Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice lacking MAOA.

Deficiency in monoamine oxidase A (MAOA), an enzyme that degrades serotonin and norepinephrine, has recently been shown to be associated with aggressive behavior in men of a Dutch family. A line of transgenic mice was isolated in which transgene integration caused a deletion in the gene encoding MAOA, providing an animal model of MAOA deficiency. In pup brains, serotonin concentrations were increased up to ninefold, and serotonin-like immunoreactivity was present in catecholaminergic neurons. In pup and adult brains, norepinephrine concentrations were increased up to twofold, and cytoarchitectural changes were observed in the somatosensory cortex. Pup behavioral alterations, including trembling, difficulty in righting, and fearfulness were reversed by the serotonin synthesis inhibitor parachlorophenylalanine. Adults manifested a distinct behavioral syndrome, including enhanced aggression in males.

A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier.

Listeria monocytogenes is responsible for severe food-borne infections, but the mechanisms by which bacteria cross the intestinal barrier are unknown. Listeria monocytogenes expresses a surface protein, internalin, that interacts with a host receptor, E-cadherin, to promote entry into human epithelial cells. Murine E-cadherin, in contrast to guinea pig E-cadherin, does not interact with internalin, excluding the mouse as a model for addressing internalin function in vivo. In guinea pigs and transgenic mice expressing human E-cadherin, internalin was found to mediate invasion of enterocytes and crossing of the intestinal barrier. These results illustrate how relevant animal models for human infections can be generated.

Both coding exons of the c-myc gene contribute to its posttranscriptional regulation in the quiescent liver and regenerating liver and after protein synthesis inhibition.

In vivo, the steady-state level of c-myc mRNA is mainly controlled by posttranscriptional mechanisms. Using a panel of transgenic mice in which various versions of the human c-myc proto-oncogene were under the control of major histocompatibility complex H-2Kb class I regulatory sequences, we have shown that the 5' and the 3' noncoding sequences are dispensable for obtaining a regulated expression of the transgene in adult quiescent tissues, at the start of liver regeneration, and after inhibition of protein synthesis. These results indicated that the coding sequences were sufficient to ensure a regulated c-myc expression. In the present study, we have pursued this analysis with transgenes containing one or the other of the two c-myc coding exons either alone or in association with the c-myc 3' untranslated region. We demonstrate that each of the exons contains determinants which control c-myc mRNA expression. Moreover, we show that in the liver, c-myc exon 2 sequences are able to down-regulate an otherwise stable H-2K mRNA when embedded within it and to induce its transient accumulation after cycloheximide treatment and soon after liver ablation. Finally, the use of transgenes with different coding capacities has allowed us to postulate that the primary mRNA sequence itself and not c-Myc peptides is an important component of c-myc posttranscriptional regulation.

The cis-acting elements known to regulate c-myc expression ex vivo are not sufficient for correct transcription in vivo.

Much of our knowledge about the regulation of the c-myc proto-oncogene expression has come from studies of c-myc gene expression in several well defined ex vivo systems, including differentiation systems and tumor cells. However, very few investigations have been performed to determine the factors and cis-acting sequences that regulate c-myc expression in vivo. In order to obtain information on the sequences required to regulate c-myc gene transcription from the two major P1 and P2 initiation sites in the mouse, we have generated several constructs containing human or murine c-myc genomic sequences with various 5' flanking sequences and derived corresponding transgenic mice. A sensitive S1 nuclease protection assay was performed to analyse and to compare transgene expression with that of the endogenous c-myc mRNA, either in adult organs, or during development. None of the transgenic mice expressed the construct appropriately, although several strains exhibited unexpected expression most probably due to position effects. Our results indicate that the cis-acting elements described to regulate c-myc expression ex vivo are not sufficient to drive the correct expression of c-myc gene in vivo and strongly suggest that additional regulatory elements located upstream from -3500 (with respect to mouse P1 promoter) and downstream 1500 bp from polyadenylation sites are required.

The 5' and 3' non-coding sequences of the c-myc gene, required in vitro for its post-transcriptional regulation, are dispensable in vivo.

