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Polarised targeting of diverse mRNAs to motile cellular protrusions is a hallmark of cell migration1–3. Although a widespread phenomenon, definitive functions for endogenous targeted mRNAs and their relevance to modulation of in-vivo tissue dynamics remain elusive. Here, using single-molecule analysis, endogenous gene-edited mRNAs and zebrafish in-vivo live-cell imaging, we report that mRNA polarisation acts as a molecular compass that orients motile cell polarity and spatially directs tissue movement. Clustering of protrusion-derived RNAseq datasets defined a core 192 bp localisation element underpinning precise mRNA targeting to incipient sites of filopodia formation at cell protrusions. Such targeting of the small GTPase, RAB13, generated tight spatial coupling of mRNA localisation, translation and protein activity, achieving precise subcellular compartmentalisation of RAB13 protein function to create a polarised domain of filopodia extension. Consequently, genomic excision of this localisation element and specific perturbation of endogenous RAB13 targeting – but not translation – depolarised filopodial dynamics in motile endothelial cells and induced miss-patterning of nascent blood vessels in-vivo. Hence, mRNA polarisation, not expression, is the primary spatial determinant of the site of RAB13 action, preventing ectopic functionality at inappropriate subcellular loci and orienting tissue morphogenesis. Considering the unexpected spatial diversity of other polarised mRNA clusters we identified, mRNA-mediated compartmentalisation of protein function at distinct subcellular sites likely coordinates broad aspects of in-vivo tissue behaviour.

The regulated generation of prostaglandins from endothelial cells is critical to vascular function. Here we identify a novel mechanism for the regulation of endothelial cell prostaglandin generation. Cytosolic phospholipase A(2)-alpha (cPLA(2)alpha) cleaves phospholipids in a Ca(2+)-dependent manner to yield free arachidonic acid and lysophospholipid. Arachidonic acid is then converted into prostaglandins by the action of cyclooxygenase enzymes and downstream synthases. By previously undefined mechanisms, nonconfluent endothelial cells generate greater levels of prostaglandins than confluent cells. Here we demonstrate that Ca(2+)-independent association of cPLA(2)alpha with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA(2)alpha and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA(2)alpha is associated with the phospholipid-binding protein annexin A1. Furthermore, the siRNA-mediated knockdown of endogenous annexin A1 significantly reverses the inhibitory effect of confluence on endothelial cell prostaglandin generation. Thus the confluence-dependent interaction of cPLA(2)alpha and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA(2)alpha activity and endothelial cell prostaglandin generation.

Cytosolic phospholipase A2-alpha (cPLA2-alpha) is a calcium-activated enzyme that plays an important role in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid can be converted subsequently into prostacyclin, a potent vasodilator and inhibitor of platelet activation, through the action of cyclooxygenase (COX) enzymes. Here we study the relocation of cPLA2-alpha in human EA.hy.926 endothelial cells following stimulation with the calcium-mobilizing agonist, A23187. Relocation of cPLA2-alpha was seen to be highly cell specific, and in EA.hy.926 cells occurred primarily to intracellular structures resembling the endoplasmic reticulum (ER) and Golgi. In addition, relocation to both the inner and outer surfaces of the nuclear membrane was observed. Colocalization studies with markers for these subcellular organelles, however, showed colocalization of cPLA2-alpha with nuclear membrane markers but not with ER or Golgi markers, suggesting that the relocation of cPLA2-alpha occurs to sites that are separate from these organelles. Colocalization with annexin V was also observed at the nuclear envelope, however, little overlap with staining patterns for the potential cPLA2-alpha interacting proteins, annexin I, vimentin, p11 or actin, was seen in this cell type. In contrast, cPLA2-alpha was seen to partially colocalize specifically with the COX-2 isoform at the ER-resembling structures, but not with COX-1. These studies suggest that cPLA2-alpha and COX-2 may function together at a distinct and novel compartment for eicosanoid signalling.

Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (VWF) are characteristic of the mammalian endothelium. We hypothesized that vascular-specific antigens such as VWF are linked to endothelial identity and proliferation in vitro. To test this idea, the cellular accumulation of VWF in WPBs was monitored as a function of cell proliferation, confluence and passage number in human umbilical vein endothelial cells (HUVECs). We found that as passage number increased the percentage of cells containing VWF in WPBs was reduced significantly, whilst the protein was still detected within the secretory pathway at all times. However, the endothelial-specific marker protein, PECAM-1, is present on all cells even when WPBs are absent, indicating partial maintenance of endothelial identity. Biochemical studies show that a significant pool of immature pro-VWF can be detected in sub-confluent HUVECs; however, a larger pool of mature, processed VWF is detected in confluent cells. Newly synthesized VWF must thus be differentially sorted and packaged along the secretory pathway in semi-confluent versus confluent endothelial cells. Our studies thus show that WPB formation is linked to the formation of a confluent endothelial monolayer.

