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Although right-sided colorectal cancer (CRC) shows a worse prognosis than left-sided CRC, the underlying mechanism remains unclear. We established patient-derived organoids (PDOs) from left- and right-sided CRCs and directly compared cell proliferation and invasion capability between them. We then analyzed the expression of numerous genes in signal transduction pathways to clarify the mechanism of the differential prognosis. Cell proliferation activity and invasion capability in right-sided cancer PDOs were significantly higher than in left-sided cancer PDOs and normal PDOs, as revealed by Cell Titer Glo and transwell assays, respectively. We then used quantitative RT-PCR to compare 184 genes in 30 pathways among right-sided and left-sided cancer and normal PDOs and found that the TIMP1 mRNA level was highest in right-sided PDOs. TIMP1 protein levels were upregulated in right-sided PDOs compared with normal PDOs but was downregulated in left-sided PDOs. TIMP1 knockdown with shRNA significantly decreased cell proliferation activity and invasion capability in right-sided PDOs but not in left-sided PDOs. Moreover, TIMP1 knockdown significantly decreased pFAK and pAkt expression levels in right-sided PDOs but not in left-sided PDOs. A database analysis of The Cancer Genome Atlas revealed that TIMP1 expression in right-sided CRCs was significantly higher than in left-sided CRCs. Kaplan-Meier survival analysis showed significantly shorter overall survival in high-TIMP1 patients versus low-TIMP1 patients with right-sided CRCs but not left-sided CRCs. Our data suggest that TIMP1 is overexpressed in right-sided CRCs and promotes cell proliferation and invasion capability through the TIMP1/FAK/Akt pathway, leading to a poor prognosis. The TIMP1/FAK/Akt pathway can be a target for therapeutic agents in right-sided CRCs.

Although many chemopreventive studies on colorectal tumors have been reported, no effective and safe preventive agent is currently available. We searched for candidate preventive compounds against colorectal tumor comprehensively from United States Food and Drug Administration (FDA)-approved compounds by using connectivity map (CMAP) analysis coupled with in vitro screening with colorectal adenoma (CRA) patient-derived organoids (PDOs). We generated CRA-specific gene signatures based on the DNA microarray analysis of CRA and normal epithelial specimens, applied them to CMAP analysis with 1309 FDA-approved compounds, and identified 121 candidate compounds that should cancel the gene signatures. We narrowed them down to 15 compounds, and evaluated their inhibitory effects on the growth of CRA-PDOs in vitro. We finally identified resveratrol, one of the polyphenolic phytochemicals, as a compound showing the strongest inhibitory effect on the growth of CRA-PDOs compared with normal epithelial PDOs. When resveratrol was administered to Apc

Intratumoral HER2 heterogeneity is a well-described gastric cancer feature and may explain many false-negative results related to this oncogene. An 81-year-old man was diagnosed at our hospital with stage IV gastric cancer with multiple lymph node metastases. Immunohistochemistry (IHC) analysis indicated that the primary tumor was HER2-negative. After a chemotherapy course, we submitted a pretreatment biopsy specimen for comprehensive cancer genome profiling (CGP) to determine the last-line therapy. This revealed HER2 amplification. The specimen was reevaluated using fluorescence in situ hybridization and IHC with deeper-cut specimens, which confirmed that the tumor was indeed HER2-positive. Therefore, the patient was treated with chemotherapy plus trastuzumab, which elicited tumor shrinkage and conferred long-term survival. Our current data underscore the CGP importance, which can provide more accurate tumor profilings and inform subsequent treatment decisions.

The vertebrate skeleton changes its shape during development through the activities of chondrocytes, osteoblasts and osteoclasts. Although much is known about the mechanisms for differentiation in these cells, it is less understood how they behave in a region-specific manner to acquire unique bone shapes. To address this question, we investigated the development of the hyomandibular (Hm) system in zebrafish. The Hm originates as cartilage carrying a single foramen (the Hm foramen), through which the facial (VII) nerve passes. We reveal that Schwann cells, which myelinate the VII nerve, regulate rearrangement of the chondrocytes to enlarge the Hm foramen. The Hm cartilage then becomes ossified in the perichondrium, where the marrow chondrocytes are replaced by adipocytes. Then, the bone matrix along the VII nerve is resorbed by osteoclasts, generating a gateway to the bone marrow. Subsequent movement of the VII nerve into the marrow, followed by deposition of new bone matrix, isolates the nerve from the jaw muscle insertion. Genetic ablation of osteoblasts and osteoclasts reveals specific roles of these cells during remodeling processes. Interestingly, the VII nerve relocation does not occur in medaka; instead, bone deposition distinct from those in zebrafish separates the VII nerve from the muscle insertion. Our results define novel mechanisms for skeletal remodeling, by which the bone shapes in a region- and species-specific manner.

Background The distribution of sensory organs is important for detecting environmental signals efficiently. The mechanosensory receptors of the lateral line system, neuromasts, are stereotypically distributed over the head and body surface of fish, although how neuromasts arise in these predetermined positions during development remains unclear. Results We investigated the development of the anterior lateral line (ALL) system in zebrafish head. The ALL neuromasts formed in the predetermined positions through proliferation and differentiation of (a) nonmigratory lateral line primordia, (b) migratory primordia, (c) interneuromast cells connecting preexisting neuromasts, and (d) budding primordia. We demonstrated that R-spondin2 (Rspo2), an activator of Wnt/β-catenin signaling, is required for the development of a particular set of neuromasts associated with hyomandibular cartilage. Further genetic analyses suggested that Rspo2, which emanates from the hyoid mesenchyme, acts on the adjacent neuromast progenitor cells to stimulate their proliferation through activating Wnt/β-catenin signaling. Conclusion This study has revealed novel mechanisms for neuromast positioning through local tissue-tissue interactions, providing insights into the development and evolution of the vertebrate head.

