The ADAM metalloprotease Kuzbanian is crucial for proper heart formation in Drosophila melanogaster

We have screened a collection of EMS mutagenized fly lines in order to identify genes involved in cardiogenesis. In the present work, we have studied a group of alleles exhibiting a hypertrophic heart. Our analysis revealed that the ADAM protein (A Disintegrin And Metalloprotease) Kuzbanian, which is the functional homologue of the vertebrate ADAM10, is crucial for proper heart formation. ADAMs are a family of transmembrane proteins that play a critical role during the proteolytic conversion (shedding) of membrane bound proteins to soluble forms. Enzymes harboring a sheddase function recently became candidates for causing several congenital diseases, like distinct forms of the Alzheimer disease. ADAMs play also a pivotal role during heart formation and vascularisation in vertebrates, therefore mutations in ADAM genes potentially could cause congenital heart defects in humans. In Drosophila, the zygotic loss of an active form of the Kuzbanian protein results in a dramatic excess of cardiomyocytes, accompanied by a loss of pericardial cells. Our data presented herein suggest that Kuzbanian acts during lateral inhibition within the cardiac primordium. Furthermore we discuss a second function of Kuzbanian in heart cell morphogenesis.     

Moleskin is essential for the formation of the myotendinous junction in Drosophila

It is the precise connectivity between skeletal muscles and their corresponding tendon cells to form a functional myotendinous junction (MTJ) that allows for the force generation required for muscle contraction and organismal movement. The Drosophila MTJ is composed of secreted extracellular matrix (ECM) proteins deposited between integrin-mediated hemi-adherens junctions on the surface of muscle and tendon cells. In this paper, we have identified a novel, cytoplasmic role for the canonical nuclear import protein Moleskin (Msk) in Drosophila embryonic somatic muscle attachment. Msk protein is enriched at muscle attachment sites in late embryogenesis and msk mutant embryos exhibit a failure in muscle–tendon cell attachment. Although the muscle–tendon attachment sites are reduced in size, components of the integrin complexes and ECM proteins are properly localized in msk mutant embryos. However, msk mutants fail to localize phosphorylated focal adhesion kinase (pFAK) to the sites of muscle–tendon cell junctions. In addition, the tendon cell specific proteins Stripe (Sr) and activated mitogen-activated protein kinase (MAPK) are reduced in msk mutant embryos. Our rescue experiments demonstrate that Msk is required in the muscle cell, but not in the tendon cells. Moreover, muscle attachment defects due to loss of Msk are rescued by an activated form of MAPK or the secreted epidermal growth factor receptor (Egfr) ligand Vein. Taken together, these findings provide strong evidence that Msk signals non-autonomously through the Vein-Egfr signaling pathway for late tendon cell late differentiation and/or maintenance.     

Rab11 is essential for fertility in Drosophila

Rab11, a small GTP binding protein involved in vesicular trafficking, has emerged as a key player in regulating various cellular events during Drosophila development and differentiation. In our earlier study a P-insertion line, Rab11(mo), was established as a new hypomorphic allele of Rab11 gene, showing degenerated eye phenotype, bristle abnormalities and sterility. We show here that Rab11 is expressed in the entire testis, more prominently in the secretory cells, and in ovary it is localized at the posterior pole. Rab11(mo) males and females are sterile. The sterility in males has been attributed to defects in the sperm individualization process, while in females, cytoskeleton disruption and reduction/loss of the posteriorly localized protein, Vasa, as a consequence of loss/mislocalization of Rab11 might be the cause of sterility. Fertility as well as the posterior localization of Rab11 and Vasa or cytoskeleton integrity was restored in pCaSpeR4-Rab11/+; Rab11(mo)/Rab11(mo) egg chambers, confirming the requirement of Rab11 in these events.     

Developmental analysis of fs(1)gastrulation defective,a dorsal-group gene of Drosophila melanogaster

Summary The gastrulation defective (gd) locus is a maternally expressed gene in Drosophila required for normal differentiation of structures along the embryonic dorso-ventral axis. Cuticular defects of the offspring from females with different combinations of gd alleles comprised a phenotypic continuum. Complementation among several alleles produced normal offspring while progressively more severe mutations produced a graded loss of structures from ventral, and then lateral, blastoderm cells. The most severely affected embryos consisted entirely of structures derived from dorsal blastoderm cells. Histological examination of staged siblings from selected allelic combinations showed that internal tissues were similarly affected. The tissues observed in amorphic embryos support new, more dorsal, assignments of fate map positions for blastoderm precursors of the cephalopharyngeal apparatus, hindgut and ventral nerve cord. The loss of ventral and lateral structures did not occur through cell death and appeared to involve a change in blastoderm cell fate. A direct effect of the mutations on blastoderm cell determination, however, was insufficient to explain the development of the dorsalized embryos. Intermediate phenotypes suggested that cell interactions or movements associated with morphogenesis are required for the determination of some cell fates in the dorsoventral axis. Thus, the developmental fate of all blastoderm cells may not be fixed at the time of blastoderm formation.     

