F-Spondin Expression in Zebrafish Figure 4. Spon1b:GFP expression in the habenular nuclei and their projections. A-C. Dorsal view of live zebrafish showing migration of ventral habenular nuclei from lateral to medial position: 3 dpf (B), 20 dpf (C), and 2 mpf (D). Asterisk indicates the position of the eye. D. Schematic of DCP in adult zebrafish showing areas of spon1b:GFP expression (E and F) and depicting the habenula (Hb), its afferent projections within the stria medullaris (sm), its efferent projection: fasciculus retroflexus (FR), and target nuclei: interpeduncular nucleus (NIn), and superior raphe (SR). E-F. Para- sagittal cut through fresh-frozen adult Tg(spon1b-GFP) brain. GFP fluorescence highlights all DCP structures, including vHb nuclei, FR and sm (E). Also, cells of the bed nucleus of the stria medullaris (BNSM, arrow in E), and projections to NIn and SR in F. Rostral to the left. G. Schematics of the relative shape and position of the spon1b-positive nuclei of the Hb, as shown in H-I. H-L. Coronal sections immunostained for GFP: rostral (H) and caudal (I) Hb, NIn (J), and rostral (K) and caudal (L) SR. Scale bars: A-B: 100 mm; D: 25 mm; H-L: 50 mm. doi:10.1371/journal.pone.0037593.g004 and continued expressing it throughout zebrafish life (Fig. 8A). In adult brain, spon1b-positive retinotectal projections of the ventral optic tract (VOT), coursed through the optic tract and innervated two distinct layers of TeO (Fig. 8D, arrows). These corresponded to the superior sublayer of the stratum fibrosum et griseum superficiale (SFGS) and stratum opticum (SO), where 80% and 15% of RGC terminate, respectively [34]. Individual spon1b-expressing cells were observed in the embry- onic TeO around 48 hpf (Fig. 1J, 2D). In adult fish, five different types of spon1b-positive neurons could be identified in the tectal layers, based on their morphology and Hu C/D staining (Fig. 8C- G). A large number of widely distributed rounded cells with thin neurites were observed in the stratum periventriculare (SPV, Fig. 8D). Also in the SPV, rhomboid-shaped neurons were located at the most rostral and lateral TeO regions, extending thin apical dendrites toward superficial layers. Their long basal axons crossed the SPV, forming the tectobulbar tract (TTB) that projected on both sides of the NIn toward the hindbrain and spinal cord (Fig. 7D, 8G). The morphology of these cells is consistent with the earlier described TeO neurons that project from the torus semicircularis in rainbow trout [35], and are similar to cells described as the type IVa/b and V in goldfish [36]. In the same area, numerous multipolar spon1b positive cells with larger perikarya demonstrated prominent dorsally oriented processes (Fig. 8D). At the boundary of SPV and stratum album centrale (SAC), a smaller population of spon1b-positive neurons extended their processes alongside the horizontal axonal bundles (Fig. 8E). In the stratum griseum centrale (SGC), a large population of neurons extended their apical dendrites toward the TeO surface, with elaborate arborizations terminating in the proximal SFGS and SO layers (Fig. 8F). Most of these neurons were unipolar, though some had short basal neurites or long descending axons that crossed inner TeO layers and contributed to the TTB (Fig. 8G). The pear-like shape of these neurons was similar to a previously described population of cells that project to the torus longitudinalis (TL) of the rainbow trout [37]. This was consistent with the dense spon1b-positive projections observed in TL of zebrafish (Fig. 8H). Moreover, TL contained granular cells organized in individual rami, strongly labeled with spon1b:GFP (Fig. 6B, 7B, 8H). Some of their long axons projected dorsolat- erally, toward the most superficial layer of the TeO, the stratum marginalis (SM), while the majority joined the tectal commissure (Ctec) or coursed along the inner layers (Fig. 7B, 8H). In the tegmentum, prominent spon1b-positive projections could be observed in the