No abstract

No abstract

We have observed that zygotic transcription does not initiate at a single point in Drosophila embryos. Rather, a gene initiates transcription in a few nuclei of a fraction of embryos. During succeeding cycles, the frequency of transcribing embryos, and of nuclei transcribing in those embryos, gradually increases. For the fushi tarazu (ftz) gene, the timing of this process is regulated by the concentration of the maternally loaded, repressing transcription factor tramtrack (ttk). Altering the dose of Ttk protein in embryos shifts the activation of ftz transcription either forward or backward during development but does not effect Kriippel (Kr) activation. We have observed that the transcription of several genes, including ftz, is triggered in embryos at a critical nuclear density; therefore, we suggest that titration of transcription factors like ttk by the nucleocytoplasmic ratio triggers zygotic transcription in Drosophila. Drosophila embryos, transcription first becomes detectable around the tenth nuclear division or "nuclear cycle" as the embryo is completing the process of nuclear migration (Zalokar 1976;Anderson and Lengyel 1979;Knipple et al. 1985;Weir and Kornberg 1985;Erickson and Cline 1993).Based on these studies, it has been generally assumed that zygotic transcription is initiated in an embryo at a specific stage during development. However, a limitation of these studies is that the assay methods available to detect transcription were either insensitive or did not allow precise staging of embryos. Tautz and Pfeifle (1989) have developed a sensitive whole-mount in situ hybridization technique. Using this technique the sites of gene transcription appear as "nuclear dots," two spheres of staining within nuclei (Shermoen and O'Farrell 1991). For several genes we have demonstrated that transcription is gradually activated. When genes first begin to transcribe during cycles 8, 9, or 10, nuclear dots are observed only in a few nuclei in a small fraction of embryos. During each succeeding nuclear cycle the frequency of nuclei showing nuclear dots gradually increases and more embryos show transcribing nuclei. Fi~Corresponding author.nally, after several nuclear cycles, most nuclei in all embryos show nuclear dots.Before cycle 8, nuclei are migrating from the center of the embryo out to the cortex and the embryo is transcriptionally silent. However, between cycles 8 and 10, the nuclei reach the cortex, complete nuclear migration, and transcription begins. One of several developmental events might trigger the initiation of transcription at this point, including the position of the nuclei (position), the age of the embryo (time), the lengthening of the cell cycle, or the embryo reaching a crucial nuclear density (nucleocytoplasmic ratio) Kimelman et al. 1987; Newport and Kirschner 1982a, b;Brown et al. 1991;Yasuda et al. 1991). To distinguish between these alternatives, we have assayed the effect of a series of experimental and genetic manipulations that alter the timing and/or pattern of nuclear migration on th...

Abstract. Drosophila embryogenesis is initiated by a series of syncytial mitotic divisions. The first nine of these divisions are internal, and are accompanied by two temporally distinct nuclear movements that lead to the formation of a syncytial blastoderm with a uniform monolayer of cortical nuclei. The first of these movemerits, which we term axial expansion, occurs during division cycles 4-6 and distributes nuclei in a hollow ellipsoid underlying the cortex. This is followed by cortical migration, during cycles 7-10, which places the nuclei in a uniform monolayer at the cortex. Here we report that these two movements differ in their geometry, velocity, cell-cycle dependence, and protein synthesis requirement. We therefore conclude that axial expansion and cortical migration are mechanistically distinct, amplifying a similar conclusion based on pharmacological data (Zalokar and Erk, 1976).We have examined microtubule organization during cortical migration and find that a network of interdigitating microtubules connects the migrating nuclei. These anti-parallel microtubule arrays are observed between migrating nuclei and yolk nuclei located deeper in the embryo. These arrays are present during nuclear movement but break down when the nuclei are not moving. We propose that cortical migration is driven by microtubule-dependent forces that repel adjacent nuclei, leading to an expansion of the nuclear ellipsoid established by axial expansion.T hE control of developmental events is often assumed to be based on regulated gene expression. In many organisms, however, the earliest events of embryogenesis proceed without transcription, and in some cases without translation (Gardet al., 1990;Sluder et al., 1986Sluder et al., , 1990). In the syncytial embryos of insects nuclei move in precise temporal and spatial patterns that are coordinated with mitotic divisions and are consistent from embryo to embryo within a species (Strasburger, 1934;Maul, 1967;Zalokar and Erk, 1976; Foe and Alberts, 1983). In Drosophila, these nuclear movements are transcription-independent processes (Edgar and Schubiger, 1986). Because these early nuclear movements are essential to normal blastoderm formation but are poorly understood, they are the subject of this investigation.In Drosophila, two temporally distinct nuclear movements, which we refer to as axial expansion and cortical migration, produce a syncytial blastoderm with a uniform monolayer of surface nuclei (see Fig. 1). Axial expansion occurs during division cycles 4 through 6, and transforms an initially spherical mass of nuclei into an ellipsoid evenly underlying the cortex (Scriba, 1964;Zalokar and Erk, 1976). Cortical migration occurs during telophase of cycles 8 and 9. During these stepwise movements the future somatic nuclei migrate from their positions in the ellipsoid toward the periphery, reaching the cortex synchronously at interphase of cycle 10. The future pole cell nuclei migrate slightly ahead of the main body of somatic nuclei (see Fig. 1 c), and arrive at the posterior ...

