F-Spondin Expression in Zebrafish Figure 1. F-spondin expression during early zebrafish development. A. Onset of spon1a and spon1b expression during embryogenesis. Representative experiment showing mRNA abundance based on real-time RT-PCR (qPCR), with 1 fold corresponding to spon1b at 9 hpf. Zebrafish eggs fertilized at the same time (n.360) were sampled at intervals over a 72 h period (n = 30 embryos/larva per time point) and mRNA abundance for both genes was quantified in each same. B. Embryo schematic, 18 hpf. C-D. Spon1b:GFP expression in the tail bud, notochord and myotomes (arrowhead), brain areas (red arrow), olfactory bulbs (OB, white arrow) and retina (asterisk) at 18 hpf (B) and 20 hpf (D). Photomontages of confocal images. E. Spon1b:GFP expression pattern at 48 hpf. Photomontage of confocal images, sagittal view, asterisk: eye. Inset: developing motor neurons of the spinal cord (red arrow), dorsal to floor plate and notochord. F-G. The floorplate and notochord highlighted by spon1b:GFP (F, live image, 4 dpf) and in situ hybridization for spon1b mRNA (G, 4 dfp). Notochord (no), floorplate (fp). H. Spon1b expression in the flexural organ (arrow), and midbrain (mb). (in situ hybridization, 4 dpf). Rostral end to the left. I. Dorsal view of the telencephalon (Tel) and TeO border at 3 dpf, showing spon1b expression in habenula (arrowhead), in the fasciculus retroflexus (FR) emerging from it (black arrow), and in individual cells of the TeO (red arrow). Confocal z-stack image. J. Dorsal view of midbrain-hindbrain area at 3 dpf showing spon1b:GFP in the developing nMLF (arrowhead), in MLF projections (arrow) and in motor neurons of the reticular formation, including Mauthner cells (red arrow). Rostral end is up in I-J. Confocal z-stack image. Scale bars: F-G: 25 mm; I-J: 100 mm. doi:10.1371/journal.pone.0037593.g001 20 hpf in the zebrafish embryo, remained prominent thereafter. In the adult olfactory bulbs (OB), spon1b-positive cells and thick projections were distributed within the glomerular (GL) cell layer, but not the internal cell layer (ICL) (Fig. 2B-C). The medial and lateral olfactory tracts (LOT/MOT), originating from these OB layers, could be traced throughout the ventral and dorsal Tel (Fig. 3A-B). A midline cluster of three large oval spon1b-positive cells was consistently present at the boundary of the OB and Tel in both larval and adult zebrafish (Fig. 2A,D red arrows). The origin of these distinct cells is unknown to us. In the telencephalon (Tel), the spon1b transgene signal and ISH revealed several F-spondin-positive regions. In the periventricular area of the ventral telencephalon (subpallium), spon1b was expressed in the ventral nucleus (Vv), which is suggested to be the teleost homolog of the mammalian septal nuclei [23,24]. The entire rostro-caudal extent of the Vv was immunopositive for spon1b:GFP, with labeled cells spanning the region from the medial periventricular zone to the most lateral boundary of the nucleus (Fig. 3A). The densely labeled LOT projections terminated in the posterior nucleus of the dorsal Tel (Dp), the primary olfactory area in zebrafish [23]. Their spon1b-positive fibers also traveled toward the medial nucleus of the dorsal Tel (Dm) and partially crossed into the contralateral hemisphere via the anterior commissure (Cant, Fig. 3B-C). In the dorsal pallium (D), spon1b was present in the cells of the medial nucleus, Dm (Fig. 3C-D), a likely homolog of the basolateral amygdala in mammals [25,26]. The spon1b-positive cells were located in the midline region of Dm, periventricularly, with their dense projections being constrained to the boundaries of the nucleus (Fig. 3B-C). A few weakly stained cells could be observed in the lateral (Dl) and posterior (Dp) nuclei of D (Fig. 3B- D). Spon1b-positive cells were observed in the dorsal nucleus of D (Dd), surrounding the sulcus ypsiloniformis (Fig. 3B-C). This nucleus is considered to be part of the processing center for somatosensory information from the lateral line [23]. In the preoptic region, spon1b