The Gridlock transcriptional repressor impedes vertebrate heart regeneration by restricting expression of lysine methyltransferase.

Teleost zebrafish and neonatal mammalian hearts exhibit the remarkable capacity to regenerate through dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). Although many mitogenic signals that stimulate zebrafish heart regeneration have been identified, transcriptional programs that restrain injury-induced CM renewal are incompletely understood. Here, we report that mutations in gridlock (grl; also known as hey2), encoding a Hairy-related basic helix-loop-helix transcriptional repressor, enhance CM proliferation and reduce fibrosis following damage. In contrast, myocardial grl induction blunts CM dedifferentiation and regenerative responses to heart injury. RNA sequencing analyses uncover Smyd2 lysine methyltransferase (KMT) as a key transcriptional target repressed by Grl. Reduction in Grl protein levels triggered by injury induces smyd2 expression at the wound myocardium, enhancing CM proliferation. We show that Smyd2 functions as a methyltransferase and modulates the Stat3 methylation and phosphorylation activity. Inhibition of the KMT activity of Smyd2 reduces phosphorylated Stat3 at cardiac wounds, suppressing the elevated CM proliferation in injured grl mutant hearts. Our findings establish an injury-specific transcriptional repression program in governing CM renewal during heart regeneration, providing a potential strategy whereby silencing Grl repression at local regions might empower regeneration capacity to the injured mammalian heart.

Improved early development potence of in vitro fertilization embryos by treatment with tubacin increasing acetylated tubulin of matured porcine oocytes.

To improve the developmental potential of in vitro embryos is a long-term concern field for human assisted reproduction and animal in vitro embryo production practice. In the current study, we examined the effects and mechanism of an HDAC6 inhibitor, tubacin, on the maturation of porcine oocytes and in vitro development of porcine IVF embryos. It has been demonstrated the effect of tubacin on the acetylation level of α-tubulin in porcine oocytes. As a result, the maturation rate of porcine oocytes was significantly improved (P < 0.05), and the following development potent of blastocysts forming rate was also significantly increased (P < 0.05). We found that the increased acetylation of α-tubulin significantly reduced the abnormal rate of microtubule, furthermore, the proportion of mitochondria in the vicinity of in vitro fertilization (IVF) nucleus was significantly enhanced in Metaphase I (MI) and Metaphase II (MII) stages. The expression levels of microtubule assembly genes (TUBA1A, αTAT1 and MAP2) significantly up-regulated in MI and MII stages. Together, these results suggest that treatment of porcine oocytes during maturation with tubacin could promote their IVF embryos developmental competence by altering spindle formation, mitochondrial concentration and genes expression patterns of matured porcine oocytes.

Histone hyperacetylation may improve the preimplantation development and epigenetic status of cloned embryos.

The current study investigated the mechanism of mini pig fetal fibroblasts in improving the epigenetic modification and preimplantation development of cloned embryos. The results showed that the increased AcH3K14 level was dose- and time-dependent. Histone hyperacetylation had no significant effect on cell morphology, cell viability, cell cycle, and relative gene (HDAC1, HAT1, DNMT3A, and BAX) expression. The treated cloned embryos had significantly higher development rates and the total nuclei number than the control (27.62 ± 6.94 % vs. 16.14 ± 10.55 %; 43.90 ± 18.39 vs. 33.06 ± 15.87; P < 0.05). The AcH3K14 level in the treated cloned blastocysts was close to that of IVF blastocysts (5.17 ± 0.93 vs. 5.45 ± 1.91, P > 0.05). The gene transcription (CDX2 and OCT4) of the treated cloned blastocysts was significantly up-regulated than the control (3.32 ± 0.51 vs. 2.05 ± 0.30; 1.21 ± 0.18 vs. 0.81 ± 0.09; P < 0.05). The improvement in the cloned embryo development and the partial correction of abnormal acetylation modification were not necessarily related to the cellular characteristics. This could be caused by histone hyperacetylation of mini pig fetal fibroblasts.

