Germ Disc Research Articles

Extraembryonic tissue in chelicerates: a review and outlook.

The formation of extraembryonic membranes (EEMs) contributes to the proper development of many animals. In arthropods, the formation and function of EEMs have been studied best in insects. Regarding the development of extraembryonic tissue in chelicerates (spiders and relatives), most information is available for spiders (Araneae). Especially two populations of cells have been considered to represent EEMs in spiders. The first of these potential EEMs develops shortly after egg deposition, opposite to a radially symmetrical germ disc that forms in one hemisphere of the egg and encloses the yolk. The second tissue, which has been described as being extraembryonic is the so-called dorsal field, which is required to cover the dorsal part of the developing spider germ rudiment before proper dorsal closure. In this review, we summarize the current knowledge regarding the formation of potential extraembryonic structures in the Chelicerata. We describe the early embryogenesis of spiders and other chelicerates, with a special focus on the formation of the potential extraembryonic tissues.This article is part of the theme issue ‘Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom’.

Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

BackgroundThe process of early development varies across the species-rich phylum Arthropoda. Owing to the limited research strategies for dissecting lineage-specific processes of development in arthropods, little is known about the variations in early arthropod development at molecular resolution. The Theridiidae spider, Parasteatoda tepidariorum, has its genome sequenced and could potentially contribute to dissecting early embryonic processes.ResultsWe present genome-wide identification of candidate genes that exhibit locally restricted expression in germ disc forming stage embryos of P. tepidariorum, based on comparative transcriptomes of isolated cells from different regions of the embryo. A subsequent pilot screen by parental RNA interference identifies three genes required for body axis formation. One of them is a GATA-like gene that has been fast evolving after duplication and divergence from a canonical GATA family gene. This gene is designated fuchi nashi (fuchi) after its knockdown phenotypes, where the cell movement toward the formation of a germ disc was reversed. fuchi expression occurs in cells outside a forming germ disc and persists in the endoderm. Transcriptome and chromatin accessibility analyses of fuchi pRNAi embryos suggest that early fuchi activity regulates chromatin state and zygotic gene activation to promote endoderm specification and pattern formation. We also show that there are many uncharacterized genes regulated by fuchi.ConclusionsOur genome-based research using an arthropod phylogenetically distant from Drosophila identifies a lineage-specific, fast-evolving gene with key developmental roles in one of the earliest, genome-wide regulatory events, and allows for molecular exploration of the developmental variations in early arthropod embryos.

Novel staining procedures for whole mount preparations of greater wax moth (Galleria mellonella) embryos: toluidine blue-rhodamine B, plus calcofluor white

ABSTRACTAlthough hematoxylin and eosin (H & E) staining of sectioned embryonic insect material is widely used, it is time-consuming and may not provide sufficient information. We evaluated new staining procedures for embryonic whole mounts of the greater wax moth, Galleria mellonella. We compared a combination of toluidine blue and rhodamine B (TB-RB) to H & E; we also investigated calcofluor white (CFW) staining. TB-RB staining produced staining similar to H & E. TB-RB staining was less time-consuming and improved visualization of the blastoderm and its differentiation into the germ disk and serosa membrane. CFW enhanced details of mitosis in nuclei post-fertilization and stained the primary serosal membrane. Staining of whole mounts with TB-RB and CFW enabled embryonic staging that was more rapid, convenient and effective than the routine approach using H & E and fluorescent probes.

Early segmentation in the mite Archegozetes longisetosus reveals conserved and derived aspects of chelicerate development.

The arthropod body plan is comprised of several repeating segments along the anteroposterior body axis. This high degree of conservation, however, obfuscates the wide degree of underlying developmental variation present across and within arthropod groups. In chelicerates, the arthropod clade containing mites, spiders, scorpions, and horseshoe crabs, development is the most similar at the stages following early germ band segmentation. Comparative studies of chelicerate segmentation prior to these events, however, remain scarce. In order to elucidate and identify possible shared and derived aspects of chelicerate segmentation, we followed the early prosomal (anterior) segmentation in the model mite Archegozetes longisetosus using the expression of the conserved segmental marker hedgehog (hh). Our data indicate that the ancestral chelicerate likely utilized the gene hedgehog in a group of cells surrounding the germ disc. We also provide evidence that chelicerate segmentation, albeit via the conserved "short/intermediate germ" mode, progresses differently in the prosoma between Archegozetes and spiders and thus early, anterior segmentation in chelicerates is heterochronic.

