Mesendodermal Cells Research Articles

SDF-1α regulates mesendodermal cell migration during frog gastrulation

During frog gastrulation, mesendodermal cells become apposed to the blastocoel roof (BCR) by endoderm rotation, and migrate towards the animal pole. The leading edge of the mesendodermal cells (LEM) contributes to the directional migration of involuting marginal zone (IMZ) cells, but the molecular mechanism of this process is not well understood. Here we show that CXCR4/SDF-1 signaling mediates the directional movement of the LEM in Xenopus embryos. Expression of xCXCR4 was detected in the IMZ, and was complemented by xSDF-1α expression in the inner surface of the BCR. Over-expression of xCXCR4 and xSDF-1α caused gastrulation defects. An xCXCR4 N-terminus deletion construct and xSDF-1α-MO also inhibited gastrulation. Furthermore, explants of LEM migrate towards the dorsal BCR in the presence of xSDF-1α, and altered xCXCR4 expression in the LEM inhibited LEM migration. These results suggest that CXCR4/SDF-1 signaling is necessary for the migrations of massive numbers of cells during gastrulation.

Rho kinase inhibitor Y27632 affects initial heart myofibrillogenesis in cultured chick blastoderm

During early vertebrate development, Rho-associated kinases (ROCKs) are involved in various developmental processes. Here, we investigated spatiotemporal expression patterns of ROCK1 protein and examined the role of ROCK during initial heart myofibrillogenesis in cultured chick blastoderm. Immunohistochemistry showed that ROCK1 protein was distributed in migrating mesendoderm cells, visceral mesoderm of the pericardial coelom (from which cardiomyocytes will later develop), and cardiomyocytes of the primitive heart tube. Pharmacological inhibition of ROCK by Y27632 did not alter the myocardial specification process in cultured posterior blastoderm. However, Y27632 disturbed the formation of striated heart myofibrils in cultured posterior blastoderm. Furthermore, Y27632 affected the formation of costamere, a vinculin/integrin-based rib-like cell adhesion site. In such cardiomyocytes, cell-cell adhesion was disrupted and N-cadherin was distributed in the perinuclear region. Pharmacological inactivation of myosin light chain kinase, a downstream of ROCK, by ML-9 perturbed the formation of striated myofibrils as well as costameres, but not cell-cell adhesion. These results suggest that ROCK plays a role in the formation of initial heart myofibrillogenesis by means of actin-myosin assembly, and focal adhesion/costamere and cell-cell adhesion.

Canonical NF-κ B Pathway Is Required for Early Mesodermal Differentiation from the Murine Embryonic Stem Cells.

