Patrick H. O'Farrell

Genetic control of cell division patterns in the Drosophila embryo

In Drosophila embryogenesis, mitotic control undergoes a significant transition during the 14th interphase. Mitoses before interphase 14 run on maternal products, and occur in metasynchronous waves. Mitoses after interphase 14 require zygotic transcription, and occur asyncronously in an intricate, highly ordered spatio-temporal pattern. Mutations at the string (stg) locus cause cell-cycle arrest during this transition, in G2 of interphase 14, yet do not arrest other aspects of development. This phenotype suggests that stg is required specifically for initiating mitosis. We describe the cloning of stg, and show that its predicted amino acid sequence is homologous to that of cdc25, a regular of mitotic initiation in the yeast S. pombe. In addition, we show that zygotic expression of stg mRNA occurs in a dynamic series of spatial patterns which anticipate the patterns of the zygotically driven cell divisions. Therefore we suggest that regulated expression of stg mRNA controls the timing and location of these embryonic cell divisions.

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing

Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.

The engrailed locus of drosophila: In situ localization of transcripts reveals compartment-specific expression

The engrailed locus plays a unique and critical role in organizing the segmented body plan of Drosophila. Embryos lacking engrailed function die with fused, abnormal segments. Adult mosaics with patches of engrailed cells similarly suffer defects in all of their segments, but only with mutant cells that are in the posterior developmental compartment of each segment. The non-uniform requirement for engrailed function reflects the position-dependent expression of the engrailed locus and we demonstrate it here unambiguously by directly visualizing engrailed transcripts in frozen sections of embryos and larvae and in whole imaginal discs. These results demonstrate that developmental compartments subdivide the embryonic insect segments. In these and in the compartments of the later developmental stages the engrailed locus is expressed in the posterior but not the anterior compartments. With its role in controlling the developmental pathway of the posterior compartment cells, the engrailed locus may be an example of a binary developmental switch.

The Three Postblastoderm Cell Cycles of Drosophila Embryogenesis Are Regulated in G2 by string

The string (stg) locus of Drosophila encodes a factor that is thought to trigger mitosis by activating the p34cdc2 protein kinase. stg is required for mitosis early in development and is transcribed in a dynamic pattern that anticipates the pattern of embryonic cell divisions. Here we show that differential cell cycle regulation during postblastoderm development (cell cycles 14-16) occurs in G2. We demonstrate that stg mRNA expressed from a heat shock promotor triggers mitosis, and an associated S phase, in G2 cells during these cycles. Hence, differential cell cycle timing at this developmental stage is controlled by stg. Finally, we use heat-induced stg expression to alter the normal pattern of embryonic mitoses. Surprisingly, the complex mitotic pattern evident during normal development is not essential for many features of pattern formation or for viability.

The Roles of Drosophila Cyclins A and B in Mitotic Control

We have cloned, sequenced, and characterized the expression of a Drosophila cyclin B gene. The independent evolutionary conservation of A- and B-type cyclins implies that they have distinct roles. Indeed, in mutant embryos deficient in cyclin A, cells that accumulate only cyclin B do not enter mitosis. Thus, in vivo, cyclin B is not sufficient for mitosis. Furthermore, we find that the two cyclins are coexpressed in all proliferating cells throughout development. Though lacking a formal demonstration that cyclin B is essential as it is in other organisms, we propose that each of these proteins fulfills a distinct and essential role in the cell cycle.

Expression and Function of Drosophila Cyclin A during Embryonic Cell Cycle Progression

Cyclin proteins are thought to trigger entry into mitosis. During mitosis they are rapidly degraded. Therefore, mitosis and consequently cyclin degradation might be triggered at a time when cyclins have reaccumulated to a critical level. We cloned and sequenced a Drosophila cyclin A homolog and identified mutations in the corresponding gene. Immunofluorescent staining revealed that cyclin A accumulates in the interphase cytoplasm of cellularized embryos, but relocates to the nuclear region early in prophase and is completely degraded within metaphase. Cyclin A was expressed in dividing cells throughout development, and a functional cyclin A gene was required for continued division after exhaustion of maternally contributed cyclin A. Importantly, the timing of post cellularization divisions was not governed by the rate of accumulation or level of cyclin A.

