Gastrulation Defects Research Articles

Cell signaling facilitates apical constriction by basolaterally recruiting Arp2/3 via Rac and WAVE.

Apical constriction is a critical cell shape change that bends tissues. How precisely-localized actomyosin regulators drive apical constriction remains poorly understood. C. elegans gastrulation provides a valuable model to address this question. The Arp2/3 complex is essential in C. elegans gastrulation. To understand how Arp2/3 is locally regulated, we imaged embryos with endogenously-tagged Arp2/3 and its nucleation-promoting factors (NPFs). The three NPFs - WAVE, WASP, and WASH - colocalized with Arp2/3 and controlled Arp2/3 localization at distinct subcellular locations. We exploited this finding to study distinct populations of Arp2/3 and found that only WAVE depletion caused penetrant gastrulation defects. WAVE localized basolaterally with Arp2/3 at cell-cell contacts, dependent on CED-10/Rac. Establishing ectopic cell contacts recruited WAVE and Arp2/3, identifying contact as a symmetry-breaking cue for localization of these proteins. These results suggest that cell-cell signaling via Rac activates WAVE and Arp2/3 basolaterally, and that basolateral Arp2/3 is important for apical constriction.

Reversion induced LIM domain protein (RIL) is a Daam1-interacting protein and regulator of the actin cytoskeleton during non-canonical Wnt signaling

The Daam1 protein regulates Wnt-induced cytoskeletal changes during vertebrate gastrulation though its full mode of action and binding partners remain unresolved. Here we identify Reversion Induced LIM domain protein (RIL) as a new interacting protein of Daam1. Interaction studies uncover binding of RIL to the C-terminal actin-nucleating portion of Daam1 in a Wnt-responsive manner. Immunofluorescence studies showed subcellular localization of RIL to actin fibers and co-localization with Daam1 at the plasma membrane. RIL gain- and loss-of-function approaches in Xenopus produced severe gastrulation defects in injected embryos. Additionally, a simultaneous loss of Daam1 and RIL synergized to produce severe gastrulation defects indicating RIL and Daam1 may function in the same signaling pathway. RIL further synergizes with another novel Daam1-interacting protein, Formin Binding Protein 1 (FNBP1), to regulate gastrulation. Our studies altogether show RIL mediates Daam1-regulated non-canonical Wnt signaling that is required for vertebrate gastrulation.

Formin Binding Protein 1 (FNBP1) regulates non-canonical Wnt signaling and vertebrate gastrulation

The Formin protein Daam1 is required for Wnt-induced cytoskeletal changes during gastrulation, though how it accomplishes this remains unresolved. Here we report the characterization of Formin Binding Protein 1 (FNBP1) as a binding partner of Daam1. The interaction of Daam1 with FNBP1 and its domains required for this interaction were delineated. Immunofluorescence studies showed FNBP1 co-localizes with Daam1, and is an integral component of the actin cytoskeletal complex that is responsive to Wnt stimulation. Specifically, FNBP1 can induce intracellular tubule-like structures and localize to focal adhesions suggesting a role for FNBP1 in cell migration. Functional FNBP1 studies in Xenopus embryos uncover a critical role for FNBP1 in regulating vertebrate gastrulation. Additionally, suboptimal doses of Daam1 and FNBP1 synergize to produce severe gastrulation defects, indicating FNBP1 and Daam1 may function within the same signaling pathway. These results together show FNBP1 is an integral component of Daam1-regulated non-canonical Wnt signaling required for vertebrate gastrulation.

Sizzled (Frzb3) physically interacts with noncanonical Wnt ligands to inhibit gastrulation cell movement

