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Wnt/β-catenin signaling is required for embryonic stem cell (ESC) pluripotency by inducing mesodermal differentiation and inhibiting neuronal differentiation; however, how β-catenin counter-regulates these differentiation pathways is unknown. Here, we show that lysine 49 (K49) of β-catenin is trimethylated (β-catMe3) by Ezh2 or acetylated (β-catAc) by Cbp. Significantly, β-catMe3 acts as a transcriptional co-repressor of the neuronal differentiation genes sox1 and sox3, whereas β-catAc acts as a transcriptional co-activator of the key mesodermal differentiation gene t-brachyury (t-bra). Furthermore, β-catMe3 and β-catAc are alternatively enriched on repressed or activated genes, respectively, during ESC and adult stem cell differentiation into neuronal or mesodermal progenitor cell lineages. Importantly, expression of a β-catenin K49A mutant results in major defects in ESC differentiation. We conclude that β-catenin K49 trimethylation and acetylation are key elements in regulating ESC pluripotency and differentiation potential.

Embryonic stem cells (ESCs) are commonly used for the analysis of gene function in embryonic development and provide valuable models for human diseases. In recent years, ESCs have also become an attractive tool for toxicological testing, in particular for the identification of teratogenic compounds. We have recently described a Bmp-reporter ESC line as a new tool to identify teratogenic compounds and to characterize the molecular mechanisms mediating embryonic toxicity. Here we describe the use of a Wnt/β-Catenin-reporter ESC line isolated from a previously described mouse line that carries the LacZ reporter gene under the control of a β-Catenin responsive promoter. The reporter ESC line stably differentiates into cardiomyocytes within 12 days. The reporter was endogenously induced between day 3-5 of differentiation reminiscent of its expression in vivo, in which strong LacZ activity is detected around gastrulation. Subsequently its expression becomes restricted to mesodermal cells and cells undergoing an epithelial to mesenchymal transition. The Wnt/β-Catenin-dependent expression of the reporter protein allowed quantification of dose- and time-dependent effects of teratogenic chemicals. In particular, valproic acid reduced reporter activity on day 7 whereas retinoic acid induced reporter activity on day 5 at concentrations comparable to the ones inhibiting the formation of functional cardiomyocytes, the classical read-out of the embryonic stem cell test (EST). In addition, we were also able to show distinct effects of teratogenic chemicals on the Wnt/β-Catenin-reporter compared with the previously described Bmp-reporter ESCs. Thus, different reporter cell lines provide complementary tools for the identification and analysis of potentially teratogenic compounds.

Embryogenesis relies on the precise interplay of signaling cascades to activate tissue-specific differentiation programs. An important player in these morphogenetic processes is β-catenin, which is a central component of adherens junctions and canonical Wnt signaling. Lack of β-catenin is lethal before gastrulation, but mice heterozygous for β-catenin (Ctnnb1) develop as wild type. Here, we confine β-catenin amounts below the heterozygous expression level to study the functional consequences for development. We generate embryonic stem (ES) cells and embryos expressing β-catenin only from the ubiquitously active ROSA26 promoter and thereby limit β-catenin expression to ~12.5% (ROSA26β/+) or ~25% (ROSA26β/β) of wild-type levels. ROSA26β/+ is sufficient to maintain ES cell morphology and pluripotent characteristics, but is insufficient to activate canonical target genes upon Wnt stimulation. This Wnt signaling deficiency is incompletely restored in ROSA26β/β ES cells. We conclude that even very low β-catenin levels are able to sustain cell adhesion, but not Wnt signaling. During development, ROSA26β/β as well as ROSA26β/+ partially rescues the knockout phenotype, yet proper gastrulation is absent. These embryos differentiate according to the neural default hypothesis, indicating that gastrulation depends on high β-catenin levels. Strikingly, if ROSA26β/+ or ROSA26β/β is first activated after gastrulation, subsequent development correlates with the dosage of β-catenin. Moreover, molecular evidence indicates that the amount of β-catenin controls the induction of specific Wnt target genes. In conclusion, by restricting its expression we determine the level of β-catenin required for adhesion or pluripotency and during different morphogenetic events.

β-Catenin/Wnt signaling is essential during early inductive stages of kidney development, but its role during postinductive stages of nephron development and maturation is not well understood. In this study, we used Pax8Cre mice to target β-catenin deficiency to renal epithelial cells at the late S-shaped body stage and the developing collecting ducts. The conditional β-catenin knockout mice formed abnormal kidneys and had reduced renal function. The kidneys were hypoplastic with a thin cortex; a superficial layer of tubules was missing. A high proportion of glomeruli had small, underdeveloped capillary tufts. In these glomeruli, well differentiated podocytes replaced parietal epithelial cells in Bowman’s capsule; capillaries toward the outer aspect of these podocytes mimicked the formation of glomerular capillaries. Tracing nephrogenesis in embryonic conditional β-catenin knockout mice revealed that these “parietal podocytes” derived from precursor cells in the parietal layer of the S-shaped body by direct lineage switch. Taken together, these findings demonstrate that β-catenin/Wnt signaling is important during the late stages of nephrogenesis and for the lineage specification of parietal epithelial cells.

