The ventral median floor plate (FP) is a well-examined embryonic structure, which is involved in neuron differentiation and axon outgrowth. The FP of different vertebrates expresses the glycoprotein Reissner's substance (RS). This glycoprotein is also produced by the dorsal median subcommissural organ (SCO). We examined if the dorsal SCO and the ventral FP are interdependent for the expression of RS and looked for indications for a role of RS in axon outgrowth. Therefore, we examined zebrafish embryos of wildtype (wt) and the mutants cyclops(tf219) (cyc) and one-eyed pinhead(tz257) (oep), which both lack the FP. Our studies demonstrate that the FP is not necessary in order to induce the expression of RS in the SCO. The pattern of the anti-RS immunolabelling in the mutants is, however, changed compared to wt zebrafish embryos. As a consequence of the lacking FP and the degenerated ventricle system in cyc and oep mutants, a Reissner's fibre (RF) is not formed. Our studies confirm earlier results about the axon growth in cyc mutants, and provide the first detailed data about the aberrant axon growth in oep mutants. The modified outgrowth of the medial longitudinal fascicle in both mutants could be associated with the lack of RS/RF in the rhombencephalon and spinal cord. The neurites of the posterior commissure follow the aberrant position of the SCO in oep mutants. Our results suggest that both the RS of the ventral FP/flexural organ (FO) and the RS of the dorsal SCO have an influence on the outgrowth of axons and formation of commissures.