A quantitative study of the diversity of stripe-forming processes in an arthropod cell-based field undergoing axis formation and growth

One of the conserved traits of arthropod embryonic development is striped expression of homologs of Drosophila segment polarity genes, including hedgehog (hh). Although a diversity of stripe-forming processes is recognized among arthropod embryos, such varied stripe-forming processes have not been well characterized from cellular and quantitative perspectives. The spider Parasteatoda tepidariorum embryo, which has a hh-dependent mechanism of axis formation, offers a cell-based field where the stripes of Pt-hh (a hh homolog) expression dynamically develop in accordance with axis formation and growth, with the patterning processes varying among the regions of the field. In this study, using cell labeling, we mapped the future body subdivisions to the germ disc in the spider embryo and provided substantial evidence for the occurrence of kinetic waves of Pt-hh expression in the presumptive head and opisthosomal (or abdominal) regions of the embryonic field. Notably, combined with cell tracking, we showed that surface cells at and near the center of the germ disc persist in the posterior portion of the field from where Pt-hh stripes sequentially arise, suggesting the operation of ordered oscillations of Pt-hh expression. We then conducted a quantitative analysis of forming/formed Pt-hh stripes using serially timed fixation of sibling embryos. By utilizing length measurements that reflect the axis growth of the embryonic field, we reconstructed the pattern dynamics, which captured repeated splitting of Pt-hh stripes and oscillations of Pt-hh expression in the presumptive head and opisthosomal regions, respectively. In the intermediate thoracic region, three stripes of Pt-hh expression showed a late appearance, with the segmental units specified much earlier by another mechanism. Analyses provided quantitative estimates related to axis growth and stripe-splitting and oscillation events, including the periods of the patterning cycles. This work characterizes the diversity of stripe-forming processes in a cell-based field in a common spatiotemporal framework and highlights the contrasting dynamics of splitting versus oscillation. The cellular and quantitative data presented here provide the foundation for experimental, theoretical and evolutionary studies of cell-based pattern formation, especially body axis segmentation in arthropods.

Formation of the germ-disc in spider embryos by a condensation-like mechanism.

BackgroundDetermination of the embryonic body axes is a crucial developmental process in all animals. The establishment of the embryonic axes of spiders has been best studied in the common-house-spider Parasteatoda tepidariorum. Here, anteroposterior (AP) polarity arises during germ disc formation; the centre of the germ-disc marks the future posterior pole, and the rim of the disc the future anterior pole of the spider embryo. The centre of the germ disc is also needed for the formation of the cumulus, a group of migratory cells needed to establish dorsoventral (DV) polarity. Thus, both body axes depend on proper germ disc formation and patterning. However, these processes have not been fully analysed at the cellular and molecular level.ResultsHere I present new techniques to stain the cell membranes/outlines in live and fixed spider embryos. I show that the germ-disc is formed from a regular and contiguous blastoderm and that co-ordinated cell shape changes, rather than migration of single cells, are required to drive germ-disc formation in P. tepidariorum embryos. Furthermore, I show that the rate of cell divisions within the embryonic and extra-embryonic region is not involved in the rapid establishment of the germ-disc. Finally, I show that the process of germ-disc formation is dependent on the initiation of zygotic transcription.ConclusionsThe presented data provide new insights in to the formation of the germ-disc in spider embryos. The establishment of the germ-disc in Parasteatoda embryos is a highly dynamic process that involves wide scale cell-shape changes. While most of the blastodermal cells become cuboidal to form the dense germ-disc, the remaining blastodermal cells stay squamous and develop into huge extra-embryonic, yolk rich cells. In addition, this study shows that the onset of zygotic transcription is needed to establish the germ-disc itself, and that the mid-blastula transition of Parasteatoda tepidariorum embryos is prior to any overt axis establishment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-016-0166-9) contains supplementary material, which is available to authorized users.

Gastrulation occurs in multiple phases at two distinct sites in Latrodectus and Cheiracanthium spiders.

