The dynamics of neurogenic signalling underlying bristle development in Drosophila melanogaster

We have examined expression of the neurogenic gene, Delta (Dl), and the regulatory relationships between the Delta-Notch signalling pathway and the proneural gene, achaete, during microchaeta development in Drosophila. Delta is expressed in all microchaeta proneural cells and microchaeta sensory organ precursors (SOPs) and is expressed dynamically in SOP progeny. We find that Delta expression in microchaeta proneural cells is detected prior to the onset of achaete expression and arises normally in the absence of achaete/scute function, indicating that initial Delta expression in the notum is not dependent on proneural gene function. Activation of the Delta-Notch pathway results in loss of Delta protein accumulation, suggesting that Delta expression is regulated, in part, by Delta-Notch signalling activity. We find that Delta signalling is required for correct delineation of early proneural gene expression in developing nota. Within microchaeta proneural stripes, we demonstrate that Delta-Notch signalling prohibits adoption of the SOP fate by repressing expression of proneural genes.     

Multiple functions of the EGF receptor in Drosophila eye development

BACKGROUND: During animal development, cells need to make spatially and temporally regulated fate decisions. These decisions are largely controlled by intercellular signalling, often through receptor tyrosine kinases. One of these, the epidermal growth factor receptor (EGFR), regulates multiple cell fate decisions. Its importance in the recruitment of photoreceptors in the developing fly eye, a useful model for neural development, has already been reported. Other EGFR functions in the eye have not been characterised. RESULTS: We have examined the consequences of removing or activating the EGFR at different stages of eye development. The earliest stages of assembly occurred normally within EGFR- clones--the morphogenetic furrow was unimpeded and the R8 photoreceptor was specified. All subsequent photoreceptor recruitment was blocked. EGFR- clones had a characteristic shape indicating that they had undergone substantial cell death posterior to the furrow, where the differentiation program is normally activated; consistent with this, excess apoptosis was detected. We found that the receptor also regulates cell proliferation in the disc, has an early function at the disc margin (where the morphogenetic furrow initiates) and contributes to the regulation of spacing of the R8 precursors. Finally, we found that activation of the receptor is sufficient to trigger non-R8 photoreceptor development, even in cells in front of the furrow or in the absence of the proneural gene atonal. CONCLUSION: At least five distinct functions of EGFR signalling need to be integrated during fly eye development. These include roles in cell proliferation, survival and differentiation.     

Isolation of a Drosophila homolog of the vertebrate homeobox gene Rx and its possible role in brain and eye development

Vertebrate and invertebrate eye development require the activity of several evolutionarily conserved genes. Among these the Pax-6 genes play a major role in the genetic control of eye development. Mutations in Pax-6 genes affect eye development in humans, mice, and Drosophila, and misexpression of Pax-6 genes in Drosophila can induce ectopic eyes. Here we report the identification of a paired-like homeobox gene, DRx, which is also conserved from flies to vertebrates. Highly conserved domains in the Drosophila protein are the octapeptide, the identical homeodomain, the carboxyl-terminal OAR domain, and a newly identified Rx domain. DRx is expressed in the embryo in the procephalic region and in the clypeolabrum from stage 8 on and later in the brain and the central nervous system. Compared with eyeless, the DRx expression in the embryo starts earlier, similar to the pattern in vertebrates, where Rx expression precedes Pax-6 expression. Because the vertebrate Rx genes have a function during brain and eye development, it was proposed that DRx has a similar function. The DRx expression pattern argues for a conserved function at least during brain development, but we could not detect any expression in the embryonic eye primordia or in the larval eye imaginal discs. Therefore DRx could be considered as a homolog of vertebrate Rx genes. The Rx genes might be involved in brain patterning processes and specify eye fields in different phyla.     

decapentaplegic is required for arrest in G1 phase during Drosophila eye development

