Germ Band Formation Research Articles

Early embryonic development of the German cockroach Blattella germanica

BackgroundEarly embryogenesis is characterized by dramatic cell proliferation and movement. In most insects, early embryogenesis includes a phase called the uniform blastoderm, during which cells evenly cover the entirety of the egg. However, the embryo of the German cockroach, Blattella germanica, like those of many insects within the super order Polyneoptera, does not have a uniform blastoderm; instead, its first cells condense rapidly at the site of a future germband. We investigated early development in this species in order to understand how early gene expression is or is not conserved in these insect embryos with distinct early cell behaviors.ResultsWe present a detailed time series of nuclear division and distribution from fertilization through germband formation and report patterns of expression for the early patterning genes hunchback, caudal, and twist in order to understand early polarization and mesoderm formation. We show a detailed time course of the spatial expression of two genes involved in the segmentation cascade, hedgehog and even-skipped, and demonstrate two distinct dynamics of the segmentation process.ConclusionsDespite dramatic differences in cell distribution between the blastoderms of many Polyneopteran insects and those of more well-studied developmental models, expression patterns of early patterning genes are mostly similar. Genes associated with axis determination in other insects are activated relatively late and are probably not maternally deposited. The two phases of segmentation—simultaneous and sequential—might indicate a broadly conserved mode of morphological differentiation. The developmental time course we present here should be of value for further investigation into the causes of this distinct blastoderm type.

Influence of Parental Age on Reproductive Potential and Embryogenesis in the Pepper Weevil, Anthonomus eugenii (Cano) (Col.: Curculionidae).

The pepper weevil (Anthonomus eugenii Cano) is a devastating pest that inflicts severe damage to pepper crops, leading to substantial economic losses. This study investigated the impact of aging on the reproductive success of the pepper weevil. Pepper weevil-infested fruit were harvested from pepper fields and subsequently transferred into an insect cage to facilitate the emergence of adults. The emerged adults were housed in separate cages and allowed to mature until they reached specified ages: 10 days old (young), 20 days old (middle-aged), and 30 days old (old) individuals. Eggs laid by each age group were carefully collected and incubated under controlled laboratory conditions (28 ± 1.5 °C). Several reproductive variables including the number of eggs laid, the percentage of hatched eggs, and the egg incubation period were recorded for each age group. Embryonic development was also monitored daily using a VHX digital microscope at a magnification of 200×. Differences in developmental stages such as the blastoderm, germ band, gastrulation, segmentation, and appendage formation were observed, and the time span of every stage was recorded. The results show that the 10-day-old weevils laid the most eggs and had the highest hatching rate and the shortest developmental time. The 30-day-old weevils laid the fewest eggs and had the lowest hatching rate and longest developmental time. Thus, the pepper weevil age significantly influenced the fecundity, length of time for each embryonic development stage, hatching rate, and incubation period, and should be considered when studying the reproductive biology of this pest insect. This first report of the effect of aging on the reproductive potential of the pepper weevil should enable pepper growers to adopt cultural practices aimed at reducing the pepper weevil populations, thereby helping to protect their crop from this important pest.

Visualisation of early embryos of the wandering glider, Pantala flavescens (Fabricius, 1798) (Odonata: Libellulidae)

The embryonic development of Pantala flavescens (Fabricius, 1798) is documented for the first time. In contrast to previously reported insect embryonic development, we describe the embryological development of P. flavescens in living conditions by focusing on its externally recognisable features, i.e., we conducted the study in a purely non-destructive manner. The entire embryonic development was completed within six to seven days. We pictured the eggs at successive embryonic developmental stages: embryogenesis starts, germband formation, segmentation, blastokinesis, appendage formation, and dorsal closure. These findings can be helpful in future studies regarding the impacts of pollutants and climate change on the early embryonic development of dragonflies.

Nuclear receptors EcR-A/RXR and HR3 control early embryogenesis in the short-germ hemimetabolous insect Blattella germanica

The role of steroid hormone signaling during insect post-embryonic development has been extensively characterized. However, its function during embryonic development is less understood, particularly in short-germ band hemimetabolous insects. To solve this, we have used Blattella germanica to analyze the embryonic functions of the heterodimeric ecdysone receptor, BgEcR-A and BgRXR. Here, we show that both transcripts are present from the beginning of embryogenesis, and that are required for proper germband formation. We also show that they regulate an early induction of a stereotypical cascade of nuclear receptors. Notably, we found that one of these, BgHR3, controls the formation of the cephalic region of the germband. Finally, spatial expression of two other receptors, BgE75-A and BgFTZ-F1, suggest their involvement in the formation of the nervous system and in germband segmentation, respectively. In summary, our results highlight the critical roles of ecdysone-signaling during early embryogenesis in short-germ band hemimetabolous insects.

