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Christine E Beattie - One of the best experts on this subject based on the ideXlab platform.
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in vivo assessment of contractile strength distinguishes differential gene function in skeletal muscle of zebrafish larvae
Journal of Applied Physiology, 2015Co-Authors: Brit L Martin, Thomas L Gallagher, Neha Rastogi, Christine E Beattie, Sharon L Amacher, Jonathan P Davis, Paul M L JanssenAbstract:The accessible genetics and extensive skeletal musculature of the zebrafish make it a versatile and increasingly used model for studying muscle contraction. We here describe the development of an in vivo assay for measuring the contractile force of intact zebrafish at the larval stage. In addition, as proof of applicability, we have used this assay to quantify contractile strength of zebrafish larvae in a Morphant model of deranged rbfox function. Average maximum tetanic (180 Hz) whole body forces produced by wild-type larvae at 2, 3, 4, and 5 days postfertilization amounted to 3.0, 7.2, 9.1, and 10.8 mN, respectively. To compare at potentially different stages of muscle development, we developed an immunohistological assay for empirically determining the cross-sectional area of larval trunk skeletal muscle to quantify muscle-specific force per cross-sectional area. At 4-5 days postfertilization, specific force amounts to ∼300 mN/mm2, which is similar to fully developed adult mammalian skeletal muscle. We used these assays to measure contractile strength in zebrafish singly or doubly deficient for two rbfox paralogs, rbfox1l and rbfox2 , which encode RNA-binding factors shown previously to modulate muscle function and muscle-specific splicing. We found rbfox2 Morphants produce maximal tetanic forces similar to wild-type larvae, whereas rbfox1l Morphants demonstrate significantly impaired function. rbfox1l/rbfox2 Morphants are paralyzed, and their lack of contractile force production in our assay suggests that paralysis is a muscle-autonomous defect. These quantitative functional results allow measurement of muscle-specific phenotypes independent of neural input.
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direct assessment of skeletal muscle contractile strength in live wildtype and rbfox Morphant zebrafish larvae
Biophysical Journal, 2015Co-Authors: Brit L Martin, Thomas L Gallagher, Neha Rastogi, Christine E Beattie, Sharon L Amacher, Paul M L JanssenAbstract:The accessible genetics and abundant skeletal musculature of the zebrafish make it an ideal model for studies of muscle contraction. Current functional assays for quantifying the effect of experimental modification of zebrafish muscle are indirect and observational, or they underestimate contractile force. Therefore, we developed an in vivo assay for measuring muscle contractile force. As proof of concept, we have measured contractile strength of skeletal muscle in wildtype larvae and in a Morphant model from 2-5 days post fertilization (dpf). Mean maximum tetanic whole-body forces produced by wildtype 2, 3, 4, and 5 dpf larvae amounted to 3.03 mN, 7.31 mN, 8.67 mN, and 10.94 mN, respectively. Mean twitch forces produced by larvae were 0.90 mN, 5.58 mN, 7.08 mN, and 9.08 mN for 2, 3, 4, and 5 dpf respectively. The Morphants we have analyzed are knockdowns of two zebrafish paralogs of rbfox, rbfox1l and rbfox2, which regulate muscle-specific splicing programs. We assessed the contractile force developed during contraction and found that our assay is clearly able to differentiate between Morphant phenotypes. rbfox2 Morphants appear to produce maximal tetanic forces similar to wildtype larvae, while rbfox1l Morphants demonstrate significantly impaired function by producing decreased forces at the same developmental time points. This supports the conclusion that rbfox1l regulates the majority of splicing events in larval skeletal muscle. rbfox1l/rbfox2 Morphants are paralyzed and their lack of significant contractile force production in our assay indicated a muscle-specific defect, not just a motoneural defect, causing the characteristic paralysis. We have also developed an immunohistological assay for empirically determining the cross-sectional area of larval trunk skeletal muscle in order quantify muscle-specific force per cross-sectional area. These functional results quantify muscle-specific phenotypes sans neural input.
