Nathan D. Lawson

sonic hedgehog and vascular endothelial growth factor Act Upstream of the Notch Pathway during Arterial Endothelial Differentiation

The appearance of molecular differences between arterial and venous endothelial cells before circulation suggests that genetic factors determine these cell types. We find that vascular endothelial growth factor (vegf) acts downstream of sonic hedgehog (shh) and upstream of the Notch pathway to determine arterial cell fate. Loss of Vegf or Shh results in loss of arterial identity, while exogenous expression of these factors causes ectopic expression of arterial markers. Microinjection of vegf mRNA into embryos lacking Shh activity can rescue arterial differentiation. Finally, activation of the Notch pathway in the absence of Vegf signaling can rescue arterial marker gene expression. These studies reveal a complex signaling cascade responsible for establishing arterial cell fate and suggest differential effects of Vegf on developing endothelial cells.

Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases

Direct genomic manipulation at a specific locus is still not feasible in most vertebrate model organisms. In vertebrate cell lines, genomic lesions at a specific site have been introduced using zinc-finger nucleases (ZFNs). Here we adapt this technology to create targeted mutations in the zebrafish germ line. ZFNs were engineered that recognize sequences in the zebrafish ortholog of the vascular endothelial growth factor-2 receptor, kdr (also known as kdra). Co-injection of mRNAs encoding these ZFNs into one-cell-stage zebrafish embryos led to mutagenic lesions at the target site that were transmitted through the germ line with high frequency. The use of engineered ZFNs to introduce heritable mutations into a genome obviates the need for embryonic stem cell lines and should be applicable to most animal species for which early-stage embryos are easily accessible.

A Novel miRNA Processing Pathway Independent of Dicer Requires Argonaute2 Catalytic Activity

No Dicer for Me MicroRNAs (miRNAs) are small noncoding RNAs found in most eukaryotes. Most are processed from primary transcripts in the nucleus by the microprocessor enzyme complex, which includes the nuclease Drosha, with a small number being generated by the messenger RNA splicing machinery. All pre-miRNAs are then exported into the cytoplasm where they are cleaved further by a second nuclease, Dicer, into the mature, functional miRNA. Cifuentes et al. (p. 1694, published online 6 May), now show that in a Dicer mutant fish at least one miRNA, miR-451, is still formed from pre-miR-451. The processing of pre-miR-451 requires the slicing activity of another protein in the miRNA pathway, Argonaute2. The unusual secondary structure of the pre-miR-451 determines its noncanonical processing pathway, which suggests that other miRNAs might also be processed in this way. The unusual secondary structure of a precursor microRNA determines its noncanonical processing. Dicer is a central enzyme in microRNA (miRNA) processing. We identified a Dicer-independent miRNA biogenesis pathway that uses Argonaute2 (Ago2) slicer catalytic activity. In contrast to other miRNAs, miR-451 levels were refractory to dicer loss of function but were reduced in MZago2 (maternal-zygotic) mutants. We found that pre-miR-451 processing requires Ago2 catalytic activity in vivo. MZago2 mutants showed delayed erythropoiesis that could be rescued by wild-type Ago2 or miR-451-duplex but not by catalytically dead Ago2. Changing the secondary structure of Dicer-dependent miRNAs to mimic that of pre-miR-451 restored miRNA function and rescued developmental defects in MZdicer mutants, indicating that the pre-miRNA secondary structure determines the processing pathway in vivo. We propose that Ago2-mediated cleavage of pre-miRNAs, followed by uridylation and trimming, generates functional miRNAs independently of Dicer.

Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries

Recent evidence indicates that growing blood-vessel sprouts consist of endothelial cells with distinct cell fates and behaviours; however, it is not clear what signals determine these sprout cell characteristics. Here we show that Notch signalling is necessary to restrict angiogenic cell behaviour to tip cells in developing segmental arteries in the zebrafish embryo. In the absence of the Notch signalling component Rbpsuh (recombining binding protein suppressor of hairless) we observed excessive sprouting of segmental arteries, whereas Notch activation suppresses angiogenesis. Through mosaic analysis we find that cells lacking Rbpsuh preferentially localize to the terminal position in developing sprouts. In contrast, cells in which Notch signalling has been activated are excluded from the tip-cell position. In vivo time-lapse analysis reveals that endothelial tip cells undergo a stereotypical pattern of proliferation and migration during sprouting. In the absence of Notch, nearly all sprouting endothelial cells exhibit tip-cell behaviour, leading to excessive numbers of cells within segmental arteries. Furthermore, we find that flt4 (fms-related tyrosine kinase 4, also called vegfr3) is expressed in segmental artery tip cells and becomes ectopically expressed throughout the sprout in the absence of Notch. Loss of flt4 can partially restore normal endothelial cell number in Rbpsuh-deficient segmental arteries. Finally, loss of the Notch ligand dll4 (delta-like 4) also leads to an increased number of endothelial cells within segmental arteries. Together, these studies indicate that proper specification of cell identity, position and behaviour in a developing blood-vessel sprout is required for normal angiogenesis, and implicate the Notch signalling pathway in this process.

