Zebrafish thrombosis models according to the location of thrombus formation.

Background: Ischemic stroke becomes a major cause of death and disability. It can develop due to intravascular or cardiac thromboemboli. Animal models that reflect diverse stroke mechanisms remain under development. Using photochemical thrombosis, we developed a feasible zebrafish model according to the thrombus location (intracerebral vs. intracardiac). We validated the model using real-time imaging and thrombolytic agent. Methods: We used transgenic zebrafish larvae (flk:gfp), which express specific fluorescence in endothelial cells. We injected Rose Bengal, a photosensitizer as a mixture of photosensitizer, and a fluorescent agent into the cardinal vein of the larvae. We then evaluated real-time thrombosis in vivo by inducing thrombosis through exposure to a confocal laser (560 nm) and staining the blood flow (RITC-dextran). We validated intracerebral and intracardiac thrombotic models with checking the activity of tissue plasminogen activator (tPA). Results: The photochemical agent induced the formation of intracerebral thrombi in transgenic zebrafish. Real-time imaging techniques confirmed the formation of the thrombi. The damage and apoptosis of the vessel's endothelial cells were seen in the in vivo model. An intracardiac thrombosis model was developed by the same method using photothrombosis, and the model was validated through thrombolysis by tPA. Conclusions: We developed and validated two zebrafish thrombosis models that are readily available, cost-effective, and intuitive for assessing the efficacy of thrombolytic agents. These models can be used for a broad spectrum of future studies, such as screening and efficacy assessment of new antithrombotic agents.

Puromycin-induced kidney injury and subsequent regeneration in adult zebrafish.

Puromycin treatment can cause glomerular injury to the kidney, leading to proteinuria. However, the pathogenesis of acute kidney injury and subsequent regeneration after puromycin administration in animal models remain unclear. In this work, we examined the characteristics of kidney injury and subsequent regeneration following puromycin treatment in adult zebrafish. We intraperitoneally injected 100 µg of puromycin into zebrafish; sacrificed them at 1, 3, 5, 7, or 14 days post-injection (dpi); and examined the morphological, functional, and molecular changes in the kidney. Puromycin-treated zebrafish presented more rapid clearance of rhodamine dextran than control animals. Morphological changes were observed immediately after the puromycin injection (1-7 dpi) and had recovered by 14 dpi. The mRNA production of lhx1a, a renal progenitor marker, increased during recovery from kidney injury. Levels of NFκB, TNFα, Nampt, and p-ERK increased significantly during nephron injury and regeneration, and Sirt1, FOXO1, pax2, and wt1b showed an increasing tendency. However, TGF-ß1 and smad5 production did not show any changes after puromycin treatment. This study provides evidence that puromycin-induced injury in adult zebrafish kidneys is a potential tool for evaluating the mechanism of nephron injury and subsequent regeneration.

Radioprotective effect of mistletoe extract on intestinal toxicity: in vivo study using adult zebrafish.

PURPOSE: The intestine is a dose-limiting organ in the treatment of intra-abdominal cancer. We previously reported that the extract of mistletoe parasites on Quercus had a more potent radioprotective effect than amifostine in reducing the developmental toxicities of zebrafish embryos. In this study, radioprotection against intestinal toxicity was investigated in adult zebrafish. METHODS: Wild-type adult AB zebrafish were exposed to 45-50 Gy of photon beam irradiation and/or treated with mistletoe extract orally 1 h before. The main endpoints of the study were survival and degree of deformation of the intestinal villi. RESULTS: The median follow-up period was 10 d post-irradiation (range: 7-11 d). A total of 105 zebrafish were used, including 42 in the radiation alone, 42 in the radiation and mistletoe arms, and 21 control subjects (mistletoe alone, mock-irradiated arm). The rate of both significant deformity and death was 53% in the radiation-alone arm, whereas the corresponding rate was 30% in the radiation and mistletoe arms. Significant deformity-free survival rates at 10 d post-irradiation in the radiation alone, and radiation and mistletoe arms were 44.7% (95% confidence interval [CI]:20-54.3) and 68.4% (95% CI:53.8-86.8), respectively (p=.046). The radiation and mistletoe arms showed decreased expression of two of three inflammatory genes (IL-1ß and IL-6) compared to the radiation alone group (p<.05). CONCLUSION: The radioprotective effect against intestinal toxicity was successfully shown in an adult zebrafish model. This result suggests the possibility of clinical use of mistletoe extract for the treatment of abdominal cancers.

