Heat shock during the development of brain structures of Drosophila: the memory development in the l(1)ts403 mutant of Drosophila melanogaster

The structures and functions of many genes are homologous in Drosophila and humans. Therefore, studying pathological processes in Drosophila, in particular neurogenerative processes accompanied by progressive memory loss, helps to understand the ethiology of corresponding human disorders and to develop therapeutic strategies. It is believed that the development of neurogenerative diseases might result from alterations in the functioning of the heat shock/chaperone machinery. In view of this, we used Drosophila mutant l(1)ts403 with defective synthesis of heat shock proteins for studying learning and memory in a test of conditioned courtship suppression following a heat shock given at different developmental stages. High learning indices were registered immediately and 30 min after training both in the intact controls and in flies subjected to different developmental heat shocks. This indicated normal learning and memory acquisition in the mutant. At the same time, memory retention (3 h after training) suffered to different extent depending on the developmental stage. The remote effects of heat shock given during the formation of the mushroom bodies indicated the important role of this brain structure in the memory formation. The observed memory defects may result from alterations both in mRNA transport and in the functions of molecular chaperones in the l(1)ts403 mutant.     

Heartbeat patterns during the postembryonic development of Drosophila melanogaster

Pulsations of the dorsal vessel were recorded in vivo during the whole postembryonic development of D. melanogaster, by means of a newly invented, pulse-light opto-cardiographic method. The young larvae of the 1st and 2nd instars submerged in the feeding medium exhibited extremely high rates of heartbeat, 7Hz at room temperature. These values are among the highest rates of heartbeat ever recorded in the animal kingdom. The fully grown larvae of the 3rd instar showed approximately half of the maximum heartbeat rate (3.5-4Hz), which became stabilized after pupariation to 2.5-2.7Hz.The larval heartbeat was always uni-directional, in the forward-oriented or anterograde direction and it was almost continuous. The slowly disintegrating, old larval heart used to beat at the constant frequency of 2.5-2.7Hz until complete cessation of all cardiac functions in 1-day-old puparium. In spite of the persisting constant heartbeat frequency, the transformation process of the larval heart was associated with successively decreasing amplitude of the systolic contractions and with the prolongation of the resting periods. The newly formed heart of the pupal-adult structure exhibited a qualitatively new pattern of heartbeat activity, which was manifested by periodic reversal of the heartbeat with the faster anterograde and slower retrograde phases. The frequencies of both of these reciprocal cardiac pulsations gradually increased during the advanced pharate adult period, reaching the values of 4-5Hz at the time of adult eclosion. Adult males and females also exhibited a perfect pattern of heartbeat reversal, with still very high rates of the anterograde heartbeat, in the range of 5-6Hz. In addition to the cardiac functions, we have recorded several kinds of extracardiac pulsations, which often interfered severely with the recordings of the heartbeat. There were strong, irregular extracardiac pulsations of a neurogenic nature (somatic muscles, oral armature) and relatively slow extracardiac pulsations of a myogenic nature (intestinal peristaltics, 0.2-0.3Hz). The extracardiac and cardiac pulsations were independent, their functions were not correlated. A possibility of creating new challenges in combination of molecular biology with the functional physiology of the heart have been discussed.     

Ecdysteroid-regulated heat-shock gene expression during Drosophila melanogaster development

Peaks in hsp 26, 28, and 83 RNA levels are correlated with peaks in ecdysteroid titers during mid-embryogenesis, pupariation, and mid-pupation, and with a peak in the level of RNA from the 74EF ecdysone puff at pupariation. Inhibition of the ecdysteroid peak at pupariation by temperature shift of the conditionally ecdysteroid-deficient strain ecd-1 was followed by a disappearance of hsp 26 RNA and a decline in hsp 83 RNA level; subsequent addition of exogeneous 20-OH-ecdysone to the temperature-shifted strain resulted in a severalfold increase in hsp 83 RNA level, and a dramatic increase in that of hsp 26. These results are consistent with the induction of the hsp 83, 28, and 26 genes by ecdysteroid at several developmental stages.     

