Lectin binding sites during Drosophila embryogenesis

The spectrum of lectin binding sites as it emerges during embryonic development of Drosophila was analysed by means of fluorescein-labelled lectins. As development and morphogenesis proceed, the reaction pattern becomes more and more complex. Mannose/glucose-, mannose-, N-acetylglucosamine- and poly-N-ace-tylglucosamine-specific lectins bind ubiquitously. Nuclear envelopes only have binding sites for wheat germ agglutinin. N-acetylgalactosamine-binding lectins are specific for ectodermal derivatives. Ga-3-N-acetylgalac-tosamine-binding lectins are highly selective markers for neural structures, haemocytes and Garland cells. It is also shown that Drosophila laminin is differentially glycosylated. The possible implications of differential and germ layer-specific glycosylation are discussed.Dedicated to the memory of Jan Callaerts     

Enhancer-trap detection of expression patterns corresponding to the polar coordinate system in the imaginal discs of Drosophila melanogaster

We have isolated three classes of enhancertrap lines of Drosophila in which lacZ expression patterns in the imaginal discs are consistent with the idea of a polar (radial and angular) coordinate system of positional information. In the first class (HZ76), a circular pattern was expressed transiently during the mid-third instar larval stage when the radial components of the coordinate are probably generated. The expression patterns of the second class (HZ84) were sector-shaped and circular in the leg disc, suggesting a correlation with both radial and angular coordinate values. The expression patterns found in the third class (PZ63 and PZ22) were circular and appeared to reflect radial positional values. Expression in the latter two classes always began in the presumptive dorsal region of the leg disc and gradually spread to the ventral region. These developmental profiles of expression suggested the existence of a centre that initiates patterned gene expression in the presumptive dorsal region of the leg disc. The PZ22 line showed transient expression during tarsal segmentation, suggesting its involvement in tarsal segment formation. We have cloned the PZ22 gene and partially determined its sequence. The deduced amino acid sequence contained a zinc finger motif found in DNA/RNA binding proteins. By in situ hybridization, we determined that the PZ22 gene was transcribed in the leg disc in a pattern identical to that of the lacZ expression. In addition, it was expressed transiently in the embryonic mesoderm during mesoderm segmentation. The PZ22 gene, therefore, may function both in mesodermal segmentation in the embryo and in tarsal segmentation in the imaginal disc.     

A simple and efficient method to identify replacements of P-lacZ by P-Gal4 lines allows obtaining Gal4 insertions in the bithorax complex of Drosophila

The functional replacement of one gene product by another one is a powerful method to study specificity in development and evolution. In Drosophila, the Gal4/UAS method has been used to analyze in vivo such functional substitutions. To this aim, Gal4 lines that inactivate a gene and reproduce its expression pattern are required, and they can be frequently obtained by replacing pre-existing P-lacZ lines with such characteristics. We have devised a new method to quickly identify replacements of P-lacZ lines by P-Gal4 lines, and applied it successfully to obtain Gal4 insertions in the Ultrabithorax and Abdominal-B Hox genes. We have used these lines to study the functional replacement of a Hox gene by another one. Our experiments confirm that the abdominal-A gene can replace Ultrabithorax in haltere development but that it cannot substitute for Abdominal-B in the formation of the genitalia.     

Establishment of an enhancer trap system with<Emphasis Type="Italic"> Ds</Emphasis> and GUS for functional genomics in rice

To develop an efficient means of enhancer trapping, a two-element system employing Ds and an Ac transposase (AcTPase) gene was tested in rice. We generated 263 transgenic rice plants, each of which harboured the maize transposable element Ds together with a GUS coding sequence under the control of a minimal promoter ( Ds-GUS), and a gene that confers resistance to the herbicide chlorsulfuron. Among the 263 lines generated, 42 were shown to have a single copy of the Ds-GUS element. Four single-copy lines were crossed with each of six transgenic plants that carried the AcTPase gene. Excision of the Ds-GUS in leaves of F₁ plants was detected in eight combinations out of seventeen examined. The frequency of transposition of Ds-GUS in germ cells in the F₁ plants was examined using 10,524 F₂ plants, and 675 (6%) were judged to be transposants. Their frequencies differed among F₁ plants depending on the AcTPase x Ds-GUS cross considered, and also among panicles on the same F₁ plant. This suggests that Ds-GUS tends to transpose during panicle development. Southern analysis with a GUS probe showed different band patterns among transposants derived from different panicles. Therefore, the transposants derived from different panicles must have arisen independently. Transposants showing tissue-specific GUS activities were obtained, and enhancers thus trapped by the Ds-GUS element were identified. These results demonstrate that the system is suitable for the isolation of large numbers of independent Ds-GUS transposants, and for the identification of various tissue-specific enhancers. The Ds-GUS lines generated in this study offer a potentially powerful tool for studies on the functional genomics of rice.Communicated by M.-A. Grandbastien     

