Developing Xenopus Embryos Research Articles

Tbx12 regulates eye development in Xenopus embryos

The regulation of vertebrate eye development requires the activity of many transcription factors. In this report, we demonstrate that the T-box factor Tbx12 is necessary for normal development of the retina. Tbx12 is expressed during early stages of retinal development in multiple species of vertebrate embryos. We injected mRNAs encoding wild type and mutant forms of Tbx12 into Xenopus embryos. The Tbx12 injected embryos exhibit multiple defects in eye development including reduced eye size and disruption of normal retinal laminar organization. Tbx12 appears to function as a repressor of transcription during eye development. Our results indicate that Tbx12 activity is required for the proper generation and organization of retinal cells in the vertebrate eye.

Ectopic EphA4 Receptor Induces Posterior Protrusions via FGF Signaling inXenopusEmbryos

The Eph family of receptor tyrosine kinases regulates numerous biological processes. To examine the biochemical and developmental contributions of specific structural motifs within Eph receptors, wild-type or mutant forms of the EphA4 receptor were ectopically expressed in developing Xenopus embryos. Wild-type EphA4 and a mutant lacking both the SAM domain and PDZ binding motif were constitutively tyrosine phosphorylated in vivo and catalytically active in vitro. EphA4 induced loss of cell adhesion, ventro-lateral protrusions, and severely expanded posterior structures in Xenopus embryos. Moreover, mutation of a conserved SAM domain tyrosine to phenylalanine (Y928F) enhanced the ability of EphA4 to induce these phenotypes, suggesting that the SAM domain may negatively regulate some aspects of EphA4 activity in Xenopus. Analysis of double mutants revealed that the Y928F EphA4 phenotypes were dependent on kinase activity; juxtamembrane sites of tyrosine phosphorylation and SH2 domain-binding were required for cell dissociation, but not for posterior protrusions. The induction of protrusions and expansion of posterior structures is similar to phenotypic effects observed in Xenopus embryos expressing activated FGFR1. Furthermore, the budding ectopic protrusions induced by EphA4 express FGF-8, FGFR1, and FGFR4a. In addition, antisense morpholino oligonucleotide-mediated loss of FGF-8 expression in vivo substantially reduced the phenotypic effects in EphA4Y928F expressing embryos, suggesting a connection between Eph and FGF signaling.

LKB1 (XEEK1) regulates Wnt signalling in vertebrate development.

Germline LKB1/STK11 mutations are associated with the cancer-prone Peutz-Jeghers syndrome (PJS) in humans, and nullizygosity provokes a poorly understood constellation of developmental perturbations in the mid-gestational mouse. To gain a better understanding of the processes regulated by LKB1, we have exploited the experimental merits of the developing Xenopus embryo. Here, specific inhibition of XEEK1, the Xenopus orthologue of LKB1, engendered developmental anomalies - shortened body axis and defective dorsoanterior patterning - associated previously with aberrant Wnt signalling. In line with this, LKB1/XEEK1 cooperates with the Wnt-beta-catenin signalling in axis induction and modulates the expression of Wnt-responsive genes in both Xenopus embryos and mammalian cells. We establish that LKB1/XEEK1 acts upstream of beta-catenin in the Wnt-beta-catenin pathway in vivo. LKB1/XEEK1 regulates glycogen synthase kinase (GSK)3beta phosphorylation and it is physically associated in vivo with GSK3beta and protein kinase C (PKC)-zeta, a known GSK3 kinase. These studies show that LKB1/XEEK1 is required for Wnt-beta-catenin signalling in frogs and mammals and provides novel insights into its role in vertebrate developmental patterning and carcinogenesis.

Participation of proteasomes in Xenopus embryogenesis.

We examined the effects of various protease substrates on Xenopus laevis embryogenesis. Thirty-three peptidyl-MCA substrates were added to the culture medium in which Xenopus embryos were developing. Five of the 33 substrates were found to inhibit embryogenesis at the early gastrula stage or much earlier ones. These results suggest that proteases that hydrolyze these substrates are involved in embryonic development. We found that the developmental stage of embryos is crucial for these substrates to inhibit their development. We purified a protease that hydrolyzes Pyr-Arg-Thr-Lys-Arg-MCA, a substrate that inhibits embryogenesis, from Xenopus embryos. This protease turned out to be a component of proteasomes. We found that 4 of the 5 substrates that inhibit embryogenesis are among the proteasome substrates. Thus, we concluded that proteasomes play a crucial role in the development of Xenopus embryos. Possibly, various catalytic subunits in proteasomes function independently, in stage-specific manners.

