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Second-site noncomplementation identifies genomic regions required for Drosophila nonmuscle myosin function during morphogenesis.

Drosophila is an ideal metazoan model system for analyzing the role of nonmuscle myosin-II (henceforth, myosin) during development. In Drosophila, myosin function is required for cytokinesis and morphogenesis driven by cell migration and/or cell shape changes during oogenesis, embryogenesis, larval development and pupal metamorphosis. The mechanisms that regulate myosin function and the supramolecular structures into which myosin incorporates have not been systematically characterized. The genetic screens described here identify genomic regions that uncover loci that facilitate myosin function. The nonmuscle myosin heavy chain is encoded by a single locus, zipper. Contiguous chromosomal deficiencies that represent approximately 70% of the euchromatic genome were screened for genetic interactions with two recessive lethal alleles of zipper in a second-site noncomplementation assay for the malformed phenotype. Malformation in the adult leg reflects aberrations in cell shape changes driven by myosin-based contraction during leg morphogenesis. Of the 158 deficiencies tested, 47 behaved as second-site noncomplementors of zipper. Two of the deficiencies are strong interactors, 17 are intermediate and 28 are weak. Finer genetic mapping reveals that mutations in cytoplasmic tropomyosin and viking (collagen IV) behave as second-site noncomplementors of zipper during leg morphogenesis and that zipper function requires a previously uncharacterized locus, E3.10/J3.8, for leg morphogenesis and viability.

Animals↗

Phylogenetic relationships and developmental expression of three sea urchin Wnt genes.

The Wnt genes comprise a family of secreted glycoproteins involved in cell-cell signaling and pattern formation during the development of a variety of organisms. We have begun to examine Wnt gene expression in sea urchins that exhibit alternative modes of larval development but produce similar adults. Here we describe the isolation of five Wnt sequences from indirect- and direct-developing sea urchin species using a PCR-based strategy and library screening. Phylogenetic and distance analyses indicate that the five sequences represent sea urchin Wnt-1, -4, and -5 orthologs. Wnt-5 sequences were isolated from three sea urchin species and show a significantly faster rate of evolution than do their counterparts in jawed vertebrates. The genomic structure of the Wnt-5 locus was also examined, and its organization is similar to that of Wnt genes from insects and vertebrates. The temporal expression of all three sea urchin Wnt orthologs during sea urchin development was examined by RNA gel blots or RNase protection assays. Transcripts from all three sea urchin Wnts are detected at various developmental stages of both indirect- and direct-developing species. These data support the view that sea urchin Wnt genes exhibit many conserved aspects and at least three orthologs are developmentally regulated in both indirect- and direct-developing sea urchin embryos.

Amino Acid Sequence↗

Requirements for integrins during Drosophila development.

The common beta subunit of the PS antigens of Drosophila is homologous with vertebrate integrins and is encoded by the lethal(1)myospheroid gene. We have generated flies mosaic for wild-type and mutant alleles of lethal(1)myospheroid using adult gynandromorphs and radiation-induced somatic crossing over. The defects observed in the gynandromorphs demonstrate widespread requirements for PS integrins during development especially in ventrally derived structures, which also show strong expression of PS beta integrin. Smaller lethal(1)myospheroid clones induced during larval development result in blister and vein defects in the wings and aberrant development of photoreceptor cells, demonstrating roles for PS integrins during development of both wings and eyes. PS integrins are required for the close apposition of the dorsal and ventral wing epithelia and for the proper arrangement of photoreceptor cells. However, many other adhesive and morphogenetic processes proceed normally in the absence of integrins containing the beta subunit encoded by lethal(1)myospheroid.

Animals↗

An essential Drosophila glutamate receptor subunit that functions in both central neuropil and neuromuscular junction.

A Drosophila forward genetic screen for mutants with defective synaptic development identified bad reception (brec). Homozygous brec mutants are embryonic lethal, paralyzed, and show no detectable synaptic transmission at the glutamatergic neuromuscular junction (NMJ). Genetic mapping, complementation tests, and genomic sequencing show that brec mutations disrupt a previously uncharacterized ionotropic glutamate receptor subunit, named here "GluRIID." GluRIID is expressed in the postsynaptic domain of the NMJ, as well as widely throughout the synaptic neuropil of the CNS. In the NMJ of null brec mutants, all known glutamate receptor subunits are undetectable by immunocytochemistry, and all functional glutamate receptors are eliminated. Thus, we conclude that GluRIID is essential for the assembly and/or stabilization of glutamate receptors in the NMJ. In null brec mutant embryos, the frequency of periodic excitatory currents in motor neurons is significantly reduced, demonstrating that CNS motor pattern activity is regulated by GluRIID. Although synaptic development and molecular differentiation appear otherwise unperturbed in null mutants, viable hypomorphic brec mutants display dramatically undergrown NMJs by the end of larval development, suggesting that GluRIID-dependent central pattern activity regulates peripheral synaptic growth. These studies reveal GluRIID as a newly identified glutamate receptor subunit that is essential for glutamate receptor assembly/stabilization in the peripheral NMJ and required for properly patterned motor output in the CNS.

