The HERC2 ubiquitin ligase is essential for embryonic development and regulates motor coordination.

A mutation in the HERC2 gene has been linked to a severe neurodevelopmental disorder with similarities to the Angelman syndrome. This gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p53 and is involved in important cellular processes such as DNA repair, cell cycle, cancer, and iron metabolism. Despite the critical role of HERC2 in these physiological and pathological processes, little is known about its relevance in vivo. Here, we described a mouse with targeted inactivation of the Herc2 gene. Homozygous mice were not viable. Distinct from other ubiquitin ligases that interact with p53, such as MDM2 or MDM4, p53 depletion did not rescue the lethality of homozygous mice. The HERC2 protein levels were reduced by approximately one-half in heterozygous mice. Consequently, HERC2 activities, including ubiquitin ligase and stimulation of p53 activity, were lower in heterozygous mice. A decrease in HERC2 activities was also observed in human skin fibroblasts from individuals with an Angelman-like syndrome that express an unstable mutant protein of HERC2. Behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. This effect was not observed in p53 knockout mice, indicating that a mechanism independent of p53 activity is involved. Morphological analysis showed the presence of HERC2 in Purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. In these animals, an increase of autophagosomes and lysosomes was observed. Our findings establish a crucial role of HERC2 in embryonic development and motor coordination.

Increased Susceptibility to Skin Carcinogenesis Associated with a Spontaneous Mouse Mutation in the Palmitoyl Transferase Zdhhc13 Gene.

Here we describe a spontaneous mutation in the Zdhhc13 (zinc finger, DHHC domain containing 13) gene (also called Hip14l), one of 24 genes encoding palmitoyl acyltransferase (PAT) enzymes in the mouse. This mutation (Zdhhc13luc) was identified as a nonsense base substitution, which results in a premature stop codon that generates a truncated form of the ZDHHC13 protein, representing a potential loss-of-function allele. Homozygous Zdhhc13luc/Zdhhc13luc mice developed generalized hypotrichosis, associated with abnormal hair cycle, epidermal and sebaceous gland hyperplasia, hyperkeratosis, and increased epidermal thickness. Increased keratinocyte proliferation and accelerated transit from basal to more differentiated layers were observed in mutant compared with wild-type (WT) epidermis in untreated skin and after short-term 12-O-tetradecanoyl-phorbol-13-acetate treatment and acute UVB exposure. Interestingly, this epidermal phenotype was associated with constitutive activation of NF-κB (RelA) and increased neutrophil recruitment and elastase activity. Furthermore, tumor multiplicity and malignant progression of papillomas after chemical skin carcinogenesis were significantly higher in mutant mice than WT littermates. To our knowledge, this is the first report of a protective role for PAT in skin carcinogenesis.

Stress-induced sphingolipid signaling: role of type-2 neutral sphingomyelinase in murine cell apoptosis and proliferation.

BACKGROUND: Sphingomyelin hydrolysis in response to stress-inducing agents, and subsequent ceramide generation, are implicated in various cellular responses, including apoptosis, inflammation and proliferation, depending on the nature of the different acidic or neutral sphingomyelinases. This study was carried out to investigate whether the neutral Mg(2+)-dependent neutral sphingomyelinase-2 (nSMase2) plays a role in the cellular signaling evoked by TNFalpha and oxidized LDLs, two stress-inducing agents, which are mitogenic at low concentrations and proapoptotic at higher concentrations. METHODOLOGY AND PRINCIPAL FINDINGS: For this purpose, we used nSMase2-deficient cells from homozygous fro/fro (fragilitas ossium) mice and nSMase2-deficient cells reconstituted with a V5-tagged nSMase2. We report that the genetic defect of nSMase2 (in fibroblasts from fro/fro mice) does not alter the TNFalpha and oxidized LDLs-mediated apoptotic response. Likewise, the hepatic toxicity of TNFalpha is similar in wild type and fro mice, thus is independent of nSMase2 activation. In contrast, the mitogenic response elicited by low concentrations of TNFalpha and oxidized LDLs (but not fetal calf serum) requires nSMase2 activation. CONCLUSION AND SIGNIFICANCE: nSMase2 activation is not involved in apoptosis mediated by TNFalpha and oxidized LDLs in murine fibroblasts, and in the hepatotoxicity of TNFalpha in mice, but is required for the mitogenic response to stress-inducing agents.

Progressive Purkinje cell degeneration in tambaleante mutant mice is a consequence of a missense mutation in HERC1 E3 ubiquitin ligase.