BackgroundThe longstanding canonical model of spider gastrulation posits that cell internalization occurs only at a unitary central blastopore; and that the cumulus (dorsal organizer) arises from within the early deep layer by cell–cell interaction. Recent work has begun to challenge the canonical model by demonstrating cell internalization at extra-blastoporal sites in two species (Parasteatodatepidariorum and Zygiella x-notata); and showing in Zygiella that the prospective cumulus internalizes first, before other cells are present in the deep layer. The cell behaviors making up spider gastrulation thus appear to show considerable variation, and a wider sampling of taxa is indicated.ResultsWe evaluated the model in three species from two families by direct observation of living embryos. Movements of individual cells were traced from timelapse recordings and the origin and fate of the cumulus determined by CM-DiI labeling. We show that there are two distinct regions of internalization: most cells enter the deep layer via the central blastopore but many additional cells ingress via an extra-blastoporal ring, either at the periphery of the germ disc (Latrodectus spp.) or nearer the central field (Cheiracanthium mildei). In all species, the cumulus cells internalize first; this is shown by tracing cells in timelapse, histology, and by CM-DiI injection into the deep layer. Injection very early in gastrulation labels only cumulus mesenchyme cells whereas injections at later stages label non-cumulus mesoderm and endoderm.ConclusionsWe propose a revised model to accommodate the new data. Our working model has the prospective cumulus cells internalizing first, at the central blastopore. The cumulus cells begin migration before other cells enter the deep layer. This is consistent with early specification of the cumulus and suggests that cell–cell interaction with other deep layer cells is not required for its function. As the cumulus migrates, additional mesendoderm internalizes at two distinct locations: through the central blastopore and at an extra-blastoporal ring. Our work thus demonstrates early, cell-autonomous behavior of the cumulus and variation in subsequent location and timing of cell internalization during gastrulation in spiders.Electronic supplementary materialThe online version of this article (doi:10.1186/s13227-015-0029-z) contains supplementary material, which is available to authorized users.

The embryonic development of the central American wandering spider Cupiennius salei

BackgroundThe spider Cupiennius salei (Keyserling 1877) has become an important study organism in evolutionary and developmental biology. However, the available staging system for its embryonic development is difficult to apply to modern studies, with strong bias towards the earliest developmental stages. Furthermore, important embryonic events are poorly understood. We address these problems, providing a new description of the embryonic development of C. salei. The paper also discusses various observations that will improve our understanding of spider development.ResultsConspicuous developmental events were used to define numbered stages 1 to 21. Stages 1 to 9 follow the existing staging system for the spider Achaearanea tepidariorum, and stages 10 to 21 provide a high-resolution description of later development. Live-embryo imaging shows cell movements during the earliest formation of embryonic tissue in C. salei. The imaging procedure also elucidates the encircling border between the cell-dense embryo hemisphere and the hemisphere with much lower cell density (a structure termed 'equator' in earlier studies). This border results from subsurface migration of primordial mesendodermal cells from their invagination site at the blastopore. Furthermore, our detailed successive sequence shows: 1) early differentiation of the precheliceral neuroectoderm; 2) the morphogenetic process of inversion and 3) initial invaginations of the opisthosomal epithelium for the respiratory system.ConclusionsOur improved staging system of development in C. salei development should be of considerable value to future comparative studies of animal development. A dense germ disc is not evident during development in C. salei, but we show that the gastrulation process is similar to that in spider species that do have a dense germ disc. In the opisthosoma, the order of appearance of precursor epithelial invaginations provides evidence for the non-homology of the tracheal and book lung respiratory systems.

Cell migration that orients the dorsoventral axis is coordinated with anteroposterior patterning mediated by Hedgehog signaling in the early spider embryo

The early embryo of the spider Achaearanea tepidariorum is emerging as a model for the simultaneous study of cell migration and pattern formation. A cell cluster internalized at the center of the radially symmetric germ disc expresses the evolutionarily conserved dorsal signal Decapentaplegic. This cell cluster migrates away from the germ disc center along the basal side of the epithelium to the germ disc rim. This cell migration is thought to be the symmetry-breaking event that establishes the orientation of the dorsoventral axis. In this study, knockdown of a patched homolog, At-ptc, that encodes a putative negative regulator of Hedgehog (Hh) signaling, prevented initiation of the symmetry-breaking cell migration. Knockdown of a smoothened homolog, At-smo, showed that Hh signaling inactivation also arrested the cells at the germ disc center, whereas moderate inactivation resulted in sporadic failure of cell migration termination at the germ disc rim. hh transcript expression patterns indicated that the rim and outside of the germ disc were the source of the Hh ligand. Analyses of patterning events suggested that in the germ disc, short-range Hh signal promotes anterior specification and long-range Hh signal represses caudal specification. Moreover, negative regulation of Hh signaling by At-ptc appears to be required for progressive derepression of caudal specification from the germ disc center. Cell migration defects caused by At-ptc and At-smo knockdown correlated with patterning defects in the germ disc epithelium. We propose that the cell migration crucial for dorsoventral axis orientation in Achaearanea is coordinated with anteroposterior patterning mediated by Hh signaling.

