F-Spondin Expression in Zebrafish Figure 3. Spon1b expression in the telencephalon of adult zebrafish. Immunostaining for GFP in Tg(spon1b:GFP), coronal sections from rostral (A) to caudal (D) end. A. Spon1b expression in the central nucleus of ventral Tel (Vv), and terminal projections of the lateral olfactory tract (LOT), medial to dorsal nucleus of dorsal Tel (Dp). Inset: thick projections of spon1b-positive cells in Vv. B. Spon1b expression in Vv and the lateral (Dl), posterior (Dp) and dorsal (Dd) nuclei of the dosal Tel. Inset: small positive cells in Dd surrounding sulcus ypsiloniformis. Cant: anterior commissure. C. Robust spon1b expression in the medial nucleus of the dorsal Tel (Dm) and in the medial olfactory tract (MOT). Inset C’: Thick and long projections, originating from LOT area. Inset C’’: medially located spon1b positive cells in Dm with thin and dense projections extending laterally throughout the nucleus (arrow). Ppa: parvocellular preoptic area D. Spon1b expression in the preoptic area. Inset: magnocellular nucleus (PM) and bed nucleus of stria medullaris (BNSM). Strong spon1b expression in the ventral habenula (vHb) (only the right vHb is visible). Scale bars: A-D: 200 mm. Insets: 50 mm. doi:10.1371/journal.pone.0037593.g003 the diencephalic ventricle, contained a well-defined population of spon1b-positive cells. These extended their projections laterally, forming a distinct tract, coursing along the ventral surface of the hypothalamus (Fig. 7B-C). In the inferior lobe, in the caudal zone of the periventricular hypothalamus (Hc), a distinct horizontal band of the nucleus of the posterior recess (NPR) [32] displayed large and densely packed spon1b-positive cells, positioned along the ventral wall of the diencephalic ventricle (Fig. 7D-F). These CSF-contacting neurons extended their prominent processes dorsally, toward the lumen of the posterior recess of the diencephalic ventricle and stained for Hu C/D, confirming their neuronal nature (Fig. 7E-F). A dense network of spon1b positive fibers could be observed in the dorsal region of the Hc, extending dorsomedially toward the tegmental areas (Fig. 7D). Part of those projections could be seen originating from the cells in the caudal and lateral aspects of the posterior recess of the DiV (Fig. 7D, inset). A weak spon1b signal from scattered thin projections was documented in the mammillary bodies (Fig. 7G). Pretectum. In larval zebrafish, strong spon1b expression was present in the nucleus of the medial longitudinal fasciculus (nMLF), with spon1b:GFP highlighting its long projections along the ventral border of the rhomboencephalic ventricle and ventral region of the spinal cord (Fig. 1J). The distinct cells of this nucleus and its projections, which communicate multimodal sensory information and locomotor commands in zebrafish [33] continued to express spon1b in adults (Fig. 7B). Both periventricular pretectal nuclei, dorsal and ventral (PPv, PPd), contained small-size cells with strong spon1b expression (Fig. 6C). Mesencephalon. Another region of the brain with pro- nounced staining for spon1b was the TeO (Fig. 8). The retinal ganglion cells (RGC) expressed spon1b during early development, PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e37593