Genetic effects of cosmic radiation in Drosophila melanogaster]

To examine possible effects of space radiation on living organism, we have analyzedtwo types of mutations, sex-linked recessive lethal mutations and somatic mutations, in fruit fly of the species Drosophila melanogaster. Drosophila strains used were wild type strains and a radiation-sensitive strain mei-41. Two different developmental stages of samples were sent into space; young adult males to analyze sex-linked recessive lethal mutations and about 30hr-old larvae to detect somatic mutations in wing epidermal cells. For wild type and mei-41 strains each, about 200 adult male flies and about 6,000 larvae were loaded on space shuttle Endeavour. The male flies returned from space were mated to virgin female flies of a tester strain, and the presence of the lethal mutations was analyzed at F2 generation. The frequencies of sex-linked recessive lethal mutations in flight groups were 2 and 3 times higher for wild type Canton-S and mei-4 1, respectively, than those in ground control groups. Most larvae sent to space emerged as adult flies within about 10 days after the landing. The presence of wing-hair somatic mutations, which give morphological change in hairs growing on the surface of wing epidermal cells, was analyzed under microscope. In wild type strain Muller-5, the frequency of wing hair mutant spots in flight group was about 1.5-fold higher than that in ground control, and in Canton-S-derived wild type strain the frequencies were similar between the two groups. By contrast, for mei-41 strain the mutation frequency was lower in flight group than in control group. The observed higher frequency of lethal mutations in the flight group might be due to a possibility that radiation effects on reproductive cells could be greatly enhanced under micro gravity. However, if this would be the case, we do not have appropriate explanation for the apparent absence of such synergistic effects on somatic wing-hair mutation system.     

Ultrastructure, development, and homology of insect embryonic cuticles

Ultrastructure and deposition of the cuticles secreted by embryos representing eight insect orders were examined by transmission and scanning electron microscopy. Embryos of the apterygote silverfish Thermobia domestica deposit two embryonic cuticles. Deposition of the first (EC1) is initiated at the beginning of appendage development when the intercalary segment and the neural groove are clearly visible. This cuticle lacks surface microsculpture and consists of an outer epicuticle and an underlying fibrous layer, thought to represent procuticle. At the time of dorsal closure, deposition of a second embryonic cuticle (EC2) begins; this bears sensilla and functions in the first instar larva. In representative embryos of seven pterygote orders (Ephemeroptera, Odonata, Plecoptera, Neuroptera, Coleoptera, Lepidoptera, and Mecoptera), three cuticles were found to be secreted. The first cuticle in pterygotes is homologous to EC1 of T. domestica, but consists solely of outer epicuticle. EC2, the "prolarval cuticle," bears a characteristic surface microsculpture in embryos of some species and egg-teeth and other hatching devices, and consists of outer and inner epicuticles and a more or less reduced procuticle. EC2 is reduced in the embryos of derived endopterygotes, where a procuticle is lacking and the inner epicuticle is reduced. After hatching, when EC2 is shed, the first instar larva is covered by a third embryonic cuticle (EC3), whose deposition was initiated while the insect was still within the egg. Presence of only two embryonic cuticles in cyclorrhaphous flies is due to the total loss of prolarval cuticle. Investigated exopterygote and endopterygote insects excluding flies thus deposit three embryonic cuticles, and their juveniles (exopterygote "nymphs"; endopterygote "larvae") seem to hatch at equivalent stages of development. Differences between the modes of cuticulogenesis in silverfish and pterygote embryos suggest that the apterygote first larval instar was embryonized and became a fully embryonic prolarva in pterygotes.     

Hybrid Lethal Systems in the Drosophila Melanogaster Species Complex. II. the Zygotic Hybrid Rescue (Zhr) Gene of D. Melanogaster

Hybrid females from Drosophila simulans females X Drosophila melanogaster males die as embryos while hybrid males from the reciprocal cross die as larvae. We have recovered a mutation in melanogaster that rescues the former hybrid females. It was located on the X chromosome at a position close to the centromere, and it was a zygotically acting gene, in contrast with mhr (maternal hybrid rescue) in simulans that rescues the same hybrids maternally. We named it Zhr (Zygotic hybrid rescue). The gene also rescues hybrid females from embryonic lethals in crosses of Drosophila mauritiana females X D. melanogaster males and of Drosophila sechellia females X D. melanogaster males. Independence of the hybrid embryonic lethality and the hybrid larval lethality suggested in a companion study was confirmed by employing two rescue genes, Zhr and Hmr (Hybrid male rescue), in doubly lethal hybrids. A model is proposed to explain the genetic mechanisms of hybrid lethalities as well as the evolutionary pathways.     

