Jeffrey J. Essner

In vivo genome editing using a high-efficiency TALEN system

The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.

CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature

Chemokines and chemokine receptors have been posited to have important roles in several common malignancies, including breast and lung cancer. Here, we demonstrate that CXCR7 (RDC1, CCX-CKR2), recently deorphanized as a chemokine receptor that binds chemokines CXCL11 and CXCL12, can regulate these two common malignancies. Using a combination of overexpression and RNA interference, we establish that CXCR7 promotes growth of tumors formed from breast and lung cancer cells and enhances experimental lung metastases in immunodeficient as well as immunocompetent mouse models of cancer. These effects did not depend on expression of the related receptor CXCR4. Furthermore, immunohistochemistry of primary human tumor tissue demonstrates extensive CXCR7 expression in human breast and lung cancers, where it is highly expressed on a majority of tumor-associated blood vessels and malignant cells but not expressed on normal vasculature. In addition, a critical role for CXCR7 in vascular formation and angiogenesis during development is demonstrated by using morpholino-mediated knockdown of CXCR7 in zebrafish. Taken together, these data suggest that CXCR7 has key functions in promoting tumor development and progression.

Conserved function for embryonic nodal cilia

How left–right handedness originates in the body plan of the developing vertebrate embryo is a subject of considerable debate. In mice, a left–right bias is thought to arise from a directional extracellular flow (nodal flow) that is generated by dynein-dependent rotation of monocilia on the ventral surface of the embryonic node. Here we show that the existence of node monocilia and the expression of a dynein gene that is implicated in ciliary function are conserved across a wide range of vertebrate classes, indicating that a similar ciliary mechanism may underlie the establishment of handedness in all vertebrates.

Left–right development: Conserved function for embryonic nodal cilia

How left–right handedness originates in the body plan of the developing vertebrate embryo is a subject of considerable debate. In mice, a left–right bias is thought to arise from a directional extracellular flow (nodal flow) that is generated by dynein-dependent rotation of monocilia on the ventral surface of the embryonic node. Here we show that the existence of node monocilia and the expression of a dynein gene that is implicated in ciliary function are conserved across a wide range of vertebrate classes, indicating that a similar ciliary mechanism may underlie the establishment of handedness in all vertebrates.

Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis

Endothelial tubulogenesis is a crucial step in the formation of functional blood vessels during angiogenesis and vasculogenesis. Here, we use in vivo imaging of living zebrafish embryos expressing fluorescent fusion proteins of β-Actin, α-Catenin, and the ERM family member Moesin1 (Moesin a), to define a novel cord hollowing process that occurs during the initial stages of tubulogenesis in intersegmental vessels (ISVs) in the embryo. We show that the primary lumen elongates along cell junctions between at least two endothelial cells during embryonic angiogenesis. Moesin1-EGFP is enriched around structures that resemble intracellular vacuoles, which fuse with the luminal membrane during expansion of the primary lumen. Analysis of silent heart mutant embryos shows that initial lumen formation in the ISVs is not dependent on blood flow; however, stabilization of a newly formed lumen is dependent upon blood flow. Zebrafish moesin1 knockdown and cell transplantation experiments demonstrate that Moesin1 is required in the endothelial cells of the ISVs for in vivo lumen formation. Our analyses suggest that Moesin1 contributes to the maintenance of apical/basal cell polarity of the ISVs as defined by adherens junctions. Knockdown of the adherens junction protein Ve-cadherin disrupts formation of the apical membrane and lumen in a cell-autonomous manner. We suggest that Ve-cadherin and Moesin1 function to establish and maintain apical/basal polarity during multicellular lumen formation in the ISVs.

Expression of VE-cadherin in Zebrafish Embryos: A New Tool to Evaluate Vascular Development

We have identified the zebrafish homologue of VE‐cadherin and documented its expression in the developing vascular system. The zebrafish VE‐cadherin gene is specifically expressed in the vascular endothelial cell lineage beginning with the differentiation and migration of angioblasts and persists throughout vasculogenesis, angiogenesis, and endocardium development. Staining zebrafish embryos by whole‐mount in situ hybridization with the VE‐cadherin probe provides a method to screen embryos for vascular defects. To illustrate this utility, we used VE‐cadherin expression to demonstrate a conservation of vascular endothelial growth factor‐A (VEGF‐A) function. The morpholino antisense oligonucleotide knockdown of VEGF‐A function in zebrafish embryos results in a loss of angiogenic blood vessels, as indicated by the lack of VE‐cadherin expression in the intersegmental vasculature. This loss can be restored in embryos supplemented with either zebrafish or human VEGF‐A, the latter indicating that genes crucial to angiogenesis have highly conserved functional activities in vertebrates. Developmental Dynamics 231:204–213, 2004.