The lipid phosphatase LPP3 regulates extra-embryonic vasculogenesis and axis patterning.

Bioactive phospholipids, which include sphingosine-1-phosphate, lysophosphatidic acid, ceramide and their derivatives regulate a wide variety of cellular functions in culture such as proliferation, apoptosis and differentiation. The availability of these lipids and their products is regulated by the lipid phosphate phosphatases (LPPs). Here we show that mouse embryos deficient for LPP3 fail to form a chorio-allantoic placenta and yolk sac vasculature. A subset of embryos also show a shortening of the anterior-posterior axis and frequent duplication of axial structures that are strikingly similar to the phenotypes associated with axin deficiency, a critical regulator of Wnt signaling. Loss of LPP3 results in a marked increase in beta-catenin-mediated TCF transcription, whereas elevated levels of LPP3 inhibit beta-catenin-mediated TCF transcription. LPP3 also inhibits axis duplication and leads to mild ventralization in Xenopus embryo development. Although LPP3 null fibroblasts show altered levels of bioactive phospholipids, consistent with loss of LPP3 phosphatase activity, mutant forms of LPP3, specifically lacking phosphatase activity, were able to inhibit beta-catenin-mediated TCF transcription and also suppress axis duplication, although not as effectively as intact LPP3. These results reveal that LPP3 is essential to formation of the chorio-allantoic placenta and extra-embryonic vasculature. LPP3 also mediates gastrulation and axis formation, probably by influencing the canonical Wnt signaling pathway. The exact biochemical roles of LPP3 phosphatase activity and its undefined effect on beta-catenin-mediated TCF transcription remain to be determined.

Overexpression of receptor-type protein tyrosine phosphatase β causes abnormal development of the cranial nerve in Xenopus embryos

Roles of receptor-type protein tyrosine phosphatase β (RPTPβ, also called PTPζ) were investigated in the nervous system development of Xenopus embryos. We previously showed that Xenopus embryos express mRNAs for 11 receptor-type (XRPTPβ.1–XRPTPβ.11) and two secretory (sXRPTPβ.1 and sXRPTPβ.2) variants generated by alternative RNA splicing. Whole-mount in situ hybridization analyzes demonstrated central nervous system-specific gene transcription in tailbud embryos. Distributions of mRNAs for receptor-type and secretory variants partially differ in the hindbrain. Overexpression of XRPTPβ.4 or sXRPTPβ.2, which was brought about by microinjection of the recombinant plasmid vectors, caused abnormal development of the cranial nerve X. Deletion of the cytoplasmic segment from XRPTPβ.4 did not affect the ability to cause the abnormality, but deletion of the extracellular segment abolished it. These results suggest that normal development of the cranial nerve X requires regulated expression of the XRPTPβ gene products.

Effects of fluoride on Xenopus embryo development

Fluoride was first associated with fetal malformation shortly after water fluoridation was initiated in the 1940s. Since many chemicals can interact directly with the embryo to cause malformation, the effects of fluoride on embryonic and fetal development were investigated. The effects of sodium fluoride on the development of frog embryos were studied under conditions described by the Frog Embryo Teratogenesis Assay— Xenopus (FETAX), a screening assay for teratogens. The most prominent malformations caused by sodium fluoride are reduction in the head-tail lengths and dysfunction of the neuromuscular system of the tadpoles. The values for LC 50, EC 50, and minimal concentration to inhibit growth (MCIG) of sodium fluoride met the limits established for a teratogen in frog embryos, showing that sodium fluoride is a direct acting teratogen on developing embryos. Since FETAX has a high degree of success in identifying mammalian teratogens, the observed teratogenic action of sodium fluoride on frog embryos would indicate a strong possibility that sodium fluoride may also act directly on developing mammalian fetuses to cause malformation.

Distinct molecular and morphogenetic properties of mutations in the human HNF1beta gene that lead to defective kidney development.

