REGULATORY MECHANISMS CONTROLLING ENGRAILED GENE EXPRESSION IN THE ZEBRAFISH MYOTOME

Abstract

Signaling by secreted proteins of the Hedgehog (Hh) family of morphogens is crucial for the development and patterning of a wide variety of tissue types in metazoans. Different levels and timing of Hh signaling 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 characterized by eng expression and require the highest levels of Hh signaling for their specification. We have defined a minimal enhancer element of eng2a that is sufficient to drive reporter gene expression specifically in MPs and MFFs. Using a tissue culture based screen we identified a number of trans-factors that can regulate the activity of this enhancer. One of the strongest negative regulators identified was Smad5, a transcription factor whose activity is controlled by the BMP signaling pathway. Using an antibody specific to activated/phosphorylated Smads, 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 signaling is attenuated. Conversely, we show that driving nuclear accumulation of pSmad suppresses the induction of eng expression even when Hh pathway activity is maximal. We also demonstrate that the nuclear accumulation of pSmads is depleted by maximal Hh pathway activation. Using a Bimolecular Fluorescence Complementation assay, we show that a synthetic form of the Gli2 repressor can interact with Smad1 specifically in the nuclei of myotomal cells in the developing embryo and that this interaction depends upon BMP signaling 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 the MPs and MFFs; we suggest a novel basis for cross talk 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.