The coordinated movement of germ layer progenitor cells reaches its peak at the dorsal side, where the Bmp signaling gradient is low, and minimum at the ventral side, where the Bmp gradient is high. This dynamic cell movement is regulated by the interplay of various signaling pathways. The non-canonical Wnt signaling cascade serves as a pivotal regulator of convergent and extension cellular movement, facilitated by the activation of small GTPases such as Rho, Rab, and Rac. However, the underlying cause of limited cell movement at the ventral side remains elusive. To explore the functional role of a key regulator in constraining gastrulation cell movement at the ventral side, we investigated the Bmp4-direct target gene, sizzled, to assess its potential role in inhibiting non-canonical Wnt signaling. In our current study, we demonstrated that ectopic expression of sizzled led to gastrulation defects in a dose-dependent manner, without altering cell fate specification. Overexpression of sizzled resulted in decreased elongation of Activin-treated animal cap and Keller explants. Furthermore, our immunoprecipitation assay unveiled the physical interaction of Sizzled with non-canonical Wnt ligand proteins (Wnt5 and Wnt11). Additionally, the activation of small GTPases involved in Wnt signaling mediation (RhoA and Rac1) was diminished upon sizzled overexpression. In summary, our findings suggest that Bmp4 signaling negatively modulates cell movement from the ventral side of the embryo by inducing sizzled expression during early Xenopus gastrulation.

Conserved enhancers control notochord expression of vertebrate Brachyury

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

Recognition of H2AK119ub plays an important role in RSF1-regulated early Xenopus development.

Polycomb group (PcG) proteins are key regulators of gene expression and developmental programs via covalent modification of histones, but the factors that interpret histone modification marks to regulate embryogenesis are less studied. We previously identified Remodeling and Spacing Factor 1 (RSF1) as a reader of histone H2A lysine 119 ubiquitination (H2AK119ub), the histone mark deposited by Polycomb Repressive Complex 1 (PRC1). In the current study, we used Xenopus laevis as a model to investigate how RSF1 affects early embryonic development and whether recognition of H2AK119ub is important for the function of RSF1. We showed that knockdown of Xenopus RSF1, rsf1, not only induced gastrulation defects as reported previously, but specific targeted knockdown in prospective neural precursors induced neural and neural crest defects, with reductions of marker genes. In addition, similar to knockdown of PRC1 components in Xenopus, the anterior-posterior neural patterning was affected in rsf1 knockdown embryos. Binding of H2AK119ub appeared to be crucial for rsf1 function, as a construct with deletion of the UAB domain, which is required for RSF1 to recognize the H2AK119ub nucleosomes, failed to rescue rsf1 morphant embryos and was less effective in interfering with early Xenopus development when ectopically expressed. Furthermore, ectopic deposition of H2AK119ub on the Smad2 target gene gsc using a ring1a-smad2 fusion protein led to ectopic recruitment of RSF1. The fusion protein was inefficient in inducing mesodermal markers in the animal region or a secondary axis when expressed in the ventral tissues. Taken together, our results reveal that rsf1 modulates similar developmental processes in early Xenopus embryos as components of PRC1 do, and that RSF1 acts at least partially through binding to the H2AK119ub mark via the UAB domain during development.

Gastrulation and Split Cord Malformation.

Split cord malformation (SCM) is a rare form of closed spinal dysraphism, in which two hemi-cords are present, instead of a single spinal cord. SCM is categorised into type 1 and type 2. Type 1 SCM is defined by the presence of a bony or osseocartilaginous spur between the hemi-cords, whereas type 2 SCM has no bony spur, and the two hemi-cords are contained within a single dura. In this chapter, we present the putative mechanisms by which SCM arises, including gastrulation defects and Pang's unified theory. The typical and rare clinical presentations and variations are described. Finally, we outline the step-by-step surgical approach to both SCM 1 and 2 and the overall prognosis of both conditions.

Dorsal lip maturation and initial archenteron extension depend on Wnt11 family ligands

Wnt11 family proteins are ligands that activate a type of Dishevelled-mediated, non-canonical Wnt signaling pathway. Loss of function causes defects in gastrulation and/or anterior-posterior axis extension in all vertebrates. Non-mammalian vertebrate genomes encode two Wnt11 family proteins whose distinct functions have been unclear. We knocked down Wnt11b and Wnt11, separately and together, in Xenopus laevis. Single morphants exhibited very similar phenotypes of delayed blastopore closure, but they had different phenotypes during the tailbud period. In response to their very similar gastrulation phenotypes, we chose to characterize dual morphants. Using dark field illuminated time-lapse imaging and kymograph analysis, we identified a failure of dorsal blastopore lip maturation that correlated with slower blastopore closure and failure to internalize the endoderm at the dorsal blastopore lip. We connected these externally visible phenotypes to cellular events in the internal tissues by imaging intact fixed embryos stained for anillin and microtubules. We found that the initial extension of the archenteron is correlated with blastopore lip maturation, and archenteron extension is dramatically disrupted by decreased Wnt11 family signaling. We were aided in our interpretation of the immunofluorescence by the novel, membrane proximal location of the cleavage furrow protein anillin in the epithelium of the blastopore lip and early archenteron.