Animals↗

Aedes albopictus (Diptera: Culicidae): physiological aspects of development and reproduction.

Basic ecophysiological data are presented on the development and reproduction of Aedes albopictus (Skuse) that were reared and maintained at four temperatures between 12 and 32 degrees C. Median larval developmental time from hatching to pupation was correlated inversely with temperature, lasting 7 d at 32 degrees C and up to 28 d at 12 degrees C. Duration of the pupal period also varied from 2-3 d at 32 degrees C to 7-12 d at 12 degrees C. This extension of larval development elongated the phagoperiod and gave rise to larger imagoes. Based on wing length measurements, body sizes varied from 10 to 57 mm3 for females and from 10 to 30 mm3 for males. The caloric protein content at emergence showed a linear and significant regression with body size, independent of sex, treatment, or temperature. Teneral lipid content also followed a linear relationship with body size at warmer temperatures, whereas at low temperatures it increased exponentially with body size. Glycogen was always below 10% of the protein or lipid levels. Reserves at emergence determined median adult survival times, which ranged from 16 d at 32 degrees C to 100 d at 17 degrees C. Access to sucrose solution allowed females to increase their teneral glycogen up to fourfold within 1 wk, and their lipids up to 10-fold within 2 wk. Despite abroad variation, the number of mature oocytes (15-110 eggs per female) was correlated positively with body size, but inversely with the rearing and maintenance temperature. Utilization of the blood meal protein for oogenesis ranged between 35 and 50%, again inversely correlated with temperature; absolute compositions per oocyte were 6.3-6.5 mcal of protein but ranged from 5 to 7 mcal of lipid.

Aedes↗

An overview of Culicoides control.

Although most of the research on ceratopogonid control has been directed at these biting flies as human pests, sufficient information is available to indicate the value of certain techniques in the suppression or control of bluetongue (BT) through Culicoides control. In areas where there is a limited number of larval development sites, larviciding or sanitation can be practiced by individual farmers or ranchers or by a small group of contiguous landowners. Where larval sites are more numerous or where large areas and many landowners are involved, vector control would need to be coordinated by a central agent or agency and would have to be coupled with other techniques aimed at the host animals or the virus into a totally integrated system of disease management.

Animals↗

Cadherin-17 is required to maintain pronephric duct integrity during zebrafish development.

We have isolated a zebrafish cadherin that is orthologous to human LI-cadherin (CDH17). Zebrafish cdh17 is expressed exclusively in the pronephric ducts during embryogenesis, and in the mesonephros during larval development and adulthood. Like its mammalian ortholog, cdh17 is also expressed in liver and intestine in adult zebrafish. We show that cdh17-positive mesodermal cells do not contribute to the hematopoietic system. Consistent with a cell adhesion role for Cdh17, depletion of Cdh17 function using antisense morpholino oligonucleotides compromised cell cohesion during pronephric duct formation. Our results indicate that Cdh17 is necessary for maintaining the integrity of the pronephric ducts during zebrafish embryogenesis. This finding contrasts with the role of mammalian CDH17, which does not appear to be involved in nephric development.

Amino Acid Sequence↗

The effect of gamma and microwave irradiation of wheat grain on development parameters of some stored grain pests.

Wheat grain was treated with different doses of gamma irradiation or different times of microwaves. Gamma rays (0.05-10 kGy) caused statistically significant differences in soluble protein contents and kernel hardness. Microwave treatment (15-180 s) resulted in some statistically significant changes in moisture and soluble protein contents. Mortality, intensity of feeding, fertility of adults, progeny number and development time of larvae of the granary weevil (Sitophilus granarius L.) on treated grain were observed. On the basis of progeny number and larval development time the coefficient of food value (CFV) was calculated. Some statistical differences were stated in studied parameters, however no one in all calculated coefficients of food values. The same treated grain was also used as a food for larvae of the confused flour beetle (Tribolium confusum Duv.) and Mediterranean flour moth (Ephestia kuehniella Zell.). Mortality of larvae and their development time were studied. Only gamma irradiated wheat grain at the dose 0.1 kGy used as a food for larvae of T. confusum Duv. caused statistically significant extension of development time by 7 days, when comparing to the control food. On the other hand, microwave treatment of wheat grain used as food significantly shorten the development time of larvae in both species.