During eye development in Drosophila, cell cycle progression is coordinated with differentiation. Prior to differentiation, cells arrest in G1 phase anterior to and within the morphogenetic furrow. We show that Decapentaplegic (Dpp), a TGF-&bgr; family member, is required to establish this G1 arrest, since Dpp-unresponsive cells located in the anterior half of the morphogenetic furrow show ectopic S phases and ectopic expression of the cell cycle regulators Cyclins A, E and B. Conversely, ubiquitous over-expression of Dpp in the eye imaginal disc transiently inhibits S phase without affecting Cyclin E or Cyclin A abundance. This Dpp-mediated inhibition of S phase occurs independently of the Cyclin A inhibitor Roughex and of the expression of Dacapo, a Cyclin E-Cdk2 inhibitor. Furthermore, Dpp-signaling genes interact genetically with a hypomorphic cyclin E allele. Taken together our results suggest that Dpp acts to induce G1 arrest in the anterior part of the morphogenetic furrow by a novel inhibitory mechanism. In addition, our results provide evidence for a Dpp-independent mechanism that acts in the posterior part of the morphogenetic furrow to maintain G1 arrest.     

The Homothorax homeoprotein activates the nuclear localization of another homeoprotein, extradenticle, and suppresses eye development in Drosophila

The Extradenticle (Exd) protein in Drosophila acts as a cofactor to homeotic proteins. Its nuclear localization is regulated. We report the cloning of the Drosophila homothorax (hth) gene, a homolog of the mouse Meis1 proto-oncogene that has a homeobox related to that of exd. Comparison with Meis1 finds two regions of high homology: a novel MH domain and the homeodomain. In imaginal discs, hth expression coincides with nuclear Exd. hth and exd also have virtually identical, mutant clonal phenotypes in adults. These results suggest that hth and exd function in the same pathway. We show that hth acts upstream of exd and is required and sufficient for Exd protein nuclear localization. We also show that hth and exd are both negative regulators of eye development; their mutant clones caused ectopic eye formation. Targeted expression of hth, but not of exd, in the eye disc abolished eye development completely. We suggest that hth acts with exd to delimit the eye field and prevent inappropriate eye development.     

Naturally occurring variation in bristle number and DNA polymorphisms at the scabrous locus of Drosophila melanogaster

The association between quantitative genetic variation in bristle number and molecular variation at a candidate neurogenic locus, scabrous, was examined in Drosophila melanogaster. Approximately 32 percent of the genetic variation in abdominal bristle number (21 percent for sternopleural bristle number) among 47 second chromosomes from a natural population was correlated with DNA sequence polymorphisms at this locus. Several polymorphic sites associated with large phenotypic effects occurred at intermediate frequency. Quantitative genetic variation in natural populations caused by alleles that have large effects at a few loci and that segregate at intermediate frequencies conflicts with the classical infinitesimal model of the genetic basis of quantitative variation.     

The role of eye movements in subitizing and counting.

Previous work has suggested that eye movements may be necessary for accurate enumeration beyond the subitization range of about 4 items. This study determined the frequency of eye movements normally made during enumeration, their relationship to response times, and whether they are required for accurate performance. This was achieved by monitoring eye movements and comparing performance when observers were allowed to saccade and when they were not. The results showed that (a) there was a sharp increase in saccadic frequency beyond about 4 items (from = 0.2 saccades per item to about 1 per item), and (b) enumeration of fewer than 4 items remained rapid and accurate even when eye movements were prevented, whereas enumeration beyond this became less efficient and sometimes less accurate. The results are discussed in relation to the memory and processing requirements of enumeration tasks. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Polygenic mutation in Drosophila melanogaster: genotype x environment interaction for spontaneous mutations affecting bristle number