Tracking nucleolar dynamics with GFP-Nopp140 during Drosophila oogenesis and embryogenesis

We expressed two green fluorescent protein (GFP)-tagged Nopp140 isoforms in transgenic Drosophila melanogaster to study nucleolar dynamics during oogenesis and early embryogenesis. Specifically, we wanted to test whether the quiescent oocyte nucleus stored maternal Nopp140 and then to determine precisely when nucleoli formed during embryogenesis. During oogenesis nurse cell nucleoli accumulated GFP-Nopp140 gradually such that posterior nurse cell nucleoli in egg chambers at stage 10 were usually brighter than the more anterior nurse cell nucleoli. Nucleoli within apoptotic nurse cells disassembled in stages 12 and 13, but not all GFP-Nopp140 entered the oocyte through inter-connecting cytoplasmic bridges. Oocytes, on the other hand, lost their nucleoli by stage 3, but GFP-Nopp140 gradually accumulated in oocyte nuclei during stages 8–13. Most oocyte nuclei at stage 10 stored GFP-Nopp140 uniformly, but many stage 10 oocytes accumulated GFP-Nopp140 in presumed endobodies or in multiple smaller spheres. All oocyte nuclei at stages 11-12 were uniformly labeled, and GFP-Nopp140 diffused to the cytoplasm upon nuclear disassembly in stage 13. GFP-Nopp140 reappeared during embryogenesis; initial nucleologenesis occurred in peripheral somatic nuclei during embryonic stage 13, one stage earlier than reported previously. These GFP-Nopp140-containing foci disassembled at the 13th syncytial mitosis, and a second nucleologenesis occurred in early stage 14. The resulting nucleoli occupied nuclear regions closest to the periphery of the embryos. Pole cells contained GFP-Nopp140 during the syncytial embryonic stages, but their nucleologenesis started at gastrulation. This work was supported by the National Science Foundation (grant MCB-0234245). O'Keith Dellafosse was supported by the Louisiana Alliance for Minority Participation (LAMP).     

Lineage analysis of transplanted individual cells in embryos of Drosophila melanogaster

Summary Some aspects of neural and epidermal cell lineages during embryogenesis of Drosophila melanogaster were studied by transplanting horseradish-peroxidase-(HRP-) labelled ectodermal cells from young gastrula donors into host embryos of similar ages. Heterotopic transplantations permitted us to assess the degree of commitment already attained by the transplanted cells. The resulting cell clones showed normal characteristics of cytodifferentiation and cell number. The results indicate that epidermal progenitors perform a maximum of three mitoses during embryonic development, whereas neuroblasts may perform more than ten mitoses. Clone size distribution is in both cases scattered, suggesting either a rather irregular mitotic pattern or cell death. As indicated by heterotopic transplantations, the neurogenic ectoderm for the ventral nervous system exhibits different neurogenic abilities in its different regions, decreasing from medial to lateral; we discuss the hypothesis that some medially located cells of the young gastrulating embryo could be committed towards the neural fate before segregating from the ectoderm. On the other hand, the cells of the dorsal ectodermal regions at the same stage seem to be indifferent with respect to commitment, for they are able to give rise to central neural lineages following their transplantation in the neurogenic region.     

