Xiaojun Zhu

Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos

Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrpy11 mutant embryos or cardia bifida phenotypes in fautm236a mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.

Egr-1 target genes in human endothelial cells identified by microarray analysis.

Early growth response factor 1 (Egr-1) is a key transcriptional factor to mediate gene expression after vascular injury. To better understand the role of Egr-1 in vasculature, we globally profiled Egr-1 target genes in human endothelial cells using adenoviral gene transfer and Affymetrix oligonucleotide-based microarray technology. More than 300 genes regulated by >/=3-fold with Egr-1 overexpression were identified and, partially, confirmed by Northern and Western blotting, including genes for transcriptional regulators, signaling proteins, cell cycle regulatory proteins, growth factors, and cytokines. Among them, thymus-expressed chemokine (TECK) and IP-30 were dramatically induced by Egr-1, but TNFalpha-related apoptosis inducing ligand (TRAIL) was significantly repressed by Egr-1, suggesting that Egr-1 is a key mediator of inflammation and apoptosis in vascular cells. These data provide novel Egr-1 target genes and contribute to the understanding of the role of Egr-1 in vasculature.

Rad As a Novel Regulator of Excitation–Contraction Coupling and β-Adrenergic Signaling in Heart

Rationale: Rad (Ras associated with diabetes) GTPase, a monomeric small G protein, binds to Cav&bgr; subunit of the L-type Ca2+ channel (LCC) and thereby regulates LCC trafficking and activity. Emerging evidence suggests that Rad is an important player in cardiac arrhythmogenesis and hypertrophic remodeling. However, whether and how Rad involves in the regulation of excitation–contraction (EC) coupling is unknown. Objective: This study aimed to investigate possible role of Rad in cardiac EC coupling and &bgr;-adrenergic receptor (&bgr;AR) inotropic mechanism. Methods and Results: Adenoviral overexpression of Rad by 3-fold in rat cardiomyocytes suppressed LCC current (ICa), [Ca2+]i transients, and contractility by 60%, 42%, and 38%, respectively, whereas the “gain” function of EC coupling was significantly increased, due perhaps to reduced “redundancy” of LCC in triggering sarcoplasmic reticulum release. Conversely, ≈70% Rad knockdown by RNA interference increased ICa (50%), [Ca2+]i transients (52%) and contractility (58%) without altering EC coupling efficiency; and the dominant negative mutant RadS105N exerted a similar effect on ICa. Rad upregulation caused depolarizing shift of LCC activation and hastened time-dependent LCC inactivation; Rad downregulation, however, failed to alter these attributes. The Na+/Ca2+ exchange activity, sarcoplasmic reticulum Ca2+ content, properties of Ca2+ sparks and propensity for Ca2+ waves all remained unperturbed regardless of Rad manipulation. Rad overexpression, but not knockdown, negated &bgr;AR effects on ICa and Ca2+ transients. Conclusion: These results establish Rad as a novel endogenous regulator of cardiac EC coupling and &bgr;AR signaling and support a parsimonious model in which Rad buffers Cav&bgr; to modulate LCC activity, EC coupling, and &bgr;AR responsiveness.

Hydrogen peroxide primes heart regeneration with a derepression mechanism

While the adult human heart has very limited regenerative potential, the adult zebrafish heart can fully regenerate after 20% ventricular resection. Although previous reports suggest that developmental signaling pathways such as FGF and PDGF are reused in adult heart regeneration, the underlying intracellular mechanisms remain largely unknown. Here we show that H2O2 acts as a novel epicardial and myocardial signal to prime the heart for regeneration in adult zebrafish. Live imaging of intact hearts revealed highly localized H2O2 (∼30 μM) production in the epicardium and adjacent compact myocardium at the resection site. Decreasing H2O2 formation with the Duox inhibitors diphenyleneiodonium (DPI) or apocynin, or scavenging H2O2 by catalase overexpression markedly impaired cardiac regeneration while exogenous H2O2 rescued the inhibitory effects of DPI on cardiac regeneration, indicating that H2O2 is an essential and sufficient signal in this process. Mechanistically, elevated H2O2 destabilized the redox-sensitive phosphatase Dusp6 and hence increased the phosphorylation of Erk1/2. The Dusp6 inhibitor BCI achieved similar pro-regenerative effects while transgenic overexpression of dusp6 impaired cardiac regeneration. H2O2 plays a dual role in recruiting immune cells and promoting heart regeneration through two relatively independent pathways. We conclude that H2O2 potentially generated from Duox/Nox2 promotes heart regeneration in zebrafish by unleashing MAP kinase signaling through a derepression mechanism involving Dusp6.