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. We analyzed the expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate our methods for expression profiling as a way to analyze developmental programs, and show that modifications to chromatin structure are key to changes in cell fate. Our findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells.

Cells employ a diverse array of signaling mechanisms to establish spatial patterns during development. Nowhere is this better understood than in Drosophila, where the limbs and eyes arise from discrete epithelial sacs called imaginal discs. Molecular-genetic analyses of pattern formation have generally treated discs as single epithelial sheets. Anatomically, however, discs comprise a columnar cell monolayer covered by a squamous epithelium known as the peripodial membrane. Here we demonstrate that during development, peripodial cells signal to disc columnar cells via microtubule-based apical extensions. Ablation and targeted gene misexpression experiments demonstrate that peripodial cell signaling contributes to growth control and pattern formation in the eye and wing primordia. These findings challenge the traditional view of discs as monolayers and provide foundational evidence for peripodial cell function in Drosophila appendage development.

Drosophila imaginal discs are sac-like appendage primordia comprising apposed peripodial and columnar cell layers. Cell survival in disc columnar epithelia requires the secreted signal Decapentaplegic (DPP), which also acts as a gradient morphogen during pattern formation. The distribution mechanism by which secreted DPP mediates global cell survival and graded patterning is poorly understood. Here we report detection of DPP in the lumenal cavity between apposed peripodial and columnar cell layers of both wing and eye discs. We show that peripodial cell survival hinges upon DPP signal reception and implicate DPP-dependent viability of the peripodial epithelium in growth of the entire disc. These results are consistent with lumenal transmission of the DPP survival signal during imaginal disc development.

Abstract. We show here using time-lapse video tapes that cytoplasmic streaming causes nuclear migration along the anterior-posterior axis (axial expansion) in the early syncytial embryo of Drosophila melanogaster. Using confocal microscopy and labeled phalloidin we explore the distribution of F,actin during axial expansion. We find that a network of F-actin fibers fills the cytoplasm in the embryo. This actin network partially disassembles around the nuclei during axial expansion. Our observations of normal development, fixed embryos, and drug injection experiments indicate that disassembly of the actin network generates cytoplasmic movements. We suggest that the cell cycle regulates disassembly of the actin network, and that this process may be mediated directly or indirectly by the microtubules. The cytoplasmic movements we observe during axial expansion are very similar to fountain streaming in the pseudopod of amoebae, and by analogy with the pseudopod we propose a working hypothesis for axial expansion based on solationcontraction coupling within the actin network.p ROGRESS in understanding the role of the actin cytoskeleton in cell crawling and cell shape change includes the identification of many proteins that regulate the state of actin (for review see Stossel, 1993) and the development of several models of actin-dependent cell shape changes including cytokinesis (White and Borisy, 1983) and amoeboid motion (Allen, 1973;Odell, 1977;Taylor and Fechheimer, 1982). All these models make use of the contractile and dynamic nature of actin/myosin networks. Despite these advances, it remains largely unknown what stimuli initiate reorganization of the actin cytoskeleton or how reorganization is transmitted to organelles and the plasma membrane.Many of the concepts regarding the function of actin/myosin networks in nonmuscle cells originate with studies of amoeboid motion. Allen (1961) defined amoeboid motion as any cellular movement caused by active cytoplasmic streaming, and outlined the importance of cytoplasmic streaming to cell motility and to morphogenesis of nonmotile cells such as blastomeres of animal embryos. Experimental study of cytoplasmic streaming during pseudopod extension in giant amoebae such as Chaos carolinensis indicates that the presence of contractile elements and the transition between "gel" and "sol" states of the cytoplasm are essential for explaining amoeboid motion (Allen, 1961). It has been possible to directly integrate observations on the living pseudopod with the biochemical and physical properties of actin, myosin,