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Calcium binding is essential for plastin 3 function in Smn-deficient motoneurons
Human Molecular Genetics, 2013Co-Authors: Alison N. Lyon, Ricardo Pineda, Le Thi Hao, Elena Kudryashova, Dmitri S. Kudryashov, Christine E BeattieAbstract:The actin-binding and bundling protein, plastin 3 (PLS3), was identified as a protective modifier of spinal muscular atrophy (SMA) in some patient populations and as a disease modifier in animal models of SMA. How it functions in this process, however, is not known. Because PLS3 is an actin-binding/bundling protein, we hypothesized it would likely act via modification of the actin cytoskeleton in axons and neuromuscular junctions to protect motoneurons in SMA. To test this, we examined the ability of other known actin cytoskeleton organizing proteins to modify motor axon outgrowth phenotypes in an smn Morphant zebrafish model of SMA. While PLS3 can fully compensate for low levels of smn, cofilin 1, profilin 2 and α-actinin 1 did not affect smn Morphant motor axon outgrowth. To determine how PLS3 functions in SMA, we generated deletion constructs of conserved PLS3 structural domains. The EF hands were essential for PLS3 rescue of smn Morphant phenotypes, and mutation of the Ca2+-binding residues within the EF hands resulted in a complete loss of PLS3 rescue. These results indicate that Ca2+ regulation is essential for the function of PLS3 in motor axons. Remarkably, PLS3 mutants lacking both actin-binding domains were still able to rescue motor axons in smn Morphants, although not as well as full-length PLS3. Therefore, PLS3 function in this process may have an actin-independent component.
Sharon L Amacher - One of the best experts on this subject based on the ideXlab platform.
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in vivo assessment of contractile strength distinguishes differential gene function in skeletal muscle of zebrafish larvae
Journal of Applied Physiology, 2015Co-Authors: Brit L Martin, Thomas L Gallagher, Neha Rastogi, Christine E Beattie, Sharon L Amacher, Jonathan P Davis, Paul M L JanssenAbstract:The accessible genetics and extensive skeletal musculature of the zebrafish make it a versatile and increasingly used model for studying muscle contraction. We here describe the development of an in vivo assay for measuring the contractile force of intact zebrafish at the larval stage. In addition, as proof of applicability, we have used this assay to quantify contractile strength of zebrafish larvae in a Morphant model of deranged rbfox function. Average maximum tetanic (180 Hz) whole body forces produced by wild-type larvae at 2, 3, 4, and 5 days postfertilization amounted to 3.0, 7.2, 9.1, and 10.8 mN, respectively. To compare at potentially different stages of muscle development, we developed an immunohistological assay for empirically determining the cross-sectional area of larval trunk skeletal muscle to quantify muscle-specific force per cross-sectional area. At 4-5 days postfertilization, specific force amounts to ∼300 mN/mm2, which is similar to fully developed adult mammalian skeletal muscle. We used these assays to measure contractile strength in zebrafish singly or doubly deficient for two rbfox paralogs, rbfox1l and rbfox2 , which encode RNA-binding factors shown previously to modulate muscle function and muscle-specific splicing. We found rbfox2 Morphants produce maximal tetanic forces similar to wild-type larvae, whereas rbfox1l Morphants demonstrate significantly impaired function. rbfox1l/rbfox2 Morphants are paralyzed, and their lack of contractile force production in our assay suggests that paralysis is a muscle-autonomous defect. These quantitative functional results allow measurement of muscle-specific phenotypes independent of neural input.