Angiogenic network formation in the developing vertebrate trunk.

We have used time-lapse multiphoton microscopy of living Tg(fli1:EGFP)y1 zebrafish embryos to examine how a patterned, functional network of angiogenic blood vessels is generated in the early vertebrate trunk. Angiogenic vascular sprouts emerge from the longitudinal trunk axial vessels (the dorsal aorta and posterior cardinal vein) in two spatially and temporally distinct steps. Dorsal aorta-derived sprouts form an initial primary network of vascular segments, followed by emergence of vein-derived secondary vascular sprouts that interact and interconnect dynamically with the primary network to initiate vascular flow. Using transgenic silent heart mutant embryos, we show that the gross anatomical patterning of this network of vessels does not require blood circulation. However, our results suggest that circulatory flow dynamics play an important role in helping to determine the pattern of interconnections between the primary network and secondary sprouts, and thus the final arterial or venous identity of the vessels in the functional network. We discuss a model to explain our results combining genetic programming of overall vascular architecture with hemodynamic determination of circulatory flow patterns.

Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish.

The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.

MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis.

Within the circulatory system, blood flow regulates vascular remodelling, stimulates blood stem cell formation, and has a role in the pathology of vascular disease. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.

ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data

BackgroundChromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) or ChIP followed by genome tiling array analysis (ChIP-chip) have become standard technologies for genome-wide identification of DNA-binding protein target sites. A number of algorithms have been developed in parallel that allow identification of binding sites from ChIP-seq or ChIP-chip datasets and subsequent visualization in the University of California Santa Cruz (UCSC) Genome Browser as custom annotation tracks. However, summarizing these tracks can be a daunting task, particularly if there are a large number of binding sites or the binding sites are distributed widely across the genome.ResultsWe have developed ChIPpeakAnno as a Bioconductor package within the statistical programming environment R to facilitate batch annotation of enriched peaks identified from ChIP-seq, ChIP-chip, cap analysis of gene expression (CAGE) or any experiments resulting in a large number of enriched genomic regions. The binding sites annotated with ChIPpeakAnno can be viewed easily as a table, a pie chart or plotted in histogram form, i.e., the distribution of distances to the nearest genes for each set of peaks. In addition, we have implemented functionalities for determining the significance of overlap between replicates or binding sites among transcription factors within a complex, and for drawing Venn diagrams to visualize the extent of the overlap between replicates. Furthermore, the package includes functionalities to retrieve sequences flanking putative binding sites for PCR amplification, cloning, or motif discovery, and to identify Gene Ontology (GO) terms associated with adjacent genes.ConclusionsChIPpeakAnno enables batch annotation of the binding sites identified from ChIP-seq, ChIP-chip, CAGE or any technology that results in a large number of enriched genomic regions within the statistical programming environment R. Allowing users to pass their own annotation data such as a different Chromatin immunoprecipitation (ChIP) preparation and a dataset from literature, or existing annotation packages, such as GenomicFeatures and BSgenom e, provides flexibility. Tight integration to the biomaRt package enables up-to-date annotation retrieval from the BioMart database.

Arteries and veins: making a difference with zebrafish

Arteries and veins are structurally different and have long been functionally defined by the direction of blood flow that they carry. However, a growing body of evidence indicates that the identity of the endothelial cells that line these vessels is determined in the developing embryo, before circulation begins. Recent work on the zebrafish has led to the identification of signals that are responsible for arterial and venous differentiation of endothelial cells, and highlights the unique benefits of this model organism in the study of vascular development.

Gateway compatible vectors for analysis of gene function in the zebrafish

The recent establishment of recombination‐based cloning systems has greatly facilitated the analysis of gene function by allowing rapid and high‐efficiency generation of plasmid constructs. However, the use of such an approach in zebrafish requires the availability of recombination‐compatible plasmids that are appropriate for functional studies in zebrafish embryos. In this work, we describe the construction and validation of Gateway compatible vectors based on commonly used zebrafish plasmids. We have generated pCS‐based plasmids that allow rapid generation of both N‐terminal and C‐terminal fusion proteins, and we demonstrate that mRNA synthesized from these plasmids encodes functional native or fusion proteins in injected zebrafish embryos. In parallel, we have established similar Gateway plasmids containing Tol2 cis elements that promote efficient integration into the zebrafish genome and allow expression of native or fusion proteins in a tissue‐specific manner in the zebrafish embryo. Finally, we demonstrate the use of this system to rapidly identify tissue‐specific cis elements to aid the establishment of blood vessel‐specific transgenic constructs. Taken together, this work provides an important platform for the rapid functional analyses of open reading frames in zebrafish embryos. Developmental Dynamics 236:3077–3087, 2007.