We have previously shown that in vivo the steady-state level of c-myc mRNA in different quiescent organs and its induction in the early stages of hepatic regeneration and after inhibition of protein synthesis are mainly controlled by post-transcriptional mechanisms. In order to localize the target sequences for these mechanisms, transgenic lines expressing various versions of the human c-myc proto-oncogene have been constructed. To avoid all possible transcriptional controls due to the c-myc 5' regulatory region, the c-myc genomic sequences were fused to MHC H-2Kb class I regulatory sequences, which have previously been shown to be able to drive reporter gene expression in most adult tissues. The transgenes contained either all human c-myc genomic sequences or were deleted for one of the sequences which have been shown in in vitro experiments to play a role in c-myc mRNA stabilization, in particular exon 1, intron 1 and the 3' non-coding region. Several independent transgenic lines were derived for each construct. Using S1 nuclease protection analysis, we have monitored H-2K, mouse c-myc and transgene mRNA expression in several quiescent adult organs, at the start of liver regeneration and after inhibition of protein synthesis in each transgenic line. Our results indicate that the 5' non-coding sequences, including exon 1 and intron 1, and the 3' untranslated region are all dispensable in the different aspects of c-myc post-transcriptional regulation.

Healthy mice with an altered c-myc gene: role of the 3' untranslated region revisited.

c-Myc is a protooncogene involved in the control of cellular proliferation, differentiation and apoptosis. Like many other early response genes, regulation of c-myc expression is mainly controlled at the level of mRNA stability. Multiple cis-acting destabilizing elements have been described that are located both in the protein-coding region and in the 3' untranslated region (3' UTR). However, it is not known when they function during development and whether they act as partly redundant or independent elements to regulate c-myc mRNA level of expression. To begin to address these questions, we created a series of c-myc alleles modified in the 3' UTR, using homologous recombination and the Cre/loxP system, and analysed the consequences of these modifications in ES cells and transgenic animals. We found that deletion of the complete 3' UTR, including runs of Us and AU-rich elements proposed, on the basis of cell-culture assays, to be involved in the control of c-myc mRNA stability, did not alter the steady-state level of c-myc mRNA in any of the various situations analysed in vivo. Moreover, mice homozygous for the 3' UTR-deleted gene were perfectly healthy and fertile. Our results therefore strongly suggest that the 3' UTR of c-myc mRNA does not play a major role in the developmental control of c-myc expression.

BALB/c alleles at modifier loci increase the severity of the maternal effect of the "DDK syndrome".

The Om locus was first described in the DDK inbred mouse strain: DDK mice carry a mutation at Om resulting in a parental effect lethality of F(1) embryos. When DDK females are mated with males of other (non-DDK) inbred strains, e.g., BALB/c, they exhibit a low fertility, whereas the reciprocal cross, non-DDK females x DDK males, is fertile (as is the DDK intrastrain cross). The low fertility is due to the death of (DDK x non-DDK)F(1) embryos at the late-morula to blastocyst stage, which is referred to as the "DDK syndrome." The death of these F(1) embryos is caused by an incompatibility between a DDK maternal factor and the non-DDK paternal pronucleus. Previous genetic studies showed that F(1) mice have an intermediate phenotype compared to parental strains: crosses between F(1) females and non-DDK males are semisterile, as are crosses between DDK females and F(1) males. In the present studies, we have examined the properties of mice heterozygous for BALB/c and DDK Om alleles on an essentially BALB/c genetic background. Surprisingly, we found that the females are quasi-sterile when mated with BALB/c males and, thus, present a phenotype similar to DDK females. These results indicate that BALB/c alleles at modifier loci increase the severity of the DDK syndrome.

lacZ sequences prevent regulated expression of housekeeping genes.

In order to dissect the MHC class I H-2K gene regulatory sequences, we p reviously generated transgenic mice containing various H-2K/lacZ fusion genes. However contrary to transgenes where H-2K sequences were fused to other coding sequences, none of the lacZ fusion transgenes was widely ex pressed like H-2K gene. We now show that this silencing also occurs when lacZ is inserted into a larger H-2K genomic construct including promoter and other regulatory elements. Because the 5'H-2K region contains a CpG island, we suspected that the presence of lacZ coding sequences was inte rfering with the mechanism by which the H-2K promoter region is normally unmethylated and transcriptionally active. Indeed, we show that in high ( >10) copy number transgenic mice, insertion of lacZ sequences in the v icinity of the H-2K promoter results in partial or complete methylation of the H-2K CpG island. However, in low (1-3) copy number transgenic mic e no methylation was observed but the transgene was still silent, sugges ting that the silencing effect of lacZ does not only rely on abnormal CpG methylation. Intriguingly, when the H -2/lacZ construct was introduced via embryonic stem (ES) cells, regulate d transgene expression was observed in several chimaeric embryos derived from independent ES clones, but never in adult chimeras. Combined with t he fact that, despite much effort, it has been very difficult to generat e 'blue' mice, our results highlight the transcription-silencing effect of lacZ sequences when they are associated with regulatory sequences of ubiquitously expressed genes.

