The alignment of the animal—vegetal axis of the mouse zygote with the axis of bilateral symmetry in blastocysts and the proximal—distal axis in egg cylinders suggests that some degree of regional specification or polarity might already exist in the egg cytoplasm Gardner, ; Weber et al. Cortical Rotation A critical step in determining the prospective dorsal region of the embryo is triggered by cortical rotation. The mechanism and consequences of such movement has attracted considerable attention. An unfertilized Xenopus egg is radially symmetrical along the animal—vegetal axis. Upon fertilization, the dorsal—ventral axis is defined by the site of sperm entry in the animal region.
The sperm entry site marks the future ventral side of the embryo and overlaps with the first cleavage plane, which divides the egg bilaterally into right and left halves. In fact, axis-inducing activity has also been detected above the equatorial region in the animal dorsal sector Gallagher et al. A possible explanation for the apparent discrepancy between the degree of cortical rotation and the localization of dorsal activity comes from studies of microtubule-dependent movement in the vegetal cortical region Elinson and Rowning, ; Rowning et al.
Early Xenopus Development 9 core Larabell et al. These multiple layers of microtubules align with the direction of rotation Elinson and Rowning, Since the rotation movement precedes the formation of the microtubule arrays, it has been suggested that the microtubules are not responsible for the rotation of the cortex Larabell et al.
However, small organelles can be propelled along the parallel array of microtubules that function independently of the cortex. Small organelles may therefore move along the microtubules by motor molecules toward the plus-end to the dorsal side of the embryo. Transport by microtubules thus accounts for the apparent differences in the localization of dorsalizing activity as predicted from the degree of rotation of the egg cortex.
In keeping with the microtubule transport model, a downstream component of the Wnt pathway, Dishevelled Dsh , has been shown to associate with small vesicle-like organelles that are translocated to the prospective dorsal side by microtubules during cortical rotation Miller et al. Microtubule transport is not only specific for dorsal—ventral specification in Xenopus embryos. A dynamic distribution of microtubules has also been observed in the yolk cells of zebrafish embryos Jesuthasan and Stahle, In zebrafish embryos, a set of parallel microtubules at the vegetal pole region is required for setting up initial asymmetry at the one-cell stage.
At the eight-cell stage, microtubule tracks originating from the dorsal equatorial blastomeres extend toward the vegetal pole. These microtubule tracks may function to mediate directional transport of organelles or determinants required for dorsal development. Nieuwkoop Center After cortical rotation takes place, it is thought that a signaling center—the Nieuwkoop center—is activated in the dorsal vegetal region that subsequently induces the formation of the organizer in the overlying cells in a non-cell-autonomous manner.
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In a series of tissue recombination experiments, different regions of the yolky vegetal mass of Urodele embryos were tested for their inductive capacity on animal caps Boterenbrood and Nieuwkoop, The dorsal vegetal region induced dorsal axial structures, whereas the lateral and ventral vegetal regions induced only ventral structures. This tissue recombination assay has been referred to as the Nieuwkoop recombinant assay.
The dorsal vegetal region carrying a dorsal endomesoderm-inducing property is commonly referred to as the Nieuwkoop center Gerhart et al. This region is a signaling center required for specifying the dorsal—ventral axis. The inductive effect of the dorsal vegetal cells is active between the early cleavage stage and the late blastula stage Boterenbrood and Nieuwkoop, Cell progenies from the Nieuwkoop center do not contribute to the dorsal lip or axial structures formed during gastrulation.
The progenies are located vegetal to the dorsal lip, in the endoderm, and are fated to become part of the anterior gut endoderm, as shown by lineage labeling Bauer et al. Etkin Vodicka and Gerhart, The proposed function of the Nieuwkoop center is to induce the cells immediately above in the equatorial region to form the organizer. However, the requirement of the dorsal vegetal cells for dorsal development has not been directly demonstrated by studies in which all tier C and tier D dorsal blastomeres are removed. In fact, all blastomeres in tier D can be removed without affecting axis formation Gimlich, Transplantations of cytoplasm, cortical peels, and blastomeres have demonstrated that dorsalizing activity is distributed broadly on the dorsal sector of the embryo, with the highest activities around the vegetal and equatorial regions before and after cortical rotation Kageura, ; Yuge et al.
Thus, the Nieuwkoop center may overlap physically with the region where the prospective organizer forms. Studies of axis induction by transplantation of blastomeres from cell embryos showed that both tier C and tier D dorsal blastomeres are active in the induction assay Gimlich and Gerhart, ; Gimlich, ; Kageura, The dorsalizing activity of tier C dorsal blastomeres and their participation in organizer formation during normal development also support the idea that the region that produces the early dorsal inductive signal overlaps with the organizer.
