Mutation in the Trapalpha/Ssr1 gene, encoding translocon-associated protein alpha, results in outflow tract morphogenetic defects.

Translocon-associated protein complex (TRAP) is thought to be required for efficient protein-specific translocation across the endoplasmic reticulum membrane. We created a mutation in the Trapalpha gene that leads to the synthesis of a truncated TRAPalpha protein fused to ShBle-beta-galactosidase. Analysis of Trapalpha cDNAs reveals that among three different messenger RNAs expressed in the mouse, one of them encodes a slightly larger protein that differs in its C-terminal end. This mRNA, specific for skeletal muscle and heart, is only expressed after birth. Homozygous Trapalpha mutant pups die at birth, likely as a result of severe cardiac defects. Indeed, the septation of the proximal part of the outflow tract is absent, resulting in a double-outlet right ventricle. Studies of protein secretion in transfected embryonic fibroblasts reveal that the TRAP complex does not function properly in homozygous mutant cells and confirm, in vivo, the involvement of TRAP in substrate-specific translocation. Our results provide the first in vivo demonstration that a member of the TRAP complex plays a crucial role in mammalian heart development and suggest that TRAPalpha could be involved in translocation of factors necessary for maturation of endocardial cushions.

Genome engineering via homologous recombination in mouse embryonic stem (ES) cells: an amazingly versatile tool for the study of mammalian biology.

The ability to introduce genetic modifications in the germ line of complex organisms has been a long-standing goal of those who study developmental biology. In this regard, the mouse, a favorite model for the study of the mammals, is unique: indeed not only is it possible since the late seventies, to add genes to the mouse genome like in several other complex organisms but also to perform gene replacement and modification. This has been made possible via two technological breakthroughs: 1) the isolation and culture of embryonic stem cells (ES), which have the unique ability to colonize all the tissues of an host embryo including its germ line; 2) the development of methods allowing homologous recombination between an incoming DNA and its cognate chromosomal sequence (gene "targeting"). As a result, it has become possible to create mice bearing null mutations in any cloned gene (knock-out mice). Such a possibility has revolutionized the genetic approach of almost all aspects of the biology of the mouse. In recent years, the scope of gene targeting has been widened even more, due to the refinement of the knock-out technology: other types of genetic modifications may now be created, including subtle mutations (point mutations, micro deletions or insertions, etc.) and chromosomal rearrangements such as large deletions, duplications and translocations. Finally, methods have been devised which permit the creation of conditional mutations, allowing the study of gene function throughout the life of an animal, when gene inactivation entails embryonic lethality. In this paper, we present an overview of the methods and scenarios used for the programmed modification of mouse genome, and we underline their enormous interest for the study of mammalian biology.

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.

Gene trap insertion reveals two open reading frames in the mouse SSeCKS gene: the form predominantly detected in the nervous system is suppressed by the insertion while the other, specific of the testis, remains expressed.

Scaffold proteins play an important role in regulating signal transduction by targeting kinases and phosphatases in close proximity to their relevant substrates. SSeCKS protein has been described as a protein kinase C and A (PKC/PKA) anchoring protein as well as a PKC substrate with a tumor suppressor activity. In this study, we report the generation, via gene trapping in embryonic stem cells of mice carrying an insertion in the mouse SSeCKS gene. Through the molecular analysis of the insertion site, we show that SSeCKS contains two alternative promoters directing the synthesis of mRNAs (P1- and P2-mRNA), encoding two different proteins, one of which would be a truncated form of the other. Interestingly, these RNAs are differentially expressed, P2 being found exclusively in the male germ line, while P1 exhibits a dynamic and wider pattern of expression during embryonic development and in the adult; its expression is predominant in the nervous system. Finally, we show that P1- but not P2-mRNA expression is abolished by the insertion and furthermore that mice homozygous for the mutation lack SSeCKS in all tissues except the male germ cells. Nevertheless and surprisingly, these mice do not exhibit any obvious phenotype. The functional implications of these observations are discussed.

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.

Transgenic mice: an irreplaceable tool for the study of mammalian development and biology.

Stable integration into the mouse genome of exogenous genetic information, i.e., the creation of transgenic mice, has become a privileged way of analyzing gene function in normal development and pathology. Both gene addition and gene replacement may be performed. This has allowed, in particular, the creation of mice in which precise mutations are introduced into a given gene. Furthermore, in recent years, strategies that induce the expression of a mutation in a given type of cell and/or at a given time in development have been developed. Thus, the transgenic methodology affords a unique and irreplaceable tool for the study of mammalian development and biology and for the creation of animal models for human genetic diseases.

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.

Introducing a null mutation in the mouse K6alpha and K6beta genes reveals their essential structural role in the oral mucosa.

