Apoptosis and differentiation commitment: novel insights revealed by gene profiling studies in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF starvation leads to apoptosis of some of the ES-derived differentiated cells, together with p38alpha mitogen-activated protein kinase (MAPK) activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at 3 days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene, which prevents apoptosis of early differentiated cells.

Knockout mice as model systems for studying nm23/NDP kinase gene functions. Application to the nm23-M1 gene.

Mice carrying a homozygous germ-line mutation in the nm23-M1 gene that eliminates its protein expression and drives expression of beta-galactosidase by nm23-M1 promoter have been generated. nm23-M1 gene inactivation is not teratogenic and the pups can grow to adult age without apparent health problems. However, they undergo a growth retardation and knocked out females cannot feed their pups. Both effects are background dependent. Beta-galactosidase mapping of nm23-M1 promoter activation during embryogenesis shows that the nm23-M1 gene is principally expressed in epithelial layer of tissues which require inductive epithelial-mesenchymal interactions for their formation. In conclusion, invalidated mice could be interesting models to analyze the role of nm23-M1 on signal transduction pathway regulation, or cancer induction and proliferation.

The ribosomal S6 kinases, cAMP-responsive element-binding, and STAT3 proteins are regulated by different leukemia inhibitory factor signaling pathways in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase.

Altered behavioral rhythms and clock gene expression in mice with a targeted mutation in the Period1 gene.

A group of specialized genes has been defined to govern the molecular mechanisms controlling the circadian clock in mammals. Their expression and the interactions among their products dictate circadian rhythmicity. Three genes homologous to Drosophila period exist in the mouse and are thought to be major players in the biological clock. Here we present the generation of mice in which the founding member of the family, Per1, has been inactivated by homologous recombination. These mice present rhythmicity in locomotor activity, but with a period almost 1 h shorter than wild-type littermates. Moreover, the expression of clock genes in peripheral tissues appears to be delayed in Per1 mutant animals. Importantly, light-induced phase shifting appears conserved. The oscillatory expression of clock genes and the induction of immediate-early genes in response to light in the master clock structure, the suprachiasmatic nucleus, are unaffected. Altogether, these data demonstrate that Per1 plays a distinct role within the Per family, as it may be involved predominantly in peripheral clocks and/or in the output pathways of the circadian clock.

Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes.

The functions of estrogen receptors (ERs) in mouse ovary and genital tracts were investigated by generating null mutants for ERalpha (ERalphaKO), ERbeta (ERbetaKO) and both ERs (ERalphabetaKO). All ERalphaKO females are sterile, whereas ERbetaKO females are either infertile or exhibit variable degrees of subfertility. Mast cells present in adult ERalphaKO and ERalphabetaKO ovaries could participate in the generation of hemorrhagic cysts. Folliculogenesis proceeds normally up to the large antral stage in both ERalphaKO and ERbetaKO adults, whereas large antral follicles of ERalpha+/-ERbetaKO and ERalphabetaKO adults are markedly deficient in granulosa cells. Similarly, prematurely developed follicles found in prepubertal ERalphaKO ovaries appear normal, but their ERalphabetaKO counterparts display only few granulosa cell layers. Upon superovulation treatment, all prepubertal ERalphaKO females form numerous preovulatory follicles of which the vast majority do not ovulate. The same treatment fails to elicit the formation of preovulatory follicles in half of the ERbetaKO mice and in all ERalpha+/-/ERbetaKO mice. These and other results reveal a functional redundancy between ERalpha and ERbeta for ovarian folliculogenesis, and strongly suggest that (1) ERbeta plays an important role in mediating the stimulatory effects of estrogens on granulosa cell proliferation, (2) ERalpha is not required for follicle growth under wild type conditions, while it is indispensable for ovulation, and (3) ERalpha is also necessary for interstitial glandular cell development. Our data also indicate that ERbeta exerts some function in ERalphaKO uterus and vagina. ERalphabetaKO granulosa cells localized within degenerating follicles transform into cells displaying junctions that are unique to testicular Sertoli cells. From the distribution pattern of anti-Müllerian hormone (AMH) in ERalphabetaKO ovaries, it is unlikely that an elevated AMH level is the cause of Sertoli cell differentiation. Our results also show that cell proliferation in the prostate and urinary bladder of old ERbetaKO and ERalphabetaKO males is apparently normal.

Serotonin 2B receptor is required for heart development.