The homeobox transcription factor hepatocyte nuclear factor 1beta (HNF1beta) is a tissue-specific regulator that plays an essential role in early vertebrate development. In humans, heterozygous mutations in the HNF1beta gene are associated with young-onset diabetes as well as a variety of disorders of renal development with cysts as the most consistent feature. This report compares and classifies nine different HNF1beta mutations that lead in humans to distinct renal diseases, including solitary functioning kidney, renal dysplasia, glomerulocystic kidney disease, and oligomeganephronia. Analysis of these mutants in vitro identifies mutants that either retain or lack DNA binding. Investigation of the transactivation potential in transfected cell lines reveals a strict correlation between DNA binding and transactivation. Introduction of these mutants into developing Xenopus embryos shows that these mutants interfere with pronephros development, the first kidney form in amphibian. Whereas three mutants lead in Xenopus to a reduction or agenesis of the pronephric tubules and the anterior part of the duct, six mutants generate an enlargement of the pronephric structures. The differential morphogenetic potential in the developing embryo does not strictly correlate with the properties observed in vitro or in transfected cell lines. This suggests that the functional test in the developing embryo defines features of the HNF1beta protein that cannot be assessed in cell cultures. The distinct properties observed in the various HNF1beta mutants may guide the classification of the phenotypes observed in patients with a mutated HNF1beta gene.

Lef/Tcf-dependent Wnt/β-catenin signaling during Xenopus axis specification

Though the Wnt/β-catenin signaling pathway is known to play key roles during Xenopus axis specification, whether it signals exclusively through Lef/Tcf transcription factors in this process remains unclear. To investigate this issue, we generated transgenic frog embryos expressing green fluorescent protein (GFP) driven by a Lef/Tcf-dependent and Wnt/β-catenin-responsive promoter. This promoter is highly sensitive and even detects maternal β-catenin activity prior to the large-scale transcription of zygotic genes. Unexpectedly, GFP expression was observed only in some, but not all, known Wnt/β-catenin-positive territories in Xenopus early development. Furthermore, ubiquitous expression of dominant Lef-1 protein variants from transgenes revealed that zygotic Lef/Tcf activity is required for the ventroposterior development of Xenopus embryos. In summary, our results suggest that endogenous Wnt/β-catenin activity does not result in obligatory Lef/Tcf-dependent gene activation, and that the ventroposteriorizing activity of zygotic Wnt-8 signaling is mediated by Lef/Tcf proteins.

Adult and embryonic blood and endothelium derive from distinct precursor populations which are differentially programmed by BMP in Xenopus.

Blood and blood vessels develop in close association in vertebrate embryos and loss-of-function mutations suggest common genetic regulation. By the criteria of co-expression of blood and endothelial genes, and lineage tracing of progeny, we locate two distinct populations of progenitors for blood and endothelial cells in developing Xenopus embryos. The first population is located immediately posterior to the cement gland during neurula stages and gives rise to embryonic blood and vitelline veins in the anterior ventral blood island (aVBI), and to the endocardium of the heart. The second population resides in the dorsal lateral plate mesoderm, and contains precursors of adult blood stem cells and the major vessels. Both populations differentiate into endothelial cells in situ but migrate to new locations to differentiate into blood, suggesting that their micro-environments are unsuitable for haematopoietic differentiation. Both require BMP for their formation, even the Spemann organiser-derived aVBI, but individual genes are affected differentially. Thus, in the embryonic population, expression of the blood genes, SCL and GATA2, depend on BMP signalling while expression of the endothelial gene, Xfli1, does not. By contrast, Xfli1 expression in the adult, DLP population does require BMP. These results indicate that both adult and the anterior component of embryonic blood in Xenopus embryos derive from populations of progenitors that also give rise to endothelial cells. However, the two populations give rise to distinct regions of the vasculature and are programmed differentially by BMP.