The key long non-coding RNA screening and validation between germinal vesicle and metaphase II of porcine oocyte in vitro maturation.

Oocyte maturation plays a vitally important role in the reproduction of pigs. However, the roles of messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) in the developmental process of porcine oocyte maturation are still largely unclear. In this study, a transcriptome analysis of germinal vesicle (GV) and metaphase II (MII) of oocytes from Chinese Duroc pigs was performed. A total of 1,753,030 and 2,486 differentially expressed (DE) mRNAs, 22,811 and 9,868 DE lncRNAs were identified between GV and MII stages, respectively. Furthermore, functional enrichment analysis showed that the common DE mRNAs and DE lncRNAs during the process of maturation were mainly involved in biological process and cellular components. Our study provides new insights of the expression changes of mRNAs and lncRNAs during GV and MII stages, which might contribute to the maturation of oocytes. These results greatly improve our understanding of the molecular mechanisms regulating the maturation of oocytes in pigs.

Disruption of Abcc6 Transporter in Zebrafish Causes Ocular Calcification and Cardiac Fibrosis.

Pseudoxanthoma elasticum (PXE), caused by ABCC6/MRP6 mutation, is a heritable multisystem disorder in humans. The progressive clinical manifestations of PXE are accompanied by ectopic mineralization in various connective tissues. However, the pathomechanisms underlying the PXE multisystem disorder remains obscure, and effective treatment is currently available. In this study, we generated zebrafish abcc6a mutants using the transcription activator-like effector nuclease (TALEN) technique. In young adult zebrafish, abcc6a is expressed in the eyes, heart, intestine, and other tissues. abcc6a mutants exhibit extensive calcification in the ocular sclera and Bruch's membrane, recapitulating part of the PXE manifestations. Mutations in abcc6a upregulate extracellular matrix (ECM) genes, leading to fibrotic heart with reduced cardiomyocyte number. We found that abcc6a mutation reduced levels of both vitamin K and pyrophosphate (PPi) in the serum and diverse tissues. Vitamin K administration increased the gamma-glutamyl carboxylated form of matrix gla protein (cMGP), alleviating ectopic calcification and fibrosis in vertebrae, eyes, and hearts. Our findings contribute to a comprehensive understanding of PXE pathophysiology from zebrafish models.

Early development of porcine parthenogenetic embryos and reduced expression of primed pluripotent marker genes under the effect of lysophosphatidic acid.

To further promote the early development of porcine embryos and capture "naïve" pluripotent state within blastocyst, the experiment explored the effects of lysophosphatidic acid (LPA) on the early development of porcine parthenogenetic embryos and the expression of pluripotency relevant genes. The results showed that the addition of 50 µM LPA significantly improved parthenogenetic embryo cleavage rate (82.7% vs. 74.7%, p < 0.05), blastocyst rate (24.5% vs. 11.3%, p < 0.05) and blastocyst cell count (56 ± 7.9 vs. 42 ± 1.0, p < 0.05) than that of the control group. In addition, immunostaining experiment determined that the fluorescence intensity of OCT4 was also significantly higher than that of the control group. The quantitative real-time polymerase chain reaction (qRT-PCR) test revealed that addition of 50 µM LPA could significantly enhance the expression level of pluripotent gene OCT4 and trophoblast marker genes CDX2, however, decrease the expression of primitive hypoblast marker gene GATA4. The results also indicated that LPA might decrease the expression of GATA4 through the ROCK signalling pathway. For further investigating the effect of the addition of LPA on the expression of "primed" and "naïve" genes, we also detected the expression of those pluripotency-related genes by qRT-PCR. The results showed addition of LPA had no significant effect on the expression of "naïve" pluripotent genes, but it was able to significantly decrease the expression of "primed" pluripotent genes, NODAL and Activin-A; furthermore, it also could significantly improve the expression of OCT4 and c-Myc which act as two important ES cell renewal factors. Above all, the addition of LPA can facilitate the early development of porcine parthenogenetic embryos, which may be able to benefit for capturing "naïve" pluripotency in vitro through inhibiting "primed" pluripotency.