Identification of the fate and regenerative mechanism of zebrafish melanocyte progenitor cells and melanocytes after laser-induced pigment ablation.

BACKGROUND AND OBJECTIVES: Lasers are known to be the most effective treatment modality for pigmentary skin diseases. However, melanocytes and melanin pigment often recur or leave post-inflammatory hyperpigmentation after the laser procedure. Studies have reported on the role of progenitor cells in pigment cell regeneration, which can be constantly replenished through mitosis. However, the response of unpigmented melanocyte progenitor cells to laser treatment is poorly understood. In this study, we used adult zebrafish skin as the melanocyte regenerative system and examined the response of melanocyte progenitor cells to laser photothermolysis. MATERIALS AND METHODS: The two groups of adult zebrafish were irradiated with 1064 nm wavelength laser system of Q-switched neodymium:yttrium-aluminum-garnet (Nd:YAG) laser with 0.3 or 0.7 J·cm-2 . We compared the regeneration of pigment at different energy levels by measuring new melanocyte counts and pigment area. We traced and quantitatively compared the melanocyte lineage cells by immunohistochemical staining using specific markers such as sox10, mitfa, and dct during the regeneration process. Three repetitive laser ablations were also held to test the postinflammatory hyperpigmentation. RESULTS: After the laser ablation of melanocytes, most of the new melanocytes appeared between Days 5 and 10. In high-energy irradiation of 0.7 J·cm-2 , the unpigmented mitfa-expressing cells showed significant decrease (p < 0.05) and showed delay in the differentiation process of melanocyte lineage cells. After repeated laser irradiation, hyperpigmentation did not appear and the final recovery ratio of the pigmented area was 87.5% and 75.3% at the 0.3 and 0.7 J·cm-2 energy levels, respectively. CONCLUSION: We suggest that laser treatment overcoming the recurrence should be planned based on the adequate energy level targeting the melanocyte progenitor cells. High-energy irradiation may induce apoptosis of progenitor cells and delay their process of differentiation. Short-term repetitive sessions of laser therapy can reduce the pigmentation in the long-term observation.

Prokinetic effects of diatrizoate meglumine (Gastrografin®) in a zebrafish for opioid-induced constipation model.

Constipation is a common disease that reduces life quality. Drugs of various mechanisms are being developed to resolve this affliction. Intestinal motility can be easily monitored in zebrafish, and so we selected this organism to develop a constipation model to measure drug-induced prokinetic effects. In this study, intestinal motility was monitored in zebrafish by tracking intestinal transit using fluorescence, after which an opioid-induced constipation model was established using loperamide. We then evaluated the prokinetic effect of diatrizoate meglumine (Gastrografin®), which has been empirically used to treat post-operative ileus or adhesive small bowel obstructions. Diatrizoate meglumine was effective in promoting bowel movements in an opioid-induced zebrafish constipation model and its prokinetic effect was associated with an increased expression of interstitial cells of Cajal (ICC) markers. Therefore, the loperamide-induced zebrafish constipation model developed herein is a promising tool to evaluate novel constipation therapies.

Development of an experimental model for ocular toxicity screening in Zebrafish.