Oocyte-specific inactivation of Omcg1 leads to DNA damage and c-Abl/TAp63-dependent oocyte death associated with dramatic remodeling of ovarian somatic cells.

Aberrant loss of oocytes following cancer treatments or genetic mutations leads to premature ovarian insufficiency (POI) associated with endocrine-related disorders in 1% of women. Therefore, understanding the mechanisms governing oocyte death is crucial for the preservation of female fertility. Here, we report the striking reproductive features of a novel mouse model of POI obtained through oocyte-specific inactivation (ocKO) of Omcg1/Zfp830 encoding a nuclear zinc finger protein involved in pre-mRNA processing. Genetic ablation of OMCG1 in early growing oocytes leads to reduced transcription, accumulation of DNA double-strand breaks and subsequent c-Abl/TAp63-dependent oocyte death, thus uncovering the key role of OMCG1 for oocyte genomic integrity. All adult Omcg1(ocKO) females displayed complete elimination of early growing oocytes and sterility. Unexpectedly, mutant females exhibited a normal onset of puberty and sexual receptivity. Detailed studies of Omcg1(ocKO) ovaries revealed that the ovarian somatic compartment underwent a dramatic structural and functional remodeling. This allowed the cooperation between oocyte-depleted follicles and interstitial tissue to produce estradiol. Moreover, despite early folliculogenesis arrest, mutant mice exhibited sexual cyclicity as shown by cyclical changes in estrogen secretion, vaginal epithelium cytology and genital tract weight. Collectively, our findings demonstrate the key role of Omcg1 for oocyte survival and highlight the contribution of p63 pathway in damaged oocyte elimination in adulthood. Moreover, our findings challenge the prevailing view that sexual cyclicity is tightly dependent upon the pace of folliculogenesis and luteal differentiation.

Ribosome biogenesis dysfunction leads to p53-mediated apoptosis and goblet cell differentiation of mouse intestinal stem/progenitor cells.

Ribosome biogenesis is an essential cellular process. Its impairment is associated with developmental defects and increased risk of cancer. The in vivo cellular responses to defective ribosome biogenesis and the underlying molecular mechanisms are still incompletely understood. In particular, the consequences of impaired ribosome biogenesis within the intestinal epithelium in mammals have not been investigated so far. Here we adopted a genetic approach to investigate the role of Notchless (NLE), an essential actor of ribosome biogenesis, in the adult mouse intestinal lineage. Nle deficiency led to defects in the synthesis of large ribosomal subunit in crypts cells and resulted in the rapid elimination of intestinal stem cells and progenitors through distinct types of cellular responses, including apoptosis, cell cycle arrest and biased differentiation toward the goblet cell lineage. Similar observations were made using the rRNA transcription inhibitor CX-5461 on intestinal organoids culture. Importantly, we found that p53 activation was responsible for most of the cellular responses observed, including differentiation toward the goblet cell lineage. Moreover, we identify the goblet cell-specific marker Muc2 as a direct transcriptional target of p53. Nle-deficient ISCs and progenitors disappearance persisted in the absence of p53, underlying the existence of p53-independent cellular responses following defective ribosome biogenesis. Our data indicate that NLE is a crucial factor for intestinal homeostasis and provide new insights into how perturbations of ribosome biogenesis impact on cell fate decisions within the intestinal epithelium.

Gene targeting the myf-5 locus with nlacZ reveals expression of this myogenic factor in mature skeletal muscle fibres as well as early embryonic muscle.

