Dorsal Mesoderm Induction Research Articles

NSrp70 is significant for embryonic growth and development, being a crucial factor for gastrulation and mesoderm induction

NSrp70 (nuclear speckle-related protein 70), a recently discovered protein and it belongs to the serine/arginine (SR) rich related protein family. NSrp70 is recognized as an important splicing factor comprising RNA recognition motif (RRM) and arginine/serine (RS)-like regions at the N- and C-terminus respectively, along with two coiled coil domains at each terminus. However, other functions of NSrp70 remain unelucidated. In this study, we investigated the role of NSrp70 in Xenopus embryogenesis and found that its maternal expression plays a critical role in embryonic development. Knockdown of NSrp70 resulted in dramatic reduction in the length of developing tadpoles and mild to severe malformation in Xenopus embryos. In addition, knockdown of NSrp70 resulted in an extremely short axis by blocking gastrulation and convergent extension. Further, animal cap assays along with activin A treatment revealed that NSrp70 is an essential factor for dorsal mesoderm induction as knockdown of NSrp70 caused a dramatic down-regulation of dorsal mesoderm specific genes and its loss significantly shortened the elongation region of animal caps. In conclusion, NSrp70 is crucial for early embryonic development, influencing gastrulation and mesoderm induction.

Blastopore formation and dorsal mesoderm induction are independent events in early Cynops embryogenesis.

The independent roles of blastopore formation and dorsal mesoderm induction in dorsal axis formation of the Cynops pyrrhogaster embryo were attempted to be clarified. The blastopore-forming (bottle) cells originated mainly from the progeny of the mid-dorsal C and/or D blastomeres of the 32-cell embryo, but were not defined to a fixed blastomere. It was confirmed that the isolated dorsal C and D blastomeres autonomously formed a blastopore. Ultraviolet-irradiated eggs formed an abnormal blastopore and then did not form a dorsal axis, although the lower dorsal marginal zone (LDMZ) still had dorsal mesoderm-inducing activity. Involution of the dorsal marginal zone was disturbed by the abnormal blastopore. These embryos were rescued by artificially facilitating involution of the dorsal marginal zone. Suramin-injected and nocodazole-treated blastulae did not have involution of the dorsal marginal zone, although the blastopore was formed. Neither embryos formed the dorsal axis. The dorsal mesoderm-inducing activity of the LDMZ in the nocodazole-treated gastrulae was still active. In contrast, the LDMZ of the suramin-injected embryos lost its dorsal mesoderm-inducing activity. bra expression was activated in the nocodazole-treated embryos but not in the suramin-injected embryos. The present study suggested that (i) the dorsal determinants consist of blastopore-forming and dorsal mesoderm-inducing factors, which are not always mutually dependent; (ii) both factors are activated during the late blastula stage; (iii) the dorsal marginal zone cannot specify to an organized notochord and muscle without the involution that blastopore formation leads to; and (iv) the localization of both factors in the same place is prerequisite for dorsal axis formation.

Hex is a transcriptional repressor that contributes to anterior identity and suppresses Spemann organiser function.

One of the earliest markers of anterior asymmetry in vertebrate embryos is the transcription factor Hex. We find that Hex is a transcriptional repressor that can be converted to an activator by fusing full length Hex to two copies of the minimal transcriptional activation domain of VP16 together with the flexible hinge region of the (lambda) repressor (Hex-(lambda)VP2). Retention of the entire Hex open reading frame allows one to examine Hex function without disrupting potential protein-protein interactions. Expression of Hex-(lambda)VP2 in Xenopus inhibits expression of the anterior marker Cerberus and results in anterior truncations. Such embryos have multiple notochords and disorganised muscle tissue. These effects can occur in a cell non-autonomous manner, suggesting that one role of wild-type Hex is to specify anterior structures by suppressing signals that promote dorsal mesoderm formation. In support of this idea, over-expression of wild-type Hex causes cell non-autonomous dorso-anteriorization, as well as cell autonomous suppression of dorsal mesoderm. Suppression of dorsal mesoderm by Hex is accompanied by the down-regulation of Goosecoid and Chordin, while induction of dorsal mesoderm by Hex-(lambda)VP2 results in activation of these genes. Transient transfection experiments in ES cells suggest that Goosecoid is a direct target of Hex. Together, our results support a model in which Hex suppresses organiser activity and defines anterior identity.

