Qinghua Tao

Maternal Wnt11 Activates the Canonical Wnt Signaling Pathway Required for Axis Formation in Xenopus Embryos

Wnt signaling pathways play essential roles in patterning and proliferation of embryonic and adult tissues. In many organisms, this signaling pathway directs axis formation. Although the importance of intracellular components of the pathway, including beta-catenin and Tcf3, has been established, the mechanism of their activation is uncertain. In Xenopus, the initiating signal that localizes beta-catenin to dorsal nuclei has been suggested to be intracellular and Wnt independent. Here, we provide three lines of evidence that the pathway specifying the dorsal axis is activated extracellularly in Xenopus embryos. First, we identify Wnt11 as the initiating signal. Second, we show that activation requires the glycosyl transferase X.EXT1. Third, we find that the EGF-CFC protein, FRL1, is also essential and interacts with Wnt11 to activate canonical Wnt signaling.

Wnt5a and Wnt11 interact in a maternal Dkk1-regulated fashion to activate both canonical and non-canonical signaling in Xenopus axis formation

Wnt signaling in development and adult tissue homeostasis requires tight regulation to prevent patterning abnormalities and tumor formation. Here, we show that the maternal Wnt antagonist Dkk1 downregulates both the canonical and non-canonical signaling that are required for the correct establishment of the axes of the Xenopus embryo. We find that the target Wnts of Dkk activity are maternal Wnt5a and Wnt11, and that both Wnts are essential for canonical and non-canonical signaling. We determine that Wnt5a and Wnt11 form a previously unrecognized complex. This work suggests a new aspect of Wnt signaling: two Wnts acting in a complex together to regulate embryonic patterning.

N- and E-cadherins in Xenopus are specifically required in the neural and non-neural ectoderm, respectively, for F-actin assembly and morphogenetic movements

Transmembrane cadherins are calcium-dependent intercellular adhesion molecules. Recently, they have also been shown to be sites of actin assembly during adhesive contact formation. However, the roles of actin assembly on transmembrane cadherins during development are not fully understood. We show here, using the developing ectoderm of the Xenopus embryo as a model, that F-actin assembly is a primary function of both N-cadherin in the neural ectoderm and E-cadherin in the non-neural (epidermal) ectoderm, and that each cadherin is essential for the characteristic morphogenetic movements of these two tissues. However, depletion of N-cadherin and E-cadherin did not cause dissociation in these tissues at the neurula stage, probably owing to the expression of C-cadherin in each tissue. Depletion of each of these cadherins is not rescued by the other, nor by the expression of C-cadherin, which is expressed in both tissues. One possible reason for this is that each cadherin is expressed in a different domain of the cell membrane. These data indicate the combinatorial nature of cadherin function, the fact that N- and E-cadherin play primary roles in F-actin assembly in addition to roles in cell adhesion, and that this function is specific to individual cadherins. They also show how cell adhesion and motility can be combined in morphogenetic tissue movements that generate the form and shape of the embryonic organs.

Wnt11/β-catenin signaling in both oocytes and early embryos acts through LRP6-mediated regulation of axin

Current models of canonical Wnt signaling assume that a pathway is active if β-catenin becomes nuclearly localized and Wnt target genes are transcribed. We show that, in Xenopus, maternal LRP6 is essential in such a pathway, playing a pivotal role in causing expression of the organizer genes siamois and Xnr3, and in establishing the dorsal axis. We provide evidence that LRP6 acts by degrading axin protein during the early cleavage stage of development. In the full-grown oocyte, before maturation, we find that axin levels are also regulated by Wnt11 and LRP6. In the oocyte, Wnt11 and/or LRP6 regulates axin to maintain β-catenin at a low level, while in the embryo, asymmetrical Wnt11/LRP6 signaling stabilizesβ -catenin and enriches it on the dorsal side. This suggests that canonical Wnt signaling may not exist in simple off or on states, but may also include a third, steady-state, modality.

Jun NH2-terminal kinase (JNK) prevents nuclear β-catenin accumulation and regulates axis formation in Xenopus embryos

Jun NH2-terminal kinases (JNKs) regulate convergent extension movements in Xenopus embryos through the noncanonical Wnt/planar cell polarity pathway. In addition, there is a high level of maternal JNK activity spanning from oocyte maturation until the onset of gastrulation that has no defined functions. Here, we show that maternal JNK activation requires Dishevelled and JNK is enriched in the nucleus of Xenopus embryos. Although JNK activity is not required for the glycogen synthase kinase-3-mediated degradation of β-catenin, inhibition of the maternal JNK signaling by morpholino-antisense oligos causes hyperdorsalization of Xenopus embryos and ectopic expression of the Wnt/β-catenin target genes. These effects are associated with an increased level of nuclear and nonmembrane-bound β-catenin. Moreover, ventral injection of the constitutive-active Jnk mRNA blocks β-catenin-induced axis duplication, and dorsal injection of active Jnk mRNA into Xenopus embryos decreases the dorsal marker gene expression. In mammalian cells, activation of JNK signaling reduces Wnt3A-induced and β-catenin-mediated gene expression. Furthermore, activation of JNK signaling rapidly induces the nuclear export of β-catenin. Taken together, these results suggest that JNK antagonizes the canonical Wnt pathway by regulating the nucleocytoplasmic transport of β-catenin rather than its cytoplasmic stability. Thus, the high level of sustained maternal JNK activity in early Xenopus embryos may provide a timing mechanism for controlling the dorsal axis formation.

