Hiroetsu Suzuki

A spontaneous mutation of the Wwox gene and audiogenic seizures in rats with lethal dwarfism and epilepsy

The lde/lde rat is characterized by dwarfism, postnatal lethality, male hypogonadism, a high incidence of epilepsy and many vacuoles in the hippocampus and amygdala. We used a candidate approach to identify the gene responsible for the lde phenotype and assessed the susceptibility of lde/lde rats for audiogenic seizures. Following backcross breeding of lethal dwarfism with epilepsy (LDE) to Brown Norway rats, the lde/lde rats with an altered genetic background showed all pleiotropic phenotypes. The lde locus was mapped to a 1.5‐Mbp region on rat chromosome 19 that included the latter half of the Wwox gene. Sequencing of the full‐length Wwox transcript identified a 13‐bp deletion in exon 9 in lde/lde rats. This mutation causes a frame shift, resulting in aberrant amino acid sequences at the C‐terminal. Western blotting showed that both the full‐length products of the Wwox gene and its isoform were present in normal testes and hippocampi, whereas both products were undetectable in the testes and hippocampi of lde/lde rats. Sound stimulation induced epileptic seizures in 95% of lde/lde rats, with starting as wild running (WR), sometimes progressing to tonic–clonic convulsions. Electroencephalogram (EEG) analysis showed interictal spikes, fast waves during WR and burst of spikes during clonic phases. The Wwox protein is expressed in the central nervous system (CNS), indicating that abnormal neuronal excitability in lde/lde rats may be because of a lack of Wwox function. The lde/lde rat is not only useful for understanding the multiple functions of Wwox but is also a unique model for studying the physiological function of Wwox in CNS.

A familial spontaneous epileptic feline strain: A novel model of idiopathic/genetic epilepsy

A spontaneous epileptic model of cats has not been described previously. Recently, we identified familial epileptic cats and investigated their clinical features. These epileptic cats are healthy except for the presence of recurrent seizures that are typically a focal limbic seizure with secondary generalization. Furthermore, generalized seizures were induced by vestibular stimulation in some cats. This spontaneous epileptic cat strain may be a valuable model for idiopathic/genetic epilepsy.

A role for the Golgi matrix protein giantin in ciliogenesis through control of the localization of dynein-2

Summary The correct formation of primary cilia is central to the development and function of nearly all cells and tissues. Cilia grow from the mother centriole by extension of a microtubule core, the axoneme, which is then surrounded with a specialized ciliary membrane that is continuous with the plasma membrane. Intraflagellar transport moves particles along the length of the axoneme to direct assembly of the cilium and is also required for proper cilia function. The microtubule motor, cytoplasmic dynein-2 mediates retrograde transport along the axoneme from the tip to the base; dynein-2 is also required for some aspects of cilia formation. In most cells, the Golgi lies adjacent to the centrioles and key components of the cilia machinery localize to this organelle. Golgi-localized proteins have also been implicated in ciliogenesis and in intraflagellar transport. Here, we show that the transmembrane Golgi matrix protein giantin (GOLGB1) is required for ciliogenesis. We show that giantin is not required for the Rab11–Rabin8–Rab8 pathway that has been implicated in the early stages of ciliary membrane formation. Instead we find that suppression of giantin results in mis-localization of WDR34, the intermediate chain of dynein-2. Highly effective depletion of giantin or WDR34 leads to an inability of cells to form primary cilia. Partial depletion of giantin or of WDR34 leads to an increase in cilia length consistent with the concept that giantin acts through dynein-2. Our data implicate giantin in ciliogenesis through control of dynein-2 localization.

Dysplastic Development of Seminiferous Tubules and Interstitial Tissue in Rat Hypogonadic (hgn/hgn) Testes

Abstract The hypogonadic rat is characterized by male sterility, reduced female fertility, and renal hypoplasia controlled by a single recessive allele (hgn) on chromosome 10. Plasma testosterone is low and levels of gonadotropins are high in adult male hgn/hgn rats, indicating that the cause of hypogonadism lies within the testis itself. We found that the postnatal growth of the seminiferous tubules was severely affected. Here we describe the details of postnatal testicular pathogenesis of the hgn/ hgn rats. In these rats, gonadal sex determination and initial differentiation of each type of testicular cell occur, but proliferation, differentiation, and maturation of these cells during postnatal testicular development is severely affected. Postnatal pathological changes include reduced proliferation and apoptotic cell death of Sertoli cells, abnormal mitosis and cell death of gonocytes, reduced deposition of extracellular matrix proteins into the basal lamina, lack of the formation of an outer basal lamina, formation of multiple layers of undifferentiated peritubular cells, and the delayed appearance and islet conformation of adult-type Leydig cells. Apoptotic cell death of Sertoli cells and disappearance of FSH receptor mRNA expression indicate that this mutant rat is a useful model for Sertoli cell dysfunction. The abnormalities listed above might be caused by defective interactions between Sertoli cells and other types of testicular cells. Because the results presented here strongly indicate that a normal allele for hgn encodes a factor playing a critical role in testicular development, the determination of the gene responsible for hgn and the analysis of early alterations of gene expression caused by mutations in this gene would provide important information on the mechanisms of testicular development.