Several lines of evidence suggest that the serotonin (5-hydroxytryptamine, 5-HT) regulates cardiovascular functions during embryogenesis and adulthood. 5-HT binds to numerous cognate receptors to initiate its biological effects. However, none of the 5-HT receptor disruptions in mice have yet resulted in embryonic defects. Here we show that 5-HT(2B) receptor is an important regulator of cardiac development. We found that inactivation of 5-HT(2B) gene leads to embryonic and neonatal death caused by heart defects. 5-HT(2B) mutant embryos exhibit a lack of trabeculae in the heart and a specific reduction in the expression levels of a tyrosine kinase receptor, ErbB-2, leading to midgestation lethality. These in vivo data suggest that the Gq-coupled receptor 5-HT(2B) uses the signaling pathway of tyrosine kinase receptor ErbB-2 for cardiac differentiation. All surviving newborn mice display a severe ventricular hypoplasia caused by impaired proliferative capacity of myocytes. In adult mutant mice, cardiac histopathological changes including myocyte disarray and ventricular dilation were consistently observed. Our results constitute genetic evidence that 5-HT via 5-HT(2B) receptor regulates differentiation and proliferation of developing and adult heart. This mutation provides a genetic model for cardiopathy and should facilitate studies of both the pathogenesis and therapy of cardiac disorders in humans.

Inactivation of the Friedreich ataxia mouse gene leads to early embryonic lethality without iron accumulation.

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is caused in almost all cases by homozygous intronic expansions resulting in the loss of frataxin, a mitochondrial protein conserved through evolution, and involved in mitochondrial iron homeostasis. Yeast knockout models, and histological and biochemical data from patient heart biopsies or autopsies indicate that the frataxin defect causes a specific iron-sulfur protein deficiency and mitochondrial iron accumulation leading to the pathological changes. Affected human tissues are rarely available to further examine this hypothesis. To study the mechanism of the disease, we generated a mouse model by deletion of exon 4 leading to inactivation of the Frda gene product. We show that homozygous deletions cause embryonic lethality a few days after implantation, demonstrating an important role for frataxin during early development. These results suggest that the milder phenotype in humans is due to residual frataxin expression associated with the expansion mutations. Surprisingly, in the frataxin knockout mouse, no iron accumulation was observed during embryonic resorption, suggesting that cell death could be due to a mechanism independent of iron accumulation.

neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas.

In the mammalian pancreas, the endocrine cell types of the islets of Langerhans, including the alpha-, beta-, delta-, and pancreatic polypeptide cells as well as the exocrine cells, derive from foregut endodermal progenitors. Recent genetic studies have identified a network of transcription factors, including Pdx1, Isl1, Pax4, Pax6, NeuroD, Nkx2.2, and Hlxb9, regulating the development of islet cells at different stages, but the molecular mechanisms controlling the specification of pancreatic endocrine precursors remain unknown. neurogenin3 (ngn3) is a member of a family of basic helix-loop-helix transcription factors that is involved in the determination of neural precursor cells in the neuroectoderm. ngn3 is expressed in discrete regions of the nervous system and in scattered cells in the embryonic pancreas. We show herein that ngn3-positive cells coexpress neither insulin nor glucagon, suggesting that ngn3 marks early precursors of pancreatic endocrine cells. Mice lacking ngn3 function fail to generate any pancreatic endocrine cells and die postnatally from diabetes. Expression of Isl1, Pax4, Pax6, and NeuroD is lost, and endocrine precursors are lacking in the mutant pancreatic epithelium. Thus, ngn3 is required for the specification of a common precursor for the four pancreatic endocrine cell types.

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.

The net repressor is regulated by nuclear export in response to anisomycin, UV, and heat shock.

The ternary complex factors (TCFs) are targets for Ras/mitogen-activated protein kinase signalling pathways. They integrate the transcriptional response at the level of serum response elements in early-response genes, such as the c-fos proto-oncogene. An important aim is to understand the individual roles played by the three TCFs, Net, Elk1, and Sap1a. Net, in contrast to Elk1 and Sap1a, is a strong repressor of transcription. We now show that Net is regulated by nuclear-cytoplasmic shuttling in response to specific signalling pathways. Net is mainly nuclear under both normal and basal serum conditions. Net contains two nuclear localization signals (NLSs); one is located in the Ets domain, and the other corresponds to the D box. Net also has a nuclear export signal (NES) in the conserved Ets DNA binding domain. Net is apparently unique among Ets proteins in that a particular leucine in helix 1, a structural element, generates a NES. Anisomycin, UV, and heat shock induce active nuclear exclusion of Net through a pathway that involves c-Jun N-terminal kinase kinase and is inhibited by leptomycin B. Nuclear exclusion relieves transcriptional repression by Net. The specific induction of nuclear exclusion of Net by particular signalling pathways shows that nuclear-cytoplasmic transport of transcription factors can add to the specificity of the response to signalling cascades.

