1. Retinal Axon Response to Ephrin-As Shows a Graded, Concentration-Dependent Transition from Growth Promotion to Inhibition 
Michael J Hansen, Gerard E Dallal, John G Flanagan 
Neuron 
Volume 42, Issue 5, Pages (June 2004)
DOI: /j.neuron

2. Figure 1 In Vitro Axon Outgrowth Assay
(A) Schematic diagram of the assay. To vary ephrin concentration, ephrin DNA-transfected cell membranes were mixed with mock-transfected membranes in varying ratios. This combined membrane preparation was then mixed at a constant ratio with anterior tectal membranes. Explants from varying positions across the retinal nasal-temporal axis were grown on the membrane carpets, and outgrowth was digitally photographed and quantitated.
(B and C) Topographically specific response of both nasal and temporal axons to tectal membranes. Retinal explants were cultured on membrane carpets from anterior or posterior tectum. (B) shows explants that were labeled with fluorescent vital dye and photographed. (C) shows axon outgrowth quantitation. Outgrowth was measured as the number of pixels above background per unit length of explant (see the Experimental Procedures) and is expressed here relative to nasal axon outgrowth on anterior tectal membranes, which is given a value of 1. For each outgrowth condition, n = 11–14 explants. Error bars show SEM.
Neuron  , DOI: ( /j.neuron )

3. Figure 2
Response Profile of Varying Retinal Positions, on Carpets Containing Ephrin-A2 or Ephrin-A5
The response profiles that were elicited by ephrin-A2 and -A5 are different and show shapes comparable to their respective expression profiles in the SC. (A) Outgrowth response of retinal explants taken from across the nasal-temporal axis of the retina and cultured on membrane carpets containing ephrin-A5 or ephrin-A2. Outgrowth is graphed here relative to the retinal position that had maximal mean outgrowth: position 1 for ephrin-A5 and position 3 for ephrin-A2. For ephrin-A5, mouse retinas were divided into six contiguous 300 μm wide explants; n = 8–10 for each retinal position. For ephrin-A2, retinas were divided into eight contiguous 225 μm wide explants; n = 5–8 for each retinal position. Error bars show SEM. (B) Expression patterns of ephrin-A5 and ephrin-A2 in mouse SC. Upper panels show RNA in situ hybridization on parasagittal E18 mouse brain sections, and lower panels show densitometric scans through the SC. N, nasal; T, temporal; SC, superior colliculus; IC, inferior colliculus; PT, pretectum; A, anterior; P, posterior.
Neuron  , DOI: ( /j.neuron )

4. Figure 3
Retinal Axon Outgrowth, with Variation in Both Retinal Position and Ephrin-A2 Concentration
Representative photographs showing outgrowth from the eight contiguous explant positions (numbered 1 through 8) across the nasal-temporal axis of the retina, grown on substrates containing different proportions of membranes from ephrin-A2 DNA-transfected and untransfected cells. Outgrowth varies with both retinal position and ephrin concentration. Responses to ephrin-A2 membranes vary from total outgrowth inhibition (at higher concentrations and more temporal positions) to several-fold outgrowth promotion (at lower concentrations and nasal positions).
Neuron  , DOI: ( /j.neuron )

5. Figure 4 Quantitation of Retinal Axon Outgrowth Response to Ephrin-A2
Explants from across the nasal-temporal axis of the retina (numbered 1–8) were grown on substrate containing different proportions of membranes from ephrin-A2 DNA-transfected and untransfected cells. Outgrowth was on (A and D) 15% ephrin, (B and E) 50% ephrin, (C and F) 100% ephrin, and (G) 0% ephrin membranes. (A–C and G) Outgrowth from each position was quantitated and graphed. (D–F) Relative outgrowth, divided by outgrowth of the same retinal position on 0% ephrin membranes; on these graphs, a value of one indicates no net effect of ephrin-A2, higher values represent fold promotion, and zero represents complete inhibition. For each outgrowth condition, n = 5–8 assays. Error bars are SEM.
Neuron  , DOI: ( /j.neuron )