Insertional mutation in the Golgb1 gene is associated with osteochondrodysplasia and systemic edema in the OCD rat

Homozygous rats (ocd/ocd) of a mutant inbred strain, OCD (osteochondrodysplasia), show osteochondrodysplasia, systemic edema, cleft palate, protruding tongue, disproportionate dwarfism, and lethality immediately after birth. Their epiphyses show decreased levels of glycosaminoglycans and weak staining for extracellular matrix proteins. The epiphyseal chondrocytes have large vesicles and expanded endoplasmic reticulum and Golgi apparatus. These phenotypic features are inherited in an autosomal recessive manner, and the ocd locus responsible for these phenotypes has been mapped close to D11Mgh3 on rat chromosome 11. In the present study, we characterized the embryonic pathogenesis of ocd/ocd rats and identified the mutant gene. Subcutaneous edema in the dorsal portion was found at embryonic day (E) 16.5, and the other anomalies described above were apparent after E18.5 in ocd/ocd. Whole mount immunohistochemistry for Sox9 revealed that mesenchymal condensation was delayed in limb bud in ocd/ocd, and skeletal preparation showed that the progression of whole-body chondrogenesis was delayed in ocd/ocd. Histological and immunohistological analyses of the femur showed that cell proliferations of resting and proliferative zones of growth plate were significantly reduced in ocd/ocd embryos. Fine linkage mapping localized the ocd locus within 84kb of positions 65,584-65,668kb containing a part of Golgb1 gene on chromosome 11. Expression of Golgb1 mRNA was found in limb buds, somite derivatives and calvaria. Sequence analysis identified a 10-bp insertion in exon 13 of the Golgb1 gene in ocd/ocd rats. The Golgb1 gene encodes the COPI vesicle tethering factor, giantin. This insertion mutation causes a frame shift, and introduces a premature termination codon at codon 1082, leading to truncation of the C-terminal two thirds of giantin. By in-gel Western analysis using anti-giantin antibody that recognizes an epitope within 200 aa of the C-terminus, the expression of giantin was not detected in ocd/ocd embryos. As the C-terminal region of giantin is required for localization to the Golgi apparatus, these results strongly suggested that giantin is functionally defective in ocd/ocd rats. Therefore, we concluded that mutation of the Golgb1 gene is responsible for the phenotypic characteristics including osteochondrodysplasia of ocd/ocd, and that giantin plays a pivotal role in multiple aspects of chondrogenesis.

A locus responsible for hypogonadism (hgn) is located on rat Chromosome 10.

