Late Blastula Stage Research Articles

Bozozok directly represses bmp2b transcription and mediates the earliest dorsoventral asymmetry of bmp2b expression in zebrafish.

Formation of the gastrula organizer requires suppression of ventralizing signals and, in fish and frog, the need to counteract the effect of ubiquitously present maternal factors that activate the expression of Bmps. How the balance between dorsalizing and ventralizing factors is shifted towards organizer establishment at late blastula stages is not well understood. Mutations in zebrafish bozozok (boz) cause severe defects in axial mesoderm and anterior neurectoderm and affect organizer formation. The boz gene encodes the homeodomain protein Bozozok/Dharma and its expression in the region of the organizer is activated through beta-catenin signaling. Here, we investigate the molecular mechanism by which boz contributes to the establishment of the organizer. We demonstrate that the homeodomain protein Boz acts as a transcriptional repressor in zebrafish: overexpression of an En-Boz fusion protein can rescue the boz phenotype, whereas a VP16-Boz fusion protein acts as an antimorph. Expression analysis of bmp2b indicates that Boz negatively regulates bmp2b in the prospective organizer. We demonstrate that this Boz activity is independent of that of other zygotic genes, because it also occurs when translation of zygotic genes is suppressed by cycloheximide (CHX). We identify two high-affinity binding sites for Boz within the first intron of the bmp2b gene. Deletion of these control elements abolishes Boz-dependent repression of bmp2b in the early blastula. Thus, Boz directly represses bmp2b by binding to control elements in the bmp2b locus. We propose that early transcriptional repression of bmp2b by Boz is one of the first steps toward formation of a stable organizer, whereas the later-acting Bmp antagonists (e.g. Chordin, Noggin) modulate Bmp activity in the gastrula to induce patterning along the dorsoventral axis. Thus, similar to Drosophila Dpp, asymmetry of Bmp expression in zebrafish is initiated at the transcriptional level, and the shape of the gradient and its function as a morphogen are later modulated by post-transcriptional mechanisms.

Regional specification during embryogenesis in Rhynchonelliform brachiopods

Fate maps have been constructed for embryos of Hemithiris and Terebratulina, representatives of two orders in the class Rhynchonellata that have been separated since the middle Ordovician. These fate maps are identical. The animal region of the egg forms the ectodermal covering of the apical lobe and the vegetal region is the site of gastrulation; the vegetal region forms the ectoderm of the ventral and posterior regions of the larva, endoderm and mesoderm. The cells that make up the animal region shift 90° with respect to the vegetal pole during gastrulation. The timing and mode of regional specification in these two species are also identical. In each case, the animal region of the unfertilized egg has the intrinsic ability to form apical lobe ectoderm, while the vegetal region has the ability to form a normal larva. During embryogenesis, the vegetal region interacts with the animal region to suppress apical tuft differentiation in the apical lobe and to promote mantle lobe ectodermal differentiation, while the ability of the vegetal half to regulate by forming apical lobe structures is lost. The plane of bilateral symmetry of the larva begins to be set up between the late blastula and early gastrula stage. The fate maps and the processes of regional specification are compared in the four subphyla that make up the Brachiopoda and used to test a developmental model that provides an explanation for the variety of different body plans generated during the Cambrian.

The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm.