Effects of spaceflight conditions on fertilization and embryogenesis in the sea urchin Lytechinus pictus

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. Calcium and cytoskeletal events were investigated within sea urchin embryos which were cultured in space under both microgravity and 1 g conditions. Embryos were fixed at time-points ranging from 3 h to 8 days after fertilization. Investigative emphasis was placed upon: (1) sperm-induced calcium-dependent exocytosis and cortical granule secretion, (2) membrane fusion of cortical granule and plasma membranes; (3) microfilament polymerization and microvilli elongation; and (5) embryonic development into morula, blastula, gastrula, and pluteus stages. For embryos cultured under microgravity conditions, the processes of cortical granule discharge, fusion of cortical granule membranes with the plasma membrane, elongation of microvilli and elevation of the fertilization coat were reduced in comparison with embryos cultured at 1 g in space and under normal conditions on Earth. Also, 4% of all cells undergoing division in microgravity showed abnormalities in the centrosome-centriole complex. These abnormalities were not observed within the 1 g flight and ground control specimens, indicating that significant alterations in sea urchin development processes occur under microgravity conditions.     

Characterization of X-Linked Recessive Lethal Mutations Affecting Embryonic Morphogenesis in Drosophila Melanogaster

This study attempted to assay the zygotic contribution of X chromosome genes to the genetic control of embryonic morphogenesis in Drosophila melanogaster. A systematic screen for X-linked genes which affect the morphology of the embryo was undertaken, employing the phenotype of whole mount embryos as the major screening criterion. Of 800 EMS-induced lethal mutations analyzed, only 14% were embryonic lethal, and of these only a minority affected embryonic morphogenesis. By recombination and complementation analyses, the mutations that affected embryonic morphogenesis were sequestered into 26 complementation groups. Fourteen of the loci correspond to genes previously identified in a large-scale screen in which fixed cuticles were examined, and 12 new loci have been identified. Most of the mutations which disrupt embryonic morphology had specific and uniform mutant phenotypes. Mutations were recovered which disrupt major morphogenetic events such as gastrulation, germ band retraction and head involution. No mutations were found which arrest the embryos prior to blastoderm formation. However, a novel class was found, one comprised of mutations which interfere with the development of internal structures but not cuticular structures. Nevertheless, saturation of the X chromosome for genes important for embryonic morphogenesis is probably incomplete.     

An infrared system for monitoring Drosophila motility during microgravity

Presently, the precise mechanisms of the aging process are unknown. Examination and comprehension of the aging process in other species could lead to significant advances in the understanding of human aging. Drosophila melanogaster (fruit fly), commonly used for aging studies, is a widely studied organism in terms of behavior, development, and genetics. Previous microgravity experiments have shown a significant decrease in the life span of young male Drosophila after microgravity exposure. This decrease in lifespan may be related to locomotor activity, a convenient measure of overall physiological performance. This study describes the design and performance of a Drosophila Infrared Motility Monitoring System (DIMMS). The DIMMS uses a unique design of two infrared (IR) beams per fly to measure the locomotor activity of 240 flies. Locomotor activity is measured in terms of number of IR crossings per unit time, instantaneous velocity, and continuous velocity. Ground-based results using the DIMMS equipment agree well with previous values for Drosophila locomotor velocity. DIMMS is an improvement over equipment previously used due to its ability to continuously monitor locomotor activity throughout short-duration microgravity exposure. DIMMS is also lightweight, compact, and power efficient. DIMMS has been flight tested onboard NASA's KC-135 reduced gravity research aircraft and a Nike-Orion sounding rocket.     

Myosin heavy chain isoforms regulate muscle function but not myofibril assembly.

