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.     

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.     

Postgastrular zen expression is required to develop distinct amniotic and serosal epithelia in the scuttle fly Megaselia

The amnioserosa is an extraembryonic epithelium that evolved in higher cyclorrhaphan flies from distinct serosal and amniotic epithelia. The underlying genetic mechanism of this evolutionary transition is unknown. Amnioserosa development of Drosophila correlates with novel expression characteristics of the homeobox gene zerknüllt (zen), including a broad zen expression domain in the syncytial blastoderm and the complete absence of postgastrular zen expression. Here we examine the functional significance of these features by altering the activity profile of zen in Megaselia (a lower cyclorrhaphan fly with distinct serosal and amniotic epithelia) and Drosophila, and by examining in Megaselia the function of u-shaped group (ush-group) genes, which in Drosophila maintain the amnioserosa after gastrulation when zen is no longer expressed. In Megaselia, loss of postgastrular zen expression abrogates serosa development but allows amnion development. Ectopic expression of zen in early Megaselia embryos allows serosa formation but perturbs amnion development. Megaselia homologues of u-shaped group genes are not essential for serosa formation but mediate germband retraction and dorsal closure. Finally, ectopic postgastrular zen expression in Drosophila causes an enlargement of amnioserosa cells and interferes with the morphogenetic functions of the amnioserosa. Our results suggest that the origin of the amnioserosa involved the loss of postgastrular zen expression from extraembryonic tissue, that the early broad expression domain of Drosophila zen evolved afterwards, and that the ush-group genes ancestrally played a role in morphogenetic functions of the amnion.     

A scanning electron microscope study of the developing embryo of Manduca sexta (L.) (Lepidoptera : Sphingidae)

Scanning electron microscopy of the developing Manduca sexta (Lepidoptera : Sphingidae) embryo reveals that the body wall of the insect undergoes considerable morphogenesis beginning at 20 hr post-oviposition. The elongated 19 hr embryo contracts in length, which gives rise to the formation of rudimentary segments. By 33 hr, many of the appendage anlagen are visible, the presumptive spiracles appear as bifurcate pits and the proctodeum begins invagination. During this same period, prior to katatrepsis, the body walls become established, and the segments and appendages develop. Between 50 and 60 hr post-oviposition, involution of the oral cavity and reorientation of the associated gnathal appendages occurs. During this same period, katatrepsis and provisional dorsal closure take place. Developmental polarity is evident as a distinctive wave of specialization proceeding posterior to anterior in the thorax/abdomen, and anterior to posterior in the head. Configuration of the oral cavity is strikingly prognathous until just prior to eclosion. Two embryonic molts are apparent, as determined by the remnants of ecdysed “embryonic cuticles”.     

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.     

Embryonic development of the hemipteran insect Rhodnius prolixus

The embryonic development of the hemipteran insect Rhodnius prolixus was studied by use of contemporary light and electron microscopy. Embryos were staged according to days postoviposition. Eggs laid on day one complete blastoderm formation and anatrepsis, the first phase of blastokinesis, by day 5. The embryo develops in a cephalocaudal orientation which is 180° to the anteroposterior axis of the egg. Subsequent development, prior to the second phase of blastokinesis (katatrepsis), leads to segmentation of the germ band, evagination of appendages, and histogenesis of germ layers. Concomitantly with these events, the amnion undergoes dramatic change. By day 7 the embryo begins a 180° revolution while migrating to the ventral surface of the yolk. This restores its polarity with respect to that of the egg and facilitates hatching. The serosa contracts, pulling the amnion and embryo anteriorly. Eventually the serosa is internalized at a point dorsal to the head and the lateral walls of the embryo grow up and surround the yolk. Development continues until day 15 when the embryo hatches as a first instar larva.     