BACKGROUND: To investigate the efficacy of a novel experimental model for exploring visual function using a contrast-optomotor response (C-OMR) assay made by applying the contrast sensitivity test to the OMR assay in zebrafish. METHODS: Zebrafish larvae were treated with 0 (control), 5, 10, or 15 µM gentamicin and digoxin for 24 h at four days post-fertilization (dpf). Zebrafish larvae were assessed using the C-OMR assay with graded contrast gray-white stripes at 5 dpf, and the results were expressed as the percentage of larvae that finished swimming for 30 s (n = 20 per each group). The same C-OMR assay was repeated four times using different larvae. RESULTS: The percentage of larvae that finished swimming within 30 s was significantly reduced in larvae treated with 5, 10, and 15 µM gentamicin and 10 and 15 µM digoxin as compared to the Control groups. The C-OMR assay could distinguish that the decrease in visual function was different depending on the concentration of gentamicin and digoxin (5, 10, and 15 µM), whereas the OMR test with one contrast gray-white stripe could not. CONCLUSIONS: The method of analyzing zebrafish OMR using graded contrast gray-white stripes is more sensitive than the OMR assay alone and may be more useful for assessing the drug toxicity and eye-related diseases to improve the understanding of drug-induced ocular side effects in the clinic.

Analysis of behavioral changes in zebrafish (Danio rerio) larvae caused by aminoglycoside-induced damage to the lateral line and muscles.

Zebrafish behavior is influenced by the lateral line hair cells and muscles. Drug-induced behavioral changes can serve as indicators in the evaluation of drug toxicity. The aminoglycoside family of antibiotics comprise a number of agents, including neomycin (NM) and gentamicin (GM). We hypothesized that NM and GM exert different effects on zebrafish larvae through their action on the lateral line and muscle fibers, inducing different swimming behavioral patterns such as locomotor behavior and the startle response. In this study, 125 µM NM and 5, 10, 20 µM GM induced hair cell damage in the anterior and posterior lateral lines of zebrafish larvae. However, unlike GM, 125 µM NM also caused muscle damage. Locomotor behavior was decreased in the 125 µM NM-exposed group compared to the group exposed to GM. Furthermore, 125 µM NM exposure induced significantly different patterns of various indices of startle behavior compared with the GM exposure groups. Additionally, the larvae exhibited different startle responses depending on the concentration of GM. These results suggest that GM may be the drug-of-choice for analyzing behavioral changes in zebrafish caused by damage to the lateral line alone. Our study highlights the importance of confirming muscle damage in behavioral analyses using zebrafish.

Assessment of hair cell damage and developmental toxicity after fine particulate matter 2.5 µm (PM 2.5) exposure using zebrafish (Danio rerio) models.

OBJECTIVES: Particulate matter (PM) exposure has become one of the most serious problems. The aim of the present study was to evaluate the hair cell damage and possible developmental toxicity caused by PM2.5 exposure using a zebrafish model. METHODS: Zebrafish embryos were exposed to various concentrations of PM2.5. Developmental toxicity was evaluated based on general morphology score (GMS) system and Panzica-Kelly score, and by measurement of body length and heart rate. To evaluate hair cell damage, the average number of total hair cells within four neuromasts exposed to various concentrations of PM2.5 was compared with that of the control group. RESULTS: Morphological abnormalities evaluated by the GMS system and Panzica-Kelly score were rare and body length tended to be shorter in the PM2.5-exposed groups. Heart rate decreased significantly in the PM2.5-exposed group. Additionally, significant hair cell damage was observed after PM2.5 exposure. It was dose-dependent and more severe after a longer period exposure (10 dpf). CONCLUSIONS: In zebrafish embryos, exposure of PM2.5 in the early stages of life decreased heart rate and caused significant hair cell damage in a dose-dependent manner.

Evaluation of Ototoxicity of an Antifog Agent and the Suspected Underlying Mechanisms: An Animal Study.

