Whole-embryo culture of Drosophila : development of embryonic tissues in vitro

Summary We have devised techniques to culture whole, dissected embryos of Drosophila melanogaster. We examine multiple aspects of the morphological and physiological development of the epidermis, musculature, nervous system, and internal organs in this cultured preparation, and show that in vitro development closely parallels normal embryogenesis. These techniques permit a wide range of experimental manipulations during embryogenesis and allow us to extend observations through late embryonic stages, after cuticle deposition. Applications of this technique are presented.     

The serosa of Manduca sexta (Insecta, Lepidoptera): ontogeny, secretory activity, structural changes, and functional considerations

In Manduca sexta, the blastoderm forms successively and becomes immediately cellularized as the cleavage energids reach the surface of the oocyte. Presumptive serosal cells are large and contain 2 or 4 large polyploid nuclei; presumptive embryonic cells are small and mononuclear. All parts of the blastoderm participate in the uptake and digestion of yolk material. About 10 h post-oviposition, the blastoderm breaks at the amnioserosal fold and the extraembryonic part closes above the germ band and constitutes the serosa (12 h post-oviposition, i.e. 10% development completed). At once, the serosa starts to secrete a cuticle consisting of an epi- and a lamellated endocuticle. Detachment of the serosal cuticle, 22h post-oviposition, is reminiscent of apolysis of larval cuticle. Thereafter, the serosa deposits a membranous structure, the serosal membrane. The sercretory process lasts from 23h to 44h post-oviposition. At first a fine granular layer, then an amorphous, spongy-like, fibrillar layer is secreted via microvilli. This persisting membrane is tough, rubbery and very elastic. It may serve to bolster the serosa during katatrepsis (48h post-oviposition) and later embryonic movements. After detachment of the serosal membrane, 44h post-oviposition, a distinct subcellular reorganization of the serosa takes place. The nuclei become still larger and more irregular. Uptake of yolk granules, but not of lipid droplets, ceases, although interaction of serosa and yolk cells are intense. Serosal cells include many mitochondria, large areas of rER, besides some sER, increasing amounts of lysosomal bodies and prominent Golgi complexes. Most conspicuous is the assembly of spindle-shaped, electron-lucent vesicles below the apical surface. These vesicles may contain metabolic products which are released into the peripheral space. The studies show that the serosa assumes changing functions during embryogenesis: digestion of yolk substances, synthesis of a serosal cuticle and a serosal membrane, which may have a protective function, and excretion.     

Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogaster embryogenesis

Background  

Morphogenetic events that shape the Drosophila melanogaster embryo are tightly controlled by a genetic program in which specific sets of genes are up-regulated. We used a suppressive subtractive hybridization procedure to identify a group of developmentally regulated genes during early stages of D. melanogaster embryogenesis. We studied the spatiotemporal activity of these genes in five different intervals covering 12 stages of embryogenesis.     

Expression of developmental genes during early embryogenesis of<Emphasis Type="Italic"> Hydra</Emphasis>

Hydra is a classical model to study key features of embryogenesis such as axial patterning and stem cell differentiation. In contrast to other organisms where these mechanisms are active only during embryonic development, in Hydra they can be studied in adults. The underlying assumption is that the machinery governing adult patterning mimics regulatory mechanisms which are also active during early embryogenesis. Whether, however, Hydra embryogenesis is governed by the same mechanisms which are controlling adult patterning, remains to be shown. In this paper, in precisely staged Hydra embryos, we examined the expression pattern of 15 regulatory genes shown previously to play a role in adult patterning and cell differentiation. RT-PCR revealed that most of the genes examined were expressed in rather late embryonic stages. In situ hybridization, nuclear run-on experiments, and staining of nucleolar organizer region-associated proteins indicated that genes expressed in early embryos are transcribed in the engulfed "nurse cells" (endocytes). This is the first direct evidence that endocytes in Hydra not only provide nutrients to the developing oocyte but also produce maternal factors critical for embryogenesis. Our findings are an initial step towards understanding the molecular machinery controlling embryogenesis of a key group of basal metazoans and raise the possibility that in Hydra there are differences in the mechanisms controlling embryogenesis and adult patterning.Edited by D. Tautz     

Evolution of the larval peripheral nervous system in <Emphasis Type="Italic">Drosophila</Emphasis> species has involved a change in sensory cell lineage