Dolichol levels during development and ageing of Drosophila melanogaster

1. Dolichol levels in the fruit fly, Drosophila melanogaster were determined at the larva and pupa stages and in 1, 10, 20 and 30-day-old flies. 2. Free dolichol increased from 1.2 micrograms/g wet weight in the larvae to 14.9 micrograms/g in 30-day-old flies, while total dolichol increased from 3.4 micrograms/g in the larvae to 21.2 micrograms/g at 30-days-old. 3. Dolichol released after saponification is primarily from dolichyl fatty acid ester, which accounts for up to 65% of the total dolichol. 4. The major dolichol homologs, which remain relatively constant throughout development and ageing in D. melanogaster, are C-80, C-85 and C-90, which represent approximately 7%, 60% and 33%, respectively.     

Ecdysone titers during postembryonic development of Drosophila melanogaster.

The level of ecdysone in Drosophila melanogaster was determined by a radioimmune assay in organisms selected between the second larval instar and maturity. Maxima in the titer of the hormone were observed at puparium formation and 38 hr later, just prior to the secretion of the adult cuticle. The level of ecdysone was very low in adults of either sex. However, adult females had significantly more ecdysone per organism than did males. The magnitude of this difference could be correlated with ovarian development, suggesting a possible role for ecdysone in ovarian maturation in this organism.     

Expression of the disconnected gene during development of Drosophila melanogaster.

Proper development of the larval visual nerve, Bolwig's nerve, of Drosophila melanogaster requires the wild type function of the disconnected (disco) gene. In disco mutants, the nerve does not make stable connections with its targets in the larval brain. We have begun to explore the role of disco in the formation of the nervous system by examining the distribution of disco mRNA and protein in embryos and third instar larvae using in situ hybridization and antibody staining respectively. No differences between the distribution patterns of the two products are detected; disco is expressed in many tissues including both neural and non-neural cells. Many of the cells which express disco undergo extensive movement during development as they participate in major morphogenetic movements. Antibody staining shows that the protein is found in the cell nucleus. Products of the disco gene are detected in cells near the terminus of the growing Bolwig's nerve. In embryos homozygous for either of two mutant alleles of disco, the disco protein is absent near the nerve terminus, although protein distribution elsewhere is indistinguishable from wild type.     

Drosophila homolog of APP-BP1 (dAPP-BP1) interacts antagonistically with APPL during Drosophila development

beta-Amyloid precursor protein binding protein 1 (APP-BP1) was previously identified based on its binding to the carboxyl terminal of beta-amyloid precursor protein. In this report, we have discovered that a mutation of dAPP-BP1 (Drosophila ortholog of APP-BP1) hinders tissue development, causes apoptosis in imaginal disc cells, and blocks the NEDD8 conjugation pathway. We show that dAPP-BP1 specifically binds the intracellular domain of APP-like protein (APPL). The dAPP-BP1 mutation partially suppresses the abnormal macrochaete phenotype of Appl(d), while overexpression of dAPP-BP1 causes abnormal macrochaetes. When APPL is overexpressed, the normal bristle pattern in the fly thorax is disturbed and apoptosis is induced in wing imaginal discs. APPL overexpression phenotypes are enhanced by reducing the level of dAPP-BP1. APPL overexpression is shown to inhibit the NEDD8 conjugation pathway. APPL-induced apoptosis is rescued by overexpression of dAPP-BP1. Our data suggest that APPL and dAPP-BP1 interact antagonistically during Drosophila development.     

