F-Spondin Expression in Zebrafish Figure 2. Expression of spon1b:GFP in the olfactory bulbs (OB) and telencephalon (Tel) of larval and adult zebrafish. A. Dorsal Tel of 3 dpf embryo, showing individual cells of the OB (red arrowheads), and a cluster of three oval midline cells at the OB-Tel boundary (red arrow). Confocal z-stack image. B. Latero-dorsal view of live larva, at 3 dpf, with robust spon1b:GFP expression in the oval cells of the Tel-OB boundary (red arrow), in the paired habenular nuclei (white arrows), in individual cells of the TeO, and in the eye, with retinal ganglion cells visible through the lens (asterisk). C-D. Sagittal (B) and coronal (C) sections of the adult OB showing spon1b-positive cells in the outer glomerular cell layers (GL), but not in the inner cell layers (ICL). R: rostral end, in A & C. Scale bars: A: 200 mm; C-D: 50 mm. doi:10.1371/journal.pone.0037593.g002 Unlike in the vast majority of brain areas where ISH for spon1b recapitulated spon1b:GFP expression, the spon1b mRNA signal was detected in additional nuclei of the dorsal Hb (data not shown) that were not revealed by the transgene. This discrepancy is unlikely to result from a positional effect of the transgene integration site, since our two independent transgenic founder lines had similar patterns of Hb expression, highlighting only laterally-positioned Hb nuclei in larvae. Potentially, this result could indicate that additional promoter or enhancer regions, beyond the 10.3 kb upstream fragment, are required for the expression of spon1b in dorsal Hb nuclei. Thalamus. The ventrolateral thalamic nucleus, which is a relay station for visual and other sensory inputs to the optic tectum (TeO) [32], contained large, widely distributed cells with strong spon1b:GFP expression (Fig. 6A). The central and dorsal posterior thalamic nuclei (CP/DP), the medial and lateral preglomerular nuclei (PGm, PGl), the paraventricular organ (PVO) and the posterior tuberal nucleus (PTN) contained spon1b expressing cells (Fig. 6–7). Large spon1b-positive CSF-contacting cells of the posterior tuberculum (TPp) extended their neurites laterally, while numerous thinner projections were visible throughout the parenchyma of the thalamus, especially in its anterior region (Fig. 6C). Hypothalamus. Strong spon1b expression was revealed in two distinct periventricular areas of the rostral and caudal hypothalamus (H, Fig. 7). Their extreme ventral location allowed for the observation of the spon1b:GFP positive cells on the ventral side of the whole brain dissection, using fluorescence microscopy (Fig. 7A). The rostral area, corresponding to the ventral nucleus of the hypothalamus (Hv) that surrounds the mediobasal region of PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e37593