Although pancreatic cancer often invades peripancreatic adipose tissue, little information is known about cancer‐adipocyte interaction. We first investigated the ability of adipocytes to de‐differentiate to cancer‐associated adipocytes (CAAs) by co‐culturing with pancreatic cancer cells. We then examined the effects of CAA‐conditioned medium (CAA‐CM) on the malignant characteristics of cancer cells, the mechanism underlying those effects, and their clinical relevance in pancreatic cancer. When 3T3‐L1 adipocytes were co‐cultured with pancreatic cancer cells (PANC‐1) using the Transwell system, adipocytes lost their lipid droplets and changed morphologically to fibroblast‐like cells (CAA). Adipocyte‐specific marker mRNA levels significantly decreased but those of fibroblast‐specific markers appeared, characteristic findings of CAA, as revealed by real‐time PCR. When PANC‐1 cells were cultured with CAA‐CM, significantly higher migration/invasion capability, chemoresistance, and epithelial‐mesenchymal transition (EMT) properties were observed compared with control cells. To investigate the mechanism underlying these effects, we performed microarray analysis of PANC‐1 cells cultured with CAA‐CM and found a 78.5‐fold higher expression of SAA1 compared with control cells. When the SAA1 gene in PANC‐1 cells was knocked down with SAA1 siRNA, migration/invasion capability, chemoresistance, and EMT properties were significantly attenuated compared with control cells. Immunohistochemical analysis on human pancreatic cancer tissues revealed positive SAA1 expression in 46/61 (75.4%). Overall survival in the SAA1‐positive group was significantly shorter than in the SAA1‐negative group (P = .013). In conclusion, we demonstrated that pancreatic cancer cells induced de‐differentiation in adipocytes toward CAA, and that CAA promoted malignant characteristics of pancreatic cancer via SAA1 expression, suggesting that SAA1 is a novel therapeutic target in pancreatic cancer., Cancer‐associated adipocyte (CAA) promotes migration/invasion of pancreatic cancer cells. CAA also induced pancreatic cancer cells drug resistance, epithelial‐mesenchymal transition.

Bone is a connective tissue composed of many cell types, including osteoblasts. How bones acquire their unique size and shape during development remains poorly understood. Herein we investigated the molecular and cellular mechanisms of bone morphogenesis in the zebrafish scale by using transgenic lines to enable visualization of specific types of osteoblasts. We demonstrate that the zebrafish scale contains three distinct types of osteoblasts: (i) a monolayer of central osteoblasts along the inner surface of scales; (ii) marginal osteoblasts elongated along the scale edge; and (iii) submarginal osteoblasts located between the central and marginal osteoblast populations. The size of the central osteoblasts increases progressively during development, suggesting that scale growth is mediated primarily by cell growth rather than the recruitment of new osteoblasts. In addition, the total number of central osteoblasts increases in regenerated scales and is correlated with scale size, possibly allowing for the rapid growth of regenerating scales. Moreover, osteoblast proliferation is not detected during regeneration, suggesting that the osteoblasts originate from post-mitotic precursor cells. Sonic hedgehog a (shha) is expressed in the epidermal cells that make contact with the marginal osteoblasts. Pharmacological inhibition of Hedgehog (Hh) signaling during regeneration reduces the number of marginal osteoblasts and interferes with scale growth, indicating that epidermis-derived Shh regulates scale regeneration. Finally, genetic inhibition of Wnt/planar cell polarity (PCP) signaling in the epidermis results in misorientation of scales with regard to the body axis. These results reveal a novel role for the epidermis in the regulation of bone patterning, namely the regeneration of osteoblasts and directional bone growth.

The mechanism of resistance to sorafenib in hepatocellular carcinoma (HCC) remains unclear. We analyzed miRNA expression profiles in sorafenib-resistant HCC cell lines (PLC/PRF5-R1/R2) and parental cell lines (PLC/PRF5) to identify the miRNAs responsible for resistance. Drug sensitivity, migration/invasion capabilities, and epithelial-mesenchymal transition (EMT) properties were analyzed by biochemical methods. The clinical relevance of the target genes to survival in HCC patients were assessed using a public database. Four miRNAs were significantly upregulated in PLC/PRF5-R1/-R2 compared with PLC/PRF5. Among them, miR-125b-5p mimic-transfected PLC/PRF5 cells (PLC/PRF5-miR125b) and showed a significantly higher IC50 for sorafenib compared with controls, while the other miRNA mimics did not. PLC/PRF5-miR125b showed lower E-cadherin and higher Snail and vimentin expression—findings similar to those for PLC/PRF5-R2—which suggests the induction of EMT in those cells. PLC/PRF5-miR125b exhibited significantly higher migration and invasion capabilities and induced sorafenib resistance in an in vivo mouse model. Bioinformatic analysis revealed ataxin-1 as a target gene of miR-125b-5p. PLC/PRF5 cells transfected with ataxin-1 siRNA showed a significantly higher IC50, higher migration/invasion capability, higher cancer stem cell population, and an EMT phenotype. Median overall survival in the low-ataxin-1 patient group was significantly shorter than in the high-ataxin-1 group. In conclusion, miR-125b-5p suppressed ataxin-1 and consequently induced Snail-mediated EMT and stemness, leading to a poor prognosis in HCC patients., The mechanism of resistance to multikinase inhibitors in hepatocellular carcinoma (HCC) remains unclear. We analyzed miRNA expression profiles in sorafenib-resistant HCC cell lines (PLC/PRF5-R1/R2) and parental cell lines (PLC/PRF5) to identify the responsible miRNAs and target genes involved in the mechanism of resistance. Four miRNAs were significantly upregulated. Among them, we found that miR-125-5p induced sorafenib resistance in HCC cells and in a mouse model. We also revealed that miR-125-5p suppressed ataxin-1 as a target gene and consequently induced Snail-mediated epithelial-mesenchymal transition (EMT) and cancer stemness. Moreover, we demonstrated that ataxin-1 expression has an impact on the prognosis of patients with HCCs. In the future, by comparing the expression status of miR-125b-5p/ataxin-1 and the effect of sorafenib in the clinical setting, it is expected that miR-125b-5p will be established as an effective drug selection marker for treatment selection in patients with HCC.

Although domesticated goldfish strains exhibit highly diversified phenotypes in morphology, the genetic basis underlying these phenotypes is poorly understood. Here, based on analysis of transposable elements in the allotetraploid goldfish genome, we found that its two subgenomes have evolved asymmetrically since a whole-genome duplication event in the ancestor of goldfish and common carp. We conducted whole-genome sequencing of 27 domesticated goldfish strains and wild goldfish. We identified more than 60 million genetic variations and established a population genetic structure of major goldfish strains. Genome-wide association studies and analysis of strain-specific variants revealed genetic loci associated with several goldfish phenotypes, including dorsal fin loss, long-tail, telescope-eye, albinism, and heart-shaped tail. Our results suggest that accumulated mutations in the asymmetrically evolved subgenomes led to generation of diverse phenotypes in the goldfish domestication history. This study is a key resource for understanding the genetic basis of phenotypic diversity among goldfish strains.

