Chikako Inoue

Immunofluorescence analysis of neutrophil nonmuscle myosin heavy chain-A in MYH9 disorders: association of subcellular localization with MYH9 mutations.

The autosomal dominant macrothrombocytopenia with leukocyte inclusions, May-Hegglin anomaly, Sebastian syndrome, and Fechtner syndrome, are rare human disorders characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like cytoplasmic inclusions in granulocytes. Epstein syndrome is another autosomal dominant macrothrombocytopenia associated with Alport syndrome but without leukocyte inclusions. These disorders are caused by mutations in the same gene, the MYH9, which encodes the nonmuscle myosin heavy chain-A (NMMHCA). The term, MYH9 disorders, has been proposed, but the clinicopathologic basis of MYH9 mutations has been poorly investigated. In this study, a total of 24 cases with MYH9 disorders and suspected cases were subjected to immunofluorescence analysis by a polyclonal antibody against human platelet NMMHCA. Abnormal subcellular localization of NMMHCA was observed in every neutrophil from individuals with MYH9 mutations. Comparison with May-Grünwald-Giemsa staining revealed that the NMMHCA always coexisted with the neutrophil inclusion bodies, suggesting that NMMHCA is associated with such bodies. In three cases, neutrophil inclusions were not detected on conventional May-Grünwald-Giemsa-stained blood smears but immunofluorescence analysis revealed the abnormal NMMHCA localization. In contrast, cases with Epstein syndrome and the isolated macrothrombocytopenia with normal NMMHCA localization had no MYH9 mutations. An antibody that recognizes the C-terminal 12 mer peptides showed similar immunoreactivity from the patients heterozygous for truncated mutations that abolished the C-terminal epitope, suggesting that normal NMMHCA dimerizes with abnormal NMMHCA to form inclusion bodies. We further propose that the localization pattern can be classified into three groups according to the number, size, and shape of the fluorescence-labeled NMMHCA granule. Immunofluorescence analysis of neutrophil NMMHCA is useful as a screening test for the clear hematopathologic classification of MYH9 disorders.

The Tomita collection of medaka pigmentation mutants as a resource for understanding neural crest cell development

All body pigment cells in vertebrates are derived from the neural crest. In fish the neural crest can generate up to six different types of pigment cells, as well as various non-pigmented derivatives. In mouse and zebrafish, extensive collections of pigmentation mutants have enabled dissection of many aspects of pigment cell development, including fate specification, survival, proliferation and differentiation. A collection of spontaneous mutations collected from wild medaka (Oryzias latipes) populations and maintained at Nagoya University includes more than 40 pigmentation mutations. The descriptions of their adult phenotypes have been previously published by Tomita and colleagues (summarised in Medaka (Killifish) Biology and Strains, 1975), but the embryonic phenotypes have not been systematically described. Here we examine these embryonic phenotypes, paying particular attention to the likely defect in pigment cell development in each, and comparing the spectrum of defects to those in the zebrafish and mouse collections. Many phenotypes parallel those of identified zebrafish mutants, although pigment cell death phenotypes are largely absent, presumably due to the different selective pressures under which the mutants were isolated. We have identified mutant phenotypes that may represent the Mitf/Kit pathway of melanophore specification and survival. We use in situ hybridisation with available markers to confirm a key prediction of this hypothesis. We also highlight a set of novel phenotypes not seen in the zebrafish collection. These mutants will be a valuable resource for pigment cell and neural crest studies and will strongly complement the mutant collections in other vertebrates.

Kidney regeneration through nephron neogenesis in medaka

Although renal regeneration is limited to repair of the proximal tubule in mammals, some bony fish are capable of renal regeneration through nephron neogenesis in the event of renal injury. We previously reported that nephron development in the medaka mesonephros is characterized by four histologically distinct stages, generally referred to as condensed mesenchyme, nephrogenic body, relatively small nephron, and the mature nephron. Developing nephrons are positive for wt1 expression during the first three of these stages. In the present study, we examined the regenerative response to renal injury, artificially induced by the administration of sublethal amounts of gentamicin in adult medaka. Similar to previous reports in other animals, the renal tubular epithelium and the glomerulus of the medaka kidney exhibited severe damage after exposure to this agent. However, kidneys showed substantial recovery after gentamicin administration, and a significant number of developing nephrons appeared 14 days after gentamicin administration (P < 0.01). Similarly, the expression of wt1 in developing nephrons also indicated the early stages of nephrogenesis. These findings show that medaka has the ability to regenerate kidney through nephron neogenesis during adulthood and that wt1 is a suitable marker for detecting nephrogenesis.

Polycystic Kidney Disease in the Medaka (Oryzias latipes) pc Mutant Caused by a Mutation in the Gli-Similar3 (glis3) Gene

Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients.

