Xenopus laevis POU91 protein, an Oct3/4 homologue, regulates competence transitions from mesoderm to neural cell fates

Cellular competence is defined as a cell's ability to respond to signaling cues as a function of time. In Xenopus laevis, cellular responsiveness to fibroblast growth factor (FGF) changes during development. At blastula stages, FGF induces mesoderm, but at gastrula stages FGF regulates neuroectoderm formation. A Xenopus Oct3/4 homologue gene, XLPOU91, regulates mesoderm to neuroectoderm transitions. Ectopic XLPOU91 expression in Xenopus embryos inhibits FGF induction of Brachyury (Xbra), eliminating mesoderm, whereas neural induction is unaffected. XLPOU91 knockdown induces high levels of Xbra expression, with blastopore closure being delayed to later neurula stages. In morphant ectoderm explants, mesoderm responsiveness to FGF is extended from blastula to gastrula stages. The initial expression of mesoderm and endoderm markers is normal, but neural induction is abolished. Churchill (chch) and Sip1, two genes regulating neural competence, are not expressed in XLPOU91 morphant embryos. Ectopic Sip1 or chch expression rescues the morphant phenotype. Thus, XLPOU91 epistatically lies upstream of chch/Sip1 gene expression, regulating the competence transition that is critical for neural induction. In the absence of XLPOU91 activity, the cues driving proper embryonic cell fates are lost.     

Expression of activated MAP kinase in Xenopus laevis embryos: evaluating the roles of FGF and other signaling pathways in early induction and patterning

FGF signaling has been implicated in germ layer formation and axial determination. An antibody specific for the activated form of mitogen-activated protein kinase (MAPK) was used to monitor FGF signaling in vivo during early Xenopus development. Activation of MAPK in young embryos is abolished by injection of a dominant negative FGF receptor (XFD) RNA, suggesting that MAPK is activated primarily by FGF in this context. A transition from cytoplasmic to nuclear localization of activated MAPK occurs in morula/blastula stage embryo animal and marginal zones coinciding with the proposed onset of mesodermal competence. Activated MAPK delineates the region of the dorsal marginal zone before blastopore formation and persists in this region during gastrulation, indicating an early role for FGF signaling in dorsal mesoderm. Activated MAPK was also found in posterior neural tissue from late gastrulation onward. Inhibition of FGF signaling does not block posterior neural gene expression (HoxB9) or activation of MAPK; however, inhibition of FGF signaling does cause a statistically significant decrease in the level of activated MAPK. These results point toward the involvement of other receptor tyrosine kinase signaling pathways in posterior neural patterning.     

Antagonistic role of XESR1 and XESR5 in mesoderm formation in Xenopus laevis

The notch signaling pathway is widely conserved from vertebrates to invertebrates and mediates the specification of numerous cell fates during developmental processes. In the Xenopus gastrula embryo, Xdelta1, one of the Notch ligands, is expressed in the prospective mesoderm prior to Xbra expression. Here, we examined the role of Notch signaling in mesoderm formation. Embryos injected with Xdelta1 morpholino oligo DNA showed a severe gastrulation defect and suppression of Xbra expression, which were completely rescued by co-injection with the active form of Notch. In order to fully understand the role of Notch signaling, we examined the expression of the Notch target genes XESR1 and XESR5. RT-PCR and whole-mount in situ hybridization analyses showed that XESR5 was highly expressed in the marginal zone of the early gastrula embryo, whereas expression of XESR1 was not detected. Animal cap assays indicated that expression of XESR5 was not induced by Notch signaling but by nodal signaling. To clarify the role of XESR5 in the gastrula embryo, a dominant negative form of XESR5 was injected into the prospective mesoderm. The truncated form of XESR5 induced the ectopic expression of XESR1, which caused a decrease in Xbra expression and defective gastrulation. In contrast, the truncated form of XESR1 caused an upregulation of XESR5 resulting in an increase in Xbra expression. The antagonistic effect of XESR1 and XESR5 suggests a dual regulation in which XESR5 produces a competent area for mesoderm formation by suppressing the gene expression of XESR1, while XESR1 sharpens the boundary of Xbra expression.     

