Lansoprazole inhibits the development of sessile serrated lesions by inducing G1 arrest via Skp2/p27 signaling pathway.

BACKGROUND: Although the serrated-neoplasia pathway reportedly accounts for 15-30% of colorectal cancer (CRC), no studies on chemoprevention of sessile serrated lesions (SSLs) have been reported. We searched for effective compounds comprehensively from a large series of compounds by employing Connectivity Map (CMAP) analysis of SSL-specific gene expression profiles coupled with in vitro screening using SSL patient-derived organoids (PDOs), and validated their efficacy using a xenograft mouse model of SSL. METHODS: We generated SSL-specific gene signatures based on DNA microarray data, and applied them to CMAP analysis with 1309 FDA-approved compounds to select candidate compounds. We evaluated their inhibitory effects on SSL-PDOs using a cell viability assay. SSL-PDOs were orthotopically transplanted into NOG mice for in vivo evaluation. The signal transduction pathway was evaluated by gene expression profile and protein expression analysis. RESULTS: We identified 221 compounds by employing CMAP analysis of SSL-specific signatures, which should cancel the gene signatures, and narrowed them down to 17 compounds. Cell viability assay using SSL-PDOs identified lansoprazole as having the lowest IC50 value (47 µM) among 17 compounds. When SSL-PDO was orthotopically transplanted into murine intestinal tract, the tumor grew gradually. Administration of lansoprazole to mice inhibited the growth of SSL xenograft whereas the tumor in control mice treated with vehicle alone grew gradually over time. The Ki67 index in xenograft lesions from the lansoprazole group was significantly lower compared with the control group. Cell cycle analysis of SSL-PDOs treated with lansoprazole exhibited a significant increase in G1 phase cell population. Microarray and protein analysis revealed that lansoprazole downregulated Skp2 expression and upregulated p27 expression in SSL-PDOs. CONCLUSIONS: Our data strongly suggest that lansoprazole is the most effective chemopreventive agent against SSL, and that lansoprazole induces G1 cell cycle arrest by downregulating Skp2 and upregulating p27 in SSL cells.

[Gastric cancer genome profiling reveals HER2 false-negative status and informs a successful trastuzumab treatment strategy].

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.

TIMP1 promotes cell proliferation and invasion capability of right-sided colon cancers via the FAK/Akt signaling pathway.

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.

Resveratrol inhibits development of colorectal adenoma via suppression of LEF1; comprehensive analysis with connectivity map.

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 ApcMin/+ mice at 15 or 30 mg/kg, the number of polyps (adenomas) was significantly reduced in both groups compared with control mice. Similarly, the number of polyps (adenomas) was significantly reduced in azoxymethane-injected rats treated with 10 or 100 mg/resveratrol compared with control rats. Microarray analysis of adenomas from resveratrol-treated rats revealed the highest change (downregulation) in expression of LEF1, a key molecule in the Wnt signaling pathway. Treatment with resveratrol significantly downregulated the Wnt-target gene (MYC) in CRA-PDOs. Our data demonstrated that resveratrol can be the most effective compound for chemoprevention of colorectal tumors, the efficacy of which is mediated through suppression of LEF1 expression in the Wnt signaling pathway.

Remodeling of the hyomandibular skeleton and facial nerve positioning during embryonic and postembryonic development of teleost fish.

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.

MiR-125b-5p Is Involved in Sorafenib Resistance through Ataxin-1-Mediated Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma.

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.

Development of the anterior lateral line system through local tissue-tissue interactions in the zebrafish head.

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/ß-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/ß-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.

Cancer-associated adipocytes promote pancreatic cancer progression through SAA1 expression.

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.

The Genetic Basis of Morphological Diversity in Domesticated Goldfish.

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.

De novo assembly of the goldfish (Carassius auratus) genome and the evolution of genes after whole-genome duplication.

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.

Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish.

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.

Epidermal regulation of bone morphogenesis through the development and regeneration of osteoblasts in the zebrafish scale.

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.

Multiple small intestinal ulcers associated with protein-losing enteropathy secondary to cholesterol crystal embolism:a case report.

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 99mTc-HSA scintigraphy was positive, and alpha-1 antitrypsin clearance was high;therefore, the hypoalbuminemia was thought to be secondary to a protein-losing enteropathy. A small bowel series revealed multiple, ring-shaped, longitudinal ulcers in the ileum. Balloon-assisted enteroscopy from the anus showed severe stenosis with an ileal ulcer. Since we were not able to diagnose the ulcers, mesalazine and supplemental nutritional care were provided. Four years after the hypoalbuminemia had been diagnosed, the patient died because of pulmonary congestion secondary to renal failure. An autopsy revealed severe atherosclerosis in his aorta and multiple cholesterol embolisms in his small intestine, kidney, stomach, colon, liver, and spleen. The multiple ulcers in the small intestine were thought to be caused by cholesterol crystal embolism, which should be considered in the differential diagnosis of small intestinal ulcers in elderly men or patients after cardiovascular intervention.

