Competence modifiers synergize with growth factors during mesoderm induction and patterning in Xenopus.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
R T Moon
Publications and source records attributed to R T Moon.
Purification and characterization of recombinant Xenopus poly(A)(+)-binding protein expressed in a baculovirus system.
The poly(A)(+)-binding protein (PABP) is a highly conserved protein that binds to the poly(A)+ tail of mRNAs. PABP has been shown to regulate message stability and translational efficiency, yet the mechanisms remain unknown. To facilitate further dissection of the functions of this protein, we have expressed and purified Xenopus PABP using a baculovirus expression system. At 48 h after infection of insect Spodoptera frugiperda (Sf9) cells with recombinant virus, approx. 3% of cell protein was PABP. Purification of PABP was achieved by affinity chromatography on poly(A)(+)-Sepharose. The purified protein was indistinguishable from Xenopus PABP with respect to its immunoreactivity and electrophoretic mobility. Furthermore, the recombinant PABP was expressed and purified as a functional protein as indicated by its ability to bind to poly(A)(+)-Sepharose and its ability to enhance the translation of adenylated messages in vitro. By comparing protein extracts from various developmental stages of Xenopus embryos with known amounts of purified PABP, we determined the amount of PABP per embryo. This analysis suggested that there is less than one PABP molecule available per PABP-binding site at early stages of development, and only a slight excess of PABP at later stages.
Animals↗
Protein kinase C isozymes have distinct roles in neural induction and competence in Xenopus.
The restricted ability of embryonic tissue to respond to inductive signals is controlled by a poorly understood phenomenon, termed competence. In Xenopus, dorsal ectoderm is more competent than ventral ectoderm to become induced to neural tissue. We tested whether the Xenopus protein kinase C (PKC) isozymes alpha and beta have a role in neural induction and competence. We found that PKC alpha is predominantly localized in dorsal ectoderm, whereas PKC beta is uniformly distributed. Overexpression of PKC beta conveys a higher propensity for neural differentiation to both dorsal and ventral ectoderm, but their difference in competence remains. However, ectopic expression of PKC alpha elevates the level of neural competence of ventral ectoderm to that of dorsal ectoderm. These data indicate that different PKC isozymes have distinct roles in mediating both neural induction and competence.
Animals↗
Xwnt-8 modifies the character of mesoderm induced by bFGF in isolated Xenopus ectoderm.
In Xenopus, growth factors of the TGF-beta, FGF and Wnt oncogene families have been proposed to play a role in generating embryonic pattern. In this paper we examine potential interactions between the bFGF and Xwnt-8 signaling pathways in the induction and dorsal-ventral patterning of mesoderm. Injection of Xwnt-8 mRNA into 2-cell Xenopus embryos does not induce mesoderm formation in animal cap ectoderm isolated from these embryos at the blastula stage, but alters the response of this tissue to mesoderm induction by bFGF. While animal cap explants isolated from non-injected embryos differentiate to form ventral types of mesoderm and muscle in response to bFGF, explants from Xwnt-8 injected embryos form dorsal mesodermal and neural tissues in response to the same concentration of bFGF, even if the ectoderm is isolated from the prospective ventral sides of embryos or from UV-ventralized animals. Our results support a model whereby dorso-ventral mesodermal patterning can be attained by a single mesoderm inducing agent, possibly bFGF, which is uniformly distributed across the prospective dorsal-ventral axis, and which acts in concert with a dorsally localized signal, possibly a Wnt protein, which either alters the response of ectoderm to induction or modifies the character of mesoderm after its induction.
Animals↗
The armadillo homologs beta-catenin and plakoglobin are differentially expressed during early development of Xenopus laevis.
