[Mathematical Modeling of the Stretching-induced Elongation of an Embryonic Epithelium Layer in the Absence of External Load].

A clue to understanding the deformation of a plane embryonic epithelium layer unloaded after a short time uniaxial stretch and fixation in a stretched state over different time periods is found. The first steps in the understanding of this process come from the knowledge about the uniform stretching of the tissue fragment (explantate) with the subsequent stretching at a fixed length. In this study we used the earlier developed continuum model that describes the stress-strain state of the epithelial tissue taking into account the parameters that characterize the shape of the cells and their stress state, and also the active stresses they exert when interact with one another. The experimentally observed continuation of deformation of the stretched tissue after the cessation of action of the external force is described theoretically as a result of active cell reactions to the mechanical stress. The strong effect of the duration of explantate fixation on its further elongation and the cell activity pattern is demonstrated.

Mechanisms associated with cellular desiccation tolerance of Artemia encysted embryos from locations around the world.

Using differential scanning calorimetry we demonstrated the presence of biological glasses and measured the glass transition temperatures (Tg) in dry encysted gastrula embryos (cysts) of the brine shrimp, Artemia, from eleven different locations, two of which provided cysts from parthenogenetic animals. Values for Tg were highest, by far, in Artemia franciscana cysts from the Mekong Delta, Vietnam (VN), these cysts having been produced from previous sequential inoculations into growth ponds of cysts from the San Francisco Bay, California, USA. Tg values for three groups of A. franciscana cysts were significantly higher than those of other cysts (except those of Artemia persimilis) studied here, as well as all other desiccation-tolerant animal systems studied to date. We also measured three stress proteins (hsc70, artemin and p26) in all these cysts as well as the total alcohol soluble carbohydrates (ASC), about 90% of which is the disaccharide trehalose, a known component of biological glasses. We interpret the results in terms of mechanisms involved with desiccation tolerance and, to some extent, with thermal conditions at the sites of cyst collection.

Engineering Ethics and Self-Organizing Models of Human Development: Opportunities and Challenges.

Incorporating engineering ethics early during the planning stages of organoid and gastruloid research may help prevent future confusion about the moral status of complex models of human development. However, the intrinsic self-organizing behavior of organoids and gastruloids may pose a slight challenge to this novel ethical approach.

Expression of Panza, an alpha2-macroglobulin, in a restricted dorsal domain of the primitive gut in Xenopus laevis.

Alpha2-macroglobulin is a major serum protein with diverse functions, including inhibition of protease activity and binding of growth factors, cytokines, and disease factors. We have cloned and characterized Panza, a new Xenopus laevis alpha2-macroglobulin. Panza has 56-60% amino acid similarity with previously identified Xenopus, mouse, rat and human alpha2-macroglobulins, indicating that Panza is a new member of the alpha2-macroglobulin family. Panza mRNA is first detected at the beginning of neurulation in the dorsal endoderm lining the primitive gut (archenteron roof). At the completion of neurulation and continuing through the late tadpole stage, Panza is restricted to a dorsal domain of the gut endoderm adjacent to the notochord and extending along the entire anterior-posterior axis. With outgrowth of the tailbud, Panza expression persists in the chordaneural hinge at the posterior end of the differentiating notochord and extends into the floor plate of the posterior neural tube. As gut coiling commences, Panza expression is initiated in the liver, and the dorsal domain of Panza expression becomes limited to the midgut and hindgut. With further gut coiling, strong Panza expression persists in the liver, but is lost from other regions of the gut. The expression of Panza in endodermal cells adjacent to the notochord points to a potential role for Panza in signal modulation and/or morphogenesis of the primitive gut.

mRNAs for activin receptors II and IIB are expressed in mouse oocytes and in the epiblast of pregastrula and gastrula stage mouse embryos.

Activin is a potent inducer of mesoderm in frog embryos. We showed previously that in the mouse, activin beta A is expressed in the uterine decidua near the embryo before and during the first appearance of mesoderm (E4.5-E6.5). Here, using Northern blotting and in situ hybridization, we show that mouse oocytes, E6.5 and E7.5 embryos, and E6.5 and E7.5 decidua contain mRNAs for both activin receptors type II and IIB. The expression of activin receptor type IIB is particularly strong in embryonic ectoderm apparent at E5.5 and continuing through E8.5. These results support the hypothesis that activin derived from the decidua promotes development of mesoderm in the period E5.5-E6.5.