NF-κB family of transcription factors play important roles in cell growth and survival as well as stress and immune responses through the target gene expression. We previously reported that NF-κB family proteins participate in both primitive and definitive hematopoiesis by preventing reactive oxygen species (ROS)-mediated apoptosis. However, their functions in mesodermal development, which is the earlier process to produce hematopoietic cells, has not been elucidated. So, in the present study, we inducibly expressed IκBSR with a Tet-off system in murine embryonic stem (ES) cells and evaluated its effects on mesodermal differentiation. In this system, IκBSR can inhibit the function of canonical NF-κB pathway as a dominant negative mutant in response to tetracycline removal from the culture medium. After 4.0–4.5-day cultures on the OP9 cell layer with differentiation medium deprived of leukemia inhibitory factor (LIF), about 30% of cultured cells developed into the vascular endothelial growth factor receptor 2 (VEGFR2)+ lateral mesodermal cells, which have the potential to differentiate into endothelial cells, hematopoietic cells, and cardiomyocytes. When Tet was deprived from the culture medium, the induced IκBSR inhibited the development of VEGFR2+ cells by inducing apoptosis via the ROS accumulation. However, even if this apoptosis was prevented by the anti-oxidants, the cultured cells did not express VEGFR2. As for this mechanism, we found that although the VEGFR2 promoter was activated by p65, c-Rel, and p50 in response to LIF deprivation in ES cells, IκBSR suppressed this induction almost completely using luciferase assays and ChIP assays. These results suggest that IκBSR blocks the development of lateral mesodermal cells from undifferentiated ES cells through the down-regulated expression of VEGFR2 in concurrence with the induction of apoptosis. To further analyze the effects of IκBSR on mesodermal development, we performed cDNA microarray analysis using differentiated ES cells deprived of Tet for 2 days. Among 4,277 genes, 123 genes were significantly down-regulated and 105 were up-regulated by induction of IκBSR. Among known essential genes for mesodermal development, the expression of platelet derived growth factor receptor α (PDGFRα), which is induced in paraxial mesoderm, was also suppressed by IκBSR significantly. Furthermore, in the prolonged cultures, we found that, although the development of E-cadherin (ECD)+/PDGFRα+ bi-potent mesendodermal cells at day 3.0–3.5 was not affected by IκBSR, it inhibited the subsequent development of ECD−/PDGFRα+ paraxial mesodermal cells at day 5.0–6.0. In accord with this result, we found that IκBSR sustained the expression of Mixl1, one of the intrinsic mesendodermal markers in differentiated ES cells, by semi-quantitive RT-PCR analysis. Together, these results suggest that the canonical NF-κB pathway is required for early mesodermal differentiation from mesendoderm stage through the expression of various essential molecules, thereby regulating their morphogenetic behaviors and cell-fate decisions. We are now evaluating the in vivo effects of IκBSR on the mesodermal development using the tetraploid chimeric mice model, and would like to further disclose the role of NF-κB pathway in our presentation.

Regulation of Xenopus gastrulation by ErbB signaling

During Xenopus gastrulation, mesendodermal cells are internalized and display different movements. Head mesoderm migrates along the blastocoel roof, while trunk mesoderm undergoes convergent extension (C&E). Different signals are implicated in these processes. Our previous studies reveal that signals through ErbB receptor tyrosine kinases modulate Xenopus gastrulation, but the mechanisms employed are not understood. Here we report that ErbB signals control both C&E and head mesoderm migration. Inhibition of ErbB pathway blocks elongation of dorsal marginal zone explants and activin-treated animal caps without removing mesodermal gene expression. Bipolar cell shape and cell mixing in the dorsal region are impaired. Inhibition of ErbB signaling also interferes with migration of prechordal mesoderm on fibronectin. Cell–cell and cell–matrix interaction and cell spreading are reduced when ErbB signaling is blocked. Using antisense morpholino oligonucleotides, we show that ErbB4 is involved in Xenopus gastrulation morphogenesis, and it partially regulates cell movements through modulation of cell adhesion and membrane protrusions. Our results reveal for the first time that vertebrate ErbB signaling modulates gastrulation movements, thus providing a novel pathway, in addition to non-canonical Wnt and FGF signals, that controls gastrulation. We further demonstrate that regulation of cell adhesive properties and cell morphology may underlie the functions of ErbBs in gastrulation.

ADMP2 is essential for primitive blood and heart development in Xenopus

We describe here the cloning of a new member of the TGF-β family with similarity to the anti-dorsalizing morphogenetic proteins (ADMPs). This new gene, ADMP2, is expressed in a broad band of mesendoderm cells that appear to include the progenitors of the endoderm and the ventral mesoderm. Antisense morpholino oligonucleotide knockdown of ADMP2 results in near-complete disruption of primitive blood and heart development, while the development of other mesoderm derivatives, including pronephros, muscle and lateral plate is not disrupted. Moreover, the development of the primitive blood in ADMP2 knockdown embryos cannot be rescued by BMP. These results suggests that ADMP2 plays an early role in specifying presumptive ventral mesoderm in the leading edge mesoderm, and that ADMP2 activity may be necessary to respond to BMP signaling in the context of ventral mesoderm induction.