Distinct functions of the Tribolium zerknüllt genes in serosa specification and dorsal closure

BACKGROUND: In the long-germ insect Drosophila, a single extraembryonic membrane, the amnioserosa, covers the embryo at the dorsal side. In ancestral short-germ insects, an inner membrane, the amnion, covers the embryo ventrally, and an outer membrane, the serosa, completely surrounds the embryo. An early differentiation step partitions the uniform blastoderm into the anterior-dorsal serosa and the posterior-ventral germ rudiment giving rise to amnion and embryo proper. In Drosophila, amnioserosa formation depends on the dorsoventral patterning gene zerknüllt (zen), a derived Hox3 gene. RESULTS: The short-germ beetle Tribolium castaneum possesses two zen homologs, Tc-zen1 and Tc-zen2. Tc-zen1 acts early and specifies the serosa. The loss of the serosa after Tc-zen1 RNAi is compensated by an expansion of the entire germ rudiment toward the anterior. Instead of the serosa, the amnion covers the embryo at the dorsal side, and later size regulation normalizes the early fate shifts, revealing a high degree of plasticity of short-germ development. Tc-zen2 acts later and initiates the amnion and serosa fusion required for dorsal closure. After Tc-zen2 RNAi, the amnion and serosa stay apart, and the embryo closes ventrally, assuming a completely everted (inside-out) topology. CONCLUSIONS: In Tribolium, the duplication of the zen genes was accompanied by subfunctionalization. One of the paralogues, Tc-zen1, acts as an early anterior-posterior patterning gene by specifying the serosa. In absence of the serosa, Tribolium embryogenesis acquires features of long-germ development with a single extraembryonic membrane. We discuss implications for the evolution of insect development including the origin of the zen-derived anterior determinant bicoid.     

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.     

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.     

The embryonic development of the primitive moth,Neomicropteryx nipponensis Issiki (lepidoptera,micropterygidae): Morphogenesis of the embryo by external observation

The micropterygid moth Neomicropteryx nipponensis belongs to the most primitive suborder Zeugloptera of the Lepidoptera. During embryogenesis the small circular germ disk formed on the ventral egg surface invaginates deeply into the yolk. It finally separates from the egg periphery or rudimentary serosa, and becomes a sac-shaped germ rudiment. Its anterior part later develops into the germ band, while its posterior part is the future amnion. Just before revolution of the embryo, the embryo assumes a completely superficial position beneath the yolk. Neither amnion nor serosa rupture during revolution; by completion of dorsal closure they have been incorporated into the yolk to form the secondary dorsal organ. The formation of the germ rudiment and embryonic membranes in N. nipponensis resembles those of swift moths, Endoclyta (suborder Monotrysia) and of the caddisflies, Stenopsyche (Trichoptera), but differs from those of ditrysian Lepidoptera. The secondary dorsal organ has never been found in any other lepidopteran embryos; however, it is formed in N. nipponensis and in the Trichoptera. The results of the present study strongly support the general phylogenetic views that the Zeugloptera have a close affinity to the Trichoptera.     

Dynamic analysis of actin cable function during Drosophila dorsal closure

Throughout development, a series of epithelial movements and fusions occur that collectively shape the embryo. They are dependent on coordinated reorganizations and contractions of the actin cytoskeleton within defined populations of epithelial cells. One paradigm morphogenetic movement, dorsal closure in the Drosophila embryo, involves closure of a dorsal epithelial hole by sweeping of epithelium from the two sides of the embryo over the exposed extraembryonic amnioserosa to form a seam where the two epithelial edges fuse together. The front row cells exhibit a thick actin cable at their leading edge. Here, we test the function of this cable by live analysis of GFP-actin-expressing embryos in which the cable is disrupted by modulating Rho1 signaling or by loss of non-muscle myosin (Zipper) function. We show that the cable serves a dual role during dorsal closure. It is contractile and thus can operate as a "purse string," but it also restricts forward movement of the leading edge and excess activity of filopodia/lamellipodia. Stripes of epithelium in which cable assembly is disrupted gain a migrational advantage over their wild-type neighbors, suggesting that the cable acts to restrain front row cells, thus maintaining a taut, free edge for efficient zippering together of the epithelial sheets.     

