Genome-wide, whole mount in situ analysis of transcriptional regulators in zebrafish embryos.

Transcription is the primary step in the retrieval of genetic information. A substantial proportion of the protein repertoire of each organism consists of transcriptional regulators (TRs). It is believed that the differential expression and combinatorial action of these TRs is essential for vertebrate development and body homeostasis. We mined the zebrafish genome exhaustively for genes encoding TRs and determined their expression in the zebrafish embryo by sequencing to saturation and in situ hybridisation. At the evolutionary conserved phylotypic stage, 75% of the 3302 TR genes encoded in the genome are already expressed. The number of expressed TR genes increases only marginally in subsequent stages and is maintained during adulthood suggesting important roles of the TR genes in body homeostasis. Fewer than half of the TR genes (45%, n=1711 genes) are expressed in a tissue-restricted manner in the embryo. Transcripts of 207 genes were detected in a single tissue in the 24h embryo, potentially acting as regulators of specific processes. Other TR genes were expressed in multiple tissues. However, with the exception of certain territories in the nervous system, we did not find significant synexpression suggesting that most tissue-restricted TRs act in a freely combinatorial fashion. Our data indicate that elaboration of body pattern and function from the phylotypic stage onward relies mostly on redeployment of TRs and post-transcriptional processes.

The words of the regulatory code are arranged in a variable manner in highly conserved enhancers.

The cis-regulatory regions of many developmental regulators and transcription factors are believed to be highly conserved in the genomes of vertebrate species, suggesting specific regulatory mechanisms for these gene classes. We functionally characterized five notochord enhancers, whose sequence is highly conserved, and systematically mutated two of them. Two subregions were identified to be essential for expression in the notochord of the zebrafish embryo. Synthetic enhancers containing the two essential regions in front of a TATA-box drive expression in the notochord while concatemerization of the subregions alone is not sufficient, indicating that the combination of the two sequence elements is required for notochord expression. Both regions are present in the five functionally characterized notochord enhancers. However, the position, the distance and relative orientation of the two sequence motifs can vary substantially within the enhancer sequences. This suggests that the regulatory grammar itself does not dictate the high evolutionary conservation between these orthologous cis-regulatory sequences. Rather, it represents a less well-conserved layer of sequence organization within these sequences.

Sequential and cooperative action of Fgfs and Shh in the zebrafish retina.

The signaling molecule Sonic hedgehog (Shh) is required for differentiation of the vertebrate retina. In the developing zebrafish retina, shh expression is initiated at the ventronasal region, from where it spreads as a wave through the retina. To investigate the molecular mechanism underlying this coordinated expression of shh, we mapped the cis-regulatory region and identified a novel regulatory sequence in the first intron of the shh locus. This sequence contains binding sites for the transcription factors Erm and Pea3 that are known transducers of Fgf signaling. Mutation of the binding sites or knockdown of Pea3 and Erm abolishes transgene expression, indicating that Fgf signaling regulates shh expression in the retina. We provide evidence that Fgf3 and -8 control initiation of expression, while Fgf19 is crucial for the propagation of transgene expression through the retina. Inhibitor experiments indicate a continued requirement of FGF and Hedgehog (Hh) signaling for transgene expression after initiation at the ventronasal aspect of the retina. We propose a model, in which Fgf3 and -8 initiate expression and Fgf19 and Shh signals cooperate subsequently to promote establishment of expression throughout the retina.

Functional diversification of sonic hedgehog paralog enhancers identified by phylogenomic reconstruction.

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.

Cooperation of sonic hedgehog enhancers in midline expression.

In zebrafish, as in other vertebrates, the secreted signalling molecule Sonic hedgehog (Shh) is expressed in organiser regions such as the embryonic midline and the zona limitans intrathalamica (zli). To investigate the regulatory mechanisms underlying the pattern of shh expression, we carried out a systematic analysis of the intronic regulatory sequences of zebrafish shh using stable transgenesis. Deletion analysis identified the modules responsible for expression in the embryonic shield, the hypothalamus and the zli and confirmed the activities of previously identified notochord and floor plate enhancers. We detected a strong synergism between regulatory regions. The degree of synergy varied over time in the hypothalamus suggesting different mechanisms for initiation and maintenance of expression. Our data show that the pattern of shh expression in the embryonic central nervous system involves an intricate crosstalk of at least 4 different regulatory regions. When compared to the enhancer activities of the mouse Shh gene, we observed a remarkable divergence of function of structurally conserved enhancer sequences. The activating region ar-C (61% identical to SFPE2 in mouse Shh), for example, mediates floor plate expression in the mouse embryo while it directs expression in the forebrain and the notochord and only weakly in the floor plate in the zebrafish embryo. This raises doubts on the predictive power of phylogenetic footprinting and indicates a stunning divergence of function of structurally conserved regulatory modules during vertebrate evolution.

Mutation in the delta-subunit of the nAChR suppresses the muscle defects caused by lack of Dystrophin.

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 delta-subunit of the nAChR, showing this mutant to be a new allele of the previously named sofa potato (sop). The mutant allele carries a missense mutation in the extracellular domain altering the cysteine at position 150 to an arginine. The delta-subunit is expressed in all striated muscles in embryonic and early larval stages together with the alpha1, beta1, epsilon, and gamma-subunits of nAChR. In contrast to mammals that show switching from the gamma embryonic to the adult epsilon-subunit, the two subunits are coexpressed in zebrafish embryos. We, furthermore, demonstrated that the sop/delta-nAChR mutation 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.

Vertebrate floor-plate specification: variations on common themes.

Situated at the ventral-most part of the vertebrate neural tube, the floor plate (FP) is an important signalling centre that controls the regional differentiation of neurons in the nervous system. It secretes guidance molecules that direct ventrally navigating axons crucial for the correct wiring of neuronal circuits. Although the function of the FP is well-conserved from fish to humans, discrepancies exists with respect to both the signalling system involved in FP induction, and the origin of the FP in various vertebrate species. Recent findings from the embryos of zebrafish, chicken and mouse provide insights that reconcile previous results and suggest common themes in vertebrate FP specification.