A key challenge in evolutionary biology is to identify developmental events responsible for morphological changes. To determine the cellular basis that underlies changes in the larval peripheral nervous system (PNS) of flies, we first described the PNS pattern of the abdominal segments A1–A7 in late embryos of several fly species using antibody staining. In contrast to the many variations reported previously for the adult PNS pattern, we found that the larval PNS pattern has remained very stable during evolution. Indeed, our observation that most of the analysed Drosophilinae species exhibit exactly the same pattern as Drosophila melanogaster reveals that the pattern observed in D. melanogaster embryos has remained constant for at least 40 million years. Furthermore, we observed that the PNS pattern in more distantly related flies (Calliphoridae and Phoridae) is only slightly different from the one in D. melanogaster. A single difference relative to D. melanogaster was identified in the PNS pattern of the Drosophilinae fly D. busckii, the absence of a specific external sensory organ. Our analysis of sensory organ development in D. busckii suggests that this specific loss resulted from a transformation in cell lineage, from a multidendritic-neuron-external-sensory-organ lineage to a multidendritic-neuron-solo lineage.Edited by P. Simpson     

Changes in the Intensity of Respiration during Ontogenesis of Some Invertebrates

We studied the dynamic of respiration intensity during ontogenesis of flat worms (Dugesia tigrina), molluscs (Anodonta piscinalis and Viviparus viviparus, and insects (Leptinotarsa decemlineata). In planarians that reproduce vegetatively, the intensity of respiration increases just after fission and decreases at the subsequent phases of growth. In A. piscinalis, this index of metabolism increases during embryonic and early larval development and decreases at the later developmental stages. In V. viviparus, which develops in the female genital tract, the intensity of respiration remains unchanged during embryogenesis and decreases during late embryogenesis and subsequent phases of growth. In L. decemlineata, the intensity of respiration increases during embryonic and early larval development and then decreases to undergo cyclic changes times to molts. This index markedly decreases in the pupae, increases in the beginning of imaginal period, and then again decreases.     

Expression of photosynthesis-related genes and their regulation by light during somatic embryogenesis in<Emphasis Type="Italic"> Daucus carota</Emphasis>

To clarify the spatial and temporal pattern of gene expression for photosynthesis-associated proteins during somatic embryogenesis in Daucus carota L., the localization of mRNAs for three genes, rbcL, Lhcb and por, was examined in dark-grown and light-irradiated somatic embryos by in situ hybridization. The three mRNAs were expressed in common in the mesophyll precursor cells of light-irradiated embryos at the late torpedo and plantlet stages, but characteristic expression patterns of each photosynthesis-related gene were also observed. Expression of rbcL mRNA first occurred throughout the embryo but gradually became localized in the mesophyll precursor cells and cortex during early embryogenesis. Localization of Lhcb mRNA in the mesophyll precursor cells and shoot apical meristem became clear in the early torpedo stage. Expression of Lhcb mRNA was not affected by light during early embryogenesis, but could be induced by light in the torpedo stage, suggesting that light-inducible expression of Lhcb mRNA arises within the torpedo stage. At the late torpedo stage, clear localization of por mRNA started in mesophyll precursor cells of the cotyledon in light-irradiated embryos. Greening potency of the embryo also appeared first at this stage. Therefore, greening and initial differentiation of photosynthetic tissues during somatic embryogenesis seem to be associated with coordinated expression of mRNA for rbcL, Lhcb and por in late torpedo-shaped embryos.Abbreviations DIG Digoxigenin - Lhcb3 Gene encoding a type-III light-harvesting chlorophyll a/b-binding protein of photosystem II - LHCII Light-harvesting chlorophyll a/b-binding protein of photosystem II - POR Protochlorophyllide oxidoreductase - rbcL Gene encoding the large subunit of Rubisco - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase     

A maize defective-kernel mutant (longcell) characterized by tubular cells, severe morphological alterations and induction of cell death

Seeds of the longcell mutant in maize (Zea mays L) have a defective-kernel phenotype: the embryo aborts at the early coleoptilar stage and the endosperm is reduced in size. Mutant embryos have severe alterations in morphogenesis. They have a suspensor-, an embryo axis- and a scutellum-like structure, but the shoot apical meristem (SAM) is not formed. Scanning electron microscopy showed that most of the cells in longcell embryos are tubular and abnormally enlarged. The level of expression of several genes involved in basic metabolism is not severely affected during early and mid embryogenesis, but storage molecule accumulation is reduced. Genes which in normal conditions are only expressed after germination, are expressed during kernel development in the longcell seeds. Mutant embryos undergo cell death in late embryogenesis. Nuclei in dying embryos are TUNEL positive, and different genes coding for hydrolytic enzymes are up-regulated. The expression of genes related to oxidative stress is also altered in longcell embryos. These results lead us to suggest that the longcell mutant may be cytokinesis-defective.     