Differential expression and function of cadherin-like proteins in the sea urchin embryo.

Cadherins are Ca(+2)-dependent cell surface proteins involved in the specification of the adhesive properties of cells. They are supposed to play a critical role in morphogenesis and pattern formation. In this paper we show that in the sea urchin embryo there are at least two different cadherins of relative molecular masses 140 and 125 kDa. The 140 kDa cadherin is already present in the fertilized egg and is the sea urchin equivalent of E-cadherin. The 125 kDa cadherin, which can be detected using a broad-spectrum anti-cadherin antibody, appears only at later stages of development. In later embryos these two molecules are distributed differently: E-cadherin is present predominantly in the invaginating endoderm of the gastrula while the 125 kDa protein is present on the cell surface of most epithelia. Consistently with the observed differences in expression and in distribution, antibodies directed against these two cadherins differently perturb sea urchin development. For example, when these antibodies are added to early gastrulas only the antibodies against the 125 kDa component can induce a complete disaggregation of the ectoderm, while anti E-cadherin antibodies induce an abnormal development of the endoderm while the embryo maintains its basic integrity. These results are discussed in view of the need for multiple adhesion receptors during pattern formation and embryogenesis.

OTX2 homeoprotein in the developing central nervous system and migratory cells of the olfactory area.

We analyzed the distribution of OTX2 during mouse development. OTX2 is a homeoprotein encoded by Otx2, a vertebrate homeobox gene expressed in the developing brain and anterior head regions. The protein is already detectable in pre-streak embryos, in nuclei of embryonic ectoderm or epiblast and primitive endoderm or hypoblast. Its distribution is uniform along the entire epiblast, while showing an antero-posterior gradient along the hypoblast at the time when primitive streak first forms. Between embryonic day 7 (E7) and E7.5 there is a progressive confinement of the protein to the anterior ectoderm corresponding to the forming headfold. At E7.5-E7.8, the protein is mainly confined in this region but is still present, though at lower level, in more posterior ectoderm. Starting from day 8 of development it is essentially confined to anterior neuroectoderm corresponding to presumptive fore- and midbrain. Its subsequent distribution in forebrain, midbrain, developing isthmo-cerebellum and posterior central nervous system is analyzed in detail. Of particular interest is the presence of OTX2 in nuclei of cells of the olfactory system starting from its origin in the olfactory placode. OTX2 protein is present in some cells of the olfactory epithelium, in both the major olfactory epithelium and the vomero-nasal organ, and in scattered migratory cells present in the mesenchyme outside it. These cells surround the axon bundles of the olfactory nerve along its path from the olfactory epithelium in the nasal cavities to the olfactory bulb in rostral telencephalon and include both ensheathing glial cells and luteinizing hormone-releasing hormone (LHRH)-positive cells.

Identification of a Spindlin homolog in gibel carp (Carassius auratus gibelio).

Spindlin has been suggested to play an important role during the transition from oocyte maturation to embryo development in mouse, but its homolog similar to the mouse Spindlin in molecular and expression characterization has not been identified up to now in other vertebrates. In this study, a full length of cDNA sequence is cloned and sequenced from the gibel carp (Carassius auratus gibelio). It contains 1240 nucleotides with an open reading frame of 771 nt encoding 257 amino acids. Based on its amino acid sequence alignment and comparison analysis with the known Spin family proteins, the newly cloned Spin is named Carassius auratus gibelio Spindlin (CagSpin). Its product could be detected from mature eggs to blastula embryos, but its content decreased from the two-cell stage, and could not be detected after the gastrula stage. It suggests that the CagSpin should be a maternal protein that is expressed during oocyte maturation, and plays a crucial role in early cleavage of embryogenesis. CagSpin is the first homolog similar to mouse spindlin identified in fish, and also in other vertebrates. GST pull-down assay reveals the first biochemical evidence for the association of CagSpin and beta-tubulin, the microtubule component. Therefore, CagSpin may play important functions by interacting with beta-tubulin and other spindle proteins during oocyte maturation and egg fertilization.

