F-Spondin Expression in Zebrafish Figure 10. Spon1b is expressed in neurogenic niches. A. Schematic of neurogenic niches in zebrafish brain based on BrdU immunostaining and modified from Grandel et. al. (2006). Cerebellar corpus (CCe), Habenula (Hb), hypothalamic areas (H), parvocellular preoptic area (Ppa), thalamic area (T), ventral nucleus of ventral telencephalon (Vv). B. Mid-sagittal of adult zebrafish brain showing spon1b mRNA expression (in situ hybridization). Note the overlap of the spon1b-positive areas with the neurogenic niches. C-E. BrdU-positive nuclei in the habenular niche in rostral (D), and mid (E) and caudal (F) areas, 2 hours post-BrdU injection. F-I. Double immunostaining of the habenular niche, showing spon1b- (green) and BrdU- (red) positive cells, at 4d (F), 30d (G-H) and 60d (I) post BrdU-injection, showing migration of the BrdU nuclei away from the niche and into the surrounding tissue. J-K. BrdU nuclei in the ventral (J) and caudal (K) hypothalamus, Hv and Hc, respectively, 2 hours post-injection. L-M. Double immunostaining for BrdU (red) and spon1b:GFP (green) 30 days post BrdU injection in ventral hypothalamus (Hv, L), and in the nucleus of the posterior recess (NPR) in Hc (M) showing lack of co-localization. N. Double immunostaining for BrdU (red) and spon1b:GFP (green) 30 days post BrdU injection in the cerebellum. Note the BrdU-positive nuclei among the spon1b-positive fibers. O. Co-localization of BrdU (red) and spon1b:GFP (green) in cells extending long projections in the tegmentum 30 days post-BrdU injection. Scale bar C: 200 mm, D-L,N: 50 mm, M: 20 mm. doi:10.1371/journal.pone.0037593.g010 spon1b and BrdU signals were found in the cerebellum for up to 2 months post-BrdU injection. Upon close examination, the only area of the brain where we found BrdU-positive cells co-localizing with spon1b:GFP was the tegmentum (Fig. 10O). One month after the division occurred, these new cells had extended relatively long projections, highlighted by a GFP signal, and their morphology was consistent with a neuronal phenotype. Most likely, these new cells migrated from the posterior mesencephalic lamina niche or from the dorsal tectal proliferation zone [41]. Expression of spon1b in Peripheral Tissues We have documented that F-spondin expression is not limited to CNS but also present in a number of peripheral tissues in developing and adult zebrafish. Unlike in the brain or eye of adult zebrafish, very low levels of spon1b mRNA abundance could be detected by qPCR in zebrafish muscles and skin (28.8 and 11.9 fold less than in zebrafish brain, respectively), with no apparent transgene expression found. Nevertheless, as in the CNS, in both of these tissues the abundance of spon1b mRNA was consistently higher than that of spon1a (n = 224; unequal variance t-test, muscle vs. brain t(2) = 9.22 p,0.005, skin vs. brain t(4) = 3.23 p,0.001). The zebrafish have only pharyngeal teeth, located posteriorly to the fifth pharyngeal arch, and these showed strong spon1b:GFP expression during development (Fig. 11A). The teeth, known to regenerate throughout the zebrafish lifespan [45], remained strongly labeled with the transgene in adult fish (Fig. 11B-C), and this was consistent with high spon1b mRNA levels determined by qPCR (data not shown). Spon1b expression was restricted to the soft tissue surrounding the mature teeth (Fig. 11C), which corresponds to the tooth-associated lamina and the replacement tooth developing from it [45]. The expression of spon1b:GFP in zebrafish enteric neurons was evident during early development (Fig. 11D). These cells, regulating gut motility, are located between the circular and longitudinal smooth muscle layers [46] and retain robust F- spondin expression throughout zebrafish life. An especially high density of spon1b:GFP positive neurons around the anal pore allowed us to easily observe them in live adult fish (data not shown). The distinct spon1b:GFP positive neurite extensions could be documented in adult gut using confocal microscopy (Fig. 11E). Spon1b was also present in developing zebrafish fins. Early massive spon1b expression in the tail bud and caudal fin rays (Fig. 1C-E) was followed by a strong spon1b:GFP signal in the pectoral and abdominal fin buds in larval fish. The larval caudal fin in zebrafish is replaced by the adult fin through a well- characterized process [47]. Consistent with this, around 8 dpf spon1b:GFP expression was detected in the adult caudal fin primordium (Fig. 11F-H). Later, the primordium gives rise to the endoskeleton of the adult tail [47] and, in adult zebrafish, we documented a thin layer of cells covering the endoskeleton that continued expressing spon1b (Fig. 11I-J). Otherwise, in adult zebrafish, the caudal fin rays were devoid of spon1b:GFP expression. Zebrafish are known to regenerate their caudal fin following partial amputation. This complex process involves reprogramming and cell migration, with extracellular matrix remodeling being essential for this process [48]. To determine whether F-spondin might be involved in fin regeneration, we conducted partial fin amputation and documented spon1b:GFP expression over a 2-week period of regeneration. There was no upregulation in spon1b expression during the first day post-trauma, which corresponds to the wound healing and epithelialization period. However, by the end of the second day of regeneration, qPCR measurements in fin tissue indicated initiation of spon1b mRNA production (data not shown). This coincided with the appearance of spon1b:GFP fluorescence at the distal ends of the fin rays (lepidotrichia) (Fig. 11K-L), in an area called the blastema, a zone of actively proliferating mesenchymal cells responsible for fin patterning and re-growth [49]. The fluorescence signal was typically restricted to paired domains at the tip of the growing rays of each lepidotrichium (Fig. 11L). Spon1b expression was observed in these areas throughout the fin regeneration process and disappeared after its completion. Discussion The goal of the present study was to investigate F-spondin expression patterns in developing and adult zebrafish, in order to compare them to the regions of F-spondin expression reported in mammals, and to relate these patterns to the functional role of F- spondin suggested by in vitro studies. Unlike mammals, which have one F-spondin gene, zebrafish have two F-spondin homologs, spon1a and spon1b. Our systematic qPCR-based quantification of relative differences between these two homologs documented that spon1b expression is initiated much earlier, within hours post fertilization, and that mRNA abundance for spon1b in embryonic, larval or adult zebrafish tissues is consistently and substantially higher than that for spon1a. An earlier study also reported lower spon1a expression in zebrafish embryos, as determined by in situ hybridization [12]. We thus focused on characterizing the patterns of expression for spon1b and, for the first time, established a transgenic vertebrate model with stable spon1b:GFP expression. The localization of cells expressing the spon1b:GFP transgene in developing and adult zebrafish proved to be consistent with the areas highlighted by the complementary spon1b in situ hybridization method, with minor exceptions (see Results). The major advantage of the transgenic animal was that it permitted visualization of both the cell bodies and the projections originating from them. Here we demonstrate that, following the onset of spon1b expression in structures involved in early patterning and polari- zation of zebrafish CNS, F-spondin-positive neurons are present in specific regions of the telencephalon, diencephalon, mesenceph- PLoS ONE | www.plosone.org 15 June 2012 | Volume 7 | Issue 6 | e37593