Summary Tissue branching morphogenesis requires the hierarchical organization of sprouting cells into leading “tip” and trailing “stalk” cells [1, 2]. During new blood vessel branching (angiogenesis), endothelial tip cells (TCs) lead sprouting vessels, extend filopodia, and migrate in response to gradients of the secreted ligand, vascular endothelial growth factor (Vegf) [3]. In contrast, adjacent stalk cells (SCs) trail TCs, generate the trunk of new vessels, and critically maintain connectivity with parental vessels. Here, we establish that h2.0-like homeobox-1 (Hlx1) determines SC potential, which is critical for angiogenesis during zebrafish development. By combining a novel pharmacological strategy for the manipulation of angiogenic cell behavior in vivo with transcriptomic analyses of sprouting cells, we identify the uniquely sprouting-associated gene, hlx1. Expression of hlx1 is almost entirely restricted to sprouting endothelial cells and is excluded from adjacent nonangiogenic cells. Furthermore, Hlx1 knockdown reveals its essential role in angiogenesis. Importantly, mosaic analyses uncover a cell-autonomous role for Hlx1 in the maintenance of SC identity in sprouting vessels. Hence, Hlx1-mediated maintenance of SC potential regulates angiogenesis, a finding that may have novel implications for sprouting morphogenesis of other tissues., Highlights ► Expression of hlx1 is associated with angiogenic cell behavior in vivo ► hlx1 selectively marks sprouting endothelial cells during zebrafish development ► Hlx1 is required for intersegmental vessel angiogenesis in zebrafish embryos ► Hlx1 cell-autonomously maintains endothelial stalk cell potential

The vertebrate vasculature forms an extensive branched network of blood vessels that supplies tissues with nutrients and oxygen. During vascular development, coordinated control of endothelial cell behaviour at the levels of cell migration, proliferation, polarity, differentiation and cell-cell communication is critical for functional blood vessel morphogenesis. Recent data uncover elaborate transcriptional, post-transcriptional and post-translational mechanisms that fine-tune key signalling pathways (such as the vascular endothelial growth factor and Notch pathways) to control endothelial cell behaviour during blood vessel sprouting (angiogenesis). These emerging frameworks controlling angiogenesis provide unique insights into fundamental biological processes common to other systems, such as tissue branching morphogenesis, mechanotransduction and tubulogenesis.

Making Split Decisions Development of the vertebrate vasculature has been thought to involve just two mechanisms of blood vessel formation. Herbert et al. (p. 294 ; see the Perspective by Benedito and Adams ) identified a third mechanism in zebrafish in which two distinct, unconnected vessels can be derived from a single precursor vessel. Several vascular endothelial growth factors and signaling pathways, including ephrin and notch signaling, coordinated the sorting and segregation of a mixture of arterial and venous-fated precursor cells into distinct arterial and venous vessels. These findings provide a mechanistic framework for how mixed populations of cells can coordinate their behavior to segregate and form distinct blood vessels.

Release of endothelial cells from contact-inhibition and cell cycle re-entry is required for the induction of new blood vessel formation by angiogenesis. Using a combination of chemical inhibition, loss of function, and gain of function approaches, we demonstrate that endothelial cell cycle re-entry, S phase progression, and subsequent angiogenic tubule formation are dependent upon the activity of cytosolic phospholipase A(2)-alpha (cPLA(2)alpha). Inhibition of cPLA(2)alpha activity and small interfering RNA (siRNA)-mediated knockdown of endogenous cPLA(2)alpha reduced endothelial cell proliferation. In the absence of cPLA(2)alpha activity, endothelial cells exhibited retarded progression from G(1) through S phase, displayed reduced cyclin A/cdk2 expression, and generated less arachidonic acid. In quiescent endothelial cells, cPLA(2)alpha is inactivated upon its sequestration at the Golgi apparatus. Upon the stimulation of endothelial cell proliferation, activation of cPLA(2)alpha by release from the Golgi apparatus was critical to the induction of cyclin A expression and efficient cell cycle progression. Consequently, inhibition of cPLA(2)alpha was sufficient to block angiogenic tubule formation in vitro. Furthermore, the siRNA-mediated retardation of endothelial cell cycle re-entry and proliferation was reversed upon overexpression of an siRNA-resistant form of cPLA(2)alpha. Thus, activation of cPLA(2)alpha acts as a novel mechanism for the regulation of endothelial cell cycle re-entry, cell cycle progression, and angiogenesis.

The association of cytosolic phospholipase A(2)-alpha (cPLA(2)alpha) with intracellular membranes is central to the generation of free arachidonic acid and thromboxane A(2) in activated platelets. Despite this, the site and nature of this membrane association has not been fully characterised upon platelet activation. High resolution imaging showed that cPLA(2)alpha was distributed in a partly structured manner throughout the resting platelet. Upon glass activation or thrombin stimulation, cPLA(2)alpha relocated to a peripheral region corresponding to the platelet plasma membrane. Upon thrombin stimulation of platelets a major pool of cPLA(2)alpha was associated with the plasma membrane in an EGTA-resistant manner. EGTA-resistant membrane binding was abolished upon de-polymerisation of actin filaments by DNase I and furthermore, cPLA(2)alpha co-immunoprecipitated with actin upon thrombin stimulation of platelets. Immunofluorescence microscopy studies revealed that, upon platelet activation, cPLA(2)alpha and actin co-localised at the plasma membrane. Thus we have identified a novel mechanism for the interaction of cPLA(2)alpha with its membrane substrate via interaction with actin.

Abstract SCI-43 Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from pre-existing vessels (angiogenesis). Using high resolution imaging of zebrafish vascular development we discovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. These processes were regulated in part by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial/venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development. Disclosures: No relevant conflicts of interest to declare.