Plakoglobin and beta-catenin are cytoplasmic proteins associated with the intracellular plaques of cell adhesive junctions. While plakoglobin is present in both adherens junctions and desmosomes, beta-catenin is associated with the cadherins that accumulate only in adherens junctions. Both beta-catenin and plakoglobin are homologs of armadillo, a Drosophila segment polarity gene that is considered to be in the wingless signaling pathway. We have characterized the expression and distribution of both plakoglobin and beta-catenin in Xenopus embryos. As shown by RNA blot analysis, beta-catenin and plakoglobin transcripts are present in fertilized eggs and in embryos through to tadpole stage. Whole-mount in situ hybridization indicates that both genes are expressed in the dorsal ectoderm and mesoderm of tailbud- and tadpole-stage embryos and that beta-catenin is expressed in the midbrain. Both plakoglobin and beta-catenin polypeptides are present during early Xenopus development; however, differences exist in the timing of maximal expression. Plakoglobin is present in the fertilized egg, increases in abundance by neurula stage, then declines at the tailbud and tadpole stages. beta-Catenin, recognized by an anti-arm antibody, is also present in the fertilized egg and in blastula-stage embryos. However, beta-catenin continues to be detected at the neurula, tailbud, and tadpole stages when levels of plakoglobin decline. The presence of multiple homologs of armadillo in Xenopus embryos and the differences in their patterns of expression suggest distinct roles for these proteins in processes affected by cell adhesion.
Amino Acid Sequence↗
Ectopic induction of dorsal mesoderm by overexpression of Xwnt-8 elevates the neural competence of Xenopus ectoderm.
The ectoderm of early Xenopus gastrula is competent to become induced to neural tissue, but dorsal ectoderm is more neural competent than ventral ectoderm. It is a tenable, but as yet untested possibility that the higher neural competence of dorsal gastrula ectoderm is dependent on the presence of the dorsal mesoderm. To test this hypothesis we overexpressed Xwnt-8 in order to ectopically induce dorsal mesoderm in the ventral side of the embryo. We found that this elevated the level of neural competence of ventral ectoderm to that of dorsal ectoderm. The effect of Xwnt-8 on neural competence of ventral ectoderm was strictly correlated with its ability to enhance the amount of dorsal structures. The data indicate that the presence of dorsal mesoderm is a prerequisite for establishing the differences in neural competence between gastrula dorsal and ventral ectoderm.
Animals↗
Distinct effects of ectopic expression of Wnt-1, activin B, and bFGF on gap junctional permeability in 32-cell Xenopus embryos.
A polarity in gap junctional permeability normally exists in 32-cell stage Xenopus embryos, in that dorsal cells are relatively more coupled than ventral cells, as measured by transfer of Lucifer yellow dye. The current study extends our analysis of whether gap junctional permeability at this stage can be modulated by secreted factors, and whether the polarity in gap junctional permeability correlates with the effects of ectopic expression of these secreted factors on the subsequent phenotype of the developing embryo. Following ectopic expression of activin B or Wnt-1, but not bFGF, the transfer of Lucifer yellow between ventral animal pole cells is detected in a greater percentage of 32-cell stage embryos. This increased incidence of dye transfer between ventral cells correlates with axial duplications later in development. However, there are differences in the extent of Lucifer yellow transfer between animal and vegetal hemisphere blastomeres which is dependent on whether activin B or Wnt-1 RNA had previously been injected. These results suggest that enhanced gap junctional permeability between ventral cells of 32-cell Xenopus embryos correlates with subsequent defects in the dorsoventral axis, although there are at present no direct data demonstrating a role for gap junctions in establishment or maintenance of this axis. Moreover, while both activin B and bFGF are mesoderm-inducing growth factors, only activin B has effects on gap junctional permeability in 32-cell embryos following ectopic expression, demonstrating an interesting difference in physiological responses to expression of these factors.
Activins↗
Analysis of Xwnt-4 in embryos of Xenopus laevis: a Wnt family member expressed in the brain and floor plate.
