Generated Mutant Mice Research Articles

Anti-STAT6 CTL activity inStat6−/−mice

The generation of germline gene mutations in mice has been an invaluable tool for experimental biology. However, studying immune responses that develop in the absence of a specific protein that could alter thymic selection complicates experimental interpretations. We observed that CD8+ T cells from Stat6−/− mice displayed “autoreactivity” to STAT6-expressing cells, associated with specific STAT6 peptides binding to MHC class I molecules. These results suggest caution in interpreting experiments where STAT6-expressing cells are transferred into Stat6−/− mice, or where adoptive transfer of Stat6−/− lymphocytes is performed. Our results further highlight additional considerations when studying immune responses involving cell transfer into gene-deficient mice.

β2 Adrenergic Receptor, Protein Kinase A (PKA) and c-Jun N-terminal Kinase (JNK) Signaling Pathways Mediate Tau Pathology in Alzheimer Disease Models

Alzheimer disease (AD) is characterized by neurodegeneration marked by loss of synapses and spines associated with hyperphosphorylation of tau protein. Accumulating amyloid β peptide (Aβ) in brain is linked to neurofibrillary tangles composed of hyperphosphorylated tau in AD. Here, we identify β2-adrenergic receptor (β2AR) that mediates Aβ-induced tau pathology. In the prefrontal cortex (PFC) of 1-year-old transgenic mice with human familial mutant genes of presenilin 1 and amyloid precursor protein (PS1/APP), the phosphorylation of tau at Ser-214 Ser-262 and Thr-181, and the protein kinases including JNK, GSK3α/β, and Ca(2+)/calmodulin-dependent protein kinase II is increased significantly. Deletion of the β2AR gene in PS1/APP mice greatly decreases the phosphorylation of these proteins. Further analysis reveals that in primary PFC neurons, Aβ signals through a β2AR-PKA-JNK pathway, which is responsible for most of the phosphorylation of tau at Ser-214 and Ser-262 and a significant portion of phosphorylation at Thr-181. Aβ also induces a β2AR-dependent arrestin-ERK1/2 activity that does not participate in phosphorylation of tau. However, inhibition of the activity of MEK, an upstream enzyme of ERK1/2, partially blocks Aβ-induced tau phosphorylation at Thr-181. The density of dendritic spines and synapses is decreased in the deep layer of the PFC of 1-year-old PS1/APP mice, and the mice exhibit impairment of learning and memory in a novel object recognition paradigm. Deletion of the β2AR gene ameliorates pathological effects in these senile PS1/APP mice. The study indicates that β2AR may represent a potential therapeutic target for preventing the development of AD.

Characterization and Modeling of Intermittent Locomotor Dynamics in Clock Gene-Deficient Mice

The scale-invariant and intermittent dynamics of animal behavior are attracting scientific interest. Recent findings concerning the statistical laws of behavioral organization shared between healthy humans and wild-type mice (WT) and their alterations in human depression patients and circadian clock gene (Period 2; Per2) mutant mice indicate that clock genes play functional roles in intermittent, ultradian locomotor dynamics. They also claim the clinical and biological importance of the laws as objective biobehavioral measures or endophenotypes for psychiatric disorders. In this study, to elucidate the roles of breakdown of the broader circadian regulatory circuit in intermittent behavioral dynamics, we studied the statistical properties and rhythmicity of locomotor activity in Per2 mutants and mice deficient in other clock genes (Bmal1, Clock). We performed wavelet analysis to examine circadian and ultradian rhythms and estimated the cumulative distributions of resting period durations during which locomotor activity levels are continuously lower than a predefined threshold value. The wavelet analysis revealed significant amplification of ultradian rhythms in the BMAL1-deficient mice, and instability in the Per2 mutants. The resting period distributions followed a power-law form in all mice. While the distributions for the BMAL1-deficient and Clock mutant mice were almost identical to those for the WT mice, with no significant differences in their parameter (power-law scaling exponent), only the Per2 mutant mice showed consistently and significantly lower values of the scaling exponent, indicating the increased intermittency in ultradian locomotor dynamics. Furthermore, based on a stochastic priority queuing model, we explained the power-law nature of resting period distributions, as well as its alterations shared with human depressive patients and Per2 mutant mice. Our findings lead to the development of a novel mathematical model for abnormal behaviors in psychiatric disorders.

