Interdisciplinary Program in Brain Science Eye movement & Vision Lab. Hwang, JaeWon

1. Genetic Analysis of Ephrin-A2 and Ephrin-A5Show Their Requirement in Multiple Aspectsof Retinocollicular Mapping Interdisciplinary Program in Brain Science Eye movement & Vision Lab. Hwang, JaeWon

2. Introduction • Topographic Maps • Chemoaffinity Theory • Eph receptor and Ephrin • Gradient vs Competition Model • Neuraxis

3. 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.

4. 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)

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

6. 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.

7. Neuraxis

8. Experiment • Methods • Results

9. 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.

10. 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

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

12. Result – Double Mutant 2

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

14. Result - Ephrin Expression 2

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

16. Result – Stripe Assays A C D B strong preference no preference Axon preference for anterior SC lanes

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

18. Discussion • Discussion 1 • Discussion 2 • Discussion 3 • Discussion 4 • Discussion 5

19. 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.

20. 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.

21. 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.

22. 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.

23. 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)

24. 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.