In summary, the Nieuwkoop center is an important concept in defining early inductive signaling during dorsal—ventral specification. The Nieuwkoop center has been regarded as a physical entity spatially and temporally distinct from the gastrula organizer. However, it is also possible that the cytoplasmic dorsal determinants, after translocation to the dorsal side by cortical rotation and interaction with components in the dorsal region, activate the Nieuwkoop center at the dorsal equatorial region during cleavage stage Kodjabachian and Lemaire, ; Moon and Kimelman, The Nieuwkoop center in turn directly induces formation of the organizer at the equatorial region during the blastula stage Kodjabachian and Lemaire, ; Moon and Kimelman, In this model, the Nieuwkoop center and the organizer essentially occupy the same region in the embryo but remain temporally distinct.
Molecular analysis of the Nieuwkoop center will help to clearly define its role in inducing the formation of the gastrula organizer. Siamois Lemaire et al. Siamois can be activated directly by Xwnt-8 and also by vegetal cortical cytoplasm Carnac et al.
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On the other hand, Xwnt-8 is not maternally expressed and cannot be the maternal dorsal determinant. Endogenous Xwnt-8 is expressed in the ventral—lateral marginal zone after the MBT and is involved in a Wnt pathway required for patterning of the mesoderm Christian and Moon, ; Hoppler et al. The basic components of the Wnt pathway are largely conserved between different developmental processes found in C.
The wingless pathway involved in epidermal cell differentiation in Drosophila has been characterized Klingensmith and Nusse, ; Siegfried and Perrimon, and provides a basis for study of the Wnt pathway in other systems. In contrast, when a secreted Wnt molecule is recognized by a transmembrane receptor of the frizzled family, a cytoplasmic component, Dsh, is activated. Etkin Figure 1 Components of the Wnt signaling pathway.
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Early Xenopus Development 13 transactivation domain, but no nuclear localization signal. Tcf-1, -3, and -4 and Lef1 have been identified in both mouse and human. XTcf3 is the only frog homolog identified so far Molenaar et al. Tcf proteins also contain a HMG domain that recognizes a DNA consensus sequence within the regulatory sequence of target genes. Groucho and CBP are corepressors that associate with Tcf proteins to repress transcription van de Wetering et al. Groucho has been suggested to function with a histone deacetylase complex to regulate histone acetylation on chromatin Choi et al.
In Drosophila, dCBP lowers the affinity of armadillo to dTcf by the acetylation of a conserved lysine residue in the armadillo-binding domain Waltzer and Bienz, Tcf-3 and Tcf-4 contain two conserved sites at the C terminus for the binding of C-terminal binding protein, which is also involved in transcriptional repression. Studies with antimorphic protein have shown that the Xenopus homolog of C-terminal binding protein is involved in the regulation of dorsoanterior development Brannon et al.
The Tcf proteins have been proposed to be architectual factors controlling chromatin structure and transcription van Houte et al. The Tcf binding sites in the siamois promoter are required for the activation of siamois expression in the 14 Agnes P. Etkin dorsal region Brannon et al. They are also necessary for the repression of siamois expression on the ventral side of the embryo, since removal of Tcf binding sites from the siamois regulatory sequence results in ectopic expression in the ventral side of the embryo Brannon et al.
Mice deficient in both Tcf1 and Lef1 show defects in the formation of axial structures Galceran et al. Overexpression of a membrane-tethered form or a wild-type cytoplasmic form of plakaglobin can result in axis duplication Merriam et al. Activation of the Wnt pathway suppresses the activity of Xgsk-3 and results 1.
Overexpression of wild-type Xgsk-3 on the dorsal side of the embryo suppresses Wnt signaling and inhibits endogenous axis formation He et al. Two distinct modes of Xgsk-3 regulation have been reported Dominguez and Green, It is shown that an endogenous mechanism exists on the dorsal side of Xenopus embryos to deplete Xgsk-3 protein. A second mode of regulation occurs as a consequence of Wnt or Dsh expression, which causes a reduction in Xgsk-3 activity rather than depletion.
APC was originally identified as a tumor suppressor gene because loss of APC function was observed in colon cancer Polakis, A mouse mutation, fused, produces a truncated gene product of axin Zeng et al. Mutant embryos exhibit duplicate axis formation implicating Axin as a negative regulator of axis formation. Etkin ventralization of endogenous axis by axin.
