A trans-Regulatory Code for the Forebrain Expression of Six3.2 in the Medaka Fish.

A well integrated and hierarchically organized gene regulatory network is responsible for the progressive specification of the forebrain. The transcription factor Six3 is one of the central components of this network. As such, Six3 regulates several components of the network, but its upstream regulators are still poorly characterized. Here we have systematically identified such regulators, taking advantage of the detailed functional characterization of the regulatory region of the medaka fish Six3.2 ortholog and of a time/cost-effective trans-regulatory screening, which complemented and overcame the limitations of in silico prediction approaches. The candidates resulting from this search were validated with dose-response luciferase assays and expression pattern criteria. Reconfirmed candidates with a matching expression pattern were also tested with chromatin immunoprecipitation and functional studies. Our results confirm the previously proposed direct regulation of Pax6 and further demonstrate that Msx2 and Pbx1 are bona fide direct regulators of early Six3.2 distribution in distinct domains of the medaka fish forebrain. They also point to other transcription factors, including Tcf3, as additional regulators of different spatial-temporal domains of Six3.2 expression. The activity of these regulators is discussed in the context of the gene regulatory network proposed for the specification of the forebrain.

Integration of Hedgehog and BMP signalling by the engrailed2a gene in the zebrafish myotome.

Different levels and timing of Hedgehog (Hh) signalling activity have been proposed to specify three distinct cell types in the zebrafish myotome. Two of these, the medial fast-twitch fibres (MFFs) and the slow-twitch muscle pioneers (MPs) are characterised by expression of eng1a, -1b and -2a and require the highest levels of Hh for their specification. We have defined a minimal eng2a element sufficient to drive reporter expression specifically in MPs and MFFs. This element binds both Gli2a, a mediator of Hh signalling, and activated Smads (pSmads), mediators of bone morphogenic protein (BMP) signalling, in vivo. We found a strict negative correlation between nuclear accumulation of pSmad, and eng2a expression in myotomal cells and show that abrogation of pSmad accumulation results in activation of eng2a, even when Hh signalling is attenuated. Conversely, driving nuclear accumulation of pSmad suppresses the induction of eng expression even when Hh pathway activity is maximal. Nuclear accumulation of pSmads is depleted by maximal Hh pathway activation. We show that a synthetic form of the Gli2 repressor interacts with Smad1 specifically in the nuclei of myotomal cells in the developing embryo and that this interaction depends upon BMP signalling activity. Our results demonstrate that the eng2a promoter integrates repressive and activating signals from the BMP and Hh pathways, respectively, to limit its expression to MPs and MFFs. We suggest a novel basis for crosstalk between the Hh and BMP pathways, whereby BMP-mediated repression of Hh target genes is promoted by a direct interaction between Smads and truncated Glis, an interaction that is abrogated by Hh induced depletion of the latter.

A global survey identifies novel upstream components of the Ath5 neurogenic network.

BACKGROUND: Investigating the architecture of gene regulatory networks (GRNs) is essential to decipher the logic of developmental programs during embryogenesis. In this study we present an upstream survey approach, termed trans-regulation screen, to comprehensively identify the regulatory input converging on endogenous regulatory sequences. RESULTS: Our dual luciferase-based screen queries transcriptome-scale collections of cDNAs. Using this approach we study the regulation of Ath5, the central node in the GRN controlling retinal ganglion cell (RGC) specification in vertebrates. The Ath5 promoter integrates the input of upstream regulators to enable the transient activation of the gene, which is an essential step for RGC differentiation. We efficiently identified potential Ath5 regulators that were further filtered for true positives by an in situ hybridization screen. Their regulatory activity was validated in vivo by functional assays in medakafish embryos. CONCLUSIONS: Our analysis establishes functional groups of genes controlling different regulatory phases, including the onset of Ath5 expression at cell-cycle exit and its down-regulation prior to terminal RGC differentiation. These results extent the current model of the GRN controlling retinal neurogenesis in vertebrates.

The discovery, positioning and verification of a set of transcription-associated motifs in vertebrates.

We have developed several new methods to investigate transcriptional motifs in vertebrates. We developed a specific alignment tool appropriate for regions involved in transcription control, and exhaustively enumerated all possible 12-mers for involvement in transcription by virtue of their mammalian conservation. We then used deeper comparative analysis across vertebrates to identify the active instances of these motifs. We have shown experimentally in Medaka fish that a subset of these predictions is involved in transcription.

Early development of functional spatial maps in the zebrafish olfactory bulb.

In the adult olfactory bulb (OB), particular chemical classes of odorants preferentially activate glomeruli within loosely defined regions, resulting in a coarse and fractured "chemotopic" map. In zebrafish, amino acids and bile acids predominantly stimulate glomeruli in the lateral and medial OB, respectively. We studied the development of these spatial response maps in zebrafish. At 3 d postfertilization (dpf), the OB contained protoglomerular structures that became refined and more numerous during subsequent days. In a transgenic zebrafish line expressing the Ca2+ indicator protein inverse pericam, mainly in mitral cells, odor responses in the OB were first detected at 2.5-3 dpf. Already at this stage, amino acids and bile acids evoked activity predominantly in the lateral and medial OB, respectively. Two-photon Ca2+ imaging using a synthetic indicator was used to reconstruct activity patterns at higher resolution in three dimensions. Responses to amino acids and bile acids were detected predominantly in the lateral and medial OB, respectively, with little overlap. Between 2.5 and 6 dpf, the number of odor-responsive units increased, but the overall spatial organization of activity persisted. Hence, a coarse spatial organization of functional activity maps is established very early during OB development when glomeruli are not yet differentiated. This spatial organization is maintained during development and growth of neuronal circuits and may have important functions for odor processing in larvae, for the differentiation of glomeruli, and for the refinement of activity maps at later developmental stages.