Medial-lateral Topography is Established in the Chick Retinotectalprojection Through Topographic Axon Ingrowth and Topographically Directed Branch Extension

Abstract

Abstract: : Purpose: To determine the molecular mechanisms that control the development of topographic axonal connections in the retinotectal projection along the medial-lateral (M-L) axis. Methods: Retinal ganglion cell (RGC) axons were labeled with DiI injections in peripheral temporal retina of embryonic chick at multiple stages of development of the retinotectal projection (33 tectal wholemounts in total, embryonic day 10 to 13). Labeled axons were then imaged in tectal wholemounts using confocal microscopy. The composite projections were traced, digitized, and quantitatively analyzed with respect to axon and branch position and length relative to the location of the topographically correct termination zone (TZ). Results: Our analyses of topographic mapping of retinal ganglion cell axons in chick optic tectum indicate the map is established by interstitial branches that form along the axon shaft. These interstitial branches are distributed topographically along both M-L and anterior-posterior (A-P) axes, however the M-L axis distribution is due to a bias of axons to enter the tectum at topographically correct M-L positions, rather than through de-novo topographic specific branching as occurs along the A-P axis. Branch density is equal across the M-L axis regardless of axon location. However, branches originating at M-L locations outside of the correct termination zone (TZ) demonstrate a preference to grow directionally towards the TZ and this behavior correlates with branch size. Branches less than 25 microns show no bias in directionality, while branches greater than 250 microns show 3 fold higher extension towards, than away from, the TZ. The topographic M-L bias in branch distribution and extension is further enhanced through preferential arborization of those branches terminating within the TZ, along with elimination of ectopic branches that terminate outside the TZ. Conclusions: There are distinct mechanisms for establishing topography along medial-lateral and anterior-posterior axes. Although M-L branching exhibits no de-novo topographic specificity, mispositioned branches appear to be able to connect to the topographically correct TZ through directed branch growth. This suggests EphB/ephrin-B, which regulates M-L map development, does so through unique molecular controls relative to EphA/ephrin-A regulation of A-P development.

Keywords: topography • visual development • microscopy: confocal/tunneling