Extraembryonic Serosa Research Articles

Fog signaling has diverse roles in epithelial morphogenesis in insects.

The Drosophila Fog pathway represents one of the best-understood signaling cascades controlling epithelial morphogenesis. During gastrulation, Fog induces apical cell constrictions that drive the invagination of mesoderm and posterior gut primordia. The cellular mechanisms underlying primordia internalization vary greatly among insects and recent work has suggested that Fog signaling is specific to the fast mode of gastrulation found in some flies. On the contrary, here we show in the beetle Tribolium, whose development is broadly representative for insects, that Fog has multiple morphogenetic functions. It modulates mesoderm internalization and controls a massive posterior infolding involved in gut and extraembryonic development. In addition, Fog signaling affects blastoderm cellularization, primordial germ cell positioning, and cuboidal-to-squamous cell shape transitions in the extraembryonic serosa. Comparative analyses with two other distantly related insect species reveals that Fog's role during cellularization is widely conserved and therefore might represent the ancestral function of the pathway.

Decoupling from yolk sac is required for extraembryonic tissue spreading in the scuttle fly Megaselia abdita.

Extraembryonic tissues contribute to animal development, which often entails spreading over embryo or yolk. Apart from changes in cell shape, the requirements for this tissue spreading are not well understood. Here, we analyze spreading of the extraembryonic serosa in the scuttle fly Megaselia abdita. The serosa forms from a columnar blastoderm anlage, becomes a squamous epithelium, and eventually spreads over the embryo proper. We describe the dynamics of this process in long-term, whole-embryo time-lapse recordings, demonstrating that free serosa spreading is preceded by a prolonged pause in tissue expansion. Closer examination of this pause reveals mechanical coupling to the underlying yolk sac, which is later released. We find mechanical coupling prolonged and serosa spreading impaired after knockdown of M. abdita Matrix metalloprotease 1. We conclude that tissue-tissue interactions provide a critical functional element to constrain spreading epithelia.

Darker eggs of mosquitoes resist more to dry conditions: Melanin enhances serosal cuticle contribution in egg resistance to desiccation in Aedes, Anopheles and Culex vectors.

Mosquito vectors lay their white eggs in the aquatic milieu. During early embryogenesis water passes freely through the transparent eggshell, which at this moment is composed of exochorion and endochorion. Within two hours the endochorion darkens via melanization but even so eggs shrink and perish if removed from moisture. However, during mid-embryogenesis, cells of the extraembryonic serosa secrete the serosal cuticle, localized right below the endochorion, becoming the third and innermost eggshell layer. Serosal cuticle formation greatly reduces water flow and allows egg survival outside the water. The degree of egg resistance to desiccation (ERD) at late embryogenesis varies among different species: Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus eggs can survive in a dry environment for ≥ 72, 24 and 5 hours, respectively. In some adult insects, darker-body individuals show greater resistance to desiccation than lighter ones. We asked if egg melanization enhances mosquito serosal cuticle-dependent ERD. Species with higher ERD at late embryogenesis exhibit more melanized eggshells. The melanization-ERD hypothesis was confirmed employing two Anopheles quadrimaculatus strains, the wild type and the mutant GORO, with a dark-brown and a golden eggshell, respectively. In all cases, serosal cuticle formation is fundamental for the establishment of an efficient ERD but egg viability outside the water is much higher in mosquitoes with darker eggshells than in those with lighter ones. The finding that pigmentation influences egg water balance is relevant to understand the evolutionary history of insect egg coloration. Since eggshell and adult cuticle pigmentation ensure insect survivorship in some cases, they should be considered regarding species fitness and novel approaches for vector or pest insects control.

Transcriptome sequencing reveals maelstrom as a novel target gene of the terminal system in the red flour beetle Tribolium castaneum.

