Uwe Strähle

The zebrafish has become a new model for adult neurogenesis, owing to its abundant neurogenic areas in most brain subdivisions. Radial glia‐like cells, actively proliferating cells, and label‐retaining progenitors have been described in these areas. In the telencephalon, this complexity is enhanced by an organization of the ventricular zone (VZ) in fast and slow‐dividing domains, suggesting the existence of heterogeneous progenitor types. In this work, we studied the expression of various transgenic or immunocytochemical markers for glial cells (gfap:gfp, cyp19a1b:gfp, BLBP, and S100β), progenitors (nestin:gfp and Sox2), and neuroblasts (PSA‐NCAM) in cycling progenitors of the adult zebrafish telencephalon (identified by expression of proliferating cell nuclear antigen (PCNA), MCM5, or bromodeoxyuridine incorporation). We demonstrate the existence of distinct populations of dividing cells at the adult telencephalic VZ. Progenitors of the overall slow‐cycling domains express high levels of Sox2 and nestin:gfp as well as all glial markers tested. In contrast, domains with an overall fast division rate are characterized by low or missing expression of glial markers. PCNA‐positive cells in fast domains further display a morphology distinct from radial glia and co‐express PSA‐NCAM, suggesting that they are early neuronal precursors. In addition, the VZ contains cycling progenitors that express neither glial markers nor nestin:gfp, but are positive for Sox2 and PSA‐NCAM, identifying them as committed neuroblasts. On the basis of the marker gene expression and distinct cell morphologies, we propose a classification for the dividing cell states at the zebrafish adult telencephalic VZ.

Adult neurogenesis arises from niches that harbor neural stem cells (NSC). Although holding great promise for regenerative medicine, the identity of NSC remains elusive. In mammals, a key attribute of NSC is the expression of the filamentous proteins glial fibrillary acidic protein (GFAP) and NESTIN. To assess whether these two markers are relevant in the fish model, two transgenic zebrafish lines for gfap and nestin were generated. Analysis of adult brains showed that the fusion GFAP–green fluorescent protein closely mimics endogenous GFAP, while the nestin transgene recapitulates nestin at the ventricular zones. Cells expressing the two reporters display radial glial morphology, colocalize with the NSC marker Sox2, undergo proliferation, and are capable of self‐renewal within the matrix of distinct thickness in the telencephalon. Together, these two transgenic lines reveal a conserved feature of putative NSC in the adult zebrafish brain and provide a means for the identification and manipulation of these cells in vivo. Developmental Dynamics 238:475–486, 2009.

The formation of thick filaments in striated muscle involves the chaperones Hsp90a and Unc45. We show that Unc45b and Hsp90a, two zebrafish orthologues, colocalize with myosin during myofibrillogenesis and associate with the Z line when myofibril assembly is completed. In response to stress or damage to the myofiber, Unc45b and Hsp90a dissociate from the Z line and transiently associate with myosin. Although chaperone activity of Unc45b requires the full-length protein, only the central and Unc45-Cro1p-She4p domains are required to anchor it to the Z line, and multiple subdomains mediate association with nascent myosin. We propose that the Z line serves as a reservoir for chaperones, allowing a rapid mobilization in response to muscle damage. Our data are consistent with a differential affinity model as an explanation for the shuttling of the chaperones between the Z line and myosin.

Background: Early life stages are generally most sensitive to toxic effects. Our knowledge on the action of manmade chemicals on the developing vertebrate embryo is, however, rather limited. We addressed the toxicogenomic response of the zebrafish embryo in a systematic manner by asking whether distinct chemicals would induce specific transcriptional profiles. Results: We exposed zebrafish embryos to a range of environmental toxicants and measured the changes in gene-expression profiles by hybridizing cDNA to an oligonucleotide microarray. Several hundred genes responded significantly to at least one of the 11 toxicants tested. We obtained specific expression profiles for each of the chemicals and could predict the identity of the toxicant from the expression profiles with high probability. Changes in gene expression were observed at toxicant concentrations that did not cause morphological effects. The toxicogenomic profiles were highly stage specific and we detected tissue-specific gene responses, underscoring the sensitivity of the assay system. Conclusion: Our results show that the genome of the zebrafish embryo responds to toxicant exposure in a highly sensitive and specific manner. Our work provides proof-of-principle for the use of the zebrafish embryo as a toxicogenomic model and highlights its potential for systematic, large-scale analysis of the effects of chemicals on the developing vertebrate embryo.

