Ekker, Stephen C

Vasculogenesis and angiogenesis are controlled by vascular permeability factor/vascular endothelial growth factor A (VEGF). Dysregulation of these physiological processes contributes to the pathologies of heart disease, cancer and stroke. Rho GTPase proteins play an integral role in VEGF-mediated formation and maintenance of blood vessels. The regulatory functions of RhoA and RhoB in vasculogenesis and angiogenesis are well defined, whereas the purpose of RhoC remains poorly understood. Here, we describe how RhoC promotes vascular homeostasis by modulating endothelial cell migration, proliferation, and permeability. RhoC stimulates proliferation of human umbilical vein endothelial cells (HUVEC) by stabilizing nuclear β-catenin, which promotes transcription of cyclin D1 and subsequently drives cell cycle progression. RhoC negatively regulates endothelial cell migration through MAPK and downstream MLC-2 signaling and decreases vascular permeability through downregulation of the phospholipase Cγ (PLCγ)/Ca2+/eNOS cascade in HUVEC. Using a VEGF-inducible zebrafish (Danio rerio) model, we observed significantly less vascular permeability in RhoC morpholino (MO)-injected zebrafish than control MO-injected zebrafish. Taken together, our findings suggest RhoC is a key regulator of vascular homeostasis in endothelial cells.

Recent advances in the targeted modification of complex eukaryotic genomes have unlocked a new era of genome engineering. From the pioneering work using zinc-finger nucleases (ZFNs), to the advent of the versatile and specific TALEN systems, and most recently the highly accessible CRISPR/Cas9 systems, we now possess an unprecedented ability to analyze developmental processes using sophisticated designer genetic tools. In this Review, we summarize the common approaches and applications of these still-evolving tools as they are being used in the most popular model developmental systems. Excitingly, these robust and simple genomic engineering tools also promise to revolutionize developmental studies using less well established experimental organisms.

ABSTRACT HIV-1 utilizes the cellular protein LEDGF/p75 as a chromosome docking and integration cofactor. The LEDGF/p75 gene, PSIP1 , is a potential therapeutic target because, like CCR5, depletion of LEDGF/p75 is tolerated well by human CD4 + T cells, and knockout mice have normal immune systems. RNA interference (RNAi) has been useful for studying LEDGF/p75, but the potent cofactor activity of small protein residua can be confounding. Here, in human cells with utility for HIV research (293T and Jurkat), we used transcription activator-like effector nucleases (TALENs) to completely eradicate all LEDGF/p75 expression. We performed two kinds of PSIP1 knockouts: whole-gene deletion and deletion of the integrase binding domain (IBD)-encoding exons. HIV-1 integration was inhibited, and spreading viral replication was severely impaired in PSIP1 −/− Jurkat cells infected at high multiplicity. Furthermore, frameshifting the gene in the first coding exon with a single TALEN pair yielded trace LEDGF/p75 levels that were virologically active, affirming the cofactor's potency and the value of definitive gene or IBD exon segment deletion. Some recent studies have suggested that LEDGF/p75 may participate in HIV-1 assembly. However, we determined that assembly of infectious viral particles is normal in PSIP1 −/− cells. The potency of an allosteric integrase inhibitor, ALLINI-2, for rendering produced virions noninfectious was also unaffected by total eradication of cellular LEDGF/p75. We conclude that HIV-1 particle assembly and the main ALLINI mechanism are LEDGF/p75 independent. The block to HIV-1 propagation in PSIP1 −/− human CD4 + T cells raises the possibility of gene targeting PSIP1 combinatorially with CCR5 for HIV-1 cure. IMPORTANCE LEDGF/p75 dependence is universally conserved in the retroviral genus Lentivirus . Once inside the nucleus, lentiviral preintegration complexes are thought to attach to the chromosome when integrase binds to LEDGF/p75. This tethering process is largely responsible for the 2-fold preference for integration into active genes, but the cofactor's full role in the lentiviral life cycle is not yet clear. Effective knockdowns are difficult because even trace residua of this tightly chromatin-bound protein can support integration cofactor function. Here, in experimentally useful human cell lines, we used TALENs to definitively eradicate LEDGF/p75 by deleting either all of PSIP1 or the exons that code for the integrase binding domain. HIV-1 replication was severely impaired in these PSIP1 knockout cells. Experiments in these cells also excluded a role for LEDGF/p75 in HIV-1 assembly and showed that the main ALLINI mechanism is LEDGF/p75 independent. Site-specific gene targeting of PSIP1 may have therapeutic potential for HIV-1 disease.

