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PubMed2005

Identification of genes expressed during Drosophila melanogaster gastrulation by using subtractive hybridization.

A subtractive hybridization approach was used to identify genes that are expressed at the beginning of gastrulation. We used tester DNA complimentary to RNA (cDNA) prepared from stages 6-7 embryos (gastrula) and excess driver cDNA from stages 2-4 embryos (syncytial blastoderm) to generate a gastrula-subtracted cDNA library. A reverse Northern blot procedure used to analyze 105 subtracted clones showed that 65% had a level of expression at least 2.5-fold higher in stages 6-7 versus stages 2-4 embryos. We determined the nucleotide sequence of these clones and identified 49 individual sequences, including 33 previously uncharacterized genes. We verified the level of expression of 24 genes during Drosophila melanogaster embryogenesis using a semiquantitative polymerase chain reaction (PCR) approach. As expected, all of the selected clones showed their highest level of expression after stages 2-4 of embryogenesis, including several that displayed peaks of expression during gastrulation. Three genes that were expressed at their highest levels in stages 6-7 were further analyzed by 5'-rapid amplification of cDNA ends (RACE) analysis, Northern blot assays and in situ hybridization. Our results indicate that these genes exhibited temporal and spatially restricted patterns of expression in developing embryos, and moreover, their transcripts were detected in cells that undergo morphological changes during the gastrulation stage. Characterizing the role of these genes will be important to increase our understanding of the molecular mechanisms that regulate cellular activities during D. melanogaster gastrulation.

Animals↗
PubMed2006

Essential role for Csk upstream of Fyn and Yes in zebrafish gastrulation.

Morphogenetic cell movements during gastrulation shape the vertebrate embryo bodyplan. Non-canonical Wnt signaling has been established to regulate convergence and extension cell movements that mediate anterior-posterior axis elongation. In recent years, many other factors have been implicated in the process by modulation of non-canonical Wnt signaling or by different, unknown mechanisms. We have found that the Src family kinases, Fyn and Yes, are required for normal convergence and extension cell movements in zebrafish embryonic development and they signal in parallel to non-canonical Wnts, eventually converging on a common downstream factor, RhoA. Here, we report that Csk, a negative regulator of Src family kinases has a role in gastrulation cell movements as well. Csk knock down induced a phenotype that was similar to the defects observed after knock down of Fyn and Yes, in that gastrulation cell movements were impaired, without affecting cell fate. The Csk knock down phenotype was rescued by simultaneous partial knock down of Fyn and Yes. We conclude that Csk acts upstream of Fyn and Yes to control vertebrate gastrulation cell movements.

Journal Article↗
PubMed2005

WNT8 and BMP2B co-regulate non-axial mesoderm patterning during zebrafish gastrulation.

During vertebrate mesoderm formation, fates are established according to position in the dorsoventral (D/V) axis of the embryo. Initially, maternal signaling divides nascent mesoderm into axial (dorsal) and non-axial (ventral) domains. Although the subsequent subdivision of non-axial mesoderm into multiple D/V fate domains is known to involve zygotic Wnt8 and BMP signaling as well as the Vent/Vox/Ved family of transcriptional repressors, how levels of signaling activity are translated into differential regulation of fates is not well understood. To address this question, we have analyzed zebrafish embryos lacking Wnt8 and BMP2b. Zebrafish wnt8; swr (bmp2b) double mutants display a progressive loss of non-axial mesoderm and a concomitant expansion of axial mesoderm during gastrulation. Mesoderm induction and specification of the axial domain occur normally in wnt8; swr mutants, but dorsal mesoderm genes eventually come to be expressed throughout the mesoderm, suggesting that the establishment of non-axial mesoderm identity requires continual repression of dorsal mesoderm factors, including repressors of ventral genes. Loss-of-function for Vent, Vox, and Ved phenocopies the wnt8; swr mutant phenotype, consistent with Wnt8 and BMP2b maintaining non-axial mesoderm identity during gastrulation through the regulation of these three transcriptional repressors. We postulate that timely differentiation of the mesoderm requires the maintenance of non-axial mesoderm identity by Wnt8 and BMP2b at the onset of gastrulation followed by subdivision of the non-axial mesoderm into different functional domains during gastrulation.

Animals↗
PubMed2000

Patterning and nuclear beta-catenin expression in the colonic adenoma-carcinoma sequence. Analogies with embryonic gastrulation.