We conditionally substituted E-cadherin (E-cad; cadherin 1) with N-cadherin (N-cad; cadherin 2) during intestine development by generating mice in which an Ncad cDNA was knocked into the Ecad locus. Mutant mice were born, demonstrating that N-cad can structurally replace E-cad and establish proper organ architecture. After birth, mutant mice gradually developed a mutant phenotype in both the small and large intestine and died at ~2-3 weeks of age, probably due to malnutrition during the transition to solid food. Molecular analysis revealed an extended domain of cells from the crypt into the villus region, with nuclear localization of β-catenin (β-cat; Ctnnb1) and enhanced expression of several β-cat target genes. In addition, the BMP signaling pathway was suppressed in the intestinal epithelium of the villi, suggesting that N-cad might interfere with BMP signaling in the intestinal epithelial cell layer. Interestingly, mutant mice developed severe dysplasia and clusters of cells with neoplastic features scattered along the crypt-villus axis in the small and large intestine. Our experimental model indicates that, in the absence of E-cad, the sole expression of N-cad in an epithelial environment is sufficient to induce neoplastic transformations.

We have previously demonstrated that differentiation of embryonic stem (ES) cells is associated with downregulation of cell surface E-cadherin. In this study, we assessed the function of E-cadherin in mouse ES cell pluripotency and differentiation. We show that inhibition of E-cadherin-mediated cell–cell contact in ES cells using gene knockout (Ecad−/−), RNA interference (EcadRNAi), or a transhomodimerization-inhibiting peptide (CHAVC) results in cellular proliferation and maintenance of an undifferentiated phenotype in fetal bovine serum-supplemented medium in the absence of leukemia inhibitory factor (LIF). Re-expression of E-cadherin in Ecad−/−, EcadRNAi, and CHAVC-treated ES cells restores cellular dependence to LIF supplementation. Although reversal of the LIF-independent phenotype in Ecad−/− ES cells is dependent on the β-catenin binding domain of E-cadherin, we show that β-catenin null (βcat−/−) ES cells also remain undifferentiated in the absence of LIF. This suggests that LIF-independent self-renewal of Ecad−/− ES cells is unlikely to be via β-catenin signaling. Exposure of Ecad−/−, EcadRNAi, and CHAVC-treated ES cells to the activin receptor-like kinase inhibitor SB431542 led to differentiation of the cells, which could be prevented by re-expression of E-cadherin. To confirm the role of transforming growth factor β family signaling in the self-renewal of Ecad−/− ES cells, we show that these cells maintain an undifferentiated phenotype when cultured in serum-free medium supplemented with Activin A and Nodal, with fibroblast growth factor 2 required for cellular proliferation. We conclude that transhomodimerization of E-cadherin protein is required for LIF-dependent ES cell self-renewal and that multiple self-renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context. Disclosure of potential conflicts of interest is found at the end of this article.

Transgenic mice with a defined cell- or tissues-specific expression of Cre-recombinase are essential tools to study gene function. Here we report the generation and analysis of a transgenic mouse line (Cdx1::Cre) with restricted Cre-expression from Cdx1 regulatory elements. The expression of Cre-recombinase mimicked the endogenous expression pattern of Cdx1 at midgastrulation (from E7.5 to early headfold stage) inducing recombination in the three germlayers of the primitive streak region throughout the posterior embryo and caudal to the heart. This enables gene modifications to investigate patterning of the caudal embryo during and after gastrulation. Interestingly, we identified Cdx1 expression in the trophectoderm (TE) of blastocyst stage embryos. Concordantly, we detected extensive Cre-mediated recombination in the polar TE and, although to lesser extent, in the mural TE. In E7.5 postimplantation embryos, almost all cells of the extraembryonic ectoderm (ExE), which are derived from the polar TE, are recombined although the ExE itself is negative for Cdx1 and Cre at this stage. These results indicate that Cdx1::Cre mice are also a valuable tool to study gene function in tissues essential for placental development.

Hematopietic stem cells (HSCs) maintain life-long hematopoiesis in the bone marrow via their ability to self-renew and to differentiate into all blood lineages. Although a central role for the canonical wnt signaling pathway has been suggested in HSC self-renewal as well as in the development of B and T cells, conditional deletion of beta-catenin (which is considered to be essential for Wnt signaling) has no effect on hematopoiesis or lymphopoiesis. Here, we address whether this discrepancy can be explained by a redundant and compensatory function of gamma-catenin, a close homolog of beta-catenin. Unexpectedly, we find that combined deficiency of beta- and gamma-catenin in hematopoietic progenitors does not impair their ability to self-renew and to reconstitute all myeloid, erythroid, and lymphoid lineages, even in competitive mixed chimeras and serial transplantations. These results exclude an essential role for canonical Wnt signaling (as mediated by beta- and/or gamma-catenin) during hematopoiesis and lymphopoiesis.