Nodal signals, a subclass of the TGFbeta superfamily of secreted factors, induce formation of mesoderm and endoderm in vertebrate embryos. We have examined the possible dorsoventral and animal-vegetal patterning roles for Nodal signals by using mutations in two zebrafish nodal-related genes, squint and cyclops, to manipulate genetically the levels and timing of Nodal activity. squint mutants lack dorsal mesendodermal gene expression at the late blastula stage, and fate mapping and gene expression studies in sqt(-/-); cyc(+/+) and sqt(-/-); cyc(+/-) mutants show that some dorsal marginal cells inappropriately form hindbrain and spinal cord instead of dorsal mesendodermal derivatives. The effects on ventrolateral mesendoderm are less severe, although the endoderm is reduced and muscle precursors are located nearer to the margin than in wild type. Our results support a role for Nodal signals in patterning the mesendoderm along the animal-vegetal axis and indicate that dorsal and ventrolateral mesoderm require different levels of squint and cyclops function. Dorsal marginal cells were not transformed toward more lateral fates in either sqt(-/-); cyc(+/-) or sqt(-/-); cyc(+/+) embryos, arguing against a role for the graded action of Nodal signals in dorsoventral patterning of the mesendoderm. Differential regulation of the cyclops gene in these cells contributes to the different requirements for nodal-related gene function in these cells. Dorsal expression of cyclops requires Nodal-dependent autoregulation, whereas other factors induce cyclops expression in ventrolateral cells. In addition, the differential timing of dorsal mesendoderm induction in squint and cyclops mutants suggests that dorsal marginal cells can respond to Nodal signals at stages ranging from the mid-blastula through the mid-gastrula.

The medaka midblastula transition as revealed by the expression of the paternal genome

At midblastula transition (MBT), zygotic gene transcription is activated, cells become motile and cell division becomes asynchronous. The onset of the medaka ( Oryzias latipes) MBT was examined using expressed sequence tag (EST) markers. Among 187 randomly chosen medaka EST markers, 33 EST markers and two genes (eIF-4C and hsc70) showed polymorphisms in terms of insertion/deletions or restriction sites between the two parental inbred strains, one from the northern Japanese population and the other from the southern Japanese population. There was no evidence of zygotic expression of these EST markers before Stage 10 (early blastula stage), whereas expression of 12 genes was found from Stage 11 on. These results suggest that the medaka MBT in terms of first time transcription of paternal genes in the life of the embryo begins at Stage 11 (late blastula stage).

Acute toxicity effects of tributyltin chloride and triphenyltin chloride on gilthead seabream, Sparus aurata L., embryos

Acute toxicity studies, using two organotin compounds, tributyltin chloride (TBTCl) and triphenyltin chloride (TPhTCl), were performed on gilthead seabream, Sparus aurata, fertilized eggs. The tests were static and run for 24 h (temperature = 19±1°C, salinity = 39.5 g/L). Three replicate egg groups were incubated in solutions containing different concentrations of the toxicants. The (24 h)LC 50 value for TBTCl was 28.3 μg/L and for TPhTCl was 34.2 μg/L, when the embryos were at the morula and late blastula stage at the start of the experiment. The value for TBTCl was 38.6 μg/L, when the embryos were at the gastrula stage at the start of the experiment. The results reconfirm the expected toxicity of TBT to this euryhaline fish species and introduce TPhT as a toxicant of potentially similar impact on teleosts.

Establishment of the organizing activity of the lower endodermal half of the dorsal marginal zone is a primary and necessary event for dorsal axis formation in Cynops pyrrhogaster.

The formation of the head and trunk-tail organizers in the dorsal marginal zone (DMZ) of an amphibian embryo is thought to require spatial and temporal interactions between the Nieuwkoop center and the DMZ. Recent studies of the Xenopus embryo suggested that intra-DMZ interaction is also needed to establish the regional specificity of the DMZ. However, it is not yet clarified when and how the final pattern of the head and trunk-tail organizers is established. To analyze the intra-DMZ interactions, we injected suramin into the blastocoel of the mid-blastula of the urodele, Cynops pyrrhogaster, at 6 h prior to the onset of gastrulation. The pigmented blastopore formed normally, but the convergent extension and involution of the DMZ and dorsal axis formation of the embryo were completely inhibited. Expression of gsc, chd and Lim-1 were not maintained, but noggin was unaffected in the suramin-treated embryos. Dorsal axis formation and the expression of these genes of the suramin-treated embryos were rescued by replacing the lower endodermal half of the DMZ (LDMZ) with normal LDMZ. The present results of embryological and molecular examinations indicate that organizing activity of the early Cynops gastrula DMZ is restricted to the LDMZ, and that the organizing activity of the LDMZ is established during the late blastula stages. The results also indicate that LDMZ triggers the sequential interaction within the DMZ that establishes the final pattern of the regional specificity of the DMZ, and that the formation of the LDMZ is a primary and necessary event for dorsal axis formation.

