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.

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.

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.

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.

A 2-Mb YAC/BAC-based physical map of the ovum mutant (Om) locus region on mouse chromosome 11.

The embryonic lethal phenotype observed when DDK females are crossed with males from other strains results from a deleterious interaction between the egg cytoplasm and the paternal pronucleus soon after fertilization. We have previously mapped the Om locus responsible for this phenotype, called the DDK syndrome, to an approximately 2-cM region of chromosome 11. Here, we report the generation of a physical map of 28 yeast and bacterial artificial chromosome clones encompassing the entire genetic interval containing the Om locus. This contig, spanning approximately 2 Mb, was used to map precisely genes and genetic markers of the region. We determined the maximum physical interval for Om to be 1400 kb. In addition, 11 members of the Scya gene family were found to be organized into two clusters at the borders of the Om region. Two other genes (Rad51l3 and Schlafen 2) and one EST (D11Wsu78e) were also mapped in the Om region. This integrated map provides support for the identification of additional candidate genes for the DDK syndrome.

Teratocarcinomas induced by embryonic stem (ES) cells lacking vimentin: an approach to study the role of vimentin in tumorigenesis.

Vimentin is a class III intermediate filament protein widely expressed in the developing embryo and in cells of mesenchymal origin in the adult. Vimentin knock-out mice develop and reproduce without any obvious defect. This is an unexpected finding in view of the high degree of conservation of the vimentin gene among vertebrates. However, it does not exclude the possibility of a role for vimentin in pathological conditions, like tumorigenesis. To address this question directly, we have used a teratocarcinoma model involving the injection of ES cells into syngeneic mice. ES cells lacking vimentin were generated from 129/Sv Vim-/- mice with high efficiency. The absence of vimentin did not affect ES cell morphology, viability or growth rate in vitro. Tumours were induced by subcutaneous injection of either Vim-/- or Vim+/+ ES cells into Vim+/+ and Vim-/- mice, in order to analyse the effect of the absence of vimentin in either the tumorigenic cells or the host mice. No significant differences were found in either tumour incidence, size or vascularization of teratocarcinomas obtained with all possible combinations. Vim-/- ES-derived tumours showed the same cellular composition typical of teratocarcinomas induced by wild-type ES cells together with a very similar apoptotic pattern. Taken together, these results demonstrate that in this model vimentin is not essential for efficient tumour growth and differentiation in vivo.

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.

Nonpermissiveness for mouse embryonic stem (ES) cell derivation circumvented by a single backcross to 129/Sv strain: establishment of ES cell lines bearing the Omd conditional lethal mutation.

The inbred mouse strain DDK carries a conditional early embryonic lethal mutation that is manifested when DDK females are crossed to males of other inbred strains but not in the corresponding reciprocal crosses. It has been shown that embryonic lethality could be assigned to a single genetic locus called Ovum mutant (Om), on Chromosome (Chr) 11 near Syca 1. In the course of our study of the molecular mechanisms underlying the embryonic lethality, we were interested in deriving an embryonic stem cell bearing the Om mutation in the homozygous state (Omd/Omd). However, it turned out that DDK is nonpermissive for ES cell establishment, with a standard protocol. Here we show that permissiveness could be obtained using Omd/Omd blastocysts with a 75% 129/Sv and 25% DDK genetic background. Several germline-competent Omd/Omd ES cell lines have been derived from blastocysts of this genotype. Such a scenario could be extended to the generation of ES cell lines bearing any mutation present in an otherwise nonpermissive mouse strain.