β-Catenin plays a key role in cadherin-mediated cell adhesion as well as in canonical Wnt signaling. To study the role of β-catenin during eye development, we used conditional Cre/loxP system in mouse to inactivate β-catenin in developing lens and retina. Inactivation of β-catenin does not suppress lens fate, but instead results in abnormal morphogenesis of the lens. Using BAT-gal reporter mice, we show that β-catenin-mediated Wnt signaling is notably absent from lens and neuroretina throughout eye development. The observed defect is therefore likely due to the cytoskeletal role of β-catenin, and is accompanied by impaired epithelial cell adhesion. In contrast, inactivation of β-catenin in the nasal ectoderm, an area with active Wnt signaling, results in formation of crystallin-positive ectopic lentoid bodies. These data suggest that, outside of the normal lens, β-catenin functions as a coactivator of canonical Wnt signaling to suppress lens fate. genesis 45:157–168, 2007. Published 2007 Wiley-Liss, Inc.

Embryonic stem cells (ESCs) were first isolated from mouse embryos more than 30 years ago. They have proven invaluable not only in generating genetically modified mice that allow for analysis of gene function in tissue development and homeostasis but also as models for genetic disease. In addition, ESCs in vitro are finding inroads in pharmaceutical and toxicological testing, including the identification of teratogenic compounds. Here, we describe the use of a bone morphogenetic protein (Bmp)-reporter ESC line, isolated from a well-characterized transgenic mouse line, as a new tool for the identification of chemical teratogens. The Bmp-mediated expression of the green fluorescent protein enabled the quantification of dose- and time-dependent effects of valproic acid as well as retinoic acid. Significant effects were detectable at concentrations that were comparable to the ones observed in the classical embryonic stem cell test, despite the fact that the reporter gene is expressed in distinct cell types, including endothelial and endodermal cells. Thus these cells provide a valuable new tool for the identification and characterization of relevant mechanisms of embryonic toxicity.

Endothelial cells express two classical cadherins, VE-cadherin and N-cadherin. VE-cadherin is absolutely required for vascular morphogenesis, but N-cadherin is thought to participate in vessel stabilization by interacting with periendothelial cells during vessel formation. However, recent data suggest a more critical role for N-cadherin in endothelium that would regulate angiogenesis, in part by controlling VE-cadherin expression. In this study, we have assessed N-cadherin function in vascular development using an in vitro model derived from embryonic stem (ES) cell differentiation. We show that pluripotent ES cells genetically null for N-cadherin can differentiate normally into endothelial cells. In addition, sprouting angiogenesis was unaltered, suggesting that N-cadherin is not essential for the early events of angiogenesis. However, the lack of N-cadherin led to an impairment in pericyte covering of endothelial outgrowths. We conclude that N-cadherin is necessary neither for vasculogenesis nor proliferation and migration of endothelial cells but is required for the subsequent maturation of endothelial sprouts by interacting with pericytes.

Cadherins have been known for a long time to be key elements in many important biological processes. In particular, the role of classical cadherins in mediating adhesion has been examined in great detail. Over recent years, the accumulation of experimental tools and mice mutants has allowed more refined analysis of cadherin functions, and new aspects such as signaling and synapse dynamics have become the center of interest. In addition, the study of mice lacking the entire protocadherin-gamma cluster shed the first light on a possible novel function of members of this cadherin family in synapse formation and cell survival during development.

Differential gene regulation integrated in time and space drives developmental programs during embryogenesis. To understand how the program of gastrulation is regulated by Wnt/β-catenin signaling, we have used genome-wide expression profiling of conditional β-catenin mutant embryos. Known Wnt/β-catenin target genes, known components of other signaling pathways, as well as a number of uncharacterized genes were downregulated in these mutants. To further narrow down the set of differentially expressed genes, we used whole-mount in situ screening to associate gene expression with putative domains of Wnt activity. Several potential novel target genes were identified by this means and two, Grsf1 and Fragilis2, were functionally analyzed by RNA interference (RNAi) in completely embryonic stem (ES) cell-derived embryos. We show that the gene encoding the RNA-binding factor Grsf1 is important for axial elongation, mid/hindbrain development and axial mesoderm specification, and that Fragilis2, encoding a transmembrane protein, regulates epithelialization of the somites and paraxial mesoderm formation. Intriguingly, the knock-down phenotypes recapitulate several aspects of Wnt pathway mutants, suggesting that these genes are components of the downstream Wnt response. This functional genomic approach allows the rapid identification of functionally important components of embryonic development from large datasets of putative targets.