Animals↗

Response to selection and evolvability of invasive populations.

While natural selection might in some cases facilitate invasions into novel habitats, few direct measurements of selection response exist for invasive populations. This study examined selection response to changes in salinity using the copepod Eurytemora affinis. This copepod has invaded fresh water from saline habitats multiple times independently throughout the Northern Hemisphere. Selection response to a constant intermediate salinity (5 PSU) was measured in the laboratory for saline source and freshwater invading populations from the St. Lawrence drainage (North America). These populations were reared under three conditions: (1) native salinities (0 or 15 PSU) for at least two generations, (2) 5 PSU for two generations, and (3) 5 PSU for six generations. Full-sib clutches taken from populations reared under these three conditions were split across four salinities (0, 5, 15, and 25 PSU) to determine reaction norms for survival and development time. Contrasts in survival and development time across the three rearing conditions were treated as the selection response. Selection at 5 PSU resulted in a significant decline in freshwater (0 PSU) tolerance for both the saline and freshwater populations. Yet, evolutionary differences in freshwater tolerance persisted between the saline and freshwater populations. The saline and freshwater populations converged in their high-salinity (25 PSU) tolerance, with an increase in the freshwater population and decline in the saline population. Development time did not shift greatly in response to selection at 5 PSU. For all three rearing conditions, the freshwater population exhibited retarded larval development and accelerated juvenile development relative to the saline population. Results from this study indicate that both the saline and freshwater populations exhibit significant responses to selection for a fitness-related trait critical for invasions into a novel habitat.

Animals↗

Krüppel, a gene whose activity is required early in the zygotic genome for normal embryonic segmentation.

Embryos homozygous for Krüppel die as late embryos with an altered segmentation pattern. In strong alleles the normal thoracic and anterior abdominal segments are replaced by a partial mirror image duplication of the posterior abdomen. Weak alleles cause smaller pattern deletions in the thorax and abdomen and are not associated with mirror image duplications. The altered segmentation pattern can be traced back to 12 min after the onset of gastrulation, when the shorter germ bands in homozygous Kr embryos provide a first indication of abnormal patterning. The mutant was mapped to position 107.6 at the tip of the right arm of the second chromosome, cytologically to bands 60F2-5. Analysis of homozygous deficiency embryos indicate that the phenotype produced by strong point mutations probably represents the amorphic condition. The requirement for Kr+ gene activity is strictly zygotic. Maternal dosage of Kr+ has no effect on the embryonic phenotype, nor does homozygosity for Kr prevent germ cells from making normal eggs capable of normal embryonic development when fertilized by wild-type sperm. The requirement for Kr+ seems restricted to embryogenesis. Homozygous clones induced in imaginal discs during larval development survive and develop normally and in vivo cultures established from homozygous embryos proliferate normally and metamorphose into adult structures of normal morphology.

Abdomen↗

Insulin-like immunoreactivity in serum and pancreas of metamorphosing tadpoles.

Insulin-like immunoreactivity (ILI) in pancreatic extracts and serum of Rana catesbeiana tadpoles of various developmental stages was determined by radioimmunoassay. Serum ILI levels increased from 2.4 +/- 0.4 microU/ml in limb bud stage tadpoles (I-V) to 13.9 +/- 2.2 microU/ml in foot stage animals (XI-XVII). During climax, serum ILI levels declined reaching 5.0 microU/ml in postmetamorphic juveniles. Embryonic pancreases (stage 25) contained sizeable amounts of ILI (115 +/- 14.4 microU). A fourfold increase in total pancreatic ILI occurred during the limb bud stages, and a twelvefold increase occurred by the end of the foot development stages. Total pancreatic ILI declined throughout climax stages to 410 microU in postmetamorphic juveniles. Serum glucose increased slightly over the course of larval development; the peak concentration occurred in foot stage tadpoles.

Animals↗

The Drosophila melanogaster LEM-domain protein MAN1.