Blastopore formation and dorsal mesoderm induction are independent events in early Cynops embryogenesis.

The independent roles of blastopore formation and dorsal mesoderm induction in dorsal axis formation of the Cynops pyrrhogaster embryo were attempted to be clarified. The blastopore-forming (bottle) cells originated mainly from the progeny of the mid-dorsal C and/or D blastomeres of the 32-cell embryo, but were not defined to a fixed blastomere. It was confirmed that the isolated dorsal C and D blastomeres autonomously formed a blastopore. Ultraviolet-irradiated eggs formed an abnormal blastopore and then did not form a dorsal axis, although the lower dorsal marginal zone (LDMZ) still had dorsal mesoderm-inducing activity. Involution of the dorsal marginal zone was disturbed by the abnormal blastopore. These embryos were rescued by artificially facilitating involution of the dorsal marginal zone. Suramin-injected and nocodazole-treated blastulae did not have involution of the dorsal marginal zone, although the blastopore was formed. Neither embryos formed the dorsal axis. The dorsal mesoderm-inducing activity of the LDMZ in the nocodazole-treated gastrulae was still active. In contrast, the LDMZ of the suramin-injected embryos lost its dorsal mesoderm-inducing activity. bra expression was activated in the nocodazole-treated embryos but not in the suramin-injected embryos. The present study suggested that (i) the dorsal determinants consist of blastopore-forming and dorsal mesoderm-inducing factors, which are not always mutually dependent; (ii) both factors are activated during the late blastula stage; (iii) the dorsal marginal zone cannot specify to an organized notochord and muscle without the involution that blastopore formation leads to; and (iv) the localization of both factors in the same place is prerequisite for dorsal axis formation.

Sequence and expression of FoxB2 (XFD-5) and FoxI1c (XFD-10) in Xenopus embryogenesis

We report on the temporal and spatial expression pattern of two novel genes of the Xenopus fork head/winged helix family, xFoxB2 and xFoxI1c. xFoxB2 is activated at the late blastula stage and first expressed within the dorsolateral ectoderm except for the organiser territory. During gastrulation, xFoxB2 is found in two ectodermal stripes adjacent to the dorsal midline. Expression is completely down-regulated during neurulation. However, two distinct sets of cells expressing xFoxB2 re-appear in the rhombencephalon of swimming tadpoles. xFoxI1c is initially expressed at the early neurula stage in an epidermal ring around the neural field. Subsequent expression is found to be increased, and is exclusively localised to placodal precursor cells. The placodal expression remains until stage 40, when it is restricted to a distinct region in the lateral body wall behind the gills.

Linker histone H1M transcripts mark the developing germ line in zebrafish

Maternally synthesised factors contribute to the establishment of the germ cell lineage in lower vertebrates. In zebrafish, germ-soma segregation appears to be completed by the late blastula stage of development. To search for new germ cell factors in the zebrafish, we have used subtractive cDNA cloning. Here we report that linker histone H1M transcripts mark the germ line from the early gastrulation up to 18 h post-fertilisation.

The germ cell lineage identified by vas-mRNA during the embryogenesis in goldfish.

vas RNA has been identified in germ-line cells and its precursors in zebrafish, with the result that the germ-line lineage can be traced throughout embryogenesis. In the present study, we described vas localization and the migration of vas-positive cells in goldfish, using whole mount in situ hybridization. The signals of vas mRNA localization appeared at the marginal part of the first to third cleavage planes. The eight signals were detected during the period from the 8- cells to the 512-cell stage. At the late-blastula stage, additional numbers of vas-positive cells were observed, suggesting the proliferation of these cells. At the segmentation period, vas-positive cells showed a long extended distribution along the embryonic axis, but did not form any clusters. vas-positive cells were occasionally distributed at the head region, especially around the future otic vesicle. These signals were inherited to the primordial germ cells, suggesting that vas-positive cells were primordial germ cells (PGCs) in goldfish.