Cadherin-mediated cell-cell adhesion plays important roles in mouse embryonic development, and changes in cadherin expression are often linked to morphogenetic events. For proper embryonic development and organ formation,the expression of E-cadherin must be tightly regulated. Dysregulated expression during tumorigenesis confers invasiveness and metastasis. Except for the E-box motifs in the E-cadherin promoter, little is known about the existence and location of cis-regulatory elements controlling E-cadherin gene expression. We have examined putative cis-regulatory elements in the E-cadherin gene and we show a pivotal role for intron 2 in activating transcription. Upon deleting the genomic intron 2 entirely, the E-cadherin locus becomes completely inactive in embryonic stem cells and during early embryonic development. Later in development, from E11.5 onwards, the locus is activated only weakly in the absence of intron 2 sequences. We demonstrate that in differentiated epithelia, intron 2 sequences are required both to initiate transcriptional activation and additionally to maintain E-cadherin expression. Detailed analysis also revealed that expression in the yolk sac is intron 2 independent, whereas expression in the lens and the salivary glands absolutely relies on cis-regulatory sequences of intron 2. Taken together, our findings reveal a complex mechanism of gene regulation, with a vital role for the large intron 2.

The recently described protocadherin gene clusters encode cadherin-related proteins, which are highly expressed in the vertebrate nervous system. Here, we report biochemical studies addressing proteolytic processing of gamma-protocadherins. These type-I transmembrane proteins are cleaved by a metalloproteinase in vivo, generating a soluble extracellular fragment and a carboxyl-terminal fragment associated with the cellular membrane. In addition, we show that the carboxyl-terminal fragment is a substrate for further cleavage mediated by presenilin. Consequently, accumulation of the fragment is found when gamma-secretase is inactivated either by the specific presenilin-inhibitor L685,458 or in double mutant murine embryonic fibroblasts lacking both presenilin genes. The gamma-secretase-generated carboxyl-terminal fragment is largely unstable but accumulates when proteasomal degradation is inhibited. Interestingly, the proteolytic fragment generated by gamma-secretase can localize to the nucleus. This is the first report providing experimental evidence for a cell surface receptor signaling function of protocadherins regulated by proteolytic events.

Many components of the Wnt/beta-catenin signaling pathway are expressed during mouse pre-implantation embryo development, suggesting that this pathway may control cell proliferation and differentiation at this time. We find no evidence for a functional activity of this pathway in cleavage-stage embryos using the Wnt-reporter line, BAT-gal. To further probe the activity of this pathway, we activated beta-catenin signaling by mating a zona pellucida3-cre (Zp3-cre) transgenic mouse line with a mouse line containing an exon3-floxed beta-catenin allele. The result is expression of a stabilized form of beta-catenin, resistant to degradation by the GSK3beta-mediated proteasome pathway, expressed in the developing oocyte and in each cell of the resulting embryos. Nuclear localization and signaling function of beta-catenin were not observed in cleavage-stage embryos derived from these oocytes. These results indicate that in pre-implantation embryos, molecular mechanisms independent of the GSK3beta-mediated ubiquitination and proteasome degradation pathway inhibit the nuclear function of beta-catenin. Although the mutant blastocysts initially developed normally, they then exhibited a specific phenotype in the embryonic ectoderm layer of early post-implantation embryos. We show a nuclear function of beta-catenin in the mutant epiblast that leads to activation of Wnt/beta-catenin target genes. As a consequence, cells of the embryonic ectoderm change their fate, resulting in a premature epithelial-mesenchymal transition.

Wnt signaling has recently emerged as a key factor in controlling stem cell expansion. In contrast, we show here that Wnt/β-catenin signal activation in emigrating neural crest stem cells (NCSCs) has little effect on the population size and instead regulates fate decisions. Sustained β-catenin activity in neural crest cells promotes the formation of sensory neural cells in vivo at the expense of virtually all other neural crest derivatives. Moreover, Wnt1 is able to instruct early NCSCs (eNCSCs) to adopt a sensory neuronal fate in a β-catenin–dependent manner. Thus, the role of Wnt/β-catenin in stem cells is cell-type dependent.

β-catenin–mediated Wnt signaling has been suggested to be critically involved in hematopoietic stem cell maintenance and development of T and B cells in the immune system. Unexpectedly, here we report that inducible Cre-loxP–mediated inactivation of the β-catenin gene in bone marrow progenitors does not impair their ability to self-renew and reconstitute all hematopoietic lineages (myeloid, erythroid, and lymphoid), even in competitive mixed chimeras. In addition, both thymocyte survival and antigen-induced proliferation of peripheral T cells is β-catenin independent. In contrast to earlier reports, these data exclude an essential role for β-catenin during hematopoiesis and lymphopoiesis.

E-cadherin is thought to mediate intercellular adhesion in the mammalian epidermis and in hair follicles as the adhesive component of adherens junctions. We have tested this role of E-cadherin directly by conditional gene ablation in the mouse. We show that postnatal loss of E-cadherin in keratinocytes leads to a loss of adherens junctions and altered epidermal differentiation without accompanying signs of inflammation. Overall tissue integrity and desmosomal structures were maintained, but skin hair follicles were progressively lost. Tumors were not observed and beta-catenin levels were not strongly altered in the mutant skin. We conclude that E-cadherin is required for maintaining the adhesive properties of adherens junctions in keratinocytes and proper skin differentiation. Furthermore, continuous hair follicle cycling is dependent on E-cadherin.

