vps25 mosaics display non-autonomous cell survival and overgrowth, and autonomous apoptosis

Appropriate cell-cell signaling is crucial for proper tissue homeostasis. Protein sorting of cell surface receptors at the early endosome is important for both the delivery of the signal and the inactivation of the receptor, and its alteration can cause malignancies including cancer. In a genetic screen for suppressors of the pro-apoptotic gene hid in Drosophila, we identified two alleles of vps25, a component of the ESCRT machinery required for protein sorting at the early endosome. Paradoxically, although vps25 mosaics were identified as suppressors of hid-induced apoptosis, vps25 mutant cells die. However, we provide evidence that a non-autonomous increase of Diap1 protein levels, an inhibitor of apoptosis, accounts for the suppression of hid. Furthermore, before they die, vps25 mutant clones trigger non-autonomous proliferation through a failure to downregulate Notch signaling, which activates the mitogenic JAK/STAT pathway. Hid and JNK contribute to apoptosis of vps25 mutant cells. Inhibition of cell death in vps25 clones causes dramatic overgrowth phenotypes. In addition, Hippo signaling is increased in vps25 clones, and hippo mutants block apoptosis in vps25 clones. In summary, the phenotypic analysis of vps25 mutants highlights the importance of receptor downregulation by endosomal protein sorting for appropriate tissue homeostasis, and may serve as a model for human cancer.     

The CARD-carrying caspase Dronc is essential for most, but not all, developmental cell death in Drosophila

The initiator caspase Dronc is the only Drosophila caspase that contains a caspase activation and recruitment domain (CARD). Although Dronc has been implicated as an important effector of apoptosis, the genetic function of dronc in normal development is unclear because dronc mutants have not been available. In an EMS mutagenesis screen, we isolated four point mutations in dronc that recessively suppress the eye ablation phenotype caused by eye-specific overexpression of hid. Homozygous mutant dronc animals die during pupal stages; however, at a low frequency we obtained homozygous adult escapers. These escapers have additional cells in the eye and wings that are less transparent and slightly curved down. We determined that this is due to lack of apoptosis. Our analyses of dronc mutant embryos suggest that dronc is essential for most apoptotic cell death during Drosophila development, but they also imply the existence of a dronc-independent cell death pathway. We also constructed double mutant flies for dronc and the apoptosis inhibitor diap1. dronc mutants can rescue the ovarian degeneration phenotype caused by diap1 mutations, confirming that dronc acts genetically downstream of diap1.     

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins ^1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references ^{1-6,7 ,8}).     

A pathway of signals regulating effector and initiator caspases in the developing Drosophila eye

Regulated cell death and survival play important roles in neural development. Extracellular signals are presumed to regulate seven apparent caspases to determine the final structure of the nervous system. In the eye, the EGF receptor, Notch, and intact primary pigment and cone cells have been implicated in survival or death signals. An antibody raised against a peptide from human caspase 3 was used to investigate how extracellular signals controlled spatial patterning of cell death. The antibody crossreacted specifically with dying Drosophila cells and labelled the activated effector caspase Drice. It was found that the initiator caspase Dronc and the proapoptotic gene head involution defective were important for activation in vivo. Dronc may play roles in dying cells in addition to activating downstream effector caspases. Epistasis experiments ordered EGF receptor, Notch, and primary pigment and cone cells into a single pathway that affected caspase activity in pupal retina through hid and Inhibitor of Apoptosis Proteins. None of these extracellular signals appeared to act by initiating caspase activation independently of hid. Taken together, these findings indicate that in eye development spatial regulation of cell death and survival is integrated through a single intracellular pathway.     

A screen for mutations that suppress the phenotype of Drosophila armadillo, the beta-catenin homolog

During development signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed cell death. The well-conserved Wingless/Wnt pathway is required for many developmental decisions in all animals. One transducer of the Wingless/Wnt signal is Armadillo/beta-catenin. Drosophila Armadillo not only transduces Wingless signal, but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the Wingless/Wnt signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown components influence signaling and junctions. We carried out a genetic modifier screen to identify some of these components by screening for mutations that can suppress the armadillo mutant phenotype. We identified 12 regions of the genome that have this property. From these regions and from additional candidate genes tested we identified four genes that suppress arm: dTCF, puckered, head involution defective (hid), and Dpresenilin. We further investigated the interaction with hid, a known regulator of programmed cell death. Our data suggest that Wg signaling modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion.     