For over a thousand years, the common goldfish (Carassius auratus) was raised throughout Asia for food and as an ornamental pet. As a very close relative of the common carp (Cyprinus carpio), goldfish share the recent genome duplication that occurred approximately 14 million years ago in their common ancestor. The combination of centuries of breeding and a wide array of interesting body morphologies provides an exciting opportunity to link genotype to phenotype and to understand the dynamics of genome evolution and speciation. We generated a high-quality draft sequence and gene annotations of a “Wakin” goldfish using 71X PacBio long reads. The two subgenomes in goldfish retained extensive synteny and collinearity between goldfish and zebrafish. However, genes were lost quickly after the carp whole-genome duplication, and the expression of 30% of the retained duplicated gene diverged substantially across seven tissues sampled. Loss of sequence identity and/or exons determined the divergence of the expression levels across all tissues, while loss of conserved noncoding elements determined expression variance between different tissues. This assembly provides an important resource for comparative genomics and understanding the causes of goldfish variants.

A genomic assembly of the goldfish (Carrassius auratus) increases the utility of goldfish in research and agriculture., For over a thousand years, the common goldfish (Carassius auratus) was raised throughout Asia for food and as an ornamental pet. As a very close relative of the common carp (Cyprinus carpio), goldfish share the recent genome duplication that occurred approximately 14 million years ago in their common ancestor. The combination of centuries of breeding and a wide array of interesting body morphologies provides an exciting opportunity to link genotype to phenotype and to understand the dynamics of genome evolution and speciation. We generated a high-quality draft sequence and gene annotations of a “Wakin” goldfish using 71X PacBio long reads. The two subgenomes in goldfish retained extensive synteny and collinearity between goldfish and zebrafish. However, genes were lost quickly after the carp whole-genome duplication, and the expression of 30% of the retained duplicated gene diverged substantially across seven tissues sampled. Loss of sequence identity and/or exons determined the divergence of the expression levels across all tissues, while loss of conserved noncoding elements determined expression variance between different tissues. This assembly provides an important resource for comparative genomics and understanding the causes of goldfish variants.

SummaryFor over a thousand years throughout Asia, the common goldfish (Carassius auratus) was raised for both food and as an ornamental pet. Selective breeding over more than 500 years has created a wide array of body and pigmentation variation particularly valued by ornamental fish enthusiasts. As a very close relative of the common carp (Cyprinus carpio), goldfish shares the recent genome duplication that occurred approximately 14-16 million years ago (mya) in their common ancestor. The combination of centuries of breeding and a wide array of interesting body morphologies is an exciting opportunity to link genotype to phenotype as well as understanding the dynamics of genome evolution and speciation. Here we generated a high-quality draft sequence of a “Wakin” goldfish using 71X PacBio long-reads. We identified 70,324 coding genes and more than 11,000 non-coding transcripts. We found that the two sub-genomes in goldfish retained extensive synteny and collinearity between goldfish and zebrafish. However, “ohnologous” genes were lost quickly after the carp whole-genome duplication, and the expression of 30% of the retained duplicated gene diverged significantly across seven tissues sampled. Loss of sequence identity and/or exons determined the divergence of the expression across all tissues, while loss of conserved, non-coding elements determined expression variance between different tissues. This draft assembly also provides an important resource for comparative genomics with the very commonly used zebrafish model (Danio rerio), and for understanding the underlying genetic causes of goldfish variants.

Background Fear conditioning is a form of learning essential for animal survival and used as a behavioral paradigm to study the mechanisms of learning and memory. In mammals, the amygdala plays a crucial role in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amygdala by anatomical and ablation studies, showing a role in conditioned avoidance response. However, the neuronal populations required for a conditioned avoidance response via the Dm have not been functionally or genetically defined. Results We aimed to identify the neuronal population essential for fear conditioning through a genetic approach in zebrafish. First, we performed large-scale gene trap and enhancer trap screens, and created transgenic fish lines that expressed Gal4FF, an engineered version of the Gal4 transcription activator, in specific regions in the brain. We then crossed these Gal4FF-expressing fish with the effector line carrying the botulinum neurotoxin gene downstream of the Gal4 binding sequence UAS, and analyzed the double transgenic fish for active avoidance fear conditioning. We identified 16 transgenic lines with Gal4FF expression in various brain areas showing reduced performance in avoidance responses. Two of them had Gal4 expression in populations of neurons located in subregions of the Dm, which we named 120A-Dm neurons. Inhibition of the 120A-Dm neurons also caused reduced performance in Pavlovian fear conditioning. The 120A-Dm neurons were mostly glutamatergic and had projections to other brain regions, including the hypothalamus and ventral telencephalon. Conclusions Herein, we identified a subpopulation of neurons in the zebrafish Dm essential for fear conditioning. We propose that these are functional equivalents of neurons in the mammalian pallial amygdala, mediating the conditioned stimulus–unconditioned stimulus association. Thus, the study establishes a basis for understanding the evolutionary conservation and diversification of functional neural circuits mediating fear conditioning in vertebrates. © Kawakami et al. 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/)

Retinoic acid (RA) signaling is important to normal development. However, the function of the different RA receptors (RARs)--RARα, RARβ, and RARγ--is as yet unclear. We have used wild-type and transgenic zebrafish to examine the role of RARγ. Treatment of zebrafish embryos with an RARγ-specific agonist reduced somite formation and axial length, which was associated with a loss of hoxb13a expression and less-clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist also disrupted formation of tissues arising from cranial neural crest, including cranial bones and anterior neural ganglia. There was a loss of Sox 9-immunopositive neural crest stem/progenitor cells in the same anterior regions. Pectoral fin outgrowth was blocked by RARγ agonist treatment. However, there was no loss of Tbx-5-immunopositive lateral plate mesodermal stem/progenitor cells and the block was reversed by agonist washout or by cotreatment with an RARγ antagonist. Regeneration of the caudal fin was also blocked by RARγ agonist treatment, which was associated with a loss of canonical Wnt signaling. This regenerative response was restored by agonist washout or cotreatment with the RARγ antagonist. These findings suggest that RARγ plays an essential role in maintaining stem/progenitor cells during embryonic development and tissue regeneration when the receptor is in its nonligated state.