The presence of an RGS domain suggests a possible involvement of G proteins in the regulation of the Wnt pathway. Antisense oligonucleotide knockout experiments have demonstrated that GBP is required for the establishment of the dorsal—ventral axis in Xenopus embryos Yost et al. Evidence indicates that GBP can also induce depletion Xgsk-3 proteins, similar to the endogenous activity existed on the dorsal side of the embryo Dominguez and Green, It appears that GBP is a regulator of Wnt signaling but is not a linear component of the pathway.
CKI has been shown to phosphorylate Dsh. It has been suggested that such phosphorylation is not required for the functioning of CKI. Further studies on the regulation of Dsh by these two kinases will be required to determine their roles in transducing the Wnt signal. Early Xenopus Development 17 8. Dsh is phosphorylated by active Wnt signaling Yanagawa et al. Dsh has been shown to translocate from the vegetal pole toward the prospective dorsal side Miller et al.
However, depletion of the maternal gene products will be required to provide definitive evidence for a role of Dsh in the establishment of dorsal—ventral axis in Xenopus.
Frizzled Frizzled proteins are receptors for Wnt ligands Bhanot et al. Members of the Frizzled family contain a cysteine-rich domain, seven transmembrane domains, and sometimes a PDZ-binding domain. Overexpression of Xfrizzled-8 Xfz8 alone on the ventral side of an embryo induces a secondary axis in the absence of exogenously supplied Wnt ligands Deardorff et al.
However, since the endogenous expression of Xfz8 is zygotically activated in the organizer region during gastrulation, Xfz8 is unlikely to function as the endogenous Wnt receptor for the dorsal specification pathway during the early cleavage of Xenopus embryos. A maternally encoded frizzled protein, Xfz7, has recently been isolated Sumanas et al. Embryos depleted of Xfz7 mRNA are deficient in dorsal mesoderm formation.
This study provides experimental evidence for the functioning of the Frizzled proteins upstream of other components of the Wnt pathway in dorsalventral mesoderm specification. Several Wnt ligands have been studied, and these can be divided into two classes according to their axis-inducing ability. In contrast, Xwnt-4 Du et al.
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However, the Wnt molecules that have strong axis-inducing activity 18 Agnes P. Etkin are not expressed at the right time and right place to be the dorsalizing signal Moon and Kimelman, Overexpression of Xwnt-8 can induce a complete secondary axis, but endogenous expression is not detected until after the MBT in the ventral-lateral mesoderm. Although Xwnt-8b is maternally expressed and has strong-axis-inducing activity, it is localized to the animal region of cleavage-stage embryos Cui et al. Xwnt mRNA is localized to the vegetal cortex of the oocyte Kloc and Etkin, and the protein is differentially translated along the dorsal—ventral axis Schroeder et al.
This result demonstrates the divergence of signal transduction cascades that Wnt molecules can elicit. Treatment of embryos with lithium chloride during early cleavage stage produces a dorsalized phenotype. It was originally suggested that the dorsalizing effect of lithium is due to an activation of the phosphatidylinositol cycle Maslanski et al.
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A dominant negative form of dsh, Xdd1, although 1. Early Xenopus Development 19 able to block ectopic axis formation by the activation of Wnt signaling, has no effect on endogenous axis formation Sokol, Similar results have been obtained for Wnt molecules in dominant negative forms Hoppler et al. Evidence supporting this model came from the study in which Dsh is shown to translocate from the vegetal pole toward the prospective dorsal side Miller et al.
However, the possibility that translocation of dsh toward the future dorsal side is preceded by the signaling of a Wnt ligand at the vegetal cortex of the egg should not be ruled out. The effect of Xdd1 overexpression has also been examined in prospective ectoderm transplanted with VCC Marikawa and Elinson, In keeping with the findings in embryos, overexpression of Xdd1 in prospective ectoderm has no effect on the activity of VCC, as demonstrated by the expression of target genes siamois and Xnr3. Overexpression of different components of the Wnt pathway has shown that the activity of VCC is not inhibited by Xdd1 or Xgsk-3 but is inhibited by Axin.
On the basis of these findings, it has been proposed that VCC may in fact act on Axin instead of Xgsk-3 to mediate its dorsalizing activity. Furthermore, transplantation of vegetal cortical cytoplasm, or overexpression of siamois, only induces ectopic axis formation when the recipient site is the equatorial region but not when the recipient site is the animal region Kageura, Overexpression of Vg1 alone induces dorsal mesoderm but no notochord, and Xwnt-8b alone does not induce mesoderm formation.
Etkin response elements are present in the promoter of the organizer gene gsc. For dorsal development to occur, the endogenous ligand has to be active in the embryo at the right time and place.