Scalloped (Sd) and Vestigial (Vg) are each needed for Drosophila wing development. We show that Sd is required for Vg function and that altering their relative cellular levels inhibits wing formation. In vitro, Vg binds directly to both Sd and its human homolog, Transcription Enhancer Factor-1. The interaction domains map to a small region of Vg that is essential for Vg-mediated gene activation and to the carboxy-terminal half of Sd. Our observations indicate that Vg and Sd function coordinately to control the expression of genes required for wing development, which implies that Vg is a tissuespecific transcriptional intermediary factor of Sd. Received August 24, 1998; revised version accepted November 3, 1998. The Drosophila vestigial (vg) and scalloped (sd) genes are expressed in similar patterns during wing development, and mutations in either gene lead to loss of wing tissue (Campbell et al. 1991(Campbell et al. , 1992Williams et al. 1991Williams et al. , 1993. Vg is a developmentally regulated nuclear protein of previously unknown function and is required principally for the development of the wing and haltere (Williams et al. 1991). Sd is part of a highly conserved family of transcription factors, the TEA/ATTS domain proteins and is an essential protein with a wider developmental role (Campbell et al. 1991(Campbell et al. , 1992.Expression of vg in cells of the developing wing primordia is established by a number of conserved signaling pathways and is required for subsequent cell proliferation and patterning. Expression of wingless (wg), as well as interactions between dorsal and ventral cells that activate the Notch receptor, initially directs limited vg expression along the dorsal-ventral (D/V) wing boundary (Williams et al. 1994;Kim et al. 1995Kim et al. , 1996; for review, see Irvine and Vogt 1997). Subsequent vg expression in the wing primordia occurs in response to both the D/V Wg signal and Decapentaplegic (Dpp), a member of the Transforming Growth Factor-␤ (TGF-␤) protein family, secreted by cells along the anterior-posterior (A/P) border. (Blair 1994;Kim et al. 1996Kim et al. , 1997Zecca et al. 1996;Neumann and Cohen 1997). By the late third larval instar, maximal amounts of Vg are seen in cells at the D/V wing disc boundary, whereas cells located farther from this border produce progressively less Vg (Williams et al. 1991). vg is also required to maintain sd expression in the wing progenitor cells, and sd is similarly required for the maintenance of elevated vg expression (Williams et al. 1993).A cellular role for Sd can be inferred from studies of its human homolog Transcription Enhancer Factor-1 (TEF-1). TEF-1 binds to SV40 enhancer sequences via a TEA/ ATTS class DNA-binding domain, and has been shown to require transcriptional intermediary factors (TIFs) for proper function (Xiao et al. 1991;Ishiji et al. 1992;Hwang et al. 1993;Gupta et al. 1997). Interestingly, it has been reported that the Sd TEA/ATTS domain does not bind the same enhancer DNA sequences in vitro as TEF-1, although TEF-1...

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The smoking of tobacco continues to be the leading cause of premature death worldwide and is linked to the development of a number of serious illnesses including heart disease, respiratory diseases, stroke and cancer. Currently, cell line based toxicity assays are typically used to gain information on the general toxicity of cigarettes and other tobacco products. However, they provide little information regarding the complex disease-related changes that have been linked to smoking. The ethical concerns and high cost associated with mammalian studies have limited their widespread use for in vivo toxicological studies of tobacco. The zebrafish has emerged as a low-cost, high-throughput, in vivo model in the study of toxicology. In this study, smoke condensates from 2 reference cigarettes and 6 Canadian brands of cigarettes with different design features were assessed for acute, developmental, cardiac, and behavioural toxicity (neurotoxicity) in zebrafish larvae. By making use of this multifaceted approach we have developed an in vivo model with which to compare the toxicity profiles of smoke condensates from cigarettes with different design features. This model system may provide insights into the development of smoking related disease and could provide a cost-effective, high-throughput platform for the future evaluation of tobacco products.

The Drosophila melanogaster genes midline and H15 encode predicted T-box transcription factors homologous to vertebrate Tbx20 genes. All identified vertebrate Tbx20 genes are expressed in the embryonic heart and we find that both midline and H15 are expressed in the cardioblasts of the dorsal vessel, the insect organ equivalent to the vertebrate heart. The midline mRNA is first detected in dorsal mesoderm at embryonic stage 12 in the two progenitors per hemisegment that will divide to give rise to all six cardioblasts. Expression of H15 mRNA in the dorsal mesoderm is detected first in four to six cells per hemisegment at stage 13. The expression of midline and H15 in the dorsal vessel is dependent on Wingless signaling and the transcription factors tinman and pannier. We find that the selection of two midline-expressing cells from a pool of competent progenitors is dependent on Notch signaling. Embryos deleted for both midline and H15 have defects in the alignment of the cardioblasts and associated pericardial cells. Embryos null for midline have weaker and less penetrant phenotypes while embryos deficient for H15 have morphologically normal hearts, suggesting that the two genes are partially redundant in heart development. Despite the dorsal vessel defects, embryos mutant for both midline and H15 have normal numbers of cardioblasts, suggesting that cardiac cell fate specification is not disrupted. However, ectopic expression of midline in the dorsal mesoderm can lead to dramatic increases in the expression of cardiac markers, suggesting that midline and H15 participate in cardiac fate specification and may normally act redundantly with other cardiogenic factors. Conservation of Tbx20 expression and function in cardiac development lends further support for a common ancestral origin of the insect dorsal vessel and the vertebrate heart.

SUMMARYThe emergence of multidrug-resistant cancers and the lack of targeted therapies for many cancers underscore an unmet need for new therapeutics with novel modes of action towards cancer cells. Host-defense peptides often exhibit selective cytotoxicity towards cancer cells and show potential as anti-cancer therapeutics. Here, we screen 26 naturally occurring variants of the peptide pleurocidin for cytotoxic and anti-cancer activities, and investigate the underlying mechanism of action. Cytotoxicities were assessed in vitro using cell-based assays and in vivo using zebrafish embryos. Morphological changes were assessed by both transmission and scanning electron microscopy, and functional assays were performed on zebrafish embryos to investigate the mechanism of cell death. A total of 14 peptides were virtually inactive against HL60 human leukemia cells, whereas 12 caused >50% death at ≤32 μg/ml. Morphological changes characteristic of oncosis were evident by electron microscopy after only 1 minute of treatment with 32 μg/ml of variant NRC-03. Only two peptides were hemolytic. Four peptides showed no toxicity towards zebrafish embryos at the highest concentration tested (25 μM; ∼64 μg/ml) and one peptide was highly toxic, killing 4-hour-post-fertilization (hpf) embryos immediately after exposure to 1 μM peptide. Four other peptides killed embryos after 24 hours of exposure at 1 μM. Most peptides caused mortality at one or more developmental stages only after continuous exposure (24 hours) with higher lethal doses (≥5 μM). Pleurocidin NRC-03 bound to embryos and induced the release of superoxide, caused an increase in the number of TUNEL-positive nuclei, and caused membrane damage and the loss of embryonic epithelial integrity, marked by the exclusion of cells from the outer epithelium and the appearance of F-actin within the circumferential cells of the repair site. Our results indicate that specific pleurocidin variants are attractive cancer-selective agents that selectively induce cell death in target cells but leave non-target cells such as erythrocytes and non-transformed cells unaffected.