Patterning is a process by which ordered arrangements of cells and tissue structure are attained. The term derived from developmental biology is also useful for the study of colonic carcinogenesis, in which the patterning of neoplastic tubules is necessary for properties of growth, invasion, and metastasis. Interestingly the nuclear expression and transcriptional activity of beta-catenin, a major oncoprotein in colonic carcinogenesis, is decisive for the first patterning of a tubule in embryogenesis, which creates the primitive gut and is called the gastrulation. Thus, basic patternings of embryogenesis and carcinogenesis might be linked. To test this hypothesis we compared morphological patterns and immunohistochemical beta-catenin stainings in colonic adenomas and adenocarcinomas with the gastrulation steps. Two analogies were found: 1) the patterning of invasion with reconstruction in adenocarcinomas corresponded to the epithelio-mesenchymal transition, ingression, and rearrangement of cells during the first phase of gastrulation; and 2) the patterning of tubular branching in adenomas and adenocarcinomas resembled the endodermal invagination during the second phase. The intratumorous distribution and intensity of nuclear beta-catenin expression was significantly correlated with the two patternings, similar to the findings in gastrulation. The results indicate microenvironmental regulations of nuclear beta-catenin expression and a return of neoplastic cells to embryonic transcriptional susceptibilities during colonic carcinogenesis.

Adenocarcinoma↗
PubMed1997

Dorsal or ventral: similarities in fate maps and gastrulation patterns in annelids, arthropods and chordates.

The idea that chordates, during their evolution, have inverted their dorsoventral body axis has recently gained substantial support. It has been shown that various dorsoventral patterning genes that are evolutionarily conserved between insects and vertebrates are expressed dorsally in insects, and ventrally in vertebrates, or vice versa. The ventral body side of insects thus seems to correspond to the dorsal body side of vertebrates, and these are nerve cord-bearing, neural body sides in both groups. In order to exclude that the inverted polarity of gene patterning activity is purely accidental, we compare here vertebrate and invertebrate blastula fate maps and their gastrulation patterns in the framework of early gene expression. From this comparison it appears that the neural body sides, 'ventral' in annelids or arthropods, and 'dorsal' in chordates, develop at similar positions with respect to the initial egg asymmetry. In addition, the formation of the neural body sides involves similar movements during gastrulation. We further suggest that the deuterostome gastrulation seen in today's chordates can be derived from a more ancestral gastrulation pattern seen in today's annelids and arthropods, and that the ventral midline cells of insects correspond to the dorsal midline cells of vertebrates.

Animals↗
PubMed1997

Mouse gastrulation: the formation of a mammalian body plan.

The process of gastrulation is a pivotal step in the formation of the vertebrate body plan. The primary function of gastrulation is the correct placement of precursor tissues for subsequent morphogenesis. There is now mounting evidence that the body plan is established through inductive interactions between germ layer tissues and by the global patterning activity emanating from embryonic organizers. An increasing number of mouse mutants have been described that have gastrulation defects, providing important insights into the molecular mechanisms that regulate this complex process. In this review, we explore the mouse embryo before and during gastrulation, highlighting its similarities with other vertebrate embryos and its unique characteristics.

Animals↗
PubMed2003

The prickle-related gene in vertebrates is essential for gastrulation cell movements.

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis [1,2]. Recently, it was reported that a noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila [3,4], participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo [5-8]. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal [6-8]. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK [9,10] and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, we show that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene [11-13], is an essential component for gastrulation cell movement. Both gain-of-function and loss-of-function of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrate that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Zebrafish gastrulation movements: bridging cell and developmental biology.

During vertebrate gastrulation, large cellular rearrangements lead to the formation of the three germ layers, ectoderm, mesoderm and endoderm. Zebrafish offer many genetic and experimental advantages for studying vertebrate gastrulation movements. For instance, several mutants, including silberblick, knypek and trilobite, exhibit defects in morphogenesis during gastrulation. The identification of the genes mutated in these lines together with the analysis of the mutant phenotypes has provided new insights into the molecular and cellular mechanisms that underlie vertebrate gastrulation movements.

Animals↗
PubMed2002

Possible role of the 38 kDa protein, lacking in the gastrula-arrested Xenopus mutant, in gastrulation.