Protein synthesis patterns following stage-specific heat shock in early Drosophila embryos

Summary Very short heat shocks are administered to carefully staged early embryos of Drosophila melanogaster, and the effects on protein synthesis pattern investigated. A shock as short as 2 min will induce the heat shock response (reduction of normal protein synthesis, increased synthesis of the heat shock proteins) in syncytial blastoderm or later stages. Thus the initial events of the heat shock response must occur within 2 min, and not reverse upon rapid return to 22° C. A low level of synthesis of the 70 kDa heat shock protein is sometimes visible in unshocked animals, but may be induced by the labeling procedure. Survival following a short shock is not strictly correlated with a high level of heat shock response. Pre-blastoderm embryos do not produce significant heat shock protein, but survive a 2 min 43°C heat shock better than do heat shock response competent blastoderm embryos. The protein synthesis pattern prior to the blastoderm stage is very stable, possibly enhancing survival following a short shock. Shocks of 3 min or longer are more detrimental to pre-blastoderm embryos than to later stages, confirming the role of the heat shock response in survival following a longer shock. Stage-specific developmental defects (phenocopies) may be induced by heat shock at the blastoderm or later stages. Induction of these defects may require disruption of the normal protein synthesis pattern. Use of very short heat shocks to induce the heat shock response will be valuable in identifying the precise time at which a specific defect can be induced.     

Analysis of twin of eyeless regulation during early embryogenesis in Drosophila melanogaster

The Drosophila Pax6 homolog twin of eyeless (toy) is so far the first zygotically expressed gene involved in eye morphogenesis in Drosophila. The study of its expression during embryogenesis is therefore informative of the initial events of eye development in Drosophila. We have analyzed how the initial expression domain of toy at cellular blastoderm is regulated. We show that the three maternal patterning systems active in the cephalic region (the anterior, terminal and dorsal-ventral systems) cooperate with zygotically activated gap genes to shape the initial expression domain of toy. Whereas Bicoid, Dorsal and Torso signaling synergistically act as activators, Hunchback, Knirps and Decapentaplegic act as repressors.     

The effects of the glucocorticoid,dexamethasone, on the development of the Drosophila embryo

We have investigated the effects of the glucocorticoid, dexamethasone, and five structural analogs on Drosophila development in an effort to identify steroid ligands that may play a role in the embryogenesis of this organism. Embryos were exposed to glucocorticoids either by direct culture in supplemented medium, or by examining embryos from adult flies raised on supplemented fly food. After exposure, embryos were examined for developmental defects. At a morphological level, exposure to dexamethasone disrupts the dorsolateral folding of the amnioserosa during germ band extension. In addition, germ band retraction and dorsal closure is also disrupted. The phenocritical period of these effects is within the first 4 h of embryogenesis. This response is dosage sensitive, with embryos responding to concentrations of dexamethasone ranging from 10^–6 to 10^–3M. Furthermore, glucocorticoids which are closely related structural analogs of dexamethasone also disrupt germ band retraction and dorsal closure, while other tested steroids had no effect on embryonic development. At a molecular level, expression of the gene, Krüppel, is absent from the amnioserosa of dexamethasone-treated embryos. The cuticular phenocopy resulting from exposure to dexamethasone and related glucocorticoids is morphologically similar to the mutant phenotype associated with four genes required for germ band retraction, namely hindsight, serpent, tail-up and u-shaped. The results of this study represent the first association of a glucocorticoid with dose, stage and tissue specific effects on Drosophila development at both morphological and molecular levels.     

Lineage analysis of transplanted individual cells in embryos of Drosophila melanogaster

Summary We describe the results of cell transplantation experiments performed to investigate mesodermal lineages in Drosophila melanogaster, particularly the lineages of the somatic muscles, the visceral muscles and the fat body. Cells to be transplanted were labelled by injecting a mixture of horseradish peroxidase (HRP) and fluorescein-dextran (FITC) in wild-type embryos at the syncytial blastoderm stage. For transplantation cells were removed from the ventral furrow, 8–12 min after the start of gastrulation, and individually transplanted into homotopic or heterotopic locations of unlabelled wild-type hosts of the same age. HRP labelling in the resulting cell clones was demonstrated histochemically in the fully developed embryo; histotypes could be distinguished without ambiguity. Mesodermal cells were already found to be committed to mesodermal fates at the time of transplantation. They developed only into mesodermal derivatives and did not integrate in non-mesodermal organs upon heterotopical transplantation. No evidence was found for commitment to any particular mesodermal organ at the time of transplantation. The majority of somatic muscle clones contributed cells to only one segment. However, clones were not infrequently distributed through two or even three segments. Clones of fat body cells were generally restricted to a small region. However, cells of clones of visceral musculature were widely distributed. With respect to the proliferative abilities of transplanted cells the clones were difficult to interpret due to the syncytial character of the somatic musculature and the fact that the organization of the other organs is poorly understood. Evidence from histological observations of developing normal embryos indicates only three mitoses for mesodermal cells. Clones larger than seven cells were not found when embryos were fixed previous to germ-band shortening; larger clones were found in the fat body and visceral musculature after fixing the embryos at the end of organogenesis. Quantitative considerations suggest that a few mesodermal cells might perform more than three mitoses.     

