alon, hindbrain, and spinal cord. The projections of these F- spondin positive neurons can be local but, more often, extend between distant CNS regions, forming long neuronal tracts. The significance of F-spondin as a secretory ECM protein is highlighted by its expression in CSF-contacting cells with projections directed toward the ventricular walls. Importantly, the distribution of spon1b in adult zebrafish brain closely coincides with the proliferative zones, suggesting that F-spondin might be essential for adult neurogenesis. Use of this new transgenic model organism also allowed for the documentation of F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults. These included the endoskeleton of zebrafish fins, as well as their continuously regenerating pharyn- geal teeth. The latter result is notable given that F-spondin was recently found to be expressed in dental follicle cells of the developing tooth germ in mice [8], and in human periodontal cementoblasts, the activity of which is essential for mature tooth stability [7]. The role of F-spondin in actively proliferating tissues is further stressed by our observation of its de novo expression in the blastema, a critical area of cell proliferation and patterning formed during zebrafish fin regeneration. Together, these findings suggest multiple roles for F-spondin in the CNS and periphery of the developing and adult vertebrate and call for further investigations into the mechanisms of its actions. F-spondin Expression during the Segmentation Period An important role of F-spondin during the segmentation period [22] is suggested by massive spon1b:GFP transgene expression in the fin buds, developing somites, optic primordia, floor plate and notochord around 10 hpf. This expression pattern is, generally, in agreement with previous in situ hybridization-based studies conducted in embryonic zebrafish, frog, chick, mouse and rat [4,10–12]. The expression of spon1b:GFP in the developing forebrain and spinal cord neurons also suggests that F-spondin is involved in early patterning and polarization of the zebrafish CNS. This is consistent with in vitro studies showing that F-spondin can guide and promote the extension of neurites and axons of dorsal spinal [4], commissural [11] and hippocampal [13] cells in mammals, while inhibiting motor neuron outgrowth [50] and neural crest cell migration [51]. Overall, the visualization of spon1b:GFP accumulation in specific areas of the developing zebrafish embryo allows for earlier detection of spon1b-producing cells. This might explain prior ISH-based reports of more restricted spon1b expression during early zebrafish development [12]. F-spondin-expressing Neurons form Long Projections In zebrafish CNS, F-spondin expression appears to be limited to neurons, indicated by consistent co-localization with Hu C/D. Many of these neurons extend long projections, including RGC axons to the TeO, medial and lateral olfactory tracts (MOT and VOT), and sensory and motor neurons of the midbrain, hindbrain and spinal cord. Moreover, F-spondin expression highlights all the components of the long dorsal conduction pathway (DCP) [31], with an especially strong spon1b:GFP signal in the FR, extending from the Hb in the dorsal diencephalon to the NIn and SR of the ventral tegmentum. Together, this expression in adult brain implies that F-spondin function is not limited to the initial patterning and direction of long axons, but that it may serve as a short-range cue that remains associated with the cell producing it, promoting maintenance of its long axon and synaptic connections. Similar roles have been previously suggested for other ECM proteins, including Reelin [52,53]. F-Spondin Expression in Zebrafish F-spondin Expression in Circumventricular Regions and CSF-contacting Neurons F-spondin is reported to be a diffusible signaling molecule [5]. Consistent with this, we observed its expression in several regions that contain CSF-contacting neurons. These cells have two principal functions. Through their dendrites, they can receive information about chemical content, pressure or flow of the CSF, then convey it to various brain regions via their axons that terminate not only in the periventricular areas but as far away as the telencephalon or the spinal cord [54]. Alternatively, the axons of the CSF-contacting neurons can produce and release neuro- transmitters, peptides and other biologically active molecules into the CSF, providing chemical signals to other brain areas lining the ventricular spaces. Accordingly, we find that F-spondin positive neurons located along the mediobasal part of the diencephalic ventricle (Hv) form distinct tracts directed away from the ventricle, thus potentially serving as chemosensory neurons. This is in contrast to other CSF-contacting cells that extend their F-spondin positive axons into the ventricular lumen and are likely to contribute soluble F-spondin protein to the CSF. Especially rich in such cells are the nuclei positioned along the wall of the diencephalic ventricle and its recesses, including the preoptic nuclei, the paraventricular organ, and the nucleus of the posterior recess (NPR) of the caudal hypothalamus (Fig. 7). The two latter nuclei are known to be strongly labeled for several catecholamin- ergic markers in zebrafish [55], indicative of serotonergic [56] and dopaminergic [57] activity. Other magnocellular neurosecretory neurons in this nucleus in lower vertebrates, including fish, show immunoreactivity to neurophysin, vasotocin, isotocin/mesotocin, somatostatin or enkephalin antibodies [54]. Together, this raises an important question as to which neurochemical systems may regulate F-spondin release into the CSF, and which ones might be affected by the presence of F-spondin. The medullo-spinal component of the CSF contacting system, which extends throughout the rhomboencephalic ventricle and central canal [54], also expresses F-spondin mRNA. The neurons of this area extend stereocilia into the CSF, which act as mechanoreceptors when they contact Reissner’s fiber [54]. This enigmatic thread-like glycoprotein fiber, spanning the entire length of the central canal in the majority of vertebrates, is produced largely by the subcommissural organ (SCO), with some contribu- tion from other circumventricular areas [21]. An earlier study in embryonic zebrafish reported the expression of F-spondin mRNA in the SCO (in situ hybridization) and the presence of spon1b protein in Reissner’s fiber (immunohistochemistry) [12]. In contrast, we could not detect spon1b in the SCO in developing or adult animals with the methods used here. Nevertheless, the spon1b:GFP transgene highlighted the flexural organ, which is also a source of Reissner’s fiber-forming proteins [21] and the habenula, located immediately dorsal to the posterior commissure and SCO. Indeed, among the circumventricular organs (CVO) identified in zebrafish [58], the Hb nuclei attracted our special attention due to the early onset and exceptionally robust nature of spon1b expression in this important structure. Although the CSF- contacting properties of Hb neurons have not yet been charac- terized in zebrafish, evidence collected in other species suggests that Hb nuclei may contain actively secreting mast cells (e.g., in doves) [59] and may receive afferent projections from the CSF- contacting PVO via the FR (e.g., in lungfish) [54]. Using in situ hybridization, the expression of F-spondin was detected in the dorsal Hb nuclei of developing zebrafish in some [29] though not other [12] studies. We have also documented broad F-spondin expression in Hb of adult zebrafish using an in situ hybridization PLoS ONE | www.plosone.org 17 June 2012 | Volume 7 | Issue 6 | e37593