Identification of regulators of germ layer morphogenesis using proteomics in zebrafish

During vertebrate gastrulation, a well-orchestrated series of morphogenetic changes leads to the formation of the three germ layers: the ectoderm, mesoderm and endoderm. The analysis of gene expression patterns during gastrulation has been central to the identification of genes involved in germ layer formation. However, many proteins are regulated on a translational or post-translational level and are thus undetectable by gene expression analysis. Therefore, we developed a 2D-gel-based comparative proteomic approach to target proteins involved in germ layer morphogenesis during zebrafish gastrulation. Proteomes of ectodermal and mesendodermal progenitor cells were compared and 35 significantly regulated proteins were identified by mass spectrometry, including several proteins with predicted functions in cytoskeletal organization. A comparison of our proteomic results with data obtained in an accompanying microarray-based gene expression analysis revealed no significant overlap, confirming the complementary nature of proteomics and transcriptomics. The regulation of ezrin2, which was identified based on a reduction in spot intensity in mesendodermal cells, was independently validated. Furthermore, we show that ezrin2 is activated by phosphorylation in mesendodermal cells and is required for proper germ layer morphogenesis. We demonstrate the feasibility of proteomics in zebrafish, concluding that proteomics is a valuable tool for analysis of early development.

Migrating anterior mesoderm cells and intercalating trunk mesoderm cells have distinct responses to Rho and Rac during Xenopus gastrulation.

Rho GTPases have been shown recently to be important for cell polarity and motility of the trunk mesoderm during gastrulation in Xenopus embryos. This work demonstrated that Rho and Rac have both distinct and overlapping roles in regulating cell shape, and the dynamic properties, polarity, and type of protrusive activity of these cells. Overexpression of activated or inhibitory versions of these GTPases also disrupts development of the head in Xenopus embryos. In this study, we have undertaken a detailed analysis of Rho and Rac function in migrating anterior mesendoderm cells. Scanning electron micrographs of these cells in situ revealed that their normal shingle arrangement is disrupted and both the cells and their lamellipodia are disoriented. Anterior mesendoderm explants plated on their natural blastocoel roof matrix, however, still migrated towards the animal pole, although the tendency to move in this direction is reduced compared to controls. Analysis of a number of parameters in time-lapse recordings of dissociated cells indicated that Rho and Rac also have both distinct and overlapping roles in the motility of the prospective head mesoderm; however, their effects differ to those previously seen in the trunk mesoderm. Both GTPases appear to modulate cell polarization, migration, and protrusive activity. Rho alone, however, regulates the retraction of the lagging edge of the cell. We propose that within the gastrulating Xenopus embryo, two types of mesoderm cells that undergo different motilities have distinct responses to Rho GTPases.

Zebrafish KLF4 Is Essential for Primitive Haematopoiesis.

Gene knockout studies of Krüppel-like factors (KLFs) in mice have demonstrated key roles in organogenesis. EKLF (Klf1) plays an essential role in definitive erythropoiesis and b-globin gene expression, but primitive (yolk sac) erythropoiesis and embryonic globin gene expression are essentially normal. Since expression of embryonic globin genes is dependent upon proximal CACC box elements, additional KLFs must function during the embryonic wave of erythropoiesis. A number of KLFs have been described in zebrafish. One of these, zKLF4, the homologue of neptune, a Xenopus Laevis KLF expressed in the ventral blood island, cement and hatching glands, is an early marker of lateral plate mesoderm (LPM). From 1 somite and prior to expression of scl and flk1, there is a continuous rim of expression of zKLF4 2–3 cells wide within the LPM extending from the postero-lateral boundary of the polster to the tail. Expression in the posterior lateral plate persists as it migrates medially in a zipper-like movement to generate the intermediate cell mass (ICM), the site of embryonic haematopoiesis. zKLF4 is also expressed in a patch of dorsal mesendodermal cells from 70% epiboly which are fated to become the polster. Morpholino knockdown of zKLF4 results in moderate anaemia which resolves as definitive erythropoiesis replaces primitive erythropoiesis at 48–72 hpf. zKLF4 morphants show a down-regulation in the expression of GATA-1 (>100 fold) and embryonic globin (>10 fold) by real time RT-PCR. Both are expressed during the primitive wave of haematopoiesis. In addition there is a dramatic up-regulation (>100 fold) in the level of cmyb expression in zKLF4 morphant compared to control embryos at 72 hpf, which is consistent with expansion of the secondary definitive wave of erythropoiesis, possibly in response to anemia. These results suggest mammalian KLF orthologs of zKLF4, either KLF4 or KLF2, may play roles in primitive erythropoiesis and embryonic globin gene expression in mice or man.

