Improving adenine and dual base editors through introduction of TadA-8e and Rad51DBD.

Base editors, including dual base editors, are innovative techniques for efficient base conversions in genomic DNA. However, the low efficiency of A-to-G base conversion at positions proximal to the protospacer adjacent motif (PAM) and the A/C simultaneous conversion of the dual base editor hinder their broad applications. In this study, through fusion of ABE8e with Rad51 DNA-binding domain, we generate a hyperactive ABE (hyABE) which offers improved A-to-G editing efficiency at the region (A10-A15) proximal to the PAM, with 1.2- to 7-fold improvement compared to ABE8e. Similarly, we develop optimized dual base editors (eA&C-BEmax and hyA&C-BEmax) with markedly improved simultaneous A/C conversion efficiency (1.2-fold and 1.5-fold improvement, respectively) compared to A&C-BEmax in human cells. Moreover, these optimized base editors catalyze efficiently nucleotide conversions in zebrafish embryos to mirror human syndrome or in human cells to potentially treat genetic diseases, indicating their great potential in broad applications for disease modeling and gene therapy.

Induction of Wnt signaling antagonists and p21-activated kinase enhances cardiomyocyte proliferation during zebrafish heart regeneration.

Heart regeneration occurs by dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). However, the signaling mechanisms by which injury induces CM renewal remain incompletely understood. Here, we find that cardiac injury in zebrafish induces expression of the secreted Wnt inhibitors, including Dickkopf 1 (Dkk1), Dkk3, secreted Frizzled-related protein 1 (sFrp1), and sFrp2, in cardiac tissue adjacent to injury sites. Experimental blocking of Wnt activity via Dkk1 overexpression enhances CM proliferation and heart regeneration, whereas ectopic activation of Wnt8 signaling blunts injury-induced CM dedifferentiation and proliferation. Although Wnt signaling is dampened upon injury, the cytoplasmic ß-catenin is unexpectedly increased at disarrayed CM sarcomeres in myocardial wound edges. Our analyses indicated that p21-activated kinase 2 (Pak2) is induced at regenerating CMs, where it phosphorylates cytoplasmic ß-catenin at Ser 675 and increases its stability at disassembled sarcomeres. Myocardial-specific induction of the phospho-mimetic ß-catenin (S675E) enhances CM dedifferentiation and sarcomere disassembly in response to injury. Conversely, inactivation of Pak2 kinase activity reduces the Ser 675-phosphorylated ß-catenin (pS675-ß-catenin) and attenuates CM sarcomere disorganization and dedifferentiation. Taken together, these findings demonstrate that coordination of Wnt signaling inhibition and Pak2/pS675-ß-catenin signaling enhances zebrafish heart regeneration by supporting CM dedifferentiation and proliferation.

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.

Tbx20 Induction Promotes Zebrafish Heart Regeneration by Inducing Cardiomyocyte Dedifferentiation and Endocardial Expansion.

Heart regeneration requires replenishment of lost cardiomyocytes (CMs) and cells of the endocardial lining. However, the signaling regulation and transcriptional control of myocardial dedifferentiation and endocardial activation are incompletely understood during cardiac regeneration. Here, we report that T-Box Transcription Factor 20 (Tbx20) is induced rapidly in the myocardial wound edge in response to various sources of cardiac damages in zebrafish. Inducing Tbx20 specifically in the adult myocardium promotes injury-induced CM proliferation through CM dedifferentiation, leading to loss of CM cellular contacts and re-expression of cardiac embryonic or fetal gene programs. Unexpectedly, we identify that myocardial Tbx20 induction activates the endocardium at the injury site with enhanced endocardial cell extension and proliferation, where it induces the endocardial Bone morphogenetic protein 6 (Bmp6) signaling. Pharmacologically inactivating endocardial Bmp6 signaling reduces expression of its targets, Id1 and Id2b, attenuating the increased endocardial regeneration in tbx20-overexpressing hearts. Altogether, our study demonstrates that Tbx20 induction promotes adult heart regeneration by inducing cardiomyocyte dedifferentiation as well as non-cell-autonomously enhancing endocardial cell regeneration.

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.

Phosphorylation of angiomotin by Lats1/2 kinases inhibits F-actin binding, cell migration, and angiogenesis.

The Hippo tumor suppressor pathway plays important roles in organ size control through Lats1/2 mediated phosphorylation of the YAP/TAZ transcription co-activators. However, YAP/TAZ independent functions of the Hippo pathway are largely unknown. Here we report a novel role of the Hippo pathway in angiogenesis. Angiomotin p130 isoform (AMOTp130) is phosphorylated on a conserved HXRXXS motif by Lats1/2 downstream of GPCR signaling. Phosphorylation disrupts AMOT interaction with F-actin and correlates with reduced F-actin stress fibers and focal adhesions. Furthermore, phosphorylation of AMOT by Lats1/2 inhibits endothelial cell migration in vitro and angiogenesis in zebrafish embryos in vivo. Thus AMOT is a direct substrate of Lats1/2 mediating functions of the Hippo pathway in endothelial cell migration and angiogenesis.