Rapalog-induced cell adhesion molecule inhibits mesoderm migration in Xenopus embryos by increasing frequency of adhesion to the ectoderm.

During the gastrula stage of Xenopus laevis, mesodermal cells migrate on the blastocoel roof (BCR) toward the animal pole. In this process, mesodermal cells directly adhere to the BCR via adhesion molecules, such as cadherins, which in turn trigger a repulsive reaction through factors such as Eph/ephrin. Therefore, the mesoderm and BCR repeatedly adhere to and detach from each other, and the frequency of this adhesion is thought to control mesoderm migration. Although knockdown of cadherin or Eph/ephrin causes severe gastrulation defects, these molecules have been reported to contribute not only to boundary formation but also to the internal function of each tissue. Therefore, it is possible that the defect caused by knockdown occurs due to tissue function abnormalities. To address this problem, we developed a method to specifically induce adhesion between different tissues using rapalog (an analog of rapamycin). When adhesion between the BCR and mesoderm was specifically enhanced by rapalog, mesoderm migration was strongly suppressed. Furthermore, we confirmed that rapalog significantly increased the frequency of adhesion between the two tissues. These results support the idea that the adhesion frequency controls mesoderm migration, and demonstrate that our method effectively enhances adhesion between specific tissues in vivo.

Retinoic Acid is Required for Normal Morphogenetic Movements During Gastrulation.

Retinoic acid (RA) is a central regulatory signal that controls numerous developmental processes in vertebrate embryos. Although activation of Hox expression is considered one of the earliest functions of RA signaling in the embryo, there is evidence that embryos are poised to initiate RA signaling just before gastrulation begins, and manipulations of the RA pathway have been reported to show gastrulation defects. However, which aspects of gastrulation are affected have not been explored in detail. We previously showed that partial inhibition of RA biosynthesis causes a delay in the rostral migration of some of the earliest involuting cells, the leading edge mesendoderm (LEM) and the prechordal mesoderm (PCM). Here we identify several detrimental gastrulation defects resulting from inhibiting RA biosynthesis by three different treatments. RA reduction causes a delay in the progression through gastrulation as well as the rostral migration of the goosecoid-positive PCM cells. RA inhibition also hampered the elongation of explanted dorsal marginal zones, the compaction of the blastocoel, and the length of Brachet’s cleft, all of which indicate an effect on LEM/PCM migration. The cellular mechanisms underlying this deficit were shown to include a reduced deposition of fibronectin along Brachet’s cleft, the substrate for their migration, as well as impaired separation of the blastocoel roof and involuting mesoderm, which is important for the formation of Brachet’s cleft and successful LEM/PCM migration. We further show reduced non-canonical Wnt signaling activity and altered expression of genes in the Ephrin and PDGF signaling pathways, both of which are required for the rostral migration of the LEM/PCM, following RA reduction. Together, these experiments demonstrate that RA signaling performs a very early function critical for the progression of gastrulation morphogenetic movements.

Bmp Signal Gradient Modulates Convergent Cell Movement via Xarhgef3.2 during Gastrulation of Xenopus Embryos.

Gastrulation is a critical step in the establishment of a basic body plan during development. Convergence and extension (CE) cell movements organize germ layers during gastrulation. Noncanonical Wnt signaling has been known as major signaling that regulates CE cell movement by activating Rho and Rac. In addition, Bmp molecules are expressed in the ventral side of a developing embryo, and the ventral mesoderm region undergoes minimal CE cell movement while the dorsal mesoderm undergoes dynamic cell movements. This suggests that Bmp signal gradient may affect CE cell movement. To investigate whether Bmp signaling negatively regulates CE cell movements, we performed microarray-based screening and found that the transcription of Xenopus Arhgef3.2 (Rho guanine nucleotide exchange factor) was negatively regulated by Bmp signaling. We also showed that overexpression or knockdown of Xarhgef3.2 caused gastrulation defects. Interestingly, Xarhgef3.2 controlled gastrulation cell movements through interacting with Disheveled (Dsh2) and Dsh2-associated activator of morphogenesis 1 (Daam1). Our results suggest that Bmp gradient affects gastrulation cell movement (CE) via negative regulation of Xarhgef3.2 expression.

