Fig. 1. Mechanisms that establish the retinotectal topographic map and lack of an effect of electroporation of a control RCAS vector on its development. (A) Normal development of the retinotopic map in the chick retinotectal projection. Initially, RGC axons extend posteriorly past the AP (anterior-posterior) location of their future TZ (circle). In addition, RGC axons originating from the same DV (dorsal-ventral) retinal location enter and extend across the tectum with a broad distribution along its LM (lateral-medial) axis. RGC axons form interstitial branches along their shafts at the AP level of their TZ; the branches are extended along the LM axis toward the TZ where they arborize. Later, overshooting segments of the primary axons are eliminated. Graded expression of Eph receptors in the retina and their ephrin ligands in the tectum are indicated. (B) Schematic of midbrain electroporation procedure on an E1.5 chick embryo. Cathode (+) and anode (–) electrodes were positioned on the opposite sides of the midbrain. (C) Dorsal view of an E12 chick brain. Between E6, when RGC axons first enter the tectum anteriorly, and E12, the tectal lobes rotate such that anterior tectum moves ventrally and away from the midline. This rotation results in the developmental AP (anterior-posterior) axis of the tectum (dashed line) being roughly perpendicular to the AP axis of the brain. For analysis, the optic tectum (ot) was removed, cut along the AP tectal axis, and the medial and lateral halves were mounted whole as shown in the drawing at right. The asterisk is in the same location in the photo and drawing. (C) In situ hybridization using an S³⁵-labeled ephrin-B1 probe on a coronal section through an E13 tectum transfected on E1.5 with an RCAS-ephrin-B1-IRES-eGFP. The transfection results in a columnar pattern of ectopic ephrin-B1 expression from the neuroepithelium (ne) to the stratum opticum (so). (D) Medial (M) half of an E13.5 tectum transfected with RCAS-eGFP at E1.5. Transfections domains express the green eGFP reporter. DiI was focally injected into NV (nasal-ventral) retina (red dot, left inset). DiI-labeled RGC axons (red) are visible in posterior tectum and arborize at the correct location for their TZ in mid-tectum. Branches are unaffected in areas of eGFP expression (right inset). RGC axons are also unaffected by the eGFP. cb, cerebellum; fb, forebrain. Scale bar in D: 300 µm and 100 µm in right inset.

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