Mammalian genomes feature multiple genes encoding highly related keratin 6 (K6) isoforms. These type II keratins show a complex regulation with constitutive and inducible components in several stratified epithelia, including the oral mucosa and skin. Two functional genes, K6alpha and K6beta, exist in a head-to-tail tandem array in mouse genomes. We inactivated these two genes simultaneously via targeting and homologous recombination. K6 null mice are viable and initially indistinguishable from their littermates. Starting at two to three days after birth, they show a growth delay associated with reduced milk intake and the presence of white plaques in the posterior region of dorsal tongue and upper palate. These regions are subjected to greater mechanical stress during suckling. Morphological analyses implicate the filiform papillae as being particularly sensitive to trauma in K6alpha/K6beta null mice, and establish the complete absence of keratin filaments in their anterior compartment. All null mice die about a week after birth. These studies demonstrate an essential structural role for K6 isoforms in the oral mucosa, and implicate filiform papillae as being the major stress bearing structures in dorsal tongue epithelium.

Impaired wound healing in embryonic and adult mice lacking vimentin.

It is generally assumed that the vimentin intermediate filament network present in most mesenchymally-derived cells is in part responsible for the strength and integrity of these cells, and necessary for any tissue movements that require the generation of significant tractional forces. Surprisingly, we have shown that transgenic KO mice deficient for vimentin are apparently able to undergo embryonic development absolutely normally and go onto develop into adulthood and breed without showing any obvious phenotype. However, fibroblasts derived from these mice are mechanically weak and severely disabled in their capacity to migrate and to contract a 3-D collagen network. To assess whether these functions are necessary for more challenging tissue movements such as those driving in vivo tissue repair processes, we have analysed wound healing ability in wild-type versus vimentin-deficient embryos and adult mice. Wounds in vimentin-deficient adult animals showed delayed migration of fibroblasts into the wound site and subsequently retarded contraction that correlated with a delayed appearance of myofibroblasts at the wound site. Wounds made to vimentin-deficient embryos also failed to heal during the 24 hour culture period it takes for wild-type embryos to fully heal an equivalent wound. By DiI marking the wound mesenchyme and following its fate during the healing process we showed that this impaired healing is almost entirely due to a failure of mesenchymal contraction at the embryonic wound site. These observations reveal an in vivo phenotype for the vimentin-deficient mouse, and challenge the dogma that key morphogenetic events occurring during development require generation of significant tractional forces by mesenchymal cells.

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.

Targeted disruption of the murine junD gene results in multiple defects in male reproductive function.

JunD is one of three mammalian Jun proteins that contribute to the AP-1 transcription factor complex. Distinct regulation and functions have been proposed for each Jun member, but less is known about the biological functions of each of these proteins in vivo. To investigate the role of JunD, we have inactivated the murine gene by replacement with a bacterial lacZ reporter gene. Embryonic JunD expression was initially detected in the developing heart and cardiovascular system. Subsequent broadening phases of JunD expression were observed during embryonic development and expression in the adult was widespread in many tissues and cell lineages. Mutant animals lack JunD mRNA and protein and showed no evidence of upregulation of c-Jun and JunB mRNA levels. In contrast to the other two Jun members, homozygous JunD-/- mutant animals were viable and appeared healthy. However, homozygous JunD-/- animals showed a reduced postnatal growth. Furthermore, JunD-/- males exhibited multiple age-dependent defects in reproduction, hormone imbalance and impaired spermatogenesis with abnormalities in head and flagellum sperm structures. No defects in fertility were observed in JunD-/- female animals. These results provide evidence for redundant functions for members of the Jun family during development and specific functions for JunD in male reproductive function.

The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression.

The assembly of eukaryotic DNA into nucleosomes and derived higher order structures constitutes a barrier for transcription, replication and repair. A number of chromatin remodeling complexes, as well as histone acetylation, were shown to facilitate gene activation. To investigate the function of two closely related mammalian SWI/SNF complexes in vivo, we inactivated the murine SNF5/INI1 gene, a common subunit of these two complexes. Mice lacking SNF5 protein stop developing at the peri-implantation stage, showing that the SWI/SNF complex is essential for early development and viability of early embryonic cells. Furthermore, heterozygous mice develop nervous system and soft tissue sarcomas. In these tumors the wild-type allele was lost, providing further evidence that SNF5 functions as a tumor suppressor gene in certain cell types.

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.

Expression of the vHNF1/HNF1beta homeoprotein gene during mouse organogenesis.