Rab35 regulates skeletogenesis and gastrulation by facilitating actin remodeling and vesicular trafficking.

Rab35 is a small GTPase that regulates plasma membrane to early endosome vesicular trafficking and mediates actin remodeling to form actin-rich cellular structures. While the function of Rab35 in the cellular context has been examined, its role during development has not been well studied. In this study, we take advantage of the sea urchin's high fecundity, external fertilization, and transparent embryos to determine the function of Rab35 during development. We found that loss of function of Rab35 results in defects in skeletogenesis and gastrulation, which were rescued by co-injection of sea urchin Rab35. The loss of Rab35's function results in decreased endocytosis and impaired exocytosis, which may be important for skeletogenesis and gastrulation. Skeletal spicules of Rab35 knockdown embryos have reduced organized actin compared to the control, supporting the notion that Rab35 regulates actin dynamics. In addition, the skeletal and gastrulation defects induced by Rab35 knockdown were rescued by co-injection with Fascin, an actin-bundling protein, indicating that proper actin dynamics play a critical role for both skeletogenesis and gastrulation. Overall, results indicate that through its role in mediating vesicular trafficking and actin remodeling, Rab35 is an important regulator of embryonic structure formation in early development.

The X-linked acrogigantism-associated gene gpr101 is a regulator of early embryonic development and growth in zebrafish

We recently described X-linked acrogigantism (X-LAG), a condition of early childhood-onset pituitary gigantism associated with microduplications of the GPR101 receptor. The expression of GPR101 in hyperplastic pituitary regions and tumors in X-LAG patients, and GPR101's normally transient pituitary expression during fetal development, suggest a role in the regulation of growth. Nevertheless, little is still known about GPR101's physiological functions, especially during development. By using zebrafish models, we investigated the role of gpr101 during embryonic development and somatic growth. Transient ectopic gpr101 expression perturbed the embryonic body plan but did not affect growth. Loss of gpr101 led to a significant reduction in body size that was even more pronounced in the absence of maternal transcripts, as well as subfertility. These changes were accompanied by gastrulation and hypothalamic defects. In conclusion, both gpr101 loss- and gain-of-function affect, in different ways, fertility, embryonic patterning, growth and brain development.

Trim-Away mediated knock down uncovers a new function for Lbh during gastrulation of Xenopus laevis.

We previously identified the protein Lbh as necessary for cranial neural crest (CNC) cell migration in Xenopus through the use of morpholinos. However, Lbh is a maternally deposited protein and morpholinos achieve knockdowns through prevention of translation. In order to investigate the role of Lbh in earlier embryonic events, we employed the new technique “Trim-Away” to degrade this maternally deposited protein. Trim-Away utilizes the E3 ubiquitin ligase trim21 to degrade proteins targeted with an antibody and was developed in mammalian systems. Our results show that Xenopus is amenable to the Trim-Away technique. We also show that early knockdown of Lbh in Xenopus results in defects in gastrulation that present with a decrease in fibronectin matrix assembly, an increased in mesodermal cell migration and decrease in endodermal cell cohesion. We further show that the technique is also effective on a second abundant maternal protein PACSIN2. We discuss potential advantages and limit of the technique in Xenopus embryos as well as the mechanism of gastrulation inhibition.

