Relationship between expression of serendipity alpha and cellularisation of the Drosophila embryo as revealed by interspecific transformation

Saad Ibnsouda 1 Franois Schweisguth 0 Grard de Billy 1 Alain Vincent 1 0 Institut Jacques Monod , Tour 43, 2, Place Jussieu, 75521 Paris Cedex 05 , France 1 Centre de Biologie du Developpement , 118 route de Narbonne, 31062 Toulouse Cedex , France - Relationship between expression of serendipity and cellularisation of A dramatic reorganization of the cytoskeleton underlies the cellularisation of the syncytial Drosophila embryo. Formation of a regular network of acto-myosin filaments, providing a structural framework, and possibly a contractile force as well, appears essential for the synchronous invagination of the plasma membrane between adjacent nuclei. The serendipity alpha (sry ) gene is required for this complete reorganization of the microfilaments at the onset of membrane invagination. We compare here the structure and expression of sry between D. pseudoobscura, D. subobscura and D. melanogaster. Interspersion of evolutionarily highly conserved and divergent regions is observed in the protein. One such highly conserved region shows sequence similarities to a motif found in proteins of the ezrin-radixin-moesin (ERM) family. Four 7-13 bp motifs are conserved in the 5 promoter region; two of these are also found, and at the same position relative to the TATA Most or all pterygote insects and at least some apterygote insects undergo a cleavage of the fertilised egg of the intralecithal kind, i.e., the yolk mass remains undivided while the zygote nucleus and its daughters divide and spread with accompanying divisions of their cytoplasmic haloes (Anderson, 1972). Formation of cells occurs after nuclei have migrated to the yolk-free periphery of the embryo to form a monolayer at the cortex. Cellularisation has been most extensively studied in the dipterans, especially D. melanogaster, where it occurs synchronously across the whole surface of the embryo at the end of mitosis 13 and during the interphase of cycle 14 (between 120 and 170 minutes after fertilisation; Foe and Alberts, 1983). During cellularisation, the plasma membrane invaginates between adjacent nuclei, forming a hexagonal array of cleavage furrows around each nucleus, and subdivides the cortex of the embryo into individual cells. A dramatic redistribution of the cytoskeletal components occurs during cellularisation (Fullilove and Jacobson, 1971; Warn and Robert-Nicoud, 1990; Young et al., 1990). At the start of cellularisation, an actin-myosin hexagonal network forms, box, in nullo, another zygotic gene recently shown to be involved in cellularisation. The compared patterns of expression of D. melanogaster sry and nullo, and D. pseudoobscura sry reveal a complex regulation of the spatiotemporal accumulation of their transcripts. The D. pseudoobscura sry gene is able to rescue the cellularisation defects associated with a complete loss of sry function in D. melanogaster embryos, even though species-specific aspects of its expression are maintained. Despite their functional homologies, the D. melanogaster and D. pseudoobscura sry RNAs have different subcellular localisations, suggesting that this specific localization has no conserved role in targeting the sry protein to the apical membranes. which provides a structural frame, and possibly the contractile force, for the synchronous invagination of membranes. During cellularisation, this network evolves into individual ring-like structures composed of filaments of actin and myosin II, at the base of the cleavage furrows. At the end of cellularisation, contraction of these rings results in the formation of the basal membrane of epithelial-like blastoderm cells (review by Warn et al., 1990). Lack of the sry zygotic gene activity results in erratic disruptions of the cytoskeleton early at mitotic cycle 14, culminating in the formation of abnormal, multinucleate cells. The sry gene therefore appears specifically required for the integrity of the actin-myosin network (Merrill et al., 1988; Schweisguth et al., 1990). It encodes a transiently expressed protein which associates with membranes at the onset of cellularisation and accumulates at the base of the cleavage furrows during membrane invagination (Schweisguth et al., 1990, 1991 and unpublished). The temporal restriction of sry accumulation is achieved both through a tight blastoderm-specific transcriptional control and by instability of both the sry mRNA and protein products (Schweisguth et al., 1989, 1990). No point mutations in the sry gene have yet been identified despite several EMS screens of the 99D4-8 chromosome interval (Crozatier et al., 1992). In order to gain further insight into the specific role of sry in cellularisation, and the control of its expression, we used an interspecific comparison to identify putative functional domains within the sry protein and mRNA, as well as transcriptional cis-regulatory elements. We report here the sequence of the sry genes from D. pseudoobscura and D. subobscura. Further, we examine the expression and functional properties of the D. pseudoobscura gene introduced into D. melanogaster. Together with a comparison of the expression of sry and the other recently described cellularisation gene nullo, (Simpson-Rose and Wieschaus, 1992), this evolutionary comparison reveals a complex temporal and spatial regulation of the expression of cellularisation genes operating at the levels of transcription and possibly of RNA stability. MATERIAL AND METHODS The Oregon R stock of wild-type flies was used for control in situ hybridizations. The Df(3R)X3F (referred to as DfX3F) stock was obtained from Dr J. Merriam, and the ry506 strain used in the transformation experiments from Dr W. Bender. The LIMDF deficiency strain uncovering the nullo gene was provided by Dr E. Wieschaus. Molecular characterization of the D. pseudoobscura and D. subobscura sry clones All molecular methods described in this and other sections were carried out using standard techniques described in Sambrook et al. (1989). M. Aguad and C. Segarra kindly provided us with DNA from the 91C region of Drosophila subobscura and the 62 region of D. Pseudoobscura in the form of overlapping clones (EMBL4 and EMBL3 phage vectors, respectively) containing the ribosomal protein 49 gene (Aguad, 1988; Segarra and Aguad, 1993). Crude maps of the serendipity gene cluster organization were obtained by Southern hybridization to phage DNA cut with BamHI, EcoRI, and HindIII restriction endonucleases, using DNA probes made from the D. melanogaster rp49 and sry , and genes. Relevant phage DNA fragments were subcloned into Bluescript (Stragagene) and sequenced in both orientations using the exonuclease directional deletion technique of Henikoff (1987). Northern blot analysis and in situ hybridization Embryos were collected from either fly cage populations (D. melanogaster) or flies raised in bottles (D. pseudoobscura). 30 minutes collections of synchronously developing embryos were obtained after two precollections of (...truncated)