Zebrafish KLF4 is essential for anterior mesendoderm/pre‐polster differentiation and hatching

Gene knockout studies of Krüppel-like factors (KLFs) in mice have shown essential roles in organogenesis. A screen for KLF family members in zebrafish identified many KLFs. One of these, zebrafish KLF4 (zKLF4) is the homologue of neptune, a Xenopus laevis KLF. zKLF4 is expressed from approximately 80% epiboly a patch of dorsal/anterior mesendodermal cells called the pre-polster and, subsequently, in the polster and hatching gland. Here we investigate the function of zKLF4 using morpholino-based antisense oligonucleotides. Knockdown of zKLF4 resulted in complete absence of hatching gland formation and subsequent hatching in zebrafish. In addition, there was early knockdown of expression of the pre-polster/anterior mesendoderm markers CatL, cap1, and BMP4. These results indicate zKLF4 is expressed within the pre-polster, an early mesendodermal site, and that it plays a critical role in the differentiation of these cells into hatching gland cells.

Cleavage and gastrulation in the shrimp Penaeus ( Litopenaeus) vannamei (Malacostraca, Decapoda, Dendrobranchiata)

While most malacostracan crustaceans develop through superficial cleavage, in the Amphipoda, Euphausiacea, and Dendrobranchiata (Decapoda) cleavage is complete. Euphausiaceans and dendrobranchiate shrimp share a similar early cleavage pattern, early cleavage arrest and ingression of mesendoderm progenitor cells, a ring of crown cells (prospective naupliar mesoderm) around the blastopore, and hatching as a nauplius larva. Yet recent phylogenies do not support a close relationship between Euphausiacea and Decapoda. In addition, some variation is reported in the timing of mesendoderm cell arrest and number of crown cells for a number of dendrobranchiates. To determine the representative pattern of development in the Dendrobranchiata, embryos of the Pacific white shrimp Penaeus ( Litopenaeus) vannamei were stained with Sytox Green to label chromosomes and nuclei and examined with confocal microscopy. The early cleavage pattern, mesendoblast arrest and subsequent ingression at the 32-cell stage, presence of 8 initial crown cells, and fates of the mesendoblasts are the same for P. vannamei (family Peneaeidae) and Sicyonia ingentis (family Sicyoniidae). The lineage of the primordial endoderm cells differs from that reported for P. kerathurus. These characters were discussed in the context of the evolution of development in the Dendrobranchiata and in comparison to the Euphausiacea.

Wnt11 Functions in Gastrulation by Controlling Cell Cohesion through Rab5c and E-Cadherin

Wnt11 plays a central role in tissue morphogenesis during vertebrate gastrulation, but the molecular and cellular mechanisms by which Wnt11 exerts its effects remain poorly understood. Here, we show that Wnt11 functions during zebrafish gastrulation by regulating the cohesion of mesodermal and endodermal (mesendodermal) progenitor cells. Importantly, we demonstrate that Wnt11 activity in this process is mediated by the GTPase Rab5, a key regulator of early endocytosis, as blocking Rab5c activity in wild-type embryos phenocopies slb/wnt11 mutants, and enhancing Rab5c activity in slb/wnt11 mutant embryos rescues the mutant phenotype. In addition, we find that Wnt11 and Rab5c control the endocytosis of E-cadherin and are required in mesendodermal cells for E-cadherin-mediated cell cohesion. Together, our results suggest that Wnt11 controls tissue morphogenesis by modulating E-cadherin-mediated cell cohesion through Rab5c, a novel mechanism of Wnt signaling in gastrulation.

Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy

During vertebrate gastrulation, progenitor cells of different germ layers acquire specific adhesive properties that contribute to germ layer formation and separation. Wnt signals have been suggested to function in this process by modulating the different levels of adhesion between the germ layers, however, direct evidence for this is still lacking. Here we show that Wnt11, a key signal regulating gastrulation movements, is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin, an abundant extracellular matrix component during gastrulation. To measure this effect, we developed an assay to quantify the adhesion of single zebrafish primary mesendodermal progenitors using atomic-force microscopy (AFM). We observed significant differences in detachment force and work between cultured mesendodermal progenitors from wild-type embryos and from slb/wnt11 mutant embryos, which carry a loss-of-function mutation in the wnt11 gene, when tested on fibronectin-coated substrates. These differences were probably due to reduced adhesion to the fibronectin substrate as neither the overall cell morphology nor the cell elasticity grossly differed between wild-type and mutant cells. Furthermore, in the presence of inhibitors of fibronectin-integrin binding, such as RGD peptides, the adhesion force and work were strongly decreased, indicating that integrins are involved in the binding of mesendodermal progenitors in our assay. These findings demonstrate that AFM can be used to quantitatively determine the substrate-adhesion of cultured primary gastrulating cells and provide insight into the role of Wnt11 signalling in modulating cell adhesion at the single cell scale.

Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture

Bipotent mesendoderm that can give rise to both endoderm and mesoderm is an established entity from C. elegans to zebrafish. Although previous studies in mouse embryo indicated the presence of bi-potent mesendoderm cells in the organizer region, characterization of mesendoderm and its differentiation processes are still unclear. As bi-potent mesendoderm is implicated as the major precursor of definitive endoderm, its identification is also essential for exploring the differentiation of definitive endoderm. In this study, we have established embryonic stem (ES) cell lines that carry GFP gene in the goosecoid (Gsc) gene locus and have investigated the differentiation course of mesendodermal cells using Gsc expression as a marker. Our results show that mesendoderm is represented as a Gsc-GFP+ E-cadherin(ECD)+ PDGFRalpha(alphaR)+ population and is selectively induced from ES cells under defined conditions containing either activin or nodal. Subsequently, it diverges to Gsc+ ECD+ alphaR- and Gsc+ ECD- alphaR+ intermediates that eventually differentiate into definitive endoderm and mesodermal lineages, respectively. The presence of mesendodermal cells in nascent Gsc+ ECD+ alphaR+ population was also confirmed by single cell analysis. Finally, we show that the defined culture condition and surface markers developed in this study are applicable for obtaining pure mesendodermal cells and their immediate progenies from genetically unmanipulated ES cells.

Initiation of convergence and extension movements of lateral mesoderm during zebrafish gastrulation

Embryonic morphogenesis is accomplished by cellular movements, rearrangements, and cell fate inductions. Vertebrate gastrulation entails morphogenetic processes that generate three germ layers, endoderm, mesoderm, and ectoderm, shaped into head, trunk, and tail. To understand how cell migration mechanistically contributes to tissue shaping during gastrulation, we examined migration of lateral mesoderm in the zebrafish. Our results illustrate that cell behaviors, different from mediolaterally oriented cell intercalation, also promote convergence and extension (C&E). During early gastrulation, upon internalization, individually migrating mesendodermal cells contribute to the elongation of the mesoderm by moving animally, without dorsal movement. Convergence toward dorsal starts later, by 70% epiboly (7.7 hpf). Depending on location along the Animal-Vegetal axis, an animal or vegetal bias is added to the dorsalward movement, so that paths fan out and the lateral mesoderm both converges and extends. Onset of convergence is independent of noncanonical Wnt signaling but is delayed when Stat3 signaling is compromised. To understand which aspects of motility are controlled by guidance cues, we measured turning behavior of lateral mesodermal cells. We show that cells exhibit directional preference, directionally-regulated speed, and turn toward dorsal when off-course. We estimate that ectoderm could supply from a fraction to all the dorsalward displacement seen in mesoderm cells. Using mathematical modeling, we demonstrate that directional preference is sufficient to account for mesoderm convergence and extension, and that, at minimum, two sources of guidance cues could orient cell paths realistically if located in the dorsal midline.