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direct assessment of skeletal muscle contractile strength in live wildtype and rbfox Morphant zebrafish larvae
Biophysical Journal, 2015Co-Authors: Brit L Martin, Thomas L Gallagher, Neha Rastogi, Christine E Beattie, Sharon L Amacher, Paul M L JanssenAbstract:The accessible genetics and abundant skeletal musculature of the zebrafish make it an ideal model for studies of muscle contraction. Current functional assays for quantifying the effect of experimental modification of zebrafish muscle are indirect and observational, or they underestimate contractile force. Therefore, we developed an in vivo assay for measuring muscle contractile force. As proof of concept, we have measured contractile strength of skeletal muscle in wildtype larvae and in a Morphant model from 2-5 days post fertilization (dpf). Mean maximum tetanic whole-body forces produced by wildtype 2, 3, 4, and 5 dpf larvae amounted to 3.03 mN, 7.31 mN, 8.67 mN, and 10.94 mN, respectively. Mean twitch forces produced by larvae were 0.90 mN, 5.58 mN, 7.08 mN, and 9.08 mN for 2, 3, 4, and 5 dpf respectively. The Morphants we have analyzed are knockdowns of two zebrafish paralogs of rbfox, rbfox1l and rbfox2, which regulate muscle-specific splicing programs. We assessed the contractile force developed during contraction and found that our assay is clearly able to differentiate between Morphant phenotypes. rbfox2 Morphants appear to produce maximal tetanic forces similar to wildtype larvae, while rbfox1l Morphants demonstrate significantly impaired function by producing decreased forces at the same developmental time points. This supports the conclusion that rbfox1l regulates the majority of splicing events in larval skeletal muscle. rbfox1l/rbfox2 Morphants are paralyzed and their lack of significant contractile force production in our assay indicated a muscle-specific defect, not just a motoneural defect, causing the characteristic paralysis. We have also developed an immunohistological assay for empirically determining the cross-sectional area of larval trunk skeletal muscle in order quantify muscle-specific force per cross-sectional area. These functional results quantify muscle-specific phenotypes sans neural input.
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Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions.
Developmental Biology, 2011Co-Authors: Thomas L Gallagher, Joshua A. Arribere, Paul A. Geurts, Cameron R.t. Exner, Kent L. Mcdonald, Kariena K. Dill, Henry L. Marr, Shaunak S. Adkar, Aaron T. Garnett, Sharon L AmacherAbstract:Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double Morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single Morphant embryos were strikingly normal; however, muscle development in double Morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double Morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 Morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions.
Paul M L Janssen - One of the best experts on this subject based on the ideXlab platform.
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in vivo assessment of contractile strength distinguishes differential gene function in skeletal muscle of zebrafish larvae
Journal of Applied Physiology, 2015Co-Authors: Brit L Martin, Thomas L Gallagher, Neha Rastogi, Christine E Beattie, Sharon L Amacher, Jonathan P Davis, Paul M L JanssenAbstract:The accessible genetics and extensive skeletal musculature of the zebrafish make it a versatile and increasingly used model for studying muscle contraction. We here describe the development of an in vivo assay for measuring the contractile force of intact zebrafish at the larval stage. In addition, as proof of applicability, we have used this assay to quantify contractile strength of zebrafish larvae in a Morphant model of deranged rbfox function. Average maximum tetanic (180 Hz) whole body forces produced by wild-type larvae at 2, 3, 4, and 5 days postfertilization amounted to 3.0, 7.2, 9.1, and 10.8 mN, respectively. To compare at potentially different stages of muscle development, we developed an immunohistological assay for empirically determining the cross-sectional area of larval trunk skeletal muscle to quantify muscle-specific force per cross-sectional area. At 4-5 days postfertilization, specific force amounts to ∼300 mN/mm2, which is similar to fully developed adult mammalian skeletal muscle. We used these assays to measure contractile strength in zebrafish singly or doubly deficient for two rbfox paralogs, rbfox1l and rbfox2 , which encode RNA-binding factors shown previously to modulate muscle function and muscle-specific splicing. We found rbfox2 Morphants produce maximal tetanic forces similar to wild-type larvae, whereas rbfox1l Morphants demonstrate significantly impaired function. rbfox1l/rbfox2 Morphants are paralyzed, and their lack of contractile force production in our assay suggests that paralysis is a muscle-autonomous defect. These quantitative functional results allow measurement of muscle-specific phenotypes independent of neural input.
