Behaviour of microtubules and actin filaments in living Drosophila embryos

We describe the preparation of novel fluorescent derivatives of rabbit muscle actin and bovine tubulin, and the use of these derivatives to study the behaviour of actin filaments and microtubules in living Drosophila embryos, in which the nuclei divide at intervals of 8 to 21 min. The fluorescently labelled proteins appear to function normally in vitro and in vivo, and they allow continuous observation of the cytoskeleton in living embryos without perturbing development. By coinjecting labelled actin and tubulin into the early syncytial embryo, the spatial relationships between the distinct filament networks that they form can be followed second by second. The dynamic rearrangements of actin filaments and microtubules observed confirms and extends results obtained from previous studies, in which fixation techniques and specific staining were used to visualize the cytoskeleton in the Drosophila embryo. However, no tested fixation method produces an exact representation of the in vivo microtubule distribution.     

Notch signaling targets the Wingless responsiveness of a Ubx visceral mesoderm enhancer in Drosophila

BACKGROUND: Members of the Notch family of receptors mediate a process known as lateral inhibition that plays a prominent role in the suppression of cell fates during development. This function is triggered by a ligand, Delta, and is implemented by the release of the intracellular domain of Notch from the membrane and by its interaction with the protein Suppressor of Hairless [Su(H)] in the nucleus. There is evidence that Notch can also signal independently of Su(H). In particular, in Drosophila, there is evidence that a Su(H)-independent activity of Notch is associated with Wingless signaling. RESULTS: We report that Ubx(VM)B, a visceral mesoderm-specific enhancer of the Ubx gene of Drosophila, is sensitive to Notch signaling. In the absence of Notch, but not of Su(H), the enhancer becomes activated earlier and over a wider domain than in the wild type. Furthermore, the removal of Notch reduces the requirement for Disheveled-mediated Wingless signaling to activate this enhancer. This response to Notch is likely to be mediated by the dTcf binding sites in the Ubx(VM)B enhancer. CONCLUSIONS: Our results show that, in Drosophila, an activity of Notch that is likely to be independent of Su(H) inhibits Wingless signaling on Ubx(VM)B. A possible target of this activity is dTcf. As dTcf has been shown to be capable of repressing Wingless targets, our results suggest that this repressive activity may be regulated by Notch. Finally, we suggest that Wingless signaling is composed of two steps, a down-regulation of a Su(H)-independent Notch activity that modulates the activity of dTcf and a canonical Wingless signaling event that regulates the activity of Armadillo and its interaction with dTcf.     

Complex organization of promoter and enhancer elements regulate the tissue- and developmental stage-specific expression of the Drosophila melanogaster Gld gene

The Drosophila melanogaster Gld gene has multiple and diverse developmental and physiological functions. We report herein that interactions among proximal promoter elements and a cluster of intronically located enhancers and silencers specify the complex regulation of Gld that underlies its diverse functions. Gld expression in nonreproductive tissues is largely determined by proximal promoter elements with the exception of the embryonic labium where Gld is activated by an enhancer within the first intron. A nuclear protein, GPAL, has been identified that binds the Gpal elements in the proximal promoter region. Regulation of Gld in the reproductive organs is particularly complex, involving interactions among the Gpal proximal promoter elements, a unique TATA box, three distinct enhancer types, and one or more silencer elements. The three somatic reproductive organ enhancers each activate expression in male and female pairs of reproductive organs. One of these pairs, the male ejaculatory duct and female oviduct, are known to be developmentally homologous. We report evidence that the other two pairs of organs are developmentally homologous as well. A comprehensive model to explain the full developmental regulation of Gld and its evolution is presented.     

Epigenetic blocking of an enhancer region controls irradiation-induced proapoptotic gene expression in Drosophila embryos

Drosophila embryos are highly sensitive to gamma-ray-induced apoptosis at early but not later, more differentiated stages during development. Two proapoptotic genes, reaper and hid, are upregulated rapidly following irradiation. However, in post-stage-12 embryos, in which most cells have begun differentiation, neither proapoptotic gene can be induced by high doses of irradiation. Our study indicates that the sensitive-to-resistant transition is due to epigenetic blocking of the irradiation-responsive enhancer region (IRER), which is located upstream of reaper but is also required for the induction of hid in response to irradiation. This IRER, but not the transcribed regions of reaper/hid, becomes enriched for trimethylated H3K27/H3K9 and forms a heterochromatin-like structure during the sensitive-to-resistant transition. The functions of histone-modifying enzymes Hdac1(rpd3) and Su(var)3-9 and PcG proteins Su(z)12 and Polycomb are required for this process. Thus, direct epigenetic regulation of two proapoptotic genes controls cellular sensitivity to cytotoxic stimuli.     

Cooperative interactions between the GRP78 enhancer and promoter elements in hamster fibroblasts

A non-tissue-specific enhancer derived from the promoter of the rat 78-kDa glucose-regulated protein (GRP78)-coding gene was tested for its ability to stimulate the activity of its homologous promoter and two heterologous promoters (simian virus 40 and mouse mammary tumor virus). Single and double copies of the enhancer were inserted at positions 5' and 3' of the cat-expression vectors under the direction of the above promoters. The recombinant plasmids were transfected into hamster fibroblast K12 cells and assayed for chloramphenicol acetyl transferase activity under induced and non-induced conditions. We report that the GRP78 enhancer (i) exhibits strong cooperative interactions with its homologous promoter; (ii) can activate and confer a calcium ionophore (A23187) inducibility to heterologous promoters in an orientation-independent manner; (iii) prefers the 5' over the 3' location and; (iv) is dosage dependent in that two copies are twice as active as a single unit.     

Drosophila gastrulation: analysis of cell shape changes in living embryos by three-dimensional fluorescence microscopy.

The first event of Drosophila gastrulation is the formation of the ventral furrow. This process, which leads to the invagination of the mesoderm, is a classical example of epithelial folding. To understand better the cellular changes and dynamics of furrow formation, we examined living Drosophila embryos using three-dimensional time-lapse microscopy. By injecting fluorescent markers that visualize cell outlines and nuclei, we monitored changes in cell shapes and nuclear positions. We find that the ventral furrow invaginates in two phases. During the first 'preparatory' phase, many prospective furrow cells in apparently random positions gradually begin to change shape, but the curvature of the epithelium hardly changes. In the second phase, when a critical number of cells have begun to change shape, the furrow suddenly invaginates. Our results suggest that furrow formation does not result from an ordered wave of cell shape changes, contrary to a model for epithelial invagination in which a wave of apical contractions causes invagination. Instead, it appears that cells change their shape independently, in a stochastic manner, and the sum of these individual changes alters the curvature of the whole epithelium.     

Drosophila Adh: a promoter element expands the tissue specificity of an enhancer

Drosophila alcohol dehydrogenase (Adh) genes are expressed in the fat body and in species-specific sets of other tissues during larval and adult development. The Drosophila mulleri Adh-1 gene is expressed in the larval fat body and in three other larval tissues. In this paper, we show that Adh-1 expression in multiple cell types is the result of synergistic interactions between fat body-specific enhancers and a specific Adh-1 promoter element.