Gene expression pattern analysis of the tight junction protein, Claudin, in the early morphogenesis of Xenopus embryos

To study how epithelial layers are formed during early development in Xenopus embryos, we have focused on Claudin, the major component of the tight junction. So far, 19 claudin genes have been found in the mouse, expressed in different epithelial tissues. However, though a number of cytological studies have been done for the roles of Claudins, their expression patterns and functions during early embryogenesis are largely unknown. We found three novel Xenopus claudin genes, which are referred to as claudin-4L1, -4L2, and -7L1. At the early gastrula stage, claudin-4L1, - 4L2, and - 7L1 mRNAs were detected in the ectoderm and in the mesoderm. At the late gastrula stage, claudin mRNAs were detected in the ectoderm through the involuting archenteron roof. At the neurula stage, claudin-4L1/4L2 and -7L1 mRNAs were differentially expressed in the neural groove and the epidermal ectoderm. At the tailbud stage, the claudin mRNAs were found in the branchial arches, the otic vesicles, the sensorial layer of the epidermis, and along the dorsal midline of the neural tube. In addition, claudin-4L1/4L2 mRNAs were detected in the pronephros and the endoderm, whereas claudin-7L1 mRNA was observed in the epithelial layer of the epidermis.

Geminin deficiency causes a Chk1-dependent G2 arrest in Xenopus.

Geminin is an unstable inhibitor of DNA replication that gets destroyed at the metaphase/anaphase transition. The biological function of geminin has been difficult to determine because it is not homologous to a characterized protein and has pleiotropic effects when overexpressed. Geminin is thought to prevent a second round of initiation during S or G2 phase. In some assays, geminin induces uncommitted embryonic cells to differentiate as neurons. In this study, geminin was eliminated from developing Xenopus embryos by using antisense techniques. Geminin-deficient embryos show a novel and unusual phenotype: they complete the early cleavage divisions normally but arrest in G2 phase immediately after the midblastula transition. The arrest requires Chk1, the effector kinase of the DNA replication/DNA damage checkpoint pathway. The results indicate that geminin has an essential function and that loss of this function prevents entry into mitosis by a Chk1-dependent mechanism. Geminin may be required to maintain the structural integrity of the genome or it may directly down-regulate Chk1 activity. The data also show that during the embryonic cell cycles, rereplication is almost entirely prevented by geminin-independent mechanisms.

Glycogen Synthase Kinase-3β Mutagenesis Identifies a Common Binding Domain for GBP and Axin

Glycogen synthase kinase-3 beta (GSK-3) is a key downstream target of Wnt signaling and is regulated by its interactions with activating and inhibitory proteins. We and others have shown that GSK-3 activity toward non-primed substrates is regulated in part through a competition between its activating (Axin) and inhibitory (GBP/FRAT) binding partners. Here we use a reverse two-hybrid screen to identify mutations in GSK-3 that alter binding to GBP and Axin. We find that these mutations overlap and propose that GBP and Axin compete for binding to the same region of GSK-3. We use these mutations to examine the ability of GSK-3 to block eye development in Xenopus embryos and suggest that GSK-3 regulates eye development through a non-Wnt pathway.

Overexpression of muscle specific kinase increases the transcription and aggregation of acetylcholine receptors in Xenopus embryos

Muscle specific kinase (MuSK) mediates agrin-induced acetylcholine receptor (AChR) aggregation on muscle membrane at the neuromuscular junction (NMJ). To examine whether MuSK enhances NMJ formation during embryonic development in vivo, the level of expression of MuSK was manipulated in Xenopus embryos and the functional consequence at the NMJ was assessed. We found that overexpression of MuSK enhanced the formation of NMJ by increasing the aggregation of AChRs at innervated regions in developing embryos. The area of AChR aggregation increased by ∼2-fold in MuSK injected embryos during the critical stages of NMJ formation. Interestingly, overexpression of MuSK in Xenopus embryos was found to induce the level of AChR transcript. Deletion of the Kringle domain in the MuSK construct did not attenuate the observed induction of AChR transcription and aggregation. Taken together, our findings provide the first demonstration that increased level of MuSK expression in vivo significantly elevate the aggregation and transcription of AChR at the NMJ in developing Xenopus embryos.