BMP regulates vegetal pole induction centres in early xenopus development.

Bone morphogenetic protein (BMP) plays an important role in mesoderm patterning in Xenopus. The ectopic expression of BMP-4 protein hyperventralizes embryos, whereas embryos expressing a BMP-2/4 dominant-negative receptor (DNR) are hyperdorsalized. Mesoderm is initially induced in the marginal zone by cells in the underlying vegetal pole. While much is known about BMP's expression and role in patterning the marginal zone, little is known about its early role in regulating vegetal mesoderm induction centre formation. The role of BMP in regulating formation of vegetal mesoderm inducing centres during early Xenopus development was examined. Ectopic BMP-4 expression in vegetal pole cells inhibited dorsal mesoderm induction but increased ventral mesoderm induction when recombined with animal cap ectoderm in Nieuwkoop explants. 32-cell embryos injected with BMP-4 RNA in the most vegetal blastomere tier were not hyperdorsalized by LiCl treatment. The ectopic expression of Smad or Mix.1 proteins in the vegetal pole also inhibited dorsal mesoderm induction in explants and embryos. Expression of the BMP 2/4 DNR in the vegetal pole increased dorsal mesoderm induction and inhibited ventral mesoderm induction in explants and embryos. These results support a role for BMP signalling in regulating ventral vegetal and dorsal vegetal mesoderm induction centre formation during early Xenopus development.

Smad proteins act in combination with synergistic and antagonistic regulators to target Dpp responses to the Drosophila mesoderm.

Dorsal mesoderm induction in arthropods and ventral mesoderm induction in vertebrates are closely related processes that involve signals of the BMP family. In Drosophila, induction of visceral mesoderm, dorsal muscles, and the heart by Dpp is, at least in part, effected through the transcriptional activation and function of the homeobox gene tinman in dorsal mesodermal cells during early embryogenesis. Here we present a functional dissection of a tinman enhancer that mediates the Dpp response. We provide evidence that mesoderm-specific induction of tinman requires the binding of both activators and repressors. Screens for binding factors yielded Tinman itself and the Smad4 homolog Medea. We show that the binding and synergistic activities of Smad and Tinman proteins are critical for mesodermal tinman induction, whereas repressor binding sites prevent induction in the dorsal ectoderm and amnioserosa. Thus, integration of positive and negative regulators on enhancers of target genes appears to be an important mechanism in tissue-specific induction by TGF-beta molecules.

Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos.

The Xenopus Vg1 gene, a TGFbeta superfamily member, is expressed as a maternal mRNA localized to prospective endoderm, and mature Vg1 protein can induce both endodermal and mesodermal markers in embryonic cells. Most previous work on embryonic inducers, including activin, BMPs and Vg1, has relied on ectopic expression to assay for gene function. Here we employ a mutant ligand approach to block Vg1 signaling in developing embryos. The results indicate that Vg1 expression is essential for normal endodermal development and the induction of dorsal mesoderm in vivo.

Regulation and function of tinman during dorsal mesoderm induction and heart specification in Drosophila

Regulation and function of tinman during dorsal mesoderm induction and heart specification in Drosophila

Regulation and function of tinman during dorsal mesoderm induction and heart specification in Drosophila.