The Drosophila melanogaster Ku70 coding sequence is wild-type in mus309 mutants

Drosophila melanogaster mus309 mutations have been identified as being in the Ku70 gene, encoding one subunit of the DNA-dependent protein kinase. However, recent work has suggested that these mutations are in the Bloom's syndrome homologue. Here, we demonstrate that the coding sequence of Ku70 gene is indeed wild-type in two mus309 mutant lines.     

Ribosomal protein S14 is not responsible for the Minute phenotype associated with the M(1)7C locus in Drosophila melanogaster.

Summary A locus associated with a severe Minute effect has been mapped at 7C on the X chromosome of Drosophila melanogaster. Previous work has suggested that this Minute encodes ribosomal proteins S14A and S141B. We have made a chromosomal deficiency that removes the S14 ribosomal protein genes, yet does not display the Minute phenotype. These data suggest that the S14 genes do not actually correspond to the Minute locus.     

spalt-induced specification of distinct dorsal and ventral domains is required for Drosophila tracheal patterning

Morphogenesis of the Drosophila tracheal system relies on different signalling pathways that have distinct roles in specifying both the migration of the tracheal cells and the particular morphological features of the primary branches. The current view is that the tracheal cells are initially specified as an equivalent group of cells whose diversification depends on signals from the surrounding cells. In this work, we show that the tracheal primordia are already specified as distinct dorsal and ventral cell populations. This subdivision depends on the activity of the spalt (sal) gene and occurs prior to the activity of the signalling pathways that dictate the development of the primary branches. Finally, we show that the specification of these two distinct cell populations, which are not defined by cell lineage, are critical for proper tracheal patterning. These results indicate that tracheal patterning depends not only on signalling from surrounding cells but also in the different response of the tracheal cells depending on their allocation to the dorsal or ventral domains.     

The heterotrimeric protein Go is required for the formation of heart epithelium in Drosophila.

The gene encoding the alpha subunit of the Drosophila Go protein is expressed early in embryogenesis in the precursor cells of the heart tube, of the visceral muscles, and of the nervous system. This early expression coincides with the onset of the mesenchymal-epithelial transition to which are subjected the cardial cells and the precursor cells of the visceral musculature. This gene constitutes an appropriate marker to follow this transition. In addition, a detailed analysis of its expression suggests that the cardioblasts originate from two subpopulations of cells in each parasegment of the dorsal mesoderm that might depend on the wingless and hedgehog signaling pathways for both their determination and specification. In the nervous system, the expression of Goalpha shortly precedes the beginning of axonogenesis. Mutants produced in the Goalpha gene harbor abnormalities in the three tissues in which the gene is expressed. In particular, the heart does not form properly and interruptions in the heart epithelium are repeatedly observed, henceforth the brokenheart (bkh) name. Furthermore, in the bkh mutant embryos, the epithelial polarity of cardial cells was not acquired (or maintained) in various places of the cardiac tube. We predict that bkh might be involved in vesicular traffic of membrane proteins that is responsible for the acquisition of polarity.     

bullwinkle is required for epithelial morphogenesis during Drosophila oogenesis

Many organs, such as the liver, neural tube, and lung, form by the precise remodeling of flat epithelial sheets into tubes. Here we investigate epithelial tubulogenesis in Drosophila melanogaster by examining the development of the dorsal respiratory appendages of the eggshell. We employ a culture system that permits confocal analysis of stage 10-14 egg chambers. Time-lapse imaging of GFP-Moesin-expressing egg chambers reveals three phases of morphogenesis: tube formation, anterior extension, and paddle maturation. The dorsal-appendage-forming cells, previously thought to represent a single cell fate, consist of two subpopulations, those forming the tube roof and those forming the tube floor. These two cell types exhibit distinct morphological and molecular features. Roof-forming cells constrict apically and express high levels of Broad protein. Floor cells lack Broad, express the rhomboid-lacZ marker, and form the floor by directed cell elongation. We examine the morphogenetic phenotype of the bullwinkle (bwk) mutant and identify defects in both roof and floor formation. Dorsal appendage formation is an excellent system in which cell biological, molecular, and genetic tools facilitate the study of epithelial morphogenesis.     