The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domains have been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a G<-->A transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N-terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology.

A role for stroma-derived annexin A1 as mediator in the control of genetic susceptibility to T-cell lymphoblastic malignancies through prostaglandin E2 secretion.

Cancer susceptibility is essentially attributable to multiple low-penetrance genes. Using interspecific consomic and congenic mice between the tumor-resistant SEG/Pas and the tumor-sensitive C57BL/6J strains, a region on chromosome 19 involved in the genetic resistance to gamma-irradiation-induced T-cell lymphomas (Tlyr1) has been identified. Through the development of nonoverlapping subcongenic strains, it has been further shown that Anxa1 may be a candidate resistance gene on the basis of its differential expression in thymus stroma cells after gamma-radiation exposure. In addition, thymus stroma cells of thymic lymphomas exhibited a significant reduction in the expression levels of Anxa1. Interestingly, the activity of Anxa1 relies on prostaglandin E(2) (PGE(2)) induction that brings about apoptosis in thymocytes. In fact, in vitro transfection experiments revealed that PGE(2) production was enhanced when HEK 293 cells were transfected with full-length cDNAs of Anxa1, with PGE(2) production in the cells transfected with the allele of the resistant strain (Anxa1(Tyr)) being higher than that in cells transfected with the allele of the susceptible strain (Anxa1(Phe)). Furthermore, the presence of this compound in the medium induced apoptosis of immature CD4(+)CD8(+)CD3(low) cells in a dose-dependent manner. These results improve our knowledge of the molecular mechanisms triggering T-cell lymphoblastic lymphoma development while highlighting the relevance of the stroma in controlling genetic susceptibility and the use of PGE(2) as a new therapeutic approach in T-cell hematologic malignancies.

Alopecia and male infertility in oligotriche mutant mice are caused by a deletion on distal chromosome 9.

The recessive mutation oligotriche (olt) affects the coat and male fertility in the mouse. In homozygous (olt/olt) mutants, the coat is sparse, most notably in the inguinal and medial femoral region. In these regions, almost all hair shafts are bent and distorted in their course through the dermis and rarely penetrate the epidermis because the hair cortex is not fully keratinized. During hair follicle morphogenesis, mutant hair follicles exit from anagen one day before those of normal littermates and show a prolongation of the catagen stage. The oligotriche (olt) locus was mapped to distal chromosome 9 within a 5-Mbp interval distal to D9Mit279. Analysis of candidate gene expression revealed that olt/olt mutant mice do not express functional phospholipase C delta 1 (Plcd1) mRNA. This deficiency is the consequence of a 234-kbp deletion involving not only the Plcd1 locus but also the chromosomal segment harboring the genes Vill (villin-like), Dlec1 (deleted in lung and esophageal cancer 1), Acaa1b (acetyl-Coenzyme A acyltransferase 1B, synonym thiolase B), and parts of the genes Ctdspl (carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase-like) and Slc22a14 (solute carrier family 22 member 14). Offspring of olt/olt females, mated with Plcd1 ( -/- ) knockout males, exhibit coat defects similar to those observed in homozygous olt/olt mutant mice but the spermiogenesis in male offspring is normal. We conclude that the 234-kbp deletion from chromosome 9 harbors a gene involved in spermiogenesis and we propose that the oligotriche mutant be used as a model for the study of the putative tumor suppressor genes Dlec1, Ctdspl, and Vill. We also suggest that the oligotriche locus be named Del(9Ctdspl-Slc22a14)1Pas.

The mitochondrial protease AFG3L2 is essential for axonal development.

The mitochondrial metalloprotease AFG3L2 assembles with the homologous protein paraplegin to form a supracomplex in charge of the essential protein quality control within mitochondria. Mutations of paraplegin cause a specific axonal degeneration of the upper motoneuron and, therefore, hereditary spastic paraplegia. Here we present two Afg3l2 murine models: a newly developed null and a spontaneous mutant that we found carrier of a missense mutation. Contrasting with the mild and late onset axonal degeneration of paraplegin-deficient mouse, Afg3l2 models display a marked impairment of axonal development with delayed myelination and poor axonal radial growth leading to lethality at P16. The increased severity of the Afg3l2 mutants is explained by two main molecular features that differentiate AFG3L2 from paraplegin: its higher neuronal expression and its versatile ability to support both hetero-oligomerization and homo-oligomerization. Our data assign to AFG3L2 a crucial role by linking mitochondrial metabolism and axonal development. Moreover, we propose AFG3L2 as an excellent candidate for motoneuron and cerebellar diseases with early onset unknown etiology.