Use of rigid endoscopes has become widespread in middle ear surgeries, thereby attracting attention to the safety of antifog agents. However, few studies on the ototoxicity of antifog agents have been conducted. The purpose of this study was to evaluate hair cell damage and the underlying mechanisms caused by antifog agents using zebrafish larvae. We exposed zebrafish larvae at 3 days postfertilization to various concentrations of the antifog agent, Ultrastop (0.01, 0.02, 0.04, and 0.08%) for 72 hours. The average number of hair cells within 4 neuromasts of larvae, including supraorbital (SO1 and SO2), otic (O1), and occipital (OC1), in the control group were compared to those in the exposure groups. Significant hair cell loss was observed in the experimental groups compared to that in the control group (P < .01; control: 53.88 ± 4.85, 0.01%: 45.08 ± 11.70, 0.02%: 41.36 ± 12.00, 0.04%: 35.36 ± 16.18, and 0.08%: 15.60 ± 7.53 cells). Concentration-dependent increase in hair cell apoptosis by terminal deoxynucleotidyltransferase (TDT)-mediated dUTP-biotin nick end labeling assay (control: 0.00 ± 0.00, 0.01%: 3.48 ± 2.18, 0.02%: 9.64 ± 5.75, 0.04%: 17.72 ± 6.26, and 0.08%: 14.60 ± 8.18 cells) and decrease in the viability of hair cell mitochondria by 2-(4-[dimethylamino] styryl)-N-ethylpyridinium iodide assay (control: 9.61 ± 1.47, 0.01%: 8.28 ± 2.22, 0.02%: 8.45 ± 2.72, 0.04%: 7.25 ± 2.44, and 0.08%: 6.77 ± 3.26 percentage of total area) were observed. Antifog agent exposure can cause hair cell damage in zebrafish larvae, possibly by induction of mitochondrial damage with subsequent apoptosis of hair cells.

Radioprotective effects of mistletoe extract in zebrafish embryos in vivo.

Introduction: Radioprotectors can enhance the efficacy of cancer radiotherapy, but their clinical use remains uncommon. The present study aimed to assess the radioprotective potential of mistletoe extract (commercial name: Abnoba Viscum), a well-known complementary cancer medicine, in zebrafish larvae. Materials and methods: Wild-type AB zebrafish embryos at 4 h-post-fertilization were exposed to 5 Gy 9-MeV electron beam irradiation after being treated for 1 h with 4 mMl/L amifostine or 0.2 mg/ml Abnoba Viscum A, F, M, or Q. Primary endpoints were abnormality-free survival and abnormality-free rates among survivors at 5 days-post-fertilization. Results: The crude abnormality-free survival rates were 33.7%, 49.0%, 38.8%, 43.9%, 38.1%, and 52.6%, whereas abnormality-free rates among survivors were 36.4%, 49.6%, 37.8%, 45.6%, 52.0%, and 62.8% for the control (with no pharmacologic treatment), amifostine, Abnoba Viscum A, F, M, and Q groups, respectively. Abnormality-free survival rates in the amifostine and Abnoba Viscum Q groups were significantly different from those in the control (p = .040 and .012, respectively), with an odds ratio (OR) of 1.90 [95% confidence interval (CI): 1.03-3.51] and 2.20 (95% CI: 1.19-4.08), respectively. Abnormality-free rates among survivors in the amifostine and Abnoba Viscum M and Q groups were significantly different from those in the control group (p = .048, .042, and <.001, respectively), with an OR of 1.79 (95% CI: 1.00-3.20), 1.82 (95% CI: 1.02-3.26), and 2.98 (1.67-5.33), respectively. Conclusion: Abnoba Viscum Q has at least a similar radioprotective effect to that of amifostine. Mistletoe extracts have been clinically applied for a long time and their effectiveness and feasibility have been verified. Abnoba Viscum Q might be a new candidate radioprotectant to enhance cancer radiotherapy efficacy.

In vivo assay of the ethanol-induced embryonic hair cell loss and the protective role of the retinoic and folic acid in zebrafish larvae (Danio rerio).