Developmental expression of the Xenopus int-2 (FGF-3) gene: activation by mesodermal and neural induction.

We have used a probe specific for the Xenopus homologue of the mammalian proto-oncogene int-2 (FGF-3) to examine the temporal and spatial expression pattern of the gene during Xenopus development. int-2 is expressed from just before the onset of gastrulation through to prelarval stages. In the early gastrula, it is expressed around the blastopore lip. This is maintained in the posterior third of the prospective mesoderm and neuroectoderm in the neurula. A second expression domain in the anterior third of the neuroectoderm alone appears in the late gastrula, which later resolves into the optic vesicles, hypothalamus and midbrain-hindbrain junction region. Further domains of expression arise in tailbud to prelarval embryos, including the stomodeal mesenchyme, the endoderm of the pharyngeal pouches and the cranial ganglia flanking the otocyst. It is shown, by treatment of blastula ectoderm with bFGF and activin, that int-2 can be expressed in response to mesoderm induction. By heterotypic grafting of gastrula ectoderm into axolotl neural plate, we have also demonstrated that int-2 can be expressed in response to neural induction. These results suggest that int-2 has multiple functions in development, including an early role in patterning of the anteroposterior body axis and a later role in the development of the tail, brain-derived structures and other epithelia.     

Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction.

The Brachyury (T) gene is required for mesoderm formation in the mouse. In this paper we describe the cloning and expression of a Xenopus homolog of Brachyury, Xbra. As with Brachyury in the mouse, Xbra is expressed in presumptive mesodermal cells around the blastopore, and then in the notochord. We show that expression of Xbra occurs as a result of mesoderm induction in Xenopus, both in response to the natural signal and in response to the mesoderm-inducing factors activin A and basic FGF. Expression of Xbra in response to these factors is rapid, and will occur in dispersed cells and in the presence of a protein synthesis inhibitor, indicating that this is an "immediate-early" response to mesoderm induction.     

FGF signaling restricts the primary blood islands to ventral mesoderm

According to the three-signal model of mesoderm patterning in Xenopus, all mesoderm, with the exception of the Spemann organizer, is originally specified as ventral type, such as lateral plate and primary blood islands. It is proposed that the blood islands become restricted to the ventralmost mesoderm because they are not exposed to the BMP-inhibiting activity of the Spemann organizer. We present evidence here that, contrary to predictions of this model, the blood islands remain ventrally restricted even in the absence of Spemann organizer signaling. We further observed that inhibition of FGF signaling with a dominant negative receptor resulted in the expansion of the blood island-forming territory with a concomitant loss of somite. The requirement for FGF signaling in specifying somite versus blood island territories was observed as early as midgastrulation. The nonoverlapping expression domains of Xnr-2 and Xbra in the gastrula marginal zone appear to mark presumptive blood island and somite, respectively. Inhibition of FGF signaling with dominant negative receptor leads to an expansion of Xnr-2 expression and to a corresponding reduction in Xbra expression. On the other hand, we found no evidence that manipulation of BMP signaling, either positively or negatively, altered the expression domains of Xnr-2 and Xbra. These results suggest that FGF signaling, rather than BMP-inhibiting activity, is essential for restriction of the ventral blood islands to ventral mesoderm.     

derrière: a TGF-beta family member required for posterior development in Xenopus

TGF-beta signaling plays a key role in induction of the Xenopus mesoderm and endoderm. Using a yeast-based selection scheme, we isolated derrière, a novel TGF-beta family member that is closely related to Vg1 and that is required for normal mesodermal patterning, particularly in posterior regions of the embryo. Unlike Vg1, derrière is expressed zygotically, with RNA localized to the future endoderm and mesoderm by late blastula, and to the posterior mesoderm by mid-gastrula. The derrière expression pattern appears to be identical to the zygotic expression domain of VegT (Xombi, Brat, Antipodean), and can be activated by VegT as well as fibroblast growth factor (FGF). In turn, derrière activates expression of itself, VegT and eFGF, suggesting that a regulatory loop exists between these genes. derrière is a potent mesoderm and endoderm inducer, acting in a dose-dependent fashion. When misexpressed ventrally, derrière induces a secondary axis lacking a head, an effect that is due to dorsalization of the ventral marginal zone. When misexpressed dorsally, derrière suppresses head formation. derrière can also posteriorize neurectoderm, but appears to do so indirectly. Together, these data suggest that derrière expression is compatible only with posterior fates. In order to assess the in vivo function of derrière, we constructed a dominant interfering Derrière protein (Cm-Derrière), which preferentially blocks Derrière activity relative to that of other TGFbeta family members. Cm-derrière expression in embryos leads to posterior truncation, including defects in blastopore lip formation, gastrulation and neural tube closure. Normal expression of anterior and hindbrain markers is observed; however, paraxial mesodermal gene expression is ablated. This phenotype can be rescued by wild-type derrière and by VegT. Our findings indicate that derrière plays a crucial role in mesodermal patterning and development of posterior regions in Xenopus.     

