Hensen's Node Research Articles

Distribution of tissue progenitors within the shield region of the zebrafish gastrula

The zebrafish has emerged as an important model system for the experimental analysis of vertebrate development because it is amenable to genetic analysis and because its optical clarity allows the movements and the differentiation of individual cells to be followed in vivo. In this paper, we have sought to characterize the spatial distribution of tissue progenitors within the outer cell layers of the embryonic shield region of the early gastrula. Single cells were labeled by iontophoretic injection of fluorescent dextrans. Subsequently, we documented their position with respect to the embryonic shield and their eventual fates. Our data show that progenitor cells of the neural, notochordal, somitic and endodermal lineages were all present within the embryonic shield region, and that these progenitors were arranged as intermingled populations. Moreover, close to the midline, there was evidence for significant biases in the distribution of neural and notochord progenitors between the layers, suggesting some degree of radial organization within the zebrafish embryonic shield region. The distributions of tissue progenitors in the zebrafish gastrula differ significantly from those in amphibians; this bears not only on interpretations of mutant phenotypes and in situ staining patterns, but also on our understanding of morphogenetic movements during gastrulation and of neural induction in the zebrafish.

Early Stages of Notochord and Floor Plate Development in the Chick Embryo Defined by Normal and Induced Expression of HNF-3β

We have cloned a cDNA encoding the chick HNF-3β gene and have used RNA and antibody probes that detect HNF-3β to monitor the normal and induced expression of the gene in early embryos. HNF-3β is expressed in Koller's sickle, at the onset of primitive streak formation, and later in Hensen's node. At neural plate and neural tube stages, HNF-3 β is expressed transiently in the notochord and is then expressed by floor plate cells. Prospective floor plate cells that are located in the epiblast immediately anterior to Hensen's node prior to its regression do not express HNF-3β, providing evidence that floor plate fate is normally determined only after these cells populate the midline of the neural plate and overlie the notechord. Removal of the notochord in vivo prevents floor plate development and in this condition HNF-3β is not expressed by cells at the ventral midline of the neural tube. Notochord grafts induce ectopic floor plate development and ectopic neural expression of HNF-3 β. In vitro, neural plate explants are induced to express HNF-3β by notochord cells in a contact-dependent but cycloheximide-resistant manner, providing evidence that expression of HNF-3 β is a direct response of neural plate cells to notochord-derived inducing signals.

Fate maps of the zebrafish embryo

In the past few years, we have seen a surge of interest in the zebrafish as a model system for the study of embryonic induction and patterning. This review summarizes our current knowledge of the organization of zebrafish fate maps during early development. Recent advances have addressed the relationship between early cleavage planes and the future dorsal axis, the pattern of cell mixing during blastula and gastrula stages, and the morphogenesis of the trunk neural keel. In addition, refined fate maps have become available for the embryonic shield, the central nervous system, and the heart. In combination with recent advances in molecular and genetic manipulations, these fate maps set the stage for new, more incisive, experimental approaches.

Experimental analyses of the rearrangement of ectodermal cells during gastrulation and neurulation in avian embryos.

The rearrangement of ectodermal cells was studied in chimeras in which grafts were transplanted during late gastrula and early neurula stages to heterotopic locations in avian embryos. Three types of experiments were done. In all experiments, Hensen's node was extirpated completely and replaced with an epithelial plug derived from 1 of 3 regions of the prospective ectoderm. In type-1 experiments, Hensen's node was replaced with a plug consisting of precursor cells of the floor plate of the neural tube. In type-2 experiments, Hensen's node was replaced with a plug consisting of precursor cells of the lateral wall of the neural tube. In type-3 experiments, Hensen's node was replaced with a plug consisting of precursor cells of the epidermal ectoderm. In all experiments, the amount and direction of cell rearrangement that occurred in the transplanted ectodermal plug was essentially typical for prospective ectodermal cells normally residing within Hensen's node. That is, transplanted ectodermal cells underwent lateral-to-medial cell-cell intercalation and contributed to the ventral midline of the neural tube along its entire rostrocaudal extent. In most embryos, a notochord was reconstituted from host cells, despite the fact that Hensen's node--the prime source of prospective notochordal cells in intact embryos--was extirpated completely; however, a few embryos had long notochordal gaps. In such essentially notochordless embryos, the ventral midline of the neural tube still derived from grafted cells, but it failed to form a floor plate, providing further confirmation of the results of several previous studies that the notochord is required to induce the floor plate.(ABSTRACT TRUNCATED AT 250 WORDS)

