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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 Elk3 knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In Elk3(-/-) mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in Elk3(-/-) 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 Elk3(-/-) 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, in vitro 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 Elk3 knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.

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.

Engrailed transcription factors regulate the expression of guidance cues that pattern retinal axon terminals in the dorsal midbrain. They also act directly to guide axon growth in vitro. We show here that an extracellular En gradient exists in the tectum along the anterior-posterior axis. Neutralizing extracellular Engrailed in vivo with antibodies expressed in the tectum causes temporal axons to map aberrantly to the posterior tectum in chick and Xenopus. Furthermore, posterior membranes from wild-type tecta incubated with anti-Engrailed antibodies or posterior membranes from Engrailed-1 knockout mice exhibit diminished repulsive activity for temporal axons. Since EphrinAs play a major role in anterior-posterior mapping, we tested whether Engrailed cooperates with EphrinA5 in vitro. We find that Engrailed restores full repulsion to axons given subthreshold doses of EphrinA5. Collectively, our results indicate that extracellular Engrailed contributes to retinotectal mapping in vivo by modulating the sensitivity of growth cones to EphrinA.

Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. We show here that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. Our findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion.

Engrailed-2 (En-2), a homeodomain transcription factor, is expressed in a caudal-to-rostral gradient in the developing midbrain, where it has an instructive role in patterning the optic tectum--the target of topographic retinal input. In addition to its well-known role in regulating gene expression through its DNA-binding domain, En-2 may also have a role in cell-cell communication, as suggested by the presence of other domains involved in nuclear export, secretion and internalization. Consistent with this possibility, here we report that an external gradient of En-2 protein strongly repels growth cones of Xenopus axons originating from the temporal retina and, conversely, attracts nasal axons. Fluorescently tagged En-2 accumulates inside growth cones within minutes of exposure, and a mutant form of the protein that cannot enter cells fails to elicit axon turning. Once internalized, En-2 stimulates the rapid phosphorylation of proteins involved in translation initiation and triggers the local synthesis of new proteins. Furthermore, the turning responses of both nasal and temporal growth cones in the presence of En-2 are blocked by inhibitors of protein synthesis. The differential guidance of nasal and temporal axons reported here suggests that En-2 may participate directly in topographic map formation in the vertebrate visual system.

Axon growth is governed by the ability of growth cones to interpret attractive and repulsive guidance cues. Recent studies have shown that secreted signaling molecules known as morphogens can also act as axon guidance cues. Of the large family of Wnt signaling components, only Wnt4 and Wnt5 seem to participate directly in axon guidance. Here we show that secreted Frizzled-related protein 1 (SFRP1), a proposed Wnt signaling inhibitor, can directly modify and reorient the growth of chick and Xenopus laevis retinal ganglion cell axons. This activity does not require Wnt inhibition and is modulated by extracellular matrix molecules. Intracellularly, SFRP1 function requires G(alpha) protein activation, protein synthesis and degradation, and it is modulated by cyclic nucleotide levels. Because SFRP1 interacts with Frizzled-2 (Fz2) and interference with Fz2 expression abolishes growth cone responses to SFRP1, we propose a previously unknown function for this molecule: the ability to guide growth cone movement via the Fz2 receptor.

The topographic positioning of retinal axons in the optic tectum is regulated, at least in part, by ephrinA/EphA repulsive interactions. Temporal axons, expressing high levels of EphA receptors, project to the ephrinA5-poor anterior tectum and avoid the ephrinA5-rich posterior tectum. To examine the dynamic behavior of temporal growth cones when they first encounter ephrinA, we manipulated ephrinA-coated beads with a laser tweezer into desired positions around the growth cones of chick retinal axons in culture. At high concentrations of ephrinA5 on the beads, growth cones typically collapsed on contacting the bead. At low concentrations, however, growth cones showed heterogeneous responses with some growth cones showing repulsive turning and others showing attractive turning after contacting the bead. Experiments with two beads indicate that retinal axons integrate guidance information that is provided simultaneously at two discrete locations. When a time-delay was introduced between exposure to the first and the second bead, individual axons exhibited a stereotyped response to the repeated stimuli, either responding with attraction followed by attraction, or showing repulsion followed by repulsion or collapse. Our results suggest the existence of at least two retinal subpopulations from the temporal retina, one being attracted, another being repelled by low levels of ephrinA5. These findings demonstrate that temporal retinal axons are not universally repelled by ephrinA5 and suggest that their ability to respond differentially to low concentrations may help them to map in a continuous manner over the surface of the anterior tectum., (2004 Wiley Periodicals, Inc.)

Axons from the retina traverse different molecular territories as they navigate to the tectum. A single territory might span only a few cell diameters and harbour multiple guidance cues, many of which are beginning to be characterized. Also present in the pathway are 'modulators' that influence a growth cone's response to a coincident signal but do not guide growth directly. An emerging principle is that the growth cone, itself, changes molecularly as it journeys through the visual pathway. Growing retinal axons contain mRNAs, ubiquitinating and apoptotic enzymes, translation and degradation machinery. Guidance cues can trigger rapid and local synthesis, degradation and endocytosis of proteins, providing a fast and flexible way for growth cones to respond to cues in their microenvironment and to alter their responsiveness. The data raise the idea that the localized synthesis and downregulation of proteins might help to steer retinal axon growth and, further, might contribute to the changing character of a growth cone as it ages.

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, play important roles during development of the nervous system. Frequently they exert their functions through a repellent mechanism, so that, for example, an axon expressing an Eph receptor does not invade a territory in which an ephrin is expressed. Eph receptor activation requires membrane-associated ligands. This feature discriminates ephrins from other molecules sculpturing the nervous system such as netrins, slits and class 3 semaphorins, which are secreted molecules. While the ability of secreted molecules to guide axons, i.e. to change their growth direction, is well established in vitro, little is known about this for the membrane-bound ephrins. Here we set out to investigate--using Xenopus laevis retinal axons--the properties of substratum-bound and (artificially) soluble forms of ephrin-A5 (ephrin-A5-Fc) to guide axons. We find--as expected on the basis of chick experiments - that, when immobilised in the stripe assay, ephrin-A5 has a repellent effect such that retinal axons avoid ephrin-A5-Fc-containing lanes. Also, retinal axons react with repulsive turning or growth cone collapse when confronted with ephrin-A5-Fc bound to beads. However, when added in soluble form to the medium, ephrin-A5 induces growth cone collapse, comparable to data from chick. The analysis of growth cone behaviour in a gradient of soluble ephrin-A5 in the 'turning assay' revealed a substratum-dependent reaction of Xenopus retinal axons. On fibronectin, we observed a repulsive response, with the turning of growth cones away from higher concentrations of ephrin-A5. On laminin, retinal axons turned towards higher concentrations, indicating an attractive effect. In both cases the turning response occurred at a high background level of growth cone collapse. In sum, our data indicate that ephrin-As are able to guide axons in immobilised bound form as well as in the form of soluble molecules. To what degree this type of guidance is relevant for the in vivo situation remains to be shown.