase, linked to a bacterial cat reporter gene encoding chloramphenicol acetyltransferase (CAT). CAT activity was observed in all transgenic mouse tissues examined. The methylation state of the fusion transgene was determined. In non-transgenic mice the endogenous HMGCR promoter is devoid of methylation while flanking regions are extensively modified. In HMGCR-cat transgenic mice the fusion gene promoter was found to be similarly hypomethylated. However, the extent of hypomethylation varied with copy number: methylation-free status was progressively lost with increasing transgene copy number. Further transgenic mouse lines were constructed carrying a truncated HMGCR regulatory region linked to cat. Transgene expression and hypomethylation were observed in testis but not in any other tissue, and testis-specific methylation-free status was also lost at high copy number. Loss of hypomethylation at high copy number may indicate that saturable DNA-binding factors normally protect the HMGCR promoter from methylation.

A vector driving the expression of foreign cDNAs in the MHC class II-positive cells of transgenic mice.

We describe a plasmid vector that drives the expression of foreign cDNAs in transgenic mice, according to the dictates of an MHC class II gene promoter. Using this vector, we have often obtained mRNA and protein synthesis with a tissue and cell-type specificity indistinguishable from that of the endogenous MHC class II genes.

Developmental roles of the retinoic acid receptors.

Retinoic acid, one of the principle active metabolites of vitamin A (retinol), is believed to be essential for numerous developmental and physiological processes. Vitamin A deprivation (VAD) during development leads to numerous congenital defects. Previous studies of retinoic acid receptor (RAR) deficient mice failed to reveal any of these VAD-induced defects. This finding suggested that either the RARs are functionally redundant or that they are not critically required during development. In order to address these possibilities, we derived a number of RAR compound mutants. Unlike RAR single mutants, these compound null mutants died either in utero or shortly following birth. Histological analysis revealed essentially all of the defects characteristic of fetal VAD. A number of additional malformations, not described in previous VAD studies, were also observed. These included defects of the ocular and salivary glands and their ducts, the skeletal elements of the fore- and hindlimbs, and the cervical region of the axial skeleton. In addition, with the exception of derivatives forming within the first pharyngeal arch, most of the elements derived from mesectoderm emanating from cranial and hindbrain levels were affected. A number of these mutants also exhibited supernumerary cranial skeletal elements characteristics of the reptilian skull. A summary of the defects found in these RAR double mutants is presented.