Anna Z. Komisarczuk

Cis-regulation and chromosomal rearrangement of the fgf8 locus after the teleost/tetrapod split.

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.

Fgf signaling in the zebrafish adult brain: association of Fgf activity with ventricular zones but not cell proliferation.

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. J. Comp. Neurol. 510:422–439, 2008.

Zebrafish Hagoromo mutants up-regulate fgf8 postembryonically and develop neuroblastoma.

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)

Identification of a melanocyte-specific, microphthalmia-associated transcription factor-dependent regulatory element in the intronic duplication causing hair greying and melanoma in horses

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.

Enhancer detection and developmental expression of zebrafish sprouty1, a member of the fgf8 synexpression group.

Signaling pathways mediated by receptor tyrosine kinases (RTKs) are under positive and negative regulation, and misregulation of RTK signaling results in developmental defects and malignancy. A major class of antagonists of Fgf and Egf signaling are the Sprouty proteins. Through an enhancer detection approach, we isolated the sprouty1 (spry1) gene, expressed in multiple developing organs during embryogenesis. We analyzed expression of spry1 between tail bud stage and 10 days postfertilization. From the tail bud stage on, transcript and reporter are detected in the craniofacial region and in the mid–hindbrain boundary, where expression persists until adulthood. Further expression domains are the telencephalon, hindbrain, dorsal diencephalon and epiphysis, branchial arches, pituitary, and the tubular gill epithelium. In the trunk spry1 is also prominently expressed in pronephros, the lateral line and tail fin. Sprouty1 acts in Fgf signaling downstream of Fgfr1, as its expression is abrogated through the small molecule inhibitor of this receptor, SU5402. Developmental Dynamics 237:2594–2603, 2008.

Diversity in cell motility reveals the dynamic nature of the formation of zebrafish taste sensory organs

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. Summary: Time-lapse imaging, cell ablation and MSD plot analysis reveal relative random, directed or confined displacement of differentiating epithelial cells during taste bud development in zebrafish.

Ionotropic receptors signal host recognition in the salmon louse (Lepeophtheirus salmonis, Copepoda)

A remarkable feature of many parasites is a high degree of host specificity but the mechanisms behind are poorly understood. A major challenge for parasites is to identify and infect a suitable host. Many species show a high degree of host specificity, being able to survive only on one or a few related host species. To facilitate transmission, parasite’s behavior and reproduction has been fine tuned to maximize the likelihood of infection of a suitable host. For some species chemical cues that trigger or attract the parasite in question have been identified but how metazoan parasites themselves receive these signals remains unknown. In the present study we show that ionotropic receptors (IRs) in the salmon louse are likely responsible for identification of a specific host. By using RNAi to knock down the expression level of different co-receptors, a significant change of infectivity and settlement of lice larvae was achieved on Atlantic salmon. More remarkably, knock down of the IRs changed the host specificity of the salmon louse and lice larvae settled at a significant rate on host that the wild type lice rejected within minutes. To our knowledge, this has never before been demonstrated for any metazoan parasite. Our results show that the parasites are able to identify the host quickly upon settlement, settle and initiate the parasitic life style if they are on the right host. This novel discovery opens up for utilizing the host recognition system for future parasite control.