Xenopus Na,K-ATPase: primary sequence of the β2 subunit and in situ localization of α1, β1, and γ expression during pronephric kidney development

The osmoregulatory function of the pronephric kidney, the first excretory organ of the vertebrate embryo, is essential for embryonic survival. The transport systems engaged in pronephric osmotic regulation are however poorly understood. The Na,K-ATPase is the key component in renal solute transport and water homeostasis. In the present study, we characterized the α, β, and γ subunits of the Na,K-ATPase of the developing Xenopus embryo. In addition to the known α1, β1, β3 and γ subunits, we report here the identification of a novel cDNA encoding the Xenopus β2 subunit. We demonstrate by in situ hybridization that each Xenopus Na,K-ATPase subunit exhibits a distinct tissue-specific and developmentally regulated expression pattern. We found that the developing pronephric kidney expresses α1, β1, and γ subunits uniformly along the entire length of the nephron. Onset of pronephric Na,K-ATPase subunit expression occurred in a coordinated fashion indicating that a common regulatory mechanism may initiate pronephric transcription of these genes. The ability to engage in active Na + reabsorption appears to be established early in pronephric development, since Na,K-ATPase expression was detected well before the completion of pronephric organogenesis. Furthermore, Na,K-ATPase expression defines at the molecular level the onset of maturation phase during pronephric kidney organogenesis. Taken together, our studies reveal a striking conservation of Na,K-ATPase subunit expression between pronephric and metanephric kidneys. The pronephric kidney may therefore represent a simplified model to dissect the regulatory mechanisms underlying renal Na,K-ATPase subunit expression.

Effect of ethanol exposure on Xenopus embryo lipid composition.

Exposure to ethanol is known to alter cell membranes both chemically and biophysically; these effects may be related to the development of tolerance and physical dependence. In the present study, the effects of various concentrations of ethanol (1-250 mM) on the lipid composition after the first 6 days of Xenopus embryo development were investigated, using an in vitro fertilization technique. Lipid analysis revealed: (1) a decrease of the cholesterol/phospholipid molar ratio mainly derived from a higher content of phospholipids; (2) an increase of phospholipid unsaturated fatty acids, especially C20:4 and C20:5, with ethanol concentration of 150-250 mM; (3) a decrease of lipid-bound sialic acid with ethanol concentrations of > or =5 mM. These results underline that sialoglycoconjugates are a more sensitive target of alcohol in comparison with other lipid components. The cultured embryo method certainly represents a useful model for investigation of the direct effects of ethanol on lipid metabolism, excluding maternal interference which can lead to misinterpretation of data.

Nkx2-5 activity is essential for cardiomyogenesis.

The homeobox transcription factor tinman is essential for heart vessel formation in Drosophila. In contrast, mice lacking the murine homologue Nkx2-5 are defective in cardiac looping but not in cardiac myocyte development. The lack of an essential role for Nkx2-5 in cardiomyogenesis in mammalian systems is most likely the result of genetic redundancy with family members. In this study, we used a dominant negative mutant of Nkx2-5, created by fusing the repressor domain of engrailed 2 to the Nkx2-5 homeodomain, termed Nkx/EnR. Expression of Nkx/EnR inhibited Me(2)SO-induced cardiomyogenesis in P19 cells but not skeletal myogenesis. Nkx/EnR inhibited expression of cardiomyoblast markers, such as GATA-4 and MEF2C, but not of mesoderm markers, such as Brachyury T and Wnt5b, or of skeletal lineage markers, such as MyoD and Mox1. To identify the minimal region of Nkx2-5 that can trigger cardiomyogenesis, we analyzed the activity of various Nkx2-5 deletion mutants. The C-terminal domain was not necessary for the ability of Nkx2-5 to induce cardiomyogenesis and loss of this domain did not enhance myogenesis. Therefore, Nkx2-5 function is essential for commitment of mesoderm into the cardiac muscle lineage, and the N-terminal region, together with the homeodomain, is sufficient for cardiomyogenesis in P19 cells.