Reversible commitment of neural and epidermal progenitor cells during embryogenesis of Drosophila melanogaster

Summary Cell-cell interactions are involved in mediating developmental fate. An example is the decision of the neuroectodermal cells of Drosophila to develop as neural or epidermal progenitors, where cellular interactions participate in the process of acquisition of either cell fate. The results of heterochronic cell transplantations we describe here suggest that both neuroblasts and epidermoblasts are not irreversibly committed to a particular developmental fate. Rather, they retain the ability to interact with neighbouring cells and, under our experimental conditions, are capable of switching their fate during a relatively long period of time, i.e. until the end of embryonic stage 11.     

Asymmetrically distributed ecdysteroid-related antigens in follicles and young embryos of Drosophila melanogaster

Summary We have produced monoclonal and polyclonal antibodies against an antigen that is asymmetrically distributed in mature oocytes of Drosophila melanogaster. During late oogenesis and early embryogenesis the antigen undergoes dramatic changes in its cellular localization: until about 2.5 h before completion of oogenesis it is homogeneously distributed in the cytoplasm, then it becomes localized in granules that are more numerous in posterior than in anterior peripheral positions of the ooplasm. The germ plasm is void of the antigen. Shortly after egg deposition the antigen is released from the granules and forms a shallow temporary gradient in the egg. Later during embryogenesis the antigen is associated with the yolk-containing cytoplasm. At the syncytial blastoderm stage it is also detected in the peripheral nuclei. Preliminary evidence suggests that the antigen is an ecdysteroid-related molecule. Five different anti-ecdysone antisera were found to bind to the same antigen or to an antigen with the same localization as our monoclonal antibody. In pattern mutants affecting anteroposterior polarity, the described asymmetrical distribution of the antigen is abnormal. In the mutant BicD, for example, which leads to the formation of two abdomina of opposite polarity, the antigen-containing granules are distributed homogeneously in mature oocytes.     

Differential mutagenic response in selected lines of Drosophila melanogaster

Summary The mutagenic efficiency of ionizing radiations has been tested on different lines of Drosophila melanogaster. It has been shown that differential lethal effects are obtained when irradiated females from different lines are mated to flies carrying heterozygous lethal genes. The results seem not to be attributable to differential expression of the lethality in the various crosses performed with the irradiated flies. This might suggest that gene activity is involved in the expression of the mutagenic effects of radiations.     

An enhancer trap in the ascidian Ciona intestinalis identifies enhancers of its Musashi orthologous gene

The enhancer trap technique, established in Drosophila melanogaster, is a very sophisticated tool. Despite its usefulness, however, there have been very few reports on enhancer traps in other animals. The ascidian Ciona intestinalis, a splendid experimental system for developmental biology, provides good material for developmental genetics. Recently, germline transgenesis of C. intestinalis has been achieved using the Tc1/mariner superfamily transposon Minos. During the course of that study, one Minos insertion line that showed a different GFP expression pattern from other lines was isolated. One fascinating possibility is that an enhancer trap event occurred in this line. Here we show that a Minos insertion in the Ci-Musashi gene was responsible for the altered GFP expression. Ci-Musashi showed a similar expression pattern to GFP. In addition, introns of Ci-Musashi have enhancer activity that can alter the expression pattern of nearby genes to resemble that of GFP in this line. These results clearly demonstrate that an enhancer trap event that entrapped enhancers of Ci-Musashi occurred in C. intestinalis.     