Innexin 3, a New Gene Required for Dorsal Closure in Drosophila Embryo

Background

Dorsal closure is a morphogenetic event that occurs during mid-embryogenesis in many insects including Drosophila, during which the ectoderm migrates on the extraembryonic amnioserosa to seal the embryo dorsally. The contribution of the ectoderm in this event has been known for a long time. However, amnioserosa tension and contractibility have recently been shown also to be instrumental to the closure. A critical pre-requisite for dorsal closure is integrity of these tissues that in part is mediated by cell-cell junctions and cell adhesion. In this regard, mutations impairing junction formation and/or adhesion lead to dorsal closure. However, no role for the gap junction proteins Innexins has so far been described.

Results and Discussion

Here, we show that Innexin 1, 2 and 3, are present in the ectoderm but also in the amnioserosa in plaques consistent with gap junctions. However, only the loss of Inx3 leads to dorsal closure defects that are completely rescued by overexpression of inx3::GFP in the whole embryo. Loss of Inx3 leads to the destabilisation of Inx1, Inx2 and DE-cadherin at the plasma membrane, suggesting that these four proteins form a complex. Accordingly, in addition to the known interaction of Inx2 with DE-cadherin, we show that Inx3 can bind to DE-cadherin. Furthermore, Inx3-GFP overexpression recruits DE-cadherin from its wildtype plasma membrane domain to typical Innexin plaques, strengthening the notion that they form a complex. Finally, we show that Inx3 stability is directly dependent on tissue tension. Taken together, we propose that Inx3 is a critical factor for dorsal closure and that it mediates the stability of Inx1, 2 and DE-cadherin by forming a complex.     

Integrin-dependent apposition of Drosophila extraembryonic membranes promotes morphogenesis and prevents anoikis

BACKGROUND: Two extraembryonic tissues form early in Drosophila development. One, the amnioserosa, has been implicated in the morphogenetic processes of germ band retraction and dorsal closure. The developmental role of the other, the yolk sac, is obscure. RESULTS: By using live-imaging techniques, we report intimate interactions between the amnioserosa and the yolk sac during germ band retraction and dorsal closure. These tissue interactions fail in a subset of myospheroid (mys: betaPS integrin) mutant embryos, leading to failure of germ band retraction and dorsal closure. The Drosophila homolog of mammalian basigin (EMMPRIN, CD147)-an integrin-associated transmembrane glycoprotein-is highly enriched in the extraembryonic tissues. Strong dominant genetic interactions between basigin and mys mutations cause severe defects in dorsal closure, consistent with basigin functioning together with betaPS integrin in extraembryonic membrane apposition. During normal development, JNK signaling is upregulated in the amnioserosa, as midgut closure disrupts contact with the yolk sac. Subsequently, the amnioserosal epithelium degenerates in a process that is independent of the reaper, hid, and grim cell death genes. In mys mutants that fail to establish contact between the extraembryonic membranes, the amnioserosa undergoes premature disintegration and death. CONCLUSIONS: Intimate apposition of the amnioserosa and yolk sac prevents anoikis of the amnioserosa. Survival of the amnioserosa is essential for germ band retraction and dorsal closure. We hypothesize that during normal development, loss of integrin-dependent contact between the extraembryonic tissues results in JNK-dependent amnioserosal disintegration and death, thus representing an example of developmentally programmed anoikis.     