Key Points Zebrafish flt3 plays an important role in the initiation of definitive hematopoietic stem cells. Expression of human FLT3-ITD activates endogenous flt3 signaling and induces myeloid expansion.

Recent advances in the burgeoning field of genome engineering are accelerating the realization of personalized therapeutics for cardiovascular disease. In the postgenomic era, sequence-specific gene-editing tools enable the functional analysis of genetic alterations implicated in disease. In partnership with high-throughput model systems, efficient gene manipulation provides an increasingly powerful toolkit to study phenotypes associated with patient-specific genetic defects. Herein, this review emphasizes the latest developments in genome engineering and how applications within the field are transforming our understanding of personalized medicine with an emphasis on cardiovascular diseases.

Abstract Zebrafish ( Danio rerio ) is a popular vertebrate model organism largely deployed using outbred laboratory animals. The nonisogenic nature of the zebrafish as a model system offers the opportunity to understand natural variations and their effect in modulating phenotype. In an effort to better characterize the range of natural variation in this model system and to complement the zebrafish reference genome project, the whole genome sequence of a wild zebrafish at 39-fold genome coverage was determined. Comparative analysis with the zebrafish reference genome revealed approximately 5.2 million single nucleotide variations and over 1.6 million insertion–deletion variations. This dataset thus represents a new catalog of genetic variations in the zebrafish genome. Further analysis revealed selective enrichment for variations in genes involved in immune function and response to the environment, suggesting genome-level adaptations to environmental niches. We also show that human disease gene orthologs in the sequenced wild zebrafish genome show a lower ratio of nonsynonymous to synonymous single nucleotide variations.

Rationale: Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ, such as the heart, especially those exhibiting adult phenotypes on depletion. Objective: We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes. Methods and Results: The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an insertional mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of 1 embryonic lethal, 1 larval lethal, and 1 adult recessive mutant exhibiting cardiac hypertrophy at 1 year of age. Further characterization of these mutants uncovered an essential function of methionine adenosyltransferase II, α a (mat2aa) in cardiogenesis, an essential function of mitochondrial ribosomal protein S18B (mrps18b) in cardiac mitochondrial homeostasis, as well as a function of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) in adult cardiac hypertrophy. Conclusions: We demonstrate that transposon-based gene trapping is an efficient approach for identifying both embryonic and adult recessive mutants with cardiac expression. The generation of a zebrafish insertional cardiac mutant collection shall facilitate the annotation of a vertebrate cardiac genome, as well as enable heart-based adult screens.

Abstract Integrated Science Education Outreach (InSciEd Out) is a collaboration formed between Mayo Clinic, Winona State University, and Rochester Public Schools (MN) with the shared vision of achieving excellence in science education. InSciEd Out employs an equitable partnership model between scientists, teachers, education researchers, and the community. Teams of teachers from all disciplines within a single school experience cutting-edge science using the zebrafish model system, as well as current pedagogical methods, during a summer internship at the Mayo Clinic. Within the internship, the teachers produce new curriculum that directly addresses opportunities for science education improvement at their own school. Zebrafish are introduced within the new curriculum to support a living model of the practice of science. Following partnership with the InSciEd Out program and 2 years of implementation in the classroom, teacher-interns from a K–8 public school reported access to local scientific technology and expertise they had not previously recognized. Teachers also reported improved integration of other disciplines into the scientific curriculum and a flow of concepts vertically from K through 8. Students more than doubled selection of an Honors science track in high school to nearly 90%. 98% of students who took the Minnesota Comprehensive Assessments in their 5 th and 8 th grade year (a span that includes 2 years of InSciEd Out) showed medium or high growth in science proficiency. These metrics indicate that cooperation between educators and scientists can result in positive change in student science proficiency and demonstrate that a higher expectation in science education can be achieved in US public schools.