An acidic, 38 kDa protein that is present in Xenopus wild-type embryos has been previously shown to be lacking in gastrula-arrested mutant embryos. To gain understanding of the role of this protein, its spatio-temporal distribution and involvement in gastrulation was investigated using the monoclonal antibody (9D10) against it. The protein was prominent in the cortical cytoplasm of cells facing the outside in the animal hemisphere of embryos until the gastrula stage, and in ciliated epithelial cells of embryos at stages later than the late neurula. When the 9D10 antibody was injected into fertilized wild-type eggs, they cleaved normally, but most of them had arrested development, always at the early stage of gastrulation, as in the mutant embryos. In contrast, the majority of the control antibody-injected eggs gastrulated normally and developed further. Cytoskeletal F-actin, which was mainly observed in the area beneath the plasma membrane facing the outside of the epithelial layer of not only the dorsal involuting marginal zone but also the dorsal, vegetal cell mass of the control antibody-injected embryos at the early gastrula stage, was scarcely recognized in the corresponding area of the 9D10 antibody-injected embryos. It is likely that the paucity of the F-actin caused by the 9D10 antibody inhibition of the 38 kDa protein might lead to a failure of cell movement in gastrulation, resulting in developmental arrest.

Animals↗
PubMed2003

Timing system for the start of gastrulation in the Xenopus embryo.

This study examined which component of the egg, the nucleus or cytoplasm, is involved in the timing of the start of gastrulation in the Xenopus embryo, and when it starts to measure time. First, nuclei of cells of 256-cell stage embryos were transplanted to enucleated eggs 60 min after activation. These eggs showed first cleavage 20-30 min later than control eggs fertilized at the same time as the activation of recipient eggs, and started gastrulation 25-35 min later than control embryos (depending on the delay in the first cleavage). Second, eggs whose nuclei were temporarily isolated by the extrusion of the portion containing the nucleus out of the fertilization envelope showed first cleavage 60-90 min later than sibling control eggs, because of delayed introduction of the nucleus from the extruded portion. They started gastrulation 60-90 min later than sibling control embryos (depending on the delay in the first cleavage). The portion inside the envelope underwent two to three rounds of oscillation in cell cycle relevant activities before the first cleavage, while the portion outside underwent the same rounds of cleavage as the inside portion. From the present and previous results it is concluded that the putative timing system for the start of gastrulation in the Xenopus embryo, whether it consists of a single or of multiple clocks, starts measuring time at or around the first cleavage, and that the presence of both the nucleus and the cytoplasm in the same cell and occurrence of mitosis and/or cleavage there are indispensable for the timing system to work, although the role of the cytoplasm is superior to that of the nucleus.

Animals↗
PubMed2006

Mesodermal cell displacements during avian gastrulation are due to both individual cell-autonomous and convective tissue movements.

Gastrulation is a fundamental process in early development that results in the formation of three primary germ layers. During avian gastrulation, presumptive mesodermal cells in the dorsal epiblast ingress through a furrow called the primitive streak (PS), and subsequently move away from the PS and form adult tissues. The biophysical mechanisms driving mesodermal cell movements during gastrulation in amniotes, notably warm-blooded embryos, are not understood. Until now, a major challenge has been distinguishing local individual cell-autonomous (active) displacements from convective displacements caused by large-scale (bulk) morphogenetic tissue movements. To address this problem, we used multiscale, time-lapse microscopy and a particle image velocimetry method for computing tissue displacement fields. Immunolabeled fibronectin was used as an in situ marker for quantifying tissue displacements. By imaging fluorescently labeled mesodermal cells and surrounding extracellular matrix simultaneously, we were able to separate directly the active and passive components of cell displacement during gastrulation. Our results reveal the following: (i) Convective tissue motion contributes significantly to total cell displacement and must be subtracted to measure true cell-autonomous displacement; (ii) Cell-autonomous displacement decreases gradually after regression from the PS; and (iii) There is an increasing cranial-to-caudal (head-to-tail) cell-autonomous motility gradient, with caudal cells actively moving away from the PS faster than cranial cells. These studies show that, in some regions of the embryo, total mesodermal cell displacements are mostly due to convective tissue movements; thus, the data have profound implications for understanding cell guidance mechanisms and tissue morphogenesis in warm-blooded embryos.

Animals↗
PubMed1992

Evidence that the deep keratin filament systems of the Xenopus embryo act to ensure normal gastrulation.

