Fig. 6

Vasna and Vasnb are specifically expressed in the retinal axon pathway at 36 and 48 hpf. a Whole-mount in situ hybridization of vasna and vasnb mRNA reveals the expression of these genes (dashed circles) in the region of the RGC axon commissure (asterisk). At 36 hpf, when the optic chiasm is first established, vasna is highly expressed in the floor plate (FP) and prospective branchial arches (BA). Additionally, vasna marks mesenchymal tissue associated with the retinal axon pathway. vasnb expression appears to border anteriorly the vasna-positive ventral domain (dashed arc). vasnb mRNA level is relatively low in this region. Lateral and ventral views. Anterior is to the left. FP: floor plate. BA: branchial arches. b A polyclonal anti-Vasna antibody shows Vasna localization to the membrane of mesenchymal cells surrounding ventro-laterally the retinal projection. At 36 hpf, Vasna-positive cells of the primitive optic artery (arrow) are already associated to retinal axons in the eye (dashed outlines). These two cell types stay in contact until retinal axons enter the diencephalic floor. At this point, axons lose contact with the vasculature and encounter the opening of a tunneling structure, marked by Vasna on its surface (dashed circles). Axons always associate with the ventral side of this channel (asterisks). At its exit, retinal axons are brought in the neighborhood of the prospective optic chiasm, which is bordered ventrally by Vasna-positive mesenchymal tissue. Each picture is a single optical section from a Z-stack encompassing antero-posteriorly the whole course of retinal axons. Frontal views. POC: postoptic commissure. OC: optic chiasm. Scale bar: 50 μm. b' At 48 hpf, many axons have extended through the lateral Vasna-positive tunnels (dashed lines). Qualitatively the expression pattern of Vasna does not change from 36 hpf. Frontal view. OC: optic chiasm. Scale bar: 50 μm. c A polyclonal antibody against Vasnb marks a subset of midline cells in the ventral diencephalon. As soon as retinal axons enter the diencephalon, they are in contact with laterally-positioned Vasnb-positive cells (arrowheads). At the chiasmatic midline (arrow), retinal axons are surrounded by Vasnb-positive membranes. Vasnb-expressing cells populate the anterior-most ventral diencephalic tissue, until the forebrain ventricle opens. This location is consistent with the reported position of the glial knot (asterisk). Single optical sections through the ventral diencephalon, posterior to anterior, are shown. Frontal views. POC: postoptic commissure. OC: optic chiasm. Scale bar: 50 μm. c' Ventral view of an embryo stained for Vasnb and acetylated tubulin. The preoptic area, between the optic recess and the chiasm is populated by cells expressing Vasnb. Vasnb-positive cells enwrap retinal axons. The picture is a Z-projection of 4 consecutive optical sections. Anterior is to the top. AC: anterior commissure. POC: postoptic commissure. Scale bar: 50 μm. c'' At 48 hpf, Vasnb is still detected on cells associated to the optic nerves (arrows) and in the glial knot (asterisk). From this stage Vasnb is clearly located on membranes wrapping the optic nerves in ventral positions along their course inside the brain. Each picture is a single optical section through the preoptic area. Frontal views. OC: optic chiasm. Scale bar: 50 μm. d A subset of Vasnb-positive cells in the knot region (brackets) expresses the transgenic glial marker Tg(gfap:GFP) (arrowheads). Single optical sections from confocal Z-stacks. Ventral and anterior views. Scale bars: 50 μm. e Tridimensional model of the retinal projection in zebrafish embryos at about 36 hpf. Retinal axons (red) are always in contact with Vasna-positive cells (yellow). Their ventral diencephalic course is shaped anteriorly by the Vasnb expression domain (green). The POC is shown in blue and borders the RGC axons caudally. OC: optic chiasm. POC: postoptic commissure. RGCs: retinal ganglion cells