Spliceosomal protein eftud2 mutation leads to p53-dependent apoptosis in zebrafish neural progenitors.

Haploinsufficiency of EFTUD2 (Elongation Factor Tu GTP Binding Domain Containing 2) is linked to human mandibulofacial dysostosis, Guion-Almeida type (MFDGA), but the underlying cellular and molecular mechanisms remain to be addressed. We report here the isolation, cloning and functional analysis of the mutated eftud2 (snu114) in a novel neuronal mutant fn10a in zebrafish. This mutant displayed abnormal brain development with evident neuronal apoptosis while the development of other organs appeared less affected. Positional cloning revealed a nonsense mutation such that the mutant eftud2 mRNA encoded a truncated Eftud2 protein and was subjected to nonsense-mediated decay. Disruption of eftud2 led to increased apoptosis and mitosis of neural progenitors while it had little effect on differentiated neurons. Further RNA-seq and functional analyses revealed a transcriptome-wide RNA splicing deficiency and a large amount of intron-retaining and exon-skipping transcripts, which resulted in inadequate nonsense-mediated RNA decay and activation of the p53 pathway in fn10a mutants. Therefore, our study has established that eftud2 functions in RNA splicing during neural development and provides a suitable zebrafish model for studying the molecular pathology of the neurological disease MFDGA.

Isoform Evolution in Primates through Independent Combination of Alternative RNA Processing Events.

Recent RNA-seq technology revealed thousands of splicing events that are under rapid evolution in primates, whereas the reliability of these events, as well as their combination on the isoform level, have not been adequately addressed due to its limited sequencing length. Here, we performed comparative transcriptome analyses in human and rhesus macaque cerebellum using single molecule long-read sequencing (Iso-seq) and matched RNA-seq. Besides 359 million RNA-seq reads, 4,165,527 Iso-seq reads were generated with a mean length of 14,875 bp, covering 11,466 human genes, and 10,159 macaque genes. With Iso-seq data, we substantially expanded the repertoire of alternative RNA processing events in primates, and found that intron retention and alternative polyadenylation are surprisingly more prevalent in primates than previously estimated. We then investigated the combinatorial mode of these alternative events at the whole-transcript level, and found that the combination of these events is largely independent along the transcript, leading to thousands of novel isoforms missed by current annotations. Notably, these novel isoforms are selectively constrained in general, and 1,119 isoforms have even higher expression than the previously annotated major isoforms in human, indicating that the complexity of the human transcriptome is still significantly underestimated. Comparative transcriptome analysis further revealed 502 genes encoding selectively constrained, lineage-specific isoforms in human but not in rhesus macaque, linking them to some lineage-specific functions. Overall, we propose that the independent combination of alternative RNA processing events has contributed to complex isoform evolution in primates, which provides a new foundation for the study of phenotypic difference among primates.

RhesusBase PopGateway: Genome-Wide Population Genetics Atlas in Rhesus Macaque.

Although population genetics studies have significantly accelerated the evolutionary and functional interrogations of genes and regulations, limited polymorphism data are available for rhesus macaque, the model animal closely related to human. Here, we report the first genome-wide effort to identify and visualize the population genetics profile in rhesus macaque. On the basis of the whole-genome sequencing of 31 independent macaque animals, we profiled a comprehensive polymorphism map with 46,146,548 sites. The allele frequency for each polymorphism site, the haplotype structure, as well as multiple population genetics parameters were then calculated on a genome-wide scale. We further developed a specific interface, the RhesusBase PopGateway, to facilitate the visualization of these annotations, and highlighted the applications of this highly integrative platform in clarifying the selection signatures of genes and regulations in the context of the primate evolution. Overall, the updated RhesusBase provides a comprehensive monkey population genetics framework for in-depth evolutionary studies of human biology.

Evolutionary interrogation of human biology in well-annotated genomic framework of rhesus macaque.

With genome sequence and composition highly analogous to human, rhesus macaque represents a unique reference for evolutionary studies of human biology. Here, we developed a comprehensive genomic framework of rhesus macaque, the RhesusBase2, for evolutionary interrogation of human genes and the associated regulations. A total of 1,667 next-generation sequencing (NGS) data sets were processed, integrated, and evaluated, generating 51.2 million new functional annotation records. With extensive NGS annotations, RhesusBase2 refined the fine-scale structures in 30% of the macaque Ensembl transcripts, reporting an accurate, up-to-date set of macaque gene models. On the basis of these annotations and accurate macaque gene models, we further developed an NGS-oriented Molecular Evolution Gateway to access and visualize macaque annotations in reference to human orthologous genes and associated regulations (www.rhesusbase.org/molEvo). We highlighted the application of this well-annotated genomic framework in generating hypothetical link of human-biased regulations to human-specific traits, by using mechanistic characterization of the DIEXF gene as an example that provides novel clues to the understanding of digestive system reduction in human evolution. On a global scale, we also identified a catalog of 9,295 human-biased regulatory events, which may represent novel elements that have a substantial impact on shaping human transcriptome and possibly underpin recent human phenotypic evolution. Taken together, we provide an NGS data-driven, information-rich framework that will broadly benefit genomics research in general and serves as an important resource for in-depth evolutionary studies of human biology.

