Somatic mutation, such as by the mobilization of retrotransposons during neurogenesis (Muotri and Gage, 2006 and Singer et al., 2010) or by copy number variation in neurons (Rehen et al.,

2005), has been proposed as a source of normal neuronal diversity. However, neurogenetic disease has also been attributed to somatic, postzygotic mutations in TSC2, NF1, and DCX that are detectable in some, but not all, blood cells and appear to be present in some, but not all, brain cells ( Gleeson et al., 2000, Messiaen et al., 2011, Qin et al., 2010 and Vogt et al., 2011). On the other hand, it has been essentially impossible to study potential roles of mutations that are limited to brain cells, because such mutations are by definition absent from blood and VX-770 in vivo other tissues typically available for genetic study. Such somatic mutations could conceivably play important roles in complex neurogenetic disorders, such as epilepsy, intellectual disability, and psychiatric disease, for which prominent selleck inhibitor roles for de novo mutations have been well documented ( Awadalla et al., 2010,

Poduri and Lowenstein, 2011 and Ropers, 2008). Here we describe a highly epileptic disorder, hemimegalencephaly (HMG, literally, enlargement of one brain hemisphere), as a model to characterize the role of somatic mutation in the developing brain. HMG is a developmental brain disorder characterized by an enlarged, malformed cerebral hemisphere (Flores-Sarnat et al., 2003). The clinical presentation typically includes intellectual disability and severe, intractable epilepsy, often necessitating surgical removal or disconnection of the abnormal hemisphere for seizure control (Gowda et al., 2010). L-NAME HCl Although no specific genetic causes have been identified for isolated HMG, HMG has been reported in association with

Proteus syndrome (Griffiths et al., 1994)—another multisystem overgrowth disorder that has recently been associated with somatic activating mutations in the gene AKT1 ( Lindhurst et al., 2011)—as well as other rare neurocutaneous syndromes ( Mochida et al., 2013). There are also rare reports of HMG associated with tuberous sclerosis complex (TSC) ( Cartwright et al., 2005), a syndrome in which multiple organ systems display disordered and sometimes cancerous growths. The striking asymmetry of the brain in individuals with HMG has long suggested that HMG reflects spontaneous, somatic, clonal mutation limited to the brain, analogous to cancer but without cellular transformation and ongoing proliferation. We hypothesized that the somatic mutations causing HMG might be essentially restricted to the brain and detectable by direct study of affected brain tissue.

For instance, there are uncontentious examples of systems specialized for processing social information in the case of pheromone detection in insects and in the case of the vomeronasal system in many mammals. Examples in primates are more debated, but again we would argue that there are clear studies ranging from lesion work to neuroimaging of face processing. Although we have moved from regions to networks, the next key step is

to identify the flow of information through these networks to follow social information processing from stimulus through to response. This requires an understanding of the detailed computations implemented by the different nodes in the networks as well the dynamic interplay between them. One could make the analogy of moving from words (brain areas) to sentences (networks) to propositions (arrangements of network dynamics) to conversations (brains interacting). We are still solidly in the age of sentences Regorafenib molecular weight and are only beginning Fulvestrant mw to enter the age of propositions and conversations. Social neuroscience must include a wide selection of methods, study a wide range of species, and utilize a range of concepts and theories. It is this topical and methodological breadth, combined with its interdisciplinary approach, that generates tension in the field. Psychologists often find the methods of neuroscience impressive

but its concepts and theories impoverished. Neurobiologists find the questions of social psychology intriguing but its methods limited. No wonder there is often little agreement at faculty meetings L-NAME HCl on whom to hire in a “social neuroscience” search! We believe that the single major challenge—and exciting open terrain—for the future of social neuroscience is conceptual rather than methodological. How can we parse social behavior, to begin with, and what vocabulary of concepts should we deploy in describing central processes and relating them to neurobiological constituents? This question, we believe, is also the main source of tension among different strands of social neuroscience or between those with backgrounds in different disciplines. A large part of this tension stems

from the belief among some social scientists that the processes responsible for understanding both human and animal social behavior are very complex, are very context-dependent, and draw on many factors, including ones outside the brain—as such making these processes ill suited to neuroscientific study. It is important to understand the several facets behind this tension. One difficulty is simply to discover the processes, a query that can be approached in different ways—further development of theoretical frameworks or “discovery science” based on data mining, to name just two (see Table 1). But another important worry is reductionism, the sense that neurobiological approaches will generate concepts that displace those of social psychology, as exemplified in the quote below: …