A functional analysis of the genes Enhancer of split and HLH-m5 during early neurogenesis in Drosophila melanogaster

To assess the functional domains of the proteins encoded by E(spl) and HLH-m5, two genes of the Enhancer of split complex [E(SPL)-C] of Drosophila melanogaster, a number of variants have been made by in vitro mutagenesis, transformed into the germ line of the wild-type, and genetically combined with a chromosomal deletion lacking four of the genes of the E(SPL)-C. All constructs used attenuated the neurogenic phenotype associated with this deletion. However, constructs encoding proteins with truncated carboxy-termini exibited in all cases a higher activity than constructs encoding the full length version of the protein. Neutralization of the basic domain severely reduced, but did not completely abolish the rescuing activity of E(spl), while proteins in which a proline residue within the basic domain had been changed to either threonine or asparagine were slightly less efficient in their rescuing activity than the corresponding wild-type versions. We discuss the possible significance of these results for the function of the protein domains.     

Contamination and persistent infection ofDrosophila cell lines by reovirus type particles

Summary A new type of contaminant particles persistently infectedDrosophila cell lines. On an ultrastructural, morphogenetic and histochemical basis, they are similar to viruses of the Reoviridae group. They have been namedDrosophila K virus (DKV).     

High efficiency of a double-screening method on single P-element insertion lines to identify quantitative trait mutants in Drosophila melanogaster

Enhancer trap P-element insertion has become a common method for generating new mutations in Drosophila melanogaster. When this method is used to isolate mutants for quantitative traits, an appropriate control must be established to define normal and mutant phenotypes. Considering that enhancer-trap lines are generated by crossing several strains, usually with no homogeneous genetic background, no clear control strain can be selected. Previous reports tried to overcome this problem by homogenizing the genetic background of the original lines. However, this is not the most common scenario, especially when functional phenotypes are studied in previously generated lines. Without such caution, is it possible to identify functional mutants among P-element insertion lines? We tested this for olfactory preference, a quantitative trait. Using as control measurement the average phenotype of 30 simultaneously generated P-element insertion lines with preferential reporter-gene expression in olfactory reception organs, we found that 25 of the lines exhibited mutant phenotypes in response to one or several of 5 tested odorants. Additional tests showed that the efficiency of the method for detecting olfactory mutations exceeded 60% even for such a small number of tested odorants. According to these results this approach greatly facilitates the identification of putative abnormal phenotypes, which must be extensively confirmed afterwards.     

Prediction of heterosis in crosses between inbred lines of Drosophila melanogaster

Summary The aim of the experiment was to determine if the estimated genetic distance between two populations could be used to predict the amount of heterosis that would occur when they were crossed. Eight lines of known relatedness to each other were produced by eight generations of sib mating and sub-lining. This produced lines that varied in coefficient of coancestry from zero to 0.78. Fourteen reciprocal crosses of these lines were used to measure heterosis for larval viability and adult fecundity. Gene frequencies at six polymorphic enzyme loci were used to estimate the genetic distances between lines, which were then compared with the known degrees of coancestry. The estimated genetic differences were poorly correlated with the known coancestry coefficients (r=0.4), possibly due to the small number of loci typed. Also genetic distances were only about 1/3 of what was expected. Selection acting on blocks of genes linked to the enzyme loci probably prevented the expected increase in homozygosity. Coancestry coefficient was correlated with heterosis (r=0.44–0.71). This level of correlation implied differences in heterosis among parent lines with the same level of coancestry. This variability is expected if a small number of loci explain most of the heterosis. The average level of heterosis was less than expected after eight generations of sib mating. This is most likely due to selection opposing the increase in homozygosity caused by inbreeding. The combination of these two imperfect correlations resulted in no significant correlation between genetic distance estimated from markers and heterosis.     

The function of type IV collagen during Drosophila embryogenesis

A collagen gene (Dcg1) was characterized in Drosophila melanogaster and shown to encode a peptide related to vertebrate basement membrane type IV collagen chains. To study the function of type IV collagen during Drosophila development, we transformed flies with a partially truncated Dcg1 gene under the control of a heat-shock promotor. This construct induced synthesis of shortened pro- chains which associated with normal ones and thereby caused degradation of the shortened and normal pro- chains through a process called pro-collagen suicide. A large proportion of embryos expressing the transgene developed a phenotype exhibiting absence or partial retraction of the germ band with defects in nerve cord condensation and dorsal closure. Together these results indicated that, during embryogenesis, type IV collagen was an essential guiding factor for cell-matrix interactions in morphogenetic events.