BBS7 is required for BBSome formation and its absence in mice results in Bardet-Biedl syndrome phenotypes and selective abnormalities in membrane protein trafficking

Bardet-Biedl Syndrome (BBS) is a pleiotropic and genetically heterozygous disorder caused independently by numerous genes (BBS1-BBS17). Seven highly conserved BBS proteins (BBS1, 2, 4, 5, 7, 8 and 9) form a complex known as the BBSome, which functions in ciliary membrane biogenesis. BBS7 is both a unique subunit of the BBSome and displays direct physical interaction with a second BBS complex, the BBS chaperonin complex. To examine the in vivo function of BBS7, we generated Bbs7 knockout mice. Bbs7(-/-) mice show similar phenotypes to other BBS gene mutant mice including retinal degeneration, obesity, ventriculomegaly and male infertility characterized by abnormal spermatozoa flagellar axonemes. Using tissues from Bbs7(-/-) mice, we show that BBS7 is required for BBSome formation, and that BBS7 and BBS2 depend on each other for protein stability. Although the BBSome serves as a coat complex for ciliary membrane proteins, BBS7 is not required for the localization of ciliary membrane proteins polycystin-1, polycystin-2, or bitter taste receptors, but absence of BBS7 leads to abnormal accumulation of the dopamine D1 receptor to the ciliary membrane, indicating that BBS7 is involved in specific membrane protein localization to cilia.

Behavioral Phenotypes in Schizophrenic Animal Models With Multiple Combinations of Genetic and Environmental Factors

Schizophrenia is a multifactorial psychiatric disorder in which both genetic and environmental factors play a role. Genetic [e.g., Disrupted-in-schizophrenia 1 (DISC1), Neuregulin-1 (NRG1)] and environmental factors (e.g., maternal viral infection, obstetric complications, social stress) may act during the developmental period to increase the incidence of schizophrenia. In animal models, interactions between susceptibility genes and the environment can be controlled in ways not possible in humans; therefore, such models are useful for investigating interactions between or within factors in the pathogenesis and pathophysiology of schizophrenia. We provide an overview of schizophrenic animal models investigating interactions between or within factors. First, we reviewed gene-environment interaction animal models, in which schizophrenic candidate gene mutant mice were subjected to perinatal immune activation or adolescent stress. Next, environment–environment interaction animal models, in which mice were subjected to a combination of perinatal immune activation and adolescent administration of drugs, were described. These animal models showed interaction between or within factors; behavioral changes, which were obscured by each factor, were marked by interaction of factors and vice versa. Appropriate behavioral approaches with such models will be invaluable for translational research on novel compounds, and also for providing insight into the pathogenesis and pathophysiology of schizophrenia.

Per2 Mutation Recapitulates the Vascular Phenotype of Diabetes in the Retina and Bone Marrow

In this study, we assessed whether Per2 clock gene–mutant mice exhibit a vascular phenotype similar to diabetes. Per2 (B6.129-Per2tm1Drw/J) or wild-type control mice 4 and 12 months of age were used. To evaluate diabetes-like phenotype in Per2 mutant mice, retina was quantified for mRNA expression, and degree of diabetic retinopathy was evaluated. Bone marrow neuropathy was studied by staining femurs for tyrosine hydroxylase (TH) and neurofilament 200 (NF-200). The rate of proliferation and quantification of bone marrow progenitor cells (BMPCs) was performed, and a threefold decrease in proliferation and 50% reduction in nitric oxide levels were observed in Per2 mutant mice. TH-positive nerve processes and NF-200 staining were reduced in Per2 mutant mice. Both retinal protein and mRNA expression of endothelial nitric oxide synthase were decreased by twofold. Other endothelial function genes (VEGFR2, VEGFR1) were downregulated (1.5–2-fold) in Per2 mutant retinas, whereas there was an upregulation of profibrotic pathway mediated by transforming growth factor-β1. Our studies suggest that Per2 mutant mice recapitulate key aspects of diabetes without the metabolic abnormalities, including retinal vascular damage, neuronal loss in the bone marrow, and diminished BMPC function.