The effect of gaseous ozone treatment on egg components

Because of the salmonella problem in poultry, disinfection technologies are necessary. Ozone is a strong oxidant used for the disinfection of surfaces, drinking water and foods. However, since ozone not only destroys bacteria but may also damage eggs, it is necessary to clarify the effects of ozone treatment on hatching egg components. In this study, doses of gaseous ozone ranging from 10 to 50 mL L(-1) were tested. The vitamin A and E contents and fatty acid composition of the egg yolk were determined. To detect possible damage to the DNA of the germ disc, single-cell gel electrophoresis was used. Moreover, free SH groups were measured in the egg white. The soluble cuticula proteins were analysed by polyacrylamide gel electrophoresis. The yolk was not significantly affected by ozone treatment. However, the DNA of the germ disc was attacked and a significant decrease in free SH groups in the egg white was recorded at 50 mL L(-1) ozone. Even at low ozone doses the soluble cuticula proteins were completely destroyed. Significant alterations of egg components were caused by 50 mL L(-1) ozone. At lower ozone doses the oxidative processes occurred mainly at the egg surface and are therefore probably harmless to the developing embryo.

Gastrulation in the spider Zygiella x‐notata involves three distinct phases of cell internalization

The cell movements of gastrulation were analyzed in embryos of the spider Zygiella x-notata, using time-lapse video, cell tracing, and improved histology. Cells are internalized near the center of the germ disc in three distinct phases. First, cumulus mesenchyme cells ingress and migrate as a group beneath the superficial layer. Second, mass internalization through a blastopore yields a diffusely organized deep layer. Third, superficial cells accumulate at the center of the germ disc to form the caudal bud. The floor is internalized, and the caudal bud moves over the nascent dorsal field to form the caudal lobe. This pattern of gastrulation differs from the canonical pattern described in the historical literature: (1) the cumulus of Z. x-notata is completely formed before any other cells internalize; and (2) the caudal lobe is formed by means of the caudal bud, which is a locus of cell internalization.

Novel method for the establishment of cardiomyocytes derived from rat embryonic stem cells in vitro

Cardiomyocytes were differentiated from embryonic stem cells (ES cells) derived from spontaneous dwarf rats (SDR) in vitro. The two-cell stage embryos were cultured in alpha-MEM supplemented with 10% fetal calf serum and embryotrophic factors (ETF). ETF were isolated from the conditioned medium of the SKG-II-SF cell line derived from a human uterine cervical epidermoid carcinoma. When two-cell stage rat embryos developed into tri-laminal germ disc embryos (flat type), colonies composed of small round cells were isolated by the colonial isolation method and used to establish an ES cell line. The ES cells were cultured in DMEM/F12 medium supplemented with 10% fetal calf serum and 1 ng/mL of leukemia inhibitory factor. Embryoid bodies were made by the hanging-drop method using 1 x 10(7) ES cells/mL. The embryoid bodies differentiated and grew to form an embryonic monster in ETF-supplemented medium using Rose's circumfusion apparatus for about 1 month. The anlages of beating hearts in embryonic monsters were collected using a glass capillary. The anlages were cut into small pieces using razor blades and dissociated with trypsin-EDTA/PBS(-) solution. The resultant single cells were cultured in growth medium and used to establish a myocardial cell line. The cell line was subcultured for more than 25 passages and confirmed as showing the morphological and ultrastructural characteristics of cardiomyocytes.