Formation of tubular structures from an epithelial tissue is a process common to many morphogenetic events during organogenesis. We report here new data concerning the expression pattern of the vHNF1/HNF1beta gene during this process in the mouse. vHNF1 (variant Hepatocyte Nuclear Factor 1) is a member of the HNF1 homeoprotein family. Its expression domain includes organs such as the liver, the kidney, the lung and the pancreas, but is restricted to the epithelial cells of these organs. To follow vHNF1 expression during organogenesis, we have introduced a NLS-lacZ gene under the control of vHNF1 regulatory regions by homologous recombination. Detection of the beta-galactosidase activity in heterozygous mice demonstrates that this gene is expressed in numerous tubular epitheliums as soon as they appear and all along development.

Essential role for the homeoprotein vHNF1/HNF1beta in visceral endoderm differentiation.

vHNF1/HNF1beta, a member of the divergent HNF1/vHNF1 homeoprotein family, is expressed in polarized epithelia of several adult organs and may participate in controlling the transcription of specific genes. In addition to this late requirement, vHNF1 may play earlier roles during development, as it is first expressed in the visceral endoderm at the onset of gastrulation. In order to shed light on its function during embryogenesis, we have inactivated the murine gene by homologous recombination. The homozygous mutation results in embryonic lethality by day 7.5 of development and vHNF1(-)(/)(-) embryos display a disorganized visceral endoderm and a significantly reduced size. Studies of ES cell differentiation and aggregation with tetraploid morulae establish that vHNF1 expression is essential for visceral endoderm differentiation, both in vitro and in vivo. Analysis of differentiation markers confirms that vHNF1 is part of a genetic network that directs the expression of HNF4 and downstream endodermal genes. Furthermore, the complementation of the mutant embryos with wild-type visceral endoderm rescues the day 7.5 lethality and reveals an additional phenotype linked to vHNF1 later expression. The examination of chimeric embryos suggests that vHNF1 expression might be cell-autonomously required in the gut for the proper morphogenesis of the embryo.

In vivo, villin is required for Ca(2+)-dependent F-actin disruption in intestinal brush borders.

Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca(2+)-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca(2+) differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca(2+), whereas Ca(2+) had no effect in villin-null isolates. Moreover, increase in intracellular Ca(2+) by serosal carbachol or mucosal Ca(2+) ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 +/- 9.6%, compared with wild-type mice, 70 +/- 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury.

A new family of mouse genes homologous to the human MAGE genes.

The human MAGE genes are expressed in a wide variety of tumors but not in normal cells, with the exception of the male germ cells, placenta, and, possibly, cells of the developing embryo. These genes encode tumor-specific antigens recognized by cytolytic T lymphocytes. The MAGE genes are located on the X chromosome, in three clusters denoted MAGE-A, B, and C, mapping at q28, p21.3, and q26, respectively. The function of these genes remains unknown. Because mice offer many advantages for the study of genes that may be involved in embryonic development, we looked for the murine equivalents of the 12 human MAGE-A genes. Using a MAGE-A probe, we isolated 8 new murine genes that are homologous to the MAGE genes. On average, the open reading frames (ORFs) of these 8 closely related genes display a slightly higher degree of nucleotide identity with the MAGE-A ORFs than with the MAGE-B or MAGE-C ORFs. Furthermore, like MAGE-A genes, they encode acidic proteins, whereas the MAGE-B genes encode basic proteins. Accordingly, these 8 murine genes were named Mage-a1 to 8 (approved symbols Magea1 to 8). Mage-a genes were mapped in two different loci on the mouse X chromosome. Mage-a4 and Mage-a7 are located in a region that is syntenic to either Xp21 or Xq28. The 6 other genes are arranged in a cluster located in a region syntenic to Xp22. Like their human counterparts, Mage-a genes were found to be transcribed in adult testis, but not in other tissues. Expression of some Mage-a genes was also detected in tumor cell lines. Two Mage-a genes were found to be expressed in blastocysts.

Cerebellar defect and impaired motor coordination in mice lacking vimentin.

Vimentin belongs to the family of intermediate filament (IF) proteins. During the nervous system development in mammals, it is transiently expressed in precursor cells of neuronal and glial lineages, and then it is progressively replaced by other types of IF proteins. Surprisingly, mice knock-out for vimentin develop and reproduce without any apparent defects (Colucci-Guyon et al. Cell 79:679-694, 1994). In adult rodents, Bergmann glia (BG) of the cerebellum continue to express vimentin together with glial fibrillary acidic protein (GFAP). A careful analysis of cerebellar morphology and ultrastructure in mutants showed poorly developed and highly abnormal BG, whereas the migration of granular neurons proceeded normally. Moreover, many Purkinje cells (PC) appeared stunted with a loss of spiny branchlets, and some of them were necrotic. Finally, impaired motor coordination was evidenced by behavioral tests. These observations demonstrate a role for vimentin in contributing to the normal development and morphology of BG and reveal a hitherto unreported functional relationship between BG and PC.