Fig. 3

Nonoverlapping morpholinos targeted to neogenin mRNA produced indistinguishable morphant phenotypes. (a-i) Reduction in the number of post-mitotic neurons in Neogenin morphants. Whole mount immunohistochemistry was used to visualize post-mitotic neurons at 24 hpf in embryos injected with the antisense morpholinos (ATG-MO and UTR-MO) or control morpholino (CONT-MO). (a, d, and g) Embryos injected with 4 ng CONT-MO. (b, e, and h) Embryos injected with 4 ng of ATG-MO. (c, f, and i) Embryos injected with 4 ng of the UTR-MO. (a) The control embryos displayed a morphologically normal and readily discernable spinal cord (sc), floorplate (fp), notochord (no), and V-shaped somites (so). (b and c) The spinal cord, floorplate, and notochord of the ATG-MO and UTR-MO Neogenin morphants were not as regular or well-defined as in the controls, and the somites were U-shaped. (d and g) At 24 hpf, a continuous population of Hu-positive post-mitotic neurons existed along the rostrocaudal axis of the spinal cord of the control embryos. (e, f, h, and i) The ATG-MO and UTR-MO Neogenin morphants displayed a reduced number of Hu-positive neurons with prominent gaps along the rostrocaudal axis of the spinal cord (arrows). (j-l) Marked increase in the number of dead cells in the neuroepithelium of Neogenin morphants. Embryos (24 hpf) were stained with acridine orange (AO) to indicate cell death. The embryos were flat-mounted for imaging in the confocal microscope. Main picture is a maximum intensity projection of the z series; inset shows a single confocal section to demonstrate that the dead cells are in the same plane as the neuroepithelium. There was a marked increase in the number of AO-positive cells present in the CNS in embryos injected with 5 ng of either the ATG-MO or the UTR-MO (j and k) compared to embryos injected with 5 ng CONT-MO (l). (j-l) Dorsal views with rostral to the top.