Progressive activation of Delta-Notch signaling from around the blastopore is required to set up a functional caudal lobe in the spiderAchaearanea tepidariorum

In the development of most arthropods, the caudal region of the elongating germ band (the growth zone) sequentially produces new segments. Previous work with the spider Cupiennius salei suggested involvement of Delta-Notch signaling in segmentation. Here, we report that, in the spider Achaearanea tepidariorum, the same signaling pathway exerts a different function in the presumptive caudal region before initiation of segmentation. In the developing spider embryo, the growth zone becomes morphologically apparent as a caudal lobe around the closed blastopore. We found that, preceding caudal lobe formation, transcripts of a Delta homolog, At-Delta, are expressed in evenly spaced cells in a small area covering the closing blastopore and then in a progressively wider area of the germ disc epithelium. Cells with high At-Delta expression are likely to be prospective mesoderm cells, which later express a twist homolog, At-twist, and individually internalize. Cells remaining at the surface begin to express a caudal homolog, At-caudal, to differentiate as caudal ectoderm. Knockdown of At-Delta by parental RNA interference results in overproduction of At-twist-expressing mesoderm cells at the expense of At-caudal-expressing ectoderm cells. This condition gives rise to a disorganized caudal region that fails to pattern the opisthosoma. In addition, knockdown of Notch and Suppressor of Hairless homologs produces similar phenotypes. We suggest that, in the spider, progressive activation of Delta-Notch signaling from around the blastopore leads to stochastic cell fate decisions between mesoderm and caudal ectoderm through a process of lateral inhibition to set up a functional caudal lobe.

Origin, fate, and function of the components of the avian germ disc region and early blastoderm: Role of ooplasmic determinants

In the avian oocytal germ disc region, at the end of oogenesis, we discerned four ooplasms (alpha, beta, gamma, delta) presenting an onion-peel distribution (from peripheral and superficial to central and deep. Their fate was followed during early embryonic development. The most superficial and peripheral alpha ooplasm plays a fundamental role during cleavage. The beta ooplasm, originally localized in the peripheral region of the blastodisc, becomes mainly concentrated in the primitive streak. At the moment of bilateral symmetrization, a spatially oblique, sickle-shaped uptake of gamma and delta ooplasms occurs so that gamma and delta ooplasms become incorporated into the deeper part of the avian blastoderm. These ooplasms seem to contain ooplasmic determinants that initiate either early neurulation or gastrulation events. The early neural plate-inducing structure that forms a deep part of the blastoderm is the delta ooplasm-containing endophyll (primary hypoblast). Together with the primordial germ cells, it is derived from the superficial centrocaudal part of the nucleus of Pander, which also contains delta ooplasm. The other structure (gamma ooplasm) that is incorporated into the caudolateral deep part of the blastoderm forms Rauber's sickle. It induces gastrulation in the concavity of Rauber's sickle and blood island formation exterior to Rauber's sickle. Rauber's sickle develops by ingrowth of blastodermal cells into the gamma ooplasm, which surrounds the nucleus of Pander. Rauber's sickle constitutes the primary major organizer of the avian blastoderm and generates only extraembryonic tissues (junctional and sickle endoblast). By imparting positional information, it organizes and dominates the whole blastoderm (controlling gastrulation, neurulation, and coelom and cardiovascular system formation). Fragments of the horns of Rauber's sickle extend far cranially into the lateral quadrants of the unincubated blastoderm, so that often Rauber's sickle material forms three quarters of a circle. This finding explains the regulative capacities of isolated blastoderm parts, with the exception of the anti-sickle region and central blastoderm region, where no Rauber's sickle material is present. In avian blastoderms, there exists a competitive inhibition by Rauber's sickle on the primitive streak and neural plate-inducing effects of sickle endoblast. Avian primordial germ cells contain delta ooplasm derived from the superficial part of the nucleus of Pander. Their original deep and central ooplasmic localization has been confirmed by the use of a chicken vasa homologue. We conclude that the unincubated blastoderm consists of three elementary tissues: upper layer mainly containing beta ooplasm, endophyll containing delta ooplasm, and Rauber's sickle containing gamma ooplasm). These elementary tissues form before the three classic germ layers have developed.

Induction and improved embryonic development by the nucleus of Pander in associated avian blastoderm parts: influence of delta or gamma ooplasm.