This study characterizes the temporal and spatial expression during early Xenopus development of Xwnt-4, a member of the Wnt gene family. The Xwnt-4 protein contains all of the sequence motifs that are hallmarks of the Wnt gene family and is 84% identical to the mouse homolog, Wnt-4. The highest level of Xwnt-4 expression occurs during the early neurula stage of development although its expression persists throughout embryogenesis and can be found in the adult testis, brain and epithelium. Consistent with its localization to head and dorsal regions of microdissected embryos, the expression of Xwnt-4 is enhanced in anterodorsalized embryos resulting from treatment with LiCl, and the expression of Xwnt-4 is suppressed in UV-ventralized embryos that lack anterior neural tissue. These results suggested that expression of Xwnt-4 is dependent on the induction of neural tissue. This idea was tested using induction experiments with dorsal or ventral ectoderm from a stage 10 embryo, recombined with dorsal marginal zone mesoderm from the same embryo. Recombinant tissue and ectoderm alone were cultured until stage 14, when Xwnt-4 expression was assayed using Northern analysis. In the recombinant assay, Xwnt-4 expression does not occur in the uninduced ectoderm but is expressed in both the dorsal and ventral recombinants. Xwnt-4 expression in neural ectoderm was confirmed in isolated, induced neural ectoderm, dissected away from the dorsal mesoderm, in a stage 12.5 embryo. Whole-mount in situ hybridization confirmed the dissection studies and demonstrated that Xwnt-4 transcripts are expressed in the dorsal midline of the midbrain, hindbrain and the floor plate of the neural tube. Collectively, the data indicate that Xwnt-4 is a unique member of the Wnt family whose expression is dependent on neural induction. The specific pattern of expression following neural induction suggests that Xwnt-4 plays a role in the early patterning events responsible in the formation of the nervous system in Xenopus.
Amino Acid Sequence↗
Expression and potential functions of G-protein alpha subunits in embryos of Xenopus laevis.
During early embryonic development, many inductive interactions between tissues depend on signal transduction processes. We began to test the possibility that G-proteins participate in the signal transduction pathways that mediate neural induction. The expression during Xenopus development of three G alpha subunits, G alpha 0, G alpha i-1 and G alpha s-1, was characterized. The three maternally expressed genes showed different expression patterns during early development. Whole-mount in situ hybridization revealed that all three genes were expressed almost exclusively in the gastrula ectoderm and predominantly in the neuroectoderm in the neurula embryo. In order to investigate the involvement of these proteins in neural induction, we overexpressed the G-protein alpha subunits by injecting the G alpha mRNAs into fertilized eggs. Overexpression of G alpha s-1 increased the ability of gastrula ectoderm to become induced to neural tissue approximately four-fold. Overexpression of G alpha 0 and G alpha i-1 had less pronounced effects on neural competence, and inhibition of the G alpha 0 and G alpha i-1 proteins by pertussis toxin did not change the neural competence of the exposed gastrula ectoderm. Overexpression of the G alpha 0 and G alpha i-1 genes did, however, inhibit the normal disappearance of the blastocoel during gastrulation, suggesting a role for these G-proteins in regulating this process. The data also suggest a specific role for the G alpha s subunit in mediating the initial phases of neural induction.
Animals↗
Synergistic principles of development: overlapping patterning systems in Xenopus mesoderm induction.
The first inductive event in Xenopus development establishes the mesoderm at the equator of the developing embryo. As part of this process, the dorsal-ventral and anterior-posterior axes of the embryo are initially established. A number of signalling molecules which may play a role in mesodermal induction and patterning have been identified in the last several years, including members of the FGF, TGF-beta and Wnt gene families. A variety of experiments, using either purified factors or injection of RNA encoding these factors, have added to the wealth of classical embryological experimental data collected over the last century. We have synthesized some recent results with the classical data to provide a framework for examining the process of mesoderm induction, and to formulate putative roles for some of the different factors. We incorporate these ideas into a working model of mesoderm induction that provides a basis for future experimental directions. Finally, we suggest that mesoderm induction may not be a discrete set of well separated events, but instead may be a process involving partially overlapping signals that produce the same pattern.
