Deletion of Jdp2 enhances Slc7a11 expression in Atoh-1 positive cerebellum granule cell progenitors in vivo.

BACKGROUND: The cerebellum is the sensitive region of the brain to developmental abnormalities related to the effects of oxidative stresses. Abnormal cerebellar lobe formation, found in Jun dimerization protein 2 (Jdp2)-knockout (KO) mice, is related to increased antioxidant formation and a reduction in apoptotic cell death in granule cell progenitors (GCPs). Here, we aim that Jdp2 plays a critical role of cerebellar development which is affected by the ROS regulation and redox control. OBJECTIVE: Jdp2-promoter-Cre transgenic mouse displayed a positive signal in the cerebellum, especially within granule cells. Jdp2-KO mice exhibited impaired development of the cerebellum compared with wild-type (WT) mice. The antioxidation controlled gene, such as cystine-glutamate transporter Slc7a11, might be critical to regulate the redox homeostasis and the development of the cerebellum. METHODS: We generated the Jdp2-promoter-Cre mice and Jdp2-KO mice to examine the levels of Slc7a11, ROS levels and the expressions of antioxidation related genes were examined in the mouse cerebellum using the immunohistochemistry. RESULTS: The cerebellum of Jdp2-KO mice displayed expression of the cystine-glutamate transporter Slc7a11, within the internal granule layer at postnatal day 6; in contrast, the WT cerebellum mainly displayed Sla7a11 expression in the external granule layer. Moreover, development of the cerebellar lobes in Jdp2-KO mice was altered compared with WT mice. Expression of Slc7a11, Nrf2, and p21Cip1 was higher in the cerebellum of Jdp2-KO mice than in WT mice. CONCLUSION: Jdp2 is a critical regulator of Slc7a11 transporter during the antioxidation response, which might control the growth, apoptosis, and differentiation of GCPs in the cerebellar lobes. These observations are consistent with our previous study in vitro.

Pathogenic POGZ mutation causes impaired cortical development and reversible autism-like phenotypes.

Pogo transposable element derived with ZNF domain (POGZ) has been identified as one of the most recurrently de novo mutated genes in patients with neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), intellectual disability and White-Sutton syndrome; however, the neurobiological basis behind these disorders remains unknown. Here, we show that POGZ regulates neuronal development and that ASD-related de novo mutations impair neuronal development in the developing mouse brain and induced pluripotent cell lines from an ASD patient. We also develop the first mouse model heterozygous for a de novo POGZ mutation identified in a patient with ASD, and we identify ASD-like abnormalities in the mice. Importantly, social deficits can be treated by compensatory inhibition of elevated cell excitability in the mice. Our results provide insight into how de novo mutations on high-confidence ASD genes lead to impaired mature cortical network function, which underlies the cellular pathogenesis of NDDs, including ASD.

Melanocytes contribute to the vasculature of the choroid.

Melanocytes develop from the vertebrate embryo-specific neural crest, migrate, and localize in various organs, including not only the skin but also several extracutaneous locations such as the heart, inner ear and choroid. Little is known about the functions of extracutaneous melanocytes except for cochlear melanocytes, which are essential for hearing ability. In this study, we focused on the structure of the choroid, in which melanocytes are abundant around the well-developed blood vascular system. By comparing structural differences in the choroid of wild-type and melanocyte-deficient Mitfmi-bw/Mitfmi-bw mutant mice, our observations suggest that choroidal melanocytes contribute to the morphogenesis and/or maintenance of the normal vasculature structure of that tissue.

The parathyroid hormone regulates skin tumour susceptibility in mice.