Terminal regions of the Drosophila embryo are patterned by the localized activation of the Torso-RTK pathway, which promotes the downregulation of Capicua. In the short-germ beetle Tribolium, the function of the terminal system appears to be rather different, as the pathway promotes axis elongation and, in addition, is required for patterning the extra-embryonic serosa at the anterior. Here, we show that Torso signalling also induces gene expression by relieving Capicua-mediated repression in Tribolium Given that the majority of Torso target genes remain to be identified, we established a differential gene-expression screen. A subset of 50 putative terminal target genes was screened for functions in early embryonic patterning. Of those, 13 genes show early terminal expression domains and also phenotypes were related to terminal patterning. Among others, we found the PIWI-interacting RNA factor Maelstrom to be crucial for early embryonic polarization. Tc-mael is required for proper serosal size regulation and head morphogenesis. Moreover, Tc-mael promotes growth-zone formation and axis elongation. Our results suggest that posterior patterning by Torso may be realized through Maelstrom-dependent activation of posterior Wnt domains.

The extraembryonic serosa is a frontier epithelium providing the insect egg with a full-range innate immune response.

Drosophila larvae and adults possess a potent innate immune response, but the response of Drosophila eggs is poor. In contrast to Drosophila, eggs of the beetle Tribolium are protected by a serosa, an extraembryonic epithelium that is present in all insects except higher flies. In this study, we test a possible immune function of this frontier epithelium using Tc-zen1 RNAi-mediated deletion. First, we show that bacteria propagate twice as fast in serosa-less eggs. Then, we compare the complete transcriptomes of wild-type, control RNAi, and Tc-zen1 RNAi eggs before and after sterile or septic injury. Infection induces genes involved in Toll and IMD-signaling, melanisation, production of reactive oxygen species and antimicrobial peptides in wild-type eggs but not in serosa-less eggs. Finally, we demonstrate constitutive and induced immune gene expression in the serosal epithelium using in situ hybridization. We conclude that the serosa provides insect eggs with a full-range innate immune response.

Egg survival is reduced by grave-soil microbes in the carrion beetle, Nicrophorus vespilloides.

BackgroundNicrophorus vespilloides eggs are deposited into the soil in close proximity to the decomposing vertebrate carcasses that these insects use as an obligate resource to rear their offspring. Eggs in this environment potentially face significant risks from the bacteria that proliferate in the grave-soil environment following nutrient influx from the decomposing carcass. Our aims in this paper are twofold: first, to examine the fitness effects of grave-soil bacteria to eggs, and second, to quantify egg immunocompetence as a defence against these bacteria.ResultsOur results provide strong evidence that grave-soil microbes significantly reduce the survival of Nicrophorus eggs. Females provided with microbe rich carcasses to rear broods laid fewer eggs that were less likely to hatch than females given uncontaminated carcasses. Furthermore, we show that egg hatch success is significantly reduced by bacterial exposure. Using a split-brood design, which controlled for intrinsic differences in eggs produced by different females, we found that eggs washed free of surface-associated bacteria show increased survival compared to unwashed eggs. By contrast, eggs exposed to the entomopathogen Serratia marcescens show decreased survival compared to unexposed eggs. We next tested the immune competence of eggs under challenge from bacterial infection, and found that eggs lacked endogenous production of antimicrobial peptides, despite well-developed responses in larvae. Finally, we found that despite lacking immunity, N. vespilloides eggs produce an extraembryonic serosa, indicating that the serosa has lost its immune inducing capacity in this species.ConclusionsThe dependency on ephemeral resources might strongly select for fast developing animals. Our results suggest that Nicrophorus carrion beetles, and other species developing on ephemeral resources, face a fundamental trade-off between egg immunity and development time.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-014-0208-x) contains supplementary material, which is available to authorized users.

