MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate.

Pluripotent mouse embryonic stem cells (mESCs), maintained in the presence of the leukemia inhibitory factor (LIF) cytokine, provide a powerful model with which to study pluripotency and differentiation programs. Extensive microarray studies on cultured cells have led to the identification of three LIF signatures. Here we focus on muscle ras oncogene homolog (MRAS), which is a small GTPase of the Ras family encoded within the Pluri gene cluster. To characterise the effects of Mras on cell pluripotency and differentiation, we used gain- and loss-of-function strategies in mESCs and in the Xenopus laevis embryo, in which Mras gene structure and protein sequence are conserved. We show that persistent knockdown of Mras in mESCs reduces expression of specific master genes and that MRAS plays a crucial role in the downregulation of OCT4 and NANOG protein levels upon differentiation. In Xenopus, we demonstrate the potential of Mras to modulate cell fate at early steps of development and during neurogenesis. Overexpression of Mras allows gastrula cells to retain responsiveness to fibroblast growth factor (FGF) and activin. Collectively, these results highlight novel conserved and pleiotropic effects of MRAS in stem cells and early steps of development.

Jun N-terminal kinase activity is required for invagination but not differentiation of the sea urchin archenteron.

Although sea urchin gastrulation is well described at the cellular level, our understanding of the molecular changes that trigger the coordinated cell movements involved is not complete. Jun N-terminal kinase (JNK) is a component of the planar cell polarity pathway and is required for cell movements during embryonic development in several animal species. To study the role of JNK in sea urchin gastrulation, embryos were treated with JNK inhibitor SP600125 just prior to gastrulation. The inhibitor had a limited and specific effect, blocking invagination of the archenteron. Embryos treated with 2 µM SP600125 formed normal vegetal plates, but did not undergo invagination to form an archenteron. Other types of cell movements, specifically ingression of the skeletogenic mesenchyme, were not affected, although the development and pattern of the skeleton was abnormal in treated embryos. Pigment cells, derived from nonskeletogenic mesenchyme, were also present in SP600125-treated embryos. Despite the lack of a visible archenteron in treated embryos, cells at the original vegetal plate expressed several molecular markers for endoderm differentiation. These results demonstrate that JNK activity is required for invagination of the archenteron but not its differentiation, indicating that in this case, morphogenesis and differentiation are under separate regulation.

Thymine DNA glycosylase is a CRL4Cdt2 substrate.

The E3 ubiquitin ligase CRL4(Cdt2) targets proteins for destruction in S phase and after DNA damage by coupling ubiquitylation to DNA-bound proliferating cell nuclear antigen (PCNA). Coupling to PCNA involves a PCNA-interacting peptide (PIP) degron motif in the substrate that recruits CRL4(Cdt2) while binding to PCNA. In vertebrates, CRL4(Cdt2) promotes degradation of proteins whose presence in S phase is deleterious, including Cdt1, Set8, and p21. Here, we show that CRL4(Cdt2) targets thymine DNA glycosylase (TDG), a base excision repair enzyme that is involved in DNA demethylation. TDG contains a conserved and nearly perfect match to the PIP degron consensus. TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and PIP degron-dependent manner during DNA repair in Xenopus egg extract. The protein can also be destroyed during DNA replication in this system. During Xenopus development, TDG first accumulates during gastrulation, and its expression is down-regulated by CRL4(Cdt2). Our results expand the group of vertebrate CRL4(Cdt2) substrates to include a bona fide DNA repair enzyme.

Biochemical biomarkers of sublethal effects in Rhinella arenarum late gastrula exposed to the organophosphate chlorpyrifos.