Differential synthesis and cytolocalization of prosomes in chick embryos during development.

Prosomes, also called "multicatalytic proteinase" or proteasomes, were purified from chick embryos of different developmental stages by a simple, single-step procedure. These were characterized by their characteristic protein patterns determined by SDS polyacrylamide gel electrophoresis (SDS PAGE) and immunoblotting with four monoclonal antibodies, namely, anti-p27, -p28, -p29 and -p31, prepared against duck prosomes. In vitro labeling of embryos with 35S-methionine followed by SDS PAGE and fluorography of the purified prosomes revealed that their polypeptides are differentially synthesized at various stages during development. Among 12 polypeptides (p21 to p56), p21 is synthesized at the beginning of gastrulation (stage 2) followed by the synthesis of p24 at stage 3. Nine other polypeptides (p25 to p35) are synthesized at the head-fold stage (stage 6), while the synthesis of polypeptide p56 is only detected at stage 10 (10-somite stage). Indirect immunofluorescence studies, with the 4 monoclonal antibodies, demonstrated 3 distinct, developmental stage-specific patterns of cytodistribution of these four prosome polypeptides in the embryos. During early embryogenesis, these are uniformly nuclear in location, while at later stages (stage 4 onwards) they are also present in the cytoplasm. Interestingly, one of the antigens (p 28), although found uniformly in all types of tissues in the embryos up to the gastrulation stage, is undetectable in the neural tissues and nonuniformly distributed in other tissues of stage-10 embryos. These data suggest that there are subcomponents of prosomes which are synthesized as well as distributed in an independent manner during development, possibly reflecting subcomponent-specific multiple functions of these particles.

Differential localization of Mox-1 and Mox-2 proteins indicates distinct roles during development.

Transcript localizations for Mox genes have implicated this homeobox gene subfamily in the early steps of mesoderm formation. We have extended these studies by determining the protein expression profile of Mox-1 and Mox-2 during mouse development. The time of onset of Mox protein expression has been accurately obtained to provide clues as to their roles during gastrulation. Expression of Mox-1 protein is first detected in the newly formed mesoderm of primitive streak stage mouse embryos (7.5 days post-coitum, d.p.c.). In contrast, Mox-2 protein is first detected at 9.0 d.p.c. in thr already formed somites. Additionally, immunostaining reveals new and distinct areas of Mox expression in the branchial arches and limbs that were not reported in our previous mRNA localization analysis. Mouse Mox-2 antibodies cross-react specifically in similar embryonic tissues in chick indicating the conservation of function of Mox genes in vertebrates. These expression data suggest that the Mox genes function transiently in the formation of mesodermal and mesenchymal derivatives, after their initial specification, but before their overt differentiation. Furthermore, while there appears to be some overlap in protein expression between Mox-1 and Mox-2 during somitogenesis, unique areas of expression indicate several distinct roles for the Mox genes during development.

HrWnt-5: a maternally expressed ascidian Wnt gene with posterior localization in early embryos.

Ascidians show a highly determinate mode of development. In particular, components of the posterior-vegetal cytoplasm of fertilized eggs are responsible for the establishment of the embryonic axis. Recent studies have, however, also revealed significant roles of cell-cell interactions during embryogenesis. Proteins encoded by the Wnt family of genes act as signals and have been shown to play important roles in a wide range of developmental processes. Here we have isolated and characterized an ascidian Wnt gene, HrWnt-5, from Halocynthia roretzi. HrWnt-5 mRNA is present in the vegetal cortex in unfertilized eggs. After fertilization, HrWnt-5 mRNA moves to the equatorial region to form a crescent-like structure, after which the mRNA is concentrated in the posteriormost region of the embryo. This early pattern of HrWnt-5 mRNA localization coincides with another posterior-vegetally localized mRNA, pem, isolated from Ciona savignyi. In the gastrula, the zygotic HrWnt-5 mRNA is found in a variety of blastomeres, suggesting multiple roles of the gene.

Characterization of an ascidian maternal T-box gene, As-mT.

