Growth cone response to ephrin gradients produced by microfluidic networks.

A microfluidic network (microFN) etched into a silicon wafer was used to deliver protein solutions containing different concentrations of the axonal guidance molecule ephrinA5 onto a silicone stamp. In a subsequent microcontact printing (microCP) step, the protein was transferred onto a polystyrene culture dish. In this way, stepwise substrate-bound concentration gradients of ephrinA5 were fabricated spanning a total distance of 320 microm. We tested the response of chick retinal ganglion cell (RGC) axons, which are guided in vivo by ephrin gradients, to these in vitro gradients. Temporal, but not nasal axons stop at a distinct zone in the gradient, which is covered with a certain surface density of substrate-bound ephrinA5. Within the temporal RGC population, all axons respond uniformly to the gradients tested. The position of the stop zone depends on the slope of the gradient with axons growing further into the gradient in shallow gradients than in steep gradients. However, axons stop at lower ephrinA5 concentrations in shallow gradients than in steep gradients, indicating that the growth cone can adjust its sensitivity during the detection of a concentration gradient of ephrinA5.

Substrate-bound protein gradients for cell culture fabricated by microfluidic networks and microcontact printing.

Graded distributions of proteins are pivotal for many signaling processes during development, such as morphogenesis, cell migration, and axon guidance. Here, we describe a technique to fabricate substrate-bound stepwise protein gradients by means of a microfluidic network etched into a silicon wafer with an array of parallel 14-micrometer-wide channels, which can be filled with a series of arbitrarily chosen protein solutions. In a subsequent microcontact printing step, the protein pattern is transferred onto a surface and is used as a substrate for cell culture. Cellular responses to a defined microscopic pattern of a protein, such as guided axonal outgrowth and directed migration, cell polarization, changes in morphology, and signaling, can be thus studied in a controlled in vitro environment.

Stripe assay to examine axonal guidance and cell migration.

Stripe assays have been widely employed as in vitro test systems to study the responses of growing axons, as well as migrating cells, to established or novel guidance molecules. We provide detailed protocols for both the original and the modified version of this assay, as they allow the analysis of the 'guidance properties' of active components present in crude membrane fractions or as purified molecules. Silicon matrices are used to produce striped patterns of active molecules on a surface (referred to as 'carpet'), followed by culturing of neurons, or any other cell type, on these carpets. After 1-2 days in culture, striped outgrowth of extending neurites--indicative of guided migration of cell processes--can be observed. We also discuss potential other applications (e.g., in neuronal regeneration and development) and modifications of the assay. The preparation of 10-12 carpets takes approximately 4-5 h.

Growth cone navigation in substrate-bound ephrin gradients.

Graded distributions of ephrin ligands are involved in the formation of topographic maps. However, it is still poorly understood how growth cones read gradients of membrane-bound guidance molecules. We used microcontact printing to produce discontinuous gradients of substrate-bound ephrinA5. These consist of submicron-sized protein-covered spots, which vary with respect to their sizes and spacings. Growth cones of chick temporal retinal axons are able to integrate these discontinuous ephrin distributions and stop at a distinct zone in the gradient while still undergoing filopodial activity. The position of this stop zone depends on both the steepness of the gradient and on the amount of substrate-bound ephrin per unit surface area. Quantitative analysis of axon outgrowth shows that the stop reaction is controlled by a combination of the local ephrin concentration and the total amount of encountered ephrin, but cannot be attributed to one of these parameters alone.

Microcontact printing of axon guidance molecules for generation of graded patterns.

Microcontact printing (microCP) of proteins has been successfully used for patterning surfaces in various contexts. Here we describe a simple 'lift-off' method to print precise patterns of axon guidance molecules, which are used as substrate for growing chick retinal ganglion cell (RGC) axons. Briefly, the etched pattern of a silicon master is transferred to a protein-coated silicone cuboid (made from polydimethylsiloxane, PDMS), which is then used as a stamp on a glass coverslip. RGC explants are placed adjacent to the pattern and cultured overnight. Fluorescent labeling of the printed proteins allows the quantitative analysis of the interaction of axons and growth cones with single protein dots and of the overall outgrowth and guidance rate in variously designed patterns. Patterned substrates can be produced in 3-4 h and are stable for up to one week at 4 degrees C; the entire protocol can be completed in 3 d.

On the turning of Xenopus retinal axons induced by ephrin-A5.

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.

RGM is a repulsive guidance molecule for retinal axons.

Axons rely on guidance cues to reach remote targets during nervous system development. A well-studied model system for axon guidance is the retinotectal projection. The retina can be divided into halves; the nasal half, next to the nose, and the temporal half. A subset of retinal axons, those from the temporal half, is guided by repulsive cues expressed in a graded fashion in the optic tectum, part of the midbrain. Here we report the cloning and functional characterization of a membrane-associated glycoprotein, which we call RGM (repulsive guidance molecule). This molecule shares no sequence homology with known guidance cues, and its messenger RNA is distributed in a gradient with increasing concentration from the anterior to posterior pole of the embryonic tectum. Recombinant RGM at low nanomolar concentration induces collapse of temporal but not of nasal growth cones and guides temporal retinal axons in vitro, demonstrating its repulsive and axon-specific guiding activity.