Hypogonadic rats of the HGN inbred strain show male sterility controlled by an autosomal single recessive allele (hgn). The testis weight of adult hgn/hgn rats is about 1% that of normal rats, and only a few thin seminiferous tubules surrounded by islet-like conformations of Leydig cells are present in the fibrous interstitial tissue of the testis (Suzuki et al. 1988). Primordial germ cells do exist in the neonatal hgn/hgn testis, but cannot differentiate into spermatogonia and degenerate before entering meiosis (Suzuki et al, 1998). hgn/hgn females have reduced fertility, including a small litter size, a reduced number of oocytes, and early reproductive senescence (Suzuki et al. 1992). hgn/hgn rats of both sexes also show bilateral hypoplastic kidney (HPK) with a reduced number (only one-quarter) of glomeruli (Suzuki et al. 1991; Suzuki and Suzuki 1995). Whether the phenotypes of hypogonadism and HPK are caused by a mutation in a single gene or defects in different genes located close to each other remains unclear. No other mutant animal or human disease showing a phenotype similar to that of the hgn/hgn rat has been reported. The hgn/hgn rat would, therefore, be a useful model for investigating the development of mammalian reproductive and urinary organs. In this study, linkage analysis was performed to determine the chromosome location of the hgn locus and to obtain information about candidate genes for hgn. Thirteen hgn/hgn females were selected from the population of the HGN inbred strain by laparotomy for detection of HPK at weaning (Suzuki et al. 1991). One hgn/hgn female was mated with a BN male rat (+/+), and the resulting F1 males were mated with the remaining 12 hgn/hgn females to produce backcross progeny. After the mating, the hgn/hgn females were autopsied to confirm the hgn/hgn phenotype (Suzuki et al. 1992). All male backcross progeny were autopsied at the age of 5 days to avoid postnatal loss, and their phenotypes were determined by macroscopic observation of testicular size. Relative testis weight of the affected rats (0.49 ± 0.06g, average ± S.D.) was about half that of normal rats (1.16 ± 0.09g). The histological appearance of the testes in the affected rats was similar to that of hgn/hgn rats of the HGN strain (Suzuki et al. 1993). Since it was difficult to clearly categorize females of the backcross progeny into affected and normal phenotypes by comparison of renal size, we did not use female rats for linkage analysis. Consequently, 29 affected and 26 normal males were obtained. The segregation ratio of the affected to normal rats did not deviate significantly from the expected 1 : 1 ratio (x 4 0.16, P > 0.20). Because of physical limitations of the PCR machine with a block of 48 samples, high-molecular-weight genomic DNA was isolated from livers of 24 affected and 24 normal rats by phenol extraction. The samples of remaining seven progeny were saved for the next mapping experiment. The total of 48 progeny were initially typed for 33 microsatellite markers on rat chromosomes. Primers for the microsatellite markers were purchased from Research Genetics Inc. (Huntsville, Ala.). The PCR was carried out in 10 m1 of a reaction mixture containing 25 ng of the genomic DNA, 1.0 pmol of each of the oligonucleotide primers, 2.0 nmol of each dNTP, and 0.26 U of Taq polymerase (TaKaRa) in the reaction buffer recommended by the manufacturer. After initial denaturation for 180 s at 92°C, 35 cycles of amplification, each consisting of denaturation for 15 s at 92°C, annealing 60 s at 55°C, and extension for 120 s at 72°C, were carried out with a Thermal Cycler TP2000 (TaKaRa). The PCR products were separated by nondenatured 10% polyacrylamide gel electrophoresis and visualized by silver staining (Silver Stain Plus Kit, Bio-Rad). The recombination fractions and the lod scores among the hgn and the other loci were calculated with Map Manager ver. 2.6.5 (distributed via the World Wide Web, URL: http://mcbio.med.buffalo.edu/mapmagr.html). The order of loci was determined so that the number of recombination events was minimized. By typing the 33 rat microsatellite marker loci in the backcross progeny, significant linkage was observed between the hgn locus and the D10Mgh10 locus on rat Chromosome (Chr) 10 with a lod score (z) of 5.1 and a recombination fraction (u) of 0.167. The D10Mgh3 locus also was linked to the hgn locus (z42.7, u40.25). No significant linkage was observed between the hgn locus and the other 31 loci. Therefore, the 48 progeny were further typed for the following seven loci located on rat Chr 10: D10Mit2, D10Mgh6, D10Mgh8 (Myh3), D10Wox14 (Asgr1), D10Wox6, D10Wox16 (Ngfr), and D10Wox12 (Abp). The highest lod score (z 4 14.4) was obtained for the D10Mit2 locus with a u value of 0. Segregation of alleles of the nine microsatellite loci as well as the hgn locus in the 48 progeny (Fig. 1) and a linkage map of rat Chr 10 with those loci (Fig. 2) are shown. The order and distances of the nine loci on the linkage map are basically identical with linkage maps reported previously (Jacob et al. 1995; Bihoreau et al. 1997). We therefore concluded that the hgn locus is located in the region close to the D10Mit2 locus on rat Chr 10. The Abp gene (Shbg in mouse) encodes androgen binding proteins expressed in Sertoli cells (Sallivan et al. 1991), which are apparently affected in the testis of hgn/hgn rats, suggesting that the gene is a candidate for hgh. As shown in Fig. 1, the presence of recombinations between the hgn and Abp loci, however, ruled out the Abp gene as a candidate. As shown in Fig. 2, the order of the functional genes (Myh3/Myhse, Abp/Shbg, Asgr1, and Ngfr) on rat Chr 10 is identical to that of the corresponding region of mouse Chr 11. Thus, the mouse gene corresponding to the hgn locus might be located in the region between map positions 37 (Asgr1) and 56 (Ngfr) of mouse Chr 11 (derived from the Mouse Genome Database; MGD). Possible candidate genes for the hgn mutation, including the Tex4, Fert2, Ube2b-rs2, Gsg2, and Lhx1 genes, have been mapped to this region. The Ube2b gene, which is implicated in postreplication repair, is located on either Chr 13 (Ube2b-rs1) or Chr 11 (Ube2brs2). Inactivation of the Ube2b gene of the mouse causes male sterility associated with chromatin modification, although homoCorrespondence to: H. Suzuki Mammalian Genome 10, 1106–1107 (1999).