Segmental expression of two ecdysone pathway genes during embryogenesis of hemimetabolous insects

Signaling networks are redeployed across different developmental times and places to generate phenotypic diversity from a limited genetic toolkit. Hormone signaling networks in particular have well-studied roles in multiple developmental processes. In insects, the ecdysone pathway controls critical events in late embryogenesis and throughout post-embryonic development. While this pathway has not been shown to function in the earliest stage of embryonic development in the model insect Drosophila melanogaster, one component of the network, the nuclear receptor E75A, is necessary for proper segment generation in the milkweed bug Oncopeltus fasciatus. Published expression data from several other species suggests possible conservation of this role across hundreds of millions of years of insect evolution. Previous work also demonstrates a second nuclear receptor in the ecdysone pathway, Ftz-F1, plays a role in segmentation in multiple insect species. Here we report tightly linked expression patterns of ftz-F1 and E75A in two hemimetabolous insect species, the German cockroach Blattella germanica and the two-spotted cricket Gryllus bimaculatus. In both species, the genes are expressed segmentally in adjacent cells, but they are never co-expressed. Using parental RNAi, we show the two genes have distinct roles in early embryogenesis. E75A appears necessary for abdominal segmentation in B. germanica, while ftz-F1 is essential for proper germband formation. Our results suggest that the ecdysone network is critical for early embryogenesis in hemimetabolous insects.

Embryonic expression patterns of Hox genes in the oligochaete annelid Tubifex tubifex

We have cloned and characterized the expression of seven Hox genes (designated Ttu-lab, Ttu-Dfd, Ttu-Scr1, Ttu-Scr2, Ttu-Lox5, Ttu-Lox4 and Ttu-Lox2) from the oligochaete annelid Tubifex tubifex. RT-PCR analyses show that except for Ttu-Lox4 and Ttu-Lox2 which begin expression as early as cleavage stages, Tubifex Hox genes are expressed during stages 13–18 when embryos undergo germ band formation, segmentation and body elongation. In terms of combination of tissues (or organs) exhibiting positive cells, the Tubifex Hox genes examined in this study are classified into three groups. Ttu-lab, Ttu-Scr1 and Ttu-Lox5 are expressed only in the ventral nerve cord; Ttu-Scr2 and Ttu-Lox4 are expressed not only in the ventral nerve cord but also in distinct lateral segmental tissues; and Ttu-Dfd and Ttu-Lox2 are expressed not only in the segmental ectoderm along the length of the AP body axis but also in the prostomium. Anterior expression boundaries of Ttu-lab, Ttu-Scr1, Ttu-Lox5 and Ttu-Lox4 are at segments 3, 4, 5, and 9, respectively. Anterior expression boundary of Ttu-Scr2 is at segment 2, and Ttu-Dfd and Ttu-Lox2 are expressed even at the anteriormost portion, the prostomium. These observations suggest that as in other annelids, so-called “spatial colinearity” of anterior expression boundaries of Hox genes has been conserved in the oligochaetes. It is also evident that there are some oligochaete Hox genes which violate the spatial colinearity rule.

A study of embryonic development in eriophyoid mites (Acariformes, Eriophyoidea) with the use of the fluorochrome DAPI and confocal microscopy.

