PhD Graduate: Ashish MAURYA
Thesis Title: Regulatory mechanisms controlling engrailed gene expression in the zebrafish myotome
Different levels and timing of Hedgehog (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 their expression of the eng1a, 1b and 2a genes and require the highest levels of Hh for their specification. We have defined a minimal element of the eng2a gene that is sufficient to drive reporter gene expression specifically in MPs and MFFs. Using a tissue culture based screen we identify a number of trans-factor that can regulate the activity of this enhancer. One of the strongest negative regulators 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.
Smad activity inhibits Engrailed in the myotome
A, B – Para saggital optical sections of a 22 somite stage zebrafish embryo at the level of yolk extension, labeled for activated Smads (pSmad, purple) and Engrailed (Eng, green). Panel-A shows wildtype pattern whereas panel-B shows an embryo which has Smad activity attenuated by injection of a BMP receptor morpholino.
C, D – Para saggital optical sections of a 22 somite stage zebrafish embryo at the end of yolk extension, labeled with the antibody F59 (slow muscle marker, red) and an eGFP transgene marking Engrailed positive cells (engGFP, green). Panel-C shows the wildtype pattern and panel-D shows the pattern when Smad activity is attenuated by injection of a BMP receptor morpholino.
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