In reference to the auditory manifestation of fetal alcohol syndrome, previous work has preferentially focused on the deviant neural development of the auditory system. Changes in the sensory hair cell, the ultimate sensory organ, were not well understood. In this study, we carried out an in vivo assessment of the embryonic hair cell changes on the lateral line of zebrafish upon exposure to various ethanol concentrations (0.25%, 0.5%, 0.75%, and 1.0%). A significant decrease in the hair cell count was confirmed as the ethanol concentration increased. Long-term observation (up to 240 hours post-fertilization [hpf]) suggested an irreversible hair cell loss with little chance of a simple delayed development. For an underlying biological process, a significant increase of hair cell apoptosis and a significant decrease of cytoplasmic mitochondria were confirmed as the ethanol concentration increased. Co-treatment with retinoic (0.1 nM) or folic (0.1 mM) acid with the same concentrations of ethanol resulted in significant increases in the remaining hair cells, compared to the ethanol-only treatment group, for every ethanol concentration. The retinoic acid provided more effective protection over folic acid, resulting in no significant changes in hair cell counts for every ethanol concentration (except 1.0%), compared with that of the negative control (without chemical treatment). Hair cell counts in every ethanol concentration were significantly lower than those in negative controls without chemical treatment after folic acid co-treatment. In conclusion, gestational ethanol exposure causes developmental sensory hair cell loss. Potential underlying mechanisms include retinoic or folic acid deficiency, and mitochondrial damage with subsequent hair cell apoptosis. Hair cell loss could possibly be prevented by administering either retinoic or folic acid, with retinoic acid supplementation as the preferred treatment.

In vivo assay of the potential gadolinium-induced toxicity for sensory hair cells using a zebrafish animal model.

Recently, intratympanic injection of gadolinium-based contrast agent (GdC) is growing in use to visualize the endolymphatic hydrops. Although GdC has been quite safely used over 20 years through intravenous injection, the biological influence of GdC on sensory hair cells needs to be thoroughly assessed for wider clinical application of it through intratympanic injection. In this in vivo experimental study, the summated number of sensory hair cells (SO1, SO2, O1 and OC1 neuromasts) showed a steep decrease in the group exposed to 10% and 20% GdC (35.7 ± 7.3, 15.09 ± 10.82, respectively, P < .01) compared with the control group (47.18 ± 2.30). An increase in apoptosis was also observed in the group exposed to 20% gadolinium (7.20 ± 5.56), as compared with the control group (0.08 ± 0.72) or the group exposed to 10% gadolinium (3.48 ± 3.32). A significant reduction in the viable cytoplasmic mitochondria was observed in embryos exposed to 20% GdC (369 ± 124 µm2 , P = .01) as compared with control embryos (447 ± 118 µm2 ) or embryos exposed to 10% GdC (420 ± 108 µm2 ). GdC administration did not impact peripheral neural structures. GdC caused a significant reduction in sensory hair cell counts in response to high concentrations along with increased apoptosis and mitochondrial damage. However, it may not be likely that GdC will lead to hair cell toxicity, as the estimated concentration in the inner ear after clinically tried intratympanic injection is far more diluted than the non-toxic concentration (0.625%) that was tested in this study.

Impact of Nicotine Exposure on Hair Cell Toxicity and Embryotoxicity During Zebrafish Development.

OBJECTIVES: Nicotine has various adverse effects including negative impacts associated with maternal exposure. In the current study, we examined nicotine-induced damage of hair cells and embryotoxicity during zebrafish development. METHODS: Zebrafish embryos were exposed to nicotine at several concentrations (5, 10, 20, and 40 µM) and embryotoxicity were evaluated at 72 hours, including hatching rate, mortality, teratogenicity rate, and heart rate. Hair cells within the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) neuromasts were identified at 120 hours. Apoptosis and mitochondrial damage of hair cells were analyzed using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) and DASPEI (2-[4-(dimethylamino)styryl]-N-ethylpyridinium iodide) assays, respectively, and changes of ultrastructure were observed by scanning electron microscopy. RESULTS: The control group without nicotine appeared normal with overall mortality and teratogenicity rate <5%. The hatching rate and mortality rate was not significantly different according to nicotine concentration (n=400 each). The abnormal morphology rate (n=400) increased and heart rate (n=150) decreased with increasing nicotine concentration (P<0.05). Nicotine-induced hair cell damage significantly increased as nicotine concentration increased. A significantly greater number of TUNEL-positive cells (P<0.01) and markedly smaller DASPEI area (P<0.01) were shown as nicotine concentration increased. CONCLUSION: The current results suggest that nicotine induces dose-dependent hair cell toxicity in embryos by promoting apoptosis and mitochondrial and structural damage.