The Wiskott-Aldrich syndrome protein (WASP) is essential for myoblast fusion in Drosophila

In higher organisms, mononucleated myoblasts fuse to form multinucleated myotubes. During this process, myoblasts undergo specific changes in cell morphology and cytoarchitecture. Previously, we have shown that the actin regulator Kette (Hem-2/Nap-1) is essential for myoblast fusion. In this study, we describe the role of the evolutionary conserved Wiskott-Aldrich syndrome protein that serves as a regulator for the Arp2/3 complex for myoblast fusion. By screening an EMS mutagenesis collection, we discovered a new wasp allele that does not complete fusion during myogenesis. Interestingly, this new wasp3D3-035 allele is characterized by a disruption of fusion after precursor formation. The molecular lesion in this wasp allele leads to a stop codon preventing translation of the CA domain. Usually, the WASP protein exerts its function through the Arp2/3-interacting CA domain. Accordingly, a waspDeltaCA that is expressed in a wild-type background acts as dominant-negative during the fusion process. Furthermore, we show that the myoblast fusion phenotype of kette mutant embryos can be suppressed by reducing the gene dose of wasp3D3-035. Thus, Kette antagonizes WASP function during myoblast fusion.     

The gene fl(2)d is required for various Sxl-controlled processes in Drosophila females

Summary In Drosophila melanogaster, the gene Sex-lethal (Sxl) controls the processes of sex determination, dosage compensation, oogenesis and sexual behaviour. The control of Sxl is by alternative splicing of its primary RNA. We have identified a gene, female-lethal-2-d (fl(2)d), which is needed for the female-specific splicing of Sxl RNA and which also has a vital function independent of Sxl. Here we analyse other aspects of the gene fl(2)d. Specifically, we have analysed the effect of the temperature-sensitive mutation fl(2)d ¹ on the viability of adult flies homozygous for this mutation. We have found that the viability of the mutant females is reduced, while that of the mutant males is not affected. In addition, the capacity of the mutant females to be inseminated is considerably reduced, whilst all the mutant males are able to inseminate females. These effects on females are suppressed by Sxl ^M1. However, the fat body cells of fl(2)d ¹ homozygous females are able to synthesize yolk proteins at the restrictive temperature. We have also carried out, in males, a clonal analysis of fl(2)d ², a mutation lethal in both sexes. We have found that the clones are fully viable. We conclude that the gene fl(2)d seems to be necessary during the adult life of females for the processes that require Sxl ⁺ activity. Moreover, the Sxl-independent vital function of fl(2)d seems to be required in both sexes only during larval development. Offprint requests to: L. Sánchez     

The association between malathion resistance and acetylcholinesterase in Drosophila melanogaster

The relationship between the 50% survival time for flies feeding on a malathion-containing medium and the activity of acetylcholinesterase (AChE) was determined for 15 isofemale lines of Drosophila melanogaster. A significant correlation was found (r=0.28, P<0.05), with more resistant lines tending to have a lower level of AChE activity. An association between AChE and malathion resistance was also observed in a selection experiment. The AChE activity decreased in two of two populations selected for malathion resistance. AChE from these populations was altered in kinetic parameters (measured in crude head extracts) and electrophoretic mobility. Although the resistant AChE had a lower activity (V _m) on either a per milligram protein or a per individual basis, its apparent K _m for acetylthiocholine was lower than that of susceptible AChE. Recombination mapping of both low activity and fast electrophoretic mobility localized these traits to the region of the structural locus (Ace) on the third chromosome. The AChE activity of flies heterozygous for a variety of Ace lesions (kindly provided by Dr. W. M. Gelbart) was consistent with this location. The changes in AChE were suggested to have been caused by selection of alleles at the Ace locus.This work was supported by NSERC Grants A5857, G0183, and A0629.     