The embryonic development of four eriophyoid mite species, Cecidophyopsis ribis, Phytoptus avellanae, Oziella liroi and Loboquintus subsquamatus, has been studied with the use of fluorochrome DAPI and confocal microscopy. The first three nuclear divisions occur on the egg periphery (the groups of 2, 4, and 6 nuclei have been recorded), while the biggest part of yolk remains undivided. After four or five nuclear divisions all nuclei are situated only in one sector of the embryo, while other sectors contain only yolk suggesting possible meroblastic cleavage. Later, the formation of superficial blastoderm takes place. A few large yolk cells are situated inside the embryo. Germ band formation initiates as funnel-like cell invagination and leads to formation of a typical stage with four paired prosomal buds (chelicerae, palps, legs I and II). Each palp contains two lobes (anterior and posterior), the adult subcapitulum is presumably a fusion product of the anterior pair of the lobes. Neither rudiments of legs III and IV, traces of opisthosomal segments nor remnants of the prelarval exuvium under the egg shell were detected. Overall, the pattern of embryonic development in eriophyoids re-emphasizes the peculiarity of this ancient group of miniaturized phytoparasitic animals, and invites researches to pursue a deeper investigation of various fundamental aspects of this aberrant group of Acari. Further studies using various fluorescent dyes and transmission electron microscopy are needed to visualize plasma membranes and clarify the pattern of early cleavage of eriophyoids.

Embryonic development of the cricket Gryllus bimaculatus

Extensive research into Drosophila melanogaster embryogenesis has improved our understanding of insect developmental mechanisms. However, Drosophila development is thought to be highly divergent from that of the ancestral insect and arthropod in many respects. We therefore need alternative models for arthopod development that are likely to be more representative of basally-branching clades. The cricket Gryllus bimaculatus is such a model, and currently has the most sophisticated functional genetic toolkit of any hemimetabolous insect. The existing cricket embryonic staging system is fragmentary, and it is based on morphological landmarks that are not easily visible on a live, undissected egg. To address this problem, here we present a complementary set of “egg stages” that serve as a guide for identifying the developmental progress of a cricket embryo from fertilization to hatching, based solely on the external appearance of the egg. These stages were characterized using a combination of brightfield timelapse microscopy, timed brightfield micrographs, confocal microscopy, and measurements of egg dimensions. These egg stages are particularly useful in experiments that involve egg injection (including RNA interference, targeted genome modification, and transgenesis), as injection can alter the speed of development, even in control treatments. We also use 3D reconstructions of fixed embryo preparations to provide a comprehensive description of the morphogenesis and anatomy of the cricket embryo during embryonic rudiment assembly, germ band formation, elongation, segmentation, and appendage formation. Finally, we aggregate and schematize a variety of published developmental gene expression patterns. This work will facillitate further studies on G. bimaculatus development, and serve as a useful point of reference for other studies of wild type and experimentally manipulated insect development in fields from evo–devo to disease vector and pest management.

Functional analysis of centipede development supports roles for Wnt genes in posterior development and segment generation.

The genes of the Wnt family play important and highly conserved roles in posterior growth and development in a wide range of animal taxa. Wnt genes also operate in arthropod segmentation, and there has been much recent debate regarding the relationship between arthropod and vertebrate segmentation mechanisms. Due to its phylogenetic position, body form, and possession of many (11) Wnt genes, the centipede Strigamia maritima is a useful system with which to examine these issues. This study takes a functional approach based on treatment with lithium chloride, which causes ubiquitous activation of canonical Wnt signalling. This is the first functional developmental study performed in any of the 15,000 species of the arthropod subphylum Myriapoda. The expression of all 11 Wnt genes in Strigamia was analyzed in relation to posterior development. Three of these genes, Wnt11, Wnt5, and WntA, were strongly expressed in the posterior region and, thus, may play important roles in posterior developmental processes. In support of this hypothesis, LiCl treatment of S. maritima embryos was observed to produce posterior developmental defects and perturbations in AbdB and Delta expression. The effects of LiCl differ depending on the developmental stage treated, with more severe effects elicited by treatment during germband formation than by treatment at later stages. These results support a role for Wnt signalling in conferring posterior identity in Strigamia. In addition, data from this study are consistent with the hypothesis of segmentation based on a "clock and wavefront" mechanism operating in this species.

Spatiotemporal expression profile of the Pumilio gene in the embryonic development of silkworm.

We previously identified a pumilio gene in silkworm (Bombyx mori L.), designated BmPUM, which was specifically expressed in the ovary and testis. To further characterize this gene's involvement in silkworm development, we have determined the spatiotemporal expression pattern of BmPUM during all embryonic stages. Real-time polymerase chain reaction (RT-PCR) analysis revealed that BmPUM was expressed in all stages of silkworm embryos and that its transcript levels displayed two distinct peaks. The first was observed at the germ-band formation stage (1 d after oviposition) and dropped to a low level at the gonad formation stage (5 d after oviposition). The second was detected at the stage of bristle follicle occurrence (6 d after oviposition), which was confirmed by Western blot analysis and immunohistochemistry. Nanos (Nos), functioning together with Pum in abdomen formation of Drosophila embryos, was also highly expressed at the beginning (0 h to 1 d after oviposition) of embryogenesis, but its transcript levels were very low after the stage of germ-band formation. These results suggest that BmPUM functions with Bombyx mori nanos (Bm-nanos) at the early stages of silkworm embryonic development, and may then play a role in gonad formation and the occurrence of bristle follicles. Our data thus provide a foundation to uncover the role of BmPUM during silkworm development.