Cyst Development in the Conchostracan Shrimp, Eulimnadia Texana(Crustacea: Spinicaudata)

The fertilized egg (or cyst) of branchiopods is a highly resistant stage in the life cycle of these aquatic crustaceans. Previous examinations of these cysts have determined that early embryonic development arrests at a late blastula stage, resulting in a small, crescent-shaped body within the egg shell of these shrimp. Herein, we examine the early development of these embryos by sectioning eggs in the ovotestis, brood chamber, and several time periods after exit from the brood chamber in the clam shrimp Eulimnadia texana Packard. The early sections find no evidence of internal fertilization in the ovotestis. Eggs in the ovotestis showed no signs of cell division, whereas eggs sectioned from the brood chamber were found to be undergoing early embryonic development. A number of empty egg shells and the lack of unfertilized eggs in the brood chamber suggested that egg yolks quickly degrade after egg extrusion from the ovotestis. Cysts that were allowed to develop for 24, 48, 72, and 96 h, 1 week and 1.5 years were sectioned, and embryonic development did not change after the 48 h time period. Thus, embryos appear to arrest development somewhere between 24 and 48 h after exiting the brood chamber.

Heads or Tails? Amphioxus and the Evolution of Anterior–Posterior Patterning in Deuterostomes

In Xenopus, the canonical Wnt-signaling pathway acting through β-catenin functions both in establishing the dorso-ventral axis and in patterning the anterior–posterior axis. This pathway also acts in patterning the animal–vegetal axis in sea urchins. However, because sea urchin development is typically indirect, and adult sea urchins have pentamerous symmetry and lack a longitudinal nerve cord, it has not been clear how the roles of the canonical Wnt-signaling pathway in axial patterning in sea urchins and vertebrates are evolutionarily related. The developmental expression patterns of Notch, brachyury, caudal, and eight Wnt genes have now been determined for the invertebrate chordate amphioxus, which, like sea urchins, has an early embryo that gastrulates by invagination, but like vertebrates, has a later embryo with a dorsal hollow nerve cord that elongates posteriorly from a tail bud. Comparisons of amphioxus with other deuterostomes suggest that patterning of the ancestral deuterostome embryo along its anterior–posterior axis during the late blastula and subsequent stages involved a posterior signaling center including Wnts, Notch, and transcription factors such as brachyury and caudal. In tunicate embryos, in which cell numbers are reduced and cell fates largely determined during cleavage stages, only vestiges of this signaling center are still apparent; these include localization of Wnt-5 mRNA to the posterior cytoplasm shortly after fertilization and localization of β-catenin to vegetal nuclei during cleavage stages. Neither in tunicates nor in amphioxus is there any evidence that the canonical Wnt-signaling pathway functions in establishment of the dorso-ventral axis. Thus, roles for Wnt-signaling in dorso-ventral patterning of embryos may be a vertebrate innovation that arose in connection with the evolution of yolky eggs and gastrulation by extensive involution.

The Effect of IQGAP1 on Xenopus Embryonic Ectoderm Requires Cdc42

IQGAP1 contains a number of protein recognition motifs through which it binds to targets. Several in vitro studies have documented that IQGAP1 interacts directly with calmodulin, actin, E-cadherin, beta-catenin, and the small GTPases Cdc42 and Rac. Nevertheless, direct demonstration of in vivo function of mammalian IQGAP1 is limited. Using a novel assay to evaluate in vivo function of IQGAP1, we document here that microinjection of IQGAP1 into early Xenopus embryos generates superficial ectoderm lesions at late blastula stages. This activity was retained by the mutated variants of IQGAP1 in which the calponin homology domain or the WW domain was deleted. By contrast, deletion of the IQ (IQGAP1-DeltaIQ), Ras-GAP-related (IQGAP1-DeltaGRD), or C-terminal (IQGAP1-DeltaC) domains abrogated the effect of IQGAP1 on the embryos. None of the latter mutants bound Cdc42, suggesting that the binding of Cdc42 by IQGAP1 is critical for its function. Moreover, overexpression of IQGAP1, but not IQGAP1-DeltaGRD, significantly increased the amount of active Cdc42 in embryonic cells. Co-injection of wild type IQGAP1 with dominant negative Cdc42, but not the dominant negative forms of Rac or Rho, blocked the effect of IQGAP1 on embryonic ectoderm. Together these data indicate that the activity of IQGAP1 in embryonic ectoderm requires Cdc42 function.

