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.

Retinoic acid (RA) generated by Raldh2 in paraxial mesoderm is required for specification of the posterior hindbrain, including restriction of Hoxb1 expression to presumptive rhombomere 4 (r4). Hoxb1 expression requires 3′ and 5′ RA response elements for widespread induction up to r4 and for r3/r5 repression, but RA has previously been detected only from r5-r8, and vHnf1 is required for repression of Hoxb1 posterior to r4 in zebrafish. We demonstrate in mouse embryos that an RA signal initially travels from the paraxial mesoderm to r3, forming a boundary next to the r2 expression domain of Cyp26a1 (which encodes an RA-degrading enzyme). After Hoxb1 induction, the RA boundary quickly shifts to r4/r5, coincident with induction of Cyp26c1 in r4. A functional role for Cyp26c1 in RA degradation was established through examination of RA-treated embryos. Analysis of Raldh2–/– and vHnf1–/– embryos supports a direct role for RA in Hoxb1 induction up to r4 and repression in r3/r5, as well as an indirect role for RA in Hoxb1 repression posterior to r4 via RA induction of vHnf1 up to the r4/r5 boundary. Our findings suggest that Raldh2 and Cyp26 generate shifting boundaries of RA activity, such that r3-r4 receives a short pulse of RA and r5-r8 receives a long pulse of RA. These two pulses of RA activity function to establish expression of Hoxb1 and vHnf1 on opposite sides of the r4/r5 boundary.

The inactivation of the Hnf1beta gene identified an essential role in epithelial differentiation of the visceral endoderm and resulted in early embryonic death. In the present study, we have specifically inactivated this gene in hepatocytes and bile duct cells using the Cre/loxP system. Mutant animals exhibited severe jaundice caused by abnormalities of the gallbladder and intrahepatic bile ducts (IHBD). The paucity of small IHBD was linked to a failure in the organization of duct structures during liver organogenesis, suggesting an essential function of Hnf1b in bile duct morphogenesis. Mutant mice also lacked interlobular arteries. As HNF1beta is not expressed in these cells, it further emphasizes the link between arterial and biliary formation. Hepatocyte metabolism was also affected and we identified hepatocyte-specific HNF1beta target genes involved in bile acids sensing and in fatty acid oxidation.

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.

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.

PRL, a hormone secreted essentially by the pituitary and other extrapituitary sources such as decidua, has been attributed regulatory roles in reproduction and cell growth in mammals. These effects are mediated by a membrane PRL receptor belonging to the cytokine receptor superfamily. Null mutation of the PRL receptor gene leads to female sterility due to a severely compromised preimplantation development and a complete failure of the implantation of the few embryos reaching the blastocyst stage, strongly implicating PRL in the maternal control of implantation. We measured the hormonal status of -/- mice, which confirmed that the corpus luteum is unable to produce progesterone. Progesterone administration to -/- mice completely rescued the development of preimplantatory eggs and embryo implantation. Pregnancy could be maintained to 19.5 days postcoitum, with about 22% of resulting embryos reaching adulthood. Although progesterone and perhaps PRL appear to facilitate mouse preembryo development throughout the preimplantation stages, other factors as well as a possible direct effect of PRL on the uterus are probably necessary to fully maintain pregnancy. Finally, reduced ductal side-branching in the mammary gland can be rescued by progesterone treatment, but females exhibit reduced alveolar formation. Our model establishes the PRL receptor as a key regulator of reproduction and provides novel insights into the function of lactogenic hormones and their receptor.

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.

AbstractvHNF1/HNF1β, 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.

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.

The availability of mouse embryonic stem (ES) cells,which have the, remarkable ability to colonize the germline of an host embryo has opened the opportunity to transfer genetic modifications, selected in cultured cells, to the living animal. In this paper, is described a strategy of gene identification which uses:es particular constructs called trapping vectors basically made;with a reporter gene, generally beta-galactosidase devoid of regulatory sequences, and with a cassette containing a selection marker. After transfer into ES cells, the reporter gene may be expressed only when integrated in the correct orientation into a transcription unit.beta-galactosidase expression can then be monitored not only in ES cells but also in chimeric mice, thereby uncovering the pattern of expression in vivo of the "trapped" endogenous gene. Thus the trapping strategy has, several virtues: (1) the reporter gene reveals the expression profile of the gene in which the construct has been integrated; (2) the construct, which interrupts the transcription unit, behaves as a mutagen; (3) the synthesis of a fusion transcript made of endogenous and reporter gene sequences; facilitates the cloning of the cDNA of the trapped gene. Due to these virtues, this strategy has become a privileged way to identify genes, whose expressions developmentally regulated and therefore likely candidates to be important in development. More generally, this strategy could serve to tag any endogenous gene, and therefore provide a way of creating whole genome EST libraries.

