B-type Eph receptors and ephrins induce growth cone collapse through distinct intracellular pathways

Forward and reverse signaling mediated by EphB tyrosine kinase receptors and their transmembrane ephrin-B ligands play important roles in axon pathfinding, yet little is known about the intracellular pathways involved. Here we have used growth cones from the ventral (EphB receptor-bearing) and dorsal (ephrin-B-bearing) embryonic Xenopus retina to investigate the signaling mechanisms in both forward and reverse directions. We report that unclustered, but not clustered, EphB2 ectodomains trigger fast (5-10 min) transient collapse responses in growth cones. This collapse response is mediated by low levels of intracellular cyclic GMP and requires proteasome function. In contrast, clustered, but not unclustered, ephrin-B1 ectodomains cause slow (30-60 min) growth cone collapse that depends on high cGMP levels and is insensitive to inhibition of the proteasomal pathway. Upon receptor-ligand binding, endocytosis occurs in the reverse direction (EphB2-Fc into dorsal retinal growth cones), but not the forward direction, and is also sensitive to proteasomal inhibition. Endocytosis is functionally important because blocking of EphB2 internalization inhibits growth cone collapse. Our data reveal that distinct signaling mechanisms exist for B-type Eph/ephrin-mediated growth cone guidance and suggest that endocytosis provides a fast mechanism for switching off signaling in the reverse direction. (C) 2003 Wiley Periodicals, Inc

Characterization of the guidance activity of ephrinA5 in vitro in different species (frog, fish, chick)

In dieser Arbeit sollte die Rolle von EphrinA5 beim Aufbau der retinotektalen Projektion charakterisiert werden. Seit langem sind Lenkungsmoleküle bekannt, die Axonen bei der Wegfindung in ihre korrekten Terminationszonen helfen. 1995 wurde EphrinA5 isoliert, das an Lenkungsprozessen beteiligt zu sein scheint. In der vorliegenden Arbeit wurde untersucht, ob EphrinA5 in vitro Axone in ihrer Wachstumsrichtung beeinflussen kann. Beobachtet wurden retinale Ganglienzellwachstumskegel von Huhn, Xenopus und Zebrafisch. In allen drei Spezies konnte EphrinA5 eine Richtungsänderung der Axone induzieren. Nachgewiesen werden konnte diese Lenkungsfunktion in verschiedenen Experimenten: im Streifentest, im Lenkungstest mit beschichteten Kügelchen und im Diffusionsgradienten löslicher Proteine. Zur Aufklärung des Lenkungsmechanismus wurden Experimente mit EphrinA5-beschichteten Kügelchen durchgeführt. Es ergab sich eine konzentrations- und substratabhängige Lenkung. Weitere Versuche zeigten, dass der Axonschaft bei der Lenkung des Wachstumskegels kaum beteiligt zu sein scheint, denn eine Reizung des Axonschaftes war nicht ausreichend, eine Richtungsänderung zu induzieren. Eine gleichzeitige Reizung mit zwei Kügelchen auf der gleichen oder auf verschiedenen Seiten des Wachstumskegels führte jeweils zu einer stark erhöhten Kollapsreaktion, Lenkung konnte nicht beobachtet werden. In allen Testsystemen traten sowohl Kollaps als auch Lenkung auf. Aufgrund dieser Beobachtung wurde ein Lenkungsmechanismus für EphrinA5 bei der Bildung der retinotektalen Projektion des Hühnchens vorgeschlagen. Er beruht auf einer Bifunktionalität des EphrinA5: es kann Lenkungs- und Stoppfunktion ausüben, je nach Ephrinkonzentration und der zeitlichen Dauer der Exposition. Mit Hilfe dieses Modells können viele der in vitro gewonnenen Ergebnisse erklärt werden. Eine Relevanz dieses Modells in vivo muss noch bestätigt werden. Die anfängliche Entstehung der Projektion kann jedoch damit erklärt werden.The aim of this work was to characterize the role of ephrinA5 in the establishment of the retinotectal projection. Some years ago guidance molecules were found, which help the axons to find the pathway to their correct termination zones. In 1995 ephrinA5 was isolated which seemed to be such a guidance molecule. In this work we want to test if ephrinA5 could induce a turning behaviour in vitro. We observed reactions of retinal ganglion cell growth cones of three different species: chick, Xenopus and zebrafish. We found that ephrinA5 could induce a turning reaction in axons of all three species. We could confirm this guidance function in three different in vitro assays: in the stripe-assay, with ephrinA5-coated beads and in the diffusion gradient of soluble proteins. The turning of axons was substrate-dependent, attraction could be converted into repulsion by changing the substrate. To learn more about the guidance mechanism of ephrinA5 we performed different experiments with ephrinA5-coated beads. We could see a substrate- and dose-dependent turning behaviour of axons. Other experiments showed that the axon shaft does not participate in guiding axons because placing the beads in contact with the axon shaft did not result in turning of the growth cones. Simultaneous irritation with two beads either on the same or on different sides of the growth cones revealed high collapse rates, but no turning. In all assays both collapse and turning reaction could be observed. Therefore we propose a guidance mechanism for ephrinA5 in the retinotectal projection in chick. EphrinA5 is a bifunctional protein: it could either exert a guidance function or a stop function depending on the ephrinA5-concentration and/or the temporal exposition to the protein. With this model many results of in vitro experiments could be explained, although the relevance of this model in vivo has to be proven. The initial projection of the axons however could be explained with this model

