Late extraembryonic morphogenesis and its zen-induced failure in the milkweed bug Oncopeltus fasciatus

Many insects undergo katatrepsis, essential reorganization by the extraembryonic membranes that repositions the embryo. Knockdown of the zen gene by RNA interference (RNAi) prevents katatrepsis in the milkweed bug Oncopeltus fasciatus. However, the precise morphogenetic defect has been uncertain, and katatrepsis itself has not been characterized in detail. The dynamics of wild type and zen^RNAi eggs were analyzed from time-lapse movies, supplemented by analysis of fixed specimens. These investigations identify three zen^RNAi defects. First, a reduced degree of tissue contraction implies a role for zen in baseline compression prior to katatrepsis. Subsequently, a characteristic ‘bouncing’ activity commences, leading to the initiation of katatrepsis in wild type eggs. The second zen^RNAi defect is a delay in this activity, suggesting that a temporal window of opportunity is missed after zen knockdown. Ultimately, the extraembryonic membranes fail to rupture in zen^RNAi eggs: the third defect. Nevertheless, the outer serosal membrane manages to contract, albeit in an aberrant fashion with additional phenotypic consequences for the embryo. These data identify a novel epithelial morphogenetic event – rupture of the ‘serosal window’ structure – as the ultimate site of defect. Overall, Oncopeltus zen seems to have a role in coordinating a number of pre-katatreptic events during mid embryogenesis.     

Ovicide-induced serosa degeneration and its impact on embryonic development in Manduca sexta (Insecta: Lepidoptera)

Eggs of Manduca sexta treated with the ovicide Ov. 165049 turn orange, and the embryos later die. The orange pigmentation is at first confined to the serosa, and is accompanied by pathological changes of serosal cells. Lipid vesicles aggregate and spindle-shaped electron-lucent vesicles-normally forming a single layer below the apical cell surface-greatly accumulate. The mitochondria swell considerably, and their matrices become electron-lucent. Subsequently, the serosal cells develop additional features of necrosis. They form many autophagic vacuoles which contain mostly degradating mitochondria, but also segregated rough endoplasmic reticulum (rER) and glycogen granules. The whole cytoplasm vesiculates, and the cells shrink considerably. The nuclei become less irregular in shape, the chromatin disperses rather evenly whereas the nucleoli persist. Neither chromatin condensation nor the production of apoptotic bodies was observed-further evidence, that the serosal cells die by necrosis rather than apoptosis. At some stage of development the damaged serosa ruptures, retracts from the embryo and forms a sphere beneath it. It is only after the rupture of the serosa, that the embryo also turns orange and disintegrates rapidly. This shows impressively the protective function which the serosa plays for the embryo. Our physiological tests indicate, that the orange pigmentation of the serosa induced by the ovicide results from a disturbance of the tryptophan/ommochrome pathway serving the excretion of potentially toxic metabolites of tryptophan-rich proteins. The results demonstrate first that the serosa represents an important target for ovicide pesticides and second that it plays a vital role as an excretory organ during embryogenesis.     

Characterization of interstitial cells of Cajal in the subserosal layer of the guinea-pig colon

Interstitial cells of Cajal in the subserosa (ICC-SS) of the guinea-pig proximal colon were studied by immunohistochemistry for c-Kit receptors and by transmission electron microscopy. These cells were distributed within a thin layer of connective tissue space immediately beneath the mesothelium and were multipolar with about five primary cytoplasmic processes that divided further into secondary and tertiary processes to form a two-dimensional network. Ultrastructural observations revealed that ICC-SS were connected to each other via gap junctions. They also formed close contacts and peg-and-socket junctions with smooth muscle cells. Three-dimensional analysis of confocal micrographs revealed that the cytoplasmic processes of ICC-SS had contacts with interstitial cells in the longitudinal muscle layer. Taking account of the location and peculiar arrangement of the ICC-SS and the main functions of the proximal colon, i.e. the absorption and transport of fluids, we suggest that the superficial network of ICC-SS acts as a stretch receptor to detect circumferential expansion and swelling of the colon wall and triggers the contraction of the longitudinal muscle to accelerate the drainage of fluids from the colon.     