Knickkopf and retroactive proteins are required for formation of laminar serosal procuticle during embryonic development of Tribolium castaneum

Chitin, a homopolymer of β-1-4-linked N-acetylglucosamine synthesized by chitin synthase A (Chs-A), is organized in the procuticle of the postembryonic cuticle or exoskeleton, which is composed of laminae stacked parallel to the cell surface to give stability and integrity to the underlying insect epidermal and other tissues. Our previous work has revealed an important role for two proteins from Tribolium castaneum named Knickkopf (TcKnk) and Retroactive (TcRtv) in postembryonic cuticular chitin maintenance. TcKnk and TcRtv were shown to be required for protection and organization of newly synthesized procuticular chitin. To study the functions of TcKnk and TcRtv in serosal and larval cuticles produced during embryogenesis in T. castaneum, dsRNAs specific for these two genes were injected into two week-old adult females. The effects of dsRNA treatment on ovarial integrity, oviposition, egg hatching and adult survival were determined. Insects treated with dsRNA for chitin synthase-A (TcChs-A) and tryptophan oxygenase (TcVer) were used as positive and negative controls for these experiments, respectively. Like TcChs-A RNAi, injection of dsRNA for TcKnk or TcRtv into adult females exhibited no adult lethality and oviposition was normal. However, a vast majority of the embryos did not hatch. The remaining (∼10%) of the embryos hatched into first instar larvae that died without molting to the second instar. Chitin content analysis following TcKnk and TcRtv parental RNAi revealed approximately 50% reduction in chitin content of eggs in comparison with control TcVer RNAi, whereas TcChs-A dsRNA-treatment led to >90% loss of chitin. Furthermore, transmission electron microscopic (TEM) analysis of serosal cuticle from TcChs-A, TcKnk and TcRtv dsRNA-treated insects revealed a complete absence of laminar organization of serosal (and larval) procuticle in comparison with TcVer dsRNA-treated controls, which exhibited normal laminar organization of procuticular chitin. The results of this study demonstrate that in addition to their essential roles in maintenance and organization of chitin in epidermal cuticle in larval and later stages of insect development, TcKnk and TcRtv also are required for egg hatch, chitin maintenance and laminar organization of both serosal and larval cuticle during embryonic development of T. castaneum.     

Larval RNAi in <Emphasis Type="Italic">Tribolium</Emphasis> (Coleoptera) for analyzing adult development

We report here on the use of RNA interference (RNAi) to create pupal and adult loss-of-function phenotypes in the red flour beetle, Tribolium castaneum, by injection of double-stranded RNA (dsRNA) into late instar larvae (we refer to this method as larval RNAi). RNAi is well-established as a useful method to mimic loss-of-function phenotypes in many organisms including insects. However, with a few exceptions (such as in the fruit fly Drosophila melanogaster), RNAi analysis has usually been limited to studies of embryogenesis. Here we demonstrate that injection of green fluorescent protein (GFP) dsRNA into the larval body cavity can inhibit GFP expression beginning shortly after injection and continuing through pupal and adult stages. RNAi analysis of the Tc-achaete-scute-homolog (Tc-ASH) revealed that larval RNAi can induce morphological defects in adult beetles, and also that larval RNAi affects the entire body rather than being localized near the site of injection. The larval RNAi technique will be useful to analyze gene functions in post-embryonic development, giving us the opportunity to study the molecular basis of adult morphological diversity in various organisms.Edited by D. Tautz     