Organization and development of the tail bud analyzed with the quail-chick chimaera system

After closure of the posterior neuropore, the caudal part of the embryo designated as the ‘tail bud’ forms a mass of undifferentiated cells from which the lumbosacral and caudal parts of the body develop. It has been proposed that the tail bud is a homogeneous structure comparable to a blastema (Holmdahl, 1925; Griffith et al., 1992). Another view is that morphogenesis of the tail bud is merely the continuation of the gastrulation process (Pasteels, 1937, 1943). In order to try to solve this controversy, we have studied the fate of definite and discrete regions of the tail bud at the 25-somite stage by using the quail-chick marker system. We found that the tail bud is composed of different domains endowed with a definite fate. A ventro-rostral region equivalent to the chordo-neural hinge defined by Pasteels gives rise to the notochord and floor plate and thus corresponds to the Hensen's node which in the tail bud pursues its rostrocaudal movement. The presumptive territory of the lateral walls of the lumbo-sacro-caudal neural tube is located caudally to the Hensen's node as it stands at the 25-somite stage. Material destined to form the sacral and caudal somites is still located in the dorsal midline in the caudalmost part of the tail bud. We thus show that the movements of invagination and divergence which characterize gastrulation are still going on in the tail bud after the 25-somite stage. Thus the somitic material located medio-dorsally diverges laterally and contributes by apposition to the growth of the trunco-caudal part of the body. The parallel between tail bud development in Amniotes and Amphibians as described recently by Gont et al. (1993) is striking and points to the unity in the developmental mechanisms within the Vertebrate phylum.

Anti-retinoic Acid Monoclonal Antibody Localizes All-trans-retinoic Acid in Target Cells and Blocks Normal Development in Early Quail Embryo

Avian cardiovascular development is vitamin A-dependent, and retinoic acid has been suggested to be active in this important developmental event. We report here that a monoclonal antibody against all-trans-retinoic acid blocks normal embryonic development in the quail causing cardiovascular abnormalities typical of avian vitamin A deficiency. In whole-mount preparations of stage 5 normal quail embryos the fluorescence associated with the anti-retinoic acid monoclonal antibody localizes in Hensen's node and in caudal area. In stage 7-8 embryos fluorescence localizes in heart-forming areas as well as in head mesenchyme, in Hensen's node, in nephrotome, and in caudal area. These studies are the first to localize endogenous all-trans-retinoic acid during very early stages of normal avian development. We propose that all-trans-retinoic acid is biosynthesized in its target cells during early avian embryo-genesis and that the availability of this signal molecule is spatiotemporally regulated. We conclude that all-trans-retinoic acid or a closely related metabolite is the physiological form of vitamin A required for normal cardiovascular development and for other very early developmental events in the quail embryo.

C-otx2 is expressed in two different phases of gastrulation and is sensitive to retinoic acid treatment in chick embryo

We cloned the chick homologue of the mouse Otx2 gene, c-otx2, and analyzed its expression pattern during gastrulation. During mouse embryogenesis, Otx2 expression is first detected in the entire epiblast and after the formation of the primitive streak becomes confined to the most anterior region of the embryo corresponding to presumptive fore- and mid-brain. Similarly, two distinst phases of c-otx2 expression were observed in the chick. c-otx2 transcripts were first detected in the unincubated egg and up to stage XIII, in all epiblast, and forming hypoblast and mesoblast cells. During primitive streak progression, c-otx2 expression becomes progressively restricted to anterior regions and is mainly associated with Hensen's node. When the extension of the streak is maximal, transcripts are only found in Hensen's node. A second phase of c-otx2 expression starts during streak regression. c-otx2 transcripts are lost from the node and present in higher abundance in anterior neuroectoderm and mesendoderm, with the exception of forming notochord and floor plate. The first phase of expression bears strong similarity with that of c-gsc, a gene shown to be a marker for cells that have organizer activity in the chick. Therefore, we compared the expression of the two genes by double staining on the same embryo. This analysis demonstrated that c-otx2 is transcribed first and its expression in the hypoblast precedes that of c-gsc. On the other hand, c-gsc is an earlier marker of primitive streak cells. The expression domains of the two genes transiently overlap in Hensen's node and anterior mesendoderm, whereas only c-otx2 is expressed in neuroectodermal areas. The second phase of c-otx2 expression is sensitive to an early treatment with retinoic acid. This treatment abolishes c-otx2 expression in mesendoderm and restricts it to most anterior regions in the forming neural plate. In conclusion, our results suggest that c-otx2 expression is first associated with cells with an anterior mesendoderm fate and subsequently extends to anterior neuroectoderm.