After placing in vitro, central subgerminal ooplasm (containing a central nucleus of Pander) from a quail germ disc of a prelaid egg (before symmetrization) on the upper layer of an isolated chicken antisickle, we observed the induction of a radially oriented preneural plate (without interference of chordamesoblast). This observation suggests the primary existence during the period of symmetrization in utero of an until now unknown temporospatially linked "vertical" effect, emanating from the nucleus of Pander, on the parallel (pre)neural plate anlage forming part of the area centralis in the overlying blastoderm. For comparison, we "sandwiched" in vitro a quail sickle endoblast fragment between the deep side of the upper layer of an isolated chicken antisickle region and a central subgerminal ooplasmic mass. This resulted in a colonization of the subgerminal ooplasmic mass by quail sickle endoblast cells followed by improved neurulation and/or gastrulation phenomena. The latter never occurs in the absence of central subgerminal ooplasm. In both types of experiments there seems to exist a common link between the observed induction phenomena: the presence of delta ooplasm in the involved deep structures. Indeed, the nucleus of Pander contains delta ooplasm as well as the structures derived from it, i.e., endophyll with primordial germ cells and sickle endoblast-derived cells after colonization of the neighboring central ooplasm (present study). Therefore, we think that the preneural plate-inducing effect observed after placing a nucleus of Pander on the antisickle region is due to the presence of a factor in the delta ooplasm that diffuses in the neighborhood. The appearance of gastrulation phenomena in the second type of experiment seems to be due to colonization of the more peripheral part of the central subgerminal ooplasm containing the more superficial and peripheral gamma ooplasm in which Rauber's sickle material can develop. This suggests that the kind of involved ooplasm (delta or gamma) can predetermine the inductive activity of the deep structures that contain it: the central part of the nucleus of Pander and/or endophyll for preneurulation phenomena and sickle endoblast (in the presence of central subgerminal ooplasm) for gastrulation and/or neurulation phenomena.

Egg Fertility in Domestic Fowl Gallus gallus domesticus as a Function of the Yolk Surface Area

The increase of egg mass and reliable decrease of egg fertility were observed when the mass surface area of yolk (egg) increased. The results obtained suggest that, in addition to the number of viable spermatozoa penetrating across the perivitelline membrane within 15–20 min after ovulation, the probability of fertilization depends on the area of egg surface, which approximately corresponds to the area of perivitelline membrane. Apparently, the ratio of receptors′ numbers and spermatozoa, which contact with them on the germ disc surface, to their number on the non-germ zone decreases with the increase of yolk size. The fertility depression associated with the increased area of perivitelline membrane suggests that the egg yolk size is one of the factors of fertility of the female gametes in terms of both egg content variability and aging.

Early patterning of the spider embryo: a cluster of mesenchymal cells at the cumulus produces Dpp signals received by germ disc epithelial cells.

In early embryogenesis of spiders, the cumulus is characteristically observed as a cellular thickening that arises from the center of the germ disc and moves centrifugally. This cumulus movement breaks the radial symmetry of the germ disc morphology, correlating with the development of the dorsal region of the embryo. Classical experiments on spider embryos have shown that a cumulus has the capacity to induce a secondary axis when transplanted ectopically. In this study, we have examined the house spider, Achaearanea tepidariorum, on the basis of knowledge from Drosophila to characterize the cumulus at the cellular and molecular level. In the cumulus, a cluster of about 10 mesenchymal cells, designated the cumulus mesenchymal (CM) cells, is situated beneath the epithelium, where the CM cells migrate to the rim of the germ disc. Germ disc epithelial cells near the migrating CM cells extend cytoneme-like projections from their basal side onto the surface of the CM cells. Molecular cloning and whole-mount in situ hybridization showed that the CM cells expressed a spider homolog of Drosophila decapentaplegic (dpp), which encodes a secreted protein that functions as a dorsal morphogen in the Drosophila embryo. Furthermore, the spider Dpp signal appeared to induce graded levels of the phosphorylated Mothers against dpp (Mad) protein in the nuclei of germ disc epithelial cells. Adding data from spider homologs of fork head, orthodenticle and caudal, we suggest that, in contrast to the Drosophila embryo, the progressive mesenchymal-epithelial cell interactions involving the Dpp-Mad signaling cascade generate dorsoventral polarity in accordance with the anteroposterior axis formation in the spider embryo. Our findings support the idea that the cumulus plays a central role in the axial pattern formation of the spider embryo.