Using a forward genetics approach to map loci in a mouse skin cancer model, we previously identified a genetic locus, Skin tumour modifier of MSM 1 (Stmm1) on chromosome 7, conferring strong tumour resistance. Sub-congenic mapping localized Parathyroid hormone (Pth) in Stmm1b. Here, we report that serum intact-PTH (iPTH) and a genetic polymorphism in Pth are important for skin tumour resistance. We identified higher iPTH levels in sera from cancer-resistant MSM/Ms mice compared with susceptible FVB/NJ mice. Therefore, we performed skin carcinogenesis experiments with MSM-BAC transgenic mice (Pth MSM-Tg) and Pth knockout heterozygous mice (Pth +/-). As a result, the higher amounts of iPTH in sera conferred stronger resistance to skin tumours. Furthermore, we found that the coding SNP (rs51104087, Val28Met) localizes in the mouse Pro-PTH encoding region, which is linked to processing efficacy and increased PTH secretion. Finally, we report that PTH increases intracellular calcium in keratinocytes and promotes their terminal differentiation. Taken together, our data suggest that Pth is one of the genes responsible for Stmm1, and serum iPTH could serve as a prevention marker of skin cancer and a target for new therapies.

New insights into the role of Jmjd3 and Utx in axial skeletal formation in mice.

Jmjd3 and Utx are demethylases specific for lysine 27 of histone H3. Previous reports indicate that Jmjd3 is essential for differentiation of various cell types, such as macrophages and epidermal cells in mice, whereas Utx is involved in cancer and developmental diseases in humans and mice, as well as Hox regulation in zebrafish and nematodes. Here, we report that Jmjd3, but not Utx, is involved in axial skeletal formation in mice. A Jmjd3 mutant embryo (Jmjd3Δ18/Δ18), but not a catalytically inactive Utx truncation mutant (Utx-/y), showed anterior homeotic transformation. Quantitative RT-PCR and microarray analyses showed reduced Hox expression in both Jmjd3Δ18/Δ18 embryos and tailbuds, whereas levels of Hox activators, such as Wnt signaling factors and retinoic acid synthases, did not decrease, which suggests that Jmjd3 plays a regulatory role in Hox expression during axial patterning. Chromatin immunoprecipitation analyses of embryo tailbud tissue showed trimethylated lysine 27 on histone H3 to be at higher levels at the Hox loci in Jmjd3Δ18/Δ18 mutants compared with wild-type tailbuds. In contrast, trimethylated lysine 4 on histone H3 levels were found to be equivalent in wild-type and Jmjd3Δ18/Δ18 tailbuds. Demethylase-inactive Jmjd3 mutant embryos showed the same phenotype as Jmjd3Δ18/Δ18 mice. These results suggest that the demethylase activity of Jmjd3, but not that of Utx, affects mouse axial patterning in concert with alterations in Hox gene expression.-Naruse, C., Shibata, S., Tamura, M., Kawaguchi, T., Abe, K., Sugihara, K., Kato, T., Nishiuchi, T., Wakana, S., Ikawa, M., Asano, M. New insights into the role of Jmjd3 and Utx in axial skeletal formation in mice.

A spontaneous and novel Pax3 mutant mouse that models Waardenburg syndrome and neural tube defects.

BACKGROUND: Genes responsible for reduced pigmentation phenotypes in rodents are associated with human developmental defects, such as Waardenburg syndrome, where patients display congenital deafness along with various abnormalities mostly related to neural crest development deficiency. OBJECTIVE: In this study, we identified a spontaneous mutant mouse line Rwa, which displays variable white spots on mouse bellies and white digits and tail, on a C57BL/6N genetic background. Curly tail and spina bifida were also observed, although at a lower penetrance. These phenotypes were dominantly inherited by offspring. We searched for the genetic mechanism of the observed phenotypes. METHODS: We harnessed a rapid mouse gene mapping system newly developed in our laboratories to identify a responsible gene. RESULTS: We detected a region within chromosome 1 as a probable locus for the causal mutation. Dense mapping using interval markers narrowed the locus down to a 670-kbp region, containing four genes including Pax3, a gene known to be implicated in the types I and III Waardenburg syndrome. Extensive mutation screening of Pax3 detected an 841-bp deletion, spanning the promoter region and intron 1 of the gene. The defective allele of Pax3, named Pax3Rwa, lacked the first coding exon and co-segregated perfectly with the phenotypes, confirming its causal nature. The genetic background of Rwa mice is almost identical to that of inbred C57BL/6N. CONCLUSION: These results highlight Pax3Rwa mice as a beneficial tool for analyzing biological processes involving Pax3, in particular the development and migration of neural crest cells and melanocytes.