45 ± 9.86 min−1), VT (10.10 ± 0.45 μl ⋅ g−1), and VE (2.97 ± 0.19 ml ⋅ min−1 ⋅ g−1) ( Figure 6A). The compromised hypercapnic response might be due to the inability of the RTN neurons to detect changes in pCO2 and trigger respiration owing to their failed migration. At the same time, the

partially preserved hypercapnic response implies that the carotid bodies are spared. To test if the carotid bodies are functionally intact, we challenged Atoh1Phox2bCKO mice (n = 9) and their littermates (WT, n = 21) with hypoxic gas http://www.selleckchem.com/products/gsk1120212-jtp-74057.html (10% O2). Interestingly, Atoh1Phox2bCKO mice displayed a stronger hypoxia-evoked ventilatory response than WT (RF: 346.63 ± 14.36 versus 286.53 ± 4.75 min−1; VT: 12.8 ± 0.74 versus 11.05 ± 0.34 μl ⋅ g−1; VE: 4.5 ± 0.33 versus 3.11 ± 0.11 ml ⋅ min−1 ⋅ g−1) ( Figure 6B), suggesting that the O2-sensing carotid bodies could provide compensatory feedback. Overall, our results demonstrate that transient Atoh1 expression in postmitotic RTN neurons is critical for mediating respiratory Proteasome inhibitor chemoresponsiveness in free-moving adult mice,

most likely through promoting their ventral localization. This study has yielded three important findings. First, Atoh1 expression in the RTN neurons is critical for neonatal survival. Second, expression of Atoh1 in the postmitotic RTN neurons directs their migration through the embryonic hindbrain and establishes the connectivity that provides excitatory drive crucial for commencing

inspiratory rhythm at birth. This cell-autonomous role for Atoh1 in RTN migration provides a mechanism by which derailed hindbrain development can result in disordered neonatal breathing and highlights the importance of the RTN neurons at this stage. Third, Atoh1-mediated RTN development at an early embryonic stage is necessary for normal respiratory chemosensitivity in the adult. Genetic removal of Atoh1 from the Phox2b neurons results in nearly 50% neonatal lethality and indicates that even transient Atoh1 embryonic expression plays a major role in neonatal respiration. Given that the glutamatergic RTN neurons have been hypothesized whatever to entrain the embryonic preBötC ( Bochorishvili et al., 2012; Thoby-Brisson et al., 2009), we proposed that the migration defect of the Atoh1Phox2bCKO mice and the consequent loss of synaptic contact dramatically decreases excitatory input, thereby challenging the neonatal respiratory rhythm-generating network ( Feldman et al., 2003; Mellen et al., 2003). Support for this contention comes from the ability of Atoh1Phox2bCKO en bloc preparations to still generate respiratory rhythm (albeit depressed), which confirms the participation of RTN neurons in neonatal respiratory rhythm modulation. Once the conditional mutants survive past P0, they do not show additional lethality, similar to the partially penetrant neonatal lethality of the Egr-2 null mice (∼50% at P0) ( Jacquin et al., 1996).

Our finding that enhanced coupling occurs with attention only between FEF visual neurons and V4 suggests that V4 neurons have preferential

connections with FEF visual neurons rather than any other FEF cell type. The pattern of anatomical connections between FEF and V4 supports this conclusion. The majority of FEF projections to V4 arise from the supragranular layers (Barone et al., 2000 and Pouget et al., 2009), and neurons in the supragranular layers of the FEF subserve visual selection (Thompson et al., 1996). With attention, an increase in gamma synchrony between FEF supragranular-layer visual cells and V4 with AZD8055 chemical structure the appropriate phase relationships may increase effective communication between the two areas to enhance processing of signals related to the attended location (Fries, 2005, Gregoriou et al., 2009a and Gregoriou et al., 2009b). Moreover, the absence of any effect of attention on synchrony

