Leonie M. Quinn

DECAY, a Novel Drosophila Caspase Related to Mammalian Caspase-3 and Caspase-7

Caspases are key effectors of programmed cell death in metazoans. In Drosophila, four caspases have been described so far. Here we describe the identification and characterization of the fifth Drosophila caspase, DECAY. DECAY shares a high degree of homology with the members of the mammalian caspase-3 subfamily, particularly caspase-3 and caspase-7. DECAY lacks a long prodomain and thus appears to be a class II effector caspase. Ectopic expression of DECAY in cultured cells induces apoptosis. Recombinant DECAY exhibited substrate specificity similar to the mammalian caspase-3 subfamily. Low levels of decaymRNA are ubiquitously expressed in Drosophila embryos during early stages of development but its expression becomes somewhat spatially restricted in some tissues. During oogenesisdecay mRNA was detected in egg chambers of all stages consistent with a role for DECAY in apoptosis of nurse cells. Relatively high levels of decay mRNA are expressed in larval salivary glands and midgut, two tissues which undergo histolysis during larval/pupal metamorphosis, suggesting that DECAY may play a role in developmentally programmed cell death inDrosophila.

Buffy, a Drosophila Bcl‐2 protein, has anti‐apoptotic and cell cycle inhibitory functions

Bcl‐2 family proteins are key regulators of apoptosis. Both pro‐apoptotic and anti‐apoptotic members of this family are found in mammalian cells, but only the pro‐apoptotic protein Debcl has been characterized in Drosophila. Here we report that Buffy, the second Drosophila Bcl‐2‐like protein, is a pro‐survival protein. Ablation of Buffy by RNA interference leads to ectopic apoptosis, whereas overexpression of buffy results in the inhibition of developmental programmed cell death and γ irradiation‐induced apoptosis. Buffy interacts genetically and physically with Debcl to suppress Debcl‐induced cell death. Genetic interactions suggest that Buffy acts downstream of Rpr, Grim and Hid, and upstream of the apical caspase Dronc. Furthermore, overexpression of buffy inhibits ectopic cell death in diap1 (th5) mutants. Taken together these data suggest that Buffy can act downstream of Rpr, Grim and Hid to block caspase‐dependent cell death. Overexpression of Buffy in the embryo results in inhibition of the cell cycle, consistent with a G1/early‐S phase arrest. Our data suggest that Buffy is functionally similar to the mammalian pro‐survival Bcl‐2 family of proteins.

Isolation and identification of homeobox genes from the human placenta including a novel member of the Distal-less family, DLX4

We have carried out a DNA binding site screen of a 32-week human placental cDNA library using a consensus homeodomain binding site as a probe. This study represents the first library screen carried out to isolate homeobox genes from the human placenta. We have shown that three homeobox genes known to be expressed in the embryo, HB24, GAX and MSX2 are also expressed in the placenta. We have also identified a novel homeobox gene, DLX4, that shows 85% sequence identity with the homeodomain encoded by the Drosophila Distal-less (Dll) gene. DLX4 therefore represents a new member of the Distal-less family of homeobox genes. This is the first evidence that members of the Distal-less family of homeobox genes are expressed in the placenta. Using fluorescence in situ hybridisation (FISH), DLX4 has been assigned to human chromosome 17q21-q22. This places DLX4 in the same region of chromosome 17 as another member of the Distal-less family, DLX3 (Scherer et al., 1995), and the HOX-B homeobox gene cluster (Acampora et al., 1989: Boncinelli et al., 1991). Members of the Distal-less family (DLX1 and DLX2; DLX5 and DLX6) are found as closely linked pairs on human chromosomes (Simeone et al., 1994). We predict that DLX3 and DLX4 are closely linked and have arisen through gene duplication and divergence from a common ancestral precursor.

STRICA, a novel Drosophila melanogaster caspase with an unusual serine/threonine-rich prodomain, interacts with DIAP1 and DIAP2.

