MIDLINE SIGNALING IS REQUIRED FOR PAX GENE-REGULATION AND PATTERNING OF THE EYES

Pax6 and Pax2 are members of the Pax family of transcription factors t hat are both expressed in the developing visual system of zebrafish em bryos, Pax6 protein is present in all cells that form the neural retin a and pigment epithelium, whereas Pax2 is located primarily in cells t hat will give rise to the optic stalk, In this study, we have addresse d the role of midline signalling in the regulation of Pax2 and Pax6 di stributions and in the subsequent morphogenesis of the eyes, Midline s ignalling is severely perturbed in cyclops mutant embryos resulting in an absence of ventral midline CNS tissue and fusion of the eyes, Muta nt embryos ectopically express Pax6 in a bridge of tissue around the a nterior pole of the neural keel in the position normally occupied by c ells that form the optic stalks, In contrast, Pax2 protein is almost c ompletely absent from this region in mutant embryos, Concommitant with the changes in Pax protein distribution, cells in the position of the optic stalks differentiate as retina, These results suggest that a si gnal emanating from the midline, which is absent in cyclops mutant emb ryos, may be required to promote Pax2 and inhibit Pax6 expression in c ells destined to form the optic stalks. Sonic hedgehog (Shh also known as Vhh-1 and Hhg-1) is a midline signalling molecule that is absent f rom the neuroepithelium of cyclops mutant embryos at early development al stages, To test the possibility that Shh might be able to regulate the spatial expression of Pax6 and Pax2 in the optic primordia, it was overexpressed in the developing CNS. The number of cells containing P ax2 was increased following shh overexpression and embryos developed h ypertrophied optic stalk-like structures, Complimentary to the changes in Pax2 distribution, there were fewer Pax6-containing cells and pigm ent epithelium and neural retina were reduced. Our results suggest tha t Shh or a closely related signalling molecule emanating from midline tissue in the ventral forebrain either directly or indirectly induces the expression of Pax2 and inhibits the expression of Pax6 and thus ma y regulate the partitioning of the optic primordia into optic stalks a nd retinal tissue.