Dusp1 modulates activin/smad2 mediated germ layer specification via FGF signal inhibition in Xenopus embryos

ABSTRACT Activin, a member of the transforming growth factor (TGF-β) superfamily, induces mesoderm, endoderm and neuro-ectoderm formation in Xenopus embryos. Despite several previous studies, the complicated gene regulatory network and genes involved in this induction await more elaboration. We identified expression of various fibroblast growth factor (FGF) genes in activin/smad2 treated animal cap explants (AC) of Xenopus embryos. Activin/smad2 increased fgf3/8 expression, which was reduced by co-injection of dominant negative activin receptor (DNAR) and dominant negative Fgf receptor (DNFR). Interestingly, activin/smad2 also increased expression of dual specificity phosphatase 1 (dusp1) which has been known to inhibit Fgf signaling. Dusp1 overexpression in dorsal marginal zone caused gastrulation defect and decreased Jnk/Erk phosphorylation as well as Smad1 linker region phosphorylation. Dusp1 decreased neural and organizer gene expression with increasing of endodermal and ventral gene expression in smad2 treated AC, indicating that dusp1 modulates germ layer specification. Dusp1 decreased neural gene expression in fgf8 treated AC, suggesting that Erk and/or Jnk phosphorylation may be involved in fgf8 induced neural induction. In addition, dusp1 decreased the reporter gene activities of activin response element (ARE) and increased it for bmp response element (BRE), indicating that dusp1 modulates two opposite morphogen signaling of dorsal (activin/Smad2) and ventral (bmp/Smad1) tracks, acting to fine tune the Fgf/Erk pathway.

Dynamic optima in cell sizes during early development enable normal gastrulation in zebrafish embryos

Cell migration is the main driver of the evolutionarily conserved process of gastrulation, which shapes metazoan embryo morphology. The molecular and cellular mechanisms of cell migration during gastrulation though well researched lacks an understanding of the contribution of cell sizes to collective cell migration. This is especially important during the early phase of metazoan development, which is dominated by constantly changing cell sizes in the background of which cells migrate en mass to shape the embryo. Here we investigate this phenomenon in zebrafish embryos, a model system in which early cell divisions causes cell sizes to decrease naturally over time as cells migrate collectively to sculpt the embryonic body plan. Because mutations that can perturb cell sizes so early in development do not exist, we generate haploid and tetraploid zebrafish embryos and show that cell sizes in such embryos are smaller and larger than the diploid norm, respectively. Cells in embryos made of smaller or larger than normal cells migrate sub-optimally, leading to gastrulation defects. Gene expression analysis suggests that the observed defects originate from altered cell size, and not from pleiotropic effects of altered ploidy. This interpretation is strengthened when gastrulation defects are rescued by increasing cell sizes in embryos wherein cell sizes are smaller than normal. We show that the migration defects are cell-autonomous by live imaging migrating haploid and tetraploid cells during gastrulation in chimeric diploid embryos. Analysis of membrane protrusion dynamics in single cells shows that cells normally extend protrusions non-uniformly during migration, a phenomenon which is perturbed when cell sizes deviate from the norm. Thus, an optimal range of developmental stage-specific cell sizes appears necessary for collective cell migration to correctly position cells in space and time to shape an amorphous ball of blastoderm into an embryo.

Embryonic ethanol exposure alters expression of sox2 and other early transcripts in zebrafish, producing gastrulation defects

Ethanol exposure during prenatal development causes fetal alcohol spectrum disorder (FASD), the most frequent preventable birth defect and neurodevelopmental disability syndrome. The molecular targets of ethanol toxicity during development are poorly understood. Developmental stages surrounding gastrulation are very sensitive to ethanol exposure. To understand the effects of ethanol on early transcripts during embryogenesis, we treated zebrafish embryos with ethanol during pre-gastrulation period and examined the transcripts by Affymetrix GeneChip microarray before gastrulation. We identified 521 significantly dysregulated genes, including 61 transcription factors in ethanol-exposed embryos. Sox2, the key regulator of pluripotency and early development was significantly reduced. Functional annotation analysis showed enrichment in transcription regulation, embryonic axes patterning, and signaling pathways, including Wnt, Notch and retinoic acid. We identified all potential genomic targets of 25 dysregulated transcription factors and compared their interactions with the ethanol-dysregulated genes. This analysis predicted that Sox2 targeted a large number of ethanol-dysregulated genes. A gene regulatory network analysis showed that many of the dysregulated genes are targeted by multiple transcription factors. Injection of sox2 mRNA partially rescued ethanol-induced gene expression, epiboly and gastrulation defects. Additional studies of this ethanol dysregulated network may identify therapeutic targets that coordinately regulate early development.

Wnt5b integrates Fak1a to mediate gastrulation cell movements via Rac1 and Cdc42.