Here we describe the Drosophila melanogaster LEM-domain protein encoded by the annotated gene CG3167 which is the putative ortholog to vertebrate MAN1. MAN1 of Drosophila (dMAN1) and vertebrates have the following properties in common. Firstly, both molecules are integral membrane proteins of the inner nuclear membrane (INM) and share the same structural organization comprising an N-terminally located LEM motif, two transmembrane domains in the middle of the molecule, and a conserved RNA recognition motif in the C-terminal region. Secondly, dMAN1 has similar targeting domains as it has been reported for the human protein. Thirdly, immunoprecipitations with dMAN1-specific antibodies revealed that this Drosophila LEM-domain protein is contained in protein complexes together with lamins Dm0 and C. It has been previously shown that human MAN1 binds to A- and B-type lamins in vitro. During embryogenesis and early larval development LEM-domain proteins dMAN1 and otefin show the same expression pattern and are much more abundant in eggs and the first larval instar than in later larval stages and young pupae whereas the LEM-domain protein Bocksbeutel is uniformly expressed in all developmental stages. dMAN1 is detectable in the nuclear envelope of embryonic cells including the pole cells. In mitotic cells of embryos at metaphase and anaphase, LEM-domain proteins dMAN1, otefin and Bocksbeutel were predominantly localized in the region of the two spindle poles whereas the lamin B receptor and lamin Dm0 were more homogeneously distributed. Downregulation of dMAN1 by RNA interference (RNAi) in Drosophila cultured Kc167 cells has no obvious effect on nuclear architecture, viability of RNAi-treated cells and the intracellular distribution of the LEM-domain proteins Bocksbeutel and otefin. In contrast, the localization of dMAN1, Bocksbeutel and otefin at the INM is supported by lamin Dm0. We conclude that the dMAN1 protein is not a limiting component of the nuclear architecture in Drosophila cultured cells.

Amino Acid Motifs↗

Mild mutations in the pan neural gene prospero affect male-specific behaviour in Drosophila melanogaster.

The fruitfly Drosophila melanogaster is one of the most appropriate model organisms to study the genetics of behaviour. Here, we focus on prospero (pros), a key gene for the development of the nervous system which specifies multiple aspects from the early formation of the embryonic central nervous system to the formation of larval and adult sensory organs. We studied the effects on locomotion, courtship and mating behaviour of three mild pros mutations. These newly isolated pros mutations were induced after the incomplete excision of a transposable genomic element that, before excision, caused a lethal phenotype during larval development. Strikingly, these mutant strains, but not the strains with a clean excision, produced a high frequency of heterozygous flies, after more than 50 generations in the lab. We investigated the factors that could decrease the fitness of homozygotes relatively to heterozygous pros mutant flies. Flies of both genotypes had slightly different levels of fertility. More strikingly, homozygous mutant males had a lower sexual activity than heterozygous males and failed to mate in a competitive situation. No similar effect was detected in mutant females. These findings suggest that mild mutations in pros did not alter vital functions during development but drastically changed adult male behaviour and reproductive fitness.

Alleles↗

Essential amino acids and other essential components for development of Angiostrongylus costaricensis from third-stage larvae to young adults.

Third-stage larvae of Angiostrongylus costaricensis were cultured to the young adult stage in Waymouth's chemically defined medium MB 752/1, which contained 18 amino acids, 11 vitamins, glutathione, hypoxanthine, and glucose in a balanced salt solution. Nutritional requirements were examined by deletion of single components from Waymouth's medium. Ten amino acids, namely L-arginine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, and L-valine, were shown to be essential for the parasite's development. L-Aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, glycine, L-proline, and L-tyrosine were not essential. Among the 11 vitamins, only choline chloride was essential for the development. The deletion of pyridoxine from the medium adversely affected parasite development. Glucose was also required by the worms, but glutathione and hypoxanthine were not required for their development. When the 10 essential L-amino acids were replaced individually by D-isomers of the same amino acids, none supported larval development in the manner of the L-amino acids.

Amino Acids, Essential↗

Seasonality and genetic architecture of development time and body size of the birch feeding sawfly Priophorus pallipes.