Rad GTPase inhibits cardiac fibrosis through connective tissue growth factor

AIMS Our previous studies documented that Rad (Ras associated with diabetes), a member of the RGK (Rad, Gem, and Kir) family of Ras-related small G protein, is significantly decreased in human failing hearts and plays an important role in attenuating cardiac hypertrophy. The goal of this study is to identify the effect of Rad on cardiac fibrosis and the underlying mechanisms. METHODS AND RESULTS Rad knockout (KO) mice showed more severe cardiac fibrosis compared with wild-type littermate controls as detected by Sirius Red staining. Western blot analyses demonstrated that the expression of connective tissue growth factor (CTGF), a key mediator of fibrosis, increased dramatically in Rad KO mice. Overexpression of Rad in cultured neonatal cardiomyocytes suppressed both basal and transforming growth factor-β1-induced CTGF expression. Elevated CTGF expression was observed in cardiomyocytes when Rad was reduced by RNA interference. Moreover, cardiac fibroblasts produced greater extracellular matrix (ECM) when stimulated with conditioned medium from Rad-knockdown cardiomyocytes. ECM production was completely abolished by adding a CTGF-neutralizing antibody into the medium. CCAAT/enhancer-binding protein δ (C/EBP-δ) was demonstrated to activate CTGF in cardiomyocytes. Chromatin immunoprecipitation assay and co-immunoprecipitation further demonstrated that Rad inhibited the binding of C/EBP-δ to the CTGF promoter via direct interaction with C/EBP-δ. CONCLUSION Our data reveal that Rad deficiency can lead to cardiac fibrosis. Rad inhibits CTGF expression through binding with C/EBP-δ, thus regulating ECM production in the heart. This study suggests a potential link between decreased Rad levels and increased cardiac fibrosis in human failing hearts.

Interferon Regulatory Factor-1 Mediates PPARγ-Induced Apoptosis in Vascular Smooth Muscle Cells

Objective—Peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;) possesses general beneficial effects on the cardiovascular system, such as inhibition of vascular lesion formation and atherosclerosis. However, molecular mechanisms for these effects are yet to be fully defined. The aim of this study is to elucidate whether interferon regulatory factor-1 (IRF-1), a transcriptional factor with anti-proliferative and pro-apoptotic properties, mediates PPAR&ggr;-induced apoptosis in vascular smooth muscle cells (VSMCs). Methods and Results—Using Northern and Western blot analyses, we documented that PPAR&ggr; ligands, including ciglitazone, troglitazone, and GW7845, significantly increased IRF-1 expression in VSMCs; however, the PPAR&agr; ligand (Wy14643) and PPAR&dgr; ligand (GW0742) did not affect its expression. PPAR&ggr;-induced IRF-1 expression was abrogated by pretreatment with the PPAR&ggr; antagonist GW9662. In contrast, adenoviral expression of PPAR&ggr; in VSMCs dramatically increased IRF-1 level. Furthermore, PPAR&ggr; activation increased IRF-1 promoter activity but did not affect IRF-1 mRNA stability. Finally, reducing IRF-1 expression by antisense technology attenuated PPAR&ggr;-induced VSMC apoptosis through decreasing cyclin-dependent kinase inhibitor p21 cip1 and caspase-3 activity. Conclusion—Our data demonstrate that IRF-1 is a novel PPAR&ggr; target gene and mediates PPAR&ggr;-induced VSMC apoptosis.

Interferon regulatory factor-1 mediates PPARgamma-induced apoptosis in vascular smooth muscle cells.

Objective—Peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;) possesses general beneficial effects on the cardiovascular system, such as inhibition of vascular lesion formation and atherosclerosis. However, molecular mechanisms for these effects are yet to be fully defined. The aim of this study is to elucidate whether interferon regulatory factor-1 (IRF-1), a transcriptional factor with anti-proliferative and pro-apoptotic properties, mediates PPAR&ggr;-induced apoptosis in vascular smooth muscle cells (VSMCs). Methods and Results—Using Northern and Western blot analyses, we documented that PPAR&ggr; ligands, including ciglitazone, troglitazone, and GW7845, significantly increased IRF-1 expression in VSMCs; however, the PPAR&agr; ligand (Wy14643) and PPAR&dgr; ligand (GW0742) did not affect its expression. PPAR&ggr;-induced IRF-1 expression was abrogated by pretreatment with the PPAR&ggr; antagonist GW9662. In contrast, adenoviral expression of PPAR&ggr; in VSMCs dramatically increased IRF-1 level. Furthermore, PPAR&ggr; activation increased IRF-1 promoter activity but did not affect IRF-1 mRNA stability. Finally, reducing IRF-1 expression by antisense technology attenuated PPAR&ggr;-induced VSMC apoptosis through decreasing cyclin-dependent kinase inhibitor p21 cip1 and caspase-3 activity. Conclusion—Our data demonstrate that IRF-1 is a novel PPAR&ggr; target gene and mediates PPAR&ggr;-induced VSMC apoptosis.

