Experimental phenocopy of a minute maternal-effect mutation alters blastoderm determination in embryos of Drosophila melanogaster

Maternal haploinsufficiency for a third chromosome Minute, M(3)i55, lowers rates of protein synthesis by approximately 30% during the syncytial nuclear cycles of early embryogenesis. The maternal effect of Mi55 also produces segmentation defects (denticle belt fusions) in the posterior abdomen of larvae. Furthermore, embryos from Minute mothers show abnormal expression patterns of the segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage of embryogenesis. We developed a computer-aided analysis to describe the deviations in ftz expression which demonstrates that abnormally narrow ftz stripes occur in segment primordia that become fused in the larva. Unexpectedly, an abnormally wide ftz stripe occurs in segment primordia which do not develop abnormally. In addition, Mi55 produces a general narrowing of all ftz- interstripes. We phenocopied the Minute mutation by injecting wild-type embryos with cycloheximide concentrations which decreased protein synthesis rates to levels comparable with those of Minute embryos. Thus, a general decrease in protein synthesis during early embryogenesis leads to abnormal determination of posterior abdominal segment primordia.     

Drop out: a third chromosome maternal-effect locus required for formation of the Drosophila cellular blastoderm.

drop out (dop) is a recessive maternal-effect locus identified in a screen for female-sterile mutations in Drosophila polytene region 71C-F. Phenotypic analyses of the dop mutation indicate that the gene is required for proper formation of the cellular blastoderm. In embryos derived from either homozygous or hemizygous dop mothers, cytoplasmic clearing, nuclear migration and division, and pole cell formation appear normal. However, developmental defects are observed prior to and during cellularization of the blastoderm. At the beginning of nuclear cycle 14, the distinct separation of the internal yolk mass and the cortical cytoplasm breaks down. Subsequently, a population of somatic nuclei located at the periphery of the syncytial blastoderm becomes irregularly spaced and nonuniform in their distribution. Despite a somewhat regular formation of the cortical actin network, cellularization in mutant embryos is extremely variable. Such embryos fail to gastrulate normally and produce variable amounts of defective cuticle. Overall, our analyses suggest that the dop gene functions in maintaining the separation of yolk and cortical cytoplasm and in stabilizing the distribution of somatic nuclei in the Drosophila syncytial blastoderm.     

daughterless-abo-like, a Drosophila maternal-effect mutation that exhibits abnormal centrosome separation during the late blastoderm divisions

daughterless-abo-like (dal) is a maternal-effect semilethal mutation in Drosophila. The nuclear divisions of embryos derived from homozygous dal females are normal through nuclear cycle 10. However, during nuclear cycles 11, 12 and 13, a total of about half of the nuclei in each embryo either fail to divide or fuse with a neighboring nucleus during telophase. These abnormal nuclei eventually sink into the interior of the embryo, leaving their centrosomes behind on the surface. The loss of about one-half of the peripheral nuclei into the interior of the embryo results in these embryos cellularizing during nuclear cycle 14 with about one-half the normal number of cells. Surprisingly, many of these embryos develop a nearly normal larval cuticle and 8% develop to adulthood. Observations of live embryos doubly injected with tubulin and histones that have been fluorescently labeled allows nuclear and centrosomal behavior to be directly followed as the embryo develops. We find that the abnormal nuclei arise from nuclei whose centrosomes have failed to separate normally in the previous interphase. These incompletely separated centrosomes can cause a non-functional spindle to form, leading to a nuclear division failure. Alternatively, they can form an abnormal spindle with a centrosome from a neighboring nucleus, causing two nuclei to share a common spindle pole. Such nuclei with a shared centrosome will undergo telophase fusions, unequal divisions, or division failures later in mitosis. These findings have helped us to understand the function of the centrosome in the Drosophila embryo.     

Drosophila mutant with a transducer defect

The trp is a conditional phototransduction mutant of Drosophila. Direct electrical measurements and shot noise analysis suggest that a prolonged intense light causes in the mutant a reduction in the quantum efficiency for quantum bump production that does not arise from bleaching of the visual pigment. This effect depends on the duration of the light and only weakly on its intensity. In the normal fly, an intense blue light that shifts the visual pigment from rhodopsin to metarhodopsin, induces an excitatory process manifested by a prolonged depolarizing after potential (PDA). In the mutant, the PDA has a small amplitude and bump noise is superimposed on the response. It can thus be shown that the excitatory process underlying the PDA is also present in those trp mutants where the PDA voltage response is small or absent. It is suggested that the absence of the PDA voltage response in the mutant is probably due to a defect in an intermediate process, which links the excitatory process to the membrane conductance change.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Diacylglycerol kinase defect in a Drosophila retinal degeneration mutant rdgA

The Drosophila visual mutant rdgA is known to show age-dependent retinal degeneration with defective diacylglycerol (DG) kinase activity. In this study we examined DG kinase activity of several visual mutants and found that only rdgA mutant eyes showed the lack of DG kinase activity in a gene dosage-dependent manner. The enzyme activity is already absent at the time of eclosion from pupal case when the degeneration is not yet apparent. To examine whether rdgA gene dosage effect holds for other enzymes related to the phosphatidylinositol turnover, phospholipase C was analyzed which did not show any gene dosage effect. Therefore, it is strongly suggested that rdgA gene correlates closely with DG kinase activity, and the defect of DG kinase activity is a primary cause of retinal degeneration in rdgA mutant.     

