Recently, messenger RNAs in eukaryotes have shown to associate with antisense (AS) transcript partners that are often referred to as long noncoding RNAs (lncRNAs) whose function is largely unknown. Here, we have identified a natural AS transcript for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1), tie-1AS lncRNA in zebrafish, mouse, and humans. In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript and in additional locations (ear and brain). The tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1 transcript levels, resulting in specific defects in endothelial cell con- IntroductionOver the past few years, intensive unbiased analysis of transcriptome species has revealed that eukaryotic genomes contain a variety of RNA species. RNA molecules are essentially classified into 2 types, protein coding and nonprotein coding. The protein-coding transcripts or messenger RNA (mRNA) account for only approximately 2.3% of the human genome. 1 The majority of transcription appears to be nonprotein coding or noncoding, and the function of these noncoding transcripts is largely unknown. 2 Of the noncoding RNAs, the regulatory short noncoding RNAs, such as microRNAs, are well studied. The long noncoding RNAs (lncRNAs), which compose the largest portion of the mammalian noncoding transcriptome, are the least understood, especially its function. 3,4 lncRNAs are oriented in sense or antisense (AS) direction with respect to a protein coding locus, and located in intronic or intergenic regions. 5 In humans and mice, 61% to 72% of all transcribed regions possess lncRNAs in AS orientation, 2,6 and AS lncRNA transcripts play important roles in pathogenesis. For instance, the BACE1-AS transcript was elevated in subjects with Alzheimer disease and in amyloid precursor protein transgenic mice. 7 A growing body of evidence suggests that lncRNAs for most critical physiologic processes will be identified. Angiogenesis, the development of new vasculature from existing vasculature, is one of the fundamental developmental physiologic processes regulated in a developing vertebrate embryo. 8 Here, we identify a natural AS transcript for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1), tie-1AS lncRNA in zebrafish, mouse, and humans. tie-1 is a cell-surface tyrosine kinase receptor for angiopoietin ligands that is known to play a role in vascular development in vertebrates. [9][10][11][12] In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript, and in additional locations (ear and brain). Its expression is controlled by a 3-kb genomic fragment in the 3Ј region of tie-1, and the bioinformatic predicted hybrid structure between tie-1:tie-1AS was detected in vivo. Capped or uncapped tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1...

The extracellular matrix plays a critical role in neural crest (NC) cell migration. In this study, we characterize the contribution of the novel GPI-linked matrix metalloproteinase (MMP) zebrafish mmp17b. Mmp17b is expressed post-gastrulation in the developing NC. Morpholino inactivation of mmp17b function, or chemical inhibition of MMP activity results in aberrant NC cell migration with minimal change in NC proliferation or apoptosis. Intriguingly, a GPI anchored protein with metalloproteinase inhibitor properties, Reversion-inducing-Cysteine-rich protein with Kazal motifs (RECK), which has previously been implicated in NC development, is expressed in close apposition to NC cells expressing mmp17b, raising the possibility that these two gene products interact. Consistent with this possibility, embryos silenced for mmp17b show defective development of the dorsal root ganglia (DRG), a crest-derived structure affected in RECK mutant fish sensory deprived (sdp). Taken together, this study has identified the first pair of MMP, and their putative MMP inhibitor RECK that functions together in NC cell migration.