Mecp2 regulates neural cell differentiation by suppressing the Id1 to Her2 axis in zebrafish

ABSTRACT Rett syndrome (RTT) is a progressive neurological disorder caused by mutations in the X-linked protein methyl-CpG-binding protein 2 (MeCP2). The endogenous function of MeCP2 during neural differentiation is still unclear. Here, we report that mecp2 is required for brain development in zebrafish. Mecp2 was broadly expressed initially in embryos and enriched later in the brain. Either morpholino knockdown or genetic depletion of mecp2 inhibited neuronal differentiation, whereas its overexpression promoted neuronal differentiation, suggesting an essential role of mecp2 in directing neural precursors into differentiated neurons. Mechanistically, her2 (the zebrafish ortholog of mammalian Hes5) was upregulated in mecp2 morphants in an Id1-dependent manner. Moreover, knockdown of either her2 or id1 fully rescued neuronal differentiation in mecp2 morphants. These results suggest that Mecp2 plays an important role in neural cell development by suppressing the Id1–Her2 axis, and provide new evidence that embryonic neural defects contribute to the later motor and cognitive dysfunctions in RTT. Summary: Mecp2 suppresses neural cell development by suppressing the Id1–Her2 (Hes5 in mammals) axis, which is a mechanism that might contribute to the later motor and cognitive dysfunctions in Rett syndrome.

Chromatin-remodelling factor Brg1 regulates myocardial proliferation and regeneration in zebrafish.

The zebrafish possesses a remarkable capacity of adult heart regeneration, but the underlying mechanisms are not well understood. Here we report that chromatin remodelling factor Brg1 is essential for adult heart regeneration. Brg1 mRNA and protein are induced during heart regeneration. Transgenic over-expression of dominant-negative Xenopus Brg1 inhibits the formation of BrdU+/Mef2C+ and Tg(gata4:EGFP) cardiomyocytes, leading to severe cardiac fibrosis and compromised myocardial regeneration. RNA-seq and RNAscope analyses reveal that inhibition of Brg1 increases the expression of cyclin-dependent kinase inhibitors such as cdkn1a and cdkn1c in the myocardium after ventricular resection; and accordingly, myocardial-specific expression of dn-xBrg1 blunts myocardial proliferation and regeneration. Mechanistically, injury-induced Brg1, via its interaction with Dnmt3ab, suppresses the expression of cdkn1c by increasing the methylation level of CpG sites at the cdkn1c promoter. Taken together, our results suggest that Brg1 promotes heart regeneration by repressing cyclin-dependent kinase inhibitors partly through Dnmt3ab-dependent DNA methylation.

Remodeling of Mitochondrial Flashes in Muscular Development and Dystrophy in Zebrafish

Mitochondrial flash (mitoflash) is a highly-conserved, universal, and physiological mitochondrial activity in isolated mitochondria, intact cells, and live organisms. Here we investigated developmental and disease-related remodeling of mitoflash activity in zebrafish skeletal muscles. In transgenic zebrafish expressing the mitoflash reporter cpYFP, in vivo imaging revealed that mitoflash frequency and unitary properties underwent multiphasic and muscle type-specific changes, accompanying mitochondrial morphogenesis from 2 to 14 dpf. In particular, short (S)-type mitoflashes predominated in early muscle formation, then S-, transitory (T)- and regular (R)-type mitoflashes coexisted during muscle maturation, followed by a switch to R-type mitoflashes in mature skeletal muscles. In early development of muscular dystrophy, we found accelerated S- to R-type mitoflash transition and reduced mitochondrial NAD(P)H amidst a remarkable cell-to-cell heterogeneity. This study not only unravels a profound functional and morphological remodeling of mitochondria in developing and diseased skeletal muscles, but also underscores mitoflashes as a useful reporter of mitochondrial function in milieu of live animals under physiological and pathophysiological conditions.