beneath the floor plate, the N-terminal fragment binds to several members of the low density lipoprotein receptor family (ApoER2, LRP2/megalin, and LRP4), inhibiting neuronal outgrowth and preventing these neurons from growing through the floor plate [9]. These effects of F-spondin are likely to be conserved, since it is present in the embryonic floor plate in the frog, mouse, chick, and zebrafish [4,10–12]. The expression of F-spondin in other brain regions of embryonic rat or zebrafish and, at lower levels, in the neocortex and hippocampus of adult rats, suggests its broader role in CNS development and maintenance [12,13]. Indeed, studies in vitro found that F-spondin accumulates in the ECM that ensheaths the developing peripheral nerves [14], promotes neurite outgrowth from embryonic hippocampal and commissural neurons [2,13], and potentiates nerve precursor differentiation [5]. In vitro studies also suggest that the effects of F-spondin depend on its concentration and presence in substrate-attached or soluble form [5]. Thus, the exact sites of F-spondin production and its release into the cerebrospinal fluid (CSF) compartments of the brain might determine its availability and efficacy. This is especially interesting in view of recent evidence that F-spondin is a putative ligand for the Alzheimer disease-related amyloid precursor protein (APP), and may exert its neurotrophic effects in part via a specific interaction with APP [15–17]. Since F- spondin binds ApoER2 via its thrombospondin domain, and APP via its reelin and spondin domains, a single F-spondin molecule could form a complex with APP and ApoER2 [17]. Indeed, an interaction between F-spondin and APP and/or ApoEr2, affecting the APP-downstream signaling molecule disabled-1 (DAB-1), has been suggested as one of the mechanisms by which F-spondin might control neuron survival and neuroblast migration [16,18]. Together, these findings underscore a need for systematic characterization of F-spondin expression patterns in the CNS and peripheral tissues during development and adulthood, which is lacking at present. To address this, we employed zebrafish as a model. This choice reflects the outstanding qualities of the zebrafish as an in vivo model for studying vertebrate development [19], the documented presence of F-spondin in this species [12], and its striking capacity for active tissue renewal and regeneration throughout life [20]. Thus, the zebrafish provides an excellent opportunity to investigate the role of F-spondin in dynamic CNS and peripheral tissue modifications throughout the life-span of a vertebrate animal. Results Expression of the F-spondin Homologs in Zebrafish The two F-spondin homologs in zebrafish, Spon1a and Spon1b have an identity of 73% and 70%, respectively, with the human SPON1 protein. The identity between the two zebrafish homologs is 74%. Using real time quantitative RT-PCR (qPCR), the mRNA abundance for the two F-spondin homologs spon1a and spon1b was measured in developing embryos. The onset of spon1b mRNA expression occurred at 9–10 hours post fertilization (hpf), increasing 6.8 fold by the end of the first day of development, and reaching 16.3 fold by 72 hpf (Fig. 1A). In contrast, spon1a mRNA expression was initiated only after 48 hpf, and increased 4.0 fold by 72 hpf. Similarly, in adult zebrafish, mRNA abundance for spon1b was 2.0 fold and 72.3 fold higher than for spon1a assessed in the same brain and eye tissue samples, respectively (n = 3–4 fish; for brain, paired t(3) = 2.90, p,0.03; for eye, paired t(2) = 10.06, p,0.0005. The higher abundance of spon1b mRNA in larval and adult zebrafish suggested a potentially more important role for this homolog. We, thus, proceeded to characterize the expression patterns for spon1b in developing and adult zebrafish using three complementary F-Spondin Expression in Zebrafish approaches. These included the imaging of a fluorescent transgene signal (in vivo or in fresh-frozen brain tissue), immunohistochemical (IHC) staining for GFP, and in situ hybridization (ISH) to localize spon1b mRNA expression. The first two techniques allowed for visualizing both the F-spondin positive cells and their projections, while ISH highlighted the cell bodies only. The results were typically consistent among the methods used and are referred to below as spon1b or F-spondin expression, with any inconsistencies between methods discussed, where applicable. The Transgenic spon1b:GFP Zebrafish Reveals in vivo Patterns of F-spondin Expression During Vertebrate Development To study the anatomical distribution of spon1b expression throughout zebrafish development and maturation, we used the 10.3 kb upstream promoter region of the spon1b gene (Accession# NM_131517) to drive expression of a fluorescent marker, the enhanced green Yuorescent protein (EGFP, also referred to here as GFP). This construct was used to establish a stable spon1b:GFP transgenic zebrafish line. In Tg(spon1b:GFP) fish, GFP expression was first detected around 15–16 hpf, being visible along the developing body axis, with a stronger signal in the head and tail regions (data not shown). This is consistent with the onset of spon1b mRNA production at 10 hpf, as per qPCR, and allowing several hours for the accumulation of newly-synthesized GFP. Thereafter, rapid increases in spon1b:GFP expression were documented in the embryonic tail bud, notochord and myotomes (Fig. 1B-D). By 18 hpf, the spon1b:GFP signal was clearly visible in the brain and eye regions, with robust expression in the developing retina and olfactory bulb by 20 hpf (Fig. 1C-D). During this period, F- spondin expression intensified along the developing notochord, and strongly labeled the entire row of individual cells of the floor plate (Fig. 1E-G). Although both the transgene and ISH revealed this F-spondin localization during development, the expression in the notochord was more robust in vivo, in the Tg(spon1b:GFP) fish (Fig. 1F-H). The spon1b:GFP signal in the most anterior end of the notochord, known as the flexural organ, was particularly pronounced (Fig. 1H). This region has been previously identified as a source of Reissner’s fiber-related proteins, including F- spondin [21]. As development proceeded, a gradual increase in F- spondin expression along the developing spinal cord was associated with the early developing Rohon-Beard sensory neurons, and with the development and extension of primary motor neurons (Fig. 1E, inset). The axons of the latter were strongly labeled in the Tg(spon1b:GFP) zebrafish, highlighting the descending pathways. These are known to innervate restricted domains within each myotome and contribute to the first muscular contractions initiated around 17 hpf [22]. The developing midbrain and diencephalic regions were rich in F-spondin starting at 24 hpf, and this could be observed in vivo in Tg(spon1b:GFP), and using whole mount in situ hybridization (Fig. 1H-J). By 72 hpf, an especially strong spon1b:GFP signal was localized to the neurons of the laterally-positioned habenula (Hb) nulclei and their descending projections forming the fasciculus retroflexus (FR; Fig. 1I). In the midbrain, F-spondin positive cells were documented in the optic tectum (TeO) and in the nucleus of the medial longitudinal fasciculus (nMLF) (Fig. 1I-J). The distinct motor neurons of the hindbrain were also rich in F-spondin expression, including the large paired Mauthner cells (Fig. 1J). F-spondin Expression in Adult Zebrafish Brain Telencephalon. The robust spon1b expression in the olfactory area and telencephalon (Tel, Fig. 1D, 2A-D), that was apparent at PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e37593