Serotonin hyperinnervation and upregulated 5-HT2A receptor expression and motor-stimulating function in nigrostriatal dopamine-deficient Pitx3 mutant mice

The striatum receives serotonin (5-hydroxytryptamine, 5-HT) innervation and expresses 5-HT2A receptors (5-HT2ARs) and other 5-HT receptors, raising the possibility that the striatal 5-HT system may undergo adaptive changes after chronic severe dopamine (DA) loss and contribute to the function and dysfunction of the striatum. Here we show that in transcription factor Pitx3 gene mutant mice with a selective, severe DA loss in the dorsal striatum mimicking the DA denervation in late Parkinson's disease (PD), both the 5-HT innervation and the 5-HT2AR mRNA expression were increased in the dorsal striatum. Functionally, while having no detectable motor effect in wild type mice, the 5-HT2R agonist 2,5-dimethoxy-4-iodoamphetamine increased both the baseline and l-dopa-induced normal ambulatory and dyskinetic movements in Pitx3 mutant mice, whereas the selective 5-HT2AR blocker volinanserin had the opposite effects. These results demonstrate that Pitx3 mutant mice are a convenient and valid mouse model to study the compensatory 5-HT upregulation following the loss of the nigrostriatal DA projection and that the upregulated 5-HT2AR function in the DA deficient dorsal striatum may enhance both normal and dyskinetic movements.

Mutant laboratory mice with abnormalities in hair follicle morphogenesis, cycling, and/or structure: An update

Human hair disorders comprise a number of different types of alopecia, atrichia, hypotrichosis, distinct hair shaft disorders as well as hirsutism and hypertrichosis. Their causes vary from genodermatoses (e.g. hypotrichoses) via immunological disorders (e.g. alopecia areata, autoimmune cicatrical alopecias) to hormone-dependent abnormalities (e.g. androgenetic alopecia). A large number of spontaneous mouse mutants and genetically engineered mice develop abnormalities in hair follicle morphogenesis, cycling, and/or hair shaft formation, whose analysis has proven invaluable to define the molecular regulation of hair growth, ranging from hair follicle development, and cycling to hair shaft formation and stem cell biology. Also, the accumulating reports on hair phenotypes of mouse strains provide important pointers to better understand the molecular mechanisms underlying human hair growth disorders. Since numerous new mouse mutants with a hair phenotype have been reported since the publication of our earlier review on this matter a decade ago, we present here an updated, tabulated mini-review. The updated annotated tables list a wide selection of mouse mutants with hair growth abnormalities, classified into four categories: Mutations that affect hair follicle (1) morphogenesis, (2) cycling, (3) structure, and (4) mutations that induce extrafollicular events (for example immune system defects) resulting in secondary hair growth abnormalities. This synthesis is intended to provide a useful source of reference when studying the molecular controls of hair follicle growth and differentiation, and whenever the hair phenotypes of a newly generated mouse mutant need to be compared with existing ones.

