Mutations in Rab3a alter circadian period and homeostatic response to sleep loss in the mouse

Rab3a is the most abundant Rab (ras-associated binding) protein in the brain and has a regulatory role in synaptic vesicle trafficking. Mice with a targeted loss-of-function mutation in Rab3a have defects in Ca(2+)-dependent synaptic transmission: the number of vesicles released in response to an action potential is greater than in wildtype mice, resulting in greater synaptic depression and the abolishment of CA3 mossy-fiber long term potentiation. The effect of these changes on behavior is unknown. In a screen for mouse mutants with abnormal rest-activity and sleep patterns, we identified a semidominant mutation, called earlybird, that shortens the circadian period of locomotor activity. Sequence analysis of Rab3a identified a point mutation in the conserved amino acid (Asp77Gly) within the GTP-binding domain of this protein in earlybird mutants, resulting in significantly reduced levels of Rab3a protein. Phenotypic assessment of earlybird mice and a null allele of Rab3a revealed anomalies in circadian period and sleep homeostasis, providing evidence that Rab3a-mediated synaptic transmission is involved in these behaviors.     

Alteration of the serine protease PRSS56 causes angle-closure glaucoma in mice and posterior microphthalmia in humans and mice

Angle-closure glaucoma (ACG) is a subset of glaucoma affecting 16 million people. Although 4 million people are bilaterally blind from ACG, the causative molecular mechanisms of ACG remain to be defined. High intraocular pressure induces glaucoma in ACG. High intraocular pressure traditionally was suggested to result from the iris blocking or closing the angle of the eye, thereby limiting aqueous humor drainage. Eyes from individuals with ACG often have a modestly decreased axial length, shallow anterior chamber and relatively large lens, features that predispose to angle closure. Here we show that genetic alteration of a previously unidentified serine protease (PRSS56) alters axial length and causes a mouse phenotype resembling ACG. Mutations affecting this protease also cause a severe decrease of axial length in individuals with posterior microphthalmia. Together, these data suggest that alterations of this serine protease may contribute to a spectrum of human ocular conditions including reduced ocular size and ACG.     

Trans allele methylation and paramutation-like effects in mice

In mammals, imprinted genes have parent-of-origin-specific patterns of DNA methylation that cause allele-specific expression. At Rasgrf1 (encoding RAS protein-specific guanine nucleotide-releasing factor 1), a repeated DNA element is needed to establish methylation and expression of the active paternal allele. At Igf2r (encoding insulin-like growth factor 2 receptor), a sequence called region 2 is needed for methylation of the active maternal allele. Here we show that replacing the Rasgrf1 repeats on the paternal allele with region 2 allows both methylation and expression of the paternal copy of Rasgrf1, indicating that sequences that control methylation can function ectopically. Paternal transmission of the mutated allele also induced methylation and expression in trans of the normally unmethylated and silent wild-type maternal allele. Once activated, the wild-type maternal Rasgrf1 allele maintained its activated state in the next generation independently of the paternal allele. These results recapitulate in mice several features in common with paramutation described in plants.     

Toward simpler and faster genome-wide mutagenesis in mice

Here we describe a practical Cre-loxP and piggyBac transposon-based mutagenesis strategy to systematically mutate coding sequences and/or the vast noncoding regions of the mouse genome for large-scale functional genomic analysis. To illustrate this approach, we first created loxP-containing loss-of-function alleles in the protocadherin alpha, beta and gamma gene clusters (Pcdha, Pcdhb and Pcdhg). Using these alleles, we show that, under proper guidance, Cre-loxP site-specific recombination can mediate efficient trans-allelic recombination in vivo, facilitating the generation of large germline deletions and duplications including deletions of Pcdha, and Pcdha to Pcdhb, simply by breeding (that is, at frequencies of 5.5%-21.6%). The same breeding method can also generate designed germline translocations between nonhomologous chromosomes at unexpected frequencies of greater than 1%. By incorporating a piggyBac transposon to insert and to distribute loxP sites randomly throughout the mouse genome, we present a simple but comprehensive method for generating genome-wide deletions and duplications, in addition to insertional loss-of-function and conditional rescue alleles, again simply by breeding.     

Tissue-specific and reversible RNA interference in transgenic mice

Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice. Transgenic mice harboring a tet-responsive RNA polymerase II promoter driving a microRNA-based short hairpin RNA targeting the tumor suppressor Trp53 reversibly express short hairpin RNA when crossed with existing mouse strains expressing general or tissue-specific 'tet-on' or 'tet-off' transactivators. Reversible Trp53 knockdown can be achieved in several tissues, and restoring Trp53 expression in lymphomas whose development is promoted by Trp53 knockdown leads to tumor regression. By leaving the target gene unaltered, this approach permits tissue-specific, reversible regulation of endogenous gene expression in vivo, with potential broad application in basic biology and drug target validation.     