A 78-year-old man with hypertension, nephrosclerosis, and angina pectoris visited his family doctor with a history of fatigue and leg edema. He had a history of percutaneous coronary intervention 5 years prior, and was taking low-dose aspirin. Blood tests revealed hypoalbuminemia, gastrointestinal

Reactivation of hepatitis B virus (HBV) has recently been reported as a fatal complication in patients undergoing cytotoxic chemotherapy. We herein describe a case of reactivation in a 76-year-old man who had undergone pelvic exenteration for colorectal cancer (CRC). He was treated with a modified FOLFOX6 chemotherapy regimen after the operation. Thirteen months later, his laboratory data showed severe liver dysfunction. His hepatitis B surface antigen (HBsAg) test was positive, and his HBV-DNA level was elevated. We diagnosed the patient with HBV reactivation as his HBsAg test was negative before starting chemotherapy. His liver dysfunction improved after administration of entecavir. This is the first report describing HBV reactivation following chemotherapy for an HBsAg-negative CRC patient.

SummaryCorrect organ size must involve a balance between promotion and inhibition of cell proliferation. A mathematical model has been proposed in which an organ is assumed to produce its own growth activator as well as a growth inhibitor [1], but there is as yet no molecular evidence to support this model [2]. The mechanosensory organs of the fish lateral line system (neuromasts) are composed of a core of sensory hair cells surrounded by nonsensory support cells. Sensory cells are constantly replaced and are regenerated from surrounding nonsensory cells [3], while each organ retains the same size throughout life. Moreover, neuromasts also bud off new neuromasts, which stop growing when they reach the same size [4, 5]. Here, we show that the size of neuromasts is controlled by a balance between growth-promoting Wnt signaling activity in proliferation-competent cells and Wnt-inhibiting Dkk activity produced by differentiated sensory cells. This negative feedback loop from Dkk (secreted by differentiated cells) on Wnt-dependent cell proliferation (in surrounding cells) also acts during regeneration to achieve size constancy. This study establishes Wnt/Dkk as a novel mechanism to determine the final size of an organ.

Superficial mechanosensory organs (neuromasts) distributed over the head and body of fishes and amphibians form the “lateral line” system. During zebrafish adulthood, each neuromast of the body (posterior lateral line system, or PLL) produces “accessory” neuromasts that remain tightly clustered, thereby increasing the total number of PLL neuromasts by a factor of more than 10. This expansion is achieved by a budding process and is accompanied by branches of the afferent nerve that innervates the founder neuromast. Here we show that innervation is essential for the budding process, in complete contrast with the development of the embryonic PLL, where innervation is entirely dispensable. To obtain insight into the molecular mechanisms that underlie the budding process, we focused on the terminal system that develops at the posterior tip of the body and on the caudal fin. In this subset of PLL neuromasts, bud neuromasts form in a reproducible sequence over a few days, much faster than for other PLL neuromasts. We show that wingless/int (Wnt) signaling takes place during, and is required for, the budding process. We also show that the Wnt activator R-spondin is expressed by the axons that innervate budding neuromasts. We propose that the axon triggers Wnt signaling, which itself is involved in the proliferative phase that leads to bud formation. Finally, we show that innervation is required not only for budding, but also for long-term maintenance of all PLL neuromasts.

Background and aim The significance of examination time of esophagogastroduodenoscopy (EGD) for asymptomatic examinees is yet to be established. We aimed to clarify whether endoscopists who allot more examination time can detect higher numbers of neoplastic lesions among asymptomatic examinees. Methods We reviewed a database of consecutive examinees who underwent EGD in our hospital from April 2010 to September 2015. Staff endoscopists were classified into fast, moderate, and slow groups based on the mean examination time of EGD without a biopsy. The neoplastic lesion detection rate among these groups was compared using multiple logistic regression. Results Of the 55,786 consecutive examinees who underwent EGD, 15,763 asymptomatic examinees who were screened by staff doctors were analyzed. The mean examination time of 13,661 EGDs without biopsy was 6.2 min (range, 2–18 min). When cutoffs of 5 and 7 min were used, 4 endoscopists were classified into the fast (mean duration, 4.4 ± 1.0 min), 12 into the moderate (6.1 ± 1.4 min), and 4 into the slow groups (7.8 ± 1.9 min). The neoplastic lesion detection rates in the fast, moderate, and slow groups were 0.57% (13/2,288), 0.97% (99/10,180), and 0.94% (31/3,295), respectively. Compared with that in the fast group, the odds ratios for the neoplastic lesion detection rate in the moderate and slow groups were 1.90 (95% confidence interval [CI], 1.06–3.40) and 1.89 (95% CI, 0.98–3.64), respectively. Conclusion Endoscopists who do not allot adequate examination time may overlook neoplastic lesions in the upper gastrointestinal tract. This article is protected by copyright. All rights reserved.

Targeted expression by the Gal4-UAS system is a powerful genetic method to analyze the functions of genes and cells in vivo. Although the Gal4-UAS system has been extensively used in genetic studies in Drosophila, it had not been applied to genetic studies in vertebrates until the mid-2000s. This was mainly due to the lack of an efficient transgenesis tool in model vertebrates, such as the P-transposable element of Drosophila, that can create hundreds or thousands of transgene insertions in different loci on the genome and thereby enables the generation of transgenic lines expressing Gal4 in various tissues and cells via enhancer trapping. This situation was revolutionized when a highly efficient transgenesis method using the Tol2 transposable element was developed in the model vertebrate zebrafish. By using the Tol2 transposon system, we and other labs successfully performed gene trap and enhancer trap screens in combination with the Gal4-UAS system. To date, numerous transgenic fish lines that express engineered versions of Gal4 in specific cells, organs, and tissues have been generated and used for various aspects of biological studies. By constructing transgenic fish lines harboring genes of interest downstream of UAS, the Gal4-expressing cells and tissues in those transgenic fish have been visualized and manipulated via the Gal4-UAS system. In this review, we describe how the Gal4-UAS system works in zebrafish and how transgenic zebrafish that express Gal4 in specific cells, tissues, and organs have been used for the study of developmental biology, organogenesis, and neuroscience.