Embryogenesis of the glowworm Rhagophthalmus ohbai Wittmer (Insecta: Coleoptera, Rhagophthalmidae), with emphasis on the germ rudiment formation

The early embryonic development and features of the developing embryo of the glowworm Rhagophthalmus ohbai are described chiefly by light microscopy, with emphasis on the germ rudiment formation and its phylogenetic implication. The egg period is 30-34 days at about 23 degrees C. The newly laid egg is a short ellipsoid, 1.09 by 0.78 mm in size, and the size increases to 1.15 by 0.95 mm by 17 days after oviposition. Cleavage is of the typical superficial type. The germ disk is formed by cell aggregation of the embryonic area at the anterior end of the egg. The central part of the germ disk then sinks into the yolk and the spherical germ rudiment is formed by fusion of the amnioserosal folds extended from all margins of the germ disk. The inner region of the germ rudiment soon becomes slender and develops into the short embryo, whereas the outer region facing the anterior end is extended to form the thin amnion. The embryo then rapidly elongates, the elongation being accompanied by embryo segmentation and formation of appendages. The submerged condition of the embryo persists until about 17 days after oviposition (about 1 day before embryonic revolution) and thereafter the embryo becomes superficial in position. The presence of the following embryonic characters in R. ohbai supports the molecular data placing it within the Lampyridae: 1) formation of a spherical germ rudiment near the anterior end of the egg, and 2) the submerged condition of the developing embryo persists until shortly before revolution.

Dynamics of expression of apoptosis-regulatory proteins Bid, Bcl-2, Bcl-X, Bax and Bak during development of murine nervous system.

We have used immunohistochemistry and immunoblotting to examine the expression of Bid and four other Bcl-2 family proteins (Bcl-2, Bcl-X, Bax and Bak) in the developing and adult murine central nervous system (CNS). Bid protein is widespread in embryonic and postnatal brain, and its expression is maintained at a high level late into the adulthood. Bid is expressed both in the germ disc, early neural tube, proliferating stem cells of ventricular zones, and in postmitotic, differentiated neurons of the developing central and peripheral nervous system. As the differentiation proceeds, the neurons express higher levels of Bid than the stem cells of the paraventricular zone. Both in embryonic and postnatal life, Bid protein is present in the most vital regions of brain, such as the limbic system, basal ganglia, mesencephalic tectum, Purkinje cells in cerebellum, and the ventral columns of spinal cord. The p15 cleaved form of Bid was detectable in the brain specimens at fetal stages of development, consistent with caspase-mediated activation of this pro-apoptotic Bcl-2 family protein. Among the Bcl-2 family proteins only Bid and Bcl-XL continue to be expressed at high levels in the adult brain.

Cleavage, gastrulation, and germ disc formation of the amphipod Orchestia cavimana (Crustacea, Malacostraca, Peracarida)

Investigations of amphipod embryonic development have a long tradition. However, many aspects of amphipod embryology are still controversial. These concern, among others, the nature of the cleavage, the origin of the germ disc, and the mode of gastrulation. On the other hand, amphipods show the same characteristic type of invariant cell division pattern in the germ band as other malacostracans. Since amphipods seem to undergo a stereotyped pattern of early cleavage they are highly interesting for our understanding of the evolution of arthropod development. In this paper, we describe the cleavage pattern of the amphipod crustacean Orchestia cavimana from the zygote to gastrulation and the formation of the germ disc using direct observation, scanning electron microscopy, histology, video recording, and lineage tracing with a vital dye. The early development follows the mode of a total, radial, unequal cleavage with a determinate stereotyped pattern. A small transient blastocoel is formed. The 8-cell stage is characterised by 4 micromeres and 4 macromeres. One quadrant is smaller than the others. There are two kinds of eggs that show a mirror handed image. The 16-cell stage is the last regular stage after which the blastomeres divide highly asynchronously. The germ disc is formed by the descendants of the macromeres and some micromere derivatives. The other micromeres constitute the extra-embryonic region. Migration of macromere descendants is involved in germ disc formation accompanied by the extrusion of the yolk. During this process some vitellophages are formed. The gastrulation sensu stricto is initiated by the micromere derivatives of the smallest quadrant at the anterior of the forming germ disc. A true blastopore occurs which involves an invagination and the immigration of cells. Our data help to correct erroneous interpretations of former students of amphipod development. We can show that many characters of amphipod embryonic development are apomorphic supporting amphipod monophyly. With the present investigation we contribute to a complete understanding of the embryonic cell lineage of amphipods from the egg to segment formation and organogenesis.