Nucleo-cytoplasmic translocation and secretion of fibroblast growth factor-2 during avian gastrulation

The expression and distribution of the fibroblast growth factor-2 (FGF-2 or bFGF) proteins during early avian embryogenesis has been analysed in detail. Three FGF-2 protein isoforms of 18.5, 20.0 and 21.5 kDa are expressed during gastrulation of chicken embryos. Using whole mount immunohistochemistry, these proteins were found to be predominantly nuclear in prestreak blastodiscs during mesoderm induction. Distribution of positive cells in the epiblast was mosaic, whereas all cells of the forming hypoblast expressed the FGF-2 proteins. During primitive streak formation, the proteins started to translocate to the cytoplasm in epiblast cells but remained nuclear in the hypoblast. The FGF-2 proteins became predominantly cytoplasmic in all cells during the subsequent developmental stages. Their highest levels were detected in endodermal cells underlying Hensen's node and the newly formed notochord, the dorsal apex of all epiblast cells and, most interestingly, in the extra-cellular basal lamina separating the epiblast from newly formed mesoderm. Heparin and suramin treatment of these advanced embryos (stage 4) revealed a dose-dependent inhibition on the regression of Hensen's node and formation of mesodermal derivatives such as somites. The results are discussed with respect to current models on FGF-mediated functions during vertebrate mesoderm induction and regionalization.

Mesodermal patterning during avian gastrulation and neurulation: Experimental induction of notochord from non‐notochordal precursor cells

The cells that are normally fated to form notochord occupy a region at the rostral tip of the primitive streak at late gastrula/early neurula stages of avian and mammalian development. If these cells are surgically removed from avian embryos in culture, a notochord will nonetheless form in the majority of cases. The origin of this reconstituted notochord previously had not been investigated and was the objective of this study. Chick embryos at late gastrulal early neurula stages were cultured, and the rostral tip of the primitive streak including Hensen's node was removed and replaced with non-node cells from quail epiblast to ensure that the cells normally fated to be notochord would be absent and that healing of the blastoderm would occur. Embryos were allowed to develop for 24 hr, and the presence and origin (host or graft) of the notochord were assessed using antibodies against notochord or quail cells. Two notochords typically developed; both were almost exclusively of host origin. The primitive streak, and in some cases adjacent tissues, was removed from another group of embryos in an attempt to estimate the mediolateral position and extent of the cells required to form reconstituted notochord. Additional experimental embryos with and without grafts were transected at various rostrocaudal levels in an attempt to estimate the rostrocaudal extent of the cells required to form reconstituted notochord. Finally, various levels of the primitive streak either were placed in a neutral environment (the germ cell crescent) or were grafted in place of the node. Collective results from all experiments indicate that the areas lateral to the rostral portion of the primitive streak, estimated to have a rostrocaudal span of less than 500 microns and a mediolateral extent of less than 250 microns, are critical for formation of the reconstituted notochord. Fate mapping and histological examination of this region identify 4 possible precursor cell populations. Further studies are underway to determine which of the 4 possible precursor cell types forms or induces the reconstituted notochord, and which tissue interactions underlie this change in cell fate.

L5: A carbohydrate epitope involved in neural development

The L5 carbohydrate epitope is developmentally regulated in the vertebrate nervous system, both at very early stages (neural induction) and during postnatal development. Here, we review the evidence indicating that, during gastrulation, L5 may be a marker for cells competent to respond to neural inducing signals emanating from Hensen's node (the ‘organizer’) and that it may itself be involved in the response to neural induction. In postnatal cerebellar development, when L5 is found on astrocytes, it participates in the outgrowth of astrocytic processes on extracellular matrix components. Finally, we indicate the structural relatedness of the L5 carbohydrate and Le x, CD15 and SSEA-1.