The lateral line system displays highly divergent patterns in adult teleost fish. The mechanisms underlying this variability are poorly understood. Here, we demonstrate that the lateral line mechanoreceptor, the neuromast, gives rise to a series of accessory neuromasts by a serial budding process during postembryonic development in zebrafish. We also show that accessory neuromast formation is highly correlated to the development of underlying dermal structures such as bones and scales. Abnormalities in opercular bone morphogenesis, in endothelin 1-knockdown embryos, are accompanied by stereotypic errors in neuromast budding and positioning, further demonstrating the tight correlation between the patterning of neuromasts and of the underlying dermal bones. In medaka, where scales form between peridermis and opercular bones, the lateral line displays a scale-specific pattern which is never observed in zebrafish. These results strongly suggest a control of postembryonic neuromast patterns by underlying dermal structures. This dermal control may explain some aspects of the evolution of lateral line patterns.

Planar cell polarity (PCP) in epithelial cells is essential for the organization of tissues and their functions. The conserved non-canonical Wnt/PCP pathway regulates this process in both Drosophila and vertebrates. However, recent studies have revealed that a similar set of genes, which may not be related to PCP, regulates oriented cell movement during development. In the present review, recent findings on neural migration in zebrafish hindbrain and axonal guidance in mouse forebrain and spinal cord are discussed. Future analyses on defects in vertebrate PCP mutants will provide novel insights into the conserved and diverse roles of non-canonical Wnt/PCP pathway genes in vertebrate brain development.

During vertebrate gastrulation, the body axis is established by coordinated and directional movements of cells that include epiboly, involution, and convergence and extension (C&E). Recent work implicates a non-canonical Wnt/planar cell polarity (PCP) pathway in the regulation of C&E. The Drosophila atypical cadherin Flamingo (Fmi) and its vertebrate homologue Celsr, a 7-pass transmembrane protein with extracellular cadherin repeats, regulate several biological processes, including C&E, cochlear cell orientation, axonal pathfinding and neuronal migration. Fmi/Celsr can function together with molecules involved in PCP, such as Frizzled (Fz) and Dishevelled (Dsh), but there is also some evidence that it may act as a cell adhesion molecule in a PCP-pathway-independent manner. We show that abrogation of Celsr activity in zebrafish embryos results in epiboly defects that appear to be independent of the requirement for Celsr in PCP signalling during C&E. Using a C-terminal truncated form of Celsr that inhibits membrane presentation of wild-type Celsr through its putative pro-region, a hanging drop assay reveals that cells from embryos with compromised Celsr activity have different cohesive properties from wild-type cells. It is disruption of this ability of Celsr to affect cell cohesion that primarily leads to the in vivo epiboly defects. In addition, Lyn-Celsr, in which the intracellular domain of Celsr is fused to a membrane localisation signal (Lyn), inhibits Fz-Dsh complex formation during Wnt/PCP signalling without affecting epiboly. Fmi/Celsr therefore has a dual role in mediating two separate morphogenetic movements through its roles in mediating cell cohesion and Wnt/PCP signalling during zebrafish gastrulation.

In mammals, the blockade of the phototransduction cascade causes loss of vision and, in some cases, degeneration of photoreceptors. However, the molecular mechanisms that link phototransduction with photoreceptor degeneration remain to be elucidated. Here, we report that a mutation in the gene encoding a central effector of the phototransduction cascade, cGMP phosphodiesterase 6alpha'-subunit (PDE6alpha'), affects not only the vision but also the survival of cone photoreceptors in zebrafish. We isolated a zebrafish mutant, called eclipse (els), which shows no visual behavior such as optokinetic response (OKR). The cloning of the els mutant gene revealed that a missense mutation occurred in the pde6alpha' gene, resulting in a change in a conserved amino acid. The PDE6 expressed in rod photoreceptors is a heterotetramer comprising two closely related similar hydrolytic alpha and beta subunits and two identical inhibitory gamma subunits, while the PDE6 expressed in cone photoreceptors consists of two homodimers of alpha' subunits, each with gamma subunits. The els mutant displays no visual response to bright light, where cones are active, but shows relatively normal OKR to dim light, where only rods function, suggesting that only the cone-specific phototransduction pathway is disrupted in the els mutant. Furthermore, in the els mutant, cones are selectively eliminated but rods are retained at the adult stage, suggesting that cones undergo a progressive degeneration in the els mutant retinas. Taken together, these data suggest that PDE6alpha' activity is important for the survival of cones in zebrafish.

In zebrafish embryos, the axons of the posterior trigeminal (Vp) and facial(VII) motoneurons project stereotypically to a small number of target muscles derived from the first and second branchial arches (BA1, BA2). Use of the Islet1 (Isl1)-GFP transgenic line enabled precise real-time observations of the growth cone behaviour of the Vp and VII motoneurons within BA1 and BA2. Screening for N-ethyl-N-nitrosourea-induced mutants identified seven distinct mutations affecting different steps in the axonal pathfinding of these motoneurons. The class 1 mutations caused severe defasciculation and abnormal pathfinding in both Vp and VII motor axons before they reached their target muscles in BA1. The class 2 mutations caused impaired axonal outgrowth of the Vp motoneurons at the BA1-BA2 boundary. The class 3 mutation caused impaired axonal outgrowth of the Vp motoneurons within the target muscles derived from BA1 and BA2. The class 4 mutation caused retraction of the Vp motor axons in BA1 and abnormal invasion of the VII motor axons in BA1 beyond the BA1-BA2 boundary. Time-lapse observations of the class 1 mutant, vermicelli (vmc), which has a defect in the plexin A3 (plxna3) gene, revealed that Plxna3 acts with its ligand Sema3a1 for fasciculation and correct target selection of the Vp and VII motor axons after separation from the common pathways shared with the sensory axons in BA1 and BA2, and for the proper exit and outgrowth of the axons of the primary motoneurons from the spinal cord.