6. Figure 5
In Vitro Outgrowth Assay: Time Course and Test for Fasciculation Effects
(A) Time course of outgrowth. Explants from nasal retina (retinal position 2, solid lines) or temporal retina (retinal position 7, dashed lines) were grown on 0% (open squares) or 15% (filled squares) ephrin-A2 substrate. For each time point, n = 4–10 assays. Error bars are SEM. Axons appear to grow steadily until reaching a plateau after more than 48 hr.
(B) Mean pixel intensity in outgrowth assays. The mean pixel intensity above background was determined for each retinal position and ephrin substrate concentration and graphed. The trend of pixel intensities indicates that results of the outgrowth assay cannot be explained by axon fasciculation. For each outgrowth condition, n = 5–8 assays. Error bars are SEM.
Neuron  , DOI: ( /j.neuron )

7. Figure 6 Outgrowth Response of Retinal Axons to Soluble Ephrin
Retinal explants from nasal position 3 and temporal position 7 were cultured on 0% ephrin-A2 substrate carpets. Purified ephrin-A2-Fc fusion protein, either unclustered (A), preclustered with limiting amounts of anti-Fc antibody (B), or preclustered with limiting amounts of ephrin-A2-Fc (C), was included at different concentrations in the culture media. The upper panels show Western blot analysis of fractions from a gel exclusion column to measure the size of unclustered and preclustered ephrin-A2-Fc complexes. Over a range of concentrations and ligand clustering ratios, soluble ephrin-A2 caused inhibition but not promotion of outgrowth. For each outgrowth condition, n = 12–14 assays. Error bars show SEM.
Neuron  , DOI: ( /j.neuron )

8. Figure 7
A Topographic Relationship for Retinal Position versus Ephrin-A2 Concentration
The graphs show experimental results from the quantitative outgrowth assay. Values greater than one represent fold promotion of growth by ephrin-A2, values of one represent no net effect, and zero represents complete inhibition. (A) Variation of axon outgrowth with retinal position; each color represents a different ephrin-A2 concentration. (B) Variation of axon outgrowth with ephrin-A2 concentration; each line represents a different retinal position. (C) Graph relating retinal position to the corresponding ephrin-A2 concentration that has no net effect on axon outgrowth (determined from the graph in [B] as concentrations that produce relative outgrowth of 1). In (B) and (C), for clarity, the graph is limited to retinal positions 3 through 8 (see text). Given the shape and orientation of the ephrin-A2 gradient in the SC, the data produce a topographically appropriate relationship between retinal position and ephrin-A2 concentration.
Neuron  , DOI: ( /j.neuron )

9. Figure 8
Model to Explain Map Specification, Based on Concentration-Dependent Positive and Negative Effects of Ephrins
(A) Model for mapping, based on positive and negative response to ephrins. Axon growth within the target is promoted by low ephrin-A concentrations and inhibited by higher ephrin-A concentrations, with axons terminating at the neutral position between these positive and negative effects. Axons originating from different positions across the retina have different sensitivities to ephrin, presumably due to the graded distribution of EphA receptors in the retina, so that the neutral inflection point between positive and negative effects in the tectum/SC varies with retinal position. The result is the production of a smooth topographic map.
(B) Model to account for positive and negative responses, based on molecular properties of the ephrins. In this model, as ephrin concentration increases (blue triangle at base), ligand density and clustering on the cell surface increase. At low concentrations, ephrin has a positive effect on axon growth, which may be mediated by signaling or a simple adhesive interaction between surface bound ligands and receptors (green line on graph). At higher concentrations, a negative effect on axon growth increases due to increased density and clustering, triggering intracellular repellent signaling (red line on graph). Increased ligand clustering is also known to cause ligand cleavage, which may facilitate the transition from positive to negative effects, by abrogating adhesion (dotted green line). The overall effect of these positive and negative molecular influences would be a concentration-dependent transition from net positive to net negative effects on axon outgrowth. N, nasal; T, temporal; A, anterior; P, posterior.
Neuron  , DOI: ( /j.neuron )