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 Ͻ 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.

Zebrafish tbx5 expresses in the heart, pectoral fins and eyes of zebrafish during embryonic development. In zebrafish, injection of tbx5 morpholino antisense RNA caused changes of heart conformation, defect of heart looping, pericardium effusion, dropsy of ventral position and decreased heart rate. We suggested that cardiac myogenesis genes might be responsible for this phenomenon. Morpholino antisense RNA which against the initiation site of tbx5 gene was designed in order to knockdown the expression of tbx5, and the results were analyzed by whole-mount in situ hybridization and quantitative real-time PCR. Expression of cardiac myogenesis genes amhc, vmhc and cmlc2 were expressed constantly at the early embryonic development and reached its highest rate right before cardiac looping initiated. These cardiac myogenesis genes showed insufficient expressions within different heart defect embryos. Moreover, vmhc showed ectopic expression in addition to heart looping defect in heart defective embryos at 36 hpf. Our data suggests that the heart failure caused by the knockdown of tbx5 gene might result from the down-regulation of cardiac myogenesis genes.

BackgroundTbx5 deficiency in zebrafish causes several abnormal phenotypes of the heart and pectoral fins. It has been reported that exogenous human growth hormone can enhance expression of downstream mediators in the growth hormone and insulin-like growth factor I (IGF-I) pathway and partially restore dysmorphogenesis in tbx5 morphants. This study aimed to further evaluate the effects of IGF-I on cell apoptosis and dysmorphogenesis in zebrafish embryos deficient for tbx5.ResultsAmong the five studied groups of zebrafish embryos (wild-type embryos [WT], tbx5 morphants [MO], mismatched tbx5 morpholino-treated wild-type embryos [MIS], IGF-I-treated wild-type embryos [WTIGF1], and IGF-I-treated tbx5 morphants [MOIGF1]), the expression levels of the ifg1, igf1-ra, ifg-rb, erk1, and akt2 genes as well as the ERK and AKT proteins were significantly reduced in the MO group, but were partially restored in the MOIGF1 group. These expression levels remained normal in the WT, MIS, and WTIGF1 groups. Exogenous human IGF-I also reduced the incidence of phenotypic anomalies, decreased the expression levels of apoptotic genes and proteins, suppressed cell apoptosis, and improved survival of the MOIGF1 group.ConclusionsThese results suggest that IGF-I has an anti-apoptotic protective effect in zebrafish embryos with tbx5 deficiency.

Functional knockdown of zebrafish tbx5a causes hypoplasia or aplasia of pectoral fins. This study aimed to assess developmental pectoral fin anomalies in tbx5a morpholino knockdown zebrafish embryos. The expression of cartilage-related genes in the tbx5a morphant was analyzed by DNA microarray, immunostaining, and thin-section histology to examine the detailed distribution of the extracellular matrix (ECM) during different pectoral fin developmental stages. Chondrogenic condensation (CC) in the tbx5a morpholino knockdown group was barely recognizable at 37 h postfertilization (hpf); the process from CC to endoskeleton formation was disrupted at 48 hpf, and the endoskeleton was only loosely formed at 72 hpf. Microarrays identified 18 downregulated genes in tbx5a-deficient embryos, including 2 fin morphogenesis-related (cx43, bbs7), 4 fin development-related (hoxc8a, hhip, axin1, msxb), and 12 cartilage development-related (mmp14a, sec23b, tfap2a, slc35b2, dlx5a, dlx1a, tfap2b, fmr1, runx3, cdh2, lect1, acvr2a, mmp14b) genes, at 24 and 30 hpf. The increase in apoptosis-related proteins (BAD and BCL2) in the tbx5a morphant influenced the cellular component of pectoral fins and resulted in chondrocyte reduction throughout the different CC phases. Furthermore, tbx5a knockdown interfered with ECM formation in pectoral fins, affecting glycosaminoglycans, fibronectin, hyaluronic acid (HA), and N-cadherin. Our results provide evidence that the pectoral fin phenotypic anomaly induced by tbx5a knockdown is related to disruption of the mesoderm and ECM, consequently interfering with mesoderm migration, CC, and subsequent endoskeleton formation.