ATF7 ablation prevents diet-induced obesity and insulin resistance.

The activating transcription factor (ATF)2 family of transcription factors regulates a variety of metabolic processes, including adipogenesis and adaptive thermogenesis. ATF7 is a member of the ATF2 family, and mediates epigenetic changes induced by environmental stresses, such as social isolation and pathogen infection. However, the metabolic role of ATF7 remains unknown. The aim of the present study is to examine the role of ATF7 in metabolism using ATF7-dificeint mice. Atf7(-/-) mice exhibited lower body weight and resisted diet-induced obesity. Serum triglycerides, resistin, and adipose tissue mass were all significantly lower in ATF7-deficient mice. Fasting glucose levels and glucose tolerance were unaltered, but systemic insulin sensitivity was increased, by ablation of ATF7. Indirect calorimetry revealed that oxygen consumption by Atf7(-/-) mice was comparable to that of wild-type littermates on a standard chow diet, but increased energy expenditure was observed in Atf7(-/-) mice on a high-fat diet. Hence, ATF7 ablation may impair the development and function of adipose tissue and result in elevated energy expenditure in response to high-fat-feeding obesity and insulin resistance, indicating that ATF7 is a potential therapeutic target for diet-induced obesity and insulin resistance.

Novel allelic mutations in murine Serca2 induce differential development of squamous cell tumors.

Dominant mutations in the Serca2 gene, which encodes sarco(endo)plasmic reticulum calcium-ATPase, predispose mice to gastrointestinal epithelial carcinoma [1-4] and humans to Darier disease (DD) [14-17]. In this study, we generated mice harboring N-ethyl-N-nitrosourea (ENU)-induced allelic mutations in Serca2: three missense mutations and one nonsense mutation. Mice harboring these Serca2 mutations developed tumors that were categorized as either early onset squamous cell tumors (SCT), with development similar to null-type knockout mice [2,4] (aggressive form; M682, M814), or late onset tumors (mild form; M1049, M1162). Molecular analysis showed no aberration in Serca2 mRNA or protein expression levels in normal esophageal cells of any of the four mutant heterozygotes. There was no loss of heterozygosity at the Serca2 locus in the squamous cell carcinomas in any of the four lines. The effect of each mutation on Ca(2+)-ATPase activity was predicted using atomic-structure models and accumulated mutated protein studies, suggesting that putative complete loss of Serca2 enzymatic activity may lead to early tumor onset, whereas mutations in which Serca2 retains residual enzymatic activity result in late onset. We propose that impaired Serca2 gene product activity has a long-term effect on squamous cell carcinogenesis from onset to the final carcinoma stage through an as-yet unrecognized but common regulatory pathway.

Endoplasmic reticulum stress-mediated apoptosis contributes to a skeletal dysplasia resembling platyspondylic lethal skeletal dysplasia, Torrance type, in a novel Col2a1 mutant mouse line.

In humans, mutations in the COL2A1 gene encoding the α1(II) chain of type II collagen, create many clinical phenotypes collectively termed type II collagenopathies. However, the mechanisms generating this diversity remain to be determined. Here we identified a novel Col2a1 mutant mouse line by screening a large-scale N-ethyl-N-nitrosourea mutant mouse library. This mutant possessed a p.Tyr1391Ser missense mutation in the C-propeptide coding region, and this mutation was located in positions corresponding to the human COL2A1 mutation responsible for platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T). As expected, p.Tyr1391Ser homozygotes exhibited lethal skeletal dysplasias resembling PLSD-T, including extremely short limbs and severe dysplasia of the spine and pelvis. The secretion of the mutant proteins into the extracellular space was disrupted, accompanied by an abnormally expanded endoplasmic reticulum (ER) and the up-regulation of ER stress-related genes in chondrocytes. Chondrocyte apoptosis was severely induced in the growth plate of the homozygotes. These findings strongly suggest that ER stress-mediated apoptosis caused by the accumulated mutant proteins in ER contributes to skeletal dysplasia in Co12a1 mutant mice and PLSD-T patients.