The Nieuwkoop center provides signals essential for the establishment of the dorsal gastrula organizer in vertebrates. Activation of β-catenin is one of the events in the Nieuwkoop center that lead to activation of dorsal-specific genes during blastula and early gastrula stages. Zebrafish bozozok (boz) mutant embryos have severe defects in axial mesoderm and anterior neuroectoderm. The boz gene is activated in the organizer in response to β-catenin signaling, and Boz protein has been demonstrated to contribute to organizer formation by repression of ventralizing genes, including bmp2b, vega1, and vega2. Here, we investigate the timing and molecular mechanism by which boz expression is activated in the organizer. We demonstrate that boz is already expressed before midblastula transition (MBT). We further identify high-affinity binding sites for Tcf/Lef1 within the boz promoter region. These sites, together with the finding that β-catenin induces boz expression, indicate that transcription of boz may be activated directly by β-catenin/Lef1. We hypothesize that pre-MBT activation of boz may be important to build up a sufficiently strong antagonizing activity against zygotic ventralizing genes activated immediately post-MBT. Thus, the early onset of boz expression may be crucial for organizer establishment in the presence of ubiquitous maternal activators of ventralizing genes. Developmental Dynamics, 2003. © 2003 Wiley-Liss, Inc.

Using the Cre/loxP system we conditionally inactivated β-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured β-catenin −/− endothelial cells showed a different organization of intercellular junctions with a decrease in α-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell–cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of β-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages.

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in β-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. β-Catenin–null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density–enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin–β-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell–cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin–null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.

The vertebral column derives from somites generated by segmentation of presomitic mesoderm (PSM). Somitogenesis involves a molecular oscillator, the segmentation clock, controlling periodic Notch signaling in the PSM. Here, we establish a novel link between Wnt/β-catenin signaling and the segmentation clock. Axin2 , a negative regulator of the Wnt pathway, is directly controlled by Wnt/β-catenin and shows oscillating expression in the PSM, even when Notch signaling is impaired, alternating with Lfng expression. Moreover, Wnt3a is required for oscillating Notch signaling activity in the PSM. We propose that the segmentation clock is established by Wnt/β-catenin signaling via a negative-feedback mechanism and that Wnt3a controls the segmentation process in vertebrates.

The formation of the apical ectodermal ridge (AER) is critical for the distal outgrowth and patterning of the vertebrate limb. Recent work in the chick has demonstrated that interplay between theWntandFgfsignaling pathways is essential in the limb mesenchyme and ectoderm in the establishment and perhaps the maintenance of the AER. In the mouse, whereas a role forFgfsfor AER establishment and function has been clearly demonstrated, the role ofWnt/β-cateninsignaling, although known to be important, is obscure. In this study, we demonstrate thatWnt3, which is expressed ubiquitously throughout the limb ectoderm, is essential for normal limb development and plays a critical role in the establishment of the AER. We also show that the conditional removal ofβ-cateninin the ventral ectodermal cells is sufficient to elicit the mutant limb phenotype. In addition, removingβ-cateninafter the induction of the ridge results in the disappearance of the AER, demonstrating the requirement for continuedβ-cateninsignaling for the maintenance of this structure. Finally, we demonstrate thatWnt/β-cateninsignaling lies upstream of theBmpsignaling pathway in establishment of the AER and regulation of the dorsoventral polarity of the limb.

beta-Catenin is a multi-functional cellular component and a substrate for several protein kinases. Here we investigated the interaction of protein kinase CKII (casein kinase II) and beta-catenin. We show that CKII phosphorylates the N-terminal region of beta-catenin and we identified Ser29, Thr102, and Thr112 as substrates for the enzyme. We provide evidence that CKII regulates the cytoplasmic stability of beta-catenin and acts synergistically with GSK-3beta in the multi-protein complex that controls the degradation of beta-catenin. In comparing wild-type and Ser/Thr-mutant beta-catenin, a decreased affinity of the mutant protein to alpha-catenin was observed. Moreover, kinase assays in vitro demonstrate a CKII-dependent increase in the binding of wild-type beta-catenin with alpha-catenin. In line with that, cells expressing Ser/Thr-mutant beta-catenin exhibit an increased migratory potential, which correlates with an enhanced cytosolic localization and a reduced association with the cytoskeleton of the mutant protein. From these results we conclude that CKII regulates the function of beta-catenin in the cadherin adhesion complex as well as its cytoplasmic stability.

