Fig. 2

Aberrant dorsal spreading of the TPOC is observed following knockdown of Robo3. Scaffolds of 28 hpf zebrafish embryos labeled with acetylated α-tubulin. Rostral is to the left, dorsal is to the top. (A) In control MO scaffolds (5 ng), the axon tracts develop normally. (B) No significant axon guidance defects were observed following knockdown of robo3var2. Aberrant growth of axons in the TPOC was observed in robo3var1 MO1-injected embryos; TPOC axons were observed to spread out into more dorsal regions of the mesencephalon (C, arrowhead; compare D and E). (F) Coinjection with 350 pg of robo3-rescue cRNA rescued the robo3var1 MO induced TPOC defect. (G–J) Robo3 morpholinos act specifically to knockdown their targeted isoform (var1 or var2). Embryos injected with 250 pg 5′ robo3var1-GFP cRNA (G) or 250 pg 5′ robo3var2-GFP cRNA (I) show strong fluorescence at 28 hpf, which is lost following coinjection with the respective MO (robo3var1 MO1, H or robo3var2 MO1, J). (I) robo3var1 MO1 failed to abrogate GFP expression when coinjected with 52robo3var2-GFP cRNA, confirming that robo3-var1 MO1 specifically targets the var1 isoform. (K) The TPOC spreading phenotype was observed at a similar penetrance when both robo3var1 and robo3var2 were simultaneously knocked down (arrowhead). Scale bars: A–C, F, K: 50 μm; D, E: 17 μm; G–J: 375 μm.