Dorsoventral patterning of the avian mesencephalon/metencephalon: Role of the notochord and floor plate in suppressing Engrailed‐2

Transcription factors that are spatially and temporally restricted within the embryo may be used for dorsoventral and rostrocaudal positional information during development. The Engrailed-2 (En-2) gene is expressed across the mesencephalon/metencephalon (mes/met) boundary in the cerebellar primordium with strong dorsolateral expression and limited expression in the floor plate. In a previous experiment we demonstrated that, after removal of Hensen's node, embryos lacked a notochord in the head and the pattern of En-2 expression was normal rostrocaudally, but it was expanded into the ventral midline of the neural tube. This suggested that the notochord suppresses En-2 in the ventral neural tube during normal development. To test further the ability of the notochord (and floor plate) to suppress En-2, we transplanted ventral midline tissues from HH 5-9 quail embryos beneath the rostral neural plate of HH 4-6 chick embryos. After 24 hours in culture, 90% of the embryos with quail notochord or floor plate near the mes/met of the host lacked En-2 expression adjacent to the graft, and suppression was distance dependent. Enzymatically isolated notochords also suppressed En-2 (71%), but the results from isolated floor plates were inconclusive. Other grafts served as controls and included tissues from the trunk ventral midline, mes/met level dorsolateral neural plate, and trunk dorsolateral neural plate/somite. Collectively, the results suggest that during normal development the notochord and possibly the floor plate are important regulators of normal En-2 expression.

Cloning, sequencing, and expressional analysis of the chick homologue of follistatin

Follistatin, a secreted glycoprotein, has been shown to act as a potent neural inducer during early amphibian development. The function of this protein during embryogenesis in higher vertebrates is unclear, and to further our understanding of its role we have cloned, sequenced, and performed an in-depth expressional analysis of the chick homologue of follistatin. In addition we also describe the expression pattern of activin beta A and activin beta B, proteins that have previously been shown to be able to interact with follistatin. In this study we show that the expression of follistatin and the activins do not always overlap. Follistatin was first detected in Hensen's node and subsequently in the region described by Spratt [1952] as the neuralising area. In older embryos it was also expressed in a highly dynamic manner in the hindbrain as well as in the somites. We also present evidence that follistatin may have a later role in the resegmentation of the somites. We were unable to detect the expression of activin beta A during early embryogenesis, whereas activin beta B was first expressed in the extending primitive streak and subsequently in the neural folds. The results from this study are consistent with a role for follistatin in neural induction but suggest it has additional functions unrelated to its inhibitory actions on activins.

Different spatio-temporal expressions of three otx homeoprotein transcripts during zebrafish embryogenesis

Three zebrafish otx homeoproteins containing a homeodomain homologous to that of the Drosophila orthodenticle head gap gene product have been identified by cloning and sequencing of cDNAs. The zebrafish otx2 homeoprotein shares high amino-acid sequence identity with the mouse Otx2 homeoprotein, whereas the zebrafish otx1 and otx3 homeoproteins exhibit moderate homology with the mouse Otx1 and Otx2 homeoproteins. Three otx homeoprotein mRNAs show different spatio-temporal expression patterns during zebrafish embryogenesis as revealed by Northern blot and whole mount in situ hybridization analyses. Large amounts of the otx1 homeoprotein mRNA are found in fertilized uncleaving eggs. The otx3 homeoprotein mRNA appears in the embryonic shield, the site of the organizer. In the developing brain, three zebrafish otx mRNAs are distributed in the diencephalon and the midbrain, but their fine expression patterns are different. These results suggest that three zebrafish otx homeoproteins, alone or in combination, may play roles in very early embryogenesis, gastrulation, and the development and subdivision of the diencephalon and the midbrain.

Quantum mechanics on graphs

We analyse the problem of one-dimensional quantum mechanics on arbitrary graphs as idealized models for quantum systems on spaces with non-trivial topologies. In particular we argue that such models can be made to accommodate the physical characteristics of wavefunctions on a network of wires and offer several derivations of a particular junction condition. Throughout we adopt a continuity condition for the wavefunction at each primitive node in the network. Results are applied to the problem of the energy spectrum of a system containing one and infinitely many junctions.

