Hiroaki Yamanaka

Pigment Pattern Formation by Contact-Dependent Depolarization

Cell culture experiments reveal that direct interactions between pigment cells play a key role in skin pattern formation. Although recent experimental studies have suggested that the interactions among the pigment cells play a key role in the skin pattern formation, details of the mechanism remain largely unknown. By using an in vitro cell culture system, we have detected interactions between the two pigment cell types, melanophores and xanthophores, in the zebrafish skin. During primary culture, the melanophore membrane transiently depolarizes when contacted with the dendrites of a xanthophore. This depolarization triggers melanophore migration to avoid further contact with the xanthophores. Cell depolarization and repulsive movement were not observed in pigment cells with the jaguar mutant, which shows defective segregation of melanophores and xanthophores. The depolarization-repulsion of wild-type pigment cells may explain the pigment cell behaviors generating the stripe pattern of zebrafish.

Increased expression of the hedgehog signaling pathway in pediatric solid malignancies.

PURPOSEnThe activation of the hedgehog (Hh) signaling is involved in the progression of various cancers. However, the correlation between the Hh signaling and tumorigenesis of pediatric malignancies has not been well documented. The present study was undertaken to examine the expression of the Hh signaling pathway in various pediatric tumors to elucidate the role of Hh signaling in pediatric malignancies.nnnMETHODSnSurgical specimens were obtained from 68 patients with pediatric malignancies (neuroblastoma, 25; rhabdomyosarcoma, 18; hepatic tumor, 12; and renal tumor, 13). The expression of sonic hedgehog (Shh), its receptor Patched (Ptch), and downstream transcription factor Gli1 was evaluated using immunohistochemical staining.nnnRESULTSnIn neuroblastoma, 96%, 100%, and 68%; in rhabdomyosarcoma, 78%, 100%, and 78%; in Wilms tumor, 71%, 100%, and 43%; and in hepatoblastoma, 100%, 100%, and 73% of the specimens stained positive for Shh, Ptch, and Gli1, respectively. Differentiated neuroblastoma cells showed more intense Gli1 expression than in immature neuroblastoma cells. In rhabdomyosarcoma, the expression of Gli1 was higher in alveolar type than in embryonal type.nnnCONCLUSIONSnThese findings suggest that the Shh-Ptch1-Gli1 signaling pathways are frequently activated in most pediatric malignant tumors. The Hh signaling pathway may therefore play an important role in the differentiation and malignant potential of pediatric malignancies.

In vitro analysis suggests that difference in cell movement during direct interaction can generate various pigment patterns in vivo

Significance The periodic pigment patterns of organisms, such as spots and stripes, have suggested the presence of a mathematical system generating the patterns. The most famous one is the reaction–diffusion system. Although the system explains the pattern formation well, the biological substances corresponding to the diffusive factors in the system have not been elucidated. Here, we observed the behavior of zebrafish pigment cells in vitro and found that WT cells showed run-and-chase movement in the direct interaction, whereas the pattern mutant cells showed different cell movement. These results suggested that gene mutations affect the reaction–diffusion system by changing the cell movement, which corresponds to the interaction of diffusive factors in the reaction–diffusion system. Pigment patterns of organisms have invoked strong interest from not only biologists but also, scientists in many other fields. Zebrafish is a useful model animal for studying the mechanism of pigment pattern formation. The zebrafish stripe pattern is primarily two types of pigment cells: melanophores and xanthophores. Previous studies have reported that interactions among these pigment cells are important for pattern formation. In the recent report, we found that the direct contact by xanthophores induces the membrane depolarization of melanophores. From analysis of jaguar mutants, it is suggested that the depolarization affects the movements of melanophores. To analyze the cell movement in detail, we established a unique in vitro system. It allowed us to find that WT xanthophores induced repulsive movement of melanophores through direct contact. The xanthophores also chased the melanophores. As a result, they showed run-and-chase movements. We also analyzed the cell movement of pigment cells from jaguar and leopard mutants, which have fuzzy stripes and spot patterns, respectively. jaguar cells showed inhibited run-and-chase movements, and leopard melanophores scarcely showed repulsive response. Furthermore, we paired mutant and WT cells and showed which of the melanophores and xanthophores have responsibility for the altered cell movements. These results suggested that there is a correspondence relationship between the cell movements and pigment patterns. The correspondence relationship highlighted the importance of the cell movements in the pattern formation and showed that our system is a quite useful system for future study in this field.