Animals↗
Cloning and developmental expression in Xenopus laevis of seven additional members of the Wnt family.
Degenerate oligonucleotide primers encoding highly conserved regions of Wnt-related proteins were used with the polymerase chain reaction (PCR) to amplify cDNA derived from Xenopus laevis embryos. cDNA sequences partially encoding seven additional members of the Xwnt gene family were isolated using this strategy. These cDNAs have been given the designation Xwnt-2, Xwnt-6, Xwnt-7A, Xwnt-7B, Xwnt-7C, Xwnt-8B and Xwnt-10 based on their amino acid identity with previously described Wnts. With regard to the timing of expression of these Xwnts during embryonic development, Xwnt-2, the least abundant transcript, was first detected during the neurula stage, while Xwnt-8B transcripts were first detected at the gastrula stage, and decreased by the tailbud stage. Multiple transcripts of Xwnt-6 were detected at varied times during development beginning at the gastrula stage. In contrast, Xwnt-7A, -7B and -10 transcripts were not detected until the tailbud stage. With regard to expression in adult tissues, Xwnt-6, -7A, -7B, -8B and -10 were all expressed abundantly in the brain, and to a lesser extent in a variety of other tissues. Whole-mount in situ hybridization was then employed to monitor the spatial expression of selected Xwnts. Xwnt-7A and -10 transcripts were detected in distinct areas of the developing brain of tailbud-stage embryos. The temporal and spatial differences in expression suggest different roles for these new Xwnt family members in Xenopus development.
Amino Acid Sequence↗
Injected Wnt RNA induces a complete body axis in Xenopus embryos.
Studies in Xenopus have shown that growth factors of the TGF beta and Wnt oncogene families can mimic aspects of dorsal axis formation. Here we directly compare the inductive properties of two Wnt proteins by injecting synthetic mRNA into developing embryos. The results show that Wnt-1 and Xwnt-8 can induce a new and complete dorsal axis and can rescue the development of axis-deficient, UV-irradiated embryos. In contrast, activin mRNA injection induces only a partial dorsal axis that lacks anterior structures. These studies demonstrate that the mechanism of Wnt-induced axis duplication results from the creation of an independent Spemann organizer. The relationship between the properties of the endogenous dorsal inducer and the effects of Wnts and activins is discussed.
Activins↗
Identification of a calcium-dependent calmodulin-binding domain in Xenopus membrane skeleton protein 4.1.
Xenopus membrane skeleton protein 4.1 is expressed constitutively during embryonic development and accumulates to high levels within the retina during normal morphogenesis. There exists a high degree of amino acid identity between Xenopus protein 4.1 and human protein 4.1, suggesting that the mechanisms known to modulate the function(s) of human protein 4.1 may also serve to regulate Xenopus protein 4.1. Calmodulin (CaM) is one regulatory protein known to affect membrane-cytoskeletal interactions. An in vitro binding assay was used to test the ability of Xenopus protein 4.1 to bind CaM. Two independent approaches, involving protein 4.1 synthesized in vitro from synthetic RNA or a partial length protein 4.1 fusion protein expressed in Escherichia coli, demonstrate calcium-dependent, CaM binding. Both approaches demonstrate that the CaM-binding site is within the amino-terminal region of Xenopus protein 4.1. Results of this calmodulin binding activity suggest a possible regulatory mechanism by which calcium and calmodulin may affect the function of protein 4.1 during development.
Amino Acid Sequence↗
Effect of wnt-1 and related proteins on gap junctional communication in Xenopus embryos.
