Kentaro Katayama

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 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.

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 Mutation in the Nuclear Pore Complex Gene Tmem48 Causes Gametogenesis Defects in Skeletal Fusions with Sterility (sks) Mice

Background: sks is a mouse mutant showing sterility caused by defects in meiosis. Results: We found a mutation of the Tmem48 gene encoding nuclear pore complex protein. The mutation causes aberrant splicing, resulting in deletion of an exon. Conclusion: Tmem48 is essential for meiosis and gametogenesis. Significance: This is the first report to demonstrate that the nuclear pore complex has an important role in mammalian gametogenesis. Skeletal fusions with sterility (sks) is an autosomal recessive mutation of mouse that results in male and female sterility because of defects in gametogenesis. The mutants also have skeletal malformations with fused vertebrae and ribs. We examined testicular phenotypes of sks/sks mice to investigate the defects in spermatogenesis. Histological and immunocytochemical analyses and expression analyses of the marker genes demonstrated that spermatogenesis is arrested at mid to late pachytene stage of meiotic prophase with defective synapsis of the homologous chromosomes. Next, we determined the precise chromosomal localization of the sks locus on a 0.3-Mb region of mouse chromosome 4 by linkage analysis. By sequencing the positional candidate genes in this region and whole exome sequencing, we found a GG to TT nucleotide substitution in exon 6 of the Tmem48 gene that encodes a putative transmembrane protein with six transmembrane domains. The nucleotide substitution causes aberrant splicing, which deletes exon 6 of the Tmem48 transcript. Specific expression of TMEM48 was observed in germ cells of males and females. Furthermore, the phenotypes of the sks mutant were completely rescued by the transgenesis of a genomic fragment containing the wild-type Tmem48 gene. These findings indicate that the Tmem48 mutation is responsible for the gametogenesis defects and skeletal malformations in the sks mice. The TMEM48 protein is a nuclear membrane protein comprising the nuclear pore complex; its exact function in the nuclear pore complex is still unknown. Our finding suggested that the nuclear pore complex plays an important role in mammalian gametogenesis and skeletal development.

Characterization of chromosomal inversion of the mouse hairy ears ( Eh ) mutation associated with cleft palate

The hairy ears (Eh) mutation in the mouse originated from neutron irradiation experiments and is associated with chromosomal inversion on chromosome 15. Eh/+ mice have small pinna and extra hairs on the pinna but the phenotypic features of Eh/Eh mice are unclear. In this study we found that Eh/Eh mice died shortly after birth and had a cleft palate caused by impaired growth of palate shelves. Because genes located on the breakpoints of inversion are likely to be responsible for the defects associated with chromosomal inversions, we determined the breakpoints of the Eh inversion. We used a new genetic method that uses recombinant chromosomes resulting from crossing over between two overlapping inversions to determine the breakpoints. Koa is a mouse mutation associated with inversion of chromosome 15, which partially overlaps with the Eh inversion. We made Eh +/+ Koa double heterozygotes and obtained the recombinant chromosomes possessing deletion and duplication of the regions flanked by the breakpoints of both inversions, which were generated by crossing over within the overlapped region of these inversions. By defining the deleted regions we identified the breakpoints of the Eh inversion. We then examined the expression of genes in the vicinities of the breakpoints and found ectopic expression of the Hoxc5 gene and a transcript with unknown function in the developing palate of Eh/Eh mice, which is likely to be responsible for the cleft palate.