The mouse genome.

The house mouse has been used as a privileged model organism since the early days of genetics, and the numerous experiments made with this small mammal have regularly contributed to enrich our knowledge of mammalian biology and pathology, ranging from embryonic development to metabolic disease, histocompatibility, immunology, behavior, and cancer. Over the past two decades, a number of large-scale integrated and concerted projects have been undertaken that will probably open a new era in the genetics of the species. The sequencing of the genome, which will allow researchers to make comparisons with other mammals and identify regions conserved by evolution, is probably the most important project, but many other initiatives, such as the massive production of point or chromosomal mutations associated with comprehensive and standardized phenotyping of the mutant phenotypes, will help annotation of the approximately 25,000 genes packed in the mouse genome. In the same way, and as another consequence of the sequencing, the discovery of many single nucleotide polymorphisms and the development of new tools and resources, like the Collaborative Cross, will contribute to the development of modern quantitative genetics. It is clear that mouse genetics has changed dramatically over the last 10-15 years and its future looks promising.

A mouse model provides evidence that genetic background modulates anemia and liver injury in erythropoietic protoporphyria.

Erythropoietic protoporphyria is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin (PP) overproduction by erythrocytes. In humans, it is responsible for painful skin photosensitivity and, occasionally, liver failure due to accumulation of PP in the liver. The ferrochelatase deficiency mouse mutation is the best animal model available for human erythropoietic protoporphyria. The original description, based on mice with a BALB/cByJCrl genetic background, reported a disease resembling the severe form of the human disease, with anemia, jaundice, and liver failure. Using congenic strains, we investigated the effect of genetic background on the severity of the phenotype. Compared with BALB/cByJCrl, C57BL/6JCrl mice developed moderate but increasing anemia and intense liver accumulation of PP with severe hepatocyte damage and loss. Bile excretory function was not affected, and bilirubin remained low. Despite the highest PP concentration in erythrocytes, anemia was mild and there were few PP deposits in the liver in SJL/JOrlCrl homozygotes. Discriminant analysis using six hematologic and biochemical parameters showed that homozygotes of the three genetic backgrounds could be clustered in three well-separated groups. These three congenic strains provide strong evidence for independent genetic control of bone marrow contribution of PP overproduction to development of liver disease and biliary PP excretion. They provide a tool to investigate the physiological mechanisms involved in these phenotypic differences and to identify modifying genes.

Assessing the genetic component of the susceptibility of mice to viral infections.

Laboratory mice often exhibit wide differences in susceptibility when infected experimentally with viruses. Based on such observations, experiments have been designed to investigate the determinism of these differences at the molecular level, and a few genes that play a major role in the innate mechanisms of defence of the species toward viral aggressions have been characterised. For example, the extraordinary resistance of SJL mice to experimental infections with hepatitis virus strain A59 is the consequence of a structural alteration of a cell adhesion molecule which normally binds to the spikes of the virus, allowing its entry into the cells. If the virus cannot bind to the molecule, or if the molecule is absent, epithelial cells of the intestine and liver are not infected and mice are resistant. In the same way, most--not to say all--laboratory strains of mice are susceptible to infections with orthomyxoviruses or flaviviruses because essential molecules, the synthesis of which is normally triggered by interferon, are defective in these mice. Wild mice, by contrast --probably because they are constantly exposed to natural infections--are resistant. Finally, some mouse strains resist experimental infections by the mouse cytomegalovirus 1 (MCMV-1) because, once infected, these mice synthesise a molecule at the surface of infected cells which allows immediate recognition and killing by natural killer (NK) cells. With the exuberant development of mouse genetics and the constant generation of new mutant alleles, it is likely that many more genes with an impact on the phenotype of resistance or susceptibility will be identified in the forthcoming years. These genes are probably numerous, however, and many of them presumably interact with each other and/or have additive effects. This might slow down progress in our understanding of the innate mechanism of defence.

The paralysé (par) mouse neurological mutation maps to a 9 Mbp (4 cM) interval of mouse chromosome 18

The Paralysé mutation is a spontaneous neuromuscular mutation, first observed in 1980 at the Pasteur Institute, which is transmitted by the autosomal recessive par allele. Affected homozygote par/par mice rarely survive beyond 16 days of age and at the end of their life they are emaciated and completely paralyzed. Several concordant histological and physiological observations indicate that mutant mice might be good models for studying early-onset human motor neuron diseases such as spinal muscular atrophy. Linkage analysis using a set of molecular markers and two F2 crosses indicate that the mutation maps to mouse chromosome 18 in a region spanning 4 cM (or 9 megabase pairs, Mbp) between the microsatellites D18Mit140 and D18Mit33. These results positioned the par locus in a region homologous to human chromosome 18p11.22 to 18q21.32.

