Retinal ganglion cell development in rtn4a- and rtn4b-knockdown, GFP–transgenic embryos. (A) through (I)tg(brn3c:mGFP) embryos at 3 dpf exhibit labeled retinal ganglion cells (RGCs) in the eye (arrow) and RGC axons in the optic nerve and tract as well as in the optic tectum (t). (A), (D) and (G) Lateral views of the left eye and optic tectum (anterior to the left). (B), (C), (E), (F), (H) and (I) Ventral views (anterior to the top). (A) to (C) In control morpholino (Co MO)–injected embryos, RGC axons reached the optic tectum (t) and innervated the neuropil (A). The ventral view shows the RGCs in both eyes (arrow) and the RGC axons in the optic nerve, the chiasm and the optic tract (B). (C), (F) and (I) RGCs and their axons were costained with antibodies against neurolin. RGCs from both morphants, rtn4a(D) to (F) and, to a greater extent, rtn4b(G) to (I), cover smaller areas of the retina (outlined) and their axons show aberrant pathways. (J) to (L) Quantification of eye size, the area covered by RGCs and their ratio. (M) to (O) and (P) to (R) Rescue of the rtn4a and rtn4b RGC phenotypes. Arrow points to RGCs. (S) Quantification at 3 dpf showing the reduction in the area covered by RGCs after injection of rtn4a- and rtn4b-MO1 (separately and combined) and rtn4b-MO1 and rtn4b-mRNA, as indicated under each bar. Control-MO (n = 28), rtn4a-l-MO1 (5.0 ng) (n = 16), rtn4a-l-MO1 (2.5 ng) + rtn4a-l-mRNA (100 pg) (n = 32), rtn4b-MO1 (2.5 ng) (n = 22), rtn4b-MO2 (2.5 ng) (n = 24), rtn4b-MO1 (5.0 ng) (n = 19), rtn4b-MO2 (5.0 ng) (n = 24), rtn4b-MO1 and rtn4b-MO2 (2.5 ng) (n = 27), rtn4b-MO (5.0 ng) and rtn4b-mRNA (100 pg) (n = 23) and rtn4a-l-MO1 (2.5 ng) and rtn4b-MO1 (2.5 ng) (n = 27). Scale bar = 100 μm.
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