Epithelial reorganization events during late extraembryonic development in a hemimetabolous insect

As extra-embryonic tissues, the amnion and serosa are not considered to contribute materially to the insect embryo, yet they must execute an array of morphogenetic movements before they are dispensable. In hemimetabolous insects, these movements have been known for over a century, but they have remained virtually unexamined. This study addresses late extraembryonic morphogenesis in the milkweed bug, Oncopeltus fasciatus. Cell shape changes and apoptosis profiles are used to characterize the membranes as they undergo a large repertoire of final reorganizational events that reposition the embryo (katatrepsis), and eliminate the membranes themselves in an ordered fashion (dorsal closure). A number of key features were identified. First, amnion-serosa “fusion” involves localized apoptosis in the amnion and the formation of a supracellular actin purse string at the amnion-serosa border. During katatrepsis, a ‘focus’ of serosal cells undergoes precocious columnarization and may serve as an anchor for contraction. Lastly, dorsal closure involves novel modifications of the amnion and embryonic flank that are without counterpart during the well-known process of dorsal closure in the fruit fly Drosophila melanogaster. These data also address the long-standing question of the final fate of the amnion: it undergoes apoptosis during dorsal closure and thus is likely to be solely extraembryonic.     

Extraembryonic development in insects and the acrobatics of blastokinesis

Extraembryonic development is familiar to mouse researchers, but the term is largely unknown among insect developmental geneticists. This is not surprising, as the model system Drosophila melanogaster has an extremely reduced extraembryonic component, the amnioserosa. In contrast, most insects retain the ancestral complement of two distinct extraembryonic membranes, amnion and serosa. These membranes are involved in several key morphogenetic events at specific developmental stages. The events of anatrepsis and katatrepsis-collectively referred to as blastokinesis-are specific to hemimetabolous insects. Corresponding events in holometabolous insects are simplified and lack formal names. All insects retain dorsal closure, which has been well studied in Drosophila. This review aims to resurrect both the terminology and awareness of insect extraembryonic development-which were last common currency in the late nineteenth and early twentieth centuries-as a number of recent studies have identified essential components of these events, through RNA interference of developmental genes and ectopic hormonal treatments. As much remains unknown, this topic offers opportunities for research on tissue specification, the regulation of cell shape changes and tissue interactions during morphogenesis, tracing the origins and final fates of cell and tissue lineages, and ascertaining the membranes' functions between morphogenetic events.     

Oncopeltus fasciatus zen is essential for serosal tissue function in katatrepsis

Unlike most Hox cluster genes, with their canonical role in anterior-posterior patterning of the embryo, the Hox3 orthologue of insects has diverged. Here, we investigate the zen orthologue in Oncopeltus fasciatus (Hemiptera:Heteroptera). As in other insects, the Of-zen gene is expressed extraembryonically, and RNA interference (RNAi) experiments demonstrate that it is functionally required in this domain for the proper occurrence of katatrepsis, the phase of embryonic movements by which the embryo emerges from the yolk and adjusts its orientation within the egg. After RNAi knockdown of Of-zen, katatrepsis does not occur, causing embryos to complete development inside out. However, not all aspects of expression and function are conserved compared to grasshopper, beetle, and fly orthologues. Of-zen is not expressed in the extraembryonic tissue until relatively late, suggesting it is not involved in tissue specification. Within the extraembryonic domain, Of-zen is expressed in the outer serosal membrane, but unlike orthologues, it is not detectable in the inner extraembryonic membrane, the amnion. Thus, the role of zen in the interaction of serosa, amnion, and embryo may differ between species. Of-zen is also expressed in the blastoderm, although this early expression shows no apparent correlation with defects seen by RNAi knockdown.     