A highly inbred line of Drosophila melanogaster was subdivided into replicate sublines that were subsequently maintained independently with 10 pairs of parents per generation. The parents were randomly sampled for 19 'unselected' sublines and artificially selected for high or low abdominal or sternopleural bristle number for 12 'selected' sublines (with 3 replicate selection lines/trait/direction of selection). Divergence in mean bristle number among the unselected sublines, and response of the selected sublines to selection, are attributable to the accumulation of new mutations affecting bristle number. The input of mutational variance per generation, VM, can be estimated from the magnitude of response or divergence, assuming neutrality of mutations affecting the bristle traits. We reared unselected lines at generations 222 and 224, and selected lines at generations 182-184 of mutation accumulation at each of three temperatures (18 degrees C, 25 degrees C, 28 degrees C), and estimated the mutational variance common to all environments and the mutational variance from genotype x environment interaction. For sternopleural bristle number, the mutational interaction variance was 26% of the mutational variance common to all temperatures, and the interaction variance was due to temperature x line interaction. For abdominal bristle number, the mutational interaction variance was 142% of the mutational variance common to all temperatures, and the interaction variance was due to interactions of temperature x line, sex x line, and temperature x sex x line. It is possible that segregating variation for bristle number is maintained partly by genotype x environment interaction, but information on the fitness profiles of mutations affecting bristle number in each environment will be necessary to evaluate this hypothesis quantitatively.     

Synergism between platelets and neutrophils in provoking cardiac dysfunction after ischemia and reperfusion: role of selectins

BACKGROUND: Neutrophils (PMNs) are known to contribute to both cardiac dysfunction and myocardial necrosis after reperfusion of an ischemic heart. Moreover, platelets are also important blood cells that can aggravate myocardial ischemic injury. This study was designed to test the effects of PMNs and platelets separately and together in provoking cardiac dysfunction in isolated perfused rat hearts after ischemia and reperfusion. METHODS AND RESULTS: Control rat hearts not subjected to ischemia were perfused without blood cells for 80 minutes. Additional control rat hearts were perfused with 75x106 PMNs, with 100x106 platelets, or with 75x106 PMNs+100x106 platelets over a 5-minute perfusion followed by a 75-minute observation period. No significant reduction in coronary flow, left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dtmax) was observed at the end of the observation period in any nonischemic group. Similarly, global ischemia (I) for 20 minutes followed by 45 minutes of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of blood cells. However, I/R hearts perfused with either PMNs or platelets alone exhibited decreases in these variables of 10% to 12% (P<0.05 from control). Furthermore, I/R hearts perfused with both PMNs and platelets exhibited decreases of 50% to 60% in all measurements of cardiac function (P<0.001). These dual-cell-perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity, indicating a significant PMN infiltration, and enhanced P-selectin expression on the coronary microvascular endothelium. All cardiodynamic effects as well as MPO accumulation and PMN infiltration were markedly attenuated by a sialyl LewisX-oligosaccharide or a recombinant soluble P-selectin ligand, which inhibits selectin-mediated cell adhesion. CONCLUSIONS: These results provide evidence that platelets and neutrophils act synergistically in provoking postreperfusion cardiac dysfunction and that this may be largely due to cell-to-cell interactions mediated by P-selectin. These findings may help explain the reperfusion injury phenomenon.     

A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature

A two-step paradigm for leukocyte recruitment has been established in a number of tissues including the mesentery, skin, and muscle, and necessitates an initial rolling step via the selectins before firm leukocyte adhesion via the integrins. In view of the many inflammatory diseases that involve the liver, we investigated the importance of rolling and the selectins in the hepatic microvasculature and compared the responses to that of the commonly used mesentery or cremaster microvasculature. We visualized the liver microvasculature using intravital microscopy and we determined that within the liver the majority of leukocytes adhere within the sinusoids (80%) in response to a chemotactic stimulus such as FMLP (20% in postsinusoidal venules) whereas leukocytes adhere exclusively within postcapillary venules in tissue like the mouse cremaster. In the sinusoids, the adhesive response to FMLP is not dependent upon selectins inasmuch as adhesion was not reduced in the sinusoidal vessels of P-selectin-deficient mice or E-selectin/P-selectin- deficient animals in the presence or absence of L-selectin antibody. No rolling or adhesion was detected in response to FMLP in the selectin-deficient cremaster microvasculature. Immunoneutralization of selectins with fucoidan in wildtype mice eliminated rolling and adhesion in the cremaster but failed to affect adhesion in the liver sinusoids in response to FMLP. More long-term leukocyte recruitment with lipopolysaccharide (4 h) was also impaired in the cremaster but not the liver microvasculature in selectin-deficient animals. Leukocyte adhesion in the sinusoids was reduced in P-selectin-deficient mice also lacking intercellular adhesion molecule-1 (ICAM-1). This study for the first time demonstrates that selectins are not an essential step for leukocyte recruitment into the inflamed liver microvasculature.     

