A defective midline glial scaffold is in part responsible for the erroneous ipsilateral projection of RGCs in zebrafish belladona/lhx2 mutants ( Seth et al., 2006). We therefore analyzed sections through the optic chiasm of Nrp1 null mutants with two established markers for midline glia, RC2 and NrCAM ( Marcus et al., 1995 and Williams et al., 2006). However, there were no obvious differences in the arrangement of the RC2-positive glia ( Figure 2E), and NrCAM was still expressed by these cells

( Figure S2B). The CD44/SSEA-positive neurons at the posterior border of the developing optic chiasm, which are required for RGC axon extension across the midline ( Marcus et al., 1995 and Sretavan et al., 1995), were also present in Nrp1 null mutants ( Figure S2C). Finally, we looked at the expression of the ephrin B2 gene (Efnb2; ephrin-B2), which encodes the guidance cue that repels EPHB1-expressing RGC axons from the midline Autophagy Compound Library manufacturer to steer them into the ipsilateral path ( Williams et al., 2003). However, ephrin B2 expression at the chiasmatic midline was similar in mutants and wild-types ( Figure 2E). We conclude that the architecture of the optic chiasm is not obviously perturbed in

Nrp1 null mutants. We next asked if the increased ipsilateral projection in Nrp1 null mutants was due CP-868596 solubility dmso to an enlargement of the retinal domain that gives rise to ipsilaterally projecting RGCs. These neurons arise in two overlapping phases in the mouse. An early but transient

ipsilateral projection arises from RGCs in the dorsocentral retina between E12.5 and E14.5; subsequently, RGCs located predominantly in the ventrotemporal retina establish the permanent ipsilateral projection between E14.5 and E16.5 ( Godement et al., 1987, Williams et al., 2003 and Williams et al., 2006). Consistent with previous studies, Ephb1 was expressed in the E14.5 wild-type dorsocentral retina, where the RGCs forming the early ipsilateral heptaminol projection arise ( Figure 2F). This expression domain appeared similar in Nrp1 null mutants ( Figure 2F). Due to lethality at E15.5, we were not able to examine Ephb1 expression in RGCs forming the permanent ipsilateral projection in Nrp1 null mutants. ZIC2 is a transcription factor that is both necessary and sufficient to specify the permanent ipsilateral RGCs and is expressed prior to Ephb1 in these cells and by undifferentiated cells in the ciliary margin ( Figure 2F; see Herrera et al., 2003 and Tian et al., 2008). Importantly, the Zic2 expression pattern was similar in Nrp1 null mutants and controls, with no expansion of the normal expression domain within the RGC layer or ectopic expression by RGCs in other regions of the retina ( Figure 2F). We conclude that NRP1 signaling does not regulate chiasm development by affecting the specification of RGCs that give rise to the transient or permanent ipsilateral projections.