Characterization of the chromosomal inversion associated with the Koa mutation in the mouse revealed the cause of skeletal abnormalities

BackgroundKoala (Koa) is a dominant mutation in mice causing bushy muzzle and pinna, and is associated with a chromosomal inversion on the distal half of chromosome 15. To identify the gene responsible for the Koa phenotypes, we investigated phenotypes of Koa homozygous mice and determined the breakpoints of the inversion with a genetic method using recombination between two different chromosomal inversions.ResultsSkeletal preparation of Koa homozygotes showed marked deformity of the ribs and a wider skull with extended zygomatic arches, in addition to a general reduction in the lengths of long bones. They also had open eyelids at birth caused by a defect in the extension of eyelid anlagen during the embryonic stages. The proximal and distal breakpoints of the Koa inversion were determined to be 0.8-Mb distal to the Trsps1 gene and to 0.1-Mb distal to the Hoxc4 gene, respectively, as previously reported. The phenotypes of mice with the recombinant inverted chromosomes revealed the localization of the gene responsible the Koa phenotype in the vicinity of the proximal recombinant breakpoint. Expression of the Trsps1 gene in this region was significantly reduced in the Koa homozygous and heterozygous embryos.ConclusionWhile no gene was disrupted by the chromosomal inversion, an association between the Koa phenotype and the proximal recombinant breakpoint, phenotypic similarities with Trps1-deficient mice or human patients with TRSP1 mutations, and the reduced expression of the Trsps1 gene in Koa mice, indicated that the phenotypes of the Koa mice are caused by the altered expression of the Trps1 gene.

Progression of renal fibrosis in congenital CKD model rats with reduced number of nephrons

A congenital reduction in the number of nephrons is a critical risk factor for both onset of chronic kidney disease (CKD) and its progression to end-stage kidney disease (ESKD). Hypoplastic kidney (HPK) rats have only about 20% of the normal number of nephrons and show progressive CKD. This study used an immunohistological method to assess glomerular and interstitial pathogenesis in male HPK rats aged 35-210days. CD68 positive-macrophages were found to infiltrate into glomeruli in HPK rats aged 35 and 70days and to infiltrate into interstitial tissue in rats aged 140 and 210days. HPK rats aged 35 and 70days showed glomerular hypertrophy, loss of normal linear immunostaining of podocine, and increased expression of PDGFr-β, TGF-β, collagens, and fibronectin, with all of these alterations gradually deteriorating with age. α-SMA-positive myofibroblasts were rarely detected in glomerular tufts, whereas α-SMA-positive glomerular parietal epithelium (GPE) cells were frequently observed along Bowmans capsular walls. The numbers of PDGFr-β-positive fibroblasts in interstitial tissue were increased in rats aged 35days and older, whereas interstitial fibrosis, characterized by the increased expression of tubular PDGF-BB, the appearance of myofibroblasts doubly positive for PDGFr-β and α-SMA, and increased expression of collagens and fibronectin, were observed in rats aged 70 and older. These results clearly indicate that congenital CKD with only 20% of nephrons cause renal fibrosis in rats.

Heparanase Localization during Palatogenesis in Mice

Palatogenesis is directed by epithelial-mesenchymal interactions and results partly from remodeling of the extracellular matrix (ECM) of the palatal shelves. Here, we assessed heparanase distribution in developing mouse palates. No heparanase was observed in the vertically oriented palatal shelves in early stages of palate formation. As palate formation progressed, the palatal shelves were reorganized and arranged horizontally above the tongue, and heparanase localized to the epithelial cells of these shelves. When the palatal bilateral shelves first made contact, the heparanase localized to epithelial cells at the tips of shelves. Later in fusing palatal shelves, the cells of the medial epithelial seam (MES) were labeled with intense heparanase signal. In contrast, the basement membrane heparan sulfate (HS) was scarcely observed in the palatal shelves in contact. Moreover, perlecan labeling was sparse in the basement membrane of the MES, on which laminin and type IV collagen were observed. Moreover, we assessed the distribution of matrix metalloproteinase- (MMP-) 9, MMP-2, and MMP-3 in developing mouse palates and these MMPs were observed in the MES. Our findings indicated that heparanase was important for palate formation because it mediated degradation of the ECM of palatal shelves. Heparanase may, in concert with other proteases, participate in the regression of the MES.