The T-box genes encode a novel family of transcriptional factors that seem to play crucial roles in various processes of animal development, in particular, mesoderm formation of chordate embryos. The ascidian egg has been regarded as a typical mosaic egg, in which several types of embryonic cells are specified autonomously dependent on prelocalized maternal factors or determinants. In the present study, we investigated a possible role of a maternal T-box gene (As-mT) of the ascidian Halocynthia roretzi. A cDNA clone we obtained predicted As-mT protein of 891 amino acids with a distinct T-domain, which was divergent from those of other T-box genes. Expression of As-mT was exclusively maternal. Although the transcript became barely detectable by the gastrula stage, no zygotic expression was evident during embryogenesis. The maternal transcript was distributed rather evenly within eggs and early embryos without any special localization. Injection of synthetic As-mT mRNA into fertilized eggs induced retardation of embryogenesis. Although cleavage occurred normally, the initiation of gastrulation was delayed, and delay in the morphogenesis resulted in dumpy larvae. Expression of a muscle-specific actin gene, a notochord-specific Brachyury gene, and an epidermis-specific gene was not detected at the early gastrula stage, all of the three genes being expressed in normal embryos at that stage. However, the expression of these genes as well as a mesenchyme-specific gene and histochemical activity of endoderm-specific alkaline phosphatase were evident by the mid-tailbud stage.

Myocyte enhancer factor 2D regulates ectoderm specification and adhesion properties of animal cap cells in the early Xenopus embryo.

In Xenopus, animal cap (AC) cells give rise to ectoderm and its derivatives: epidermis and the central nervous system. Ectoderm has long been considered a default pathway of embryonic development, with cells that are not under the influence of vegetal Nodal signaling adopting an ectodermal program of gene expression. In the present study, we describe the involvement of the animally-localized maternal transcription factor myocyte enhancer factor (Mef) 2D in regulating the identity of AC cells. We find that Mef2D is required for the formation of both ectodermal lineages: neural and epidermis. Gain and loss of function experiments indicate that Mef2D regulates early gastrula expression of key ectodermal/epidermal genes in the animal region. Mef2D controls the activity of zygotic bone morphogenetic protein (BMP) signaling known to dictate the epidermal differentiation program. Exogenous expression of Mef2D in vegetal blastomeres was sufficient to induce ectopic expression of ectoderm/epidermal genes in the vegetal half of the embryo, when Nodal signaling was inhibited. Depletion of Mef2D caused a loss of AC cell adhesion that was rescued by the expression of E-cadherin or bone morphogenetic protein 4. In addition, expression of Mef2D in the prospective endoderm caused unusual aggregation of vegetal cells with animal cells in vitro and inappropriate segregation to other germ layers in vivo. Mef2D cooperates with another animally-expressed transcription factor, FoxI1e. Together, they regulate the expression of genes encoding signaling proteins and the transcription factors that control the regional identity of animal cells. Therefore, we describe a new role for the animally-localized Mef2D protein in early ectoderm specification, which is similar to that of the vegetally-localized VegT in endoderm and mesoderm formation.

Analysis of ATF2 gene expression during early Xenopus laevis development.

Activating transcription factor 2 (ATF2) is a cellular sequence-specific DNA-binding protein that mediates transcriptional activation by the adenovirus (Ad) E1A protein. In injected Xenopus laevis oocytes, E1A-induced transactivation requires an ATF2 recognition sequence within the responding promoters, thereby suggesting that ATF2 is present in oocytes and perhaps has a developmentally important function. As a first step in assessing this, an ATF2 cDNA was cloned and sequenced. The protein encoded by this cDNA contains 486 amino acids and is 92% identical to mammalian ATF2. ATF2 RNA and protein levels are very low in oocytes, but rise dramatically during blastulation. These high levels are maintained through gastrulation, but return to low levels during neurulation. In the blastula, ATF2 RNA and protein are virtually completely confined to cells of the animal pole. The temporal and spatial regulation of ATF2 suggests that it has an important function in early development.

Transient expression of a novel Six3-related zebrafish gene during gastrulation and eye formation.

Both the Drosophila homeobox gene sine oculis and its murine homologue Six3 have regulatory functions in eye development. In zebrafish, in addition to two previously reported homologues of murine Six3, we have identified a related gene (six7). Although the deduced Six7 protein shares less than 68% sequence identity with the other known zebrafish Six3-like proteins, the embryonic expression patterns have highly conserved features. The six7 transcripts are first detected in involuting axial mesendoderm and, subsequently, in the overlying neurectoderm from which the forebrain and optic primordia develop. Similar to the two other zebrafish Six3 homologues, the expression boundaries of six7 correspond quite closely with the edges of the optic vesicles. Hence, the partially overlapping expression domains of these three six genes probably contribute to anteroposterior specification and in defining the eye primordia.