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direct assessment of skeletal muscle contractile strength in live wildtype and rbfox Morphant zebrafish larvae
Biophysical Journal, 2015Co-Authors: Brit L Martin, Thomas L Gallagher, Neha Rastogi, Christine E Beattie, Sharon L Amacher, Paul M L JanssenAbstract:The accessible genetics and abundant skeletal musculature of the zebrafish make it an ideal model for studies of muscle contraction. Current functional assays for quantifying the effect of experimental modification of zebrafish muscle are indirect and observational, or they underestimate contractile force. Therefore, we developed an in vivo assay for measuring muscle contractile force. As proof of concept, we have measured contractile strength of skeletal muscle in wildtype larvae and in a Morphant model from 2-5 days post fertilization (dpf). Mean maximum tetanic whole-body forces produced by wildtype 2, 3, 4, and 5 dpf larvae amounted to 3.03 mN, 7.31 mN, 8.67 mN, and 10.94 mN, respectively. Mean twitch forces produced by larvae were 0.90 mN, 5.58 mN, 7.08 mN, and 9.08 mN for 2, 3, 4, and 5 dpf respectively. The Morphants we have analyzed are knockdowns of two zebrafish paralogs of rbfox, rbfox1l and rbfox2, which regulate muscle-specific splicing programs. We assessed the contractile force developed during contraction and found that our assay is clearly able to differentiate between Morphant phenotypes. rbfox2 Morphants appear to produce maximal tetanic forces similar to wildtype larvae, while rbfox1l Morphants demonstrate significantly impaired function by producing decreased forces at the same developmental time points. This supports the conclusion that rbfox1l regulates the majority of splicing events in larval skeletal muscle. rbfox1l/rbfox2 Morphants are paralyzed and their lack of significant contractile force production in our assay indicated a muscle-specific defect, not just a motoneural defect, causing the characteristic paralysis. We have also developed an immunohistological assay for empirically determining the cross-sectional area of larval trunk skeletal muscle in order quantify muscle-specific force per cross-sectional area. These functional results quantify muscle-specific phenotypes sans neural input.
Robert M. Tombes - One of the best experts on this subject based on the ideXlab platform.
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tbx5 mediated expression of ca2 calmodulin dependent protein kinase ii is necessary for zebrafish cardiac and pectoral fin morphogenesis
Developmental Biology, 2009Co-Authors: Sarah C. Rothschild, Charles A. Easley, Ludmila Francescatto, James A. Lister, Deborah M. Garrity, Robert M. TombesAbstract:Abstract Mutations in the T-box transcription factor, TBX5, result in Holt–Oram syndrome (HOS), a human condition in which cardiac development is defective and forelimbs are stunted. Similarly, zebrafish tbx5 Morphants and mutants (heartstrings; hst) lack pectoral fins and exhibit a persistently elongated heart that does not undergo chamber looping. Tbx5 is expressed in the developing atrium, ventricle and in pectoral fin fields, but its genetic targets are still being uncovered. In this study, evidence is provided that Tbx5 induces the expression of a specific member of the CaMK-II (the type II multifunctional Ca2+/calmodulin-dependent protein kinase) family; this CaMK-II is necessary for proper heart and fin development. Morphants of β2 CaMK-II (camk2b2), but not the β1 CaMK-II (camk2b1) paralog, exhibit bradycardia, elongated hearts and diminished pectoral fin development. Normal cardiac phenotypes can be restored by ectopic cytosolic CaMK-II expression in tbx5 Morphants. Like tbx5, camk2b2 is expressed in the pectoral fin and looping heart, but this expression is diminished in both tbx5 Morphant and hst embryos. Conversely, the introduction of excess Tbx5 into zebrafish embryos and mouse fibroblasts doubles CaMK-II expression. We conclude that β CaMK-II expression and activity are necessary for proper cardiac and limb morphogenesis. These findings not only identify a morphogenic target for Ca2+ during heart development, but support implied roles for CaMK-II in adult heart remodeling.