Genetic interaction between pku300 and fbn2b controls endocardial cell proliferation and valve development in zebrafish.

Abnormal cardiac valve morphogenesis is a common cause of human congenital heart disease. The molecular mechanisms regulating endocardial cell proliferation and differentiation into cardiac valves remain largely unknown, although great progress has been made on the endocardial contribution to the atrioventricular cushion and valve formation. We found that scotch tape(te382) (sco(te382)) encodes a novel transmembrane protein that is crucial for endocardial cell proliferation and heart valve development. The zebrafish sco(te382) mutant showed diminished endocardial cell proliferation, lack of heart valve leaflets and abnormal common cardinal and caudal veins. Positional cloning revealed a C946T nonsense mutation of a novel gene pku300 in the sco(te382) locus, which encoded a 540-amino-acid protein on cell membranes with one putative transmembrane domain and three IgG domains. A known G3935T missense mutation of fbn2b was also found ∼570 kb away from pku300 in sco(te382) mutants. The genetic mutant sco(pku300), derived from sco(te382), only had the C946T mutation of pku300 and showed reduced numbers of atrial endocardial cells and an abnormal common cardinal vein. Morpholino knockdown of fbn2b led to fewer atrial endocardial cells and an abnormal caudal vein. Knockdown of both pku300 and fbn2b phenocopied these phenotypes in sco(te382) genetic mutants. pku300 transgenic expression in endocardial and endothelial cells, but not myocardial cells, partially rescued the atrial endocardial defects in sco(te382) mutants. Mechanistically, pku300 and fbn2b were required for endocardial cell proliferation, endocardial Notch signaling and the proper formation of endocardial cell adhesion and tight junctions, all of which are crucial for cardiac valve development. We conclude that pku300 and fbn2b represent the few genes capable of regulating endocardial cell proliferation and signaling in zebrafish cardiac valve development.

Facilitating Clinical Trial Recruitment by Recommending Cost-Efficient Medical Exams.

Clinical trials are key and essential processes for researchers to develop new treatments as well as evaluate their effectiveness and safety, whilst more than half of all clinical trials experience delays, which leads to a considerable amount of cost. In this paper, we present a cost-effective framework to reduce the time and monetary cost in the stage of recruiting and screening eligible clinical trial participants. By leveraging patients' observed conditions and the cost of medical examinations, the proposed framework uses collaborative filtering techniques to predict the utilized cost for the to-do medical examinations and then rank patients and medical examinations. The preliminary experiment results indicate that the framework is promising to reduce the cost spent on medical examinations by three quarters or even more and accelerate the recruitment process in the screening stage.

Antimalarial drug artemisinin depletes erythrocytes by activating apoptotic pathways in zebrafish.

Despite its extraordinary efficacy, administration of the major antimalarial drug artemisinin leads to anemia, and the underlying cellular and molecular mechanisms are not well understood. Here, we report the effects of artemisinin on erythroid development and apoptosis in zebrafish and human cells. By performing a small-molecule screen with zebrafish embryos, we found that artemisinin treatment depleted red blood cells and slightly decreased definitive hematopoietic stem cells, but had no effect on primitive hematopoietic progenitors. RNA-Seq revealed that artemisinin suppressed a cluster of genes in the heme biosynthesis and globin synthesis pathways. Furthermore, artemisinin induced apoptosis in erythrocytes in zebrafish embryos, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and preferentially acted on differentiated erythrocytes by elevating caspase 8 and caspase 9 activity in differentiated human K562 cells. Consistently, artemisinin suppressed the ectopic expression of erythroid genes in jak2aV581F-injected embryos, a zebrafish model for human polycythemia vera in which the bone marrow makes too many red blood cells. Taken together, our data suggested that artemisinin, in addition to killing parasites, has a direct action on differentiated erythrocytes other than definitive hematopoietic stem cells and causes erythroid apoptosis by interfering with the heme biosynthesis pathway in zebrafish and human cells.

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.