Two sites in the Delta gene region contribute to naturally occurring variation in bristle number in Drosophila melanogaster

A restriction enzyme survey of a 57-kb region including the gene Delta uncovered 53 polymorphic molecular markers in a sample of 55 naturally occurring chromosomes. A permutation test, which assesses the significance of the molecular marker with the largest effect on bristle variation in four genetic backgrounds relative to permuted data-sets, found two sites that were independently associated with variation in bristle number. A common site in the second intron of Delta affected only sternopleural bristle number, and another common site in the fifth intron affected only abdominal bristle number in females. Under an additive genetic model, the polymorphism in the second intron may account for 12% of the total genetic variation in sternopleural bristle number due to third chromosomes, and the site in the fifth intron may account for 6% of the total variation in female abdominal bristle number due to the third chromosomes. These results suggest the following: (1) models that incorporate balancing selection are more consistent with observations than deleterious mutation-selection equilibrium models, (2) mapped quantitative trait loci of large effect may not represent a single variable site at a genetic locus, and (3) linkage disequilibrium can be used as a tool for understanding the molecular basis of quantitative variation.     

Pax proteins and eye development

Male and female rats were used to investigate the effects of type of dietary carbohydrate (CHO), copper, and ethanol consumption on lung antioxidant enzyme activities and levels of phosphorylated compounds in whole blood. Copper-deficient female rats exhibited a greater degree of copper deficiency than males, as assessed by hepatic copper concentration and hepatic copper superoxide dismutase (CuSOD) activity. However, copper-deficient male rats fed fructose-containing diets exhibited greater growth retardation, anemia, and heart hypertrophy than females consuming the same diets and males fed starch. In addition, one of 10 copper-deficient male rats that ate a fructose-based diet and drank water and one of 10 copper-deficient male rats that ate a starch-based diet and drank ethanol died. Copper-deficient, starch-fed males exhibited the highest activities of glutathione peroxidase (GSH-Px) and catalase as compared with fructose-fed rats. Ethanol consumption elevated the activities of GSH-Px and catalase. Copper-deficient female rats exhibited higher catalase but lower GSH-Px activities than males. It is suggested that in copper deficiency, the ability to increase antioxidant enzyme activities in rats consuming starch is greater than in rats consuming fructose. Rats fed starch are provided with a greater degree of protection against oxidative damage than rats fed fructose. In addition, polyphosphorylated compounds in blood were reduced in copper-deficient male rats that consumed fructose-based diets. This may impair supply of oxygen to tissues.     

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.     

skittles, a Drosophila phosphatidylinositol 4-phosphate 5-kinase, is required for cell viability, germline development and bristle morphology, but not for neurotransmitter release

The phosphatidylinositol pathway is implicated in the regulation of numerous cellular functions and responses to extracellular signals. An important branching point in the pathway is the phosphorylation of phosphatidylinositol 4-phosphate by the phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to generate the second messenger phosphatidylinositol 4,5-bis-phosphate (PIP2). PIP5K and PIP2 have been implicated in signal transduction, cytoskeletal regulation, DNA synthesis, and vesicular trafficking. We have cloned and generated mutations in a Drosophila PIP5K type I (skittles). Our analysis indicates that skittles is required for cell viability, germline development, and the proper structural development of sensory bristles. Surprisingly, we found no evidence for PIP5KI involvement in neural secretion.