The amnioserosa is an apomorphic character of cyclorrhaphan flies

In developing insect eggs the cells of the blastoderm adopt either an embryonic or an extraembryonic fate. The extraembryonic tissue consists of epithelia, termed amnion and serosa, which wrap the germ band embryo. The serosa develops directly from part of the blastoderm and surrounds the embryo as well as the yolk. The amnion develops from the margins of the germ band and in most insect species generates a transient ventral cavity for the developing embryo. The amniotic cavity and the serosa have been reduced in the course of dipteran evolution. The insect order of Diptera includes the paraphyletic Nematocera, including gnats and mosquitoes, and the more derived monophyletic Brachycera, the true flies. Nematocera develop within an amniotic cavity and the surrounding serosa, whereas cyclorrhaphan Brachycera do not. This observation implies that the amnion and serosa have been reduced before the radiation of the monophyletic cyclorrhaphan flies. Here I show that an amniotic cavity is formed during embryogenesis of the horsefly Haematopota pluvialis (Tabanidae) and the dancefly Empis livida (Empididae). The results suggest that extraembryonic tissue was reduced in the stem lineage of cyclorrhaphan flies, with consequences for the molecular basis of pattern formation along the anterior-posterior axis of the embryo. Received: 21 October 1999 / Accepted: 17 January 2000     

The significance of extracellular Ca<Superscript>2+</Superscript> in contractile responses of chick amnion

Neurotransmitter receptors are formed during chick embryo development in the amnion, an avascular extraembryonic membrane devoid of innervation. Carbachol induces phasic and tonic contractions mediated by M₃ cholinoceptors in an amniotic membrane strip isolated from 11–14-day-old chick embryo. The carbachol effect on the amnion contractile activity was studied in normal physiological salt solution, during depolarization by K⁺, exposure to nifedipine, and in calcium-free medium. Voltage-dependent and receptor-operated Ca²⁺ channels as well as calcium from intracellular stores are involved in the contractile response to carbachol. Phasic contractions of the amnion are mainly induced by calcium ions entering through voltage-dependent calcium channels, while tonic contractions are also maintained by receptor-operated channels. Ca²⁺-activated potassium channels can serve as a negative feedback factor in regulation of the amnion contractile responses.     

Embryonic development of the jumping bristletail Pedetonutus unimaculatus Machida,with special reference to embryonic membranes (Hexapoda: Microcoryphia,Machilidae)

In the machilid Pedetonutus unimaculatus, a germ disc is formed by the aggregation and proliferation of cells within a broadly defined embryonic area. Cells adjacent to the embryonic area form the serosal fold that grows beneath the embryo. Then the embryonic margin is extended to form a cell layer or amnion that lies between the embryo and serosal fold. Thus, an amnioserosal fold is formed by the addition of the amnion to the serosal fold. Serosal cells cover the entire surface of the egg and begin to secrete a serosal cuticle. Soon the amnioserosal fold is withdrawn, and the embryo is exposed to the egg surface. The spreading amnion replaces the serosal cells that finally degenerate through the formation of a secondary dorsal organ. In the areas of amnion anterior and lateral to the embryo, yolk folds form and encompass the embryo. The amnion is a provisional dorsal closure and never participates in the formation of the definitive one. The amnioserosal fold of the Microcoryphia appears to have the functional role of secreting a serosal cuticle beneath the embryo. This fold of the Microcoryphia may be regarded as an ancestral form of the amnioserosal folds of the Thysanura-Pterygota. the yolk folds may appear to be passive transformation of the yolk mass linked to positioning of the growing embryo within the egg. There is no evidence that the yolk folds and the cavity appearing between them in the Microcoryphia are homologous to the amnioserosal fold and amniotic cavity in the Thysanura-Pterygota. The yolk folds appear to be one of the embryological autapomorphies in the Microcoryphia. © 1994 Wiley-Liss, Inc.     