Shield formation at the onset of zebrafish gastrulation

During vertebrate gastrulation, the three germ layers, ectoderm, mesoderm and endoderm are formed, and the resulting progenitor cells are brought into the positions from which they will later contribute more complex tissues and organs. A core element in this process is the internalization of mesodermal and endodermal progenitors at the onset of gastrulation. Although many of the molecules that induce mesendoderm have been identified, much less is known about the cellular mechanisms underlying mesendodermal cell internalization and germ layer formation. Here we show that at the onset of zebrafish gastrulation, mesendodermal progenitors in dorsal/axial regions of the germ ring internalize by single cell delamination. Once internalized, mesendodermal progenitors upregulate E-Cadherin (Cadherin 1) expression, become increasingly motile and eventually migrate along the overlying epiblast (ectodermal) cell layer towards the animal pole of the gastrula. When E-Cadherin function is compromised, mesendodermal progenitors still internalize, but, with gastrulation proceeding, fail to elongate and efficiently migrate along the epiblast, whereas epiblast cells themselves exhibit reduced radial cell intercalation movements. This indicates that cadherin-mediated cell-cell adhesion is needed within the forming shield for both epiblast cell intercalation, and mesendodermal progenitor cell elongation and migration during zebrafish gastrulation. Our data provide insight into the cellular mechanisms underlying mesendodermal progenitor cell internalization and subsequent migration during zebrafish gastrulation, and the role of cadherin-mediated cell-cell adhesion in these processes.

STAT3 noncell-autonomously controls planar cell polarity during zebrafish convergence and extension.

Zebrafish signal transducer and activator of transcription 3 (STAT3) controls the cell movements during gastrulation. Here, we show that noncell-autonomous activity of STAT3 signaling in gastrula organizer cells controls the polarity of neighboring cells through Dishevelled-RhoA signaling in the Wnt-planar cell polarity (Wnt-PCP) pathway. In STAT3-depleted embryos, although all the known molecules in the Wnt-PCP pathway were expressed normally, the RhoA activity in lateral mesendodermal cells was down-regulated, resulting in severe cell polarization defects in convergence and extension movements identical to Strabismus-depleted embryos. Cell-autonomous activation of Wnt-PCP signaling by ΔN-dishevelled rescued the defect in cell elongation, but not the orientation of lateral mesendodermal cells in STAT3-depleted embryos. The defect in the orientation could be rescued by transplantation of shield cells having noncell-autonomous activity of STAT3 signaling. These results suggest that the cells undergoing convergence and extension movement may sense the gradient of signaling molecules, which are expressed in gastrula organizer by STAT3 and noncell-autonomously activate PCP signaling in neighboring cells during zebrafish gastrulation.

ALK4 functions as a receptor for multiple TGFβ-related ligands to regulate left–right axis determination and mesoderm induction in Xenopus

In Xenopus, several TGFβs, including nodal-related 1 (Xnr1), derriere, and chimeric forms of Vg1, elicit cardiac and visceral organ left–right (LR) defects when ectopically targeted to right mesendoderm cell lineages, suggesting that LR axis determination may require activity of one or more TGFβs. However, it is not known which, if any, of these ligands is required for LR axis determination, nor is it known which type I TGFβ receptor(s) are involved in mediating left-side TGFβ signaling. We report here that similar to effects of ectopic TGFβs, right-side expression of constitutively active activin-like kinase (ALK) 4 results in LR organ reversals as well as altered Pitx2 expression in the lateral plate mesoderm. Moreover, left-side expression of a kinase-deficient, dominant-negative ALK4 (DN-ALK4) or an ALK4 antisense morpholino also results in abnormal embryonic body situs, demonstrating a left-side requirement for ALK4 signaling. To determine which TGFβ(s) utilize the ALK4 pathway to mediate LR development, biochemical and functional assays were performed using an Activin-Vg1 chimera (AVg), Xnr1, and derriere. Whereas ALK4 can co-immunoprecipitate all of these TGFβs, including endogenous Vg1 protein from embryo homogenates, functional assays demonstrate that not all of these ligands require an intact ALK4 signaling pathway to modulate LR asymmetry. When AVg and DN-ALK4 are co-expressed, LR defects otherwise induced by AVg alone are attenuated by DN-ALK4; however, when functional assays are performed with Xnr1 or derriere, LR defects otherwise elicited by these ligands alone still occur in the presence of DN-ALK4. Intriguingly, when any of these TGFβs is expressed at a higher concentration to elicit primary axis defects, DN-ALK4 blocks gastrulation and dorsoanterior/ventroposterior defects that otherwise occur following ligand-only expression. Together, these results suggest not only that ALK4 interacts with multiple TGFβs to generate embryonic pattern, but also that ALK4 ligands differentially utilize the ALK4 pathway to regulate distinct aspects of axial pattern, with Vg1 as a modulator of ALK4 function in LR axis determination and Vg1, Xnr1, and derriere as modulators of ALK4 function in mesoderm induction during primary axis formation.

Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region

In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 ( Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals. In this study, we isolated three clones of the Xenopus ( Silurana) tropicalis nodal-related gene 3 ( Xtnr3) and analyzed their function. The Xtnr3 genes show high homology to Xnr3 and have the same activity. Southern blot and genomic PCR analyses indicate that the X. tropicalis genome has duplications in the Xtnr3 gene sequences and our three clones represent separate gene loci. We also found a partial clone of Xtnr3 that coded for the N-terminal part of its pro-region. Surprisingly, this sequence also induced neural tissue by antagonizing BMP signals, and its coded protein physically associated with BMP4 mature protein. Furthermore, we showed that the pro-region of Xnr5 has the same activity. Together, these findings indicate that the pro-region of nodal-related genes acts antagonistically towards BMP signals, which identifies a novel mechanism for the inhibition of BMP signaling.

Phosphoinositide 3-Kinase Is Required for Process Outgrowth and Cell Polarization of Gastrulating Mesendodermal Cells

Background: During vertebrate gastrulation, cell polarization and migration are core components in the cellular rearrangements that lead to the formation of the three germ layers, ectoderm, mesoderm, and endoderm. Previous studies have implicated the Wnt/planar cell polarity (PCP) signaling pathway in controlling cell morphology and movement during gastrulation. However, cell polarization and directed cell migration are reduced but not completely abolished in the absence of Wnt/PCP signals; this observation indicates that other signaling pathways must be involved.Results: We show that Phosphoinositide 3-Kinases (PI3Ks) are required at the onset of zebrafish gastrulation in mesendodermal cells for process formation and cell polarization. Platelet Derived Growth Factor (PDGF) functions upstream of PI3K, while Protein Kinase B (PKB), a downstream effector of PI3K activity, localizes to the leading edge of migrating mesendodermal cells. In the absence of PI3K activity, PKB localization and cell polarization are strongly reduced in mesendodermal cells and are followed by slower but still highly coordinated and directed movements of these cells.Conclusions: We have identified a novel role of a signaling pathway comprised of PDGF, PI3K, and PKB in the control of morphogenetic cell movements during gastrulation. Furthermore, our findings provide insight into the relationship between cell polarization and directed cell migration at the onset of zebrafish gastrulation.

Desulfurization of peroxyacid treated Indonesian coals by organic base : Ishak, M. A. M. et al. ACGC Chemical Research Communications, 2002, 14, 14–25

Cell-cell contact formation constitutes an essential step in evolution, leading to the differentiation of specialized cell types. However, remarkably little is known about whether and how the interplay between contact formation and fate specification affects development. Here, we identify a positive feedback loop between cell-cell contact duration, morphogen signaling, and mesendoderm cell-fate specification during zebrafish gastrulation. We show that long-lasting cell-cell contacts enhance the competence of prechordal plate (ppl) progenitor cells to respond to Nodal signaling, required for ppl cell-fate specification. We further show that Nodal signaling promotes ppl cell-cell contact duration, generating a positive feedback loop between ppl cell-cell contact duration and cell-fate specification. Finally, by combining mathematical modeling and experimentation, we show that this feedback determines whether anterior axial mesendoderm cells become ppl or, instead, turn into endoderm. Thus, the interdependent activities of cell-cell signaling and contact formation control fate diversification within the developing embryo.