Genetic analysis of a meiotic mutant resulting in precocious sister-centromere separation in Drosophila melanogaster

Summary It is shown that mei-S332, a semidominant mutant in Drosophila melanogaster, has the following properties: 1) It maps at about position 95 on the right arm of chromosome 2. 2) Its primary result is the precocious division of sister centromeres, which leads to nondisjunction, mostly equational, and loss for all chromosomes in both sexes. 3) Chromosome pairs behave approximately independently. 4) Gamete types for each chromosome appeared in the approximate ratios of 0.20 nullo-gametes: 0.12 diplo-gametes: 0.68 regular gametes for experiments reported here. 5) Exchange is correlated with a lower probability of reductional nondisjunction, but is independent of the probability of equational nondisjunction. 6) Since the phenotype of mei-S332 is similar in the two sexes, at least part of the meiotic events and their control for the second division and perhaps also the first division is the same in the two sexes. 7) Mosaics, resulting from mitotic chromosome loss appear among progeny of mei-S332 parents.Adapted from a dissertation presented in partial fulfillment of the degree of Doctor of Philosophy. The research was supported by a PHS Training Grant and PHS Grant RG-9965.     

The initiation of pair-rule stripes in the Drosophila blastoderm.

The interactions between the products of gap genes and pair-rule promoters results in the single most dramatic increase in the spatial complexity of gene expression during the segmentation process. We attempt to relate recent findings on the regulation of striped patterns of gene expression in the early Drosophila embryo to general strategies of gene expression and development employed by higher organisms.     

Developmental analysis of the torso-like phenotype in Drosophila produced by a maternal-effect locus

The segmental plan of the Drosophila embryo is already established at the blastoderm stage through the action of maternal effect genes which determine the polarity of the embryo and zygotically active genes involved in segmentation. We have analyzed the first example of a group of maternally acting genes which are necessary for establishing the developmental potential of the posterior 25% of the blastoderm. Females, homozygous for the X-linked maternal-effect mutation female sterile(1)Nasrat211 [fs(1)N211], produce embryos, characterized as torso-like, which lack all posterior endodermal derivatives as well as structures characteristic of abdominal segments 8 to 10. In addition, anterior endodermal derivatives are deficient and the absence of pharyngeal musculature causes a collapse of the cephalopharyngeal apparatus. The columnar blastoderm cell layer is defective at the posterior tip below the pole cells in these embryos. This defect, however, is presumably secondary to some abnormal feature of pole cell formation since in double mutants of fs(1)Nasrat211; tudor3 the blastoderm is normal but the embryos still show the torso-like phenotype. In situ hybridization with RNA probes derived from the fushi tarazu gene establishes that the cellular determination of the posterior blastoderm of embryos produced by fs(1)N211 is changed. This represents the first direct demonstration that a maternal-effect mutation alters the spatial distribution of a zygotic gene product involved in the segmental patterning of the embryo.     

Lack of an antibacterial response defect in Drosophila Toll-9 mutant

Toll and Toll-like receptors represent families of receptors involved in mediating innate immunity response in insects and mammals. Although Drosophila proteome contains multiple Toll paralogs, Toll-1 is, so far, the only receptor to which an immune role has been attributed. In contrast, every single mammalian TLR is a key membrane receptor upstream of the vertebrate immune signaling cascades. The prevailing view is that TLR-mediated immunity is ancient. Structural analysis reveals that Drosophila Toll-9 is the most closely related to vertebrate TLRs and utilizes similar signaling components as Toll-1. This suggests that Toll-9 could be an ancestor of TLR-like receptors and could have immune function. Consistently, it has been reported that over-expression of Toll-9 in immune tissues is sufficient to induce the expression of some antimicrobial peptides in flies. These results have led to the idea that Toll-9 could be a constitutively active receptor that maintain significant levels of antimicrobial molecules and therefore provide constant basal protection against micro-organisms. To test theses hypotheses, we generated and analyzed phenotypes associated with a complete loss-of-function allele of Toll-9. Our results suggest that Toll-9 is neither required to maintain a basal anti-microbial response nor to mount an efficient immune response to bacterial infection.     