The gap gene Krüppel of Rhodnius prolixus is required for segmentation and for repression of the homeotic gene sex comb-reduced

The establishment of the anterior–posterior segmentation in insects requires the concerted action of a hierarchical gene network. Here, we study the orthologue of Krüppel gap gene in the hemipteran Rhodnius prolixus (Rp-Kr). We characterized its structure, expression pattern and function. The genomic sequence upstream of the Rp-Kr transcriptional unit shows a putative regulatory region conserved in the orthologue genes from Drosophila melanogaster and Tribolium castaneum. Rp-Kr expression is zygotic and it is expressed in the anterior half of the embryo (the posterior half of the egg) during the blastoderm stage and germ band formation; later, during germ band extension, it is expressed in a central domain, from T2 to A3. The Rp-Kr loss of function phenotypes shows disrupted thoracic and abdominal segmentation. Embryos with weak segmentation phenotypes show homeotic transformations, in which an ectopic tibial comb, typical of T1 leg, appears in T2, which correlates with the ectopic expression of Rp-sex-comb reduced in this leg.

Analysis of snail genes in the crustacean Parhyale hawaiensis: insight into snail gene family evolution

The transcriptional repressor snail was first discovered in Drosophila melanogaster, where it initially plays a role in gastrulation and mesoderm formation, and later plays a role in neurogenesis. Among arthropods, this role of snail appears to be conserved in the insects Tribolium and Anopheles gambiae, but not in the chelicerates Cupiennius salei and Achaearanea tepidariorum, the myriapod Glomeris marginata, or the Branchiopod crustacean Daphnia magna. These data imply that within arthropoda, snail acquired its role in gastrulation and mesoderm formation in the insect lineage. However, crustaceans are a diverse group with several major taxa, making analysis of more crustaceans necessary to potentially understand the ancestral role of snail in Pancrustacea (crustaceans + insects) and thus in the ancestor of insects as well. To address these questions, we examined the snail family in the Malacostracan crustacean Parhyale hawaiensis. We found three snail homologs, Ph-snail1, Ph-snail2 and Ph-snail3, and one scratch homolog, Ph-scratch. Parhyale snail genes are expressed after gastrulation, during germband formation and elongation. Ph-snail1, Ph-snail2, and Ph-snail3 are expressed in distinct patterns in the neuroectoderm. Ph-snail1 is the only Parhyale snail gene expressed in the mesoderm, where its expression cycles in the mesodermal stem cells, called mesoteloblasts. The mesoteloblasts go through a series of cycles, where each cycle is composed of a migration phase and a division phase. Ph-snail1 is expressed during the migration phase, but not during the division phase. We found that as each mesoteloblast division produces one segment's worth of mesoderm, Ph-snail1 expression is linked to both the cell cycle and the segmental production of mesoderm.

Silkworm egg proteins at the germ-band formation stage and a functional analysis of BmEP80 protein

The patterning of embryos in early stages is a critical process for embryo development. In order to understand the molecular mechanism of early embryogenesis in silkworm, 2-DE combined with MALDI-TOF–MS technologies were used to analyze the proteins from diapause-destined eggs at the germ-band formation stage. From over 1000 spots, 93 were selected for analysis and data were obtained from 59 revealing 42 proteins. Gene Ontology annotation showed these proteins were involved in several biological processes at the germ-band formation stage, including cell stress response and protein folding, cell growth and migration, termination of diapause, and nutrition storage. Prominent among them was a new 80 kDa protein, named Bombyx mori egg protein 80 (BmEP80). BmEP80 was a component of the eggshell which was secreted by follicle cells during the late vitellogenesis stage to early choriogenesis stage (FCs −5 to +10). It disappears during early embryogenesis and RNAi against it resulted in the collapse of eggs, thus it is likely that BmEP80 is a new component of the silkworm vitelline membrane.