The extraembryonic serosa protects the insect egg against desiccation

Insects have been extraordinarily successful in occupying terrestrial habitats, in contrast to their mostly aquatic sister group, the crustaceans. This success is typically attributed to adult traits such as flight, whereas little attention has been paid to adaptation of the egg. An evolutionary novelty of insect eggs is the serosa, an extraembryonic membrane that enfolds the embryo and secretes a cuticle. To experimentally test the protective function of the serosa, we exploit an exceptional possibility to eliminate this membrane by zerknüllt1 RNAi in the beetle Tribolium castaneum. We analyse hatching rates of eggs under a range of humidities and find dramatically decreasing hatching rates with decreasing humidities for serosa-less eggs, but not for control eggs. Furthermore, we show serosal expression of Tc-chitin-synthase1 and demonstrate that its knock-down leads to absence of the serosal cuticle and a reduction in hatching rates at low humidities. These developmental genetic techniques in combination with ecological testing provide experimental evidence for a crucial role of the serosa in desiccation resistance. We propose that the origin of this extraembryonic membrane facilitated the spectacular radiation of insects on land, as did the origin of the amniote egg in the terrestrial invasion of vertebrates.

Immune competence in insect eggs depends on the extraembryonic serosa

Innate immunity is common to all metazoans and serves as a first line of defense against pathogens. Although the immune response of adult and larval insects has been well characterized, it remains unknown whether the insect egg is able to mount an immune response. Contrary to Drosophila, Tribolium eggs develop an extraembryonic epithelium, the serosa. Epithelia are well known for their ability to fight infection, so the serosa has the potential to protect the embryo against pathogens. To test this hypothesis we created serosa-less eggs by Tc-zen1 parental RNAi. We found that the Tribolium egg upregulates several immune genes to comparable levels as adults in response to infection. Drosophila eggs and serosa-less Tribolium eggs, however, show little to no upregulation of any of the tested immune genes. We conclude that the extraembryonic serosa is crucial for the early immune competence of the Tribolium egg.

Evidence for a composite anterior determinant in the hover flyEpisyrphus balteatus(Syrphidae), a cyclorrhaphan fly with an anterodorsal serosa anlage

Most insect embryos develop from a monolayer of cells around the yolk, but only part of this blastoderm forms the embryonic rudiment. Another part forms extra-embryonic serosa. Size and position of the serosa anlage vary between species, and previous work raises the issue of whether such differences co-evolve with the mechanisms that establish anteroposterior (AP) polarity of the embryo. AP polarity of the Drosophila embryo depends on bicoid, which is necessary and sufficient to determine the anterior body plan. Orthologs of bicoid have been identified in various cyclorrhaphan flies and their occurrence seems to correlate with a mid-dorsal serosa or amnioserosa anlage. Here, we introduce with Episyrphus balteatus (Syrphidae) a cyclorrhaphan model for embryonic AP axis specification that features an anterodorsal serosa anlage. Current phylogenies place Episyrphus within the clade that uses bicoid mRNA as anterior determinant, but no bicoid-like sequence could be identified in this species. Using RNA interference (RNAi) and ectopic mRNA injection, we obtained evidence that pattern formation along the entire AP axis of the Episyrphus embryo relies heavily on the precise regulation of caudal, and that anterior pattern formation in particular depends on two localized factors rather than one. Early zygotic activation of orthodenticle is separated from anterior repression of caudal, two distinct functions which in Drosophila are performed jointly by bicoid, whereas hunchback appears to be regulated by both factors. Furthermore, we found that overexpression of orthodenticle is sufficient to confine the serosa anlage of Episyrphus to dorsal blastoderm. We discuss our findings in a phylogenetic context and propose that Episyrphus employs a primitive cyclorrhaphan mechanism of AP axis specification.