Identification and analyses of the Xenopus TERT gene that encodes the catalytic subunit of telomerase

The Xenopus telomerase catalytic component gene, xTERT ( Xenopus telomerase reverse transcriptase), has been cloned. The production of xTERT recombinant protein together with the proposed Xenopus telomerase RNA (xTR) ( Chen et al., 2000. Cell 100, 503–514) in a rabbit reticulocyte lysate system led to the reconstitution of active telomerase, indicating that both products are functional telomerase components. Both xTERT expression and telomerase activity are high from the early to the late blastula stage. However, they are decreased at the gastrula stage and thereafter, suggesting that the xTERT expression level is the primary mechanism for regulating telomerase activity in Xenopus development. This is the first report of a non-mammalian vertebrate TERT gene. Sequence comparison of xTERT with human and mouse TERTs has uncovered four regions conserved in the amino-terminal halves of vertebrate TERT proteins, the functions of which will be discussed herein.

Spectacular Fluorescence Emission in Sea Urchin Larvae

Abstract Strong fluorescence emission occurred in sea urchin larvae when irradiated with blue light under a fluorescence microscope. The blue light irradiation first broke red granules in the pigment cells, releasing green fluorescent substance(s) into the cytoplasm of the pigment cells. The released and dispersed fluorescent substance(s) then made the entire pigment cell emit strong green fluorescence. With prolonged blue light irradiation, the pigment cell itself bursted, dispersing the fluorescent substance(s) into the body cavity or seawater. This resulted in “explosive emission” of the fluorescence. Cells that can emit such fluorescence first appeared at the late-blastula stage, proliferated with development, and changed into the red-pigmented cells. Similar fluorescence emission was observed in the larvae of Clypeaster japonicus, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus, of which C. japonicus larvae displayed the strongest fluorescence.

Developmentally-regulated interaction of a transcription factor complex containing CDP/cut with the early histone H3 gene promoter of the sea urchin Tetrapygus niger is associated with changes in chromatin structure and gene expression

During sea urchin embryogenesis the early histone genes are temporally expressed to accommodate the high demand for histone proteins during DNA replication at early cleavage stages of development. The early histone genes are transcriptionally active from the 16-cell stage, reaching a peak in expression at the 128-cell stage that gradually decreases until expression is completely inhibited at the late blastula stage. We are studying the gene regulatory mechanisms that control early histone gene expression in sea urchins to understand the interrelationships between chromatin remodeling and transcriptional activation during development. Here, we have investigated chromatin organization and transcription factor interactions by analyzing nuclease hypersensitivity and protein binding in the promoter region of the early histone H3 gene from the sea urchin Tetrapygus niger. We have found a DNase I hypersensitive domain centered at −90 in the early histone H3 gene promoter which is only detected in embryos at the 128-cell stage expressing high levels of early histone H3 mRNA. This hypersensitive site (−110 to −70) encompasses two regulatory elements (TnH3NFH3.1 and TnH3CCAAT). The −94 to −77 region of the histone H3 promoter is recognized by a transcription factor complex in nuclear extracts from 128-cell embryos. Methylation interference analysis and competition studies demonstrated a specific interaction at the CCAAT sequence. Using specific antibodies we find that the homeodomain transcription factor CDP/cut is the DNA-binding component of the complex interacting with the early histone H3 gene promoter in T. niger. Our results provide further evidence for the functional role of CDP/cut in developmental regulation of histone gene expression in phylogenetically diverse eukaryotic species.