KDEL receptor 1 regulates T-cell homeostasis via PP1 that is a key phosphatase for ISR.

KDEL receptors are responsible for retrotransporting endoplasmic reticulum (ER) chaperones from the Golgi complex to the ER. Here we describe a role for KDEL receptor 1 (KDELR1) that involves the regulation of integrated stress responses (ISR) in T cells. Designing and using an N-ethyl-N-nitrosourea (ENU)-mutant mouse line, T-Red (naïve T-cell reduced), we show that a point mutation in KDELR1 is responsible for the reduction in the number of naïve T cells in this model owing to an increase in ISR. Mechanistic analysis shows that KDELR1 directly regulates protein phosphatase 1 (PP1), a key phosphatase for ISR in naïve T cells. T-Red KDELR1 does not associate with PP1, resulting in reduced phosphatase activity against eIF2α and subsequent expression of stress responsive genes including the proapoptotic factor Bim. These results demonstrate that KDELR1 regulates naïve T-cell homeostasis by controlling ISR.

IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through tyrosine hydroxylase phosphorylation.

Inositol 1,4,5-trisphosphate receptor (IP3R) binding protein released with IP3 (IRBIT) contributes to various physiological events (electrolyte transport and fluid secretion, mRNA polyadenylation, and the maintenance of genomic integrity) through its interaction with multiple targets. However, little is known about the physiological role of IRBIT in the brain. Here we identified calcium calmodulin-dependent kinase II alpha (CaMKIIα) as an IRBIT-interacting molecule in the central nervous system. IRBIT binds to and suppresses CaMKIIα kinase activity by inhibiting the binding of calmodulin to CaMKIIα. In addition, we show that mice lacking IRBIT present with elevated catecholamine levels, increased locomotor activity, and social abnormalities. The level of tyrosine hydroxylase (TH) phosphorylation by CaMKIIα, which affects TH activity, was significantly increased in the ventral tegmental area of IRBIT-deficient mice. We concluded that IRBIT suppresses CaMKIIα activity and contributes to catecholamine homeostasis through TH phosphorylation.

Sirh7/Ldoc1 knockout mice exhibit placental P4 overproduction and delayed parturition.

Sirh7/Ldoc1 [sushi-ichi retrotransposon homolog 7/leucine zipper, downregulated in cancer 1, also called mammalian retrotransposon-derived 7 (Mart7)] is one of the newly acquired genes from LTR retrotransposons in eutherian mammals. Interestingly, Sirh7/Ldoc1 knockout (KO) mice exhibited abnormal placental cell differentiation/maturation, leading to an overproduction of placental progesterone (P4) and placental lactogen 1 (PL1) from trophoblast giant cells (TGCs). The placenta is an organ that is essential for mammalian viviparity and plays a major endocrinological role during pregnancy in addition to providing nutrients and oxygen to the fetus. P4 is an essential hormone in the preparation and maintenance of pregnancy and the determination of the timing of parturition in mammals; however, the biological significance of placental P4 in rodents is not properly recognized. Here, we demonstrate that mouse placentas do produce P4 in mid-gestation, coincident with a temporal reduction in ovarian P4, suggesting that it plays a role in the protection of the conceptuses specifically in this period. Pregnant Sirh7/Ldoc1 knockout females also displayed delayed parturition associated with a low pup weaning rate. All these results suggest that Sirh7/Ldoc1 has undergone positive selection during eutherian evolution as a eutherian-specific acquired gene because it impacts reproductive fitness via the regulation of placental endocrine function.