The homeobox gene tinman plays a key role in the specification of Drosophila heart progenitors and the visceral mesoderm of the midgut, both of which arise at defined positions within dorsal areas of the mesoderm. Here, we show that in addition to the heart and midgut visceral mesoderm, tinman is also required for the specification of all dorsal body wall muscles. Thus it appears that the precursors of the heart, visceral musculature, and dorsal somatic muscles are all specified within the same broad domain of dorsal mesodermal tinman expression. Locally restricted activities of tinman are also observed during its early, general mesodermal expression, where tinman is required for the activation of the homeobox gene buttonless in precursors of the "dorsal median" (DM) glial cells along the ventral midline. These observations, together with others showing only mild effects of ectopic tinman expression on heart development, indicate that tinman function is obligatory, but not sufficient to determine individual tissues within the mesoderm. Therefore, we propose that tinman has a role in integrating positional information that is provided by intersecting domains of additional regulators and signals, which may include Wingless, Sloppy Paired, and Hedgehog in the dorsal mesoderm and EGF-signaling at the ventral midline. Previous studies have shown that Dpp acts as an inductive signal from dorsal ectodermal cells to induce tinman expression in the dorsal mesoderm, which, in turn, is needed for heart and visceral mesoderm formation. In the present report, we show that Thickveins, a type I receptor of Dpp, is essential for the transmission of Dpp signals into the mesoderm. Constitutive activity of Tkv in the entire mesoderm induces ectopic tinman expression in the ventral mesoderm, and this results in the ectopic formation of heart precursors in a defined area of the ventrolateral mesoderm. We further show that Screw, a second BMP2/4-related gene product, Tolloid, a BMP1-related protein, and the zinc finger-containing protein Schnurri, are required to allow full levels of tinman induction during this process. It is likely that some of these functional and regulatory properties of tinman are shared by tinman-related genes from vertebrates that have similarly important roles in embryonic heart development.

Modulation of Xenopus embryo mesoderm-specific gene expression and dorsoanterior patterning by receptors that activate the phosphatidylinositol cycle signal transduction pathway.

A role for the phosphatidylinositol (PI) cycle signal transduction pathway in Xenopus mesoderm induction has been revealed by observations of PI cycle activation coincident with this process, combined with the demonstration that Li+ (a PI cycle inhibitor) blocks this response and hyperdorsalizes mesoderm induction in intact embryos or augments growth factor-mediated induction in animal caps. It has been suggested that spatially restricted PI cycle activity in the marginal zone might modulate (but not, itself, activate) mesoderm induction. To better characterize the ability of PI cycle activity to modulate the pattern of mesoderm-specific gene expression elicited by mesoderm-inducing growth factors we have expressed in the embryo exogenous 5-hydroxytryptamine receptors that activate the PI cycle. In embryos, ventral expression and activation of these receptors during mesoderm induction are without obvious effect, whereas dorsal expression and activation yield dorsoanterior-deficient tadpoles. In animal caps induced with activin, simultaneous activation of exogenous 5-hydroxytryptamine receptors inhibits both convergent extension movements associated with dorsal mesoderm induction and the expression of goosecoid, a dorsal-specific gene, but is without effect on expression of a 149 generic mesodermal marker, Xbra. All of these effects of a 149 PI cycle-stimulating receptor are the opposites of those previously reported for the PI cycle inhibitor, Li+. PI cycle activity thus proves able to modulate the dorsal/ventral character of early mesodermal gene expression elicited by growth factor, suggesting a model for mesodermal patterning.

Competition between noggin and bone morphogenetic protein 4 activities may regulate dorsalization during Xenopus development.

Bone morphogenetic protein 4 (BMP-4) induces ventral mesoderm but represses dorsal mesoderm formation in Xenopus embryos. We show that BMP-4 inhibits two signaling pathways regulating dorsal mesoderm formation, the induction of dorsal mesoderm (Spemann organizer) and the dorsalization of ventral mesoderm. Ectopic expression of BMP-4 RNA reduces goosecoid and forkhead-1 transcription in whole embryos and in activin-treated animal cap explants. Embryos and animal caps overexpressing BMP-4 transcribe high levels of genes expressed in ventral mesoderm (Xbra, Xwnt-8, Xpo, Mix.1, XMyoD). The Spemann organizer is ventralized in these embryos; abnormally high levels of Xwnt-8 mRNA and low levels of goosecoid mRNA are detected in the organizer. In addition, the organizer loses the ability to dorsalize neighboring ventral marginal zone to muscle. Overexpression of BMP-4 in ventral mesoderm inhibits its response to dorsalization signals. Ventral marginal zone explants ectopically expressing BMP-4 form less muscle when treated with soluble noggin protein or when juxtaposed to a normal Spemann organizer in comparison to control explants. Endogenous BMP-4 transcripts are downregulated in ventral marginal zone explants dorsalized by noggin, in contrast to untreated explants. Thus, while BMP-4 inhibits noggin protein activity, noggin downregulates BMP-4 expression by dorsalizing ventral marginal zone to muscle. Noggin and BMP-4 activities may control the lateral extent of dorsalization within the marginal zone. Competition between these two molecules may determine the final degree of muscle formation in the marginal zone, thus defining the border between dorsolateral and ventral mesoderm.