Mouse SCO-spondin, a gene of the thrombospondin type 1 repeat (TSR) superfamily expressed in the brain.

SCO-spondin is specifically expressed in the subcommissural organ (SCO), a secretory ependymal differentiation lining the roof of the third ventricular cavity of the brain. When released into the cerebro-spinal fluid (CSF), SCO-spondin aggregates and forms Reissner's fiber (RF), a structure present in the central canal of the spinal cord. SCO-spondin belongs to the superfamily of proteins exhibiting conserved motifs called TSRs for 'thrombospondin type 1 repeats' and involved in axonal pathfinding during development. The mouse SCO-spondin coding sequence was searched by alignement of the coding bovine SCO-spondin sequence with the mouse whole genome shotgun (WGS) supercontig (NW 000250). Compared to the bovine, mouse SCO-spondin shows 66.8% identity of amino acids. This extracellular matrix glycoprotein has a modular arrangement of several conserved domains including 25 TSRs, 10 low-density lipoprotein receptor (LDLr) type A repeats and cystein-rich regions in the -NH2 and -COOH ends. The spatio-temporal expression of SCO-spondin was analyzed using specific antisera and an homospecific SCO-spondin riboprobe. In the adult, the patterns obtained by in situ hybridization (ISH) and immunohistochemistry correlated well in the SCO, while Reissner's fiber and the ampulla caudalis were immunoreactive only. In the fetus, both the immuno and ISH reactions appeared between 14 and 15 days post coïtum (dpc) in the SCO anlage. In addition, the mouse SCO-spondin gene was located at chromosome 6, between marker D6Mit352 and D6Mit119, in a conserved syntenic region.

Infection of mouse neurones by West Nile virus is modulated by the interferon-inducible 2'-5' oligoadenylate synthetase 1b protein.

Over the past 7 years, West Nile zoonosis has been an emerging concern for public health in Europe, Middle East and more recently in North America. West Nile virus causes epidemic outbreaks in humans and infected patients may exhibit severe neurological symptoms. Because susceptibility and sensitivity to West Nile virus infections may depend on host genetic factors, a mouse model has been established to investigate the genetic determinism of host susceptibility to West Nile virus. A nonsense mutation in gene encoding the 1b isoform of the 2'-5'oligoadenylate synthetase (OAS1b) was constantly associated with the susceptibility of mouse strains to experimental West Nile virus infection. Oligoadenylate synthetase are interferon-inducible proteins playing a role in the endogeneous antiviral pathway. It was of interest to establish whether interferon-alpha and OAS 1B were sufficient to mediate resistance to West Nile virus infection. In the present study, we showed that interferon-alpha had the ability to modulate West Nile virus infection in mouse. In vitro, interferon-alpha protected mouse neuroblastoma cells against West Nile virus infection if cells have been pretreated with the cytokine for several hours. As a consequence of the presence of a stop codon, the Oas1b gene of the susceptible mice encodes a truncated and presumably inactive form, while resistant mice have a normal copy of the gene. Stable mouse neuroblastoma cell clones overexpressing mutant or wild-type OAS 1B were established. Replication of West Nile virus was less efficient in cells that produce the normal copy of OAS 1B as compared to those expressing the truncated form. Our data illustrate the notion that interferon-alpha and Oas genes may be critical for West Nile virus pathogenesis.

Wild mice: an ever-increasing contribution to a popular mammalian model.

Classical laboratory inbred strains of mice have been extremely helpful for research in immunology and oncology, and more generally, for the analysis of complex traits. Unfortunately, because they all derive from a relatively small pool of ancestors, their genetic polymorphism is rather limited. However, recently strains belonging to different species of Mus have been established from wild progenitors. These are an interesting addition to the arsenal of mouse geneticists, because they can be crossed with classical laboratory strains to produce viable and fertile offspring with a large number of polymorphisms of natural origin. These strains are helpful for making genome annotations because they permit highly refined genotype-phenotype correlations. They also allow the interpretation of molecular variation within a clear evolutionary framework. In this article, we provide examples with the aim of promoting the use of these new strains.

A new locus for resistance to gamma-radiation-induced thymic lymphoma identified using inter-specific consomic and inter-specific recombinant congenic strains of mice.