Novel retinoblastoma mutation abrogating the interaction to E2F2/3, but not E2F1, led to selective suppression of thyroid tumors.

Mutant mouse models are indispensable tools for clarifying gene functions and elucidating the pathogenic mechanisms of human diseases. Here, we describe novel cancer models bearing point mutations in the retinoblastoma gene (Rb1) generated by N-ethyl-N-nitrosourea mutagenesis. Two mutations in splice sites reduced Rb1 expression and led to a tumor spectrum and incidence similar to those observed in the conventional Rb1 knockout mice. The missense mutant, Rb1(D326V/+) , developed pituitary tumors, but thyroid tumors were completely suppressed. Immunohistochemical analyses of thyroid tissue revealed that E2F1, but not E2F2/3, was selectively inactivated, indicating that the mutant Rb protein (pRb) suppressed thyroid tumors by inactivating E2F1. Interestingly, Rb1(D326V/+) mice developed pituitary tumors that originated from the intermediate lobe of the pituitary, despite selective inactivation of E2F1. Furthermore, in the anterior lobe of the pituitary, other E2F were also inactivated. These observations show that pRb mediates the inactivation of E2F function and its contribution to tumorigenesis is highly dependent on the cell type. Last, by using a reconstitution assay of synthesized proteins, we showed that the D326V missense pRb bound to E2F1 but failed to interact with E2F2/3. These results reveal the effect of the pRb N-terminal domain on E2F function and the impact of the protein on tumorigenesis. Thus, this mutant mouse model can be used to investigate human Rb family-bearing mutations at the N-terminal region.

Identification of Stmm3 locus conferring resistance to late-stage chemically induced skin papillomas on mouse chromosome 4 by congenic mapping and allele-specific alteration analysis.

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to chemically induced skin papillomas on chromosome 4 and 7 with a large number of [(FVB/N × MSM/Ms) F1 × FVB/N] backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 4. We used linkage analysis and a congenic mouse strain, FVB.MSM-Stmm3 to refine the location of Stmm3 (Skin tumor modifier of MSM 3) locus within a physical interval of about 34 Mb on distal chromosome 4. In addition, we used patterns of allele-specific imbalances in tumors from N2 and N10 congenic mice to narrow down further the region of Stmm3 locus to a physical distance of about 25 Mb. Furthermore, immunohistochemical analysis showed papillomas from congenic mice had less proliferative activity. These results suggest that Stmm3 responsible genes may have an influence on papilloma formation in the two-stage skin carcinogenesis by regulating papilloma growth rather than development.

Congenic mapping and allele-specific alteration analysis of Stmm1 locus conferring resistance to early-stage chemically induced skin papillomas.

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to early-stage chemically induced skin papillomas on chromosome 7 with a large number of [(FVB/N×MSM/Ms)×FVB/N] F1 backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 7. We used linkage analysis and congenic mouse strains to refine the location of Stmm1 (Skin tumor modifier of MSM 1) locus within a genetic interval of about 3 cM on proximal chromosome 7. In addition, we used patterns of allele-specific imbalances in tumors from F1 backcross and N10 congenic mice to narrow down further the region of Stmm1 locus to a physical distance of about 5.4 Mb. To gain the insight into the function of Stmm1 locus, we carried out a long term BrdU labelling experiments with congenic mice containing Stmm1 locus. Interestingly, we observed a decrease of BrdU-LRCs (Label Retaining Cells) in a congenic strain heterozygous or homozygous for MSM allele of Stmm1. These results suggest that Stmm1 responsible genes may have an influence on papillomagenesis in the two-stage skin carcinogenesis by regulating epidermal quiescent stem cells.

Novel mouse model for Gardner syndrome generated by a large-scale N-ethyl-N-nitrosourea mutagenesis program.