Genetic variation in laboratory mice

Characterizing the patterns of genetic variation in an organism provides fundamental insight into the evolutionary history of the organism and defines the scope and nature of studies that must be designed to correlate genotype to phenotype. Given the pre-eminent role of the inbred mouse in biomedical research, considerable effort has been undertaken in recent years to describe more fully the nature and amount of genetic variation among the numerous strains of mice that are in widest use. Here, we discuss recent studies that have contributed to an emerging understanding of the unique variation patterns found in inbred strains of mice and how they have arisen through a combination of natural evolution and human-directed breeding. These preliminary results have ramifications for genetic research into complex biomedical traits and are the basis for the development of future variation resources.     

Meiotic arrest and aneuploidy in MLH3-deficient mice

MutL homolog 3 (Mlh3) is a member of a family of proteins conserved during evolution and having dual roles in DNA mismatch repair and meiosis. The pathway in eukaryotes consists of the DNA-binding components, which are the homologs of the bacterial MutS protein (MSH 2 6), and the MutL homologs, which bind to the MutS homologs and are essential for the repair process. Three of the six homologs of MutS that function in these processes, Msh2, Msh3 and Msh6, are involved in the mismatch repair of mutations, frameshifts and replication errors, and two others, Msh4 and Msh5, have specific roles in meiosis. Of the four MutL homologs, Mlh1, Mlh3, Pms1 and Pms2, three are involved in mismatch repair and at least two, Pms2 and Mlh1, are essential for meiotic progression in both yeast and mice. To assess the role of Mlh3 in mammalian meiosis, we have generated and characterized Mlh3(-/-) mice. Here we show that Mlh3(-/-) mice are viable but sterile. Mlh3 is required for Mlh1 binding to meiotic chromosomes and localizes to meiotic chromosomes from the mid pachynema stage of prophase I. Mlh3(-/-) spermatocytes reach metaphase before succumbing to apoptosis, but oocytes fail to complete meiosis I after fertilization. Our results show that Mlh3 has an essential and distinct role in mammalian meiosis.     

Telomere dysfunction and evolution of intestinal carcinoma in mice and humans

Telomerase activation is a common feature of advanced human cancers and facilitates the malignant transformation of cultured human cells and in mice. These experimental observations are in accord with the presence of robust telomerase activity in more advanced stages of human colorectal carcinogenesis. However, the occurrence of colon carcinomas in telomerase RNA (Terc)-null, p53-mutant mice has revealed complex interactions between telomere dynamics, checkpoint responses and carcinogenesis. We therefore sought to determine whether telomere dysfunction exerts differential effects on cancer initiation versus progression of mouse and human intestinal neoplasia. In successive generations of ApcMin Terc-/- mice, progressive telomere dysfunction led to an increase in initiated lesions (microscopic adenomas), yet a significant decline in the multiplicity and size of macroscopic adenomas. That telomere dysfunction also contributes to human colorectal carcinogenesis is supported by the appearance of anaphase bridges (a correlate of telomere dysfunction) at the adenoma-early carcinoma transition, a transition recognized for marked chromosomal instability. Together, these data are consistent with a model in which telomere dysfunction promotes the chromosomal instability that drives early carcinogenesis, while telomerase activation restores genomic stability to a level permissive for tumor progression. We propose that early and transient telomere dysfunction is a major mechanism underlying chromosomal instability of human cancer.     

Mitochondrial dysfunction and apoptosis in myopathic mice with collagen VI deficiency

Collagen VI is an extracellular matrix protein that forms a microfilamentous network in skeletal muscles and other organs. Inherited mutations in genes encoding collagen VI in humans cause two muscle diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy. We previously generated collagen VI-deficient (Col6a1-/-) mice and showed that they have a muscle phenotype that strongly resembles Bethlem myopathy. The pathophysiological defects and mechanisms leading to the myopathic disorder were not known. Here we show that Col6a1-/- muscles have a loss of contractile strength associated with ultrastructural alterations of sarcoplasmic reticulum (SR) and mitochondria and spontaneous apoptosis. We found a latent mitochondrial dysfunction in myofibers of Col6a1-/- mice on incubation with the selective F1F(O)-ATPase inhibitor oligomycin, which caused mitochondrial depolarization, Ca2+ deregulation and increased apoptosis. These defects were reversible, as they could be normalized by plating Col6a1-/- myofibers on collagen VI or by addition of cyclosporin A (CsA), the inhibitor of mitochondrial permeability transition pore (PTP). Treatment of Col6a1-/- mice with CsA rescued the muscle ultrastructural defects and markedly decreased the number of apoptotic nuclei in vivo. These findings indicate that collagen VI myopathies have an unexpected mitochondrial pathogenesis that could be exploited for therapeutic intervention.     