Mice lacking all conventional MHC class II genes.

MHC class II (MHC-II) molecules play a central role in the selection of the T cell repertoire, in the establishment and regulation of the adaptive immune response, and in autoimmune deviation. We have generated knockout mice lacking all four of the classical murine MHC-II genes (MHCII(Delta/Delta) mice), via a large (80-kilobase) deletion of the entire class II region that was engineered by homologous recombination and Cre recombinase-mediated excision. These mice feature immune system perturbations like those of Aalpha and Abeta knockout animals, notably a dearth of CD4(+) lymphocytes in the thymus and spleen. No new anatomical or physiological abnormalities were observed in MHCII(Delta/Delta) mice. Because these animals are devoid of all classical MHC-II chains, even unpaired chains, they make excellent recipients for MHC-II transgenes from other species, avoiding the problem of interspecies cross-pairing of MHC-II chains. Therefore, they should be invaluable for engineering "humanized" mouse models of human MHC-II-associated autoimmune disorders.

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.

Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells.

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+: poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents. To determine its biological function, we have inactivated both alleles by gene targeting in mice. Treatment of PARP-/- mice either by the alkylating agent N-methyl-N-nitrosourea (MNU) or by gamma-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body gamma-irradiation (8 Gy) mutant mice died rapidly from acute radiation toxicity to the small intestine. Mice-derived PARP-/- cells displayed a high sensitivity to MNU exposure: a G2/M arrest in mouse embryonic fibroblasts and a rapid apoptotic response and a p53 accumulation were observed in splenocytes. Altogether these results demonstrate that PARP is a survival factor playing an essential and positive role during DNA damage recovery.

Role of the retinoic acid receptor beta (RARbeta) during mouse development.

Homozygous RAR beta mutants are growth-deficient, but are fertile and have a normal longevity. They display homeotic transformations and malformations of cervical vertebrae and a retrolenticular membrane. This latter abnormality arises from the persistence and hyperplasia of the primary vitreous body. In contrast, we found that abnormalities of cranial nerves IX and X which were previously proposed to be specific features of the RAR beta mutant phenotype (Luo et al., Mech. Dev. 53: 61-71, 1995) occur with the same low penetrance in wildtype littermates. Although the RAR beta protein is expressed at high levels in the striatum and interdigital mesenchyme, the brain and limbs of RAR beta mutants appear morphologically normal. RAR alpha/RAR beta double mutants display numerous visceral abnormalities, most of which are incompatible with post-natal life. The majority of these abnormalities was previously detected in RAR alpha/RAR beta2 mutants with the notable exceptions of agenesis of the stapedial (2nd aortic arch-derived) artery, thymic and spleen agenesis and abnormal inferior vena cava. RAR beta/RAR gamma double mutants show major ocular defects including a shortening of the ventral retina and pre-natal retinal dysplasia, both of which represent the only abnormalities of the fetal vitamin-A deficiency (VAD) syndrome not previously detected in RAR beta2/RAR gamma compound mutants. In addition, RAR beta is apparently functionally redundant with either RAR alpha or RAR gamma for the formation of a small subset of craniofacial skeletal elements, as well as for eyelid development and digit separation. We also provide evidence that, at least in some instances, this phenomenon of functional redundancy between RARs may be an artifactual consequence of gene knock-out.

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.

Independent modes of natural killing distinguished in mice lacking Lag3.

The LAG3 protein has several features in common with CD4, suggesting that it may be important in controlling T cell reactivity. However, mice with a Lag3 null mutation have now been shown to exhibit a defect in the natural killer cell, rather than the T cell, compartment. Killing of certain tumor targets by natural killer cells from these mice was inhibited or even abolished, whereas lysis of cells displaying major histocompatibility complex class I disparities remained intact. It appears that LAG3 is a receptor or coreceptor that defines different modes of natural killing.

Specific and redundant functions of the paralogous Hoxa-9 and Hoxd-9 genes in forelimb and axial skeleton patterning.