Myosin heavy chain (MHC) is the motor protein of muscle thick filaments. Most organisms produce many muscle MHC isoforms with temporally and spatially regulated expression patterns. This suggests that isoforms of MHC have different characteristics necessary for defining specific muscle properties. The single Drosophila muscle Mhc gene yields various isoforms as a result of alternative RNA splicing. To determine whether this multiplicity of MHC isoforms is critical to myofibril assembly and function, we introduced a gene encoding only an embryonic MHC into Drosophila melanogaster. The embryonic transgene acts in a dominant antimorphic manner to disrupt flight muscle function. The transgene was genetically crossed into an MHC null background. Unexpectedly, transformed flies expressing only the embryonic isoform are viable. Adult muscles containing embryonic MHC assemble normally, indicating that the isoform of MHC does not determine the dramatic ultrastructural variation among different muscle types. However, transformed flies are flightless and show reduced jumping and mating ability. Their indirect flight muscle myofibrils progressively deteriorate. Our data show that the proper MHC isoform is critical for specialized muscle function and myofibril stability.     

Expression of Ixodes scapularis antifreeze glycoprotein enhances cold tolerance in Drosophila melanogaster

Drosophila melanogaster experience cold shock injury and die when exposed to low non-freezing temperatures. In this study, we generated transgenic D. melanogaster that express putative Ixodes scapularis antifreeze glycoprotein (IAFGP) and show that the presence of IAFGP increases the ability of flies to survive in the cold. Male and female adult iafgp-expressing D. melanogaster exhibited higher survival rates compared with controls when placed at non-freezing temperatures. Increased hatching rates were evident in embryos expressing IAFGP when exposed to the cold. The TUNEL assay showed that flight muscles from iafgp-expressing female adult flies exhibited less apoptotic damage upon exposure to non-freezing temperatures in comparison to control flies. Collectively, these data suggest that expression of iafgp increases cold tolerance in flies by preventing apoptosis. This study defines a molecular basis for the role of an antifreeze protein in cryoprotection of flies.     

Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development

We developed a simple methodology for labeling sulfated glycosaminoglycans (GAGs) in adult Drosophila melanogaster and studied some aspects of the biosynthesis and metabolism of these polymers during development. Adult D. melanogaster flies were fed with Na(2)(35)SO(4) for 72 h. During this period, (35)S-sulfate was incorporated into males and females and used to synthesize (35)S-sulfate-heparan sulfate (HS) and (35)S-sulfate-chondroitin sulfate (CS). The incorporation of (35)S-sulfate into HS was higher when compared to CS. In a pulse-chase experiment, we observed that (35)S-sulfate incorporated into adult female was recovered in embryos and used for the synthesis of new (35)S-sulfate-GAGs after 2 h of embryonic development. The synthesis of CS was higher than that of HS, indicating a change in the metabolism of these glycans from adult to embryonic and larval stages. Analysis of the CS in embryonic and larval tissues revealed the occurrence of nonsulfated and 4-sulfated disaccharide units in embryos, L1 and L2. In L3, in addition to these disaccharides, we also detected significant amount of 6-sulfated units that are reported here for the first time. Immunohistochemical analysis indicated that HS and CS were present in nonequivalent structures in adult and larval stages of the fly. Overall, these results indicate that (35)S-sulfate-precursors are transferred from adult to embryonic and larval tissues and used to assemble different morphological structures during development.     

Genetic control of cuticle formation during embryonic development of Drosophila melanogaster

The embryonic cuticle of Drosophila melanogaster is deposited by the epidermal epithelium during stage 16 of development. This tough, waterproof layer is essential for maintaining the structural integrity of the larval body. We have characterized mutations in a set of genes required for proper deposition and/or morphogenesis of the cuticle. Zygotic disruption of any one of these genes results in embryonic lethality. Mutant embryos are hyperactive within the eggshell, resulting in a high proportion reversed within the eggshell (the "retroactive" phenotype), and all show poor cuticle integrity when embryos are mechanically devitellinized. This last property results in embryonic cuticle preparations that appear grossly inflated compared to wild-type cuticles (the "blimp" phenotype). We find that one of these genes, krotzkopf verkehrt (kkv), encodes the Drosophila chitin synthase enzyme and that a closely linked gene, knickkopf (knk), encodes a novel protein that shows genetic interaction with the Drosophila E-cadherin, shotgun. We also demonstrate that two other known mutants, grainy head (grh) and retroactive (rtv), show the blimp phenotype when devitellinized, and we describe a new mutation, called zeppelin (zep), that shows the blimp phenotype but does not produce defects in the head cuticle as the other mutations do.     