In the developing vertebrate hindbrain, the characteristic trajectory of the facial (nVII) motor nerve is generated by caudal migration of the nVII motor neurons. The nVII motor neurons originate in rhombomere (r) 4, and migrate caudally into r6 to form the facial motor nucleus. In this study,using a transgenic zebrafish line that expresses green fluorescent protein(GFP) in the cranial motor neurons, we isolated two novel mutants, designated landlocked (llk) and off-road (ord), which both show highly specific defects in the caudal migration of the nVII motor neurons. We show that the landlocked locus contains the gene scribble1 (scrb1), and that its zygotic expression is required for migration of the nVII motor neurons mainly in a non cell-autonomous manner. Taking advantage of the viability of the llkmutant embryos, we found that maternal expression of scrb1 is required for convergent extension (CE) movements during gastrulation. Furthermore, we show a genetic interaction between scrb1 and trilobite(tri)/strabismus(stbm) in CE. The dual roles of the scrb1 gene in both neuronal migration and CE provide a novel insight into the underlying mechanisms of cell movement in vertebrate development.

Slit is a secreted protein known to repulse the growth cones of commissural neurons. By contrast, Slit also promotes elongation and branching of axons of sensory neurons. The reason why different neurons respond to Slit in different ways is largely unknown. Islet2 is a LIM/homeodomain-type transcription factor that specifically regulates elongation and branching of the peripheral axons of the primary sensory neurons in zebrafish embryos. We found that PlexinA4, a transmembrane protein known to be a co-receptor for class III semaphorins,acts downstream of Islet2 to promote branching of the peripheral axons of the primary sensory neurons. Intriguingly, repression of PlexinA4 function by injection of the antisense morpholino oligonucleotide specific to PlexinA4 or by overexpression of the dominant-negative variant of PlexinA4 counteracted the effects of overexpression of Slit2 to induce branching of the peripheral axons of the primary sensory neurons in zebrafish embryos, suggesting involvement of PlexinA4 in the Slit signaling cascades for promotion of axonal branching of the sensory neurons. Colocalized expression of Robo, a receptor for Slit2, and PlexinA4 is observed not only in the primary sensory neurons of zebrafish embryos but also in the dendrites of the pyramidal neurons of the cortex of the mammals, and may be important for promoting the branching of either axons or dendrites in response to Slit, as opposed to the growth cone collapse.

Many genes that play essential roles in organ growth have been identified across a range of organisms. However, the mechanisms by which growing organs can sense their sizes and stop growing when they reach their proper sizes remain poorly understood. The mechanosensory organs of the fish lateral line system (neuromasts) provide an ideal system to address this question for the following reasons. First, each superficial neuromast is composed of a small number of cells situated on the body surface, making it relatively easy to quantify organ size throughout development. Second, while the sensory cells of superficial neuromasts are continuously renewed, overall organ size is homeostatically maintained. Third, there is another type of neuromast showing an opposite mode of growth: that is, canal neuromasts increase in size in proportion to organism body size. Here, we review recent findings regarding the mechanisms that control organ size in the zebrafish lateral line.

The lateral line system of teleost fishes presents large variations of patterns and forms, usually thought of as adaptive. This raises the question of how divergent adult patterns are achieved, and how selective pressures have contributed to this divergence. Our understanding of the development of this sensory system has much improved over the past 10 years, mostly through work on the zebrafish. Because this progress is restricted to a single species, we cannot yet answer questions about the determinism of lateral line evolution, but we can at least propose plausible and testable hypotheses. Here we review the mechanisms that mediate the transition from embryonic to adult pattern in the zebrafish posterior lateral line system (PLL), and we show that the adult pattern is largely determined by developmental events that take place during early larval life. We also show that simple variations in the use of the same mechanisms account for the very different patterns observed in juvenile zebrafish and blue-fin tuna, and could potentially account for many or all of the patterns observed in other adult teleosts. We conclude that, in the case of the lateral line at least, large variations in pattern depend on minor changes in the deployment of conserved developmental programs, with uncertain adaptive value. We propose that organisms neurally adapt to whatever tools they are provided with by their own development, and use them as best as they can, thereby giving the impression that such tools were actually selected for.

Double anal fin (Da) is a medaka with an autosomal semidominant mutation that causes mirror image duplication of the ventral region concentrating on the caudal region. The chromosomal location of the Da gene and its sequence have remained unknown. We constructed a medaka linkage map as a first step to approach positional cloning of the gene. The segregation analysis was performed on the basis of genetic recombination during female meiosis using 134 random amplified polymorphic DNA (RAPD) markers, 13 sequence-tagged sites (STSs), 15 polymorphic sequences from known genes, and the Da gene. One hundred forty-six markers from the above markers segregated into 26 linkage groups. The size of the genome was estimated to be 1776 cM in length. We identified four syntenic regions between medaka and zebrafish (and human) by mapping the known genes and found one of them to be located in close proximity to theDa gene. By mapping the region surrounding the Dagene in high resolution, two markers were detected flanking theDa gene at 0.32 and 0.80 cM. The detected markers providing a vital clue to initiate chromosome walking will lead us to the definite location of the Da gene.

The lf (leucophore free) locus was previously reported autosomal recessive in the medaka fish (Oryzias latipes). However, extensive linkage analyses in this study using various strains revealed that the lf locus was closely sex-linked. The recombination frequency between lf and the male determining factor (y) was 2.2% (10 recombinants out of 464 progeny). Because the lf/lf homozygous fish do not have visible leucophores, they are distinguishable from wild type in early developmental stages. In the Qurt strain with heterozygous sex chromosomes (X(lf)/X(lf) in females and X(lf)/Y(+) in males), we can predict sex of each embryo on second day after fertilization. The strain should be a very useful material for studying sex determination or differentiation mechanisms in the medaka fish.