Drosophila sickle is a novel grim-reaper cell death activator

The Drosophila genes reaper, head involution defective (hid), and grim all reside at 75C on chromosome three and encode related proteins that have crucial functions in programmed cell death (reviewed in ). In this report, we describe a novel grim-reaper gene, termed sickle, that resides adjacent to reaper. The sickle gene, like reaper and grim, encodes a small protein which contains an RHG motif and a Trp-block. In wild-type embryos, sickle expression was detected in cells of the developing central nervous system. Unlike reaper, hid, and grim, the sickle gene is not removed by Df(3L)H99, and strong ectopic sickle expression was detected in the nervous system of this cell death mutant. sickle very effectively induced cell death in cultured Spodoptera Sf-9 cells, and this death was antagonized by the caspase inhibitors p35 or DIAP1. Strikingly, unlike the other grim-reaper genes, targeted sickle expression did not induce cell death in the Drosophila eye. However, sickle strongly enhanced the eye cell death induced by expression of either an r/grim chimera or reaper.     

Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila

Oogenesis in Drosophila involves specification of both germ cells and the surrounding somatic follicle cells, as well as the determination of oocyte polarity. We found that two neurogenic genes, Notch and Delta, are required in oogenesis. These genes encode membrane proteins with epidermal growth factor repeats and are essential in the decision of an embryonic ectodermal cell to take on the fate of neuroblast or epidermoblast. In oogenesis, mutation in either gene leads to an excess of posterior follicle cells, a cell fate change reminiscent of the hyperplasia of neuroblasts seen in neurogenic mutant embryos. Furthermore, the Notch mutation in somatic cells causes mislocalization of bicoid in the oocyte. These results suggest that the neurogenic genes Notch and Delta are involved in both follicle cell development and the establishment of anterior-posterior polarity in the oocyte.     

Bicoid Signal Extraction with a Selection of Parametric and Nonparametric Signal Processing Techniques

The maternal segmentation coordinate gene bicoid plays a significant role during Drosophila embryogenesis. The gradient of Bicoid, the protein encoded by this gene, determines most aspects of head and thorax development. This paper seeks to explore the applicability of a variety of signal processing techniques at extracting bicoid expression signal, and whether these methods can outperform the current model. We evaluate the use of six different powerful and widely-used models representing both parametric and nonparametric signal processing techniques to determine the most efficient method for signal extraction in bicoid. The results are evaluated using both real and simulated data. Our findings show that the Singular Spectrum Analysis technique proposed in this paper outperforms the synthesis diffusion degradation model for filtering the noisy protein profile of bicoid whilst the exponential smoothing technique was found to be the next best alternative followed by the autoregressive integrated moving average.     

Programmed cell death mechanisms of identifiable peptidergic neurons in Drosophila melanogaster

The molecular basis of programmed cell death (PCD) of neurons during early metamorphic development of the central nervous system (CNS) in Drosophila melanogaster are largely unknown, in part owing to the lack of appropriate model systems. Here, we provide evidence showing that a group of neurons (vCrz) that express neuropeptide Corazonin (Crz) gene in the ventral nerve cord of the larval CNS undergo programmed death within 6 hours of the onset of metamorphosis. The death was prevented by targeted expression of caspase inhibitor p35, suggesting that these larval neurons are eliminated via a caspase-dependent pathway. Genetic and transgenic disruptions of ecdysone signal transduction involving ecdysone receptor-B (EcR-B) isoforms suppressed vCrz death, whereas transgenic re-introduction of either EcR-B1 or EcR-B2 isoform into the EcR-B-null mutant resumed normal death. Expression of reaper in vCrz neurons and suppression of vCrz-cell death in a reaper-null mutant suggest that reaper functions are required for the death, while no apparent role was found for hid or grim as a death promoter. Our data further suggest that diap1 does not play a role as a central regulator of the PCD of vCrz neurons. Significant delay of vCrz-cell death was observed in mutants that lack dronc or dark functions, indicating that formation of an apoptosome is necessary, but not sufficient, for timely execution of the death. These results suggest that activated ecdysone signaling determines precise developmental timing of the neuronal degeneration during early metamorphosis, and that subsequent reaper-mediated caspase activation occurs through a novel DIAP1-independent pathway.     

