We report evidence for a mechanism for the maintenance of long-range conserved synteny across vertebrate genomes. We found the largest mammal-teleost conserved chromosomal segments to be spanned by highly conserved noncoding elements (HCNEs), their developmental regulatory target genes, and phylogenetically and functionally unrelated "bystander" genes. Bystander genes are not specifically under the control of the regulatory elements that drive the target genes and are expressed in patterns that are different from those of the target genes. Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2, rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the expression patterns of these genes even if located inside or beyond bystander genes, suggesting that the regulatory domain of a developmental regulatory gene can extend into and beyond adjacent transcriptional units. We termed these chromosomal segments genomic regulatory blocks (GRBs). After whole genome duplication in teleosts, GRBs, including HCNEs and target genes, were often maintained in both copies, while bystander genes were typically lost from one GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy GRBs of higher vertebrates intact. We show that loss of bystander genes and other mutational events suffered by duplicated GRBs in teleost genomes permits target gene identification and HCNE/target gene assignment. These findings explain the absence of evolutionary breakpoints from large vertebrate chromosomal segments and will aid in the recognition of position effect mutations within human GRBs.

Summary Greying with age in horses is an autosomal dominant trait, characterized by hair greying, high incidence of melanoma and vitiligo‐like depigmentation. Previous studies have revealed that the causative mutation for this phenotype is a 4.6‐kb intronic duplication in STX17 (Syntaxin 17). By using reporter constructs in transgenic zebrafish, we show that a construct containing two copies of the duplicated sequence acts as a strong enhancer in neural crest cells and has subsequent melanophore‐specific activity during zebrafish embryonic development whereas a single copy of the duplicated sequence acts as a weak enhancer, consistent with the phenotypic manifestation of the mutation in horses. We further used luciferase assays to investigate regulatory regions in the duplication, to reveal tissue‐specific activities of these elements. One region upregulated the reporter gene expression in a melanocyte‐specific manner and contained two microphthalmia‐associated transcription factor (MITF) binding sites, essential for the activity. Microphthalmia‐associated transcription factor regulates melanocyte development, and these binding sites are outstanding candidates for mediating the melanocyte‐specific activity of the element. These results provide strong support for the causative nature of the duplication and constitute an explanation for the melanocyte‐specific effects of the Grey allele.

The complex expression pattern of fibroblast growth factor 8 (Fgf8) and the cellular responses dependent on concentration of its mRNA in vertebrates suggest that Fgf8 should be tightly controlled at the transcriptional level. We found zebrafish conserved noncoding elements (CNEs) with pan-vertebrate as well as fish-specific orthologous sequences from across 200 kb of the zebrafish fgf8a genomic regulatory block to direct reporter expression in patterns consistent with the expression pattern of fgf8a. These included elements from inside the introns of the skin-specific slc2a15a and the ubiquitously expressed fbxw4 bystander genes. The fgf8a/fbxw4 gene pair, which has remained joined throughout three whole genome duplications in chordate evolution, is inverted in teleost genomes, but CNEs across both evolutionary breakpoints showed specific activity. While some CNEs directed highly reproducible expression patterns, others were subject to variation but showed, in a subset of transgenes, expression in the apical ectodermal ridge, the anterior boundaries of somites and the midbrain-hindbrain boundary, specific Fgf8 signaling domains, suggesting that their activity may be context specific. A human element with tetrapod-specific orthologous sequences directed reporter expression to the vasculature, possibly corresponding to a tetrapod innovation. We conclude that fgf8a transcriptional regulation employs pan-vertebrate and teleost-specific enhancers dispersed over three genes in the zebrafish genome.

The zebrafish adult brain contains numerous neural progenitors and is a good model to approach the general mechanisms of adult neural stem cell maintenance and neurogenesis. Here we use this model to test for a correlation between Fgf signaling and cell proliferation in adult progenitor zones. We report expression of Fgf signals (fgf3,4,8a,8b,17b), receptors (fgfr1-4), and targets (erm, pea3, dusp6, spry1,2,4, and P-ERK) and document that genes of the embryonic fgf8 synexpression group acquire strikingly divergent patterns in the adult brain. We further document the specific expression of fgf3, fgfr1-3, dusp6, and P-ERK in ventricular zones, which contain neural progenitors. In these locations, however, a comparison at the single-cell level of fgfr/P-ERK expression with bromo-deoxy-uridine (BrdU) incorporation and the proliferation marker MCM5 indicates that Fgf signaling is not specifically associated with proliferating progenitors. Rather, it correlates with the ventricular radial glia state, some of which only are progenitors. Together these results stress the importance of Fgf signaling in the adult brain and establish the basis to study its function in zebrafish, in particular in relation to adult neurogenesis.