Cthrc1, a Novel Circulating Hormone Regulating Metabolism

BackgroundWe discovered the gene Collagen Triple Helix Repeat Containing 1 (Cthrc1) and reported its developmental expression and induction in adventitial cells of injured arteries and dermal cells of skin wounds. The role of Cthrc1 in normal adult tissues has not yet been determined.Methodology/Principal FindingsWe generated mutant mice with a novel Cthrc1 null allele by homologues recombination. Cthrc1 null mice appeared developmentally normal. On the C57BL/6J background, livers from Cthrc1 null mice accumulated vast quantities of lipid, leading to extensive macrovesicular steatosis. Glycogen levels in skeletal muscle and liver of Cthrc1 null mice on the 129S6/SvEv background were significantly increased. However, Cthrc1 expression is not detectable in these tissues in wild-type mice, suggesting that the lipid and glycogen storage phenotype may be a secondary effect due to loss of Cthrc1 production at a distant site. To investigate potential hormonal functions of Cthrc1, tissues from adult mice and pigs were examined for Cthrc1 expression by immunohistochemistry with monoclonal anti-Cthrc1 antibodies. In pigs, Cthrc1 was detected around chromophobe cells of the anterior pituitary, and storage of Cthrc1 was observed in colloid-filled follicles and the pituitary cleft. Pituitary follicles have been observed in numerous vertebrates including humans but none of the known pituitary hormones have hitherto been detected in them. In C57BL/6J mice, however, Cthrc1 was predominantly expressed in the paraventricular and supraoptic nucleus of the hypothalamus but not in the posterior pituitary. In human plasma, we detected Cthrc1 in pg/ml quantities and studies with 125I-labeled Cthrc1 revealed a half-life of 2.5 hours in circulation. The highest level of Cthrc1 binding was observed in the liver.ConclusionsCthrc1 has characteristics of a circulating hormone generated from the anterior pituitary, hypothalamus and bone. Hormonal functions of Cthrc1 include regulation of lipid storage and cellular glycogen levels with potentially broad implications for cell metabolism and physiology.

Expression of the clock genes Per1 and Bmal1 during follicle development in the rat ovary. Effects of gonadotropin stimulation and hypophysectomy

Daily oscillations of clock genes have recently been demonstrated in the ovaries of several species. Clock gene knockout or mutant mice demonstrate a variety of reproductive defects. Accumulating evidence suggests that these rhythms act to synchronise the expression of specific ovarian genes to hypothalamo-pituitary signals and that they are regulated by one or both of the gonadotropins. The aim of this study has been to examine the spatio-temporal expression of the clock genes Per1 and Bmal1 during gonadotropin-independent and gonadotropin-dependent follicle development in the rat ovary. We have examined the ovaries of prepubertal rats, of prepubertal rats stimulated with equine chorionic gonadotropin (eCG)/human chorionic gonadotropin (hCG) and of hypophysectomised adult animals. Using quantitative reverse transcription with the polymerase chain reaction, in situ hybridisation histochemistry and immunohistochemistry, we have demonstrated that the expression of the two clock genes is low and arrhythmic in ovarian cells during early gonadotropin-independent follicle development in prepubertal animals and in hypophysectomised animals. We have also demonstrated that the expression of the clock genes becomes rhythmic following eCG stimulation in the theca interna cells and the secondary interstitial cells and that, following additional hCG stimulation, the expression of the clock genes also becomes rhythmic in the granulosa cells of preovulatory follicles. These findings link the initiation of clock gene rhythms in the rat ovary to the luteinising hormone receptor and suggest a functional link to androgen and progesterone production. In hypophysectomised animals, rhythmic clock gene expression is also observed in the corpora lutea and in secondary interstitial cells demonstrating that, in these compartments, entrainment of clock gene rhythms is gonadotropin-independent.

Highly Malignant Behavior of a Murine Oligodendrocyte Precursor Cell Line following Transplantation into the Demyelinated and Nondemyelinated Central Nervous System