The sea urchin larva, a suitable model for biomineralisation studies in space (IML-2 ESA Biorack experiment '24-F urchin')

By the ESA Biorack 'F-24 urchin' experiment of the IML-2 mission, for the first time the biomineralisation process in developing sea urchin larvae could be studied under real microgravity conditions. The main objectives were to determine whether in microgravity the process of skeleton formation does occur correctly compared to normal gravity conditions and whether larvae with differentiated skeletons do 'de-mineralise'. These objectives have been essentially achieved. Postflight studies on the recovered 'sub-normal' skeletons focused on qualitative, statistical and quantitative aspects. Clear evidence is obtained that the basic biomineralisation process does actually occur normally in microgravity. No significant differences are observed between flight and ground samples. The sub-normal skeleton architectures indicate, however, that the process of positioning of the skeletogenic cells (determining primarily shape and size of the skeleton) is particularly sensitive to modifications of environmental factors, potentially including gravity. The anatomical heterogeneity of the recovered skeletons, interpreted as long term effect of an accidental thermal shock during artificial egg fertilisation (break of climatisation at LSSF), masks possible effects of microgravity. No pronounced demineralisation appears to occur in microgravity; the magnesium component of the skeleton seems yet less stable than the calcium. On the basis of these results, a continuation of biomineralisation studies in space, with the sea urchin larva as model system, appears well justified and desirable.     

黑腹果蝇Mst84Db敲降引起的父本缺陷类似Wolbachia诱导的细胞质不亲和

【目的】Wolbachia 是广泛存在于昆虫体内的一类通过母系传递的共生菌,能够通过多种方式影响宿主的生殖。细胞质不亲和（CI）是Wolbachia 引起的最普遍的一种表型,即感染Wolbachia的雄性和未感染的雌性宿主交配后,胚胎发育停滞于早期阶段。但目前有关CI的分子机理还不清楚。本研究组前期实验表明,Wolbachia感染引起黑腹果蝇Drosophila melanogaster 3龄幼虫精巢中Mst84Db基因的表达显著下调。本研究的目的是进一步研究Mst84Db与CI的关系。【方法】我们体外合成了Mst84Db的双链RNA（dsRNA）,注射雄性果蝇,将注射过的雄果蝇与野生雌果蝇交配,检测其繁殖力。基因表达采用定量RT-PCR方法进行检测。胚胎表型采用DAPI染色进行分析。【结果】注射dsRNA 24 h后,Mst84Db基因的表达水平发生显著下调。注射后72 h,基因表达下调幅度最大。与对照组相比,基因敲降后的雄蝇繁殖能力显著下降,与雌果蝇交配后胚胎孵化率显著低于对照组,这与Wolbachia诱导的CI现象类似。未孵化胚胎的表皮上没有体节出现,说明胚胎停滞于发育的早期。部分胚胎细胞核分裂不同步,且有染色质间桥出现,这也与CI胚胎中的细胞学表型一致。【结论】Wolbachia感染可能抑制果蝇精子发生过程中Mst84Db基因的表达,从而使精子失去正常功能,最终导致与雌性果蝇交配后,胚胎发育停滞,并最终死亡。Mst84Db基因在雄性果蝇中表达下调可能是产生CI的重要原因之一。     

The embryonic expression patterns of zfh-1 and zfh-2, two Drosophila genes encoding novel zinc-finger homeodomain proteins.