The proto-oncogene wnt-1 (previously referred to as int-1) is thought to be important in embryonic pattern formation although its mechanisms of action are unknown. Premature and increased expression of the Wnt-1 protein, achieved by injection of synthetic wnt-1 RNA into fertilized Xenopus eggs, enhanced gap junctional communication between ventral cells of the developing embryo. This result is consistent with the hypothesis that Wnt proteins activate a receptor-mediated signal transduction pathway and that gap junctional communication can be a target of this pathway. The effects of two Wnt-1-related proteins on gap junctional communication were also investigated: overexpression of Xwnt-8 increased gap junctional coupling in a manner similar to Wnt-1, whereas Xwnt-5A did not. These findings are consistent with the existence of multiple receptors for Wnt proteins.
Animals↗
Isolation of cDNAs partially encoding four Xenopus Wnt-1/int-1-related proteins and characterization of their transient expression during embryonic development.
To begin to study the functions of the Wnt-1/int-1 gene family during vertebrate development, we have isolated four Xenopus laevis cDNAs encoding the partial sequence of proteins homologous to Wnt-1/int-1. Xwnt-3, Xwnt-4, Xwnt-5A, and Xwnt-8 demonstrate between 35 and 50% amino acid identity with X. laevis Wnt-1/int-1 and most cysteine residues are conserved. Xwnt-4 and Xwnt-3 transcripts are detected only during the neurula through tadpole stages of development. Expression of Xwnt-8 is observable during gastrulation, declines during neurulation, and is undetectable by the tadpole stage of development. Xwnt-5A transcripts are most prevalent in RNA from oocytes and tadpoles, although low level expression is detected at all stages examined. The temporal changes in expression of these transcripts imply a unique role for each Xwnt during embryogenesis.
Amino Acid Sequence↗
Histological preparation of Xenopus laevis oocytes and embryos.
Explore the source record for details and available documents.
Animals↗
Xwnt-8, a Xenopus Wnt-1/int-1-related gene responsive to mesoderm-inducing growth factors, may play a role in ventral mesodermal patterning during embryogenesis.
In amphibian embryos, formation of the basic body plan depends on positional differences in the mesoderm. Although peptide growth factors involved in mesoderm induction have tentatively been identified, additional signals are required to generate pattern in this tissue. We have isolated a Xenopus cDNA for a Wnt-1 related gene, designated Xwnt-8, which is activated in response to mesoderm-inducing growth factors. Xwnt-8 transcripts are transiently expressed, being most abundant during gastrulation at which time expression is confined primarily to ventral mesodermal cells. Embryos dorsoanteriorized by exposure to lithium exhibit greatly reduced levels of Xwnt-8 mRNA, supporting a correlation between Xwnt-8 expression and a ventral mesodermal cell fate. Surprisingly, ectopic expression of Xwnt-8 in embryos causes a dorsoanterior-enhanced phenotype. These findings suggest that Xwnt-8 may be a secondary signalling agent which is produced in response to mesoderm-inducing factors and is involved in the early steps of mesodermal patterning.
Animals↗
Membrane skeleton protein 4.1 in inner segments of retinal cones.
Retinal cone photoreceptors are polarized cells with discrete functional compartments. Little is known of the membrane-skeleton components in cones responsible for the maintenance of cellular morphology and compartmentalization of integral membrane proteins. The authors describe a high density of the membrane-skeleton protein 4.1 in the myoid region of cones in retinas of developing and adult frogs, and adult lizards, turtles, and chicks. In the developing retina, a discrete locus of protein 4.1 is first detected by immunocytochemistry in the cone myoid after the completion of photoreceptor synaptogenesis. Protein 4.1 immunoreactivity expands to line the plasma membrane of the myoid after cones attain their adult proportions. Nonerythroid alpha-spectrin and protein 4.1 colocalize in the cone myoid, and both are in close proximity to long bundles of f-actin that traverse the myoid and extend from the cone outer segment to the external limiting membrane. By analogy to the erythrocyte, where protein 4.1 modulates the interaction of spectrin and actin to regulate cell shape, the authors propose that protein 4.1 interacts with nonerythroid alpha-spectrin and f-actin in cones and plays a role in the maintenance of cone inner segment morphology.
Actins↗