Using gene targeting, we have produced mice with a disruption of Hoxa-9 or Hoxd-9, two paralogous Abdominal B-related genes. During embryogenesis, these genes are expressed in limb buds and along the vertebral axis with anterior expression boundaries at the level of prevertebra #20 for Hoxa-9 and #23 for Hoxd-9. Skeletal analysis revealed homeotic transformations corresponding to anteriorisations of vertebrae #21 to #25 (L1 to L5) in the lumbar region of Hoxa-9-/- mutants; vertebrae #23 to #25 (L3 to L5) in the lumbar region together with vertebrae #28, #30 and #31 (S2, S4 and Ca1) in the sacrum and tail were anteriorized in Hoxd-9-/- mutants. Thus, anteriorisation of vertebrae #23 to #25 were common to both phenotypes. Subtle forelimb (but not hindlimb) defects, corresponding to a reduction of the humerus length and malformation of its deltoid crest, were also observed in Hoxd-9-/-, but not in Hoxa-9-/-, mutant mice. By intercrosses between these two lines of mutant mice, we have produced Hoxa-9/Hoxd-9 double mutants which exhibit synergistic limb and axial malformations consisting of: (i) an increase of penetrance and expressivity of abnormalities present in the single mutants, and (ii) novel limb alterations at the level of the forelimb stylopod and additional axial skeleton transformations. These observations demonstrate that the two paralogous genes Hoxa-9 and Hoxd-9 have both specific and redundant functions in lumbosacral axial skeleton patterning and in limb morphogenesis at the stylopodal level. Taken all together, the present and previously reported results show that disruption of different Hox genes can produce similar vertebral transformations, thus supporting a combinatorial code model for specification of vertebral identity by Hox genes.

Mice lacking H2-M complexes, enigmatic elements of the MHC class II peptide-loading pathway.

We have generated mice lacking H2-M complexes, critical facilitators of peptide loading onto major histo-compatibility complex class II molecules. Ab molecules in these mice matured into stable complexes and were efficiently expressed at the cell surface. Most carried a single peptide derived from the class II-associated invariant chain; the diverse array of peptides normally displayed by class II molecules was absent. Cells from mutant mice presented both whole proteins and short peptides very poorly. Surprisingly, positive selection of CD4+ T cells was quite efficient, yielding a large and broad repertoire. Peripheral T cells reacted strongly to splenocytes from syngeneic wild-type mice, no doubt reflecting the unique peptide complement carried by class II molecules in mutant animals.

Function of retinoic acid receptor gamma in the mouse.

Null mutant mice for retinoic acid receptor gamma 2 (RAR gamma 2) or all RAR gamma isoforms were generated. RAR gamma 2 mutants appeared normal, whereas RAR gamma mutants exhibited growth deficiency, early lethality, and male sterility due to squamous metaplasia of the seminal vesicles and prostate. These defects were previously observed in vitamin A-deficient animals and could be prevented by RA administration, demonstrating that RAR gamma mediates some of the retinoid signal in vivo. Congenital defects included Harderian gland agenesis, tracheal cartilage malformations, and homeotic transformations along the rostral axial skeleton, establishing a direct link between RA and patterning of the axial skeleton. We also show that in utero RA-induced lumbosacral truncations are mediated by RAR gamma. The observed RAR gamma null phenotype suggests a high degree of functional redundancy among the RARs. The variable penetrance of some of the observed defects is discussed in light of this redundancy and stochastic variation of gene activity.

Disruption of the Hox-1.6 homeobox gene results in defects in a region corresponding to its rostral domain of expression.

The Hox-1.6 gene disrupted in embryonic stem cells by homologous recombination was introduced into the mouse germline. Heterozygous mice were normal, but homozygous mice died at birth from anoxia and had numerous defects that were centered at the level of rhombomeres 4 to 7 and included delayed hindbrain neural tube closure, absence of certain cranial nerves and ganglia, and malformed inner ears and bones of the skull. Thus, Hox-1.6 is involved in regional specification along the rostrocaudal axis, but only in its most rostral domain of expression. Hox-1.6 appears to specify neurogenic neural crest cells prior to specification of mesenchymal neural crest cells by Hox-1.5. Thus, within the same region of the presumptive hindbrain, two HOX-1 genes are involved in the patterning of two different populations of neural crest cells. The implication of these results for the function of the Hox network during mouse embryogenesis is discussed.