The zfh-1 and zfh-2 genes of D. melanogaster encode novel proteins containing both homeodomain and C2-H2 zinc-finger DNA-binding motifs. Antisera against these proteins were used to investigate their expression patterns during embryonic development. The zfh-1 gene is expressed in the mesoderm of early embryos and in a number of mesodermally-derived structures of late embryos, including the dorsal vessel, support cells of the gonads, and segment-specific arrays of adult muscle precursors. In addition, zfh-1 is expressed in the majority of identified motor neurons of the developing CNS. The mesodermal zfh-1 expression requires the products of the twist and snail genes. The zfh-2 gene displays a more limited expression pattern, largely restricted to the CNS of late embryos. Ubiquitous zfh-1 expression in transgenic flies bearing an hsp70-zfh-1 construct has specific developmental consequences, including embryonic CNS defects as well as adult eye and bristle abnormalities. The expression patterns of zfh-1 and zfh-2 suggest that both genes may be involved in Drosophila neurogenesis and that zfh-1 may have additional functions in mesoderm development.     

Sensitivity of wild type and recombination-deficient strains of Drosophila melanogaster to ultraviolet radiation.

The sensitivity of Drosophila melanogaster to ultraviolet light has been studied in wild type and recombination-deficient strains. Survival was measured as the proportion of irradiated embryos or larvae which developed to adult flies. In view of the fact that males of this species do not participate in meiotic recombination, emphasis was placed on the relative sensitivity of males and females. The results show that young wild type male larvae are more sensitive to UV radiation than are young female larvae. This difference in sensitivity, however, is not apparent in some recombination-deficient strains. In addition, young embryos of the recombination-deficient strain Df(3)sbd105/T(2;E)Xa are exceptionally sensitive to UV radiation.     

Impact of microgravity and hypergravity on free-running circadian rhythm of the desert beetle Trigonoscelis gigas Reitt

Free-running circadian rhythms of locomotor activity of Tenebrionid beetles Trigonoscelis gigas Reitt., taken from the Turkmenian sand desert, were monitored in DD. The effects of microgravity --11 days in space flight aboard the Russian BION-10 "COSMOS" satellite, and of 2G hypergravity--seven days on a centrifuge, were determined. Two kinds of effects were found. In stable 2-peak records, there was a moderate decrease of tau in microgravity and an increase of tau in 2G, both of about 0.3 hr. In unstable records, alterations of gravity caused drastic deviations of tau and phi. Remarkably, two peaks of the activity rhythm, which are supposed to be controlled by separate oscillators, responded to gravity transitions in different ways. Gravity effects on the circadian system could be explained from a direct effect on the oscillator(s) itself or from a feed-back by altered locomotion to the pacemaker. Thus, for the first time the gravity dependence of a free-running circadian rhythm was proved in a combination of real space flight and centrifuge experiments.     

Effects of microgravity on cell cytoskeleton and embryogenesis

The aim of this review is to compile, summarize and discuss the effects of microgravity on embryos, cell structure and function that have been demonstrated from data obtained during experiments performed in space or in altered gravity induced by clinostats. In cells and tissues cellular structure and genetic expression may be changed in microgravity and this has a variety of effects on embryogenesis which include death of the embryo, failure of neural tube closure, or final deformities to the overall morphology of the newborn or hatchling. Many species and protocols have been used for microgravity space experiments making it difficult to compare results. Experiments on the ways in which embryonic development and cell interactions occur in microgravity could also be performed. Experiments that have been done with cells in microgravity show changes in morphology, cytoskeleton and function. Changes in cytoskeleton have been noted and studies on microtubules in gravity have shown that they are gravity sensitive. Further study of basic chemical reactions that occur in cells should be done to shed some light on the underling processes leading to the changes that are observed in cells and embryos in microgravity.     

模拟微重力环境对昆明小鼠早期胚胎体外发育的影响（简报）

In order to investigate the development of Kunming mouse preimplantation embryos cultured in vitro under simulated microgravtiy, one-cell and 4-cell embryos of Kunming mouse (Fertilization In Situ) were cultured in CZB or KSOM media under simulated microgravity or normal gravity environment respectively. The results showed that under normal gravity, the percentage of passing 2-cell development block embryos was not different in CZB with in KSOM, but the percentage of blastocysts was lower in CZB than that in KSOM significantly. The percentages of passing 2-cell development block embryos and blastocysts in CZB or KSOM were lower under simulated microgravity than those under normal gravity. It is suggested that the frequency of early embryonic lethality is possibly increased by simulated microgravity.