between buy 17-AAG FEF movement cells and V4 further indicates that attentional mechanisms at the network level are largely independent and distinct from movement processing. If visual FEF cells subserve visual selection and provide top-down inputs to extrastriate cortex, whereas movement FEF neurons mediate saccade execution via projections to oculomotor centers what is the role of visuomovement neurons? Previous studies have indicated that the responses of visuomovement neurons do not mediate saccade preparation and have suggested that they may provide a corollary discharge to update the visual representations every time the all eyes move (Ray et al., 2009). Similar presaccadic enhancements have also been recorded in areas that are anatomically distant from the brainstem saccade generator such as area V4 and area 46 (Boch and Goldberg, 1989, Fischer and Boch, 1981 and Moore et al., 1998). It is thus possible that such a corollary discharge signal is provided by FEF visuomovement neurons once a saccade is bound to occur. Our task was not designed

to test this possibility. Given that no saccades were executed during our attention task the absence of coupling between FEF visuomovement neurons and V4 is not surprising. A very recent study showed that FEF cells mediating saccade selection are affected by activation of both D1 and D2 dopamine receptors, whereas those contributing to visual modulation of V4 are sensitive only to D1 receptor agonists (Noudoost and Moore, 2011). This is in line with the finding that in infragranular layers, source of saccade-related signals in the FEF, both D1 and D2 receptors are found, whereas in supragranular layers, source of FEF signals responsible for the enhancement of activity in V4, D2 receptors are less frequent (Lidow et al., 1991 and Santana et al., 2009).

In the present study, we have tackled this issue by the extensive use of targeted cell lineage and conditional gene manipulation in mouse, combined with in vitro live axon imaging. First, genetic manipulations that completely blocked motor projections

triggered randomized formation of either epaxial or hypaxial sensory nerves. Second, conditional or systemic removal of motor axonal EphA3/4 triggered selective loss of epaxial sensory projections, while preserving epaxial motor projections. Third, subsequent gene replacement experiments in mice revealed that, intriguingly, the requirement of EphA3/4 for determining epaxial sensory projections operates independently from the EphA3/4 repulsive forward signaling involved in sensory-motor axon segregation. LY294002 concentration Palbociclib in vivo Herein, reconstituting EphA4 extracellular domain expression on epaxial motor axons in EphA3/4-deficient mice effectively rescued epaxial sensory projections, but not the misrouting of motor axons into DRGs triggered by the loss of EphA3/4 repulsive forward signaling. Fourth, in vivo genetic interaction data and in vitro experiments indicated that motor axonal EphAs act by reverse signaling through cognate ephrin-A binding partners on sensory growth cones. Fifth, live axon imaging revealed that motor axons pre-extending in vitro induced sensory growth cones to track along their

trajectories. Sixth, these sensory growth cone tracking behaviors required EphA3/4 ectodomain expression on motor axons or ephrin-A2/5 expression on sensory axons, but did not require EphA3/4 signaling in motor axons proper. Seventh, recombinant EphA ectodomains were sufficient to induce sensory axon extension in vitro, which involved ephrin-A2/5

expressed by sensory axons. EphA3/4 therefore fulfills two diametrically opposed functions during peripheral nerve assembly. Initially, EphA3/4 repulsive forward signaling assures Fossariinae the segregation of epaxial motor axons from proximal sensory pathways (Figures 9A–9A″) (Gallarda et al., 2008). Subsequently, EphA3/4 operate through the reverse activation of ephrin-As on sensory growth cones to couple sensory projections to epaxial motor pathways (Figures 9B–9B″) (this study). What determines whether kinase-dependent EphA3/4 forward signaling or kinase-independent EphA3/4 reverse signaling are elicited between epaxial motor and sensory axons? A key factor is likely the developmental status of epaxial motor axon extension relative to sensory projections, because it dictates the specific growth cone-axon encounters possible between epaxial motor and sensory axons (Figures S8A and S8B). Herein, the initial extension of epaxial motor axons is predicted to favor interactions of epaxial motor growth cones with sensory growth cones and axons extending from DRGs within the same spinal segment (Figure S8A).