Giantin is required for coordinated production of aggrecan, link protein and type XI collagen during chondrogenesis

Extracellular matrix (ECM) constitutes a proper micro-environment for cell proliferation, migration and differentiation, as well as playing pivotal roles in developmental processes including endochondral ossification. Cartilage ECM is mainly composed of fibrous proteins, including collagen, proteoglycan, and hyaluronan. Because almost all ECM components are transported by intracellular vesicular transport systems, molecules that mediate vesicle transport are also important for endochondral ossification. Giantin, encoded by the Golgb1 gene, is a tethering factor for coatomer 1 (COPI) vesicles and functions in the cis-medial Golgi compartments. An insertion mutation in the Golgb1 gene, resulting in a lack of giantin protein expression, has been detected in ocd/ocd rats that exhibit a pleiotropic phenotype including osteochondrodysplasia. To reveal the function of giantin in chondrogenesis, the present study assessed the effects of loss of giantin expression on cartilage ECM and Golgi morphology. Giantin was expressed in normal, but not in ocd/ocd, chondrocytes in the epiphyseal areas of embryonic femurs, whereas GM130 was expressed in both normal and ocd/ocd chondrocytes. The staining intensities of safranin O and azan (aniline blue) were reduced and enhanced, respectively, in epiphyseal cartilage of ocd/ocd femurs. Immunostaining showed that levels of type II collagen and fibronectin were comparable in normal and ocd/ocd cartilage. Levels of type XI collagen were higher, while levels of aggrecan, link protein and hyaluronan were lower, in ocd/ocd than in normal cartilage, although semi-quantitative RT-PCR showed similar levels of type XI collagen, aggrecan and link protein mRNAs in normal and ocd/ocd cartilage. Isolated chondrocytes of ocd/ocd and normal rats showed similar immunostaining patterns for cis-, medial-, and trans-Golgi marker proteins, whereas monolayers of ocd/ocd chondrocytes showed reduced levels of aggrecan and link protein and increased level of type XI collagen in spite of similar transcripts levels. These findings suggest that giantin plays a pivotal role in coordinated production of aggrecan, link protein and type XI collagen in chondrocytes, and that loss of giantin causes osteochondrodysplasia with disturbance of these ECM components.

Homeobox family Hoxc localization during murine palate formation

Homeobox genes play important roles in craniofacial morphogenesis. However, the characteristics of the transcription factor Hoxc during palate formation remain unclear. We examined the immunolocalization patterns of Hoxc5, Hoxc4, and Hoxc6 in palatogenesis of cleft palate (Eh/Eh) mice. On the other hand, mutations in the FGF/FGFR pathway are exclusively associated with syndromic forms of cleft palate. We also examined the immunolocalization of Fgfr1 and Erk1/2 to clarify their relationships with Hoxc in palatogenesis. Some palatal epithelial cells showed Hoxc5 labeling, while almost no labeling of mesenchymal cells was observed in +/+ mice. As palate formation progressed in +/+ mice, Hoxc5, Hoxc4, and Hoxc6 were observed in medial epithelial seam cells. Hoxc5 and Hoxc6 were detected in the oral epithelium. The palatal mesenchyme also showed intense staining for Fgfr1 and Erk1/2 with progression of palate formation. In contrast, the palatal shelves of Eh/Eh mice exhibited impaired horizontal growth and failed to fuse, resulting in a cleft. Hoxc5 was observed in a few epithelial cells and diffusely in the mesenchyme of Eh/Eh palatal shelves. No or little labeling of Fgfr1 and Erk1/2 was detected in the cleft palate of Eh/Eh mice. These findings suggest that Hoxc genes are involved in palatogenesis. Furthermore, there may be the differences in the localization pattern between Hoxc5, Hoxc4, and Hoxc6. Additionally, Hoxc distribution in palatal cells during palate development may be correlated with FGF signaling. (228/250 words)