The calcium-dependent adhesion protein E-cadherin is present in noncompacted regions of myelin sheaths in the peripheral nervous system. There, it is localized to electron-dense structures between membranes of the same Schwann cell referred to as autotypic adherens junctions. It has been suggested that the failure of E-cadherin-mediated adhesion might cause demyelination that proceeds in certain pathological states. To test the requirement of E-cadherin in peripheral nerves, we used tissue-specific gene ablation techniques based on the Cre/LoxP system. We show that E-cadherin deficiency does not cause significant demyelination up to the age of 15 months. Immunostainings for nodal sodium channels, the paranodal protein Caspr1, and the juxtaparanodal potassium channels Kv1.1 and Kv1.2 revealed that E-cadherin is not necessary to maintain the general functional architecture of the nodal region. On the ultrastructural level, we detected a widening of the outer mesaxon accompanied by a loss of electron-dense cytoplasmic areas. We conclude that E-cadherin is required for the proper establishment and/or the maintenance of the outer mesaxon in myelinated PNS fibers but is dispensable for proper nerve function.

Protocadherins constitute the largest subgroup within the cadherin family of calcium-dependent cell-cell adhesion molecules. Recent progress in genome sequencing has enabled a refined phylogenetic analysis of protocadherins and led to the discovery of three large protocadherin clusters on human chromosome 5/mouse chromosome 18. Interestingly, many of the circa 70 protocadherins in mammals are highly expressed in the central nervous system. Roles in tissue morphogenesis and formation of neuronal circuits during early vertebrate development have been inferred. In the postnatal brain, protocadherins are possibly involved in the modulation of synaptic transmission and the generation of specific synaptic connections.

Members of the Caudal-related homeobox gene family are implicated in anteroposterior (AP) patterning and posterior axis elongation in mammals. Previously, we found that Caudal-related homeobox gene1 (Cdx1) is directly regulated by the Wnt/beta-catenin signaling pathway, and we identified Wnt/beta-catenin responsive elements in the Cdx1 promoter. Here, we have generated a series of transgenic mouse lines to determine if the Cdx1 promoter region (-3.6 kb to -0.7 kb) sufficient to drive reporter gene expression in the normal Cdx1 expression domains. A 0.7-kb Cdx1 upstream regulatory sequence is sufficient to give the early phase of Cdx1 expression in the primitive streak. However, none of the reporter constructs was able to drive expression in the definitive endoderm at later stages of development. More detailed analysis of the cis-regulatory elements of this 0.7-kb Cdx1 promoter region revealed that two Wnt/beta-catenin responsive elements are important for initiation of Cdx1 expression in the primitive streak and one retinoic acid response element for maintenance. Our analysis demonstrates that the early Cdx1 expression is regulated through the caudalizing Wnt/beta-catenin and RA activities.

Using Cre/ loxP , we conditionally inactivated the β - catenin gene in cells of structures that exhibit important embryonic organizer functions: the visceral endoderm, the node, the notochord, and the definitive endoderm. Mesoderm formation was not affected in the mutant embryos, but the node was missing, patterning of the head and trunk was affected, and no notochord or somites were formed. Surprisingly, deletion of β-catenin in the definitive endoderm led to the formation of multiple hearts all along the anterior-posterior (A/P) axis of the embryo. Ectopic hearts developed in parallel with the normal heart in regions of ectopic Bmp2 expression. We provide evidence that ablation of β-catenin in embryonic endoderm changes cell fate from endoderm to precardiac mesoderm, consistent with the existence of bipotential mesendodermal progenitors in mouse embryos.

The Ca(++)-dependent cell adhesion molecule E-cadherin is expressed throughout mouse development and in adult tissues. Classical gene targeting has demonstrated that E-cadherin-deficient embryos die at the blastocyst stage. To study the involvement of E-cadherin in organogenesis, a conditional gene inactivation scheme was undertaken using the bacteriophage P1 recombinase Cre/loxP system. Mice with homozygous loxP sites in both alleles of the E-cadherin (Cdh1) gene were generated and these mice were crossed with transgenic mice with the Cre recombinase under the control of the hormone-inducible MMTV promoter. This resulted in deletion of the E-cadherin gene in the differentiating alveolar epithelial cells of the mammary gland. The mutant mammary gland developed normally up to 16-18 days of pregnancy but exhibited a dramatic phenotype around parturition. The production of milk proteins was so drastically reduced that adult mutant mothers could not suckle their offspring. Thus, the lack of E-cadherin affected the terminal differentiation program of the lactating mammary gland. In concordance with this finding, the prolactin-dependent activation of the transcription factor Stat5a was initiated but not maintained in the mutant gland. Instead, without E-cadherin massive cell death was observed at parturition and the mutant mammary gland at this stage resembled that of the involuted gland normally seen after weaning. These results demonstrate an essential role for E-cadherin in the function of differentiated alveolar epithelial cells. No tumors were detected in mutant glands lacking E-cadherin.