We have introduced the nlacZ reporter gene into the locus of the myogenic factor gene myf-5 by homologous recombination in embryonic stem (ES) cells. Targeted ES clones were injected into precompaction morula, and the beta-galactosidase expression pattern was monitored. These mice permit the sensitive visualization of myf-5 expression throughout the embryo, and provide a standard for comparing it with that seen with different myf-5/nlacZ transgenes. Thus, in a comparison using ES cells in chimaeric embryos containing the targeted or randomly integrated myf-5/nlacZ construct, we demonstrate that 5.5 kbp of myf-5 upstream flanking sequence including exon1 and most of intron1 directs some skeletal muscle expression, but this is neither qualitatively nor quantitatively equivalent to that of the endogenous gene. Myf-5 is expressed early, before terminal myogenesis takes place in the medial half of the somite, and subsequently it is a major myogenic factor as skeletal muscle forms. All skeletal muscle shows beta-galactosidase activity, even after birth, indicating that myf-5 expression is not confined to primary myotubes, which are derived from embryonic myoblasts, but is also present in muscles containing different adult fibre types. The presence of myf-5 transcripts from the endogenous gene in older muscle was confirmed by in situ hybridization. These results suggest that the myf-5 gene is not activated in only a subset of muscle cells and are consistent with the results on the MyoD knockout mice.

Developmental expression of H-2K major histocompatibility complex class I transgenes requires the presence of proximal introns.

The pattern of expression of the H-2K major histocompatibility complex (MHC) class I gene is complex. During embryonic development H-2K mRNA, detectable from midgestation, is poorly expressed. In the adult, H-2K expression is nearly ubiquitous but transcriptional regulation occurs leading to different mRNA levels in the different organs of the mouse. In vitro studies have shown that most of the regulatory elements controlling H-2K gene transcription are located in the 5' region of the gene. However, using fusion transgenes in which reporter genes were under the control of 2 kb of H-2K 5' regulatory region, we have previously shown that this region was not sufficient to ensure correct developmental transgene expression. By contrast, a native 9.25 kb H-2K transgene was expressed appropriately both in the adult and in the embryo. In order to localise more precisely the cis-acting regulatory sequences involved in H-2K developmental expression, we have now constructed new transgenic lines containing H-2Kb transgenes that were deleted from specific parts of the H-2Kb gene. We show that deletion of either the H-2K 3' flanking region or the 5 (out of 7) distal introns results in an expression of the transgenes which is similar to that of the endogenous H-2K gene, both in the adult and during embryonic development. By contrast, deletion of all the introns or of the two proximal ones abrogates H-2K transgene expression. Our data reveal the complexity of H-2K regulation and highlight the crucial role of proximal introns in H-2K expression in the living organism.

A maternal factor affecting mouse blastocyst formation.

Normal development of the mouse embryo requires the presence of both paternal and maternal genomes. This is due to functional differences having their origin in a differential imprinting of parental genomes. Furthermore, several lines of evidence show that the very early interactions between egg cytoplasm and pronuclei may influence the programming of the embryonic genome and modulate the functional inequality of the parental contribution even during preimplantation stages. In this paper, we show that a factor present in ovulated oocytes of the mouse mutant strain DDK and therefore of maternal origin prevents the formation of the blastocyst. This factor, which acts via an interaction with the paternal genome, is present in oocytes as an RNA and is still active in preimplantation embryos. This is the first direct evidence of such a maternal control in the mouse.

Mice lacking vimentin develop and reproduce without an obvious phenotype.

To address the biological role of vimentin in the context of the living organism, we have introduced a null mutation of the vimentin gene into the germ line of mice. Surprisingly, animals homozygous for this mutation developed and reproduced without an obvious phenotype. Immunoblotting, immunofluorescence, and immunogold labeling analysis confirmed the absence of vimentin and of the corresponding filament network. Furthermore, no compensatory expression of another intermediate filament could be demonstrated. While these results leave open the question of the possible role of vimentin in unusual situations or pathological conditions, they show that a conspicuous developmental and cell-specific structure that is an integral part of the cytoskeleton can be eliminated without apparent effect on mouse reproduction and development.

The intronic immunoglobulin kappa gene enhancer acts independently on rearrangement and on transcription.

It is not known which cis-acting elements regulate kappa gene rearrangement. However, the onset of rearrangement coincides with the onset of transcription suggesting that common regulatory elements are used. We have investigated the role of the intronic enhancer on rearrangement using mice transgenic for kappa minigenes. The rabbit kappa enhancer, which is defective in activation of transcription in mouse cells, allows a high level of rearrangement. However, transgenes which possess no enhancer region at all are rearranged a hundred times less compared to transgenes which possess the rabbit enhancer. Our results suggest that while the enhancer region can activate rearrangement as well as transcription, its action on both phenomena can be uncoupled.

A high-resolution map around the locus Om on mouse Chromosome 11.