Focal adhesion kinase (FAK) mediates vital cellular pathways during development. Despite its necessity, how FAK regulates and integrates with other signals during early embryogenesis remains poorly understood. We found that the loss of Fak1a impaired epiboly, convergent extension and hypoblast cell migration in zebrafish embryos. We also observed a clear disturbance in cortical actin at the blastoderm margin and distribution of yolk syncytial nuclei. In addition, we investigated a possible link between Fak1a and a well-known gastrulation regulator, Wnt5b, and revealed that the overexpression of fak1a or wnt5b could cross-rescue convergence defects induced by a wnt5b or fak1a antisense morpholino (MO), respectively. Wnt5b and Fak1a were shown to converge in regulating Rac1 and Cdc42, which could synergistically rescue wnt5b and fak1a morphant phenotypes. Furthermore, we generated several alleles of fak1a mutants using CRISPR/Cas9, but those mutants only revealed mild gastrulation defects. However, injection of a subthreshold level of the wnt5b MO induced severe gastrulation defects in fak1a mutants, which suggested that the upregulated expression of wnt5b might complement the loss of Fak1a. Collectively, we demonstrated that a functional interaction between Wnt and FAK signalling mediates gastrulation cell movements via the possible regulation of Rac1 and Cdc42 and subsequent actin dynamics.

Role of TrkA signaling during tadpole tail regeneration and early embryonic development in Xenopus laevis.

Neurotrophic signaling regulates neural cell behaviors in development and physiology, although its role in regeneration has not been fully investigated. Here, we examined the role of neurotrophic signaling in Xenopus laevis tadpole tail regeneration. After the tadpole tails were amputated, the expression of neurotrophin ligand family genes, especially ngf and bdnf, was up-regulated as regeneration proceeded. Moreover, notochordal expression of the NGF receptor gene TrkA, but not that of other neurotrophin receptor genes TrkB and TrkC, became prominent in the regeneration bud, a structure arising from the tail stump after tail amputation. The regenerated tail length was significantly shortened by the pan-Trk inhibitor K252a or the TrkA inhibitor GW-441756, but not by the TrkB inhibitor ANA-12, suggesting that TrkA signaling is involved in elongation of regenerating tails. Furthermore, during Xenopus laevis embryonic development, TrkA expression was detected in the dorsal mesoderm at the gastrula stage and in the notochord at the neurula stage, and its knockdown led to gastrulation defects with subsequent shortening of the body axis length. These results suggest that Xenopus laevis TrkA signaling, which can act in the mesoderm/notochord, plays a key role in body axis elongation during embryogenesis as well as tail elongation during tadpole regeneration.

P120-catenin regulates WNT signaling and EMT in the mouse embryo

Epithelial-to-mesenchymal transitions (EMTs) require a complete reorganization of cadherin-based cell-cell junctions. p120-catenin binds to the cytoplasmic juxtamembrane domain of classical cadherins and regulates their stability, suggesting that p120-catenin may play an important role in EMTs. Here, we describe the role of p120-catenin in mouse gastrulation, an EMT that can be imaged at cellular resolution and is accessible to genetic manipulation. Mouse embryos that lack all p120-catenin, or that lack p120-catenin in the embryo proper, survive to midgestation. However, mutants have specific defects in gastrulation, including a high rate of p53-dependent cell death, a bifurcation of the posterior axis, and defects in the migration of mesoderm; all are associated with abnormalities in the primitive streak, the site of the EMT. In embryonic day 7.5 (E7.5) mutants, the domain of expression of the streak marker Brachyury (T) expands more than 3-fold, from a narrow strip of posterior cells to encompass more than one-quarter of the embryo. After E7.5, the enlarged T+ domain splits in 2, separated by a mass of mesoderm cells. Brachyury is a direct target of canonical WNT signaling, and the domain of WNT response in p120-catenin mutant embryos, like the T domain, is first expanded, and then split, and high levels of nuclear β-catenin levels are present in the cells of the posterior embryo that are exposed to high levels of WNT ligand. The data suggest that p120-catenin stabilizes the membrane association of β-catenin, thereby preventing accumulation of nuclear β-catenin and excessive activation of the WNT pathway during EMT.