Cardiac Endothelial Heterogeneity Defines Valvular Development as Demonstrated by the Diverse Expression of JB3, an Antigen of the Endocardial Cushion Tissue

The endothelium of the embryonic vertebrate heart evokes a regional specificity that remains an unexplained phenomenon in cardiac morphogenesis. A restricted population of endothelial cells lining the atrioventricular (AV) canal and proximal outflow tract (OT) transforms into mesenchyme, the reputed progenitor of the valves and membranous septa. The remainder of the cells lining these and other regions of the heart, in particular the ventricle, stay epithelial. At the present time there is no information regarding the determinants for endothelial cell diversity. To investigate the molecular basis for functionally distinct endothelial cell populations, we undertook a search for cell surface proteins within the endocardial cushions of Day 4 chicken embryos that might be sensitive to subtle differences in endothelial cell composition. We theorized that monoclonal antibodies raised against proteins expressed during early valve morphogenesis could provide markers for endothelial subpopulations, thereby assisting our efforts in defining, and determining the origin of, endothelial heterogeneity. In the present study, an in vitro collagen gel culture assay was employed to identify an antibody, JB3, that distinguishes between AV/OT endothelium and ventricular endothelium. Based on this assay, JB3-positive material was associated only with AV/OT endothelia or the mesenchyme derived from these epithelia. Also, a network of JB3-positive fibrillar material was observed within the collagen gel surrounding the explanted cells. The JB3 antigen showed a conspicuous distribution in pregastrulation-stage chicken embryos with immunolabeling observed in the initial primitive streak at 5 hr incubation (stage 2). Subsequent detection in the definitive primitive streak, Hensen's node, and notochord indicate a consistent relationship to midline structures. JB3 antigen also localized to the regions of presumptive precardiac mesoderm and, at later stages, neural crest, somites, and ventral mesocardium. These data suggest that the JB3 antigen may play a role in establishing cardiac endothelial diversity by defining a subpopulation of cells destined to participate in valve formation. Moreover, JB3 may also influence formation of the primary axis and mesoderm structures that form at the midline. Immunochemical analyses showed that JB3 recognizes a polypeptide that migrates near the molecular weight position of fibrillin (350-390 kDa), the extracellular matrix protein linked to the Marfan syndrome. Based on the molecular mass and similar immunostaining patterns in early embryos, we propose that the JB3 antigen is a fibrillin isotype or a fibrillin-associated protein.

Dorso-ventral polarity of the zebrafish embryo is distinguishable prior to the onset of gastrulation.

We describe a set of observations on developing zebrafish embryos and discuss the main conclusions they allow:(1) the embryonic dorso-ventral polarity axis is morphologically distinguishable prior to the onset of gastrulation; and (2) the involution of deep layer cells starts on the prospective dorsal side of the embryo. An asymmetry can be distinguished in the organization of the blastomeres in the zebrafish blastula at the 30% epiboly stage, in that one sector of the blastoderm is thicker than the other. Dye-labelling experiments with DiI and DiO and histological analysis allow us to conclude that the embryonic shield will form on the thinner side of the blastoderm. Therefore, this side corresponds to the prospective dorsal side of the embryo. Simultaneous injections of dyes on the thinner side of the blastoderm and on the opposite side show that involution of deep layer cells during gastrulation starts at the site at which the embryonic shield will form and extends from here to the prospective ventral regions of the germ ring.

Retinoic acid receptor isoform β2 is an early marker for alimentary tract and central nervous system positional specification in the chicken

In this study I describe the distribution of one variant of retinoic acid receptor-beta (RAR-beta), the RAR-beta 2 isoform, during the stages before organogenesis of the chick embryo. Unlike the situation in older embryos, at these stages its distribution does not differ qualitatively from that of all RAR-beta transcripts. During the presomite headfold stage, RAR-beta 2 transcripts are simultaneously upregulated in two different locations. These locations define positional identities within the anlage of the chick alimentary tract and within the central nervous system (CNS). As development proceeds the transcript expression maintains its spatial restriction within those two regions. At presomite stages RAR-beta 2 transcripts are enriched within the proamnion, which contains the presumptive foregut and precardiac cells; somewhat later it is present within the foregut endoderm at the site where foregut and the lateral amniocardiac vesicles fuse to form the coelom and cardiac tube. As the foregut continues its caudal extension, RAR-beta 2 expression defines an anteroposterior boundary at the level of the pharynx within the alimentary tract. The second expression site of RAR-beta 2 mRNA first appears within the posterior neural plate at the level where Hensen's node commences its caudal regression. This boundary lies at the border between the future rhombomeres 5 and 6 within the hindbrain. Expression of RAR-beta 2 transcripts is also spatially restricted within some migrating cranial neural crest cells. The expression of RAR-beta 2 in cranial neural crest cells is consistent with what is known about the mechanisms by which cranial neural crest cell fate is determined. These data support the hypothesis that retinoids may contribute to positional specification of anteroposterior body axis, and perhaps also to the formation and identity of the developing alimentary tract and heart tube.