Functional Conservation of the Wnt Signaling Pathway Revealed by Ectopic Expression of Drosophila dishevelled in Xenopus

Wnt genes encode secreted growth factors that exhibit potent effects on both embryonic and postembryonic development in vertebrates and invertebrates. Recently, the dishevelled (dsh), shaggy/zeste-white 3, and armadillo genes have been shown to participate in Wnt ( wingless; wg) signaling in Drosophila. Vertebrate genes that have sequence similarities to all of these Drosophila genes have been identified. To determine whether these structurally conserved components of insect wg signaling represent a functionally conserved Wnt signaling pathway in vertebrates, we investigated the role of Drosophila dsh in Xenopus Wnt signaling. Xenopus embryos ectopically injected with Drosophila dsh mRNA developed duplicated axes similar to those seen in embryos injected with Wnt mRNAs. The involvement of dsh function in the Wnt signaling pathway in Xenopus was demonstrated using two assays which are specifically sensitive to Wnt signaling: synergistic induction of dorsal mesoderm with bFGF and the specific induction of a Wnt-responsive reporter gene. These findings support the notion that the intracellular response to the Wnt signal has been conserved during evolution to such an extent that its components may be interchanged between distantly related species.

Induction of dorsal mesoderm by soluble, mature Vg1 protein

Mesoderm induction during Xenopus development has been extensively studied, and two members of the transforming growth factor-beta family, activin beta B and Vg1, have emerged as candidates for a natural inducer of dorsal mesoderm. Heretofore, analysis of Vg1 activity has relied on injection of hybrid Vg1 mRNAs, which have not been shown to direct efficient secretion of ligand and, therefore, the mechanism of mesoderm induction by processed Vg1 protein is unclear. This report describes injection of Xenopus oocytes with a chimeric activin-Vg1 mRNA, encoding the pro-region of activin beta B fused to the mature region of Vg1, resulting in the processing and secretion of mature Vg1. Treatment of animal pole explants with mature Vg1 protein resulted in differentiation of dorsal, but not ventral, mesodermal tissues and dose-dependent activation of both dorsal and ventrolateral mesodermal markers. At high doses, mature Vg1 induced formation of 'embryoids' with a rudimentary axial pattern, head structures including eyes and a functional neuromuscular system. Furthermore, truncated forms of the activin and FGF receptors, which block mesoderm induction in the intact embryo, fully inhibited mature Vg1 activity. To examine the mechanism of inhibition, we have performed receptor-binding assays with radiolabeled Vg1. Finally, follistatin, a specific inhibitor of activin beta B which is shown not to block endogenous dorsal mesoderm induction, failed to inhibit Vg1. The results support a role for endogenous Vg1 in dorsal mesoderm induction during Xenopus development.

Overexpression of cadherins and underexpression of β-catenin inhibit dorsal mesoderm induction in early Xenopus embryos

The cadherin-catenin complex has an important role in cell-cell adhesion and may also function in signaling pathways. We report that overexpression of three cadherin types in Xenopus embryos causes them to develop with reduced dorsal axial structures. The same phenotype is produced in embryos that have been depleted of maternal β-catenin protein by an antisense oligodeoxynucleotide complementary to β-catenin mRNA. They show an inhibition in the expression of dorsal mesodermal markers MyoD and goosecoid, but not of ventral and general mesodermal markers. They lack notochords, somites, and neural tubes and are defective in dorsal mesodermal signaling in Nieuwkoop assays. The phenotype can be rescued by the injection of β-catenin mRNA and not by the injection of Xwnt-8 mRNA. These results show that β-catenin has an important role in dorsal mesoderm induction. They directly demonstrate the activity of a maternal mRNA in axis specification.