Maternal Torso Signaling Controls Body Axis Elongation in a Short Germ Insect

In the long germ insect Drosophila, all body segments are determined almost simultaneously at the blastoderm stage under the control of the anterior, the posterior, and the terminal genetic system . Most other arthropods (and similarly also vertebrates) develop more slowly as short germ embryos, where only the anterior body segments are specified early in embryogenesis. The body axis extends later by the sequential addition of new segments from the growth zone or the tail bud . The mechanisms that initiate or maintain the elongation of the body axis (axial growth) are poorly understood . We functionally analyzed the terminal system in the short germ insect Tribolium. Unexpectedly, Torso signaling is required for setting up or maintaining a functional growth zone and at the anterior for the extraembryonic serosa. Thus, as in Drosophila, fates at both poles of the blastoderm embryo depend on terminal genes, but different tissues are patterned in Tribolium. Short germ development as seen in Tribolium likely represents the ancestral mode of how the primary body axis is set up during embryogenesis. We therefore conclude that the ancient function of the terminal system mainly was to define a growth zone and that in phylogenetically derived insects like Drosophila, Torso signaling became restricted to the determination of terminal body structures.

Developmental Evolution: Torso — a Story with Different Ends?

The Torso pathway patterns the ends of the Drosophila embryo. Now, it has been found to control axis elongation in the short germ insect Tribolium. This result raises the issue of the ancestral function of the Torso pathway and its evolution.

Regulation of the Tribolium homologues of caudal and hunchback in Drosophila: evidence for maternal gradient systems in a short germ embryo.

In short germ embryos, the germ rudiment forms at the posterior ventral side of the egg, while the anterior-dorsal region becomes the extra-embryonic serosa. It is difficult to see how an anterior gradient like that of bicoid in Drosophila could in these embryos be directly involved in patterning of the germ rudiment. Moreover, since it has not yet been possible to recover a bicoid homologue from any species outside the diptera, it has been speculated that the anterior bicoid gradient could be a late addition during insect evolution. We addressed this question by analysing the regulation of potential target genes of bicoid in the short germ embryo of Tribolium castaneum. We demonstrate that homologues of caudal and hunchback from Tribolium are regulated by Drosophila bicoid. In Drosophila, maternal caudal RNA is translationally repressed by bicoid. We find that Tribolium caudal RNA is also translationally repressed by bicoid, when it is transferred into Drosophila embryos under a maternal promoter. This strongly suggests that a functional bicoid homologue must exist in Tribolium. The second target gene, hunchback, is transcriptionally activated by bicoid in Drosophila. Transfer of the regulatory region of Tribolium hunchback into Drosophila also results in regulation by early maternal factors, including bicoid, but in a pattern that is more reminiscent of Tribolium hunchback expression, namely in two early blastoderm domains. Using enhancer mapping constructs and footprinting, we show that caudal activates the posterior of these domains via a specific promoter. Our experiments suggest that a major event in the evolutionary transition from short to long germ embryogenesis was the switch from activation of the hunchback gap domain by caudal to direct activation by bicoid. This regulatory switch can explain how this domain shifted from a posterior location in short germ embryos to its anterior position in long germ insects, and it also suggest how an anterior gradient can pattern the germ rudiment in short germ embryos, i.e. by regulating the expression of caudal.

Conserved and divergent expression aspects of the Drosophila segmentation gene hunchback in the short germ band embryo of the flour beetle Tribolium.

The segmentation gene hunchback (hb) plays a central role in determining the anterior-posterior pattern in the Drosophila embryo. We have cloned the homologue of hb from the flour beetle Tribolium and show that, on the basis of its expression pattern, most of its functions seem to be conserved between these two species. Like Drosophila, Tribolium has a maternal hb expression that appears to be under translational control by a factor at the posterior pole of the embryo. The maternal expression is followed by a zygotic expression in the region of the developing head and thoracic segments. During germ band extension, a posterior expression domain appears that is likely to be homologous to the posterior blastoderm expression of hb in Drosophila. These observations suggest that hb may have the same functions in early Drosophila and Tribolium development, despite the different types of embryogenesis in these two species (long versus short germ development). One differing aspect of hb expression in Tribolium concerns a structure that is not present in Drosophila, namely the serosa. An hb expression domain at the anterior pole precisely demarcates the border between the extraembryonic serosa and the embryonic field in the Tribolium embryo at an early stage, and hb protein remains expressed in the serosa cells until the end of embryogenesis.