Understanding the basic mechanisms that control CNS remyelination is of direct clinical relevance. Suitable model systems include the analysis of naturally occurring and genetically generated mouse mutants and the transplantation of oligodendrocyte precursor cells (OPCs) following experimental demyelination. However, aforementioned studies were exclusively carried out in rats and little is known about the in vivo behavior of transplanted murine OPCs. Therefore in the present study, we (i) established a model of ethidium bromide-induced demyelination of the caudal cerebellar peduncle (CCP) in the adult mouse and (ii) studied the distribution and marker expression of the murine OPC line BO-1 expressing the enhanced green fluorescent protein (eGFP) 10 and 17 days after stereotaxic implantation. Injection of ethidium bromide (0.025%) in the CCP resulted in a severe loss of myelin, marked astrogliosis, and mild to moderate axonal alterations. Transplanted cells formed an invasive and liquorogenic metastasizing tumor, classified as murine giant cell glioblastoma. Transplanted BO-1 cells displayed substantially reduced CNPase expression as compared to their in vitro phenotype, low levels of MBP and GFAP, prominent upregulation of NG2, PDGFRα, nuclear p53, and an unaltered expression of signal transducer and activator of transcription (STAT)-3. Summarized environmental signaling in the brain stem was not sufficient to trigger oligodendrocytic differentiation of BO-1 cells and seemed to block CNPase expression. Moreover, the lack of the remyelinating capacity was associated with tumor formation indicating that BO-1 cells may serve as a versatile experimental model to study tumorigenesis of glial tumors.

Reactivity changes in coronary artery and thoracic aorta in the gene mutant mice sgcd (B6.129 Sgcdtm1Mcn)

ObjectiveStudy of cardiovascular reactivity changes in the gene mutant mice sgcd.IntroductionDeletion of d‐sarcoglicano gene (delta‐SG) is related with structural and functional changes of squeletical, smooth and cardiac muscle which are related with limb‐girdle muscular dystrophy and cardiomiopathy. In this work we study the effect of some contractile and vasodilator agonist like angiotensin II and acethylcholine in this experimental model of dystrophy.MethodsWe used wild‐type and Knock‐out 5 months old female mice. We obtain thoracic aorta and the heart and both were immediately excised and placed in cold buffer, cleaned and freed from surrounding connective tissue. Isolated aortic rings and Langendorff system were maintained at 37° C, pH 7.4, were and bubbled with a mixture of 95% O2 and 5% CO2. After that we probe angiotnesin II (10−9 a 10−6M) with and without L‐NAME( sNOS antagonist) and indomethacin (COX‐1 and COX‐2 antagonist ).Results and conclusionsWe obtein significant differences between wild type and knock‐out mice, in the knock‐out mice we found an increase in the response of angiotensin II for that reason we suggest the participation of one contractile prostanoid in this effect. For that reason we decide to inhibit the contractile effect of angiotensin II with indometacin and the result was the inhibition of the contractile response. In other hand we analyse the vasodilator effect of acethylcholine at different concentrations and the result was less vasodilator effect in knock‐out mice, however we did not have differences in the vasodilator effect of nitroprusiate between wild type and knock‐out.

Significance of the Dynamic Expression of Pax6 and Dlx2 in the Postnatal Periventricular Zone Cells

Significance of the Dynamic Expression of Pax6 and Dlx2 in the Postnatal Periventricular Zone Cells

Period2 gene mutant mice show compromised insulin-mediated endothelial nitric oxide release and altered glucose homeostasis

Period2 (Per2) is an important component of the circadian clock. Mutation of this gene is associated with vascular endothelial dysfunction and altered glucose metabolism. The aim of this study is to further characterize whole body glucose homeostasis and endothelial nitric oxide (NO) production in response to insulin in the mPer2Brdm1 mice. We show that mPer2Brdm1 mice exhibit compromised insulin receptor activation and Akt signaling in various tissues including liver, fat, heart, and aortas with a tissue-specific heterogeneous diurnal pattern, and decreased insulin-stimulated NO release in the aortas in both active and inactive phases of the animals. As compared to wild type (WT) mice, the mPer2Brdm1 mice reveal hyperinsulinemia, hypoglycemia with lower fasting hepatic glycogen content and glycogen synthase level, no difference in glucose tolerance and insulin tolerance. The mPer2Brdm1 mice do not show increased predisposition to obesity either on normal chow or high fat diet compared to WT controls. Thus, mice with Per2 gene mutation show altered glucose homeostasis and compromised insulin-stimulated NO release, independently of obesity.