To study the role of keratin filaments in Xenopus development, fertilized eggs were injected with anti-keratin monoclonal antibodies. The anti-keratin monoclonal antibodies AE1 and AE3 induce abnormal gastrulation; in the most severely affected embryos gastrulation fails completely. In contrast, embryos injected with the anti-keratin antibody 1h5 develop normally. Immunocytochemical data indicate that injected 1h5 binds to the dense superficial keratin filament system of the embryo but not to the deeper keratin filament networks of ectodermal and subectodermal cells. Injected AE1 and AE3 do not bind to the superficial keratin system but appear to interact preferentially with the deep keratin filament systems of the embryo. We conclude that the superficial keratin filament system is not involved in the process of gastrulation per se but may protect the embryo from mechanical damage. On the other hand, our results suggest that the integrity of the deeper keratin filament systems is required for the mechanical integration of the morphogenetic movements that underlie gastrulation in Xenopus.

Animals↗
PubMed1989

Expression of cell adhesion molecule E-cadherin in Xenopus embryos begins at gastrulation and predominates in the ectoderm.

The expression of the Ca2+-dependent epithelial cell adhesion molecule E-cadherin (also known as uvomorulin and L-CAM) in the early stages of embryonic development of Xenopus laevis was examined. E-Cadherin was identified in the Xenopus A6 epithelial cell line by antibody cross-reactivity and several biochemical characteristics. Four independent mAbs were generated against purified Xenopus E-cadherin. All four mAbs recognized the same polypeptides in A6 cells, adult epithelial tissues, and embryos. These mAbs inhibited the formation of cell contacts between A6 cells and stained the basolateral plasma membranes of A6 cells, hepatocytes, and alveolar epithelial cells. The time of E-cadherin expression in early Xenopus embryos was determined by immunoblotting. Unlike its expression in early mouse embryos, E-cadherin was not present in the eggs or early blastula of Xenopus laevis. These findings indicate that a different Ca2+-dependent cell adhesion molecule, perhaps another member of the cadherin gene family, is responsible for the Ca2+-dependent adhesion between cleavage stage Xenopus blastomeres. Detectable accumulation of E-cadherin started just before gastrulation at stage 9 1/2 and increased rapidly up to the end of gastrulation at stage 15. In stage 15 embryos, specific immunofluorescence staining of E-cadherin was discernible only in ectoderm, but not in mesoderm and endoderm. The ectoderm at this stage consists of two cell layers. The outer cell layer of ectoderm was stained intensely, and staining was localized to the basolateral plasma membrane of these cells. Lower levels of staining were observed in the inner cell layer of ectoderm. The coincidence of E-cadherin expression with the process of gastrulation and its restriction to the ectoderm indicate that it may play a role in the morphogenetic movements of gastrulation and resulting segregation of embryonic germ layers.

Age Factors↗
PubMed2003

Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation.

Wnt/Frizzled (Fz) signaling controls cell polarity/movements during vertebrate gastrulation via incompletely defined mechanisms. We demonstrated previously that Wnt/Fz activation of Rho, a GTPase and regulator of cytoskeletal architecture, is essential for vertebrate gastrulation. Here we report that in mammalian cells and Xenopus embryos, Wnt/Fz signaling coactivates Rho and Rac, another GTPase and distinct regulator of cytoskeletal architecture. Wnt/Fz activation of Rac is independent of Rho and mediates Wnt/Fz activation of Jun N-terminal kinase (JNK). Dishevelled (Dvl), a cytoplasmic protein downstream of Fz, forms a Wnt-induced complex with Rac independent of the Wnt-induced Dvl-Rho complex. Depletion or inhibition of Rac function perturbs Xenopus gastrulation without affecting Wnt/Fz activation of the Rho or beta-catenin pathway. We propose that parallel activation of Rac and Rho pathways by Wnt/Fz signaling is required for cell polarity and movements during vertebrate gastrulation.

Adaptor Proteins, Signal Transducing↗
PubMed1998

DRhoGEF2 encodes a member of the Dbl family of oncogenes and controls cell shape changes during gastrulation in Drosophila.

We have identified a gene, DRhoGEF2, which encodes a putative guanine nucleotide exchange factor belonging to the Dbl family of oncogenes. DRhoGEF2 function is essential for the coordination of cell shape changes during gastrulation. In the absence of maternal DRhoGEF2 gene activity, mesodermal and endodermal primordia fail to invaginate. The phenotype seen in DRhoGEF2 mutants is more severe than the defects associated with mutations in two previously identified gastrulation genes, folded gastrulation and concertina, suggesting that DRhoGEF2 acts in a signaling pathway independent of these genes. Expression of dominant-negative DRhoA during gastrulation results in phenocopies of the DRhoGEF2 mutant, suggesting that a signaling cascade involving DRhoGEF2 and the small GTPase DRhoA is responsible for the regulation of cell shape changes during early Drosophila morphogenesis.