Intralesional sclerotherapy for subcutaneous venous malformations in children

BackgroundVenous malformations (VMs) involve multiple anatomical spaces and encase critical neuromuscular structures, making surgical treatment difficult. Recently sclerotherapy has been suggested as the primary treatment for VMs instead of surgical intervention. This report represents eight cases of children with VMs treated with direct percutaneous injections of sclerosing agents, such as ethanol, polidocanol or ethanolamine oleate.MethodsAll eight patients had large lesions (>3xa0cm) located on the head, foot, neck and face. Sclerotherapy was performed in an angiographic suite under general anesthesia. Prior to sclerotherapy, percutaneous phlebography was performed in order to visualize the dynamic situation inside the lesion and the draining flow into the adjacent venous vascular system. A 2–15xa0ml of sclerosing agent was injected into VM lesions under fluoroscopy.Results and conclusionsAn evaluation by MRI examination showed that 6 out of 8 patients had remission, and alleviation of their symptoms without major complications, furthermore one of the lesions apparently disappeared. Intralesional sclerotherapy provides a simple, safe and effective treatment for VMs in the subcutaneous lesions in children.

Increased expression of multidrug resistance-associated genes after chemotherapy in pediatric solid malignancies.

BACKGROUND/PURPOSEnThe overexpression of multidrug resistance (MDR)-associated genes in primary untreated tumors has been proven to be associated with worse prognosis in various pediatric malignancies. This study compared the expression of 3 MDR-associated genes, MDR1, MDR-associated protein 1 (MRP1), and lung resistance-related protein (LRP), in pediatric tumors before and after chemotherapy to elucidate the mechanism of MDR during chemotherapy.nnnMETHODSnSurgical specimens of both primary and chemotherapy-treated tumors were obtained from 24 patients with pediatric malignancies (neuroblastoma [NB] 8; hepatoblastoma [HB] 8; Wilms tumor [WT] 4; rhabdomyosarcoma [RB] 4). The expression of MDR1, MRP1, and LRP was evaluated using the immunohistochemical staining.nnnRESULTSnIn primary tumors, MDR1 expression was observed in 6 NBs, 8 HBs, 3 WTs, and 3 RBs. MRP1 expression was observed in 3 NBs and 1 HB. LRP expression was not detected in any of the primary tumors. After chemotherapy, MDR1 expression was observed to increase in 5 NBs, 4 HBs, 2 WTs, and 3 RBs. MRP1 expression was newly observed or increased in 7 NBs, 4 HBs, and 3 RBs. LRP expression was newly observed in 3 HBs and 2 WTs.nnnCONCLUSIONSnThese results indicate that these 3 MDR-associated genes were upregulated after chemotherapy in various pediatric malignancies. These findings may be useful to understand the mechanism of drug resistance in pediatric malignancies.

Focal intestinal perforation in extremely-low-birth-weight neonates: etiological consideration from histological findings.

In Japan, mortality due to intestinal perforation has been increasing for the last 15xa0years, as the survival rate of extremely-low-birth-weight neonates (ELBWs) has been increasing. In our NICU, although the incidence of necrotizing enterocolitis has been decreasing, that of focal intestinal perforation (FIP) has been increasing. To elucidate the pathogenesis of FIP, a histological study was performed. For the last 20xa0years, in our NICU, 11 ELBWs with FIP, including one with multiple perforations and impending rupture, underwent laparotomy and enterostomy. The intestinal wall around the perforation was resected for histological study. Histological studies revealed well-developed ganglion cells in all the cases. Although some lymphocytic infiltration and hemorrhage were observed, no apparent neutrophilic infiltration, or necrotic change was identified in any case. No evidence of microcirculatory impairment was identified. The intestinal musculature discontinued abruptly with thinning in three cases, and without thinning in the others. The histology of impending rupture showed an absence of the muscularis with preservation of the remaining components of the bowel wall. The findings that the punched-out perforations had the appearance of Hirschsprung’s disease, FIP exclusively occurred in ELBWs, and that neither mechanical obstruction nor necrotic change could be identified as the cause of the perforation suggest that immature bowel movement and congenital muscle defects may be involved in the pathogenesis of FIP.

Rotating pigment cells exhibit an intrinsic chirality

In multicellular organisms, cell properties, such as shape, size and function are important in morphogenesis and physiological functions. Recently, ‘cellular chirality’ has attracted attention as a cellular property because it can cause asymmetry in the bodies of animals. In recent in vitro studies, the left–right bias of cellular migration and of autonomous arrangement of cells under some specific culture conditions were discovered. However, it is difficult to identify the molecular mechanism underlying their intrinsic chirality because the left–right bias observed to date is subtle or is manifested in the stable orientation of cells. Here, we report that zebrafish (Danio rerio) melanophores exhibit clear cellular chirality by unidirectional counterclockwise rotational movement under isolated conditions without any special settings. The chirality is intrinsic to melanophores because the direction of the cellular rotation was not affected by the type of extracellular matrix. We further found that the cellular rotation was generated as a counter action of the clockwise movement of actin cytoskeleton. It suggested that the mechanism that directs actin cytoskeleton in the clockwise direction is pivotal for determining cellular chirality.