Hereditary retinal degeneration in Drosophila melanogaster. A mutant defect associated with the phototransduction process

Two genes in Drosophila, rdgA and rdgB, which when defective cause retinal degeneration, were discovered by Hotta and Benzer (Hotta, Y., and S. Benzer. 1970. Proc. Natl, Acad. Sci. U. S, A. 67:1156-1163). These mutants have photoreceptor cells that are histologically normal upon eclosion but subsequently degenerate. The defects in the rdgA and rdgB mutants were localized by the study of genetic mosaics to the photoreceptor cells. In rdgB mutants retinal degeneration is light induced. It can be prevented by rearing the flies in the dark or by blocking the receptor potential with a no-receptor-potential mutation, norpA. Vitamin A deprivation and genetic elimination of the lysosomal enzyme acid phosphatase alsoprotect the photoreceptors of rdgB flies against light-induced damage. The photopigment kinetics of dark-reared rdgB flies appear normal in vitro by spectrophotometric measurements, and in vivo by measurements of the M potential. In normal Drosophila, a 1-s exposure to intense 470-nm light produces a prolonged depolarizing afterpotential (PDA) which can last for several hours. In dark-reared rdgB mutants the PDA lasts less than 2 min;; it appears to initiate the degeneration process, since the photoreceptors become permanently unresponsive after a single such exposure. Another mutant was isolated which prevents degeneration in rdgB flies but which has a normal receptor potential. This suppressor of degeneration is an allele of norpA. It is proposed that the normal norpA gene codes for a product which, when activated, leads to the receptor potential, and which is inactivated by the product of the normal rdgB gene.     

A CHO-cell mutant with a defect in cytokinesis.

In a selection procedure designed to enrich for temperature-sensitive mutant cells blocked in mitosis a CHO-cell mutant was isolated which has a defect in cytokinesis as the basis of its temperature-sensitive phenotype. Cultures of the mutant had an abnormally high percentage (ie. 34%) of polyploid cells at the permissive temperature of 34 degress C and showed further increased frequencies of polyploidy as well as many multinucleated cells at 38.5 degrees 39.5 degrees. When the mutant cells were synchronized in metaphase by Colcemid arrest and then placed into fresh medium at nonpermissive temperature, they did not divide although the completion of mitosis appeared cytologically normal. Ultrastructural examination by electron microscopy of such synchronized cells at telophase revealed no specific defects in cellular components other than failure of development of a normal midbody. The sensitivity of the mutant to cytochalasin B and to Colcemid was the same as for wild-type cells. This mutation behaved as recessive in tetraploid cell hybrids constructed by fusing the mutant with a CHO strain which was wild-type with respect to temperature sensitivty.     

Death resulting from unbalanced growth in a temperature-sensitive mutant of Neurospora crassa

A temperature-sensitive mutant of Neurospora crassa was found to undergo rapid death on minimal medium at 35 degrees C. The loss of viability in this mutant was prevented by various factors which retard growth, including deprivation of carbon sources or interruption of protein synthesis. Synthesis of nucleic acids and protein in this mutant was normal at the early stages of germination and then depressed at 35 degrees C. The active transport of glucose and the respiration rate in this mutant were depressed at 35 degrees C. Phopholipid synthesis was significantly repressed at 35 degrees C. The possible significance of the characteristics of this mutant is discussed in terms of membrane biosynthesis.     

Cation permeability and cation-anion interactions in a mutant GABA-gated chloride channel from Drosophila.

To investigate the structural basis of anion selectivity of Drosophila GABA-gated Cl(-) channels, the permeation properties of wild-type and mutant channels were studied in Xenopus oocytes. This work focused on asparagine 319, which by homology is one amino acid away from a putative extracellular ring of charge that regulates cation permeation in nicotinic receptors. Mutation of this residue to aspartate reduced channel conductance, and mutation to lysine or arginine increased channel conductance. These results are consistent with an electrostatic interaction between this site and permeating anions. The lysine mutant, but not the arginine mutant, formed a channel that is permeable to cations, and this cannot be explained in terms of electrostatics. The lysine mutant had a 25-mV reversal potential in solutions with symmetrical Cl(-) and asymmetrical cations. The permeability ratio of K(+) to Cl(-) was determined as 0. 33 from reversal potential measurements in KCl gradients. Experiments with large organic cations and anions showed that cation permeation can only be seen in the presence of Cl(-), but Cl(-) permeation can be seen in the absence of permeant cations. Measurements of permeability ratios of organic anions indicated that the lysine mutant has an increased pore size. The cation permeability of the lysine-containing mutant channel cannot be accounted for by a simple electrostatic interaction with permeating ions. It is likely that lysine substitution causes a structural change that extends beyond this one residue to influence the positions of other channel-forming residues. Thus protein conformation plays an important role in enabling ion channels to distinguish between anions and cations.     

Cytochalasin induces spindle fusion in the syncytial blastoderm of the early Drosophila embryo.

Microfilament integrity is needed to maintain the regular arrangement of the spindle microtubules and to guarantee the normal progression of the last syncytial mitoses in Drosophila embryo. To investigate when and how microfilaments participate in this process, we incubated permeabilized embryos with the inhibitor of actin polymerization, cytochalasin B, at different times during the nuclear cycle. Our results suggest that the correct microfilament distribution is only required for the appropriate segregation of nuclei during the 11th, 12th and 13th syncytial mitoses rather than during the 10th mitosis when the spindles are too far apart to interact. When cytochalasin B treatment was performed during the last syncytial mitoses many spindles fuse among them and the regular mitotic progression is perturbed.