Dynamics of the endoplasmic reticulum during early development of Drosophila melanogaster

In this study, we analyze for the first time endoplasmic reticulum (ER) dynamics and organization during oogenesis and embryonic divisions of Drosophila melanogaster using a Protein Disulfide Isomerase (PDI) GFP chimera protein. An accumulation of ER material into the oocyte takes place during the early steps of oogenesis. The compact organization of ER structures undergoes a transition to an expanded reticular network at fertilization. At the syncytial stage, this network connects to the nuclear envelope as each nucleus divides. Time-lapse confocal microscopy on PDI transgenic embryos allowed us to characterize a rapid redistribution of the ER during the mitotic phases. The ER network is massively recruited to the spindle poles in prophase. During metaphase most of the ER remains concentrated at the spindle poles and shortly thereafter forms several layers of membranes along the ruptured nuclear envelope. Later, during telophase an accumulation of ER material occurs at the spindle equator. We also analyzed the subcellular organization of the ER network at the ultrastructural level, allowing us to corroborate the results from confocal microscopy studies. This dynamic redistribution of ER suggests an unexpected regulatory function for this organelle during mitosis.     

Lethal(1)fibrillardysgenesis (I(1)fdg), a mutation affecting muscle development in the embryo of Drosophila melanogaster

The I(1)fdg mutation demonstrates two separate phases of lethality, depending on developmental conditions. At 32–33°C, an embryonic lethality is expressed whereas at lower temperatures a larval-pupal lethality is observed. This larval-pupal lethality characteristically produces noncondensed, curved puparia, and since the contraction of the pupa depends on strong muscular contraction, this phase of lethality implicates some involvement of abnormal musculature. The embryonic expression of I(1)fdg at 32–33°C is the subject of this study. In these embryos, which are alive but immobile (incapable of hatching), the fibrillar organization and fiber morphology of the somatic musculature varies from being apparently normal to being grossly abnormal. While the abnormalities appear as unusual distributions of fiber organelles, abnormal convolutions of the muscle fibers, and disorganizations of fibrillar components, it seems most probable that the underlying defect ultimately responsible resides in some system essential for Z body alignment and sarcomere formation. Accompanying the embryonic lethality, certain abnormalities in midgut development are observed which at present do not appear to be related to the defects observed in the somatic muscle.     

Effect of the wingless (wg1) mutation on wing and haltere development in Drosophila melanogaster

Genetic and developmental studies of wingless (wg¹), a new second chromosome recessive mutation in Drosophila melanogaster, have shown that it affects not only wing and haltere development (giving rise to wingless and/or halterless flies), but also results in various abnormalities of the mesothorax. The larvae destined to develop into wingless and/or halterless flies possessed underdeveloped mesothoracic and/or metathoracic imaginal discs.     

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.     

Differential requirements for the neurogenic gene almondex during Drosophila melanogaster development

During early development, the neurogenic genes of Drosophila melanogaster are involved in the control of cell fates in the neurectoderm; almondex (amx) belongs to this category of genes. We have identified the amx locus and rescued the amx embryonic neurogenic phenotype with a 1.5 kb DNA fragment. Using a small deficiency, we generated a new amx mutant background called amx(m), which is a null allele. Besides the characteristic neurogenic maternal effect caused by loss of amx, amx(m) flies display a new imaginal phenotype resembling loss of function of Notch. We describe amx-induced misregulation of the Notch pathway target E(spl) m7 in embryos and genetic interactions between amx and Notch pathway mutants in adult flies. These data show that wildtype amx acts as a novel positive regulator of the Notch pathway and is required at different levels during development.     

Regulation of cell migration during tracheal development in Drosophila melanogaster

Most of the knowledge concerning the intracellular mechanisms involved in cell locomotion have been obtained from in vitro studies of cells in culture. Many of the concepts derived from these studies have been partially confirmed in in vivo systems but numerous questions regarding the developmental control of cell migration remain to be addressed. Tracheal morphogenesis in Drosophila melanogaster embryos represents an in vivo model system to study the genetic control of cell migration. We review what is known about tracheal development and regulation of tracheal cell migration. We try to link these in vivo studies and the movement of cells over two dimensional substrates and elaborate on important questions which remain to be addressed in the future.