, 2005, Madison et al., 2005, Stevens et al., 2005 and Guan et al., 2008), but the mechanisms of Munc13 function in priming, and of the inactivation of Munc13 function by homodimerization, remain unclear. One possibility is that homodimeric Munc13 is inherently unstable and becomes degraded in RIM-deficient neurons, thereby accounting for the priming phenotype and the reduced Munc13 levels in RIM-deficient neurons (Figure 1; Schoch et al., 2002). However, overexpression of wild-type Munc13 did not rescue the priming phenotype in RIM-deficient neurons, suggesting that simply increasing Munc13 levels is not sufficient to rescue priming in RIM-deficient synapses. Another possibility is that homodimeric Munc13 is not

correctly targeted to synapses and becomes degraded if it is not in the correct location (Andrews-Zwilling et al., 2006 and Kaeser et al., 2009). Although

possible, this hypothesis check details appears rather unlikely given the rescue of the RIM- and Munc13-deficiency phenotypes by N-terminally truncated Munc13 (Figure 7 and Figure 8), which suggests that Munc13 is transported to synapses without RIM proteins and without binding to RIM proteins. Independent of which explanation will turn out to be correct, the mechanism of Munc13 activation we identify here is opposite to what is classically observed for signal transduction events; dimerization Dactolisib mouse is usually activating, whereas in our case it is inhibitory, suggesting a more diverse range of biological activation mechanisms than previously envisioned. The current study identifies a molecular mechanism involved in vesicle priming by the active zone but raises new questions. At a basic level, how is an active zone generated—what protein nucleates its assembly? The fact that the RIM Zn2+ finger alone is active suggests that it acts downstream of Munc13 targeting to active zones and cannot physically tether Munc13 to them; similarly, Munc13

is not essential for targeting other proteins to active zones and thus also not secondly involved in their recruitment to active zones. Clearly, despite its central function, RIM alone does not organize the active zone, an activity that may be carried out by an overlapping set of several proteins instead of a single master regulator. Another important question is how RIM proteins contribute to long-term synaptic plasticity—is this mediated by a coordination of their various functions or by one particular aspect? With the present results, we now know of two switches at the active zone that involve RIM and regulate synaptic neurotransmitter release: the GTP-dependent interaction of Rab3 with RIMs, and the Zn2+ finger mediated RIM-dependent monomerization of Munc13. Given the central roles of RIM and Rab3 in all known forms of long-term presynaptic plasticity (e.g., Castillo et al., 1997, Castillo et al., 2002, Chevaleyre et al., 2007, Fourcaudot et al., 2008 and Kaeser et al.

Flow cytometric analysis of surface accessibility of FLAG epitope-tagged D1 DA receptors (FD1R) in HEK293 cells verified robust internalization in response to DA. Internalization was dose-dependent and rapid, approaching the steady state value with an estimated t1/2 of 3.9 min (Figure 1A). For greater temporal resolution, we employed live imaging by total internal reflection fluorescence (TIRF) microscopy and the pH-sensitive GFP variant superecliptic pHluorin (SpH, or SEP) fused to the N-terminal extracellular region of the D1 receptor (SpH-D1R). SpH is highly

fluorescent at neutral pH, facilitating detection when in contact with the extracellular media. This fluorescence is rapidly quenched in the acidic SCH727965 clinical trial environment of the endocytic pathway (Miesenböck et al., 1998 and Sankaranarayanan et al., 2000). We used these properties to observe individual endocytic events

Autophagy Compound Library screening in SpH-D1R expressing HEK293 cells. In the absence of DA, SpH-D1R fluorescence was visible on the plasma membrane (Figure 1B, left). Bath application of DA caused rapid clustering of SpH-D1Rs into puncta that subsequently endocytosed (Figure 1B, right; see Movie S1 available online). Strikingly, an initial wave of SpH-D1R clustering and endocytosis occurred as soon as 30 s after agonist addition (Movie S1). Analysis of individual puncta by fluorescence intensity tracing verified their disappearance within 30 s to 1 min after formation (Figure 1C). These properties are consistent with previous data indicating that D1 receptors endocytose primarily via clathrin-coated

pits in HEK293 cells (Vickery and von Zastrow, 1999), and with descriptions of clathrin-mediated endocytosis of signaling receptors imaged by TIRF microscopy (Puthenveedu and von Zastrow, 2006 and Yudowski Carnitine dehydrogenase et al., 2006). Rapid endocytosis was further verified by the rate of DA-dependent reduction in integrated SpH-D1R surface fluorescence intensity (Figure 1D). Given that significant endocytosis of D1 receptors occurred within ∼1 min of DA addition, we examined D1 receptor-mediated signaling over a similar time scale. We employed the FRET-based cAMP biosensor, Epac1-cAMPs, to measure DA stimulated cAMP production in real time, in individual cells, without phosphodiesterase inhibitors (Nikolaev et al., 2004). Cells expressing FD1R and Epac1-cAMPs showed a robust decrease in the normalized FRET emission ratio after DA addition, indicating elevated cytoplasmic cAMP concentration (Figure 1E). DA application produced both a rapid decrease of YFP emission and a corresponding increase in CFP emission, verifying that the observed changes were indeed due to decreased FRET (Figure S1A).