Xbra3 induces mesoderm and neural tissue in Xenopus laevis

Homologues of the murine Brachyury gene have been shown to be involved in mesoderm formation in several vertebrate species. In frogs, the Xenopus Brachyury homologue, Xbra, is required for normal formation of posterior mesoderm. We report the characterisation of a second Brachyury homologue from Xenopus, Xbra3, which has levels of identity with mouse Brachyury similar to those of Xbra. Xbra3 encodes a nuclear protein expressed in mesoderm in a temporal and spatial manner distinct from that observed for Xbra. Xbra3 expression is induced by mesoderm-inducing factors and overexpression of Xbra3 can induce mesoderm formation in animal caps. In contrast to Xbra, Xbra3 is also able to cause the formation of neural tissue in animal caps. Xbra3 overexpression induces both geminin and Xngnr-1, suggesting that Xbra3 can play a role in the earliest stages of neural induction. Xbra3 induces posterior nervous tissue by an FGF-dependent pathway; a complete switch to anterior neural tissue can be effected by the inhibition of FGF signalling. Neither noggin, chordin, follistatin, nor Xnr3 is induced by Xbra3 to an extent different from their induction by Xbra nor is BMP4 expression differentially affected.     

Zygotic Wnt activity is required for Brachyury expression in the early Xenopus laevis embryo

The canonical, beta-catenin-dependent Wnt pathway is a crucial player in the early events of Xenopus development. Dorsal axis formation and mesoderm patterning are accepted effects of this pathway, but the regulation of expression of genes involved in mesoderm specification is not. This conclusion is based largely on the inability of the Wnt pathway to induce mesoderm in animal cap explants. Using injections of inhibitors of canonical Wnt signaling, we demonstrate that expression of the general mesodermal marker Brachyury (Xbra) requires a zygotic, ligand-dependent Wnt activity throughout the marginal zone. Analysis of the Xbra promoter reveals that putative TCF-binding sites mediate Wnt activation, the first sites in this well-studied promoter to which an activation role can be ascribed. However, established mesoderm inducers like eFGF and activin can bypass the Wnt requirement for Xbra expression. Another mesoderm promoting factor, VegT, activates Xbra in a Wnt-dependent manner. We also show that the activin/nodal signaling is necessary for ectopic Xbra induction by the Wnt pathway, but not by VegT. Our data significantly change the understanding of Brachyury regulation in Xenopus, implying the existence of an unknown zygotic Wnt ligand in Spemann's organizer. Since Brachyury is considered to have a major role in mesoderm formation, it is possible that Wnts might play a role in mesoderm specification, in addition to patterning.     

FGF-8 stimulates neuronal differentiation through FGFR-4a and interferes with mesoderm induction in Xenopus embryos