Visualization of endogenous BMP signaling during Xenopus development

The TGF-β superfamily of growth factors is known to transmit signals to the nucleus mainly through the Smads, intracellular signaling components that are highly conserved from nematodes to humans. The signaling activity of the Smads is regulated by their ligand-stimulated phosphorylation through Ser/Thr kinase receptors. Here, to examine the in vivo role of BMP, we investigated the spatio-temporal activation of BMP-regulated signals during Xenopus development, using a polyclonal antibody that specifically recognizes the phosphorylated form of BMP-regulated Smads. BMP signaling was observed uniformly in embryos as early as stage 7, but was restricted to the ventral side of the embryo at the late blastula stage, supporting the proposed role of BMP4 as a ventralizing factor in Xenopus embryos. In addition, localized staining was detected in several developing organs, consistent with the predicted function of BMP family members in organogenesis.

Multiple roles for Gata5 in zebrafish endoderm formation.

Previous studies have indicated that gata5, a zinc-finger transcription factor gene, is required for the development of the zebrafish gut tube. Here, we show that gata5 mutants also display defects in the development of other endodermal organs such as the liver, pancreas, thyroid and thymus. gata5 is expressed in the endodermal progenitors from late blastula stages, suggesting that it functions early during endoderm development. We indeed find that during gastrulation stages, gata5 mutants form fewer endodermal cells than their wild-type siblings. In addition, the endodermal cells that form in gata5 mutants appear to express lower than wild-type levels of endodermal genes such as sox17 and axial/foxA2. Conversely, overexpression of gata5 leads to expanded endodermal gene expression. These data indicate that Gata5 is involved both in the generation of endodermal cells at late blastula stages and in the maintenance of endodermal sox17 expression during gastrulation. We have also analyzed the relationship of Gata5 to other factors involved in endoderm formation. Using complementary mutant and overexpression analyses, we show that Gata5 regulates endoderm formation in cooperation with the Mix-type transcription factor Bon, that Gata5 and Bon function downstream of Nodal signaling, and that cas function is usually required for the activity of Gata5 in endoderm formation. Finally, we show that fau/gata5, bon and cas exhibit dominant genetic interactions providing additional support that they function in the same pathway. Together, these data demonstrate that Gata5 plays multiple roles in endoderm development in zebrafish, and position Gata5 relative to other regulators of endoderm formation.

Immunocytochemical localization of epidermal growth factor receptor in early embryos of the Japanese medaka fish (Oryzias latipes).

This study was undertaken to localize epidermal growth factor receptor (EGFR) during early development of Japanese medaka embryos using immunocytochemistry. Specific staining was observed in all stages studied. All of the cells of the embryonic disc from the germinal disc (1 cell) through the late high blastula stages stained moderately for EGFR. Beginning with the flat blastula stage, the surface and lateral cells of the embryonic disc and the cells migrating around the yolk stained intensely for EGFR, and this continued throughout the study period. The presence of the keel at the late gastrula stage did not affect the moderate staining of the majority of the embryonic disc cells. When somites first appeared, the keel region stained less intensely than before, but scattered individual cells stained intensely for EGFR. Embryos with 12 somites had a neural tube that was lightly stained except for a few intensely stained individual cells. The neural tube, notochord and somites in 24-somite embryos lacked immunostaining. However, the surface epithelium, aorta, intestinal epithelium and pronephric duct demonstrated EGFR immunostaining. This study demonstrates that EGFR is present during medaka development and supports the hypothesis that EGFR ligands are important during cleavage, gastrulation and early organogenesis.

Artificially applied tensions normalize development of relaxed Xenopus Laevis embryos

Relaxation of tensions of the surface of Xenopus laevis embryos at the late blastula stage leads to deep and diverse developmental defects and increased variability in mutual position and volume ratios of the axial rudiments. Here, we demonstrate that the development of such embryos was markedly normalized if the relaxed tensions were restored in one of two ways: (1) isotropic stretching of the blastocoel roof induced by incubation of relaxed embryos in a hypotonic medium or (2) anisotropic stretching of embryos on two needles. In the latter case, we succeeded in restoring the morphological axis not only after longitudinal stretching, but also after transverse stretching, and the new axis had signs of anteroposterior polarity. The role of isotropic and anisotropic tensions in organization of the early amphibian development is discussed.