An analysis of the embryonic defects in Punch mutants of Drosophila melanogaster

Punch (Pu), a complex genetic locus, encodes GTP cyclohydrolase, the first enzyme in the pteridine biosynthetic pathway. In the larval and adult stages of the Drosophila life cycle, the function of the locus can be monitored by enzyme assays. Although enzyme activity cannot be detected prior to larval stages, the locus must also have earlier functions since most homozygous Pu mutants die during embryogenesis. In order to assess the role of the locus during this stage of development, morphological examinations of embryos from different classes of Pu mutants were performed. An exact correspondence has been found between genetic and morphological classes of Pu mutations. The locus is required during two periods of embryogenesis. These requirements are genetically separable as shown by mutants with defects specific to each period. An early function utilizes both maternal and zygotic components. Mutants defective for these components have abnormal segment patterns. Late in embryogenesis, a Pu product is necessary for the proper pigmentation of larval cuticle and proper orientation and differentiation of other larval structures, particularly in the head region. A cold-sensitive period corresponds to this later function as determined by temperature-shift experiments. Some of the phenotypes observed correspond to known physiological roles of pteridines; others are unexpected and unexplained.     

Cuticle differentiation in the embryo of the amphipod crustacean Parhyale hawaiensis

The arthropod cuticle is a multilayered extracellular matrix produced by the epidermis during embryogenesis and moulting. Molecularly and histologically, cuticle differentiation has been extensively investigated in the embryo of the insect Drosophila melanogaster. To learn about the evolution of cuticle differentiation, we have studied the histology of cuticle differentiation during embryogenesis of the amphipod crustacean Parhyale hawaiensis, which had a common ancestor with Drosophila about 510 million years ago. The establishment of the layers of the Parhyale juvenile cuticle is largely governed by mechanisms observed in Drosophila, e.g. as in Drosophila, the synthesis and arrangement of chitin in the inner procuticle are separate processes. A major difference between the cuticle of Parhyale and Drosophila concerns the restructuring of the Parhyale dorsal epicuticle after deposition. In contrast to the uniform cuticle of the Drosophila larva, the Parhyale cuticle is subdivided into two regions, the ventral and the dorsal cuticles. Remarkably, the boundary between the ventral and dorsal cuticles is sharp suggesting active extracellular regionalisation. The present analysis of Parhyale cuticle differentiation should allow the characterisation of the cuticle-producing and -organising factors of Parhyale (by comparison with the branchiopod crustacean Daphnia pulex) in order to contribute to the elucidation of fundamental questions relevant to extracellular matrix organisation and differentiation. This work was supported by the German Research Foundation (DFG, grant number MO 1714/1-1).     

Identification and comparisons of the yolk polypeptides and the genes which code for them in D. melanogaster sibling species

Summary The yolk proteins stored in Drosophila, oocytes for utilisation during embryogenesis are an ideal system for studying the regulation of gene expression during development. The 3 major polypeptides found in yolk in D. melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated by the oocyte during vitellogenesis. In order to understand more about their regulation and the mechanism of uptake, studies on other species are necessary.Three yolk polypeptides have previously been identified in the D. melanogaster sibling species (D. melanogaster, D. simulans, D. mauritiana, D. erecta, D. teissieri, D. orena and D. yakuba). In D. melanogaster three genes located on the X chromosome are known to code for these yolk polypeptides. in this study genomic Southern transfers and in situ hybridisation experiments were carried out on the sibling species. Using the three cloned yolk protein genes from D. melanogaster, homologous sequences could be detected in the sibling species. It is suggested that three yolk protein genes occur in each of these species, all being located on the X chromosome, and that two of the genes are very closely linked in these same species. Yolk protein gene-homologous DNA sequences have also been identified in two more distantly related species D. funebris and D. virilis.     

Ultrastructure de l'œuf de Triatoma infestans Klug

Summary The thick rigid chorion of the egg of Triatoma secreted by the follicle cells shows two porous layers: an aerial layer in the exochorion, an alveolar one in the endochorion. The anterior part of the eggshell is closed up by an operculum which is heaved up by the hatching larva. The operculum has no alveolar layer. The air enters through the numerous holes of the shell surface into the aerial layer and through the micropyles into the alveolar layer. The egg has no respiratory plastron.The follicle cells produce also a vitelline envelope whose structure shows a rapid condensation at fertilization time. During its development the embryo secretes two layers: serosal and embryonic cuticle.At high humidities, at low temperatures the egg is able to increase its weight during the early stages of embryogenesis, and this increase stops when the serosal cuticle is secreted. In a dry atmosphere the egg loses water but can develop if the temperature is higher than 20°C.The little permeability of the egg is related to the structure of its envelopes. The chorion and the vitelline envelope prevent the water from getting out of the egg. The serosal cuticle seems to be opposed to the penetration of the water into the egg. The role of the embryonic cuticle is probably limited in the transit of water.