Reduced Fertility in Female Homozygotes for hgn (Male Hypogonadism) Selected by hgn‐Associated Hypoplastic Kidney

The male hypogonadism rat (hgn/hgn) shows a characteristic male sterility as a single autosomal recessive trait. Recently, the female homozygotes for hgn, assumed to be fertile, could be detected by a /jgji‐associated hypoplastic kidney (hpk/hpk). The present study was to investigate a possible influence of the hgn gene on female reproduction. The hgn/hgn females showed a significant growth retardation as compared with the phenotypically normal ones (+/?; +/hgn or +/+). The litter size at birth and number of implantation traces were significantly less in the hgn/hgn than in the +/? females. The hgn/hgn females became anestrous and infertile much earlier than the +/? did. Histologically, there were a few corpora lutea, some atretic follicles at different stages of maturation and abundant abnormal interstitial cells with pyknotic or karyorexic nuclei in the ovaries of hgn/hgn females that have been infertile. The birth rate expressed by per cent litter size at birth against number of implantation traces was comparable between the hgn/hgn and the +/? female, suggesting that the small litter size of hgn/hgn female could not be due to the embryonic death in utero. Nevertheless, the number of the tubal ova at estrous was comparable in the hgn/hgn and +/? females. Therefore, it was suggested that the half of ova or embryos may be lost during the period from the fertilization to the implantation. Histological appearances of the neonatal ovary in the hgn/hgn seemed hypoplastic. The number of cells including oocytes and interstitial cells, enzymatically separated from neonatal ovary, was significantly less in the hgn/hgn than in the +/hgn. These results suggest that the gene product(s) coded by normal allele of hgn gene(s) involves normal gonadal development in both sexes; the defect may lead testicular dysmorphology in the male and reduced fertility in the female.

Genetic Analysis and Histology of Hypoplastic Kidneys in the Male Hypogonadic Mutant (hgn/hgn) Rat

Abstract Bilateral hypoplastic kidneys have been found to associate with the male hypogonadism rat (hgn/hgn). The hypoplastic kidney was persistent to the male homozygote for hgn and it was also seen in some females with unknown genotype. The affected kidney was apparently smaller in size than phenotypically normal one. When females with hypoplastic kidney were mated to proven heterozygous males for hypogonadism (hgn/+), the ratio of the affected testis to phenotypically normal one was 32: 20 in the Fl generation. This segregation ratio did not deviate siginificantly from the 1: 1 ratio expected from the hypothesis that the genotype of females with hypoplastic kidney in parent generation for hypogonadism would consist of hgn/hgn alone. The ratio expected from the other hypotheses was denied statistically. The ratio of the affected kidney to phenotypically normal one in both sexs of the Fi was 53: 50, fitting to 1: 1 hypothesis for a single autosomal recessive trait. There was neither a case showing the phenotypically normal kidney with the affected testis, nor the hypoplastic kidney with the phenotypically normal testis. The results suggest that the gene responsible for the hypoplastic kidney and the gene for hypogonadism would be identical or both genes reside in the close vicinity in a chromosome. The results also suggest that the homozygotes for hgn can be selected by the presence of hypoplastic kidney.

A locus responsible for osteochondrodysplasia (ocd) is located on rat chromosome 11.