G-protein-coupled signals control cortical actin assembly by controlling cadherin expression in the early Xenopus embryo

During embryonic development, each cell of a multicellular organ rudiment polymerizes its cytoskeletal elements in an amount and pattern that gives the whole cellular population its characteristic shape and mechanical properties. How does each cell know how to do this? We have used the Xenopus blastula as a model system to study this problem. Previous work has shown that the cortical actin network is required to maintain shape and rigidity of the whole embryo, and its assembly is coordinated throughout the embryo by signaling through G-protein-coupled receptors. In this paper, we show that the cortical actin network colocalizes with foci of cadherin expressed on the cell surface. We then show that cell-surface cadherin expression is both necessary and sufficient for cortical actin assembly and requires the associated catenin p120 for this function. Finally, we show that the previously identified G-protein-coupled receptors control cortical actin assembly by controlling the amount of cadherin expressed on the cell surface. This identifies a novel mechanism for control of cortical actin assembly during development that might be shared by many multicellular arrays.

Lysophosphatidic acid signaling controls cortical actin assembly and cytoarchitecture in Xenopus embryos

The mechanisms that control shape and rigidity of early embryos are not well understood, and yet are required for all embryonic processes to take place. In the Xenopus blastula, the cortical actin network in each blastomere is required for the maintenance of overall embryonic shape and rigidity. However, the mechanism whereby each cell assembles the appropriate pattern and number of actin filament bundles is not known. The existence of a similar network in each blastomere suggests two possibilities: cell-autonomous inheritance of instructions from the egg; or mutual intercellular signaling mediated by cell contact or diffusible signals. We show that intercellular signaling is required for the correct pattern of cortical actin assembly in Xenopus embryos, and that lysophosphatidic acid (LPA) and its receptors, corresponding to LPA1 and LPA2 in mammals, are both necessary and sufficient for this function.

A novel G protein-coupled receptor, related to GPR4, is required for assembly of the cortical actin skeleton in early Xenopus embryos

As the fertilized Xenopus egg undergoes sequential cell divisions to form a blastula, each cell develops a network of cortical actin that provides shape and skeletal support for the whole embryo. Disruption of this network causes loss of shape and rigidity of the embryo, and disrupts gastrulation movements. We previously showed that lysophosphatidic acid (LPA) signaling controls the change in cortical actin density that occurs at different stages of the cell cycle. Here, we use a gain-of-function screen, using an egg cDNA expression library, to identify an orphan G protein-coupled cell-surface receptor (XFlop) that controls the overall amount of cortical F-actin. Overexpression of XFlop increases the amount of cortical actin, as well as embryo rigidity and wound healing, whereas depletion of maternal XFlop mRNA does the reverse. Both overexpression and depletion of XFlop perturb gastrulation movements. Reciprocal rescue experiments, and comparison of the effects of their depletion in early embryos, show that the XLPA and XFlop signaling pathways play independent roles in cortical actin assembly, and thus that multiple signaling pathways control the actin skeleton in the blastula.

The role of maternal CREB in early embryogenesis of Xenopus laevis

In Xenopus embryos, body patterning and cell specification are initiated by transcription factors, which are themselves transcribed during oogenesis, and their mRNAs are stored for use after fertilization. We have previously shown that the T-box transcription factor VegT is both necessary and sufficient to initiate transcription of all endoderm, and most mesoderm genes. In the absence of maternal VegT, no mesodermal organs (including the heart) or endodermal organs form. A second maternal transcription factor XTcf3 acts as a global repressor of transcription of dorsal genes, whose repression is inactivated on the dorsal side by a maternally encoded Wnt signaling pathway. In the absence of beta-catenin, no mesodermal or endodermal organs form. We show here that the maternally encoded transcription factor CREB is also essential for development. It is required for the initiation of expression of several mesodermal genes, including Xbra, Xcad2, and -3 and also regulates the cardiogenic gene Nkx 2-5. We show that maternal CREB-depleted embryos develop gastrulation defects that are rescued by the reintroduction of activated CREB mRNA. We conclude that maternal CREB must be added to the list of essential maternal transcription factors regulating cell specification in the early embryo.

Regulation of Classical Cadherin Membrane Expression and F-Actin Assembly by Alpha-Catenins, during Xenopus Embryogenesis

Alpha (α)-E-catenin is a component of the cadherin complex, and has long been thought to provide a link between cell surface cadherins and the actin skeleton. More recently, it has also been implicated in mechano-sensing, and in the control of tissue size. Here we use the early Xenopus embryos to explore functional differences between two α-catenin family members, α-E- and α-N-catenin, and their interactions with the different classical cadherins that appear as tissues of the embryo become segregated from each other. We show that they play both cadherin-specific and context-specific roles in the emerging tissues of the embryo. α-E-catenin interacts with both C- and E-cadherin. It is specifically required for junctional localization of C-cadherin, but not of E-cadherin or N-cadherin at the neurula stage. α-N-cadherin interacts only with, and is specifically required for junctional localization of, N-cadherin. In addition, α -E-catenin is essential for normal tissue size control in the non-neural ectoderm, but not in the neural ectoderm or the blastula. We also show context specificity in cadherin/ α-catenin interactions. E-cadherin requires α-E-catenin for junctional localization in some tissues, but not in others, during early development. These specific functional cadherin/alpha-catenin interactions may explain the basis of cadherin specificity of actin assembly and morphogenetic movements seen previously in the neural and non-neural ectoderm.