Previously, roles for BMP signaling in the guidance of RGC axons to the optic disc were reported.
15,42 To examine whether
Smad4 is potentially involved in this BMP–dependent process, the behavior of RGC axons was analyzed by anterograde labeling of the RGC axons with the lipophilic dye DiI (
Fig. 6A). In the control retina, the RGC axons were traced from their site of origin to the optic disc in a highly stereotypic manner (
Fig. 6B). In contrast, in
Smad4-CKO embryos examined at E15.5 through E17.5, overt defects in optic disc axon trajectories were noted (
Figs. 6C,
6D). These defects were not localized to axons arising from any particular retinal quadrant. In several instances, the number of misrouting axons was large enough to obscure the optic disc itself (
Fig. 6D). At histologic levels, an apparent majority of RGC axons was fascicled to form the optic nerve at the ONH in CKO mutants at E14.5 (
Fig. 6E). Earlier in development, at E12.5, Pax2 expression marking the ventral aspects of the developing retina, including the prospective ONH, was observed in patterns comparable to that of controls (
Fig. 6F). However, by E15.5, Pax2 expression normally maintained in the ONH area (
Fig. 6G, arrow) was almost diminished in the mutant retina (
Fig. 6G, arrowheads). We also found abnormality in the expression of
netrin1 (
Ntn1), a key axon guidance gene, in the ONH (
Figs. 6H,
6I). Although
Ntn1 transcripts were properly initiated in the prospective ONH area at E12.5 in the
Smad4-CKO retina (
Fig. 6H, arrows), it was not upregulated and maintained within the retinal proper to define the ONH at E14.5 (
Fig. 6I, arrows). These defects were associated with markedly hypoplastic optic nerves (
Supplementary Fig. S1A).