Spatiotemporal expression pattern of the zebrafish aquaporin 8 family during early developmental stages.

Aquaporin 8 (Aqp8) is a transmembrane protein that is selectively permeated by water and some small solutes, and physiologically contributes to acid-base equilibrium in the gastrointestinal tract. Here, we described the characterization and spatiotemporal expression pattern of zebrafish aqp8 (zaqp8) gene family, including zaqp8a.1, zaqp8a.2, and zaqp8b, during the early developmental stages. The expression of zaqp8a.1 started first in the lateral plate mesoderm at the 12-somite stage (ss) and then expanded sequentially to the dorsal aorta, intersegmental blood vessels and then to the dorsal longitudinal anastomotic vessel at 24 h post fertilization (hpf). At 28 hpf, expression of zaqp8a.1 was also detected in the embryonic heart tube. Four days post fertilization (dpf), strong zaqp8a.1 expression was detected in the gastrointestinal tract and liver. By 72 hpf, the expression of zaqp8a.2 was first detected in the primitive gut region but not detected in the liver. The expression of zaqp8b was first detected in the intermediate mesoderm at 10 ss. From 24 hpf to 6 dpf, the proximal convoluted segment of the embryonic kidney was marked by zaqp8b expression Overall, these differential expression patterns of aqp8a.1, aqp8a.2, and aqp8b suggest that they possibly play distinct roles throughout the embryonic development by controlling or maintaining organ-specific cellular water homeostasis. Our study provides new evidence that organogenesis requires differential roles of Aqp8 proteins in zebrafish.

Proper Activity of Histone H3 Lysine 4 (H3K4) Methyltransferase Is Required for Morphogenesis during Zebrafish Cardiogenesis.

While increasing evidence indicates the important function of histone methylation during development, how this process influences cardiac development in vertebrates has not been explored. Here, we elucidate the functions of two histone H3 lysine 4 (H3K4) methylation enzymes, SMYD3 and SETD7, during zebrafish heart morphogenesis using gene expression profiling by whole mount in situ hybridization and antisense morpholino oligonucleotide (MO)-based gene knockdown. We find both smyd3 and setd7 are highly expressed within developing zebrafish heart and knock-down of these genes led to severe defects in cardiac morphogenesis without altering the expressions pattern of heart markers, including cmlc2, vmhc, and amhc. Furthermore, double knock-down by coinjection of smyd3 and setd7 MOs caused the synergistic defects in heart development. As similar to knock-down effect, overexpression of these genes also caused the heart morphogenesis defect in zebrafish. These results indicate that histone modifying enzymes, SMYD3 and SETD7, appear to function synergistically during heart development and their proper functioning is essential for normal heart morphogenesis during development.

Zebrafish Crip2 plays a critical role in atrioventricular valve development by downregulating the expression of ECM genes in the endocardial cushion.

The initial step of atrioventricular (AV) valve development involves the deposition of extracellular matrix (ECM) components of the endocardial cushion and the endocardial-mesenchymal transition. While the appropriately regulated expression of the major ECM components, Versican and Hyaluronan, that form the endocardial cushion is important for heart valve development, the underlying mechanism that regulates ECM gene expression remains unclear. We found that zebrafish crip2 expression is restricted to a subset of cells in the AV canal (AVC) endocardium at 55 hours post-fertilization (hpf). Knockdown of crip2 induced a heart-looping defect in zebrafish embryos, although the development of cardiac chambers appeared to be normal. In the AVC of Crip2-deficient embryos, the expression of both versican a and hyaluronan synthase 2 (has2) was highly upregulated, but the expression of bone morphogenetic protein 4 (bmp4) and T-box 2b (tbx2b) in the myocardium and of notch1b in the endocardium in the AVC did not change. Taken together, these results indicate that crip2 plays an important role in AV valve development by downregulating the expression of ECM components in the endocardial cushion.