The role of WT1 gene in neuroblastoma

BACKGROUND/PURPOSEnThe oncogenic properties of the Wilms tumor gene (WT1) have recently been reported in various malignancies. However, the role of WT1 in pediatric tumors is unclear. To elucidate the role of WT1 in the development of neuroblastoma (NB), we examined the WT1 expression in NB and the effect of WT1 suppression on NB cell proliferation.nnnMETHODSnWe examined the expression of the WT1 protein in 20 NBs and 5 ganglioneuromas (GNs) by performing immunohistochemical analysis. We determined WT1 messenger RNA expression in 22 NBs, 5 GNs, and 4 NB cell lines by real-time reverse transcription polymerase chain reaction. We studied the effects of WT1 suppression on cell proliferation using small interfering RNA against WT1.nnnRESULTSnExpression of WT1 was higher in mature ganglionic cells, and in the immunohistochemical analysis, the WT1 positivity for GNs was significantly higher than that for NBs (P < .01). The level of WT1 messenger RNA expression did not correlate with histologic grade, clinical stage, and prognosis of the tumor. Knockdown of WT1 gene promoted the proliferation of NB69 cells (P < .01).nnnCONCLUSIONSnThe WT1 may govern cell differentiation and suppress cell proliferation in NB. The WT1 does not act as an oncogene, but it may participate in the maturation of NB.

Forskolin, a Hedgehog signal inhibitor, inhibits cell proliferation and induces apoptosis in pediatric tumor cell lines.

The Hedgehog (Hh) signaling pathway regulates the development of many organs in mammals. Recent studies have indicated that the activation of the Hh signaling pathway contributes to the growth of various adult cancers. However, little is known about its role in the development of pediatric malignancies. The present study was undertaken to examine the expression and functional involvement of Hh signal transcription factors in pediatric tumor cells in order to determine their potential as therapeutic targets. We utilized real-time RT-PCR to investigate the expression of Glioma-associated oncogene homolog 1 (Gli1) in various pediatric tumor cell lines, including rhabdomyosarcoma, neuroblastoma and hepatoblastoma. The mRNA expression of Gli1 was markedly increased in rhabdomyosarcoma (RMS-YM, RD, RH30) cell lines, and moderately increased in neuroblastoma (NB19) and hepatoblastoma (Huh6) cell lines. The proliferation of these cell lines was dose dependently inhibited by Forskolin, a specific Hh signal inhibitor. In addition, Forskolin-induced growth suppression was associated with the down-regulation of C-Myc. Moreover, the blockade of Hh signaling with Forskolin enhanced cell apoptosis in a dose-dependent manner. These results demonstrated that Hh signal activation frequently occurs in neuroblastoma, hepatoblastoma and rhabdomyosarcoma cell lines. The inhibition of Hh signaling suppressed proliferation and increased apoptosis in these tumor cells. These findings suggest that the Hh signaling pathway plays an important role in tumorigenesis and is a potential molecular target of new treatment strategies for these pediatric malignant tumors.

Expression of Wilms tumor 1 gene in a variety of pediatric tumors

BACKGROUND/PURPOSEnWilms tumor 1 (WT1) gene is overexpressed in many types of neoplasms, thus suggesting that WT1 has oncogenic properties. Therefore, WT1 is a molecular target for cancer therapy. The objectives of this study were to evaluate the WT1 gene expression in various pediatric tumors and to elucidate that WT1 can be a target of cancer therapy in pediatric malignancies.nnnPATIENTS AND METHODSnThe expression of WT1 protein was examined in 60 cases of primary pediatric tumors. The levels of WT1 messenger RNA (mRNA) expression were examined by a quantitative real-time reverse transcriptase polymerase chain reaction analysis in frozen tissue samples from 56 cases with pediatric tumors.nnnRESULTSnImmunohistochemical staining revealed that WT1 protein was widely detected in pediatric malignancies. The alveolar subtype of rhabdomyosarcoma showed more intensive staining than the embryonal subtype. The positive rate of the alveolar type was significantly higher than that of the embryonal type. The expression of WT1 mRNA in the tumor samples varied widely. However, no significant correlation was observed between WT1 mRNA expression and clinical factors.nnnCONCLUSIONnThe WT1 expression was broadly detected in various pediatric neoplasms. These results indicate that WT1 may therefore be a potentially useful therapeutic target in most of pediatric malignancies.