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Tbx5-mediated expression of Ca2+/calmodulin-dependent protein kinase II is necessary for zebrafish cardiac and pectoral fin morphogenesis
Developmental biology, 2009Co-Authors: Sarah C. Rothschild, Charles A. Easley, Ludmila Francescatto, James A. Lister, Deborah M. Garrity, Robert M. TombesAbstract:Mutations in the T-box transcription factor, TBX5, result in Holt-Oram syndrome (HOS), a human condition in which cardiac development is defective and forelimbs are stunted. Similarly, zebrafish tbx5 Morphants and mutants (heartstrings; hst) lack pectoral fins and exhibit a persistently elongated heart that does not undergo chamber looping. Tbx5 is expressed in the developing atrium, ventricle and in pectoral fin fields, but its genetic targets are still being uncovered. In this study, evidence is provided that Tbx5 induces the expression of a specific member of the CaMK-II (the type II multifunctional Ca(2+)/calmodulin-dependent protein kinase) family; this CaMK-II is necessary for proper heart and fin development. Morphants of beta2 CaMK-II (camk2b2), but not the beta1 CaMK-II (camk2b1) paralog, exhibit bradycardia, elongated hearts and diminished pectoral fin development. Normal cardiac phenotypes can be restored by ectopic cytosolic CaMK-II expression in tbx5 Morphants. Like tbx5, camk2b2 is expressed in the pectoral fin and looping heart, but this expression is diminished in both tbx5 Morphant and hst embryos. Conversely, the introduction of excess Tbx5 into zebrafish embryos and mouse fibroblasts doubles CaMK-II expression. We conclude that beta CaMK-II expression and activity are necessary for proper cardiac and limb morphogenesis. These findings not only identify a morphogenic target for Ca(2+) during heart development, but support implied roles for CaMK-II in adult heart remodeling.
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Tbx5-mediated expression of Ca2+/calmodulin-dependent protein kinase II is necessary for zebrafish cardiac and pectoral fin morphogenesis
Developmental Biology, 2009Co-Authors: Sarah C. Rothschild, Charles A. Easley, Ludmila Francescatto, James A. Lister, Deborah M. Garrity, Robert M. TombesAbstract:Abstract Mutations in the T-box transcription factor, TBX5, result in Holt–Oram syndrome (HOS), a human condition in which cardiac development is defective and forelimbs are stunted. Similarly, zebrafish tbx5 Morphants and mutants (heartstrings; hst) lack pectoral fins and exhibit a persistently elongated heart that does not undergo chamber looping. Tbx5 is expressed in the developing atrium, ventricle and in pectoral fin fields, but its genetic targets are still being uncovered. In this study, evidence is provided that Tbx5 induces the expression of a specific member of the CaMK-II (the type II multifunctional Ca2+/calmodulin-dependent protein kinase) family; this CaMK-II is necessary for proper heart and fin development. Morphants of β2 CaMK-II (camk2b2), but not the β1 CaMK-II (camk2b1) paralog, exhibit bradycardia, elongated hearts and diminished pectoral fin development. Normal cardiac phenotypes can be restored by ectopic cytosolic CaMK-II expression in tbx5 Morphants. Like tbx5, camk2b2 is expressed in the pectoral fin and looping heart, but this expression is diminished in both tbx5 Morphant and hst embryos. Conversely, the introduction of excess Tbx5 into zebrafish embryos and mouse fibroblasts doubles CaMK-II expression. We conclude that β CaMK-II expression and activity are necessary for proper cardiac and limb morphogenesis. These findings not only identify a morphogenic target for Ca2+ during heart development, but support implied roles for CaMK-II in adult heart remodeling.
Yau-hung Chen - One of the best experts on this subject based on the ideXlab platform.
