Figure 3

Rtn4a and Rtn4b protein levels and morpholino knockdown. (A) Western blot (WB) analysis using the zebrafish α-Rtn4a antibody [15] shows that Rtn4a-l (an approximately 43-kDa band, arrow) is suppressed either by injection of a mixture of morpholinos (MOs) against each rtn4a isoform or a MO against the rtn4a-l isoform. Embryos expressing an Rtn4a-l-GFP fusion can overcome the Rtn4a MO1 downregulation. GFP antibodies detect the fusion protein at approximately 70 kDa and GFP alone at approximately 26 kDa. α-tubulin (Tub) served as a protein loading control. (B) Exogenous Rtn4al-GFP was detected at 6 hpf, but simultaneous coinjection of rtn4a-l MO1 abrogated its expression. (C) α-Rtn4b antibodies show the downregulation of Rtn4b for both MOs used in the experiments. The 180-kDa band (arrowhead), probably a dimerization band, is entirely reduced in MO-injected embryos, and the 90-kDa band (arrow) shows a strong reduction. (D) through (J) Rtn4a and Rtn4b immunostainings at various developmental stages. At 1dpf, Rtn4a is expressed in distinct neuronal structures, including the forebrain (fb), the presumptive optic tectum (t) and the hindbrain (hb). Rtn4a is also present in the eye anlage (e), otic vesicle (ov) (D), and in muscle tissue (m) (E). At 2 dpf, Rtn4a is detected in retinal ganglion cells (RGCs) (arrow), the optic nerve (arrowhead) and the olfactory system (o) (F). At 1 dpf, Rtn4b is expressed in the same structures as Rtn4a except the muscle tissue (G). Rtn4b is also detected in the spinal cord (sc) (H). At 2 dpf, the Rtn4b signal is still present in the spinal cord (sc) and can also be seen in growing primary motor neurons (arrow) (I). In the head, Rtn4b is present in RGCs (arrow) and the optic nerve (arrowhead) (J).