CONSTITUTIVE GENE EXPRESSION OF INTERLEUKIN‐5 IN KIMURA'S DISEASE

We have investigated IL-5 mRNA expression in a resected lymph node of Kimuras disease. The patient was a 54-year-old man who had been working as a driver

Sox5 functions as a fate switch in medaka pigment cell development.

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor).

Patchy haemopoiesis in long-term remission of idiopathic aplastic anaemia

ABSTRACT: 13 patients with idiopathic aplastic anaemia in remission for more than 2 years were examined to define the haemopoietic status by means of bone marrow scintigraphy, ferrokinetics and bone marrow culture for haemopoietic progenitor cells. Haemoglobin levels reached the normal range in all these patients although mild neutropenia and thrombocytopenia were still observed in 5 patients. Bone marrow scintigrams using indium‐111 showed normal distribution in 2, diffuse low accumulation in 3, patchy distribution in 7, and expanded distribution with patchy uptake in 1 patient. The defective haemopoiesis was also confirmed by ferrokinetic and bone marrow culture studies. The patchy haemopoiesis appears to characterize the residual marrow damage in remission of idiopathic aplastic anaemia.

Activated Charcoal Diminishes the Lot Difference of Fetal Bovine Sera in Erythroid Colony Formation of Human Bone Marrow Cells

Abstract Using normal bone marrow as target cells, we assayed the colony-forming efficiency of early and late erythroid progenitor cells and granulocyte-macrophage progenitor cells using several different lots of fetal bovine serum (FBS). There was a marked difference in the ability of these sera to support colony formation, particularly in erythroid colony assays. When adsorbed by activated charcoal, all these sera supported erythroid colony formation more efficiently than before adsorption. There was no significant effect of charcoal adsorption of FBS on granulocyte-macrophage colony formation. Gel-filtration study showed that charcoal adsorption diminished low-molecular-weight fractions by less than 5000 Da. The inhibitory activity of this fraction was heat labile and Pronase sensitive. Concentrated samples obtained from these fractions inhibited erythroid colony formation in a dose-dependent manner. These results suggest that low-molecular-weight inhibitors that are relatively specific to erythropoiesis play a critical role in the lot differences of FBS for erythroid colony formation.

Successful Treatment of Hemosiderosis with Regular Phlebotomy and Recombinant Human Erythropoietin

A 55-year-old female patient with hemosiderosis induced by administration of excessive doses of parenteral iron was successfully treated with regular phlebotomy combined with recombinant human erythropoietin (rHuEPO). Ferrokinetic data before therapy showed 28.0 mumol/l of serum iron, 4.1 mumol/l of unsaturated iron-binding capacity, 4,060 ng/ml of serum ferritin, 148 min of plasma iron disappearance time, 45% of red cell iron utilization and 0.4 mg/kg/day of plasma iron turnover rate. She had 300-ml phlebotomies, first every other week then weekly, and subcutaneous injections of rHuEPO twice a week. Two years later, the total volume of phlebotomized blood reached 31 liters and her ferrokinetic data showed: serum iron 8.6 mumol/l, iron-binding capacity 39.6 mumol/l, serum ferritin 277 ng/ml, plasma iron disappearance time 52 min, red cell iron utilization 100% and plasma iron turnover rate 0.5 mg/kg/day. During the phlebotomy therapy, her hemoglobin levels were maintained above 12 g/dl. No adverse effect due to rHuEPO occurred. These findings provide evidence for the efficacy of rHuEPO in multiple phlebotomy therapy for hemosiderosis and may open new avenues for its clinical application.

Medaka and zebrafish contactin1 mutants as a model for understanding neural circuits for motor coordination

A spontaneous medaka ro mutant shows abnormal wobbling and rolling swimming behaviors. By positional cloning, we mapped the ro locus to a region containing the gene encoding Contactin1b (Cntn1b), which is an immunoglobulin (Ig)‐superfamily domain‐containing membrane‐anchored protein. The ro mutant had a deletion in the cntn1b gene that introduced a premature stop codon. Furthermore, cntn1b mutants generated by the CRISPR/Cas9 system and trans‐heterozygotes of the CRISPR mutant allele and ro had abnormal swimming behavior, indicating that the cntn1b gene was responsible for the ro‐mutant phenotype. We also established zebrafish cntn1a and cntn1b mutants by transcription activator‐like effector nucleases (TALENs). Zebrafish cntn1b but not cntn1a mutants showed abnormal swimming behaviors similar to those in the ro mutant, suggesting that Cntn1b plays a conserved role in the formation or function of the neural circuits that control swimming in teleosts. Although Cntn1‐deficient mice have abnormal cerebellar neural circuitry, there was no apparent histological abnormality in the cerebellum of medaka or zebrafish cntn1b mutants. The medaka cntn1b mutants had defective optokinetic response (OKR) adaptation and abnormal rheotaxis (body positioning relative to water flow). Medaka and zebrafish cntn1b mutants are effective models for studying the neural circuits involved in motor learning and motor coordination.