The dynamic expression of extraembryonic marker genes in the beetle Tribolium castaneum reveals the complexity of serosa and amnion formation in a short germ insect

Most insect embryos develop with two distinct extraembryonic membranes, the serosa and the amnion. In the insect beetle Tribolium the early origin of the serosa within the anterior blastoderm is well established but the origin of the amnion is still debated. It is not known whether this tissue develops from a blastodermal precursor or originates de novo later from embryonic tissue during embryogenesis.We undertook an in-depth analysis of the spatio-temporal expression pattern profile of important extraembryonic membrane marker genes with emphasis on early blastoderm development in Tribolium.The amnion marker iroquois (Tc-iro) was found co-expressed with the serosa marker zerknüllt1 (Tc-zen1) during early blastoderm formation in an anterior cap domain. This domain later resolved into two adjacent domains that likely represent the precursors of the serosa and the amnion. In addition, we found the hindsight ortholog in Tribolium (Tc-hnt) to be a serosa-specific marker. Surprisingly, decapentaplegic (Tc-dpp) expression was not seen as a symmetric cap domain but detected asymmetrically first along the DV- and later also along the AP-axis. Moreover, we found a previously undescribed domain of phosphorylated MAD (pMAD) protein in anterior ventral serosal cells.This is the first study showing that the anterior-lateral part of the amnion originates from the anterior blastoderm while the precursor of the dorsal amnion develops later de novo from a dorsal-posterior region within the differentiated blastoderm.     

Stage-dependent strategies of host invasion in the egg-larval parasitoid Chelonus inanitus

Chelonus inanitus (Braconidae) is a solitary egg-larval parasitoid which lays its eggs into eggs of Spodoptera littoralis (Noctuidae); the parasitoid larva then develops in the haemocoel of the host larva. Host embryonic development lasts approx. 3.5 days while parasitoid embryonic development lasts approx. 16 h. All stages of host eggs can be successfully parasitized, and we show here that either the parasitoid larva or the wasp assures that the larva eventually is located in the host's haemocoel. (1) When freshly laid eggs, up to almost 1-day-old, are parasitized, the parasitoid hatches while still in the yolk and enters the host either after waiting or immediately through the dorsal opening. (2) When 1-2-day-old eggs are parasitized, the host embryo has accomplished final dorsal closure and is covered by an embryonic cuticle when the parasitoid hatches; in this case the parasitoid larva bores with its moving abdominal tip into the host. (3) When 2.5-3.5-day-old eggs are parasitized, the wasp oviposits directly into the haemocoel of the host embryo; from day 2 to 2.5 the embryo is still very small and the wasps, after probing, often restrain from oviposition for a few hours.     

The occurrence of a juvenile hormone binding protein and in vitro synthesis of juvenile hormone by the serosa of Locusta migratoria embryos

Summary At the end of blastokinesis, serosal epitheliae of 4- to 5-day-old embryos of Locusta migratoria contain an immunohistologically detectable cytosolic protein (M_r 240 kDa) which is related to the juvenile hormone carrier-protein in the haemolymph of the same species and which binds tritiated juvenile hormone 3 (JH3) (K_d10^–8 M). At this early stage of development the corpora allata of the embryo are not yet fully differentiated and do not synthesize JH3 in organ cultures. The earliest detectable JH3 production by corpora allata in isolated heads is on day 6. On the other hand, serosal epitheliae of 4- to 5-day-old embryos produce JH3 in organ cultures, as has been shown by methylation of (10-³H)-JH3-acid to (10-³H)-JH3, and by incorporation of tritiated CH₃ from l-(methyl-³H)-methionine into JH3. Isolated heads and abdomens of the embryos used as donors for the serosal preparations did not show methyl transferase activity responsible for JH3 biosynthesis. The serosal cells represent a hitherto unrecognized source of methyl transferase activity and of JH3 production. Degradation of JH3 to JH3-acid was also observed.Dedicated to Professor Herbert Röller on the occasion of his 60th birthday