Background: Little is known about the control of the development of vertebrate unpaired appendages such as the caudal fin, one of the key morphological specializations of fishes. Recent analysis of lamprey and dogshark median fins suggests the co-option of some molecular mechanisms between paired and median in Chondrichthyes. However, the extent to which the molecular mechanisms patterning paired and median fins are shared remains unknown. Results: Here we provide molecular description of the initial ontogeny of the median fins in zebrafish and present several independent lines of evidence that Sonic hedgehog signaling emanating from the embryonic midline is essential for establishment and outgrowth of the caudal fin primordium. However, gene expression analysis shows that the primordium of the adult caudal fin does not harbor a Sonic hedgehog-expressing domain equivalent to the Shh secreting zone of polarizing activity (ZPA) of paired appendages. Conclusion: Our results suggest that Hedgehog proteins can regulate skeletal appendage outgrowth independent of a ZPA and demonstrates an unexpected mechanism for mediating Shh signals in a median fin primordium. The median fins evolved before paired fins in early craniates, thus the patterning of the median fins may be an ancestral mechanism that controls the outgrowth of skeletogenic appendages in vertebrates.

Background: Cis-regulatory modules of developmental genes are targets of evolutionary changes that underlie the morphologic diversity of animals. Little is known about the 'grammar' of interactions between transcription factors and cis-regulatory modules and therefore about the molecular mechanisms that underlie changes in these modules, particularly after gene and genome duplications. We investigated the ar-C midline enhancer of sonic hedgehog (shh) orthologs and paralogs from distantly related vertebrate lineages, from fish to human, including the basal vertebrate Latimeria menadoensis. Results: We demonstrate that the sonic hedgehog a (shha) paralogs sonic hedgehog b (tiggy winkle hedgehog; shhb) genes of fishes have a modified ar-C enhancer, which specifies a diverged function at the embryonic midline. We have identified several conserved motifs that are indicative of putative transcription factor binding sites by local alignment of ar-C enhancers of numerous vertebrate sequences. To trace the evolutionary changes among paralog enhancers, phylogenomic reconstruction was carried out and lineage-specific motif changes were identified. The relation between motif composition and observed developmental differences was evaluated through transgenic functional analyses. Altering and exchanging motifs between paralog enhancers resulted in reversal of enhancer specificity in the floor plate and notochord. A model reconstructing enhancer divergence during vertebrate evolution was developed. Conclusion: Our model suggests that the identified motifs of the ar-C enhancer function as binary switches that are responsible for specific activity between midline tissues, and that these motifs are adjusted during functional diversification of paralogs. The unraveled motif changes can also account for the complex interpretation of activator and repressor input signals within a single enhancer.

Normal motility of the zebrafish embryo requires a large number of gene loci, many of which have human orthologues implicated in myasthenias and other myopathies. We have identified a mutation in the zebrafish that abolishes body motility. Embryos have narrower myofibrils and lack clusters of nicotinic acetylcholine receptors (nAChRs) on the surface of the somitic muscle. We mapped the mutation to the δ‐subunit of the nAChR, showing this mutant to be a new allele of the previously namedsofa potato(sop). The mutant allele carries a missense mutation in the extracellular domain altering the cysteine at position 150 to an arginine. The δ‐subunit is expressed in all striated muscles in embryonic and early larval stages together with the α1, β1, ϵ, and γ‐subunits of nAChR. In contrast to mammals that show switching from the γ embryonic to the adult ϵ‐subunit, the two subunits are coexpressed in zebrafish embryos. We, furthermore, demonstrated that thesop/δ‐nAChRmutation is a suppressor of the myopathy caused by lack of Dystrophin. The myofiber detachment phenotype of Dystroglycan‐deficient embryos was not suppressed, suggesting that Dystrophin and Dystroglycan play distinct roles in muscle formation and maintenance of muscle integrity. Developmental Dynamics 234:1016–1025, 2005.