Background We have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene trapping, enhancer trapping and the Gal4FF-UAS system. Gene trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and enhancer trap constructs contain the zebrafish hsp70l promoter and the GFP or Gal4FF gene. By performing genetic screens using these constructs, we have generated transgenic zebrafish that express GFP and Gal4FF in specific cells, tissues and organs. Gal4FF expression is visualized by creating double transgenic fish carrying a Gal4FF transgene and the GFP reporter gene placed downstream of the Gal4-recognition sequence (UAS). Further, the Gal4FF-expressing cells can be manipulated by mating with UAS effector fish. For instance, when fish expressing Gal4FF in specific neurons are crossed with the UAS:TeTxLC fish carrying the tetanus neurotoxin gene downstream of UAS, the neuronal activities are inhibited in the double transgenic fish. Thus, these transgenic fish are useful to study developmental biology and neurobiology. Description To increase the usefulness of the transgenic fish resource, we developed a web-based database named zTrap http://kawakami.lab.nig.ac.jp/ztrap/. The zTrap database contains images of GFP and Gal4FF expression patterns, and genomic DNA sequences surrounding the integration sites of the gene trap and enhancer trap constructs. The integration sites are mapped onto the Ensembl zebrafish genome by in-house Blat analysis and can be viewed on the zTrap and Ensembl genome browsers. Furthermore, zTrap is equipped with the functionality to search these data for expression patterns and genomic loci of interest. zTrap contains the information about transgenic fish including UAS reporter and effector fish. Conclusion zTrap is a useful resource to find gene trap and enhancer trap fish lines that express GFP and Gal4FF in desired patterns, and to find insertions of the gene trap and enhancer trap constructs that are located within or near genes of interest. These transgenic fish can be utilized to observe specific cell types during embryogenesis, to manipulate their functions, and to discover novel genes and cis-regulatory elements. Therefore, zTrap should facilitate studies on genomics, developmental biology and neurobiology utilizing the transgenic zebrafish resource.

The adoption of U.S.-style independent regulatory agencies (IRAs) such as the FCC and the SEC has become increasingly pervasive in advanced nations. Much recent political analysis of public regulation tends to regard this worldwide spread of the American model, or “regulatory state,” as a typical example of a policy diffusion mechanism, such as emulation or regulatory competition. After critically examining that view, this article develops an alternative framework that combines international structural forces, such as techno-economic changes and ideational factors, and domestic institutional factors, in particular, path dependence of historical institutions. The cross-national and cross-sectoral case analysis (the financial services and communications sector in Britain and Japan) demonstrates that while apparent convergence on the American model is due to international structural forces, national and sectoral differences with respect to the extent of organizational changes depend largely on domestic institutions. This finding suggests that, facing complex dynamism between international structural forces and domestic institutions, each national government chooses the feature of regulatory organization independently from other nation’s decisions, rather than interdependently as assumed in the policy diffusion approach.

The molecular mechanisms by which neurons migrate and accumulate to form the neural layers and nuclei remain unclear. The formation of vagus motor nuclei in zebrafish embryos is an ideal model system in which to address this issue because of the transparency of the embryos and the availability of established genetic and molecular biological techniques. To determine the genes required for the formation of the vagus motor nuclei, we performed N-ethyl-N-nitrosourea-based mutant screening using a zebrafish line that expresses green fluorescent protein in the motor neurons. In wild-type embryos, the vagus motor neuron progenitors are born in the ventral ventricular zone, then migrate tangentially in the dorsolateral direction, forming the nuclei. However, in towhead(twdrw685) mutant embryos, the vagus motor neuron progenitors stray medially away from the normal migratory pathway and fail to stop in the right location. The twdrw685 mutant has a defect in the GDP-mannose 4,6 dehydratase (gmds) gene, which encodes a key enzyme in the fucosylation pathway. Levels of fucosylated glycans were markedly and specifically reduced in twdrw685mutant embryos. Cell transplantation analysis revealed that GMDS is not essential in the vagus motor neuron progenitors for correct formation of the vagus motor nuclei, but is required in the neuroepithelial cells that surround the progenitors. Together, these findings suggest that fucosylated glycans expressed in neuroepithelial cells are required to guide the migration of vagus motor neuron progenitors.

Planar cell polarity (PCP) in epithelial cells is essential for the organization of tissues and their functions. The conserved non-canonical Wnt/PCP pathway regulates this process in both Drosophila and vertebrates. However, recent studies have revealed that a similar set of genes, which may not be related to PCP, regulates oriented cell movement during development. In the present review, recent findings on neural migration in zebrafish hindbrain and axonal guidance in mouse forebrain and spinal cord are discussed. Future analyses on defects in vertebrate PCP mutants will provide novel insights into the conserved and diverse roles of non-canonical Wnt/PCP pathway genes in vertebrate brain development.

Noncanonical Wnt/planar cell polarity (PCP) pathways regulate oriented cell movement during development in both Drosophila and vertebrates. Recent studies have revealed similarities and differences between these pathways in the tissues on which they act. In zebrafish, PCP pathway genes regulate the directional migration of a specific population of motor neurons in the hindbrain, as well as morphogenesis of the neuroepithelium. In the present review, neuronal and neuroepithelial defects in zebrafish PCP pathway mutants are compared, and the possible cellular and molecular mechanisms underlying these phenotypes are discussed. Future analyses of zebrafish PCP mutants will reveal the general mechanisms underlying the development of the neuroepithelium and provide novel insights into both conserved and diverse functions of PCP pathway genes in vertebrate development.

The lateral line is composed of mechanoreceptors, the neuromasts, which are distributed over the body surfaces of fish. We examine the development of neuromast patterns on the caudal fins of medaka and zebrafish. In medaka, the terminal neuromast is established just prior to the caudal fin formation. The terminal neuromast subsequently gives rise to a cluster of accessory neuromasts. In zebrafish, the terminal neuromasts vary in terms of both number and position, and they achieve their final positions relative to the caudal fin structures through migration. Subsequently, they give rise to four lines of accessory neuromasts that extend along the caudal fin. We show that developmental processes similar to those observed in medaka and zebrafish may account for a large variety of patterns in other teleost species. These results establish terminal neuromast patterning as a new model for the study of the developmental mechanisms underlying diverse lateral line patterns. Developmental Dynamics 237:2889–2902, 2008. © 2008 Wiley-Liss, Inc.

Migration of neurons from their birthplace to their final target area is a crucial step in brain development. Here, we show that expression of the off-limits/frizzled3a (olt/fz3a) and off-road/celsr2 (ord/celsr2) genes in neuroepithelial cells maintains the facial (nVII) motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. In the absence of olt/fz3a expression in the neuroepithelium, nVII motor neurons extended aberrant radial processes towards the ventricular surface and mismigrated radially to the dorsomedial part of the hindbrain. Our findings reveal a novel role for these genes, distinctive from their already known functions, in the regulation of the planar cell polarity (i.e. preventing integration of differentiated neurons into the neuroepithelial layer). This contrasts markedly with their reported role in reintegration of neuroepithelial daughter cells into the neuroepithelial layer after cell division.