, 2009), and thus should generate a measurable Ca Ibrutinib cost transient in the postsynaptic compartment (Goldberg et al., 2003b). We recorded from fast-spiking interneurons (see Experimental Procedures) with patch pipettes containing the fluorescent Ca indicator Oregon Green BAPTA-1 (150 μM) in a slice preparation that preserves much of the thalamocortical fiber bundle (Agmon and Connors,

1991 and Porter et al., 2001), allowing a stimulation electrode to be placed in this pathway slightly ventral to the fimbria. We simultaneously imaged the dendritic arbor of the recorded neuron (Figure 1D) and recorded the electrophysiological response in voltage clamp to stimulation of thalamic afferents. We used three stimulation protocols: (1) bulk stimulation, in which multiple thalamic afferents are recruited; (2) threshold single fiber stimulation, in which a single afferent impinging on the recorded neuron or imaged dendrite is stimulated just at threshold, leading PI3K inhibitor to fluctuation between recruitment successes and failures; and (3) single fiber stimulation, in which a single fiber impinging on the recorded neuron or imaged dendrite is recruited reliably,

without failures, by the stimulation electrode (see Experimental Procedures). Bulk stimulation of thalamic afferents elicited a pattern of Ca hotspots—localized, transient postsynaptic increases in Ca concentration—decorating the dendritic arbor of cortical interneurons (Figure 1E). Addition of the AMPA-R antagonist NBQX reduced hotspot intensity by 58% ± 5% (n = 5), while further addition of the NMDA-R antagonist R-CPP eliminated hotspots entirely (to −1% ± 1%, n = 3; Figure 1F). Similarly, application of R-CPP reduced hotspot intensity by 59% ± 4% (n = 5; see Figure S1 available online), suggesting that Ca-permeable see more AMPA-R and NMDA-R contribute about equally to the postsynaptic Ca signal under our recording conditions. Do hotspots mark the location

of a synaptic contact? In the absence of perfect voltage clamp, hotspots could in principle result from the activation of voltage-gated Ca channels (VGCCs) not necessarily colocalized with the synaptic contact. To test this possibility, we monitored the spatial distribution of Ca transients in response to depolarizing voltage steps (Goldberg et al., 2003b). The resulting Ca transient was spread throughout the dendritic arbor (Figure 1G), indicating that VGCCs are distributed broadly and therefore unlikely to produce hotspots at significant distance from the site of synaptic contact. Thus, thalamic stimulation generates a spatial pattern of Ca transients that corresponds to the location of glutamate receptor-mediated thalamic inputs. To ascertain whether an individual hotspot corresponded to the input of a single thalamic fiber, we used a threshold single fiber stimulation paradigm.

To investigate whether the difference in the number of transduction channels between Tmc1+/Δ;Tmc2Δ/Δ and Tmc1Bth/Δ;Tmc2Δ/Δ hair cells was due to changes in gene expression, we performed a quantitative RT-PCR analysis using cochlear tissue harvested from Tmc1+/Δ;Tmc2Δ/Δ and Tmc1Bth/Δ;Tmc2Δ/Δ mice. Neratinib mouse The analysis revealed significantly higher Tmc1 mRNA expression

in Bth mice ( Figure 4G), which may account for the larger whole-cell transduction currents in these mice. The mechanism for altered Tmc1 gene expression and eventual hair cell death in the Bth mice is unclear. However, recent evidence shows that proper calcium homeostasis is required for hair cell survival ( Esterberg et al., 2013), raising the possibility that altered calcium permeability in Bth mice may lead to hair cell degeneration and deafness ( Figure S1B). A similar mechanism may underlie dominant, progressive Trametinib supplier hearing loss in DFNA36 patients who carry a point mutation (p.G417R) in an adjacent residue in the human TMC1 ortholog ( Yang et al., 2010). Next, we measured single-channel currents