Miranda couples oskar mRNA/Staufen complexes to the bicoid mRNA localization pathway

The double-stranded RNA binding protein Staufen is required for the microtubule-dependent localization of bicoid and oskar mRNAs to opposite poles of the Drosophila oocyte and also mediates the actin-dependent localization of prospero mRNA during the asymmetric neuroblast divisions. The posterior localization of oskar mRNA requires Staufen RNA binding domain 2, whereas prospero mRNA localization mediated the binding of Miranda to RNA binding domain 5, suggesting that different Staufen domains couple mRNAs to distinct localization pathways. Here, we show that the expression of Miranda during mid-oogenesis targets Staufen/oskar mRNA complexes to the anterior of the oocyte, resulting in bicaudal embryos that develop an abdomen and pole cells instead of the head and thorax. Anterior Miranda localization requires microtubules, rather than actin, and depends on the function of Exuperantia and Swallow, indicating that Miranda links Staufen/oskar mRNA complexes to the bicoid mRNA localization pathway. Since Miranda is expressed in late oocytes and bicoid mRNA localization requires the Miranda-binding domain of Staufen, Miranda may play a redundant role in the final step of bicoid mRNA localization. Our results demonstrate that different Staufen-interacting proteins couple Staufen/mRNA complexes to distinct localization pathways and reveal that Miranda mediates both actin- and microtubule-dependent mRNA localization.     

The choice of cell fate in the epidermis of Drosophila

In Drosophila, neural precursors are formed in a spaced pattern separated by intervening epidermal cells. Segregation of neural and epidermal lineages relies on cellular interactions. Failure of this cell communication, as in the mutants Notch (N), Delta, and shaggy, results in most or all of the cells becoming neural. Cells mutant for N and shaggy, but not Delta, autonomously adopt the neural fate when adjacent to wild-type cells in mosaics. Furthermore, wild-type cells adopt the epidermal fate if adjacent cells express a lower level of N activity than themselves, but produce neural precursors if adjacent cells express a higher level of N activity. This shows that there is competition between the cells and that the N protein is required for the mechanism whereby the cells choose between alternative fates. It also suggests that N acts as a receptor for an inhibitory signal emanating from the neural precursors.     

numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos

Neurons and support cells of each sensory organ in Drosophila embryos are most likely derived from a single precursor cell. This cell lineage is affected in numb mutants. Morphological alterations of sensory structures, as well as changes in the number of cells expressing cell type-specific markers, indicate that sensory neurons in numb mutant embryos are transformed into lineage-related nonneuronal support cells. Thus the numb gene controls the fate of progeny derived from sensory organ precursors. The numb gene has been isolated by the plasmid rescue method. The structure of its predicted product is discussed.     

Vacuolar H<Superscript>+</Superscript>-translocating inorganic pyrophosphatase (Vpp1) marks partial aleurone cell fate in cereal endosperm development

Cereal endosperm is a model system for cell fate determination in plants. In wild-type plants the outermost endosperm cells adopt aleurone cell fate, while all underlying cells display starchy endosperm cell fate. Mutant analysis showed that cell fate is determined by position rather than lineage. To further characterise the precise cell fate of the outermost cells, we performed a differential screen and isolated the novel marker gene Vpp1. It encodes a vacuolar H⁺-translocating inorganic pyrophosphatase (V-PPase) and is mainly expressed in kernels, leaves and tassels. In kernels, its expression is restricted to the aleurone layer with the maximum of expression shifting from the adaxial to the abaxial side during early stages. Together with three other marker genes Vpp1 was then used to analyse the cell fate of the outermost cells in Dap3, Dap7, cr4 and dek1 mutants, all of which have aberrant aleurone layers. In the Dap3 and Dap7 mutants the Vpp1 and Ltp2 markers but not the A1 and Zein markers were expressed in patches without aleurone indicating that the outermost cells had some but not all features of aleurone cells and did not simply adopt starchy endosperm cell fate. A similar result was obtained in the cr4 mutant, although Ltp2 expression was less generalised. In other Dap7 patches characterised by multiple aleurone-like cell layers the expression of Vpp1 and Ltp2 confirmed the aleurone cell fate of the cells in the additional cell layers. The analysis of dek1 mutants confirmed the starchy endosperm cell fate of the majority but not all outermost cells. Based on these data we propose a model suggesting a stepwise commitment to aleurone cell fate. Sequential steps are marked by the expression of Vpp1, the expression of Ltp2, the acquisition of a regular shape and thick walls and finally pigmentation coupled with A1 expression.