We determined the biochemical and molecular effects of the organophosphate insecticide chlorpyrifos (CPF) in the late gastrula embryonic stage of the South American toad Rhinella arenarum continuously exposed from fertilization (24 h). Our objective was to evaluate these responses as potential biomarkers at low, sublethal levels of the toxicant. We first established the EC50 for embryo arrest in 21.3 mg/L, with a LOEC of 16 mg/L. At 4 mg/L CPF, some embryos were unable to complete the dorsal lip of the blastopore and the yolk plug became blur, probably because of abnormal cell migration. Acetylcholinesterase activity, the specific biomarker for organophosphates, was unaffected by any of the tested concentrations of CPF (2-14 mg/L). In turn, 2 mg/L CPF increased the reduced glutathione levels and inhibited glutathione-S-transferase activity, suggesting an oxidative stress and antioxidant response. Catalase was induced by CPF exposure at higher concentrations (8 and 14 mg/L). We also studied transcription factor c-Fos as a signaling event related to development in early embryogenesis. Analysis of nuclear c-Fos protein showed two bands, both enhanced in embryos exposed to 2 and 8 mg/L CPF. While nuclear Erk protein was practically unaffected, Mek protein levels were induced by the OP. Transcription factor c-Fos may be then linking oxidative stress with developmental alterations observed due to CPF exposure. These molecular and biochemical responses observed in R. arenarum gastrula at sublethal CPF exposures may replace non-responsive AChE as very early biomarkers in toad gastrula.

PtdIns(3,4,5)P(3)-dependent and -independent roles for PTEN in the control of cell migration.

BACKGROUND: Phosphatase and tensin homolog (PTEN) mediates many of its effects on proliferation, growth, survival, and migration through its PtdIns(3,4,5)P(3) lipid phosphatase activity, suppressing phosphoinositide 3-kinase (PI3K)-dependent signaling pathways. PTEN also possesses a protein phosphatase activity, the role of which is less well characterized. RESULTS: We have investigated the role of PTEN in the control of cell migration of mesoderm cells ingressing through the primitive streak in the chick embryo. Overexpression of PTEN strongly inhibits the epithelial-to-mesenchymal transition (EMT) of mesoderm cells ingressing through the anterior and middle primitive streak, but it does not affect EMT of cells located in the posterior streak. The inhibitory activity on EMT is completely dependent on targeting PTEN through its C-terminal PDZ binding site, but can be achieved by a PTEN mutant (PTEN G129E) with only protein phosphatase activity. Expression either of PTEN lacking the PDZ binding site or of the PTEN C2 domain, or inhibition of PI3K through specific inhibitors, does not inhibit EMT, but results in a loss of both cell polarity and directional migration of mesoderm cells. The PTEN-related protein TPTE, which normally lacks any detectable lipid and protein phosphatase activity, can be reactivated through mutation, and only this reactivated mutant leads to nondirectional migration of these cells in vivo. CONCLUSIONS: PTEN modulates cell migration of mesoderm cells in the chick embryo through at least two distinct mechanisms: controlling EMT, which involves its protein phosphatase activity; and controlling the directional motility of mesoderm cells, through its lipid phosphatase activity.

Comparative expression of zebrafish lats1 and lats2 and their implication in gastrulation movements.

Large tumor suppressor (Lats) is a Ser/Thr kinase, and it presents an important function in tumor suppression. lats was originally identified in Drosophila and recently in mammals. In mammals, it contains two homologues, lats1 and lats2. In the present study, lats1 and lats2 were characterized from zebrafish (Danio rerio), which is the first report of lats in a nonmammalian vertebrate. The primary structure, genomic organization, and phylogenesis of lats from different species were studied, and the results suggest that lats1 is the direct descendant of invertebrate lats, whereas lats2 is formed by genome duplication. In zebrafish, both lats genes are maternally expressed, while they show distinctly different expression profiles during gastrulation. lats1 is almost ubiquitously expressed through development, and lats2 is more prominently expressed in the non-neural ectoderm region of zebrafish gastrula. Most intriguingly, as revealed by cell tracing and gene expression analysis, morpholino-mediated knockdown of either lats1 or lats2 led to obvious defects of cell migration in gastrulation, indicating the functional significance of lats in gastrulation movements.

Homozygous disruption of the Tip60 gene causes early embryonic lethality.