We tested, using the sawfly Priophorus pallipes feeding on leaves of mountain birch, whether the expression of genetic (co)variation of larval development time and body size can be altered by exposing larvae to diets with differential seasonal changes in quality. In nature, larvae feed mainly on mature leaves, but occasionally they are forced to consume senescing leaves. Sixty families were assayed on three experimentally simulated diets: mature leaves of high quality, senescing leaves of rapidly declining quality, and senesced leaves of low quality. The intuitively obvious positive phenotypic and genetic correlations between development time and final mass were observed when the larvae consumed leaves of stable high quality, but low and declining food quality prevented long-growing individuals and families from achieving high final mass, switching the correlations to close to zero or negative in these treatments. The amount of genetic variation for body size showed a non-linear change across the diet quality gradient, whereas genetic variation for development time increased with decreasing diet quality. The among-trait difference in the degree reaction norms crossed along the diet gradient caused the changes in the expression of genetic (co)variation within the environments. Our results show that seasonally varying diet quality induces dramatic changes in the genetic (co)variation of development time and body size, and that simultaneous analysis of reaction norms and environment-specific expression of genetic (co)variation is necessary for the understanding of the genetic characteristics underlying the construction of phenotypes in heterogeneous environments.

Analysis of Variance↗

Thermal accumulation and the early development of Ixodes scapularis.

We examined the relationship between the accumulation of thermal energy and the onset of oviposition and eclosion of the northern deer tick, Ixodes scapularis, and explored the usefulness of comparing degree days (DD) required for larval emergence with area-wide National Weather Service (NWS) data to construct maps indicating where the establishment of this vector tick would be climatologically constrained. Initially, the validity of basal temperatures for egg and larval development was confirmed by prolonged incubations of gravid females and eggs at 6 degrees C and 10 degrees C respectively. Next, the number of DD accumulated in situ from the placement of gravid females to oviposition, and from oviposition to larval emergence, were measured using temperature data loggers placed next to fall- and spring-fed ticks held within individual vials under leaf litter in multiple enclosures located in diverse biophysical regions of Maine. Finally, when it was found that total DD to larval emergence, as measured in ambient air above the enclosures, compared favorably with DD accumulated simultaneously at nearby NWS stations, maps were constructed, based on archived NWS data, to demonstrate where temperatures were sufficient to allow the hatching of larvae both within one season and over the last three decades as I. scapularis has advanced into northern New England.

Animals↗

Specificity of motoneuron projection patterns during development of the bullfrog tadpole (Rana catesbeiana).

The pattern of connectivity between motoneurons of the lumbar lateral motor column (LMC) and hindlimb regions was examined in bullfrog tadpoles (Rana catesbeiana) over the course of larval development. The purpose of this study was to determine if a period of relatively imprecise connectivity, such has been found in the toad Xenopus laevis (Lamb, '76), could be identified. Patterns of connectivity were assessed by placing small amounts of the retrogradely transported enzyme horseradish peroxidase (HRP) into discrete hindlimb regions and mapping the locations of labeled motoneuron somata along the transverse and longitudinal axes of the LMC. The distribution of labeled motoneurons was as circumscribed in the youngest animals studied (st. IV of Taylor and Kollros, '46), before mesenchymal condensation into distinct myotubes, as in metamorphic or adult animals. This finding that the pattern of neuromuscular connectivity is as precise early in development as in mature animals is consistent with previous studies of chick hindlimb (Landmesser, '78). The relevance of these results to the hypothesis that naturally occurring cell death plays a substantial part in molding the mature pattern of neuromuscular connectivity (Lamb, '77) is discussed.

Animals↗

Inducibility of metallothionein mRNA expression and cadmium tolerance in larvae of a marine teleost, the turbot (Scophthalmus maximus).

An acute (48-hr) lethal toxicity study of turbot at various stages from hatch to juveniles showed that prior to first exogenous feeding, larvae of turbot were an order of magnitude more sensitive to cadmium toxicity than later developmental stages (LC50 of 0.18-0.23 ppm Cd versus 2-5 ppm Cd). To investigate the possible role of metallothionein (MT) in Cd tolerance, the concentrations of MT mRNA were determined in control and Cd-exposed animals using a plaice MT cDNA probe. MT mRNA was expressed throughout embryonic and early larval development at levels approximately twice those in juvenile turbot liver apart from the period around hatching, when there was a decreased level of expression. During endogenous feeding, despite larval drinking, MT mRNA levels did not appear to be inducible by exposure to Cd in the water within 3 days of hatching but were induced three- to fivefold after 48 hr exposure to waterborne Cd at all later stages of development. The results suggest that Cd tolerance is associated with metal induction of MT gene transcription and that the enhanced sensitivity of very early stage larvae is attributable to a deficiency in MT gene transcription.

Animals↗