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von Hippel–Lindau gene plays a role during zebrafish pronephros development
In Vitro Cellular & Developmental Biology - Animal, 2015Co-Authors: Yau-hung Chen, Yu Ju Ding, Chiung-fang Chang, Jen-ning TsaiAbstract:von Hippel-Lindau (pVHL)-mediated ubiquitination of HIF-1α plays a central role in the cellular responses to changes in oxygen availability. In the present study, using zebrafish as a model, we showed that specific knockdown of endogenous vhl leads to pronephros malformation and renal failure. Knockdown of vhl resulted in abnormal kidney development, including curved and cystic pronephric tubule or/and cystic and atrophic glomerulus. Co-injecting capped vhl messenger RNA (mRNA) partially rescued pronephros Morphant phenotype, confirming the specificity of the morpholino oligonucleotide (MO)-induced pronephric defects. In keeping with the pronephros phenotype, renal function was affected as well in vhl Morphants. Dextran clearance abilities of vhl Morphants were significantly reduced as compared with those of control embryos. Further analysis indicated that glomerular integrity is impaired in vhl Morphants, while the organization of pronephric duct was minimally affected. Vhl Morphants display global increased vegf signaling and angiogenesis. In addition, we found that vhl Morphants displayed elevated expression of vegfa in podocytes and increased angiogenesis at pronephric glomerulus and the nearby vessels. Treatment of vegf inducer to embryos also caused pronephros phenotype resembling vhl Morphants, further supporting that increased vegfa signaling contribute to the pronephros Morphant phenotype. Our study establishes the zebrafish as an alternative vertebrate model system for studying Vhl function during kidney development.
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von Hippel-Lindau gene plays a role during zebrafish pronephros development.
In Vitro Cellular & Developmental Biology - Animal, 2015Co-Authors: Yau-hung Chen, Yu Ju Ding, Chiung-fang Chang, Yen-yu Lai, Chiao-yin Sun, Jen-ning TsaiAbstract:von Hippel-Lindau (pVHL)-mediated ubiquitination of HIF-1α plays a central role in the cellular responses to changes in oxygen availability. In the present study, using zebrafish as a model, we showed that specific knockdown of endogenous vhl leads to pronephros malformation and renal failure. Knockdown of vhl resulted in abnormal kidney development, including curved and cystic pronephric tubule or/and cystic and atrophic glomerulus. Co-injecting capped vhl messenger RNA (mRNA) partially rescued pronephros Morphant phenotype, confirming the specificity of the morpholino oligonucleotide (MO)-induced pronephric defects. In keeping with the pronephros phenotype, renal function was affected as well in vhl Morphants. Dextran clearance abilities of vhl Morphants were significantly reduced as compared with those of control embryos. Further analysis indicated that glomerular integrity is impaired in vhl Morphants, while the organization of pronephric duct was minimally affected. Vhl Morphants display global increased vegf signaling and angiogenesis. In addition, we found that vhl Morphants displayed elevated expression of vegfa in podocytes and increased angiogenesis at pronephric glomerulus and the nearby vessels. Treatment of vegf inducer to embryos also caused pronephros phenotype resembling vhl Morphants, further supporting that increased vegfa signaling contribute to the pronephros Morphant phenotype. Our study establishes the zebrafish as an alternative vertebrate model system for studying Vhl function during kidney development.
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Inhibition of the P2X7 receptor reduces cystogenesis in PKD.
Journal of the American Society of Nephrology, 2011Co-Authors: Ming-yang Chang, Yau-hung Chen, Ya Chung Tian, Yung Chang Chen, Cheng Chieh Hung, Yihui Huang, Chih-wei Yang, Yi-chuan ChengAbstract:The P2X7 receptor participates in purinergic signaling, which may promote the progression of ADPKD. We examined the effects of a P2X7 receptor antagonist and a P2X7 receptor agonist on cyst development in a zebrafish model of polycystic kidney disease in which we knocked down pkd2 by morpholinos. We used live wt-1b pronephric-specific GFP-expressing zebrafish embryos to directly observe changes in the pronephros. Exposure of pkd2-Morphant zebrafish to a P2X7 receptor antagonist (oxidized ATP [OxATP]) significantly reduced the frequency of the cystic phenotype compared with either exposure to a P2X7 receptor agonist (BzATP) or with no treatment (P < 0.01). Histology confirmed improvement of glomerular cysts in OxATP-treated pkd2 Morphants. OxATP also reduced p-ERK activity and cell proliferation in pronephric kidneys in pkd2 Morphants. Inhibition of P2X7 with an additional specific antagonist (A-438079), and through morpholino-mediated knockdown of p2rx7, confirmed these effects. In conclusion, blockade of the P2X7 receptor reduces cyst formation via ERK-dependent pathways in a zebrafish model of polycystic kidney disease, suggesting that P2X7 antagonists may have therapeutic potential in ADPKD.