Mice of the C57BL/6J inbred strain develop thymic lymphomas at very high frequency after acute gamma-irradiation, while mice of several inbred strains derived from the wild progenitor of the Mus spretus species and their F1 hybrids with C57BL/6J appear extremely resistant. Analysis of the genetic determinism of the gamma-radiation-induced thymic lymphoma (RITL) resistance with the help of inter-specific consomic strains (ICS), which carry a single introgressed Mus spretus chromosome on a C57BL/6J genetic background, provide significant evidence for the existence of a thymic lymphoma resistance (Tlyr1) locus on chromosome 19. The subsequent analysis of the backcross progeny resulting from a cross between consomic mice heterozygous for the Mus spretus chromosome 19 and C57BL/6J mice, together with the study of inter-specific recombinant congenic strains (IRCS), suggest that this Tlyr1 locus maps within the D19Mit60-D19Mit40 chromosome interval. In addition to the discovery of a new locus controlling RITL development, our study emphasizes the value of ICS and IRCS for the genetic analysis of cancer predisposition.

Impaired hair follicle morphogenesis and cycling with abnormal epidermal differentiation in nackt mice, a cathepsin L-deficient mutation.

We previously described an autosomal-recessive mutation named nackt (nkt) exhibiting partial alopecia associated with CD4(+) T-cell deficiency. Also, we recently reported that nkt (now Ctsl(nkt)) comprises a deletion in the cathepsin L (Ctsl) gene. Another recent study reported that Ctsl knockout mice have CD4(+) T-cell deficiency and periodic shedding of hair, which recapitulate the nkt mutation and the old furless (fs) mutation. The current study focuses on the dermatological aspects of the nkt mutation. Careful histological analysis of skin development of homozygous nkt mice revealed a delayed hair follicle morphogenesis and late onset of the first catagen stage. The skin of Ctsl(nkt)/Ctsl(nkt) mice showed mild epidermal hyperplasia and hyperkeratosis, severe hyperplasia of the sebaceous glands, and structural alterations of hair follicles. Epidermal differentiation seems to be affected in nkt skin, with overexpression of involucrin and profilaggrin/filaggrin along with focal areas of keratin 6 expression in the interfollicular epidermis. Severe epidermal hyperplasia, acanthosis, orthokeratosis, and hyperkeratosis were only observed in mice maintained in nonpathogen-free environments. The analysis of Rag2-/- Ctsl(nkt)/Ctsl(nkt) double-mutant mice indicates that the skin defect remains under the absence of T and B cells. This animal model provides in vivo evidence that cysteine protease cathepsin L plays a critical role in hair follicle morphogenesis and cycling, as well as epidermal differentiation.

Hyporesponsiveness of SPRET/Ei mice to lethal shock induced by tumor necrosis factor and implications for a TNF-based antitumor therapy.

Tumor necrosis factor (TNF) is a central mediator in lethal shock and an interesting cytokine for anticancer therapy. To inhibit TNF-induced lethal shock, it is important to identify protective genes. Here we demonstrate that the SPRET/Ei mouse strain, derived from Mus spretus, exhibits an extremely dominant resistance to TNF-induced lethal inflammation. An interspecific backcross experiment revealed that the TNF hyporesponse is linked to loci on chromosomes 2, 6, and 11. Treatment of inoculated tumors with TNF and IFN-gamma leads to regression and a highly reduced toxicity in (C57BL/6 x SPRET/Ei)F(1) mice.

Deletion of T cells bearing the V beta8.1 T-cell receptor following mouse mammary tumor virus 7 integration confers resistance to murine cerebral malaria.

Plasmodium berghei ANKA induces a fatal neurological syndrome known as cerebral malaria (CM) in susceptible mice. Host genetic elements are among the key factors determining susceptibility or resistance to CM. Analysis of mice of the same H-2 haplotype revealed that mouse mammary tumor virus 7 (MTV-7) integration into chromosome 1 is one of the key factors associated with resistance to neurological disease during P. berghei ANKA infection. We investigated this phenomenon by infecting a series of recombinant inbred mice (CXD2), derived from BALB/c (susceptible to CM) and DBA/2 (resistant to CM) mice, with P. berghei ANKA. We observed differences in susceptibility to CM induced by this Plasmodium strain. Mice with the MTV-7 sequence in their genome were resistant to CM, whereas those without integration of this gene were susceptible. Thus, an integrated proviral open reading frame or similar genomic sequences may confer protection against neuropathogenesis during malaria, at least in mice.