The locus Om (ovum mutant) identified in the mouse strain DDK affects the viability of (DDK x non-DDK)F1 preimplantation embryos. We previously located this locus on Chromosome (Chr) 11 close to Scya2 (Baldacci et al. Mamm. Genome 2, 100-105, 1992). Here we report a high-resolution map of the region around Om based on a large number of backcross individuals. The same region has been analyzed on the EUCIB backcross, and the two maps have been compared. The results define the proximal and distal boundaries for the Om mutation as Scya2 and D11Mit36 respectively. The distance between these two markers is about 2 cM. These data should facilitate the positional cloning and molecular characterization of Om.

Cardiovascular lesions and skeletal myopathy in mice lacking desmin.

In order to further our understanding of the biological role of desmin, the muscle-specific intermediate filament protein, a null mutation in the desmin gene was introduced into the germ line of mice. Despite the complete lack of desmin, these mice developed and reproduced. Since we show that skeletal, cardiac, and smooth muscles form in the Des-/- mice, it is reasonable to propose that desmin is not essential for myogenic commitment or for myoblast fusion or differentiation in vivo. However, morphological abnormalities were observed in the diaphragm of adult mice; these were demonstrated by disorganized, distended, and nonaligned fibers. The heart presented areas of hemorrhaging in which fibrosis and ischemia were observed. We have also shown that the absence of desmin produces specific defects in smooth muscles. In conclusion, our results have demonstrated that desmin is not required for the differentiation of skeletal, cardiac, and smooth muscles but is essential to strengthen and maintain the integrity of these tissues.

Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain.

The zinc finger gene Krox-20 is transcribed in two alternate segments (rhombomeres) of the developing hindbrain. To investigate its function, we have used homologous recombination to generate mice carrying an in-frame insertion of the E. coli lacZ gene within Krox-20. Analysis of the beta-galactosidase pattern in heterozygous embryos confirmed the known profile with expression restricted to rhombomeres (r) 3 and 5. Mice homozygous for the mutation die during the first two weeks after birth. Anatomical analysis of the hindbrain and of the cranial nerves during embryogenesis, combined with the determination of the expression patterns of rhombomere-specific genes, demonstrated that Krox-20 inactivation results in a marked reduction or elimination of r3 and r5. We conclude that Krox-20, although not required for the initial delimitation of r3 and r5, plays an important role in the process of segmentation governing hindbrain development.

Immortalization of multiple cell types from transgenic mice using a transgene containing the vimentin promoter and a conditional oncogene.

Differentiated clonal cell lines were obtained from transgenic mice carrying a recombinant gene composed of DNA coding for a temperature-sensitive mutant of the simian virus large T antigen under the control of regulatory elements of the human vimentin gene. In response to mitogenic factors the vimentin promoter is activated in the presence of serum in almost all cultured cells independently of their origin. The expression of the T antigen could be controlled both at the level of transcription since the vimentin promoter is growth-regulated and at the level of the protein structure through the temperature stability of the T antigen. Indeed, the switch-off of the oncogene protein is obtained by serum deprivation of the culture and achieved with enhancement of the growth temperature. From transgenic mice several types of clonal differentiated cell lines were established and characterized including melanocytes, macrophages, mesangial, muscle, and endothelial cells. Melanocytes displayed melanin while endothelial cells from brain and heart expressed the related factor VIII and low density lipoprotein absorption capacities. Mesangial cells from kidney exhibited numerous desmosomes. Typical markers of macrophages from bone marrow were observed while skeletal muscle cells fused and contracted.

Normal and pathological expression of GFAP promoter elements in transgenic mice.

The expression of the glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. In order to characterize DNA sequences involved in such regulations, we produced transgenic mice bearing 2 kb of the 5' flanking region of the murine GFAP gene linked to the Escherichia coli beta-galactosidase (beta-gal) reporter gene. Seven transgenic lines were obtained. We observed that the regulatory elements present in the transgene GFAP-nls-LacZ direct an expression in the neural and non-neural tissue and target in vivo an unexpected subpopulation of astrocyte. In the developing brain, beta-gal activity and GFAP appeared simultaneously and in the same region, on embryonic day 18 (E18), suggesting that the 2 kb of the promoter contains the regulatory sequences responsible for the perinatal vimentin/GFAP switch. In addition, we demonstrated that the 2 kb sequence of the GFAP promoter used in the transgene possess elements which are activated after a surgical injury, thus permitting to study some aspects of reactive gliosis in these transgenic mice. These transgenic lines provide a useful tool by enabling further studies of astroglial and, probably, neuronal physiologies.