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Novel and unexpected functions of zebrafish CCAAT box binding transcription factor (NF-Y) B subunit during cartilages development.
Bone, 2009Co-Authors: Yau-hung Chen, Yung Tsang Lin, Gang Hui LeeAbstract:Abstract We used zebrafish as a model to study the biological functions of NF-YB during early development. Both RT-PCR and whole-mount in situ hybridization experiments revealed that nf-yb was a maternally inherited gene. Later, its expression was restricted in the future head cartilages as well as in the developing notochord. Embryos after injection with nf-yb -morpholino displayed reduced-head phenotypes, including smaller head (WT, length of head, L: 0.515 ± 0.019 mm, width of head, W: 0.323 ± 0.077 mm; nf-yb -Morphant, L: 0.347 ± 0.037 mm; W: 0.266 ± 0.018 mm), sharpen Meckel's cartilage, loss of ceratobranchial, and enlarged angles of ceratohyal (WT: 72.6 ± 9.4°; nf-yb -Morphant: 110.0 ± 32.5°). Subsequently, those abnormalities can be rescued after injection with capped nf-yb mRNA. TUNEL assay suggested that large amounts of cell apoptosis appeared in the head region of nf-yb -Morphants. Staining with digoxigenin-labeled dlx 2a, sox 9a, runx2b and col 2a1 riboprobes showed that nf-yb -Morphants displayed reduced amounts of cranial neural crest cells which are required for mandibular and branchial arches formation. These observations clearly indicate that knockdown of nf-yb translation induced parts of cranial neural crest cells apoptosis, affected cartilages formation and consequently caused reduced-head phenotypes. These findings uncover a novel and unexpected role for NF-YB as a critical modulator of neural crest cell's gene expression governing embryonic cartilage growth.
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foxd3 mediates zebrafish myf5 expression during early somitogenesis
Developmental Biology, 2006Co-Authors: Hungchieh Lee, Yau-hung Chen, Hsingyen Huang, Chengyung Lin, Huaijen TsaiAbstract:Myf5, one of the basic helix-loop-helix transcription factors, controls muscle differentiation and is expressed in somites during early embryogenesis. However, the transcription factors bound to the cis-elements of myf5 are poorly understood. In this study, we used the yeast one-hybrid assay and found that Forkhead box d3 (Foxd3) interacted specifically with the -82/-62 cassette, a key element directing somite-specific expression of myf5. The dual-luciferase assay revealed that the expression of Foxd3 potently transactivated the myf5 promoter. Knocking down foxd3 with morpholino oligonucleotide (MO) resulted in a dramatic down-regulation of myf5 in somites and adaxial cells but not in the presomitic mesoderm. On the other hand, myod expression remained unchanged in foxd3 Morphants. Foxd3 mediation of myf5 expression is stage-dependent, maintaining myf5 expression in the somites and adaxial cells during the 7- to 18-somite stage. Furthermore, in the pax3 Morphant, the expression of foxd3 was down-regulated greatly and the expression of myf5 was similar to that of the foxd3 Morphant. Co-injection of foxd3 mRNA and pax3-MO1 greatly restored the expression of myf5 in the somites and adaxial cells, suggesting that pax3 induces foxd3 expression, which then induces the expression of myf5. This report is the first study to show that Foxd3, a well-known regulator in neural crest development, is also involved in myf5 regulation.