Axons of retinal ganglion cells (RGCs) make a divergent choice at the optic chiasm to cross or avoid the midline in order to project to ipsilateral and contralateral targets, thereby establishing the binocular visual pathway. The zinc-finger transcription factor Zic2 and a member of the Eph family of receptor tyrosine kinases, EphB1, are both essential for proper development of the ipsilateral projection at the mammalian optic chiasm midline. Here, we demonstrate in mouse by functional experiments in vivo that Zic2 is not only required but is also sufficient to change the trajectory of RGC axons from crossed to uncrossed. In addition, our results reveal that this transcription factor regulates the expression of EphB1 in RGCs and also suggest the existence of an additional EphB1-independent pathway controlled by Zic2 that contributes to retinal axon divergence at the midline.

Background: The neural retina is a highly structured tissue of the central nervous system that is formed by seven different cell types that are arranged in layers. Despite much effort, the genetic mechanisms that underlie retinal development are still poorly understood. In recent years, largescale genomic analyses have identified candidate genes that may play a role in retinal neurogenesis, axon guidance and other key processes during the development of the visual system. Thus, new and rapid techniques are now required to carry out high-throughput analyses of all these candidate genes in mammals. Gene delivery techniques have been described to express exogenous proteins in the retina of newborn mice but these approaches do not efficiently introduce genes into the only retinal cell type that transmits visual information to the brain, the retinal ganglion cells (RGCs).

The organization of the visual system is different in birds and mammals. In both, retinal axons project topographically to the visual targets in the brain; but whereas in birds visual fibers from the entire retina decussate at the optic chiasm, in mammals, a number of axons from the temporal retina diverge at the midline to project ipsilaterally. Gain-of-function experiments in chick raised the hypothesis that the transcription factor Foxd1 specifies retinal temporal identity. However, it remains unknown whether Foxd1 is necessary for this function. In mammals, the crucial role of Foxd1 in the patterning of the optic chiasm region has complicated the interpretation of its cell-autonomous function in the retina. Furthermore, target molecules identified for Foxd1 are different in chicks and mice, leading to question the function of Foxd1 in mammals. Here we show that in the mouse, Foxd1 imprints temporal features in the retina such as axonal ipsilaterality and rostral targeting in collicular areas and that EphA6 is a Foxd1 downstream effector that sends temporal axons to the rostral colliculus. In addition, our data support a model in which the desensitization of EphA6 by ephrinA5 in cis is not necessary for the proper functioning of EphA6. Overall, these results indicate that Foxd1 functions as a conserved determinant of temporal identity but reveal that the downstream effectors, and likely their mechanisms of action, are different in mammals and birds.

Sonic Hedgehog (Shh) signaling is an important determinant of vertebrate retinal ganglion cell (RGC) development. In mice, there are two major RGC populations: (1) the Islet2-expressing contralateral projecting (c)RGCs, which both produce and respond to Shh; and (2) the Zic2-expressing ipsilateral projecting RGCs (iRGCs), which lack Shh expression. In contrast to cRGCs, iRGCs, which are generated in the ventrotemporal crescent (VTC) of the retina, specifically express Boc, a cell adhesion molecule that acts as a high-affinity receptor for Shh. In Boc Ϫ/Ϫ mutant mice, the ipsilateral projection is significantly decreased. Here, we demonstrate that this phenotype results, at least in part, from the misspecification of a proportion of iRGCs. In Boc Ϫ/Ϫ VTC, the number of Zic2-positive RGCs is reduced, whereas more Islet2/Shh-positive RGCs are observed, a phenotype also detected in Zic2 and Foxd1 null embryos. Consistent with this observation, organization of retinal projections at the dorsal lateral geniculate nucleus is altered in Boc Ϫ/Ϫ mice. Analyses of the molecular and cellular consequences of introducing Shh into the developing VTC and Zic2 and Boc into the central retina indicate that Boc expression alone is insufficient to fully activate the ipsilateral program and that Zic2 regulates Shh expression. Taking these data together, we propose that expression of Boc in cells from the VTC is required to sustain Zic2 expression, likely by regulating the levels of Shh signaling from the nearby cRGCs. Zic2, in turn, directly or indirectly, counteracts Shh and Islet2 expression in the VTC and activates the ipsilateral program.