The sequence specificity of homeodomain-DNA interaction

The Drosophila developmental gene, engrailed, encodes a sequence-specific DNA binding activity. Using deletion constructs expressed as fusion proteins in E. coli, we localized this activity to the conserved homeodomain (HD). The binding site consensus, TCAATTAAAT, is found in clusters in the engrailed regulatory region. Weak binding of the En HD to one copy of a synthetic consensus is enhanced by adjacent copies. The distantly related HD encoded by fushi tarazu binds to the same sites as the En HD, but differs in its preference for related sites. Both HDs bind a second type of sequence, a repeat of TAA. The similarity in sequence specificity of En and Ftz HDs suggests that, within families of DNA binding proteins, close relatives will exhibit similar specificities. Competition among related regulatory proteins might govern which protein occupies a given binding site and consequently determine the ultimate effect of cis-acting regulatory sites.

Development of embryonic pattern in D. melanogaster as revealed by accumulation of the nuclear engrailed protein

Engrailed is required to establish and maintain developmental compartments within each segment of the fly. To understand the role of the engrailed protein in this process, we have raised antibodies against engrailed and have visualized an engrailed protein in embryos by indirect immunofluorescence. The protein accumulates in the nucleus, supporting the notion that engrailed is a regulatory factor. The first pattern of expression is in alternating segments followed by expression in every segment, suggesting that engrailed may be responding to pair-rule segmentation gene products. Overall, engrailed protein levels peak in areas undergoing morphogenesis. Finally, the complex final form of the head and terminalia derive from earlier simple subdivision of these areas into developmental fields by engrailed.

Terminal Cytokinesis Events Uncovered after an RNAi Screen

Much of our understanding of animal cell cytokinesis centers on the regulation of the equatorial acto-myosin contractile ring that drives the rapid ingression of a deep cleavage furrow. However, the central part of the mitotic spindle collapses to a dense structure that impedes the furrow and keeps the daughter cells connected via an intercellular bridge. Factors involved in the formation, maintenance, and resolution of this bridge are largely unknown. Using a library of 7,216 double-stranded RNAs (dsRNAs) representing the conserved genes of Drosophila, we performed an RNA interference (RNAi) screen for cytokinesis genes in Schneiders S2 cells. We identified both familiar and novel genes whose inactivation induced a multi-nucleate phenotype. Using live video microscopy, we show that three genes: anillin, citron-kinase (CG10522), and soluble N-ethylmaleimide sensitive factor (NSF) attachment protein (alpha-SNAP), are essential for the terminal (post-furrowing) events of cytokinesis. anillin RNAi caused gradual disruption of the intercellular bridge after furrowing; citron-kinase RNAi destabilized the bridge at a later stage; alpha-SNAP RNAi caused sister cells to fuse many hours later and by a different mechanism. We have shown that the stability of the intercellular bridge is essential for successful cytokinesis and have defined genes contributing to this stability.

Identification of Drosophila Gene Products Required for Phagocytosis of Candida albicans

Phagocytosis is a highly conserved aspect of innate immunity. We used Drosophila melanogaster S2 cells as a model system to study the phagocytosis of Candida albicans, the major fungal pathogen of humans, by screening an RNAi library representing 7,216 fly genes conserved among metazoans. After rescreening the initial genes identified and eliminating certain classes of housekeeping genes, we identified 184 genes required for efficient phagocytosis of C. albicans. Diverse biological processes are represented, with actin cytoskeleton regulation, vesicle transport, signaling, and transcriptional regulation being prominent. Secondary screens using Escherichia coli and latex beads revealed several genes specific for C. albicans phagocytosis. Characterization of one of those gene products, Macroglobulin complement related (Mcr), shows that it is secreted, that it binds specifically to the surface of C. albicans, and that it promotes its subsequent phagocytosis. Mcr is closely related to the four Drosophila thioester proteins (Teps), and we show that TepII is required for efficient phagocytosis of E. coli (but not C. albicans or Staphylococcus aureus) and that TepIII is required for the efficient phagocytosis of S. aureus (but not C. albicans or E. coli). Thus, this family of fly proteins distinguishes different pathogens for subsequent phagocytosis.