Revisitingbicoidfunction: complete inactivation reveals an additional fundamental role inDrosophilaegg geometry specification

Abstract

AbstractIntroductionThebicoid(bcd) gene inDrosophilahas served as a paradigm for a morphogen in textbooks for decades. Discovered in 1986 as a mutation affecting anterior development in the embryo, its expression pattern as a protein gradient later confirmed the prediction from transplantation experiments. These experiments suggested that the protein fulfills the criteria of a true morphogen, with the existence of a homeodomain crucial for activation of genes along the anterior-posterior axis, based on the concentration of the morphogen. Thebcdgene undergoes alternative splicing, resulting in, among other isoforms, a small and often neglected isoform with low abundance, which lacks the homeodomain, termedsmall bicoid(smbcd). Most importantly, all known classical strongbcdalleles used in the past to determinebcdfunction apparently do not affect the function of this isoform.ResultsTo overcome the uncertainty regarding which isoform regulates what, I removed thebcdlocus entirely using CRISPR technology.bcdCRISPReggs exhibited a short and round appearance. The phenotype could be ascribed tosmbcdbecause allbcdalleles affecting the function of the major transcript, termedlarge bicoid(lgbcd) showed normally sized eggs. Several patterning genes for the embryo showed expression in the oocyte, and their expression patterns were altered inbcdCRISPRoocytes. InbcdCRISPRembryos, all downstream segmentation genes showed altered expression patterns, consistent with the expression patterns in “classical” alleles; however, due to the altered egg geometry resulting in fewer blastoderm nuclei, additional constraints came into play, further affecting their expression patterns.ConclusionsThis study unveils a novel and fundamental role ofbcdin shaping the egg’s geometry. This discovery demands a comprehensive revision of our understanding of this important patterning gene and prompts a reevaluation of past experiments conducted under the assumption thatbcdmutants werebcdnull-mutants.