Interaction of Wnt and activin in dorsal mesoderm induction in Xenopus

Both the activin and Wnt families of peptide growth factors are capable of inducing dorsal mesoderm in Xenopus embryos. Presumptive ventral ectoderm cells isolated from embryos injected with Xwnt8 mRNA were cultured in the presence of activin A to study the possible interactions between these two classes of signaling proteins. We find that overexpression of Xwnt8 RNA alters the response of ventral ectoderm to activin such that ventral explants differentiate dorsoanterior structures including notochord and eyes. This response is similar to the response of dorsal ectoderm to activin alone. When embryos are irradiated with uv light to inhibit dorsal axis formation, ectodermal explants differentiate notochord when they are induced by a combination of both signaling factors, but not when cells receive only one inducing signal (activin or Xwnt8). This result is further supported by the observation that goosecoid ( gsc) mRNA, an early marker for dorsal mesoderm, is expressed in these explants only when they are injected with Xwnt8 mRNA followed by exposure to activin. Early morphogenetic movements of the induced cells and activation of muscle-specific actin and Brachyury ( Xbra) genes also reveal a cooperation of activin A and Xwnt8 in mesoderm induction.

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.

The induction of the primordial germ cells in the urodeles.

Xenoplastic recombinates of animal ectodermal caps with the ventral vegetative yolk mass of blastulae of stage 81/2-83/4 ofA. mexicanum, T. alpestris, T. cristatus carnifex andP. waltlii have demonstrated unambiguously that in the urodeles the primordial germ cells-together with other ventro-caudal mesodermal structures-develop from the animal ectodermal moiety of the blastula under an inductive influence emanating from the ventral vegetative yolk mass. Similar recombinates of3H-labeled and unlabeled ectodermal and endodermal components fully support this conclusion.Recombinates of the ventral vegetative yolk mass with different regions of the animal ectodermal hemisphere show that primordial germ cells can be formed by any region of the animal ectodermal hemisphere, including those regions which in normal development will never form them. The number of primordial germ cells formed differs significantly among the various regions, that of the ventral peripheral region being the highest and that of the central, animal region the lowest. The capacity for primordial germ cell formation shows two increasing gradients, one animal-vegetative and the other dorse-ventral (in the peripheral zone). Although accurate measurements could not be made, there seems to be a relation between the number of primordial germ cells formed and the amount of ventro-caudal mesoderm induced.The experiments, moreover, show that notochord differentiation largely or entirely suppresses primordial germ cell formation. Notochord differentiation shows a similar animalvegetative, but an opposite ventro-dorsal increase in frequency (in the peripheral zone) as compared with the capacity for primordial germ cell formation. The notochord-forming gradient in the peripheral regions is mainly due to the inductive action already exerted by the dorsal vegetative yolk mass in the intact blastula prior to isolation and recombination (see control explants). The ventro-dorsal decline in primordial germ cell formation in the peripheral regions is very probably due only to the inhibition of primordial germ cell formation by notochord differentiation (as an expression of dorsal mesoderm induction). Therefore, in the animal ectodermal moiety of the blastula there exists only an animal-vegetative gradient in mesodermal competence.These results make it very likely that in urodeles the primordial germ cells do not arise from predetermined elements such as those demonstrated in anurans, but develop from common, totipotent animal ectodermal cells. The discrepancy in the mode of origin of the primordial germ cells between anurans and urodeles could be due only to pronounced differences in the time of appearance of the germinal cytoplasm (in anurans during oogenesis, in urodeles possibly during determination of the primordial germ cells within the ventro-caudal mesoderm).The differences in site and mode of origin of the primordial germ cells between urodeles and anurans favor a dual phylogenetic origin of the two groups.