IntroductionThe mitogen-activated protein kinase (MAPK) pathway 1 represent a key signal integration step inside the cell for various extracellular stimuli. 2 At least 4 MAPK families have been identified: the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38, and Erk5. Each MAPK is activated by specific MAPK kinases (Mkks or Meks), which in turn are regulated by Mek kinases (Mekks). To date, several members of the MAPK family, including Erk5 3 and Mekk3, 4 have been implicated in vascular development in vivo. 5 Furthermore, MAPKs are often associated with pathologic responses, such as stress, apoptosis, inflammation, and cell proliferation. 5,6 In terms of vascular conditions, differential expression of active MAPKs was noticed in several types of hemangiomas, 7 the most common vascular tumor of infancy and childhood, 8 and the presence of immunoreactive phosphorylated MAPK inversely correlate with degree of malignancy. 7 Therefore, from a drug discovery standpoint, vascular specific components of MAPKs cascade are promising drug targets. 6 MAPKs are activated by dual phosphorylation of tyrosine and threonine residues in their activation loops. To control MAPK activation, a family of proteins called dual-specificity protein tyrosine phosphatases (DUSPs) dephosphorylate both residues. 9 DUSPs come in 2 varieties: dual-specificity MAPK phosphatases and atypical DUSPs. A dual specific phosphatase, Dusp-5, was recently identified by 2 independent microarray studies as a vascular-specific gene, 10,11 and we hypothesized that dusp-5 plays a specific role in vascular development. To investigate dusp-5 function, we initiated a loss-of-function (LOF) study in zebrafish (ZF). We identified that dusp-5 LOF embryos showed enhanced etsrp ϩ angioblasts at the lateral plate mesoderm (LPM). Recently, we had identified in a gain-of-function (GOF) and LOF study for a serine-threonine kinase member of the sucrose nonfermenting kinase family, Snrk-1, that it increased or decreased, respectively, the same etsrp ϩ angioblast population at the LPM. 12 Taking these findings together, we hypothesized that, during vascular development, Dusp-5 and Snrk-1 function together in controlling angioblast numbers at the LPM. In this study, we show that dusp-5 is expressed in angioblasts in the embryonic ZF and is essential for vascular development in vivo. We show that loss of Dusp-5 function in vitro causes apoptosis of endothelial cells (ECs), and Dusp-5 and Snrk-1 target a common signaling pathway responsible for maintaining angioblast populations along the LPM. Snrk-1 ectopically induces etsrp ϩ angioblasts in the LPM, which is blocked by Dusp-5 that functions downstream of Snrk-1. In addition, we have identified mutations in dusp-5 and snrk-1 in the lesional tissue of many vascular anomaly patient samples, suggesting a critical role for this pathway in disease. Methods ZF stocksWild-type ZF (TuAB strain) were grown and maintained at 28.5°C. 13 All procedures were performed according to animal protoc...

In vertebrates, molecular mechanisms dictate angioblasts' migration and subsequent differentiation into arteries and veins. In this study, we used a microarray screen to identify a novel member of the sucrose nonfermenting related kinase

Endothelial cell-specific chemotaxis receptor (ECSCR) is a cell surface protein expressed by blood endothelial cells with roles in endothelial cell migration and signal transduction. We investigated the function of ecscr in the development of the zebrafish vasculature. Zebrafish ecscr is expressed in angioblasts and in axial vessels during angioblast migration and vasculogenesis. Morpholino-directed ecscr knockdown resulted in defective angioblast migration in the posterior lateral plate mesoderm, a process known to depend on vascular endothelial-derived growth factor (VEGF). In cultured cells, transfected ECSCR localized to actin-rich membrane protrusions, colocalizing with kinase insert domain protein receptor (KDR)/VEGF receptor 2 in these regions. ECSCR-silenced cells show reduced VEGF-induced phosphorylation of KDR but not of FMS-like tyrosine kinase 1 (FLT1)/VEGF receptor 1. Finally, chemical inhibition of VEGF receptor activity in zebrafish resulted in angioblast deficiencies that partially overlap with those seen in ecscr morphants. We propose that ecscr promotes migration of zebrafish angioblasts by enhancing endothelial kdr sensitivity to VEGF.

There was an error published in J. Cell Sci. 128, 3556-3568 (doi:10.1242/jcs.167601).In the Abstract, the penultimate sentence should read: 'Using a VEGF-inducible zebrafish (Danio rerio) model, we observed significantly increased vascular permeability in RhoC morpholino (MO)-injected zebrafish compared with control MO-injected zebrafish.'The authors apologise to the readers for any confusion that this error might have caused.