Quantitative analyses of cell behaviors underlying notochord formation and extension in mouse embryos

Formation and extension of the notochord (i.e., notogenesis) is one of the earliest and most obvious events of axis development in vertebrate embryos. In birds and mammals, prospective notochord cells arise from Hensen's node and come to lie beneath the midline of the neural plate. Throughout the period of neurulation, the notochord retains its close spatial relationship with the developing neural tube and undergoes rapid extension in concert with the overlying neuroepithelium. In the present study, we examined notochord development quantitatively in mouse embryos. C57BL/6 mouse embryos were collected at 8, 8.5, 9, 9.5, and 10 days of gestation. They were then embedded in paraffin and sectioned transversely. Serial sections from 21 embryos were stained with Schiff's reagent according to the Feulgen-Rossenbeck procedure and used for quantitative analyses of notochord extension. Quantitative analyses revealed that extension of the notochord involves cell division within the notochord proper and cell rearrangement within the notochordal plate (the immediate precursor of the notochord). In addition, extension of the notochord involves cell accretion, that is, the addition of cells to the notochord's caudal end, a process that involves considerable cell rearrangement at the notochordal plate-node interface. Extension of the mouse notochord occurs similarly to that described previously for birds (Sausedo and Schoenwolf, 1993 Anat. Rec. 237:58-70). That is, in both birds (i.e., quail and chick) and mouse embryos, notochord extension involves cell division, cell rearrangement, and cell accretion. Thus higher vertebrates utilize similar morphogenetic movements to effect notogenesis.

Forward spreading in the establishment of a vertebrate Hox expression boundary: The expression domain separates into anterior and posterior zones, and the spread occurs across implanted glass barriers

By use of wholemount in situ hybridization, we show how expression of the chicken homeobox gene Hoxd-4 commences in the posterior part of the primitive streak and then spreads forward, covering most of the primitive streak by the 2 somite stage, covering the entire primitive streak by the 5 somite stage, reaching the somite 1/somite 2 level of the neural tube by the 9 somite stage, and reaching the rhombomere 6/rhombomere 7 junction of the hindbrain by the 15 somite stage. Forward spreading does not depend upon cell migration, as was evidenced by vital dye (DiI) cell marking experiments. Furthermore, forward spreading does not apparently require tissue continuity since it could not be blocked by impermeable (glass) barriers surgically implanted to divide embryonic tissues. As forward spreading of chick Hoxd-4 proceeds, the domain of expression separates, at late primitive streak stages, into "anterior" and "posterior zones," with an intervening "intermediate zone" of weak or non-expression. Clear anterior and posterior zones were also found for Hoxa-3 and a-4 expression in late primitive streak stage mouse embryos. We present evidence that the anterior zone corresponds with the "definitive" domain of Hox gene expression, as has earlier been extensively characterized in midgestation embryos. The posterior zone is transitory, probably persisting only for the duration of the primitive streak, and it is a region of intense Hox expression in primitive streak tissue, Hensen's node, and adjacent regions of neurectoderm and mesoderm. We suggest that the posterior zone marks the source of a morphogen which is the primary activator of Hox gene expression, and we discuss possible models for the mechanism of forward spreading in expression.

The orientation of the dorsal/ventral axis of zebrafish is influenced by gravitation.

Following fertilization of eggs of Brachydanio rerio a blastodisc is formed which, by cleavage divisions and epibolic movements, gives rise to a cell cap covering the yolk. When about half of the yolk is covered by the cells, a thickening appears at the progressing margin, the embryonic shield, which gives rise to the axial organs of the future embryo. Thereby, the dorsal/ventral axis can be recognized. Our experiments show that the blastodisc is formed at the position of the micropyle, the only site where the sperm can enter the egg and oocyte. Later the head appears at this position. We show that the position of the embryonic shield on the circumference of the cell cap is influenced by gravitation. We also demonstrate that there is no correlation between the orientation of the first or second cleavage plane and the position of the embryonic shield.