We screened an existing collection of zebrafish insertional mutants for cancer susceptibility by histologic examination of heterozygotes at 2 years of age. As most mutants had no altered cancer predisposition, this provided the first comprehensive description of spontaneous tumor spectrum and frequency in adult zebrafish. Moreover, the screen identified four lines, each carrying a different dominant mutant allele of Hagoromo previously linked to adult pigmentation defects, which develop tumors with high penetrance and that histologically resemble neuroblastoma. These tumors are clearly neural in origin, although they do not express catecholaminergic neuronal markers characteristic of human neuroblastoma. The zebrafish tumors result from inappropriate maintenance of a cell population within the cranial ganglia that are likely neural precursors. These neoplasias typically remain small but they can become highly aggressive, initially traveling along cranial nerves, and ultimately filling the head. The developmental origin of these tumors is highly reminiscent of human neuroblastoma. The four mutant Hagoromo alleles all contain viral insertions in the fbxw4 gene, which encodes an F-box WD40 domain-containing protein. However, although one allele clearly reduced the levels of fbxw4 mRNA, the other three insertions had no detectable effect on fbw4 expression. Instead, we showed that all four mutations result in the postembryonic up-regulation of the neighboring gene, fibroblast growth factor 8 (fgf8). Moreover, fgf8 is highly expressed in the tumorigenic lesions. Although fgf8 overexpression is known to be associated with breast and prostate cancer in mammals, this study provides the first evidence that fgf8 misregulation can lead to neural tumors. (Mol Cancer Res 2009;7(6):841-50)

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Salmon lice (Lepeophtheirus salmonis) are parasitic copepods, living mainly on Atlantic salmon and leading to large economical losses in aquaculture every year. Due to the emergence of resistances to several drugs, alternative treatments are developed, including treatment with hydrogen peroxide, freshwater or thermal treatment. The present study gives a first overview of the thermotolerance and stress response of salmon lice. Sea lice nauplii acclimated to 10 °C can survive heat shocks up to 30 °C and are capable of hardening by a sublethal heat shock. We searched in the genome for heat shock protein (HSP) encoding genes and tested their inducibility after heat shock, changes in salinity and treatment with hydrogen peroxide, employing microfluidic qPCRs. We assessed 38 candidate genes, belonging to the small HSP, HSP40, HSP70 and HSP90 families. Nine of these genes showed strong induction after a non-lethal heat shock. In contrast, only three and two of these genes were induced after changes in salinity and incubation in hydrogen peroxide, respectively. This work provides the basis for further work on the stress response on the economically important parasite L. salmonis.

Taste buds are sensory organs in jawed vertebrates, composed of distinct cell types that detect and transduce specific taste qualities. Taste bud cells differentiate from oropharyngeal epithelial progenitors, which are localized mainly in proximity to the forming organs. Despite recent progress in elucidating the molecular interactions required for taste bud cell development and function, the cell behavior underlying the organ assembly is poorly defined. Here, we used time-lapse imaging to observe the formation of taste buds in live zebrafish larvae. We found that tg(fgf8a.dr17)-expressing cells form taste buds and get rearranged within the forming organs. In addition, differentiating cells move from the epithelium to the forming organs and can be displaced between developing organs. During organ formation, tg(fgf8a.dr17) and type II taste bud cells are displaced in random, directed or confined mode relative to the taste bud they join or by which they are maintained. Finally, ascl1a activity in the 5-HT/type III cell is required to direct and maintain tg(fgf8a.dr17)-expressing cells into the taste bud. We propose that diversity in displacement modes of differentiating cells acts as a key mechanism for the highly dynamic process of taste bud assembly.