Genetic Analysis of Ephrin-A2 and Ephrin-A5Show Their Requirement in Multiple Aspects

of Retinocollicular Mapping

Interdisciplinary Program in Brain ScienceEye 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 post

erior arborization Nasal axons- no defects in ephrin-A2-/-

mice- an additional more anter

ior arborization in ephrin-A5-/- mice

Result – Double Mutant

Homozygote- the ectopic terminati

on extended over all regions

Heterozygote- similar to single mut

ant

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

A BC D

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.

1. Nasal axons shift anteriorly.2. Axons are not respecified to a specific ecto

pic position.3. 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.

1. Ephrin acting directly as dorsoventral labels.

2. A secondary effect to the anteroposterior defect.