in wild-type inner hair cells at time points when both Tmc1 and Tmc2 were expressed ( Figure 5). During the first postnatal week we recorded a range of unitary current amplitudes from all regions of the cochlea that was significantly broader than that observed in Tmc mutant mice. Representative examples that span the range are shown in Figures 5A–5C. Unitary current values were divided by driving force (84 mV based on a 0 mV reversal potential in 50 μM Ca2+, see Figure S3) to calculate single-channel conductance (g). In wild-type mouse inner hair cells, g ranged between 60 and 330 pS during the first postnatal week. These values encompass the wide range of single-channel conductances reported for auditory hair cells of various species ( Crawford et al., 1991, Géléoc et al., 1997, Ricci et al., 2003 and Beurg et al., 2006). Figure 5D shows a scatter plot of 44 single-channel conductances measured from wild-type cells, along with measurements from

18 Tmc1+/Δ;Tmc2Δ/Δ and Tmc1Δ/Δ;Tmc2+/Δ cells. The data from the Tmc mutant mice are tightly clustered relative Sitaxentan to the wild-type data which are more broadly distributed. To examine the possibility that the wild-type data form discrete groups we performed a cluster analysis using three statistical tests ( Experimental Procedures) each of which reported that the data are best described by four clusters. The mean value for each cluster is indicated by the straight lines ( Figure 5D). Together, these data reveal that both Tmc1+/Δ;Tmc2Δ/Δ and Tmc1Δ/Δ;Tmc2+/Δ hair cells have transduction channels with relatively homogeneous single-channel properties, whereas wild-type hair cells that express both Tmc1 and Tmc2 display significant heterogeneity.

, 2010), while expression of angiopoietin-2 (Ang2), an Ang1 antagonist, was enhanced. Neural progenitors also participate in establishing the BBB by secreting Wnt ligands that activate β-catenin signaling in ECs (Figure 3). Genetic studies show that β-catenin signaling in ECs in vivo is required to induce and maintain BBB properties such as the expression of the glucose transporter Glut1 and the tight junction molecule claudin3 (Daneman et al., 2009, Liebner et al., 2008 and Stenman et al., 2008). Moreover, αvβ8 integrin-mediated adhesion

of neural progenitors and their glial progeny to the neurovascular unit are required for morphogenesis of the forebrain vasculature. Indeed, deletion of αvβ8 in neural progenitors results in the formation of misshaped EC clusters and cerebral hemorrhage despite basement membrane formation and pericyte coverage (McCarty, 2009). More PI3K cancer than five centuries ago, the Belgian anatomist Vesalius discovered that nerves and vessels track along each other to reach their target. The vascular and nervous system display intriguing parallelisms in their stereotyped architectural patterning and functional organization (Carmeliet and Tessier-Lavigne, 2005). Explanations for this copatterning mTOR inhibitor are that neurons and ECs respond to

the same (classes of) molecular cues, or that they coregulate each other’s migration. As the vascular system developed later in evolution than the nervous system, vessels are believed to have co-opted some of the genetic pathways for similar biological processes. Four classical axon guidance cue families Sclareol (netrins, slits, ephrins, semaphorins) guide growth cones of axons and regulate navigation of endothelial tip cells via similar principles of repulsion and attraction, which we will illustrate here only with a few (recent) prototypic examples. Endothelial tip cells extend filopodia that explore their surroundings for guidance cues. Neuropilin-1 (Nrp1) was discovered as a receptor for semaphorins in repulsive axon guidance but is also a coreceptor for VEGF and other

angiogenic factors on ECs (Carmeliet and Tessier-Lavigne, 2005). Nrp1 null embryos succumb due to cardiovascular malformations because of an interrupted interaction with VEGF (Fantin et al., 2009 and Rosenstein et al., 2010). Nrp1 blockade is currently being evaluated as novel anti-angiogenic strategy for the treatment of cancer (Bagri et al., 2009). Semaphorins, other ligands of Nrp1, usually inhibit angiogenesis, though some can also be stimulatory (Capparuccia and Tamagnone, 2009). By activating Plexin-D1 directly, semaphorin 3E (Sema3E) controls vessel navigation via distinct mechanisms. In intersomitic vessels in zebrafish embryos, Sema3E, produced by perivascular cells, prevents ECs from erroneous navigation in unwanted territories, presumably by reorienting the cytoskeleton of the tip cell itself.