The role of fibroblast growth factors (FGFs) in neural induction is controversial [1,2]. Although FGF signalling has been implicated in early neural induction [3-5], a late role for FGFs in neural development is not well established. Indeed, it is thought that FGFs induce a precursor cell fate but are not able to induce neuronal differentiation or late neural markers [6-8]. It is also not known whether the same or distinct FGFs and FGF receptors (FGFRs) mediate the effects on mesoderm and neural development. We report that Xenopus embryos expressing ectopic FGF-8 develop an abundance of ectopic neurons that extend to the ventral, non-neural, ectoderm, but show no ectopic or enhanced notochord or somitic markers. FGF-8 inhibited the expression of an early mesoderm marker, Xbra, in contrast to eFGF, which induced ectopic Xbra robustly and neuronal differentiation weakly. The effect of FGF-8 on neurogenesis was blocked by dominant-negative FGFR-4a (DeltaXFGFR-4a). Endogenous neurogenesis was also blocked by DeltaXFGFR-4a and less efficiently by dominant-negative FGFR-1 (XFD), suggesting that it depends preferentially on signalling through FGFR-4a. The results suggest that FGF-8 and FGFR-4a signalling promotes neurogenesis and, unlike other FGFs, FGF-8 interferes with mesoderm induction. Thus, different FGFs show specificity for mesoderm induction versus neurogenesis and this may be mediated, at least in part, by the use of distinct receptors.     

FGF4 regulates blood and muscle specification in Xenopus laevis

BACKGROUND INFORMATION: FGF (fibroblast growth factor) signalling is known to be required for many aspects of mesoderm formation and patterning during Xenopus development and has been implicated in regulating genes required for the specification of both blood and skeletal muscle lineages. RESULTS: In the present study, we have specifically knocked down the expression of FGF4 using AMO (antisense morpholino oligonucleotide)-mediated inhibition and demonstrate that FGF4 acts in the dorsal marginal zone to restrict blood development and promote the development of skeletal muscle. In addition, we used a drug inhibitor of FGF signalling and an inducible form of FGFR1 (FGF receptor 1) to identify a period of competence during late blastula and gastrula stages when FGF signalling acts to regulate blood versus muscle specification. Notably, we found that it is the dorsal activity of FGF that is required to restrict the expression of SCL (stem cell leukaemia) to the ventral blood island. CONCLUSIONS: Our data indicate that FGF4 is a key organizer-derived signal involved in the process of dorsoventral patterning of the mesoderm.     

Role of fibroblast growth factors as inducing agents in early embryonic development

To assess the potential role of a molecule in development we need to know three things: 1) what are the biological activities of the molecule, 2) what is its expression pattern, and 3) what are the consequences of removing it from the embryo? In the case of the FGF family in Xenopus embryos we have quite a lot of information about all three questions. Most members of the family can induce mesoderm from isolated animal caps, thus mimicking the natural “ventral vegetal” inducing signal operative in the blastula. This activity can be exerted on isolated, disaggregated cells and does not involve a change in division rate. When overexpressed from injected mRNA, the activity of FGFs depends largely on whether or not they possess a signal sequence, showing the importance of secretion in the inductive process. In addition to the mesoderm-inducing activity, there are effects of overexpression on whole embryos which lead to a suppression of anterior structures. Three types of FGF have so far been cloned from Xenopus: direct homologs of each of the mammalian types FGF-2 and FGF-3, and eFGF (“embryonic FGF”), which is equidistant in sequence from mammalian FGF-4 and FGF-6. Attempts to find homologs of mammalian FGF-5 and FGF-7 in Xenopus have proved unsuccessful. All three types of Xenopus FGF are expressed in early development. FGF-2 and eFGF are present in the oocyte and fertilized egg, and are thus both available at the time of mesoderm induction. FGF-3 and eFGF are both expressed from the embryonic genome during gastrulation and concentrated in the forming mesoderm. FGF-2 is expressed from the embryonic genome during neurulation in the brain, and a little later in the branchial arch mesenchyme and in the forming myotomes. These expression patterns suggest that there are several functions for the FGFs. The most successful strategy for inhibition of the FGF system has been the use of a dominant negative receptor construct introduced by Kirschner and colleagues. Overexpression of this construct can abolish the FGF responsiveness of animal caps. In whole embryos, the absence of FGF signaling causes a reduction, although not a total ablation, of mesoderm formation. There is also a severe effect on axis formation in which formation of the posterior parts is reduced consequent on an inhibition of invagination and elongation of the dorsal mesoderm. Thus, the present evidence suggests that the FGF system contributes to, although is not solely responsible for, mesoderm induction in vivo. It is also necessary for normal gastrulation movements, particularly in the dorsal mesoderm, and is likely to have several later functions, particularly in development of the central nervous system and the myotomes. © 1994 Wiley-Liss, Inc.