expression was strong and abundant in the anterior parvocellular nucleus (Ppa; Fig. 3C). Individual cells and their dorsolaterally projecting axons were evident in the most lateral regions of the Ppa. More caudally, the magnocellular preoptic nucleus (PM) and its dorsal projections were strongly immunoreactive, while fewer positive cells were present in the medial region of the posterior parvocellular nucleus (PPp, Fig. 3D). The most posterior parts of the Ppp, and the suprachismatic nucleus appeared to be free of spon1b expressing cells (Fig. 3D). More laterally, spon1b was expressed in the bed nucleus of the stria medullaris (BNSM, Fig. 3D), a recently described nucleus in adult zebrafish [27]. Diencephalon. Pronounced spon1b:GFP expression was documented in the epithalamus and Dorsal Conduction Pathway. This was especially robust in the habenula (Hb), which displayed a strong fluorescence signal in the developing and adult Tg(spon1b:GFP) fish (Fig. 2A and 4A-I). In larvae, the transgene was expressed only in the laterally-positioned Hb nuclei, located at the dorsal surface of the developing brain (Fig. 1I, 4A). As development proceeded, the spon1b:GFP positive Hb nuclei increased in size and gradually moved medially (Fig. 4A-C). By 3 months post fertilization (mpf) the Hb nuclei were adjacent to each other at the midline (Fig. 4C). In both developing and adult Tg(spon1b:GFP) zebrafish, the entire dorsal conduction pathway (DCP) was highlighted, visualizing the fasciculus retroflexus (FR), projecting from the Hb to the interpeduncular nucleus (NIn) and superior raphe (SR) (Fig. 1I, 4D-L). Consistent with the earlier findings that the laterally-positioned Hb nuclei in larvae migrate ventrally during maturation [28], the strongest spon1b labeling was present in the ventral Hb (vHb) in adult zebrafish (Fig. 4G-I, 5A). The paired symmetrical vHb nuclei each have a conical shape defined by a wide rostral end, occupying the entire anterior region of the Hb, and a narrow caudal end (Fig. 4H-I). Moreover, a region located dorsolateral to the vHb, and separated from it by the emerging FR, was also spon1b:GFP positive (Fig. 4I, 5A). Based on the localization of this additional area, we have named it the inferior subnucleus (dmHbi) of the earlier described dorsomedial nucleus of the Hb (dmHb) [29,30]. In contrast to the vHb having small densely packed cells (Fig. 5B), dmHbi had relatively large and sparsely positioned cells (Fig. 5C). Both of these cell types double-stained for spon1b:GFP and Hu C/ D, indicating their neuronal origin (Fig. 5A). It should be noted that the vHb cells stained more weakly for Hu C/D than those in the adjacent dmHbi nucleus. The rest of the dorsal Hb was also immunopositive for Hu C/D, but not for spon1b:GFP (Fig. 5A). Differentiating between the two spon1b:GFP positive areas of Hb was further assisted by the distinctly different targets of dorsal and ventral Hb nuclei. In zebrafish, the vHb projects to the raphe nuclei [28]. In contrast, the two principal nuclei of dorsal Hb have asymmetric projections. The dorsolateral Hb (dlHb) projects to the dorsal part of the interpeduncular nucleus (dNIn), while fibers originating in the dmHb are traced to ventral and intermediate NIn (vNIn, iNIn) [29,30]. Accordingly, the spon1b-positive FR fibers were present in ventral but not the dorsal NIn, consistent with the dmHb pathway (Fig. 5D), and circumvented the NIn on their way to the SR (Fig. 4J-L, 5D), consistent with the vHB projections [28]. These latter fibers were seen as densely packed spon1b-positive terminals in ventral regions of SR (Fig. 4F, K-L). Spon1b expression was present in cells of the NIn core but not in the SR (Fig. 4J-L). Moreover, projections between the Hb and BNSM, as well as some ventral projections, were seen in sagittal brain sections (Fig. 4E). Considering the conserved connectivity of the dorsal conduction pathway in vertebrates [31], these projections were, presumably, part of the stria medullaris. PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e37593