FATE SPECIFICATION ALONG THE SEA URCHIN EMBRYO A-V AXIS

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The VEB (very early blastula) genes are the first strictly zygotic genes activated in the sea urchin embryo; they are transcribed in approximately 80% of the embryo, but not in a region around the vegetal pole. The VEB message distribution thus provides one of the earliest molecular indications of the maternally specified animal-vegetal axis. Several lines of evidence suggest that the asymmetry of VEB gene activity is regulated by one or more maternal factors whose activity is similarly disposed in the egg and early embryo. Furthermore, one VEB gene, SpAN, encodes an astacin- family protease with demonstrated function in activating SpBMP4, a growth factor of the TGFb superfamily. Thus, the SpAN gene is a strong candidate for a switch point in very early development which converts prelocalized maternal gene regulatory information to spatially regulated production of a growth factor likely to have an important early function in specification of cell fates along the animal-vegetal (AV) axis, which depends on cell- cell interactions. To test this overall hypothesis we are (Aim I) studying spatial regulation of SpAN and a second VEB gene (SpHE, which encodes the hatching enzyme) in an effort to understand the molecular mechanism that creates the AV axis and (Aim II) examining the potential role of SpAN/SpBMP4 as a morphogen. The cis-acting elements and trans-acting factors which regulate SpAN and SpHE transcription are being analyze by in vivo assays of promoter transgene constructs in sea urchin embryos and by in vitro assays of protein-DNA interaction. The role of SpAN/SpBMP4 in fate specification will be studied by isolating cDNA clones encoding the receptor for SpBMP4 (SpBMP4R); by characterizing the developmental expression of SpBMP4 and SpBMP4R peptides; and by overexpression, misexpression and dominant negative interference with SpAN, SpBMP4 and SpBMP4R function. (Aim III) if suitable candidate maternal regulators of the VEB genes are identified, whose functions are spatially restricted, we shall carry out similar experiments to test the developmental effects of perturbing their function.