Ultrastructural Changes in Cerebral Capillary Pericytes in Aged Notch3 Mutant Transgenic Mice

Pericytes, the specialized vascular smooth muscle cells (VSMCs), play an important role in supporting and maintaining the structure of capillaries. Pericytes show biochemical and physiologic features similar to VSMC, usually containing smooth muscle actin fibers and rich endoplasm reticulum. Studies have indicated that degeneration of VSMCs due to Notch3 mutations is the cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). However, it remains unclear whether the Notch3 mutation also affects cerebral cortex capillary pericytes. In this ultrastructural morphologic study, the authors have observed pathological changes in the cerebral cortex capillary pericytes in aged mice that carry human mutant Notch3 genes. The number of abnormal pericytes in the cerebral cortex in Notch3 gene mutant mice was slightly increased when compared to an age-matched control group. Morphologically, the pericytes in the brains of Notch3 gene mutant mice showed more severe cellular injury, such as the presence of damaged mitochondria, secondary lysosomes, and large cytoplasmic vesicles. In addition, morphologic structures related to autophagy were also present in the pericytes of Notch3 gene mutant group. These ultrastructural morphologic alterations suggest that Notch3 mutation precipitates age-related pericytic degeneration that might result in cellular injury and trigger autophagic apoptosis. Microvascular dysfunction due to pericyte degeneration could initiate secondary neurodegenerative changes in brain parenchyma. These findings provide new insight into understanding the role of pericyte degeneration in the phathogenesis of CADASIL disease.

Circadian Variation of the Response of T Cells to Antigen

Circadian clocks regulate many important aspects of physiology, and their disturbance leads to various medical conditions. Circadian variations have been found in immune system variables, including daily rhythms in circulating WBC numbers and serum concentration of cytokines. However, control of immune functional responses by the circadian clock has remained relatively unexplored. In this study, we show that mouse lymph nodes exhibit rhythmic clock gene expression. T cells from lymph nodes collected over 24 h show a circadian variation in proliferation after stimulation via the TCR, which is blunted in Clock gene mutant mice. The tyrosine kinase ZAP70, which is just downstream of the TCR in the T cell activation pathway and crucial for T cell function, exhibits rhythmic protein expression. Lastly, mice immunized with OVA peptide-loaded dendritic cells in the day show a stronger specific T cell response than mice immunized at night. These data reveal circadian control of the Ag-specific immune response and a novel regulatory mode of T cell proliferation, and may provide clues for more efficient vaccination strategies.

Circadian and Acamprosate Modulation of Elevated Ethanol Drinking in mPer2 Clock Gene Mutant Mice

The PER2 clock gene modulates ethanol consumption, such that mutant mice not expressing functional mPer2 have altered circadian behavior that promotes higher ethanol intake and preference. Experiments were undertaken to characterize circadian-related behavioral effects of mPer2 deletion on ethanol intake and to explore how acamprosate (used to reduce alcohol dependence) alters diurnal patterns of ethanol intake. Male mPer2 mutant and WT (wild-type) mice were entrained to a 12:12 h light-dark (12L:12D) photocycle, and their locomotor and drinking activities were recorded. Circadian locomotor measurements confirmed that mPer2 mutants had an advanced onset of nocturnal activity of about 2 h relative to WTs, and an increased duration of nocturnal activity (p < .01). Also, mPer2 mutants preferred and consumed more ethanol and had more daily ethanol drinking episodes vs. WTs. Measurements of systemic ethanol using subcutaneous microdialysis confirmed the advanced rise in ethanol intake in the mPer2 mutants, with 24-h averages being ∼60 vs. ∼25 mM for WTs (p < .01). A 6-day regimen of single intraperitoneal (i.p.) acamprosate injections (300 mg/kg) at zeitgeber time (ZT) 10 did not alter the earlier onset of nocturnal ethanol drinking in the mPer2 mutants, but reduced the overall amplitude of drinking and preference (both p < .01). Acamprosate also reduced these parameters in WTs. These results suggest that elevated ethanol intake in mPer2 mutants may be a partial consequence of an earlier nighttime activity onset and increase in nocturnal drinking activity. The suppressive action of acamprosate on ethanol intake is not due to an altered diurnal pattern of drinking, but rather a decrease in the number of daily drinking bouts and amount of drinking per bout. (Author correspondence: jglass@kent.edu)

