F-Spondin Expression in Zebrafish Figure 9. Spon1b expression in the hindbrain. A. Dorsal view of a freshly-dissected Tg(spon1b:GFP) juvenile zebrafish showing relative position of the paired habenular nuclei (Hb), emerging fasciculus retroflexus (FR), optic tectum (TeO), cerebellar corpus (Cce), granular eminence (EG), and medulla (Me). B. Coronal section of adult zebrafish brain at the level of the cerebellar corpus showing spon1b:GFP positive projections within the cerebellum, and to the superior raphe (SR). Tectobulbar tract (TTB). C. Spon1b:GFP signal in cells of the secondary gustatory nucleus (SGN), in the gustatory commissure (Cgus), and in scattered cells and projections throughout the tegmentum (arrow), and nucleus isthmi (NI). (High magnification picture of area in B). D. Spon1b mRNA expression (in situ hybridization) in Purkinje cell layer (Pur), ventral tip of rhomboencephalic ventricle (rv), SGN, nucleus isthmi (NI), superior reticular nucleus (arrowhead) and mammillary bodies (CM). Note the lack of signal in the Cgus and SR, confirming that the spon1b:GFP label in these regions comes from projections and not cell bodies. E. Coronal section at the level of the caudal cerebellum showing spon1b-positive cells and thick projections from cerebellar crest (CC), the descending octaval nuclei (DON) and the commissure that joins the nuclei, the internal arcuate fibers (IAF) coursing in between the axons of the medial longitudinal fasciculus (MLF). F. Spon1b-positive FR fibers (arrows) terminating on both sides of the ventral region of SR (high magnification image of area boxed in B). G. Coronal section at the level of the caudal medulla showing spon1b:GFP expression in the vagal motor nucleus (NXm), but not in the vagal lobe (LX). Note the symmetrically labeled motor neurons (mn), and the strongly labeled axons of the MLF and motor neuron axons in the ventral funiculus (Fv). H. Coronal section of the spinal cord showing spon1b:GFP expression in the axons of MLF and the ventral funiculus (Fv). Scale bars: B, F-H: 100 mm; C-D: 50 mm; E: 200 mm. doi:10.1371/journal.pone.0037593.g009 administration (Fig. 10L-M). The spon1b-positive cells were positioned in the peri-ventricular regions and parenchyma of the hypothalamic nuclei, while the BrdU-positive cells were found along the ventricular wall (Fig. 10J-M), consistent with previous observations [41,42]. One month after BrdU injection, some of the newly formed cells had migrated away from the ventricle and into the parenchyma, although the majority remained closely associ- ated with the niche. Similar to those in the Hb niche, the BrdU- positive cells of the hypothalamus did not co-localize with spon1b:GFP positive cells one or two months after cell division had occurred (Fig. 10L-M). The cerebellar niche is one of the largest and most complex proliferation zones of the teleost brain, and the site of origin of the majority of new cells in the zebrafish brain [43]. It is also the only niche in the zebrafish that is not associated with the ventricles [43,44]. The BrdU positive nuclei were found throughout the molecular cell layer of the Val, CCe, CC and EG at 2 hours after injection, consistent with earlier findings [43]. At 1 and 2 months post injection, the new cells had migrated into the granular cell layer of the cerebellum and could be observed among the spon1b:GFP positive fibers (Fig. 10N). No cells with co-localized PLoS ONE | www.plosone.org 13 June 2012 | Volume 7 | Issue 6 | e37593