Tat-interactive protein 60 (Tip60) is a member of the MYST family, proteins of which are related by an atypical histone acetyltransferase (HAT) domain. Although Tip60 has been implicated in cellular activities including DNA repair, apoptosis, and transcriptional regulation, its function during embryonic development is unknown. We ablated the Tip60 gene (Htatip) from the mouse by replacing exons 1-9 with a neomycin resistance cassette. Development and reproduction of wild-type and heterozygous animals were normal. However, homozygous ablation of the Tip60 gene caused embryolethality near the blastocyst stage of development, as evidenced by inability of cells in Tip60-null blastocysts to hatch and survive in culture. Monitoring cell proliferation and death by detecting EdU-substituted DNA and TUNEL labeling revealed suppression of cell proliferation concomitant with increased cell death as Tip60-null cells attempted to hatch from blastocysts. These findings indicate that Tip60 is essential for cellular survival during the blastocyst-gastrula transition of embryogenesis.

Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation.

The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior-posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis.

Complementary expression of HSPG 6-O-endosulfatases and 6-O-sulfotransferase in the hindbrain of Xenopus laevis.

Heparan sulfate proteoglycans (HSPGs) are abundant cell surface molecules, consisting of glycosaminoglycan (GAG) chains bound to a protein core. There is high diversity in the sulfation pattern within each GAG chain, creating specific binding sites for many proteins including cell signalling factors, whose activities and distribution are modified by their association with HSPGs (Danesin et al., 2006; Freeman et al., 2008). Here, we describe the distinct expression of three enzymes which modify the 6-O-sulfation state of HSPGs: two 6-O-endosulfatases (Sulf1 and Sulf2), and a 6-O-sulfotransferase (6OST-1). We use in situ hybridisation to determine the spatial distribution of transcripts during tailbud stages of Xenopus laevis development, with a particular focus on neural regions where the 6-O-sulfatases are expressed ventrally and the 6-O-sulfotransferase is restricted dorsally. The complementary expression of these enzymes in the hindbrain and neural tube suggest a role for specific HSPG structure in dorsoventral patterning, possibly through modifying the activity or distribution of signalling molecules such as BMP, Sonic hedgehog, Wnt and/or FGF.

Processing by proprotein convertases is required for glypican-3 modulation of cell survival, Wnt signaling, and gastrulation movements.

Glypican (GPC)-3 inhibits cell proliferation and regulates cell survival during development. This action is demonstrated by GPC3 loss-of-function mutations in humans and mice. Here, we show that the GPC3 core protein is processed by a furinlike convertase. This processing is essential for GPC3 modulating Wnt signaling and cell survival in vitro and for supporting embryonic cell movements in zebrafish. The processed GPC3 core protein is necessary and sufficient for the cell-specific induction of apoptosis, but in vitro effects on canonical and noncanonical Wnt signaling additionally require substitution of the core protein with heparan sulfate. Wnt 5A physically associates only with processed GPC3, and only a form of GPC3 that can be processed by a convertase is able to rescue epiboly and convergence/extension movements in GPC3 morphant embryos. Our data imply that the Simpson-Golabi-Behmel syndrome may in part result from a loss of GPC3 controls on Wnt signaling, and suggest that this function requires the cooperation of both the protein and the heparan sulfate moieties of the proteoglycan.

PI3K and Erk MAPK mediate ErbB signaling in Xenopus gastrulation.

ErbB signaling regulates cell adhesion and movements during Xenopus gastrulation, but the downstream pathways involved have not been elucidated. In this study, we show that phosphatidylinositol-3 kinase (PI3K) and Erk mitogen-activated protein kinase (MAPK) mediate ErbB signaling to regulate gastrulation. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways to control cell behaviors. Activation of either PI3K or Erk MAPK rescues gastrulation defects in ErbB4 morphant embryos, and restores convergent extension in the trunk mesoderm as well as coherent cell migration in the head mesoderm. The two signals preferentially regulate different aspects of cell behaviors, with PI3K more efficient in rescuing cell adhesion and spreading and MAPK more effective in stimulating the formation of filopodia. PI3K and MAPK also weakly activate each other, and together they modulate gastrulation movements. Our results reveal that PI3K and Erk MAPK, which have previously been considered as mesodermal inducing signals, also act downstream of ErbB signaling to participate in regulation of gastrulation morphogenesis.