Transgenic animals have been routinely produced by microinjecting or electroporating naked DNA into 1-cell-stage embryos or unfertilized eggs. However, these techniques are inapplicable to live-bearing fish and many crustacean species for which unfertilized or newly fertilized eggs are not readily obtainable. In the present study, replication-defective pantropic retroviral vectors carrying a reporter gene (neo(R) or beta-gal) were used to directly transform the immature ovary or testis of a live-bearing fish (Poeciliopsis lucida) and crayfish (Procambarus clarkii). The fraction of the progeny derived from these treated individuals shown to contain the neo(R) reporter gene by an assay based on polymerase chain reaction (PCR) was significant. The PCR-positive individuals were crossed with nontransgenic individuals, and about 50% of the resulting progeny carried the transgene, suggesting that the F(1) animals are germline transgenic. Integration of the transgenes was confirmed by detecting the junction fragments of the genomic DNA associated with transgene constructs. Expression of reporter genes was detected by a reverse transcription-nested PCR assay. These results showed that transgenic live-bearing fish and crustaceans could be easily produced by directly transforming the immature gonads with replication-defective pantropic retroviral vectors.

Despite their importance as members of the Roundabout (Robo) family in the control of axonal and vascular patterning, the transcriptional regulation of these genes is poorly understood. In this study, we show that members of the Sry-related high mobility box (Sox) transcription factor family as being transcriptional regulators of roundabout4 (robo4), a Robo gene family member that participates in sprouting angiogenesis in vivo, in zebrafish. Double whole mount in situ hybridization analysis in zebrafish embryos revealed co-localization of the vascular relevant Sox factors sox7 or sox18 mRNA with robo4 transcripts in developing intersomitic vessels. A 3-kb human ROBO4 promoter element was able to drive reporter expression in zebrafish to recapitulate the endogenous temporal intersomitic vessel expression pattern of robo4. EMSA analysis confirmed binding of Sox18 to a canonical Sox binding site (from ؊1170 bp to ؊1176 bp) in the ROBO4 promoter (3 kb), and mutation analysis indicated that this site was partially responsible for ROBO4 promoter activity in ECs. A combination of gain-and loss-of-function analysis identified Sox7 and Sox18 co-regulation of robo4 but not fli1a transcripts in zebrafish. Finally, Sox-mediated robo4 transcriptional regulation is conserved across evolution. These studies imply Sox-mediated transcriptional regulation of Robo4 in the developing embryonic vasculature.In developing vertebrates, neural and vascular patterning generate intricate branching networks that share several similar features (1). However, this connectivity is governed by a limited toolkit of signaling receptor systems. These systems must therefore be subjected to exquisite control to achieve proper patterning and avoid miscues. Recently, members of the axon guidance family have shown both expression and functionality in the developing vasculature. Of the four distinct families of axon guidance signaling partners, namely Slit-Robo, Ephrin-Eph, Netrin-Unc, and Semaphorin-Plexin, our laboratory has focused on the Slit-Robo family members and their role in the vasculature.Roundabouts (Robos), 4 a class of cell surface receptors that were originally identified to function in axon guidance (2), have recently been implicated in providing critical directional information for migration of endothelial cells (ECs) (3, 4). Four mammalian Robos are known, of which the fourth member robo4 is expressed in the intersomitic vessels (ISVs) and is strikingly regulated with peak expression passing in a "wave" along the trunk axis from 19 -29 somites (3), suggesting a high degree of transcriptional control of this gene product. Recently, a 3-kb human ROBO4 promoter sequence has been identified that directs endothelial cell-specific expression pattern in vivo and in vitro (5). In addition, a guanine and adenine-binding protein-binding element in the ROBO4 promoter is necessary for endothelial expression in vivo (6). However, to date little is known regarding transcription factors that are involved in regulating robo4 expression during embryoni...