F-Spondin Expression in Zebrafish C/D (red) and spon1b:GFP (green). G. Coronal section at the level of NIn showing spon1b:GFP in vNIn (white arrow), but its absence in dorsal NIn (dNIn, red asterisk). Spon1b-positive fibers in the cerebellar corpus (CCe), FR (fibers circumventing NIn on the way to SR), mammillary bodies (arrowhead) and TTB. Scale bars B,G: 100 mm; C: 25 mm, D: 50 mm; E-F: 20 mm. doi:10.1371/journal.pone.0037593.g007 newly formed cells that migrated into the vHb or dHb expressed spon1b at any post-injection interval examined. The periventricular hypothalamic niche in zebrafish is divided into dorsal, ventral and caudal zones (Fig. 10J-K), corresponding to their location in specific periventricular hypothalamic regions (Hd, Hv and Hc, respectively [41]). Spon1b-positive cells were abundant in the Hd, Hv and Hc, being close to BrdU-positive cells but not co-localizing with them at 2 hours or 30 days post-BrdU Figure 8. Spon1b expression in areas of the visual system. A. Spon1b expression in retinal ganglion cells (RGC) forming optic nerve (on) in live zebrafish at 20 dpf. Confocal image. B. Coronal section of adult zebrafish fish brain at the level of the optic tectum (TeO) showing spon1b-positive retino-tectal projections of the ventrolateral optic tract (VOT) splitting into two TeO layers: stratum fibrosum et griseum superficiale (SFGS, arrow) and stratum opticum (SO, arrowhead). Spon1b-positive cells in the medial (PGm) and lateral (PGl) preglomerular nuclei, and in the posterior tuberal nucleus (PTN). C. Double immunostaining in TeO demonstrates co-localization of spon1b:GFP (green) and Hu C/D (red) signal, confirming the neuronal nature of spon1b-positive cells. D. Coronal section identifying six TeO layers, with spon1:GFP expression in retinotectal projections in SO and SFGS, cell types 1 (in SPV border) and 4 (in SAC). E. Type 3 TeO cell, with horizontal projections in the SAC layer. F. Type 5 TeO cells with vertical projections in the SGC layer. G. Type 2 TeO cell with vertical projections traversing all TeO layers and forming the tectobulbar tract (TTB). H: Spon1b- positive cells and rami-like neuropil in the torus longitudinalis (TL). Scale bars: A: 10 mm; B: 100 mm; E-F: 15 mm; D,H: 20 mm, G: 50 mm. doi:10.1371/journal.pone.0037593.g008 PLoS ONE | www.plosone.org 12 June 2012 | Volume 7 | Issue 6 | e37593