Mesodermal defect in late phase of gastrulation by a targeted mutation of focal adhesion kinase, FAK.

FAK is a unique non-receptor protein tyrosine kinase that was found in cellular focal adhesions. An increasing number of in vitro observations has suggested that FAK mediates signaling through integrins brought about by interactions with extracellular matrix (ECM). It is highly tyrosine-phosphorylated in v-src-transformed cells and during embryogenesis. To clarify the function of FAK in cell-ECM interactions, embryonic phenotype of its mutant was analysed. FAK-deficient embryos could implant and initiate gastrulation normally, but showed abnormalities in subsequent development. The abnormalities were characterized as a general deficiency in mesoderm, and the phenotype was quite similar to that caused by fibronectin-deficiency. The results suggest that FAK mediates fibronectin-integrin interactions uniquely at this stage of development, thereby playing an essential role in development of mesodermal cell lineages.

Purification and characterization of Artemia 2',3'-cyclic nucleotide 3'-phosphodiesterase.

This paper describes the purification and properties of a 2',3'-cyclic nucleotide 3'-phosphodiesterase which hydrolyzes nucleoside 2',3'-cyclic monophosphates to nucleoside 2'-phosphates. The enzyme is present in encysted gastrulae of Artemia and its specific activity greatly increases during larval development. The purified enzyme has a molecular weight of around 55 000 as estimated by gel filtration, does not require metals for activity, is inhibited by Zn2+ and inactivated by Cu2+ and has a pH optimum at around neutrality. Based on the relative values of V(max)/Km, the specificity of the phosphodiesterase toward the four 2',3'-cyclic nucleotides is Guo-2',3'-P > Ado-2',3'-P > Cyd-2',3'-P > Urd-2',3'-P = 45:36:20:7. The enzyme from Artemia gastrulae is competitively inhibited by the four nucleosides 2'-phosphates (Ki values around 1 mM) while the enzyme from larvae is only inhibited by the purine nucleotides. The phosphodiesterase characterized in this work is more similar in substrate specificity to the 2',3'-cyclic nucleotide 3'-phosphodiesterase from the mammalian nervous system than to the plant enzyme. The functional relationship of this enzyme with the Artemia ribonuclease VI is discussed.

Maternally localized RNA encoding a serine/threonine protein kinase in the ascidian, Halocynthia roretzi.

Maternally localized cytoplasmic determinants play important roles in the embryogenesis of many animals, including ascidians. Cytoplasmic determinants are particularly important in the determination of cell fates, and in the establishment of the embryonic axes. Ascidians, which show mosaic development, are good models for the study of maternal cytoplasmic determinants. Here we report the isolation and characterization of HrPOPK-1 (Halocynthia roretzi posterior protein kinase-1), a putative protein serine/threonine kinase. HrPOPK-1 cDNA was obtained from a Halocynthia roretzi fertilized egg cDNA library by screening for localized RNAs using whole-mount in situ hybridization. HrPOPK-1 mRNA is strongly localized at the posterior pole of embryos. The pattern of HrPOPK-1 mRNA localization during early embryogenesis is identical to that of HrWnt-5 in Halocynthia roretzi, and to those of the posterior end mark (pem) transcripts of Ciona savignyi. In addition, HrPOPK-1 shows zygotic expression in neural tissues at the tailbud stage. These results show that the temporal regulation of HrPOPK-1 transcription is complex.

Cloning of a Xenopus laevis cDNA encoding focal adhesion kinase (FAK) and expression during early development.

Focal adhesion kinase (FAK) is a widely produced nonreceptor protein-tyrosine kinase thought to participate in signalling pathways activated in response to cell interaction with the extracellular matrix. Fibronectin-dependent cell adhesion mediated by integrin receptors plays a critical role in mesodermal cell migration during amphibian gastrulation in early development. As a first step toward understanding the role of FAK in Xenopus laevis (Xl) early development, we isolated cDNAs encoding Xl FAK and deduced the entire amino acid (aa) sequence. Xl FAK has 89-91% overall identity to the homologs previously described from mouse, human and chicken sources. Within the catalytic domain, the aa identity is about 97%. Northern blot analysis revealed that abundant maternal FAK transcript is present in Xl eggs, with levels decreasing slightly through cleavage and early blastula stages. At early gastrulation, the FAK mRNA level becomes modestly elevated, followed by a steady decline through late gastrulation. The mRNA level undergoes a further drop at the neurula stage, then begins a steady increase through the tailbud and tadpole stages. These data indicate that the steady-state level of FAK mRNA is regulated during Xl early development, and are consistent with a proposed role for FAK in the process of gastrulation.

