Genetic Analysis of Ephrin-A2 and Ephrin-A5 Show Their Requirement in Multiple Aspects of Retinocollicular Mapping Interdisciplinary Program in Brain Science Eye movement & Vision Lab. Hwang, JaeWon

Introduction  Topographic Maps  Chemoaffinity Theory  Eph receptor and Ephrin  Gradient vs Competition Model  Neuraxis

Topographic Maps  Axon projections in the vertebrate nervous system are typically organized with nearest neighbor relationships of the projecting neurons maintained in their connections within the target.

Chemoaffinity Theory  There are labels in gradients across the projecting and target fields and that axons find their correct location by matching up the labels. (Sperry, 1963)

Eph receptor and Ephrin  Eph receptor - RTK - At least 14 members  Ephrin - Eph ligand - At least 8 members - A family & B family - Repellant activity

Gradient vs Competition  Dual-gradient model There may be two opposing gradients, for example a repellent and an attractant, with each axon identifying its correct place as the point where the opposing forces cancel out.  Axon-axon competition There may be axon-axon competition for limiting positive factors in the target or by direct axon-axon interactions.

Neuraxis

Experiment  Methods  Results

Methods  Mice with a disruption in the ephrin-A2 and ephrin-A5 gene. (using homologous recombination in ES cells)  A focal injection of DiI was made in one retina followed by examination of the contralateral midbrain.

Result - Single Mutant  Temporal axons - an additional more posterior arborization  Nasal axons - no defects in ephrin-A2 - /- mice - an additional more anterior arborization in ephrin-A5 -/- mice

Result – Double Mutant  Homozygote - the ectopic termination extended over all regions  Heterozygote - similar to single mutant

Result – Double Mutant 2

Result - Ephrin Expression Ephrin RNA distribution Ephrin(protein) distribution (Wild Type)

Result - Ephrin Expression 2

Result - EphA Expression EphA RNA expression Ephrin-A2 protein EphA5 protein

Result – Stripe Assays Axon preference for anterior SC lanes no preference strong preference ABCD

Result - Summary Fig 8. Schmatic illustration of retinocollicular mapping phenotypes in mice with disrupted ephrin-A2 or ephrin-A5 genes.

Discussion  Discussion 1  Discussion 2  Discussion 3  Discussion 4  Discussion 5

Discussion 1-1  Simple repulsion model and two counterbalanced gradients cannot account for the behavior of nasal axons and double mutant phenotype. ⇒ An alternative is a model involving a repellent gradient of ephrins, in combination with axon-axon competition.

Discussion 1-2  Incorporating competiton in the model can explain several aspects of data.  Nasal axons shift anteriorly.  Axons are not respecified to a specific ectopic position.  Retinal axons fill the available space in the target.

Discussion 2  The termination zones always took the form of punctate spots in the SC. But why? ⇒ There may be a mechanism that causes neighboring axons in the retina to cluster together in the SC, such as Hebbian activity-dependent refinement.

Discussion 3  Do axons detect absolute or relative ephrin levels? ⇒ Data in double heterozygotes show neither of them correct. The ability of axons to discriminate between any two points on the tectum would depend on those two points having a sufficiently great difference in ephrin concentration.

Discussion 4  Additive and Distinct function of ephrin-A2 and ephrin-A5 - Double homozygous mutant shows a synergistic phenotype. (Additive) - Ephrin-A5 is dominant in posterior tectum. (Distinct) - Ephrin-A2 -/-, ephrin-A5 -/-, and ephrin-A2 +/- ;ephrin-A5 +/- mice all show a similar temporal axon phenotype in anterior SC. (Additive)

Discussion 5  Two possible model of dorsoventral mapping errors in double homozygous mutant.  Ephrin acting directly as dorsoventral labels.  A secondary effect to the anteroposterior defect.