The recently published genome sequence of Drosophila melanogaster predicts seven caspases in the fly. Five of these caspases have been previously characterised. Here, we describe the Drosophila caspase, STRICA. STRICA is a caspase with a long amino-terminal prodomain that lacks any caspase recruitment domain or death effector domain. Instead, the prodomain of STRICA consists of unique serine/threonine stretches. Low levels of strica expression were detected in embryos, larvae, pupae and adult animals. STRICA is a cytoplasmic protein that, upon overexpression, caused apoptosis in cultured Drosophila SL2 cells that was partially suppressed by DIAP1. Interestingly, unlike other fly caspases, STRICA showed physical association with DIAP2, in cotransfection experiments. These results suggest that STRICA may have a unique cellular function. Cell Death and Differentiation (2001) 8, 387–394

Characterization of the Drosophila caspase, DAMM.

Caspases are main effectors of apoptosis in metazoans. Genome analysis indicates that there are seven caspases in Drosophila, six of which have been previously characterized. Here we describe the cloning and characterization of the last Drosophila caspase, DAMM. Similar to mammalian effector caspases, DAMM lacks a long prodomain. We show that the DAMM precursor, along with the caspases DRONC and DECAY, is partially processed in cells undergoing apoptosis. Recombinant DAMM produced inEscherichia coli shows significant catalytic activity on a pentapeptide caspase substrate. Low levels of damm mRNA are ubiquitously expressed in Drosophila embryos during early stages of development. Relatively high levels ofdamm mRNA are detected in larval salivary glands and midgut, and in adult egg chambers. Ectopic expression of DAMM in cultured cells induces apoptosis, and similarly, transgenic overexpression of DAMM, but not of a catalytically inactive DAMM mutant, in Drosophila results in a rough eye phenotype. We demonstrate that expression of the catalytically inactive DAMM mutant protein significantly suppresses the rough eye phenotype due to the overexpression of HID, suggesting that DAMM may be required in a hid-mediated cell death pathway.

A distal-less class homeobox gene, DLX4, is a candidate for regulating epithelial-mesenchymal cell interactions in the human placenta

Homeobox genes of the Distal-less (Dlx) family are expressed in the vertebrate embryo in regions where epithelial cell layers contact adjacent mesenchymal cells. This study shows that the human Dlx family member, DLX4, is expressed in the placenta, primarily in regions where epithelial and mesenchymal cell layers contact. In situ hybridization studies at first trimester human placental sections revealed that DLX4 was expressed predominantly in the cytotrophoblast stem cell layer. In term placenta, DLX4 was expressed in the syncytiotrophoblast. Northern analysis revealed two DLX4 transcripts in first trimester placenta of 2.8 and 3.0 kb. Elevated levels of DLX4 mRNA were detected in a choriocarcinoma derived cell line when compared with a cytotrophoblast cell line and normal placenta. This is the first study to show that a member of the Dlx family of homeobox genes is expressed in regions of epithelial and mesenchymal cell layer contact in the human. Accumulated evidence from several studies suggest that a combinatorial code of homeobox genes is required to regulate epithelial-mesenchymal cell interactions in the vertebrate embryo. It is predicted that a similar combination of homeobox genes, that includes DLX4, is involved in regulating epithelial-mesenchymal cell interactions in extraembryonic tissues. DLX4 may also have a role in the regulation of the genes important for trophoblast invasion since the level of expression in trophoblast cell lines reflects invasive potential.

Characterization of the DrosophilaCaspase, DAMM

Caspases are main effectors of apoptosis in metazoans. Genome analysis indicates that there are seven caspases in Drosophila, six of which have been previously characterized. Here we describe the cloning and characterization of the last Drosophila caspase, DAMM. Similar to mammalian effector caspases, DAMM lacks a long prodomain. We show that the DAMM precursor, along with the caspases DRONC and DECAY, is partially processed in cells undergoing apoptosis. Recombinant DAMM produced inEscherichia coli shows significant catalytic activity on a pentapeptide caspase substrate. Low levels of damm mRNA are ubiquitously expressed in Drosophila embryos during early stages of development. Relatively high levels ofdamm mRNA are detected in larval salivary glands and midgut, and in adult egg chambers. Ectopic expression of DAMM in cultured cells induces apoptosis, and similarly, transgenic overexpression of DAMM, but not of a catalytically inactive DAMM mutant, in Drosophila results in a rough eye phenotype. We demonstrate that expression of the catalytically inactive DAMM mutant protein significantly suppresses the rough eye phenotype due to the overexpression of HID, suggesting that DAMM may be required in a hid-mediated cell death pathway.