The complex, yet highly ordered and predictable, structure of the neural retina is one of the most conserved features of the vertebrate central nervous system. In all vertebrate classes, retinal neurons are organized into laminae with each neuronal class adopting specific morphologies and patterns of connectivity. Using genetic analyses in zebrafish, we demonstrate that N-cadherin (Ncad) has several distinct and crucial functions during the establishment of retinal organization. Although the location of cell division is disorganized in embryos with reduced or no Ncad function, different classes of retinal neurons are generated. However, these neurons fail to organize into correct laminae, most probably owing to compromised adhesion between retinal cells. In addition, amacrine cells exhibit exuberant and misdirected outgrowth of neurites that contributes to severe disorganization of the inner plexiform layer. Retinal ganglion cells also exhibit defects in process outgrowth, with axons exhibiting fasciculation defects and adopting incorrect ipsilateral trajectories. At least some of these defects are likely to be due to a failure to maintain compartment boundaries between eye, optic nerve and brain. Although in vitro studies have implicated Fgf receptors in modulating the axon outgrowth promoting properties of Ncad, most aspects of the Ncad mutant phenotype are not phenocopied by treatments that block Fgf receptor function.

Outcome of Repeat ERCP After Biliary Cannulation Failure Following Needle Knife Sphincterotomy Hiroyuki Hisai*, Yutaka Okagawa, Hironori Wada, Yutaka Koshiba, Etsu Miyazaki Department of Gastroenterology, Japan Red Cross Date General Hospital, Date, Japan Background: Needle knife sphincterotomy (NKS) is often used to gain biliary access in failed standard cannulation techniques. If selective biliary cannulation is unsuccessful, the next step may include either percutaneous or EUS-guided biliary drainage. ERCP is sometimes repeatedly performed in clinically stable patients. However, there are few reports about the outcome. The aim of this study was to assess the outcome of repeat ERCP after failure with NKS for selective biliary access. Materials & Methods: Between September 2000 and November 2012, we performed NKS in failed standard biliary cannulation in 147 pts (65 men, 82 women; mean age 78 years, range 41-97 years). Surgically altered anatomy was noticed in 3 pts. The indications for ERCP were choledocolithiasis including biliary pancreatitis in 85 pts (57.8%), malignant biliary strictures in 42 (28.6%), cholecystitis in 6 (4.1%), and miscellaneous in 14. Sixty-nine pts (46.9%) underwent prior placement of a pancreatic stent (a straight, double-barbed, 5 Fr in diameter and 3 cm in length). Success was defined as deep placement of a catheter into the common bile duct. A diagnosis and severity of complication was made according to Cotton’s classification. Success of repeat ERCP in gaining biliary access, repeat ERCP time interval, and complications were retrospectively evaluated. Results: Selective biliary cannulation was successful after the initial NKS in 99 pts (67.3%), during a second ERCP in 38 (25.9%), in a third ERCP in 3 (1.4%), and in a fourth ERCP in 1 (0.7%), achieving a total cannulation rate of 95.9% (141 of 147) with additional NKS in 4 pts. Among the 6 patients (4.1%) with biliary cannulation failure, ERCP was not attempted again in 4 pts. The median time to repeat ERCP was 1 days (range 1-13 days). Of 48 pts, common causes of failed initial NKS were biliary deep cannulation failure in 41 patients (85.4%) and blocking of the endoscopic view due to bleeding in 7 (14.6%). The success rate of the second ERCP after one day was lower than that of more than 2 days later (76% vs. 100%, P 0.064). Fifteen complications (10.2%) occurred in 14 (9.5%) pts (pancreatitis in 11, bleeding in 4) after initial NKS, however, moderate perforation developed in 1 (2.3%) after second ERCP. The use of a pancreatic stent was not related to complication rate. There was no procedure-related mortality, and all complications were resolved by conservative management. Conclusions: Repeat ERCP after failed initial NKS for biliary access is safe and effective in the majority of cases. It is more worthwhile repeating ERCP more than 2 days later such failure than one day later, if the patient’s clinical condition permits.

Evolutionary studies of complement factor B (Bf) and C2 in lower vertebrates have revealed the presence of the Bf/C2 common ancestor-like molecule in lamprey (cyclostome) and the Bf molecule encoded by the duplicated genes closely linked to the major histocompatibility complex (MHC) in Xenopus (amphibian). To further define when Bf/C2 gene duplication occurred and when linkage between the Bf/C2 gene and the MHC was established, we amplified the Bf/C2 sequences in teleost, the Japanese medaka (Oryzias latipes), by reverse transcription - polymerase chain reaction with primers corresponding to the common amino acid sequences shared by mammalian Bf and C2. Only a single molecular species has been amplified, and the corresponding cDNA clones were isolated from the liver cDNA library. The longest insert contained 2384 nucleotides with an open reading frame of 754 residues. The deduced amino acid sequence showed 33.6% and 34.1% overall identity with the human Bf and C2 sequences, respectively, hence this clone was named medaka Bf/C2. The single-copy medaka Bf/C2 gene had exactly the same exon-intron organization as the mammalian Bf and C2 genes, and spanned about 8 kilobases. The Bf/C2 locus was mapped to the close proximity (2.9 cM) of the superoxide dismutase locus on the linkage group XX by the use of a restriction site polymorphism between two inbred strains of the medaka.

The Hedgehog (Hh) signal plays a pivotal role in induction of ventral neuronal and muscle cell types around the midline during vertebrate development [1]. We report that the gene disrupted in zebrafish you mutants, in which Hh signaling is impaired, encodes the secreted matrix protein Scube2. Consistently, epistasis analyses suggested that Scube2 functions upstream of Hh ligands or through a parallel pathway. In addition, overexpression analyses suggested that Scube2 is an essential, but a permissive, mediator of Hh signaling in zebrafish embryos. Surprisingly, the you gene is expressed in the dorsal neural tube, raising the possibility that Scube2 could indirectly act via a long-range regulator of Hh signaling. The dorsal Bmps have a long-range and opposing influence on Hh signaling [2–5]. We show that neural plate patterning is affected in you mutants in a way that is consistent with the aberrant long-range action of a Bmp-dependent signal. We further show that Bmp activity can be attenuated by the coexpression of Scube2. Our data support the idea that Scube2 can modulate the long-range action of Bmp-dependent signaling in the neural tube and somites.