DNA methyltransferase activity in the early stages of a sea urchin embryo. Evidence of differential control.

The specific activity of DNA methyltransferase increases in the nuclei of Sphaerechinus granularis sea urchin embryos at increasing stages of development. The activity reaches maximal value at about 20 h of growth, when embryos are at the mesenchyme blastula stage, then abruptly decreases and is essentially zero at about 35 h of development, when embryos are at the early gastrula stage. Both the increase and the drop of the activity are faster than embryonic cell duplication indicating that the enzyme is under strict control during development and that, in the more advanced embryo, a mechanism is activated to specifically block its activity.

Sea urchin deciliation induces thermoresistance and activates the p38 mitogen-activated protein kinase pathway.

In this study, we demonstrate by a variety of approaches (ie, morphological analysis, Western blots, immunolocalization, and the use of specific antibodies) that hyperosmotic deciliation stress of sea urchin embryos induces a thermotolerant response. Deciliation is also able to activate a phosphorylation signaling cascade the effector of which might be the p38 stress-activated protein kinase because we found that the administration of the p38 inhibitor SB203580 to sea urchin deciliated gastrula embryos makes the hyperosmotic deciliation stress lethal.

The Xenopus laevis homologue to the neuronal cyclin-dependent kinase (cdk5) is expressed in embryos by gastrulation.

Phosphorylation of the neuronal cytoskeletal proteins NF-H, NF-M and tau is important for normal axonal development, and is involved in axonal injury and neurodegenerative diseases. In mammalian neurons, one kinase that phosphorylates these axonal cytoskeletal proteins is cyclin-dependent kinase 5 (cdk5). Cdk5 is a member of the family of cyclin-dependent kinases (cdks), whose other family members regulate mitosis. Unlike the other cdks, cdk5 is abundant in differentiated neurons. Embryos of the clawed frog Xenopus laevis have proved useful for studying other cyclin-dependent kinases, neurofilament proteins and tau during development. As a first step in studying the role of cdk5 and its effects on neurofilaments during Xenopus neural development, four cDNA clones were isolated by screening a frog brain cDNA library at lowered stringency with a cDNA probe to rat cdk5. The frog cdk5 clones encoded a protein of 292 amino acids that was 97% identical to rat cdk5. In situ hybridization demonstrated that the Xenopus cdk5 transcript, like that of mammals, was expressed in differentiated post-mitotic neurons. The high degree of sequence homology and shared neuronal expression suggests that the role of cdk5 in neurons is highly conserved between mammals and amphibians. Northern blot analysis indicated that during Xenopus development, cdk5 mRNA was first expressed between the midblastula transition and gastrulation, which both occur long before neuronal differentiation. These stages mark the transition from synchronous to asynchronous cell division and are the earliest stages of zygotic gene expression. This early expression of Xenopus cdk5 mRNA implies a role for cdk5 during embryogenesis that is separate from its role as an axonal cytoskeletal protein kinase. These observations provide the foundation for exploiting X. laevis embryos to study the role of cdk5 both in the early stages of axonal differentiation and also in early embryogenesis.

[Acetylcholinesterase distribution in the blastoderm of chick embryos]. / Raspredelenie atsetilkholinésterazy v blastoderme kruinykh zarodyshei.

The distribution of acetylcholine esterase (AChE; EC 3.1.1.7) was studied cytochemically in the blastoderm of chick embryos (stages 2-5 by Hamburger and Hamilton). The onset of primitive streak formation was accompanied by a sharp increase in the AChE activity in the anterior part of hypoblast. Changes in the AChE distribution during the early embryogenesis correlated fairly well with the movement of the primary hypoblast cells.