Normal ontogenic observations on the expression of Eph receptor tyrosine kinase, Cek8, in chick embryos.

The spatio-temporal pattern of expression for the Eph receptor tyrosine kinase, Cek8, was observed in normal chick embryos from H-H stage 6 to 23 by immunohistochemical techniques. Expression of Cek8 was already present in embryos at H-H stage 6, where it was located in the neural plate of the brain region, paraxial mesoderm, and the primitive streak. Regions expressing Cek8 subsequently increased during development to include the neural folds of the brain, rhombomeres 3 and 5, the caudal part of the neural plate, neural crest cells related to the formation of glossopharyngeal nerve ganglia, invaginated cells throughout the primitive groove and the epithelium of the rudiment of the gall bladder. Cek8 was also expressed in the mesenchymal cells of the pharyngeal arches, allantoic stalk and limb buds as well as in the areas surrounding the eye vesicles and nasal pits. Furthermore, cells in the tail bud progressing to the secondary neurulation expressed Cek8. Thus, the spatiotemporal patterns of Cek8 expression appears to have intimate relationships with tissue rebuilding, the maturation of differentiated cells, and the spatial organization of tissues. Consequently, it appears that Cek8 plays an integral role in the developmental events leading to the formation of a wide--though not inclusive--variety of tissues and organ systems.

Transient effects of microgravity on early embryos of Xenopus laevis.

In order to study the role of gravity on the early development of the clawed toad Xenopus laevis, we performed an experiment on the Maser-6 sounding rocket launched from Kiruna (Sweden) on 4 Nov 1993. The aim was to find out whether a short period of microgravity during fertilization and the first few minutes of development does indeed result in abnormal axis formation as was suggested by a pilot experiment on the Maser 3 in 1989. On the Maser 6 we used two new technical additions in the Fokker CIS unit, viz. a 1-g control centrifuge and a video recording unit which both worked successfully. The 1-g control centrifuge was used to discriminate between the influences of flight perturbations and microgravity. After fertilization shortly before launch, one of the first indications of successful egg activation, the cortical contraction, was registered in microgravity and on earth. Analysis of the video tapes revealed that the cortical contraction in microgravity starts earlier than at 1 g on earth. After recovery of the eggs fertilized in microgravity and culture of the embryos on earth, the morphology of the blastocoel has some consistent differences from blastulae from eggs fertilized in the 1-g centrifuge of the rocket. However from the gastrula stage onward, the microgravity embryos apparently recover and resume normal development: the XBra gene is normally expressed, and histological examination shows normal axis formation.

[Concanavalin-binding proteins and cytokeratins in different tissues of the early amphibian gastrula (Rana temporaria, Xenopus laevis)]. / Konkanavalin-sviazyvaiushchie belki i tsitokeratiny raznykh tkanei rannei gastruly amfibii (Rana temporaria, Xenopus laevis).