Mutant mouse models are indispensable tools for clarifying the functions of genes and elucidating the underlying pathogenic mechanisms of human diseases. We carried out large-scale mutagenesis using the chemical mutagen N-ethyl-N-nitrosourea. One specific aim of our mutagenesis project was to generate novel cancer models. We screened 7012 animals for dominant traits using a necropsy test and thereby established 17 mutant lines predisposed to cancer. Here, we report on a novel cancer model line that developed osteoma, trichogenic tumor, and breast cancer. Using fine mapping and genomic sequencing, we identified a point mutation in the adenomatous polyposis coli (Apc) gene. The Apc1576 mutants bear a nonsense mutation at codon 1576 in the Apc gene. Although most Apc mutant mice established thus far have multifocal intestinal tumors, mice that are heterozygous for the Apc1576 mutation do not develop intestinal tumors; instead, they develop multifocal breast cancers and trichogenic tumors. Notably, the osteomas that develop in the Apc1576 mutant mice recapitulate the lesion observed in Gardner syndrome, a clinical variant of familial adenomatous polyposis. Our Apc1576 mutant mice will be valuable not only for understanding the function of the Apc gene in detail but also as models of human Gardner syndrome.

Tysnd1 deficiency in mice interferes with the peroxisomal localization of PTS2 enzymes, causing lipid metabolic abnormalities and male infertility.

Peroxisomes are subcellular organelles involved in lipid metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids, among others. Peroxisome matrix proteins are synthesized in the cytoplasm. Targeting signals (PTS or peroxisomal targeting signal) at the C-terminus (PTS1) or N-terminus (PTS2) of peroxisomal matrix proteins mediate their import into the organelle. In the case of PTS2-containing proteins, the PTS2 signal is cleaved from the protein when transported into peroxisomes. The functional mechanism of PTS2 processing, however, is poorly understood. Previously we identified Tysnd1 (Trypsin domain containing 1) and biochemically characterized it as a peroxisomal cysteine endopeptidase that directly processes PTS2-containing prethiolase Acaa1 and PTS1-containing Acox1, Hsd17b4, and ScpX. The latter three enzymes are crucial components of the very-long-chain fatty acids ß-oxidation pathway. To clarify the in vivo functions and physiological role of Tysnd1, we analyzed the phenotype of Tysnd1(-/-) mice. Male Tysnd1(-/-) mice are infertile, and the epididymal sperms lack the acrosomal cap. These phenotypic features are most likely the result of changes in the molecular species composition of choline and ethanolamine plasmalogens. Tysnd1(-/-) mice also developed liver dysfunctions when the phytanic acid precursor phytol was orally administered. Phyh and Agps are known PTS2-containing proteins, but were identified as novel Tysnd1 substrates. Loss of Tysnd1 interferes with the peroxisomal localization of Acaa1, Phyh, and Agps, which might cause the mild Zellweger syndrome spectrum-resembling phenotypes. Our data established that peroxisomal processing protease Tysnd1 is necessary to mediate the physiological functions of PTS2-containing substrates.

Independent genetic control of early and late stages of chemically induced skin tumors in a cross of a Japanese wild-derived inbred mouse strain, MSM/Ms.

MSM/Ms is an inbred mouse strain derived from a Japanese wild mouse, Mus musculus molossinus. In this study, we showed that MSM/Ms mice exhibit dominant resistance when crossed with susceptible FVB/N mice and subjected to the two-stage skin carcinogenesis protocol using 7,12-dimethylbenz(a)anthracene (DMBA)/ 12-O-tetradecanoylphorbol-13-acetate (TPA). A series of F1 backcross mice were generated by crossing p53(+/+) or p53(+/-) F1 (FVB/N × MSM/Ms) males with FVB/N female mice. These generated 228 backcross animals, approximately half of which were p53(+/-), enabling us to search for p53-dependent skin tumor modifier genes. Highly significant linkage for papilloma multiplicity was found on chromosomes 6 and 7 and suggestive linkage was found on chromosomes 3, 5 and 12. Furthermore, in order to identify stage-dependent linkage loci we classified tumors into three categories (<2mm, 2-6mm and >6mm), and did linkage analysis. The same locus on chromosome 7 showed strong linkage in groups with <2mm or 2-6mm papillomas. No linkage was detected on chromosome 7 to papillomas >6mm, but a different locus on chromosome 4 showed strong linkage both to papillomas >6mm and to carcinomas. This locus, which maps near the Cdkn2a/p19(Arf) gene, was entirely p53-dependent, and was not seen in p53 (+/-) backcross animals. Suggestive linkage conferring susceptibility to carcinoma was also found on chromosome 5. These results clearly suggest distinct loci regulate each stage of tumorigenesis, some of which are p53-dependent.

