Heading in a new direction: implications of the revised fate map for understanding Xenopus laevis development

Amphibian embryos have served as a model system for vertebrate axial patterning for more than a century. Recent changes to the Xenopus laevis fate map revised the assignment of the embryonic dorsal/ventral (back-to-belly) axis in pre-gastrula embryos and allowed the assignment of the rostral/caudal (head-to-tail) axis for the first time. Revising the embryonic axes after many years of experimentation changes our view of axial patterning in amphibians. In this review, we discuss the revised maps and axes, and show by example how the new map alters the interpretation of three experiments that form the foundations of amphibian embryology. We compare the revised amphibian fate map to the general maps of the protochordates, and discuss which features of the maps and early development are shared by chordates and which distinguish vertebrates. Finally, we offer an explanation for the formation of both complete and incomplete axes in the rescue assays routinely used to study axial patterning in Xenopus, and a model of amphibian axial patterning.     

Glycogen synthase kinase-3beta regulates DeltaNp63 gene transcription through the beta-catenin signaling pathway

Overexpression of DeltaNp63 has been observed in a number of human cancers, suggesting a role for DeltaNp63 in carcinogenesis. In the present study, we show that inhibition of glycogen synthase kinase-3beta (GSK-3beta) by lithium chloride (LiCl) elicited a stimulatory effect on DeltaNp63 promoter activity in HEK 293T cells. Exposure to LiCl induced DeltaNp63 promoter activation as well as DeltaNp63 protein expression in the cells. The effect of GSK-3beta on DeltaNp63 expression was further confirmed by the use of two highly specific GSK-3beta inhibitors, SB216763 and SB415286. Further study showed the presence of a putative beta-catenin responsive element (beta-catenin-RE) in the DeltaNp63 promoter region, and the stimulation of DeltaNp63 promoter activity by GSK-3beta inhibitor is markedly abolished by mutation or deletion of the putative beta-catenin-RE. Data are also presented to show that beta-catenin acts together with Lef-1 to influence DeltaNp63 promoter activity and protein expression.     

Parallel microtubules and other conserved elements of dorsal axial specification in the direct developing frog, <Emphasis Type="Italic">Eleutherodactylus coqui</Emphasis>

Specification of the dorsal axis in commonly studied frogs, such as Xenopus laevis and Rana pipiens, depends on a microtubule-mediated movement of cytoplasm in the fertilized egg. The Puerto Rican tree frog, Eleutherodactylus coqui, has an egg that is twenty times the volume of that of X. laevis, raising the question as to whether the mechanism of dorsal axial specification is conserved in these large eggs. Fertilized eggs of E. coqui develop a transient array of parallel microtubules, similar to other frogs, but proportionately larger. The array persists after first cleavage, longer than in other frogs, and is gone by the third cleavage. Correlated with the longer life of the parallel microtubules, both 2- and 8-cell E. coqui embryos remain sensitive to gravity-mediated axial specification, a sensitivity lost in X. laevis before the 2-cell stage. Activation of the Wnt/beta-catenin pathway by injected Xwnt8 RNA causes axial formation as in X. laevis. The results indicate that elements of dorsal axial specification are conserved in E. coqui, but they occur later compared to in X. laevis.     

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.     

Galphaq negatively regulates the Wnt-beta-catenin pathway and dorsal embryonic Xenopus laevis development

The non-canonical Wnt/Ca2+ signaling pathway has been implicated in the regulation of axis formation and gastrulation movements during early Xenopus laevis embryo development, by antagonizing the canonical Wnt/beta-catenin dorsalizing pathway and specifying ventral cell fate. However, the molecular mechanisms involved in this antagonist crosstalk are not known. Since Galphaq is the main regulator of Ca2+ signaling in vertebrates and from this perspective probably involved in the events elicited by the non-canonical Wnt/Ca2+ pathway, we decided to study the effect of wild-type Xenopus Gq (xGalphaq) in dorso-ventral axis embryo patterning. Overexpression of xGalphaq or its endogenous activation at the dorsal animal region of Xenopus embryo both induced a strong ventralized phenotype and inhibited the expression of dorsal-specific mesoderm markers goosecoid and chordin. Dorsal expression of an xGalphaq dominant-negative mutant reverted the xGalphaq-induced ventralized phenotype. Finally, we observed that the Wnt8-induced secondary axis formation is reverted by endogenous xGalphaq activation, indicating that it is negatively regulating the Wnt/beta-catenin pathway.     

Artificially applied tensions normalize development of relaxed Xenopus Laevis embryos

Relaxation of tensions of the surface of Xenopus laevis embryos at the late blastula stage leads to deep and diverse developmental defects and increased variability in mutual position and volume ratios of the axial rudiments. Here, we demonstrate that the development of such embryos was markedly normalized if the relaxed tensions were restored in one of two ways: (1) isotropic stretching of the blastocoel roof induced by incubation of relaxed embryos in a hypotonic medium or (2) anisotropic stretching of embryos on two needles. In the latter case, we succeeded in restoring the morphological axis not only after longitudinal stretching, but also after transverse stretching, and the new axis had signs of anteroposterior polarity. The role of isotropic and anisotropic tensions in organization of the early amphibian development is discussed.     

Polar effects of lithium in the heart of the zebrafish Danio rerio

 The molecular signalling mechanisms that are believed to govern the patterning of the heart early in embryonic development are not well understood. We have investigated the events which occur during patterning of the vertebrate heart by exposing gastrula stage zebrafish embryos to lithium, which is known to affect the phosphoinositol signalling pathway. Treatment of embryos at 50% epiboly (5.25 h after fertilization at 28.5°C) with 0.3 m LiCl for 5–15 min, results in embryos with defects which range from mild to severe, depending on the length of time the embryos are exposed to lithium. In the heart, defects appear progressively in the inflow tract, the sinus venosus and atrium. By using an antibody that recognizes an atrium-specific isoform of myosin, our results show that lithium treatment at gastrulation specifically affects the atrium and sinus venosus, and has little obvious effect on the ventricle. Defects induced by lithium differ from those induced by retinoic acid (RA) treatment of similarly staged embryos, and suggest that lithium and RA may affect the patterning signals important for establishment of the vertebrate heart by acting on different populations of cells or by influencing different patterning pathways. Received: 8 December 1995 / Accepted: 11 April 1996     

Epigenetic alterations brought about by lithium treatment disrupt mouse embryo development

The commonly accepted mechanism by which LiCl corsalizes amphibian embryos is a respecification of ventral blastomeres, presumably through realignment of dorsal positional information in the embryo. An alternative mechanism, however, is an epigenetic change in the competence of cells to respond to cues they may be normally exposed to without effect. In order to test this hypothesis, we treated mouse preimplantation embryos, which do not possess any axial positional information, with LiCl, and observed axial abnormalities which must have been elaborated several days after treatment. We interpret this as support for the hypothesis that cellular competence rather than positional information is altered by LiCl, and suggest that this competence may be altered through the action of lithium sensitive enzymes that interact with chromatin. © 1996 Wiley-Liss, Inc.