Differential Effect of Two Stathmin/Op18 Phosphorylation Mutants on Xenopus Embryo Development

Stathmin/Op18 destabilizes microtubules in vitro and regulates microtubule polymerization in vivo. Both a microtubule catastrophe-promoting activity and a tubulin sequestering activity were demonstrated for stathmin in vitro, and both could contribute to microtubule depolymerization in vivo. Stathmin activity can be turned down by extensive phosphorylation on its four phosphorylatable serines, and down-regulation of stathmin activity by phosphorylation is necessary for cells to proceed through mitosis. We show here that microinjection of a nonphosphorylatable Ser to Ala (4A) quadruple mutant in Xenopus two-cell stage embryos results in cell cleavage arrest in the injected blastomeres and aborted development, whereas injection of a pseudo-phosphorylated Ser to Glu quadruple mutant (4E) does not prevent normal development. Addition of these mutants to mitotic cytostatic factor-arrested extracts in which spindle assembly was induced led to a dramatic reduction of spindle size with 4A stathmin, and to a moderate increase with 4E stathmin, but both localized to spindle poles. Interestingly, the microtubule assembly-dependent phosphorylation of endogenous stathmin was abolished in the presence of 4A stathmin, but not of 4E stathmin. Altogether, this shows that the phosphorylation-mediated regulation of stathmin activity during the cell cycle is essential for early Xenopus embryonic development.

Changing Sensitivity at the Promoter

The extracellular ligand Wnt can activate multiple signaling pathways, the classical one being through the frizzled family of receptors, activating Dishevelled and glycogen synthase kinase 3, leading to the accumulation of β-catenin that enters the nucleus and regulates gene expression along with the transcription factors of Tcf/Lef family (see the Wnt/β-catenin pathway by Moon ). However, the cellular responsiveness to Wnt changes during development: an example is the loss of dorsalizing activity of Wnt signaling after the midblastula transition (MBT) in Xenopus . Darken and Wilson explored how these different cellular outcomes can arise by manipulating Wnt signaling at different times during the development of Xenopus embryos. Their results indicate that Wnt signaling both before and after MBT is mediated by the classic Wnt pathway, despite the loss of induction of early genes, such as samois and Xnr3 , at later stages of development. Expression of an activated Tcf-glucocorticoid fusion construct (which allowed controlled activation of the transcription factor) showed that the ability of TcF to increase the expression of the early genes is lost after the MBT, suggesting that the responsiveness of the late genes' promoters changes after the MBT. A more direct test of this was performed using the promoter of the samois gene fused to a luciferase reporter gene, which also demonstrated a loss of responsiveness to the Tcf-glucocorticoid fusion protein after MBT. Thus, in this instance, the cellular responsiveness is not altered by changing the signaling pathway, but by changes in the target promoters' ability to respond to the signal. R. Moon, Wnt/beta-catenin pathway, Science's STKE (Connections Map, as seen May 2001), http://stke.sciencemag.org/cgi/cm/CMP_5533. [Online Journal] R. S. Darken, P. A. Wilson, Axis induction by Wnt signaling: Target promoter responsiveness regulates competence. De v. Biol. 234 , 42-54 (2001). [Online Journal]

Xebf3 Is a Regulator of Neuronal Differentiation during Primary Neurogenesis in Xenopus

During primary neurogenesis in Xenopus, a cascade of helix–loop–helix (HLH) transcription factors regulates neuronal determination and differentiation. While XNeuroD functions at a late step in this cascade to regulate neuronal differentiation, the factors that carry out terminal differentiation are still unknown. We have isolated a new Xenopus member of the Ebf/Olf-1 family of HLH transcription factors, Xebf3, and provide evidence that, during primary neurogenesis, it regulates neuronal differentiation downstream of XNeuroD. In developing Xenopus embryos, Xebf3 is turned on in the three stripes of primary neurons at stage 15.5, after XNeuroD. In vitro, XEBF3 binds the EBF/OLF-1 binding site and functions as a transcriptional activator. When overexpressed, Xebf3 is able to induce ectopic neurons at neural plate stages and directly convert ectodermal cells into neurons in animal cap explants. Expression of Xebf3 can be activated by XNeuroD both in whole embryos and in animal caps, indicating that this new HLH factor might be regulated by XNeuroD. Furthermore, in animal caps, XNeuroD can activate Xebf3 in the absence of protein synthesis, suggesting that, in vitro, this regulation is direct. Similar to XNeuroD, but unlike Xebf2/Xcoe2, Xebf3 expression and function are insensitive to Delta/Notch-mediated lateral inhibition. In summary, we conclude that Xebf3 functions downstream of XNeuroD and is a regulator of neuronal differentiation in Xenopus.