Concanavalin A (con A), a lectin which specifically interacts with aD-mannose and aD-glucose, has a neutralizing effect on the explants of the early gastrula ectoderm of several amphibian species. Consequently, it was interesting to study con A-binding protein spectrum of the ectoderm and compare it to those of other early gastrula tissues. Animal pole ectoderm (APE), dorsal blastopore lip (DBL) and vegetal pole endoderm (VPE) were dissected from early gastrulae of Rana temporaria and Xenopus laevis. The extracts were subjected to SDS-PAGE with subsequent immunoelectroblotting on nitrocellulose membranes. The blots were sequentially treated with con A solution, horseradish peroxidase and diaminobenzidine. Spectra of the con A-binding glycoproteins were similar in APE, DBL and VPE of R. temporaria. Ten-twelve fractions with the molecular weight in the range from 30 to 150 kDa were stained in each blot. Fractions with the molecular weight of 150, 125, 104, 94 and 42 kDa showed more prominent lectin binding. Con A-binding protein spectra remained unchanged after freezing-thawing of the studied extracts, as well as after blots were treated with neuraminidase or sulphuric acid in order to remove sialic acid residues; the only exception was 42 kDa fraction. At the same time, a-methyl-D-mannoside pyranoside completely blocked con A binding by fractions of the studied extracts. In histological sections of R. temporaria early gastrula, all cells bound FITC-labelled con A. Similar data were obtained with tissues of X. laevis early gastrula. While electrophoretic pattern of X. laevis tissues drastically differed from that of R. temporaria, there were no significant differences between con A-binding protein spectra of X. laevis APE, DBL or VPE. Thus, all studied tissues of the amphibian early gastrula contain similar set of con A-binding proteins; however, only APE is capable of neutralization in response to con A action. These data favor our earlier assumption (see Mikhailov et al., 1989) that con A reception and transmission of the corresponding signal do not determine the characteristics of the target cells response. APE, DBL and VPE extracts were assayed also for the presence of a protein similar to cytokeratin No. 8 characteristic of simple epithelia of mammals. Experiments were performed using immunoelectroblotting with monoclonal antibodies (mAB) against cytokeratin No. 8 from rat colon (mAB E2 and E7 kindly supplied by Dr. G. A. Bannikov). In R. temporaria embryos, cytokeratin 8 was detected in APE, but not in DBL or VPE. In X. laevis gastrulae all the tissues studied contained this cytokeratin.

Dickkopf-1 regulates gastrulation movements by coordinated modulation of Wnt/beta catenin and Wnt/PCP activities, through interaction with the Dally-like homolog Knypek.

Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/beta catenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a "head inducer" during gastrulation. We show here that lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and that gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements. The motility phenotype found in the morphants is not observed in embryos in which the Wnt/beta catenin pathway is overactivated, and that dominant-negative Wnt proteins are not able to rescue the gastrulation movement defect induced by absence of Dkk1. These data strongly suggest that Dkk1 is acting in a beta catenin independent fashion when modulating gastrulation movements. We demonstrate that the glypican 4/6 homolog Knypek (Kny) binds to Dkk1 and that they are able to functionally interact in vivo. Moreover, Dkk1 regulation of gastrulation movements is kny dependent. Kny is a component of the Wnt/planar cell polarity (PCP) pathway. We found that indeed Dkk1 is able to activate this pathway in both Xenopus and zebrafish. Furthermore, concomitant alteration of the beta catenin and PCP activities is able to mimic the morphant accelerated cell motility phenotype. Our data therefore indicate that Dkk1 regulates gastrulation movement through interaction with LRP5/6 and Kny and coordinated modulations of Wnt/beta catenin and Wnt/PCP pathways.

The Snail repressor is required for PMC ingression in the sea urchin embryo.

In metazoans, the epithelial-mesenchymal transition (EMT) is a crucial process for placing the mesoderm beneath the ectoderm. Primary mesenchyme cells (PMCs) at the vegetal pole of the sea urchin embryo ingress into the floor of the blastocoele from the blastula epithelium and later become the skeletogenic mesenchyme. This ingression movement is a classic EMT during which the PMCs penetrate the basal lamina, lose adherens junctions and migrate into the blastocoele. Later, secondary mesenchyme cells (SMCs) also enter the blastocoele via an EMT, but they accompany the invagination of the archenteron initially, in much the same way vertebrate mesenchyme enters the embryo along with endoderm. Here we identify a sea urchin ortholog of the Snail transcription factor, and focus on its roles regulating EMT during PMC ingression. Functional knockdown analyses of Snail in whole embryos and chimeras demonstrate that Snail is required in micromeres for PMC ingression. Snail represses the transcription of cadherin, a repression that appears evolutionarily conserved throughout the animal kingdom. Furthermore, Snail expression is required for endocytosis of cadherin, a cellular activity that accompanies PMC ingression. Perturbation studies position Snail in the sea urchin micromere-PMC gene regulatory network (GRN), downstream of Pmar1 and Alx1, and upstream of several PMC-expressed proteins. Taken together, our findings indicate that Snail plays an essential role in PMCs to control the EMT process, in part through its repression of cadherin expression during PMC ingression, and in part through its role in the endocytosis that helps convert an epithelial cell to a mesenchyme cell.