The left-right determinant inversin has highly conserved ankyrin repeat and IQ domains and interacts with calmodulin

Abstract. All vertebrates have a left-right body axis with invariant asymmetries of the heart and the positions of the abdominal viscera. Major advances have recently been made in defining molecular components of the pathway specifying the vertebrate left-right axis, but our knowledge of the early determinants is extremely limited. In the inv mouse the left-right axis is consistently reversed, unlike other vertebrate mutants where randomisation of situs is apparent. The gene disrupted in this mouse encodes a 1062-amino-acid protein, inversin. We previously reported 16 tandem ankyrin repeats, spanning amino acids 13-557, and two putative nuclear localisation sequences, but otherwise the sequence offered few clues to the possible function. In order to identify regions likely to be functionally important, we have identified and characterised orthologous sequences in several species, including chick, Xenopus and zebrafish. Sequence comparisons show strong conservation of the ankyrin repeat region and also a lysine-rich domain spanning amino acids 558–604. Further analysis identified a highly conserved IQ calmodulin-binding domain in the latter region and another such domain in an otherwise poorly conserved C-terminal region. A yeast two-hybrid screen identified calmodulin in one third of the positive clones, and we confirmed this interaction by immunoprecipitation.

Somatic Mutagenesis with a Sleeping Beauty Transposon System Leads to Solid Tumor Formation in Zebrafish

Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB) T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700–6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.

SCORE Imaging: Specimen in a Corrected Optical Rotational Enclosure

Visual data collection is paramount for the majority of scientific research. The added transparency of the zebrafish (Danio rerio) allows for a greater detail of complex biological research that accompanies seemingly simple observational tools. We developed a visual data analysis and collection approach that takes advantage of the cylindrical nature of the zebrafish allowing for an efficient and effective method for image capture that we call Specimen in a Corrected Optical Rotational Enclosure imaging. To achieve a nondistorted image, zebrafish were placed in a fluorinated ethylene propylene tube with a surrounding optically corrected imaging solution (water). By similarly matching the refractive index of the housing (fluorinated ethylene propylene tubing) to that of the inner liquid and outer liquid (water), distortion was markedly reduced, producing a crisp imagable specimen that is able to be fully rotated 360 degrees. A similar procedure was established for fixed zebrafish embryos using convenient, readily available borosilicate capillaries surrounded by 75% glycerol. The method described here could be applied to chemical genetic screening and other related high-throughput methods within the fish community and among other scientific fields.

Transient axonal glycoprotein-1 (TAG-1) and laminin-α1 regulate dynamic growth cone behaviors and initial axon direction in vivo

BackgroundHow axon guidance signals regulate growth cone behavior and guidance decisions in the complex in vivo environment of the central nervous system is not well understood. We have taken advantage of the unique features of the zebrafish embryo to visualize dynamic growth cone behaviors and analyze guidance mechanisms of axons emerging from a central brain nucleus in vivo.ResultsWe investigated axons of the nucleus of the medial longitudinal fascicle (nucMLF), which are the first axons to extend in the zebrafish midbrain. Using in vivo time-lapse imaging, we show that both positive axon-axon interactions and guidance by surrounding tissue control initial nucMLF axon guidance. We further show that two guidance molecules, transient axonal glycoprotein-1 (TAG-1) and laminin-α1, are essential for the initial directional extension of nucMLF axons and their subsequent convergence into a tight fascicle. Fixed tissue analysis shows that TAG-1 knockdown causes errors in nucMLF axon pathfinding similar to those seen in a laminin-α1 mutant. However, in vivo time-lapse imaging reveals that while some defects in dynamic growth cone behavior are similar, there are also defects unique to the loss of each gene. Loss of either TAG-1 or laminin-α1 causes nucMLF axons to extend into surrounding tissue in incorrect directions and reduces axonal growth rate, resulting in stunted nucMLF axons that fail to extend beyond the hindbrain. However, defects in axon-axon interactions were found only after TAG-1 knockdown, while defects in initial nucMLF axon polarity and excessive branching of nucMLF axons occurred only in laminin-α1 mutants.ConclusionThese results demonstrate how two guidance cues, TAG-1 and laminin-α1, influence the behavior of growth cones during axon pathfinding in vivo. Our data suggest that TAG-1 functions to allow growth cones to sense environmental cues and mediates positive axon-axon interactions. Laminin-α1 does not regulate axon-axon interactions, but does influence neuronal polarity and directional guidance.