Mutant beta-spectrin 4 causes auditory and motor neuropathies in quivering mice

The autosomal recessive mouse mutation quivering (qv), which arose spontaneously in 1953, produces progressive ataxia with hind limb paralysis, deafness and tremor. Six additional spontaneous alleles, qvJ, qv2J, qv3J, qv4J, qvlnd and qvlnd2J, have been identified. Ear twitch responses (Preyer's reflex) to sound are absent in homozygous qv/qv mice, although cochlear morphology seems normal and cochlear potentials recorded at the round window are no different from those of control mice. However, responses from brainstem auditory nuclei show abnormal transmission of auditory information, indicating that, in contrast to the many known mutations causing deafness originating in the cochlea, deafness in qv is central in origin. Here we report that quivering mice carry loss-of-function mutations in the mouse beta-spectrin 4 gene (Spnb4) that cause alterations in ion channel localization in myelinated nerves; this provides a rationale for the auditory and motor neuropathies of these mice.     

Cardiac defects and renal failure in mice with targeted mutations in Pkd2

PKD2, mutations in which cause autosomal dominant polycystic kidney disease (ADPKD), encodes an integral membrane glycoprotein with similarity to calcium channel subunits. We induced two mutations in the mouse homologue Pkd2 (ref.4): an unstable allele (WS25; hereafter denoted Pkd2WS25) that can undergo homologous-recombination-based somatic rearrangement to form a null allele; and a true null mutation (WS183; hereafter denoted Pkd2-). We examined these mutations to understand the function of polycystin-2, the protein product of Pkd2, and to provide evidence that kidney and liver cyst formation associated with Pkd2 deficiency occurs by a two-hit mechanism. Pkd2-/- mice die in utero between embryonic day (E) 13.5 and parturition. They have structural defects in cardiac septation and cyst formation in maturing nephrons and pancreatic ducts. Pancreatic ductal cysts also occur in adult Pkd2WS25/- mice, suggesting that this clinical manifestation of ADPKD also occurs by a two-hit mechanism. As in human ADPKD, formation of kidney cysts in adult Pkd2WS25/- mice is associated with renal failure and early death (median survival, 65 weeks versus 94 weeks for controls). Adult Pkd2+/- mice have intermediate survival in the absence of cystic disease or renal failure, providing the first indication of a deleterious effect of haploinsufficiency at Pkd2on long-term survival. Our studies advance our understanding of the function of polycystin-2 in development and our mouse models recapitulate the complex human ADPKD phenotype.     

Inactivation of Hdh in the brain and testis results in progressive neurodegeneration and sterility in mice

Inactivation of the mouse homologue of the Huntington disease gene (Hdh) results in early embryonic lethality. To investigate the normal function of Hdh in the adult and to evaluate current models for Huntington disease (HD), we have used the Cre/loxP site-specific recombination strategy to inactivate Hdh expression in the forebrain and testis, resulting in a progressive degenerative neuronal phenotype and sterility. On the basis of these results, we propose that huntingtin is required for neuronal function and survival in the brain and that a loss-of-function mechanism may contribute to HD pathogenesis.     

Cryptorchidism in mice mutant for Insl3.

Impaired testicular descent (cryptorchidism) is one of the most frequent congenital abnormalities in humans, involving 2% of male births. Cryptorchidism can result in infertility and increases risk for development of germ-cell tumours. Testicular descent from abdomen to scrotum occurs in two distinct phases: the trans-abdominal phase and the inguino-scrotal phase. Currently, little is known about the factors that regulate the trans-abdominal phase of testicular descent. Leydig insulin-like hormone (Insl3) is a member of the insulin hormone superfamily expressed in the developing testis. We show here that mice mutant for Insl3 are viable, but exhibit bilateral cryptorchidism due to developmental abnormalities of the gubernaculum, resulting in abnormal spermatogenesis and infertility. Female homozygotes have impaired fertility associated with deregulation of the oestrus cycle. These findings reveal roles for Insl3 in the development of the urogenital tract and in female fertility. Insl3 may act as a hormone to regulate the growth and differentiation of the gubernaculum, thereby mediating intra-abdominal testicular descent.     

Social amnesia in mice lacking the oxytocin gene

The development of social familiarity in rodents depends predominantly on olfactory cues and can critically influence reproductive success. Researchers have operationally defined this memory by a reliable decrease in olfactory investigation in repeated or prolonged encounters with a conspecific. Brain oxytocin (OT) and vasopressin (AVP) seem to modulate a range of social behaviour from parental care to mate guarding. Pharmacological studies indicate that AVP administration may enhance social memory, whereas OT administration may either inhibit or facilitate social memory depending on dose, route or paradigm. We found that male mice mutant for the oxytocin gene (Oxt-/-) failed to develop social memory, whereas wild-type (Oxt+/+) mice showed intact social memory. Measurement of both olfactory foraging and olfactory habituation tasks indicated that olfactory detection of non-social stimuli is intact in Oxt-/- mice. Spatial memory and behavioural inhibition measured in a Morris water-maze, Y-maze, or habituation of an acoustic startle also seemed intact. Treatment with OT but not AVP rescued social memory in Oxt-/- mice, and treatment with an OT antagonist produced a social amnesia-like effect in Oxt+/+ mice. Our data indicate that OT is necessary for the normal development of social memory in mice and support the hypothesis that social memory has a neural basis distinct from other forms of memory.