Endothelial SRF/MRTF ablation causes vascular disease phenotypes in murine retinae

Retinal vessel homeostasis ensures normal ocular functions. Consequently, retinal hypovascularization and neovascularization, causing a lack and an excess of vessels, respectively, are hallmarks of human retinal pathology. We provide evidence that EC-specific genetic ablation of either the transcription factor SRF or its cofactors MRTF-A and MRTF-B, but not the SRF cofactors ELK1 or ELK4, cause retinal hypovascularization in the postnatal mouse eye. Inducible, EC-specific deficiency of SRF or MRTF-A/MRTF-B during postnatal angiogenesis impaired endothelial tip cell filopodia protrusion, resulting in incomplete formation of the retinal primary vascular plexus, absence of the deep plexi, and persistence of hyaloid vessels. All of these features are typical of human hypovascularization-related vitreoretinopathies, such as familial exudative vitreoretinopathies including Norrie disease. In contrast, conditional EC deletion of Srf in adult murine vessels elicited intraretinal neovascularization that was reminiscent of the age-related human pathologies retinal angiomatous proliferation and macular telangiectasia. These results indicate that angiogenic homeostasis is ensured by differential stage-specific functions of SRF target gene products in the developing versus the mature retinal vasculature and suggest that the actin-directed MRTF-SRF signaling axis could serve as a therapeutic target in the treatment of human vascular retinal diseases

<i>Elk3</i> Deficiency Causes Transient Impairment in Post-Natal Retinal Vascular Development and Formation of Tortuous Arteries in Adult Murine Retinae

<div><p>Serum Response Factor (SRF) fulfills essential roles in post-natal retinal angiogenesis and adult neovascularization. These functions have been attributed to the recruitment by SRF of the cofactors Myocardin-Related Transcription Factors MRTF-A and -B, but not the Ternary Complex Factors (TCFs) Elk1 and Elk4. The role of the third TCF, Elk3, remained unknown. We generated a new <i>Elk3</i> knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In <i>Elk3(−/−)</i> mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in <i>Elk3(−/−)</i> mice from the age of four weeks, and persisted into late adulthood. Tortuous vessels have been observed in human pathologies, e.g. in ROP and FEVR. These human disorders were linked to altered activities of vascular endothelial growth factor (VEGF) in the affected eyes. However, in <i>Elk3(−/−)</i> mice, we did not observe any changes in VEGF or several other potential confounding factors, including mural cell coverage and blood pressure. Instead, concurrent with the post-natal transient delay of radial outgrowth and the formation of adult tortuous arteries, Elk3-dependent effects on the expression of Angiopoietin/Tie-signalling components were observed. Moreover, <i>in vitro</i> microvessel sprouting and microtube formation from P10 and adult aortic ring explants were reduced. Collectively, these results indicate that Elk3 has distinct roles in maintaining retinal artery integrity. The <i>Elk3</i> knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.</p></div

Incidence (%) of tortuous arteries.

<p>(* p<0.05 ** p<0.01 *** p<0.001, na = not analysed, ns = not significant, n = number of retinae analysed).</p><p>Incidence (%) of tortuous arteries.</p

Scanning laser ophthalmoscopy reveals abnormally shaped tortuous arteries in adult <i>Elk3(−/−)</i>, but not <i>Elk1/Elk4</i>^dKO eyes.

<p>(A) Scanning laser ophthalmoscopy (SLO) of <i>Elk3(+/+)</i> control (upper panel) and <i>Elk3(−/−)</i> knockout (lower panel) retinae by indocyanin green angiography (left) and a higher magnification of arteries in IsolectinB4 stained retinal flat-mounts of the same retina (middle; tortuous arteries are highlighted by red arrowheads). H&E staining of paraffin embedded whole eyes (right). (B) Scanning laser ophthalmoscopy (SLO) of <i>Elk1/Elk4</i> control (upper panel) and <i>Elk1/Elk4^dKO</i> (lower panel) retinae by indocyanin green angiography (left) and a higher magnification of arteries in IsolectinB4 stained retinal flat-mounts of the same retina (middle). H&E staining of paraffin embedded whole eyes (right). Scale bar in (middle) 200 µm, in (A right) 50 µm, in (B right) 100 µm.</p