Examination time as a quality indicator of screening upper gastrointestinal endoscopy for asymptomatic examinees.

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. 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. Mean examination time of 13 661 EGD without biopsy was 6.2 min (range, 2-18 min). When cut-off times of 5 and 7 min were used, four endoscopists were classified into the fast (mean duration, 4.4 ± 1.0 min), 12 into the moderate (6.1 ± 1.4 min), and four into the slow (7.8 ± 1.9 min) groups. Neoplastic lesion detection rates in the fast, moderate, and slow groups were 0.57% (13/2288), 0.97% (99/10 180), and 0.94% (31/3295), respectively. Compared with that in the fast group, 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.

Size control during organogenesis: Development of the lateral line organs in zebrafish.

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 development and growth of tissues derived from cranial neural crest and primitive mesoderm is dependent on the ligation status of retinoic acid receptor γ: evidence that retinoic acid receptor γ functions to maintain stem/progenitor cells in the absence of retinoic acid.

Retinoic acid (RA) signaling is important to normal development. However, the function of the different RA receptors (RARs)--RARα, RARß, 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.

Successful treatment with entecavir for reactivation of hepatitis B virus following systemic chemotherapy in a hepatitis B surface antigen-negative patient with colorectal cancer.

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.

Development of the lateral line canal system through a bone remodeling process in zebrafish.

The lateral line system of teleost fish is composed of mechanosensory receptors (neuromasts), comprising superficial receptors and others embedded in canals running under the skin. Canal diameter and size of the canal neuromasts are correlated with increasing body size, thus providing a very simple system to investigate mechanisms underlying the coordination between organ growth and body size. Here, we examine the development of the trunk lateral line canal system in zebrafish. We demonstrated that trunk canals originate from scales through a bone remodeling process, which we suggest is essential for the normal growth of canals and canal neuromasts. Moreover, we found that lateral line cells are required for the formation of canals, suggesting the existence of mutual interactions between the sensory system and surrounding connective tissues.

Regular heartbeat rhythm at the heartbeat initiation stage is essential for normal cardiogenesis at low temperature.

BACKGROUND: The development of blood flow in the heart is crucial for heart function and embryonic survival. Recent studies have revealed the importance of the extracellular matrix and the mechanical stress applied to the valve cushion that controls blood flow to the formation of the cardiac valve during embryogenesis. However, the events that trigger such valve formation and mechanical stress, and their temperature dependence have not been explained completely. Medaka (Oryzias latipes) inhabits a wide range of East Asia and adapts to a wide range of climates. We used medaka embryos from different genomic backgrounds and analyzed heartbeat characteristics including back-and-forth blood flow and bradyarrhythmia in embryos incubated at low temperature. We also used high-speed imaging analysis to examine the heartbeat of these animals after transient exposure to low temperature. RESULTS: Embryos of the Hd-rR medaka strain exhibited back-and-forth blood flow in the heart (blood regurgitation) after incubation at 15 °C. This regurgitation was induced by exposure to low temperature around the heartbeat initiation period and was related to abnormalities in the maintenance or pattern of contraction of the atrium or the atrioventricular canal. The Odate strain from the northern Japanese group exhibited normal blood flow after incubation at 15 °C. High-speed time-lapse analysis of the heartbeat revealed that bradyarrhythmia occurred only in Hd-rR embryos incubated at 15 °C. The coefficient of contraction, defined as the quotient of the length of the atrium at systole divided by its length at diastole, was not affected in either strain. The average heart rate after removing the effect of arrhythmia did not differ significantly between the two strains, suggesting that the mechanical stress of individual myocardial contractions and the total mechanical stress could be equivalent, regardless of the presence of arrhythmia or the heart rate. Test-cross experiments suggested that this circulation phenotype was caused by a single major genomic locus. CONCLUSIONS: These results suggest that cardiogenesis at low temperature requires a constant heartbeat. Abnormal contraction rhythms at the stage of heartbeat initiation may cause regurgitation at later stages. From the evolutionary viewpoint, strains that exhibit normal cardiogenesis during development at low temperature inhabit northern environments.

Wnt/Dkk negative feedback regulates sensory organ size in zebrafish.

Correct 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.