approach and the same probe used in an earlier study that demonstrated the presence of F-spondin in Hb [60]. Although the spon1b:GFP transgene showed prominent expression in Hb starting early in development, its localization was more restricted than that suggested by in situ hybridization, highlighting only laterally- positioned Hb nuclei in larval fish. Thereafter, the transgene allowed for detailed in vivo tracing of the migration of the two lateral Hb nuclei toward the midline during maturation, until they finally acquired their rostro-ventral location, corresponding to the ventral Hb nucleus (vHb) in adult zebrafish. This is consistent with a recent report on the complex migratory path of the lateral Hb nuclei during zebrafish ontogenesis and the identification of the lateral Hb in mammals as being homologous to the lateral Hb in larval zebrafish but the ventral Hb in adult zebrafish [28]. In addition to the distinct ventral and dorsal nuclei of the Hb, the dorsal Hb in zebrafish is further divided into the dorso-lateral (dlHb) and dorso-medial (dmHb) sub-divisions, indicated by distinct gene expression patterns and projections to the dorsal and ventral NIn, respectively [29,30,61]. In other species, it has been suggested that the Hb has even more complex morphological and functional structure [62,63]. Our data, as well as others’, suggest that the Hb in zebrafish is also likely to have more than three subdivisions [29,30], since the spon1b:GFP transgene, in addition to vHb, highlighted a previously undescribed subnucleus. We named it the inferior subnucleus of the dorso-medial Hb (dmHbi) based on its localization, cell morphology and projections to ventral NIn, consistent with dmHb. However, the dlHb and dmHb are known to keep a midline position starting with early development [60,64], while we do not detect a spon1b:GFP signal in the midline region until maturation occurs. It is thus possible that dmHbi starts expressing the transgene only later in life, in contrast to the early onset of spon1b production in the lateral, later turned ventral, Hb. Overall, in view of the major role that the Hb plays in emotional and cognitive functions in diverse species, including humans [65], specific mapping of spon1b:GFP transgene expression here could shed light on the role of the lateral Hb in CNS development and function throughout vertebrate life. F-spondin Expression in Neurogenic Zones During embryonic stages in vertebrates, ECM proteins are abundant in the developing brain. Later, in adults, they become more restricted to brain regions with germinal capacities [66]. For example, the rostral migratory stream (RMS) and granular zone of adult rodents, regions abundant in neural stem cells, are also characterized by high expression of ECM proteins [67]. In particular, F-spondin is present in the RMS of mice and was suggested to be part of the signaling cascade for chain formation, migration, and detachment of neuroblasts from the stream via its interaction with the ApoER2 receptor and intracellular adapter Dab1 [18]. Here, we report that spon1b in zebrafish is abundant not only in the areas that resemble mammalian neurogenic niches (i.e., Vv as RMS [68]), but also in all other proliferative zones along the rostro-caudal axis of the zebrafish brain. This suggests that the role of F-spondin in neurogenesis might be broad and not specific to the telencephalic niches. In vitro studies in mammalian hippocampal and cortical progenitor cell lines have identified F- spondin as a secreted protein that regulates migration and differentiation of neurons [5]. A potential role for F-spondin in these processes is also supported by our data showing that the habenular and cerebellar niches, which contain the largest migratory stem cells populations in zebrafish [41], are also the regions that contain the highest density of F-spondin-expressing cells (in Hb) or fibers (in CCe). F-Spondin Expression in Zebrafish Double staining for spon1b:GFP and the proliferative marker BrdU revealed that, in all of the stem cell niches examined, the newly divided cells do not express spon1b. Even a month later, the BrdU-positive cells that migrate to the spon1b-rich areas, e.g., vHb, remain spon1b negative. Nevertheless, we could observe a few cells positive for both BrdU and spon1b:GFP that migrated to the tegmentum within a month after cell division, and were in the process of extending long projections. Still to be elucidated is the extent to which F-spondin is involved in adult neurogenesis, and the mechanism through which this ECM protein could contribute to the microenvironment of the neurogenic niches, the migration of new cells, and their ability to form long projections. The Role of F-spondin in Peripheral Tissues In addition to the proliferative niches in the brain, the peripheral tissues involved in patterning of newly forming or regenerating structures express spon1b throughout the life of the zebrafish. These include the endoskeleton of the zebrafish fins, involved in patterning, and the unique blastema region of fin rays undergoing regeneration following partial amputation. Moreover, quite remarkable is the massive and constant expression of spon1b in the soft lamina surrounding the pharyngeal teeth, which continuously renew throughout zebrafish life. In fact, recently F- spondin was identified as being important for mammalian tooth development and maintenance, and its specific expression in the dental follicle cells of the tooth germ in mice [8], and in human periodontal cementoblasts, essential for the stabilization of the periodontal ligament and the tooth itself [7]. Similarly, the presence of F-spondin in embryonic chick and rat cartilage and its ability to stimulate chondrocyte activity and collagen degradation in cultured cartilage explants, [6] suggest additional F-spondin functions. The peripheral nervous system arises from neural crest cells that migrate to different areas of the organism. F-spondin has been found to be involved in patterning of the sympathetic nervous system by acting as a signaling molecule that prevents neural crest cells from entering the caudal end of each somite [50]. Our finding that F-spondin is expressed in enteric neurons of zebrafish, raises the interesting question of whether it might also play a role in enteric neuron differentiation, migration, or maintenance, in other vertebrates, including humans, and calls for further investigation of this issue. Together, our findings imply multiple roles for F-spondin in the CNS and periphery of the developing and adult vertebrates, making the zebrafish an attractive model to investigate the role of this ECM protein in normal and pathological conditions. Methods Animal Care and Maintenance Adult male and female zebrafish (Danio rerio, AB wild type strain, 5 fish/3-L tank) were housed in a 14 h light/10 h dark cycle, in a temperature (26.5uC) and pH (7.0–7.4) controlled multi-tank re- circulating water system (Aquaneering, San Diego, CA, USA). Animals were fed three times a day with live brine shrimp (Brine Shrimp Direct, Ogden, Utah. USA), enriched with fish pellets (Lansy NRD, Salt Lake City, UT. USA). Embryos were raised at 28.5uC in a 12 h light/12 h dark cycle before being transferred to system tanks at 21 dpf. All animal procedures were performed in accordance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Boston University School of Medicine. PLoS ONE | www.plosone.org 18 June 2012 | Volume 7 | Issue 6 | e37593