The distinct expression of R-cadherin in the induced aggregating metanephric mesenchyme suggests that it may regulate the mesenchymal-epithelial transition during kidney development. To address whether R-cadherin is required for kidney ontogeny, R-cadherin-deficient mice were generated. These mice appeared to be healthy and were fertile, demonstrating that R-cadherin is not essential for embryogenesis. The only kidney phenotype of adult mutant animals was the appearance of dilated proximal tubules, which was associated with an accumulation of large intracellular vacuoles. Morphological analysis of nephrogenesis in R-cadherin(-/-) mice in vivo and in vitro revealed defects in the development of both ureteric bud-derived cells and metanephric mesenchyme-derived cells. First, the morphology and organization of the proximal parts of the ureteric bud epithelium were altered. Interestingly, these morphological changes correlated with an increased rate of apoptosis and were further supported by perturbed branching and patterning of the ureteric bud epithelium during in vitro differentiation. Second, during in vitro studies of mesenchymal-epithelial conversion, significantly fewer epithelial structures developed from R-cadherin(-/-) kidneys than from wild-type kidneys. These data suggest that R-cadherin is functionally involved in the differentiation of both mesenchymal and epithelial components during metanephric kidney development. Finally, to investigate whether the redundant expression of other classic cadherins expressed in the kidney could explain the rather mild kidney defects in R-cadherin-deficient mice, we intercrossed R-cadherin(-/-) mice with cadherin-6(-/-), P-cadherin(-/-), and N-cadherin(+/-) mice. Surprisingly, however, in none of the compound knockout strains was kidney development affected to a greater extent than within the individual cadherin knockout strains.

Wnt signaling regulates cell fate decisions and cell proliferation during development and in adult tissues in both invertebrates and vertebrates. Here we describe the identification of Wnt genes, Wnt2a, 4, 5a, 5b, 6 and 11, expressed in mouse embryonic gut development. Each of these genes exhibits a characteristic and regional-specific expression pattern along the anterior–posterior axis of the digestive tube between embryonic day (E) 12.5 and 16.5 of embryonic development. The expression of Wnt5a is confined to the mesenchymal compartment, while expression of Wnt4 is found both in the intestinal epithelium and the mesenteric anlage. Wnt11 is expressed in the epithelium of esophagus and colon, but also in mesenchymal cells of the stomach. Wnt5b and Wnt6 exhibit restricted expression in the epithelium of the esophagus. A characteristic regionalized expression pattern is observed in the developing stomach. Wnt5a is expressed in the mesenchymal layer of the prospective gland region but becomes restricted to the tip of the gland region by E14.5. Wnt11 is highly expressed at the gastro-esophageal junctions, while Wnt4 is found in the epithelium lining the pyloric region of the stomach but not in the epithelium of the prospective gland region.

Cadherin-mediated cell–cell interactions are thought to be critical in controlling cell sorting during embryogenesis. Here, we report that chimeric embryos generated with N-cadherin-deficient (N-cadherin −/− ) embryonic stem cells develop further than embryos completely lacking N-cadherin only when the myocardium consists of N-cadherin-positive cells. Initially, the N-cadherin-negative and -positive cells mix together to form chimeric tissues; however, by embryonic day 9.5, the N-cadherin −/− cells segregate from the wild-type cells forming distinct aggregates. The chimeric embryos have large aggregates of N-cadherin −/− myocardial cells in the heart lumen, indicating that the cells are unable to maintain cell–cell contacts with N-cadherin-positive myocytes. This sorting-out phenomenon also is apparent in somites, neural tube, and developing brain where N-cadherin −/− cells form distinct lumenal structures. These studies demonstrate that N-cadherin-mediated adhesion is critical for maintaining cell–cell interactions in tissues undergoing active cellular rearrangements and increased mechanical stress associated with morphogenesis.

β-Catenin is a central component of both the cadherin- catenin cell adhesion complex and the Wnt signaling pathway. We have investigated the role of β-catenin during brain morphogenesis, by specifically inactivating the β- catenin gene in the region of Wnt1 expression. To achieve this, mice with a conditional (‘floxed’) allele of β-catenin with required exons flanked by loxP recombination sequences were intercrossed with transgenic mice that expressed Cre recombinase under control of Wnt1 regulatory sequences. β-catenin gene deletion resulted in dramatic brain malformation and failure of craniofacial development. Absence of part of the midbrain and all of the cerebellum is reminiscent of the conventional Wnt1 knockout (Wnt1−/−), suggesting that Wnt1 acts through β- catenin in controlling midbrain-hindbrain development. The craniofacial phenotype, not observed in embryos that lack Wnt1, indicates a role for β-catenin in the fate of neural crest cells. Analysis of neural tube explants shows that β-catenin is efficiently deleted in migrating neural crest cell precursors. This, together with an increased apoptosis in cells migrating to the cranial ganglia and in areas of prechondrogenic condensations, suggests that removal of β-catenin affects neural crest cell survival and/or differentiation. Our results demonstrate the pivotal role of β-catenin in morphogenetic processes during brain and craniofacial development.