Antagonistic regulation of PAF1C and p-TEFb is required for oligodendrocyte differentiation.

Oligodendrocytes are myelinating glial cells in the CNS and are essential for proper neuronal function. During development, oligodendrocyte progenitor cells (OPCs) are specified from the motor neuron precursor domain of the ventral spinal cord and differentiate into myelinating oligodendrocytes after migration to the white matter of the neural tube. Cell cycle control of OPCs influences the balance between immature OPCs and myelinating oligodendrocytes, but the precise mechanism regulating the differentiation of OPCs into myelinating oligodendrocytes is unclear. To understand the mechanisms underlying oligodendrocyte differentiation, an N-ethyl-N-nitrosourea-based mutagenesis screen was performed and a zebrafish leo1 mutant, dalmuri (dal(knu6)) was identified in the current study. Leo1 is a component of the evolutionarily conserved RNA polymerase II-associated factor 1 complex (PAF1C), which is a positive regulator of transcription elongation. The dal(knu6) mutant embryos specified motor neurons and OPCs normally, and at the appropriate time, but OPCs subsequently failed to differentiate into myelinating oligodendrocytes and were eliminated by apoptosis. A loss-of-function study of cdc73, another member of PAF1C, showed the same phenotype in the CNS, indicating that PAF1C function is required for oligodendrocyte differentiation. Interestingly, inhibition of positive transcription elongation factor b (p-TEFb), rescued downregulated gene expression and impaired oligodendrocyte differentiation in the dal(knu6) mutant and Cdc73-deficient embryos. Together, these results indicate that antagonistic regulation of gene expression by PAF1C and p-TEFb plays a crucial role in oligodendrocyte development in the CNS.

Histone deacetylase is required for the activation of Wnt/ß-catenin signaling crucial for heart valve formation in zebrafish embryos.

During vertebrate heart valve formation, Wnt/ß-catenin signaling induces BMP signals in atrioventricular canal (AVC) myocardial cells and underlying AVC endocardial cells then undergo endothelial-mesenchymal transdifferentiation (EMT) by receiving this BMP signals. Histone deacetylases (HDACs) have been implicated in numerous developmental processes by regulating gene expression. However, their specific roles in controlling heart valve development are largely unexplored. To investigate the role of HDACs in vertebrate heart valve formation, we treated zebrafish embryos with trichostatin A (TSA), an inhibitor of class I and II HDACs, from 36 to 48 h post-fertilization (hpf) during which heart looping and valve formation occur. Following TSA treatment, abnormal linear heart tube development was observed. In these embryos, expression of AVC myocardial bmp4 and AVC endocardial notch1b genes was markedly reduced with subsequent failure of EMT in the AVC endocardial cells. However, LiCl-mediated activation of Wnt/ß-catenin signaling was able to rescue defective heart tube formation, bmp4 and notch1b expression, and EMT in the AVC region. Taken together, our results demonstrated that HDAC activity plays a pivotal role in vertebrate heart tube formation by activating Wnt/ß-catenin signaling which induces bmp4 expression in AVC myocardial cells.

Transcriptome analysis of the zebrafish mind bomb mutant.

Mind bomb (Mib) facilitates Notch signaling pathway by promoting the endocytosis of Notch ligand. The zebrafish mib ( ta52b ) mutant has a defect in its ubiquitin ligase activity which is necessary to inhibit the neurogenesis, resulting in a neuronal hyperplasia. Several genes regulated in the mib ( ta52b ) mutant have been well established, however, there were relatively few reports about the transcriptome profile. To identify the genes differentially expressed in the mib ( ta52b ) mutant, genome-wide analysis was performed using serial analysis of gene expression. Three hundred and thirty-five transcripts were identified whose expressions were significantly altered in the mib ( ta52b ) mutant as compared with the wild-type. Interestingly, it was suggested that the mib ( ta52b ) mutation may affect not only neurogenesis but also mesoderm development. These results provide new insights into Notch signaling pathway.