HOW Is Required for Stem Cell Maintenance in the Drosophila Testis and for the Onset of Transit-Amplifying Divisions

The mechanisms by which germline stem cells (GSCs) in the Drosophila testis undergo asymmetric division to regenerate a stem cell as well as a daughter (gonialblast) that will only undergo a further four mitotic divisions prior to entering premeiotic S phase and differentiating into a cyst of spermatocytes are not fully resolved. Here we demonstrate that the HOW RNA-binding protein is required for maintenance of CycB and therefore mitotic progression in GSCs and gonialblasts as well as determining the timing of the spermatogonial divisions. HOW is normally expressed in a complementary pattern to Bam in the germline and bam mRNA is bound by HOW in vivo. Ectopic expression of the HOW(L) isoform is associated with a delay in accumulation of Bam to the level required for differentiation, resulting in extra mitotic divisions. Spatiotemporal regulation of HOW expression is therefore required to specify the four spermatogonial transit-amplifying divisions.

Drosophila Hfp negatively regulates dmyc and stg to inhibit cell proliferation.

Mammalian FIR has dual roles in pre-mRNA splicing and in negative transcriptional control of Myc. Here we show that Half pint (Hfp), the Drosophila orthologue of FIR, inhibits cell proliferation in Drosophila. We find that Hfp overexpression potently inhibits G1/S progression, while hfp mutants display ectopic cell cycles. Hfp negatively regulates dmyc expression and function, as reducing the dose of hfp increases levels of dmyc mRNA and rescues defective oogenesis in dmyc hypomorphic flies. The G2-delay in dmyc-overexpressing cells is suppressed by halving the dosage of hfp, indicating that Hfp is also rate-limiting for G2-M progression. Consistent with this, the cycle 14 G2-arrest of stg mutant embryos is rescued by the hfp mutant. Analysis of hfp mutant clones revealed elevated levels of Stg protein, but no change in the level of stg mRNA, suggesting that hfp negatively regulates Stg via a post-transcriptional mechanism. Finally, ectopic activation of the wingless pathway, which is known to negatively regulate dmyc expression in the wing, results in an accumulation of Hfp protein. Our findings indicate that Hfp provides a critical molecular link between the developmental patterning signals induced by the wingless pathway and dMyc-regulated cell growth and proliferation.

The Ecdysone-inducible zinc-finger transcription factor Crol regulates Wg transcription and cell cycle progression in Drosophila

The steroid hormone Ecdysone is crucial for developmental cell death, proliferation and morphogenesis in Drosophila. Herein, we delineate a molecular pathway linking Ecdysone signalling to cell cycle regulation in the Drosophila developing wing. We present evidence that the Ecdysone-inducible zinc-finger transcription factor Crol provides a crucial link between the Ecdysone steroid hormone pathway and the Wingless (Wg) signalling pathway in Drosophila. We identified Crol as a strong enhancer of a wing phenotype generated by overexpression of the Wg-inducible cell cycle inhibitor Hfp. We demonstrate that Crol is required for cell cycle progression: crol mutant clones have reduced cell cycles and are removed by apoptosis, while upregulation of Crol overrides the Wg-mediated developmental cell cycle arrest in the zone of non-proliferating cells in the wing disc. Furthermore, we show that Crol acts to repress wg transcription. We also show that overexpression of crol results in downregulation of Hfp, consistent with the identification of the crol mutant as a dominant enhancer of the Hfp overexpression phenotype. Taken together, our studies have revealed a novel mechanism for cell cycle regulation, whereby Crol links steroid hormone signals to Wg signalling and the regulation of crucial cell cycle targets.