Disappearance of chorion proteins from Bombyx mori eggs treated with HCl solution to prevent diapause

Bombyx mori eggs enter diapause immediately after completion of mesoderm segregation. HCl treatment of approximately 24-hour-old eggs (germband formation stage) is well known to be the most effective procedure to prevent entry into diapause, although the molecular mechanism remains unclear. In this study, we examined the protein composition of diapausing and nondiapausing eggs after various HCl treatments known to prevent or break diapause and found that proteins of approximately 11 and 8kDa disappeared immediately after HCl treatment. Partial amino acid sequences of these proteins indicated that they were members of the chorion class A protein L12 family synthesized in follicle cells. Under the hypothesis that the chorion provides a barrier to oxygen, dechorionation of diapausing eggs induces resumption of embryonic development. Hence, to test this and other hypotheses about the function of these proteins, we used 20% SDS-PAGE with Coomassie Brilliant Blue staining to trace their disappearance from embryos and eggshells after treatment with HCl under different conditions and on polyvoltine, univoltine, and bivoltine silkworm races. Even when 10-day-old diapausing eggs were treated with HCl, which did not break diapause, the 11 and 8kDa proteins disappeared. Our results suggest that disappearance of these proteins is not directly associated with preventing entry into or breaking a diapause state. Nevertheless, our results cannot completely rule out the possibility that the 11 and 8kDa proteins function to block permeability of O2 during the period when HCl treatment is physiologically effective to prevent diapause so that after the diapause system is established within the egg, even removing the 11 and 8kDa proteins may not affect to prevent diapause. We also discuss the role of these proteins in choriogenesis.

Apolipophorin-III expression and low density lipophorin formation during embryonic development of the silkworm, Bombyx mori

We examined the expression of apolipophorin-III (apoLp-III) during embryonic development of the silkworm Bombyx mori. ApoLp-III mRNA was first expressed 24h after oviposition, which corresponds to the time of germ band formation. The amount of apoLp-III in the eggs increased from day 2, peaked on day 4, and then gradually decreased until hatching (on day 9.5). ApoLp-III was apparently synthesized during early embryogenesis, as radioactive amino acids were incorporated into newly synthesized apoLp-III in three-day-old eggs. Moreover, radioactive apoLp-III was found only in the embryo and not in the extraembryonic tissue. KBr density gradient ultracentrifugation of egg homogenates showed that apoLp-III was associated with low-density lipophorin (LDLp). These results suggest that LDLp is required for the delivery of lipids for organogenesis during embryogenesis.

Engrailed-like immunoreactivity in the embryonic ventral nerve cord of the Marbled Crayfish (Marmorkrebs)

The homeobox transcription factor Engrailed is involved in controlling segmentation during arthropod germ band formation but also in establishing individual neuronal identities during later embryogenesis. In Crustacea, most studies analysing the expression of Engrailed so far have focussed on its function as segment polarity gene. In continuation to these previous studies, we analysed the neuronal expression of the Engrailed protein by immunohistochemistry in the embryonic nerve cord of a parthenogenetic crustacean, the Marbled Crayfish (Marmorkrebs). We paid particular attention to the individual identification of Engrailed expressing putative neuroblasts in the crayfish embryos. Engrailed positive cells in the neuroectoderm were counted, measured and mapped from 38 to 65% of embryonic development. That way, several Engrailed positive putative neuroblasts and putative neurons were identified. Our findings are compared with earlier studies on Engrailed expression during germ band formation in Crustacea. Recent data on neurogenesis in an amphipod crustacean have provided compelling evidence for the homology of several identified neuroblasts between this amphipod and insects. The present report may serve as a basis to explore the question if during crustacean neurogenesis additional communalities with insects exist.