The mammalian SWI–SNF complex is an evolutionarily conserved, multi‐subunit machine, involved in chromatin remodelling during transcriptional activation. Within this complex, the BRM (SNF2α) and BRG1 (SNF2β) proteins are mutually exclusive subunits that are believed to affect nucleosomal structures using the energy of ATP hydrolysis. In order to characterize possible differences in the function of BRM and BRG1, and to gain further insights into the role of BRM‐containing SWI–SNF complexes, the mouse BRM gene was inactivated by homologous recombination. BRM−/− mice develop normally, suggesting that an observed up‐regulation of the BRG1 protein can functionally replace BRM in the SWI–SNF complexes of mutant cells. Nonetheless, adult mutant mice were ∼15

La recherche de genes essentiels au developpement embryonnaire de la souris a ete abordee par la mise en oeuvre de strategies variees. Comparee a toutes ces approches, la strategie de piegeage de genes presente un - important avantage : elle associe l'isolement et la caracterisation moleculaire d'un gene a l'etude de son profil d'expression ainsi qu'a la determination de sa fonction in vivo. Au-dela d'un moyen d'identification des genes impliques dans le developpement de la souris, cette strategie ouvre la voie a la constitution de veritables banques de cellules, etiquetees chacune dans un gene different, base de depart necessitant ensuite une analyse detaillee gene par gene. Par ailleurs, les informations relatives au profil d'expression des genes pieges devraient permettre d'elargir l'interet du piegeage de genes a la production d'animaux chimeres ou la realisation de greffes. En outre, le piegeage d'une activite transcriptionnelle particuliere pourrait servir a l'expression ectopique de genes d'interet ou a la destruction specifique d'une structure embryonnaire ou d'un lignage cellulaire donnes.

Mice carrying a germ-line null mutation of the prolactin receptor gene have been produced by gene targeting in embryonic stem cells. Heterozygous females showed almost complete failure of lactation attributable to greatly reduced mammary gland development after their first, but not subsequent, pregnancies. Homozygous females were sterile owing to a complete failure of embryonic implantation. Moreover, they presented multiple reproductive abnormalities, including irregular cycles, reduced fertilization rates, defective preimplantation embryonic development, and lack of pseudopregnancy. Half of the homozygous males were infertile or showed reduced fertility. This work establishes the prolactin receptor as a key regulator of mammalian reproduction, and provides the first total ablation model to further study the role of the prolactin receptor and its ligands.

HNF1 is a transcriptional activator of many hepatic genes including albumin, alpha1-antitrypsin, and alpha- and beta-fibrinogen. It is related to the homeobox gene family and is predominantly expressed in liver and kidney. Mice lacking HNF1 fail to thrive and die around weaning after a progressive wasting syndrome with a marked liver enlargement. The transcription rate of genes like albumin and alpha1-antitrypsin is reduced, while the gene coding for phenylalanine hydroxylase is totally silent, giving rise to phenylketonuria. Mutant mice also suffer from severe Fanconi syndrome caused by renal proximal tubular dysfunction. The resulting massive urinary glucose loss leads to energy and water wasting. HNF1-deficient mice may provide a model for human renal Fanconi syndrome.

A new gene trap vector has been designed, comprised of a fusion between the Sh ble gene, which confers resistance to the antibiotic phleomycin, and the lacZ gene (phleal fusion gene). A synthetic splice acceptor, made of the yeast branchpoint followed by a pyrimidin-rich sequence of 27 nucleotides, is included at the 5′ extremity. The linearized gene trap vector was introduced into mouse embryonic stem cells (ES cells), and 40 phleomycin resistant (phleAL) cell lines possessing a single copy of the insert were selected. They were stable in expressing the lacZ gene. Reporter gene expression was studied at days 8.5 and 10.5 of embryonic development in chimeric embryos obtained after injection of phleoL ES clones into 8-cell stage embryos. Out of 20 phleal lines examined, 14 exhibited β-galactosidase expression at day 10.5. Use of the phleal fusion gene trap vector to select genes expressed in ES cells, therefore, is compatible with the isolation of genes expressed at midgestation. However, and most intriguingly, 10 out of these 14 cell lines (71%) displayed reporter gene expression mostly in heart and liver. Two of them exhibited, in addition, expression in central nervous system (CNS) or in CNS and limb buds, respectively. Germline chimeras were subsequently obtained and 15 mouse lines have been established. Intercrosses of animals heterozygous for the insertion revealed a mutant phenotype in several lines. © 1996 Wiley-Liss, Inc.

Despite the high efficiency of bleomycin (BLM) as a chemotherapeutic agent against various carcinomas, the potentially lethal and chronic fibrotic response of the lung is a major dose-limiting side effect, Here, we explore the possibility of a direct inhibition of lung tissue injury by in vivo expression of the actinomycetes BLM resistance protein Sh ble, Transgenic mice expressing the Sh ble gene under the control of a composite viral promoter were produced after introduction of the transgene into D3 ES cells, The protein was detected at high level in lungs, spleen, and kidney, We then assessed its ability to modulate the BLM-induced fibrotic response in the transgenic mice in comparison with C57BL/6 and 129/Sv parental mice, Cumulative doses of 300, 400, or 500 mg/kg BLM were administered either by i,p, or s,c, repeated injections in the different strains, Transgenic mice were shown to be clearly less sensitive to BLM toxicity, as assessed by lung histology, The pulmonary hydroxyproline content in the treated transgenic mice was close to its baseline level, whereas it was up to 50% higher than the control level in C57BL/6 and 129/Sv parental mice, These observations are consistent with the hypothesis that a resistance gene specifically expressed in lungs may prevent the BLM-induced inflammation.