Cu/Zn superoxide dismutase is differentially regulated in period gene-mutant mice

The circadian clock in the brain coordinates the phase of peripheral oscillators that regulate tissue-specific physiological outputs. Here we report that circadian variations in the expression and activity of Cu/Zn superoxide dismutase (SOD1; EC 1.15.1.1) are present in liver homogenates from mice. The SOD1 mRNA expression from wild-type (WT) mice peaked at Zeitgeber Time 9 (ZT9; 9h after lights-on time). While there was no rhythmicity in that from period2 (per2) gene knockout (P2K) mice, the level of SOD1 from per1/per2 double knockout (DKO) mice was significantly elevated at ZT5. The enzyme activity of SOD1 was also rhythmic in the mouse liver. Moreover, the total amount of the SOD1 exhibited a rhythmic oscillation with a peak at ZT9 in the liver from WT mice. We also found that tert-butylhydroperoxide (t-BHP)-induced oxidative damage in both WT and P2K mouse embryonic fibroblast (MEF) cells resulted in the up-regulation of SOD1 levels. Our data suggest that the expression of an important antioxidant enzyme, SOD1, is under circadian clock control and that mice are more susceptible to oxidative stress depending on the time of day.

A Mutation in Synaptojanin 2 Causes Progressive Hearing Loss in the ENU-Mutagenised Mouse Strain Mozart

BackgroundHearing impairment is the most common sensory impairment in humans, affecting 1∶1,000 births. We have identified an ENU generated mouse mutant, Mozart, with recessively inherited, non-syndromic progressive hearing loss caused by a mutation in the synaptojanin 2 (Synj2), a central regulatory enzyme in the phosphoinositide-signaling cascade.Methodology/Principal FindingsThe hearing loss in Mozart is caused by a p.Asn538Lys mutation in the catalytic domain of the inositol polyphosphate 5-phosphatase synaptojanin 2. Within the cochlea, Synj2 mRNA expression was detected in the inner and outer hair cells but not in the spiral ganglion. Synj2 N538K mutant protein showed loss of lipid phosphatase activity, and was unable to degrade phosphoinositide signaling molecules. Mutant Mozart mice (Synj2 N538K/N538K) exhibited progressive hearing loss and showed signs of hair cell degeneration as early as two weeks of age, with fusion of stereocilia followed by complete loss of hair bundles and ultimately loss of hair cells. No changes in vestibular or neurological function, or other clinical or behavioral manifestations were apparent.Conclusions/SignificancePhosphoinositides are membrane associated signaling molecules that regulate many cellular processes including cell death, proliferation, actin polymerization and ion channel activity. These results reveal Synj2 as a critical regulator of hair cell survival that is essential for hair cell maintenance and hearing function.

In vivo role of different domains and of phosphorylation in the transcription factor Nkx2-1

BackgroundThe transcription factor Nkx2-1 (also known as TTF-1, Titf1 or T/EBP) contains two apparently redundant activation domains and is post-translationally modified by phosphorylation. We have generated mouse mutant strains to assess the roles of the two activation domains and of phosphorylation in mouse development and differentiation.ResultsMouse strains expressing variants of the transcription factor Nkx2-1 deleted of either activation domain have been constructed. Phenotypic analysis shows for each mutant a distinct set of defects demonstrating that distinct portions of the protein endow diverse developmental functions of Nkx2-1. Furthermore, a mouse strain expressing a Nkx2-1 protein mutated in the phosphorylation sites shows a thyroid gland with deranged follicular organization and gene expression profile demonstrating the functional role of phosphorylation in Nkx2-1.ConclusionsThe pleiotropic functions of Nkx2-1 are not all due to the protein as a whole since some of them can be assigned to separate domains of the protein or to specific post-translational modifications. These results have implication for the evolutionary role of mutations in transcription factors.