Cloning of a novel homeobox (NK-7.1) containing gene,DmHboxNK-7.1, from Drosophila melanogaster

We have cloned a novel Drosophila melanogaster homeobox (Hbox) containing gene, NK-7.1 (Dm.HboxNK-7.1), which is located at 88B3 on the chromosome map, and is 1.5 kb downstream of the spn-B gene. The newly identified gene is expressed at high levels in the embryo, is switched off during larval and pupal stages, and is expressed again in the adult. The Hbox is highly similar to NK-1/S59 (Drosophila) and NK-3/bap (Drosophila). The amino acid (aa) identity ratios (%) were 58 between NK-7.1 and NK-1/S59, and between NK-7.1 and NK-3/bap. The other characteristic structures are the presence of homopolymeric aa stretches consisting of Q, N, and E.     

Spatial differences in patterns of modification: selection on hairy in Drosophila melanogaster wings

Artificial selection was carried out for over 45 generations to enhance and suppress expression of the mutation hairy on the Drosophila melanogaster wing. Whole chromosome mapping of X‐linked and autosomal modifiers of sense organ number displayed regional differences in magnitude and direction of their effects. Regional specificity of modifier effects was also seen in some interchromosomal interactions. Scanning electron microscopy allowed precise measurement of sense organ size and position along the L3 longitudinal wing vein. Sense organ size varied in a predictable fashion along the proximal–distal axis, and the dorsal pattern differed from the ventral pattern. The high and low selection lines differed most in the proximal portion of the L3 vein. Extra sense organs in the High line were often associated with vein fragments at locations predicted from ancestral vein patterns. Thus, regional specificity of polygenic or quantitative trait locus modifier effects was identified in several different parts of the wing. This revised version was published online in July 2006 with corrections to the Cover Date.     

An in vitro method for recording the electrical activity of the isolated heart of the adult Drosophila melanogaster

A recording chamber for monitoring the electrophysiological properties of the isolated heart of adult Drosophila melanogaster has been developed. Spontaneously generated field potentials of constant amplitude can be recorded for 6-8 h (n = 14); in very few cases, records were maintained stable for over 10 h (n = 4), and in some cases below 6 h (n = 5). The chamber consists of the tip of a micropipette, which allows for monitoring the field potential generated by the spontaneously contracting heart. The method can produce accurate information about the heart rate and the amplitude of the cardiac action potential. The preparation can be used for pharmacological studies on the heart of D. melanogaster since it responds, with an increase in the heart rate, to unusually low concentrations of octopamine, 1 nM, a compound with cardioaccelerating properties for insect heart. The recording system can be easily modified for experiments on the heart of other insects. Finally, the isolated heart of D. melanogaster provides a simple method for identifying mutations that affect heart physiology.     

The pleiohomeotic gene is required for maintaining expression of genes functioning in ventral appendage formation in Drosophila melanogaster

Polycomb group (PcG) proteins are negative regulators that maintain the expression of homeotic genes and affect cell proliferation. Pleiohomeotic (Pho) is a unique PcG member with a DNA-binding zinc finger motif and was proposed to recruit other PcG proteins to form a complex. The pho null mutants exhibited several mutant phenotypes such as the transformation of antennae to mesothoracic legs. We examined the effects of pho on the identification of ventral appendages and proximo-distal axis formation during postembryogenesis. In the antennal disc of the pho mutant, Antennapedia (Antp), which is a selector gene in determining leg identity, was ectopically expressed. The homothorax (hth), dachshund (dac) and Distal-less (Dll) genes involved in proximo-distal axis formation were also abnormally expressed in both the antennal and leg discs of the pho mutant. The engrailed (en) gene, which affects the formation of the anterior-posterior axis, was also misexpressed in the anterior compartment of antennal and leg discs. These mutant phenotypes were enhanced in the mutant background of Posterior sex combs (Psc) and pleiohomeotic-like (phol), which are another PcG genes. These results suggest that pho functions in maintaining expression of genes involved in the formation of ventral appendages and the proximo-distal axis.     

Analysis of morphogenetic movements in the development of the notum anlage of Drosophila melanogaster

Summary A comparison of the morphogenetic maps of the notum anlage of Drosophila melanogaster derived from the gynandromorph data and mosaics induced by somatic crossing-over during the first instar larval stage revealed that practically no major morphogenetic movements occur in the development of the anlage between the blastoderm and first instar larval stages and the adult stage. By comparing the morphogenetic map derived from gynandromorphs and the fate map derived from data on the transplantation of fragments of the mature wing imaginal disc, it was observed that no major morphogenetic movements occur in the notum anlage between the stages of the allocation of the disc and the mature disc. The results are consistent with the observations of other authors concerning the larval development of eye-antenna, wing and leg discs.