Structure and Expression Analysis of theCecropin-EGene from the Silkworm,Bombyx mori

Cecropins belong to the antibacterial peptides family and are induced after injection of bacteria or their cell-wall components. By silkworm cDNA microarray analysis, a novel type of Cecropin family gene was identified as a cDNA up-regulated in early embryo, 1 day after oviposition. The cDNA isolated was 394 bp with 198 ORF translating 65 amino acids, encoding BmCecropin-E (BmCec-E). Using Southern hybridization and genome search analysis, the number of BmCec-E gene was estimated to be at least two per haploid, which consisted of two exons, as in other Cecropin family members. BmCec-E mRNA was expressed transiently 1 day after egg-laying (AEL, germ-band formation stage), and was specifically expressed in the degenerating intestine during the pre-pupal and pupal stages, unlike other Cecropin family genes. Immune challenge analysis showed that BmCec-E gene expression was more strongly induced by Escherichia coli (gram-negative) than by Micrococus luteus (gram-positive), and not by virus injection. By bacterial challenge, expression of BmCec-E mRNA was induced 12 h after injection, and was maintained for 24 h. Expression of BmCec-E after immune challenge was observed strongly in excretory organs, such as hindgut and malphigian, slightly in fat body, skin, and midgut.

Two gap junction channel (innexin) genes of the Bombyx mori and their expression

Gap junctions are clusters of intercellular channels that are associated with embryonic development and neural signaling. Innexins, invertebrate gap junction proteins, have been identified in Drosophila and Caenorhabditis. Here, we report the isolation and characterization of two novel members of the insect innexin family, Bm inx2 and Bm inx4, from embryos of the silkworm, Bombyx mori, during the germ-band formation stage. Bm inx2 is a single copy gene with one exon, while Bm inx4 is a single copy gene with four exons and three introns. The predicted proteins show structural similarities with other innexin family members, including four transmembrane (TM) domains, two extracellular loops (ELs), one cytoplasmic loop (CL), and typical conserved amino acids. Bm inx2 is phylogenetically orthologous to the other insect inx2 genes, but Bm inx4 is not orthologous to any known innexin including Dm inx4. Interestingly, Northern blotting and in situ hybridization showed that Bm inx2 was variously expressed across all developmental stages and in various tissues, with high expression seen in the nervous system at the time of embryogenesis. In contrast, Bm inx4 was transiently expressed at the germ-band formation stage of embryogenesis, and was specifically expressed in the ovary and testis during the larval and pupal stages. The isolation and characterization of these novel genes should form the basis for further study of the functional events that occur during development and neuronal communication in B. mori.

A 4D-microscopic analysis of the germ band in the isopod crustacean Porcellio scaber (Malacostraca, Peracarida)—developmental and phylogenetic implications

Malacostracan crustaceans have evolved a conserved stereotyped cell division pattern in the post-naupliar germ band. This cleavage pattern is unique in arthropods investigated so far, and allows a combined analysis of gene expression and cell lineage during segmentation and organ development at the level of individual cells. To investigate the cell lineage in the germ band of the isopod Porcellio scaber, we used a 4D-microscopy system, which enables us to analyse every cell event in the living embryo. The study was combined with the analysis of the expression of the gene engrailed (en) at different stages of germ band formation. Our findings confirm the results of earlier investigations of the cell division pattern in the posterior part of the isopod germ band. Furthermore, we can show that in the anterior region, in contrast to the posterior part, cleavage directions are variable and cell sorting takes place-similar to other arthropod germ bands. Additionally, the gene expression pattern of en in this region is not as regular as in the post-naupliar germ band, and only later becomes regulated into its characteristic stripe pattern. The comparison of the cell lineage of P. scaber with that of other malacostracan crustaceans shows an enhancement in the velocity of cell divisions relative to the arrangement of these cells in rows in the isopod germ band. The striking similarity of the formation of the genealogical units in the anterior part suggests a sister group relationship between the peracarid taxa Tanaidacea and Isopoda.

Gene Expression Profiling in the Silkworm, Bombyx mori, During Early Embryonic Development

We prepared a cDNA library for a microarray from eggs of the silkworm, Bombyx mori, at the germ-band formation (24 hours after fertilization) stage. Using a microarray constructed with 2,445 ESTs, we screened gene expression profiles during germ-band formation at six specific time points in the early embryonic stages (from the unfertilized egg to the formation of abdominal leg appendages), and determined 241 of these cDNAs to represent genes that were expressed differentially during the germ-band formation stage. These differentially expressed genes grouped into two clusters. In the early and late clusters, 203 and 38 genes were upregulated, respectively. In the upregulated clusters, we isolated several genes that were associated with development and cell communication, including egalitarian, RAD23b, innexin 2, and senescence-associated protein. Northern blot hybridization revealed that the expression patterns of 14 genes had changed in each of the stages. In this study, we assessed changes in the levels of gene expression in relation to the germ-band formation stages in whole Bombyx embryos.