Specialized extraembryonic cells connect embryonic and extraembryonic epidermis in response to Dpp during dorsal closure in Drosophila

Dorsal closure in Drosophila embryogenesis involves expansion of the dorsal epidermis, followed by closure of the opposite epidermal edges. This process is driven by contractile force generated by an extraembryonic epithelium covering the yolk syncytium known as the amnioserosa. The secreted signaling molecule Dpp is expressed in the leading edge of the dorsal epidermis and is essential for dorsal closure. We found that the outermost row of amnioserosa cells (termed pAS) maintains a tight basolateral cell-cell adhesion interface with the leading edge of dorsal epidermis throughout the dorsal closure process. pAS was subject to altered cell motility in response to Dpp emanating from the dorsal epidermis, and this response was essential for dorsal closure. alphaPS3 and betaPS integrin subunits accumulated in the interface between pAS and dorsal epidermis, and were both required for dorsal closure. Looking at alphaPS3, type I Dpp receptor, and JNK mutants, we found that pAS cell motility was altered and pAS and dorsal epidermis adhesion failed under the mechanical stress of dorsal closure, suggesting that a Dpp-mediated mechanism connects the squamous pAS to the columnar dorsal epidermis to form a single coherent epithelial layer.     

Embryogenesis of the dipluran Lepidocampa weberi Oudemans (hexapoda: diplura, campodeidae): formation of dorsal organ and related phenomena

The developmental changes of embryonic membranes of a dipluran Lepidocampa weberi, with special reference to dorsal organ formation, are described in detail by light, scanning, and transmission electron microscopies. Newly differentiated germ band and serosa secrete the blastodermic cuticle at the entire egg surface beneath the chorion. Soon after, the serosal cells start to move dorsad. All the serosal cells finally concentrate at the dorsal side of the egg and form the dorsal organ. During their concentration, the serosal cells attenuate their cytoplasm to form filaments. The extensive area from which the serosa has receded is occupied by a second embryonic membrane, the amnion, which originates from the embryonic margin. The embryo and newly emerged amnion then secrete three fine cuticular layers, "cuticular lamellae I, II, and III," above which the filaments of the (developing) dorsal organ are situated. With the progression of definitive dorsal closure, the amnion reduces its extension, the dorsal organ is incorporated into the body cavity of the embryo, and the amnion and dorsal organ finally degenerate.The dorsal organ of diplurans is formed by the concentration of whole serosal cells, while that of collembolans is formed by the direct differentiation of a part of serosal cells. However, the dorsal organs of diplurans and collembolans closely resemble each other in major aspects, including that of ultrastructural features, and there is no doubt regarding their homology. The amnion, which has been regarded as being a characteristic of Ectognatha, also develops in the Diplura. This might suggest a closer affinity between the Diplura and Ectognatha than previously believed.     

Development of the subsoephageal body cells and the pericardiac cells during embryogenesis with diapause in Locusta migratoria (Linnaeus 1758) (Orthoptera: Acrididae)

During Locusta migratoria embryogenesis, the yolk is progressively degraded and the resulting metabolites are released in the haemolymph. We researched the organs possibly involved in the uptake of haemolymphatic proteins. Among organs originated from mesoderm, the SOB (suboesophageal bodies) situated in the embryonic head are remarkable by a very early acquisition of differentiated cytological characters, while most other cells of the embryo are undifferentiated. The SOB quite disappear before hatching. Just before katatrepsis stage, the other organs derived from mesoderm begin to differentiate, including the PC (pericardial cells) which take over from the SOB. These cells, situated in thorax and abdomen, are developed during the dorsal close of embryo. The development and the ultrastructural changes of the SOB cells and of the PC were studied during an embryogenesis with diapause. The morphology of embryos which enter diapause is comparable with that of a continuous development at the beginning of katatrepsis. However, the cells of SOB and PC cells suffer from remarkable changes not only physiologically but cytologically. At the beginning of diapause, the proteosynthetic activity practically disappears in the SOB cells and the lysis areas appear. Nevertheless, the exchanges between these cells and the haemolymph still remain important. For the period of cold, which is necessary to the resumption of development, the aspect of the SOB cells changes and in particular the areas of lysis become less wide. When the embryo reopens its development, the SOB cells show a proteosynthetic activity and the areas of lysis disappear. The changes of the SOB cells and of the PC cells are regularized during the resumption of the development: the SOB cells which had again taken a normal activity start to regress from the stage VII on, while the PC cells take over.