Nous remercions Messieurs les Professeurs Maillet et Folliot qui ont mis le microscope R.C.A. à notre disposition, Madame Allo et Mademoiselle Le Gac, technicienne au microscope à balayage J.S.M. S1, pour leur collaboration technique.     

The hobo transposable element has transposase-dependent and-independent excision activity in drosophilid species

Mobility of the hobo transposable element was determined for several strains of Drosophila melanogaster and several Drosophila species. Mobility was assessed by use of an in vivo transient assay in the soma of developing embryos, which monitored hobo excision from injected indicator plasmids. Excision was detected in a D. melanogaster strain (cn; ry ⁴²) devoid of endogenous hobo elements only after co-injection of a helper plasmid containing functional hobo transposase under either heat shock or normal promoter regulation. Excision was also detected in D. melanogaster without helper in strains known to contain genomic copies of hobo. In Drosophila species confirmed not to contain hobo, hobo excision occurred at significant rates both in the presence and absence of co-injected helper plasmid. In four of the seven species tested, excision frequencies were two- to fivefold lower in the presence of plasmid-borne hobo. hobo excision donor sites were sequenced in indicator plasmids extracted from D. melanogaster cn; ry ⁴² and D. virilis embryos. In the presence of hobo transposase, the predominant excision sites were identical in both species, having breakpoints at the hobo termini with an inverted duplication of proximal insertion site DNA. However, in the absence of hobo transposase in D. virilis, excision breakpoints were apparently random and occurred distal to the hobo termini. The data indicate that hobo is capable of functioning in the soma during embryogenesis, and that its mobility is unrestricted in drosophilids. Furthermore, drosophilids not containing hobo are able to mobilize hobo, presumably by a hobo-related cross-mobilizing system. The cross-mobilizing system in D. virilis is not functionally identical to hobo with respect to excision sequence specificity.     

The multi sex combs gene of Drosophila melanogaster is required for proliferation of the germline

We present a genetic analysis showing that the Drosophila melanogaster gene multi sex combs (mxc; Santamaria and Randsholt 1995) is needed for proliferation of the germline. Fertility is the feature most easily affected by weak hypomorphic mutations of this very pleiotropic locus. Pole cell formation and early steps of gonadogenesis conform to the wild-type in embryos devoid of zygotic mxc ⁺ product. mxc mutant gonad phenotypes and homozygous mxc germline clones suggest a role for mxc ⁺ in control of germ cell proliferation during the larval stages. mxc ⁺ requirement is germ cell autonomous and specific in females, whilst in males mxc ⁺ product is also needed in somatic cells of the gonads. Although mxc can be classified among the Polycomb group (Pc-G) of genes, negative trans-regulators of the ANT-C and BX-C gene complexes, germline requirement for mxc appears independent of a need for other Pc-C gene products, and mxc gonad phenotypes are different from those induced by mutations in BX-C genes. We discuss the possible functions of the mxc ⁺ product which helps to maintain homeotic genes repressed and prevents premature larval haemocyte differentiation and neoplasic overgrowth, but promotes growth and differentiation of male and female gonads.F.D. and O.S. should be considered as equal first authors     

Soldier differentiation during embryogenesis of a social aphid, Pseudoregma bambucicola

To understand the developmental process of aphid soldier differentiation, we investigated the morphological characters of normal nymphs, soldier nymphs and developing embryos of Pseudoregma bambucicola. Results of morphometric analyses showed that normal and soldier nymphs formed discrete clusters on the basis of several morphological characters, although a small number of intermediate individuals, termed ‘intercaste nymphs’, were present. In late embryonic stages, normal and soldier embryos were morphologically distinguishable. The earlier the embryonic stage, the smaller the morphological differences between them. In early embryos less than 1000 µm in length, normal and soldier embryos were not morphologically distinguishable, suggesting that the onset of soldier differentiation occurs at an early embryonic stage. Throughout embryonic development, morphological differentiation of the soldier caste proceeded gradually. Notably, several morphological characters of soldiers grew remarkably upon larviposition. Observation of embryonic leg cuticle revealed a characteristic folding structure, indicating that some morphological traits of the soldier are exaggerated upon larviposition through expansion of the folded cuticle. We suggest that morphological differentiation of the soldier caste in P. bambucicola comprises two phases: gradual growth during embryogenesis and rapid growth upon larviposition.