Rats of the congenital osteochondrodysplasia (OCD) strain display lethal dwarfing characteristics that are controlled by an autosomal single recessive allele ( ocd). External features of these ocd/ocdrats include systemic subcutaneous edema, protruded tongue, cleft plate, and shortening of the extremities, head, body, and tail (Suzuki et al. 1987, 1988). These ocd/ocd rats also exhibit renal anomalies (Kikukawa et al. 1989a) and die shortly after birth from respiratory insufficiency, resulting from both morphological and functional abnormalities of the respiratory system (Kikukawa et al. 1989b, 1989c). Irregular columnization, expansion of chondrocytes, widespread areas of necrosis, and weak stainability of glycosaminoglycans (GAGs) are observed in the epiphyseal cartilage of ocd/ocd neonates (Kikukawa et al. 1991a). Electron microscopic examination of the cartilage reveals a decrease in the size of GAG granules and the presence of large collagen bundles in the extracellular matrix (ECM) (Kikukawa et al. 1990). The total amounts of GAG, hyaluronic acid and chondroitin sulfate in the ECM are reduced (Kikukawa et al. 1991b). It has been suggested that these abnormalities of the epiphyseal cartilage may be caused by defects in the process that releases ECM components from chondrocytes (Kikukawa and Suzuki 1992). No other mutant rat or mouse has been reported to have the same phenotype as that of the ocd/ocd rat. In this study, we performed linkage analysis in order to determine the position of the ocd locus. In order to analyze the linkage between the ocd locus and microsatellite marker loci, we mated heterozygote (+/ ocd) females of the OCD strain with Brown Norway (BN/Crj) male rats (+/+). Among the resulting progeny (the first filial generation: F 1), two male rats identified as +/ ocd were mated with F 1 females. The resulting F2 progeny were extracted by cesarean section on day 21 of gestation to minimize postnatal losses. Their phenotypes were determined by their external features. Consequently, 14 affected (ocd/ocd) and 55 phenotypically normal progeny were obtained from 8 +/ocd F1 mothers. The segregation ratio of the affected: normal fetuses did not deviate significantly from the expected 1:3 (x 4 0.82,P > 0.20). High-molecular-weight genomic DNA was isolated from the livers of the 14 affected rats by phenol extraction. The rats were then typed for 33 microsatellite markers known to occur on rat chromosomes. Primers for the microsatellite markers were purchased from Research Genetics Inc. (Huntsville, Ala.). Polymerase chain reactions (PCRs) were done as previously described (H. Suzuki et al. 1999). The PCR products were separated by non-denatured 10% polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The recombination fractions and the lod scores among the ocd and the other loci were calculated with Map Manager ver. 2.6.5 (Map manager 1999). The order of the loci was determined in a way that minimized the number of recombination events. By typing of the 33 rat microsatellite marker loci in the F 2 progeny, significant linkage was observed between the ocd locus and five loci on rat Chromosome (Chr) 11. No significant linkage was observed between the ocd locus and the other 28 loci. The highest lod score (8.4) was obtained for the D11Mgh3locus with a recombination fraction of 0. Segregation of the alleles of the five microsatellite loci as well as the ocd locus in the 14 neonates (Fig. 1A) and a linkage map of rat Chr 11 with these loci (Fig. 1B) are shown. The order of and distances between the five loci on the linkage map are basically identical with those on linkage maps released from the Rat Genome Database (RGD 1999). We therefore concluded that the ocd locus was located in the region close to the D11Mgh3 locus on rat Chr 11. TheD11Mgh3 locus is located in the region between SmstandMox2 in rat Chr 11 (RGD 1999). Therefore, the mouse gene corresponding to the cd locus may be located in the region between map positions 19.0 ( Smst ) and 29.0 ( Mox2) of mouse Chr 16 (MGD 1999). At present, however, we cannot specify possible candidates for ocd in this region. In order to check whether we could determine the genotype for theocd locus by the presence of allelism of the D11Mgh3locus in the OCD strain, we isolated genomic DNA from the livers of rats of this strain. We typed the rats for the D11Mgh3locus and were then able to confirm the presence of two allelic genes at the Correspondence to: H Suzuki; E-mail: hiroetsu@ea.mbn.or.jp Fig. 1. (A) Segregation of 5 loci on rat Chr 11 in 14 [(HGN × BN)F1 × F1] intercross progeny. The clear boxes represent the homozygous OCD haplotype, and the black boxes represent the heterozygous F 1 haplotype. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. (B) A partial linkage map of rat Chr 11, showing the location of ocd in relation to the linked loci. Mammalian Genome 11, 464–465 (2000).

Genotyping of exercise-induced collapse in Labrador retrievers using an allele-specific PCR

Exercise-induced collapse (EIC) is an autosomal recessive disorder in Labrador retrievers. In this study, an allele-specific PCR was developed to detect the point mutation G767T in exon 6 of canine DNM1, previously shown to be responsible for canine EIC. Of 133 Labrador retrievers tested in Japan, 6 (4.5%) were homozygous (EIC) and 50 (37.6%) were heterozygous (carriers) for the G767T mutation.