ENU-induced missense mutation in the C-propeptide coding region of Col2a1 creates a mouse model of platyspondylic lethal skeletal dysplasia, Torrance type.

The COL2A1 gene encodes the α1(II) chain of the homotrimeric type II collagen, the most abundant protein in cartilage. In humans, COL2A1 mutations create many clinical phenotypes collectively termed type II collagenopathies; however, the genetic basis of the phenotypic diversity is not well elucidated. Therefore, animal models corresponding to multiple type II collagenopathies are required. In this study we identified a novel Col2a1 missense mutation--c.44406A>C (p.D1469A)--produced by large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis in a mouse line. This mutation was located in the C-propeptide coding region of Col2a1 and in the positions corresponding to a human COL2A1 mutation responsible for platyspondylic lethal skeletal dysplasia, Torrance type (PLSD-T). The phenotype was inherited as a semidominant trait. The heterozygotes were mildly but significantly smaller than wild-type mice. The homozygotes exhibited lethal skeletal dysplasias, including extremely short limbs, severe spondylar dysplasia, severe pelvic hypoplasia, and brachydactyly. As expected, these skeletal defects in the homozygotes were similar to those in PLSD-T patients. The secretion of the mutant proteins into the extracellular space was disrupted, accompanied by abnormally expanded rough endoplasmic reticulum (ER) and upregulation of ER stress-related genes, such as Grp94 and Chop, in chondrocytes. These findings suggested that the accumulation of mutant type II collagen in the ER and subsequent induction of ER stress are involved, at least in part in the PLSD-T-like phenotypes of the mutants. This mutant should serve as a good model for studying PLSD-T pathogenesis and the mechanisms that create the great diversity of type II collagenopathies.

Attenuated food anticipatory activity and abnormal circadian locomotor rhythms in Rgs16 knockdown mice.

Regulators of G protein signaling (RGS) are a multi-functional protein family, which functions in part as GTPase-activating proteins (GAPs) of G protein α-subunits to terminate G protein signaling. Previous studies have demonstrated that the Rgs16 transcripts exhibit robust circadian rhythms both in the suprachiasmatic nucleus (SCN), the master circadian light-entrainable oscillator (LEO) of the hypothalamus, and in the liver. To investigate the role of RGS16 in the circadian clock in vivo, we generated two independent transgenic mouse lines using lentiviral vectors expressing short hairpin RNA (shRNA) targeting the Rgs16 mRNA. The knockdown mice demonstrated significantly shorter free-running period of locomotor activity rhythms and reduced total activity as compared to the wild-type siblings. In addition, when feeding was restricted during the daytime, food-entrainable oscillator (FEO)-driven elevated food-anticipatory activity (FAA) observed prior to the scheduled feeding time was significantly attenuated in the knockdown mice. Whereas the restricted feeding phase-advanced the rhythmic expression of the Per2 clock gene in liver and thalamus in the wild-type animals, the above phase shift was not observed in the knockdown mice. This is the first in vivo demonstration that a common regulator of G protein signaling is involved in the two separate, but interactive circadian timing systems, LEO and FEO. The present study also suggests that liver and/or thalamus regulate the food-entrained circadian behavior through G protein-mediated signal transduction pathway(s).