Amino Acid Sequence↗
PubMed1998

The type I activin receptor ActRIB is required for egg cylinder organization and gastrulation in the mouse.

ActRIB is a type I transmembrane serine/threonine kinase receptor that has been shown to form heteromeric complexes with the type II activin receptors to mediate activin signal. To investigate the function of ActRIB in mammalian development, we generated ActRIB-deficient ES cell lines and mice by gene targeting. Analysis of the ActRIB-/- embryos showed that the epiblast and the extraembryonic ectoderm were disorganized, resulting in disruption and developmental arrest of the egg cylinder before gastrulation. To assess the function of ActRIB in mesoderm formation and gastrulation, chimera analysis was conducted. We found that ActRIB-/- ES cells injected into wild-type blastocysts were able to contribute to the mesoderm in chimeric embryos, suggesting that ActRIB is not required for mesoderm formation. Primitive streak formation, however, was impaired in chimeras when ActRIB-/- cells contributed highly to the epiblast. Further, chimeras generated by injection of wild-type ES cells into ActRIB-/- blastocysts formed relatively normal extraembryonic tissues, but the embryo proper developed poorly probably resulting from severe gastrulation defect. These results provide genetic evidence that ActRIB functions in both epiblast and extraembryonic cells to mediate signals that are required for egg cylinder organization and gastrulation.

Activin Receptors, Type I↗
PubMed2006

Cyclooxygenase-1-derived PGE2 promotes cell motility via the G-protein-coupled EP4 receptor during vertebrate gastrulation.

Gastrulation is a fundamental process during embryogenesis that shapes proper body architecture and establishes three germ layers through coordinated cellular actions of proliferation, fate specification, and movement. Although many molecular pathways involved in the specification of cell fate and polarity during vertebrate gastrulation have been identified, little is known of the signaling that imparts cell motility. Here we show that prostaglandin E(2) (PGE(2)) production by microsomal PGE(2) synthase (Ptges) is essential for gastrulation movements in zebrafish. Furthermore, PGE(2) signaling regulates morphogenetic movements of convergence and extension as well as epiboly through the G-protein-coupled PGE(2) receptor (EP4) via phosphatidylinositol 3-kinase (PI3K)/Akt. EP4 signaling is not required for proper cell shape or persistence of migration, but rather it promotes optimal cell migration speed during gastrulation. This work demonstrates a critical requirement of PGE(2) signaling in promoting cell motility through the COX-1-Ptges-EP4 pathway, a previously unrecognized role for this biologically active lipid in early animal development.

Animals↗
PubMed1992

Expression of a zebrafish caudal homeobox gene correlates with the establishment of posterior cell lineages at gastrulation.

This paper deals with the first identification of a caudal cDNA containing a homeobox of the Drosophila caudal family in the zebrafish. A cDNA library from late gastrula stage embryos was constructed and screened with a mouse Cdx 1 homeobox probe. A 1.6 kb cDNA clone containing a homeobox related to other caudal homeoboxes was isolated and called cdx[Zf-cad1]. Analysis of the predicted 301 amino acid translation product reveals additional regions of homology outside the homeodomain with other members of the caudal family. Particularly, the cdx[Zf-cad1] putative protein shares a conserved N-terminal region with its chicken homolog CHox-cad. Transcripts are first detected just before the onset of gastrulation. At the beginning of gastrulation, a single 1.8 kb cdx[Zf-cad1] transcript is located near the blastoderm margin with a high level of expression restricted to the epiblast. At this stage, the hypoblast is clearly negative. At the end of gastrulation, cdx[Zf-cad1] is widely expressed in vegetal (i.e. prospective posterior) epiblast and hypoblast, with a somewhat weaker expression in the dorsal hypoblast. During somitogenesis, cdx[Zf-cad1] exhibits a posterior regionalization in the neurectoderm. In contrast, no expression is detected in the mesoderm of 22 h embryos (late somitogenesis). Posterior endoderm is also positive at this stage. cdx[Zf-cad1] transcripts cease to be detected about 48 h after fertilization. They are undetectable in the adult, particularly in female gonads. The pattern of cdx[Zf-cad1] expression during and after gastrulation is consistent with its possible involvement in the regionalization of the embryo at these stages.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