ansulate commissure (Cans, Fig. 7D). Dorsal and caudal to the Cans, thick fibers of the FR and TTB could be seen projecting to the NIn/SR and hindbrain motor neurons, respectively (Fig. 7D,G). This area also contained numerous spon1b-positive projections and scattered cell bodies throughout the torus semicircularis and superior reticular formation (Fig. 7B-D). Metencephalon. By 5 dpf, spon1b expression was visible at the most rostromedial region of the hindbrain. By 40 dpf, the spon1b:GFP signal could be observed in the projections within the valvula cerebelli (Val), cerebellar corpus (CCe), cerebellar crest (CC) and granular eminence (EG, Fig. 9A). In the adult brain, cells positive for spon1b:GFP were documented in the EG and CC of the cerebellum, and in the secondary gustatory nucleus (SGN), nucleus isthmi (NI), and superior reticular nucleus (SRN) (Fig. 9B-E), which are all derived from the upper rhombic lip (URL) and known to project to all lobes of the cerebellum [38]. Other spon1b- positive afferents to zebrafish cerebellum might have originated from the pretectal nuclei, torus longitudinalis, octavolateral region [38], which were also positive for the transgene. This wide range of spon1b-positive afferents explain, in part, the strong and uniform spon1b:GFP signal throughout the cerebellum. Large spon1b- positive cells were visible along both sides of the cerebellar midline, in the ventral anterior tip of the Val and around the dorsal surface of the Val and the CCe, marking the boundary between the molecular and granular cell layers (Fig. 9D). Their location and morphology are characteristic of Purkinje cells. It should be noted that these cells had a weak spon1b:GFP signal, though were well defined by spon1b mRNA in situ hybridization. While the spon1b mRNA signal was absent from the SR (Fig. 9D), GFP labeling was strong in Tg(spon1b:GFP) zebrafish (Fig. 9F), confirming that the signal comes not from cells but from axons originating in the Hb and traveling via the FR. The most ventral tip of the rhomboencephalic ventricle (RV), medial to MLF, was strongly positive for spon1b (Fig. 6A, 9D). The spon1b mRNA expression in this area was especially well defined by ISH and extended throughout the entire RV to the level of the caudal medulla. This medullo-spinal region is characterized by the presence of CSF-contacting neurons, which are abundant in teleosts [39]. Myelencephalon. In the medulla oblongata, spon1b is expressed in multiple symmetrically positioned hindbrain neurons (Fig. 6A, 9E,G), including the large Mauthner cells (Fig. 1J), and in the neurons of the motor nucleus of X (Fig. 9G). In the spinal cord, the spon1b:GFP positive projections of these cells run along with the MLF and ventral funiculus (Fv, Fig. 9G-H). The tectobulbar tract (TTB) is also strongly labeled by spon1b:GFP (Fig. 7B-D, 8G, 9B), which sends motor output from the deeper layers of the TeO to the premotor reticulospinal system in the hindbrain [40]. Spon1b is Expressed in Neurogenic Zones of the Zebrafish Brain In zebrafish, adult neurogenesis is a continuous process, much more active than in mammals [20]. A total of 16 distinct proliferative niches have been identified in zebrafish brain. These are located mainly in periventricular areas, but also within the brain parenchyma [41]. We have documented a notable overlap of the areas of spon1b expression with proliferative zones in adult zebrafish brain (Fig. 10A-B). Spon1b expression is present within or next to BrdU-positive areas, such as the olfactory bulbs, ventral and dorsal telencephalon, parvocellular preoptic nuclei, Hb, ventral and dorsal thalamus, posterior tuberculum, posterior tuberal nucleus, hypothalamus, median optic tectum, torus longitudinalis, cerebellum, medulla and spinal cord (Fig. 10B). To understand the relationship between spon1b and stem cells, we examined cell proliferation patterns in several germinal zones in 1- PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e37593