Wnt molecules control numerous developmental processes by altering specific gene expression patterns, and deregulation of Wnt signaling can lead to cancer. Many Wnt factors employ beta-catenin as a nuclear effector. Upon Wnt stimulation, beta-catenin heterodimerizes with T-cell factor (TCF) DNA-binding proteins to form a transcriptional activator complex. As the activating subunit of this complex, beta-catenin performs dual tasks: it alleviates repression of target gene promoters and subsequently it activates them. Beta-catenin orchestrates these effects by recruiting chromatin modifying cofactors and contacting components of the basal transcription machinery. Although beta-catenin and TCFs are universal activators in Wnt signaling, their target genes display distinct temporal and spatial expression patterns. Apparently, post-translational modifications modulate the interactions between TCFs and beta-catenin or DNA, and certain transcription factors can sequester beta-catenin from TCFs while others synergize with beta-catenin-TCF complexes in a promoter-specific manner. These mechanisms provide points of intersection with other signaling pathways, and contribute to the complexity and specificity of Wnt target gene regulation.

E-cadherin is the main cell adhesion molecule of early embryonic and adult epithelial cells. Downregulation of E-cadherin is associated with epithelial-mesenchymal transition during embryonic mesoderm formation and tumor progression. To identify genes whose expression is affected by the loss of E-cadherin, we compared mRNA expression patterns between wild-type and E-cadherin null mutant embryonic stem (ES) cells. We found that expression of several Eph receptors and ephrins is dependent on E-cadherin. Rescue of E-cadherin null ES cells with E-cadherin cDNA restores the wild-type expression pattern of Eph family members. Rescue of E-cadherin null ES cells with N-cadherin cDNA does not restore the wild-type expression pattern, indicating that the regulation of differential expression of Eph family members is specific to E-cadherin. Constitutive ectopic expression of E-cadherin in non-epithelial NIH3T3 cells results in the production of the EphA2 receptor. In epithelial cells, E-cadherin is required for EphA2 receptor localization at cell-cell contacts; in the absence of functional E-cadherin, EphA2 localizes to the perinuclear region. Our results indicate that E-cadherin may be directly or indirectly required for the membrane localization of Eph receptors and their membrane-bound ligands.

To identify target genes of the Wnt/beta-catenin signaling pathway in early mouse embryonic development we have established a co-culture system consisting of NIH3T3 fibroblasts expressing different Wnts as feeder layer cells and embryonic stem (ES) cells expressing a green fluorescent protein (GFP) reporter gene transcriptionally regulated by the TCF/beta-catenin complex. ES cells specifically respond to Wnt signal as monitored by GFP expression. In GFP-positive ES cells we observe expression of Brachyury. Two TCF binding sites located in a 500 bp Brachyury promoter fragment bind the LEF-1/beta-catenin complex and respond specifically to beta-catenin-dependent transactivation. From these results we conclude that Brachyury is a target gene for Wnt/beta-catenin signaling.

Lateral clustering of E-cadherin molecules is required for the adhesive properties of this cell-cell adhesion molecule. Both the extracellular domain and the cytoplasmic region of E-cadherin were previously reported to contribute to lateral clustering, but little is known about a role of the transmembrane domain in this respect. Following our previous findings indicating self-assembly of artificial transmembrane segments based on leucine residues, we asked whether the leucine-rich transmembrane segment of E-cadherin participates in lateral clustering. Here, we demonstrate that its transmembrane domain self-assembles as analyzed using the ToxR reporter system. Certain point mutations within the transmembrane domain markedly reduced self-assembly. To study whether the same point mutations also affect E-cadherin-mediated adhesion in vivo, wild-type and mutant E-cadherin cDNAs were transfected into Ltk(-) cells. Indeed, cell aggregation assays revealed significantly reduced adhesiveness when mutations had been introduced which disrupted transmembrane segment interaction. In control experiments, cell-surface expression, interaction with catenins and the cytoskeleton as well as trypsin-resistance of the protein were unaffected. These data suggest that interactions between the transmembrane segments are important for the lateral association of E-cadherin molecules required for cell-cell adhesion.

During mouse preimplantation development, the components of the E-cadherin-catenin complex are derived from both maternal and zygotic gene activity and the adhesion complex is increasingly accumulated and stored in a nonfunctional form, ready to be used for compaction and the formation of the trophectoderm cell layer (Ohsugi et al., Dev. Dyn. 206:391–402, 1996). Here, we show that β-catenin is a major tyrosine-phosphorylated protein in oocytes and early cleavage-stage embryos and that the relative amount of phosphorylated β-catenin is greatly reduced during the morula-blastocyst transition. Peptide-specific antibodies indicate that β-catenin undergoes conformational changes and/or that the carboxy-terminal region of β-catenin is blocked during preimplantation development. Moreover, the availability of a carboxy-terminal epitope seems to depend on the tyrosine phosphorylation state of β-catenin and becomes unmasked when oocytes are treated with the tyrosine kinase inhibitor genistein. Our results suggest that tyrosine phosphorylation of β-catenin represents a molecular mechanism to keep the accumulating E-cadherin adhesion complex in a nonfunctional form. Dev Dyn 1999;216:168–176. © 1999 Wiley-Liss, Inc.

Cadherins and other cell-substrate and cell-cell adhesion molecules play an essential role during development. Through their cytoplasmic interaction with the cytoskeleton, cell adhesion molecules physically link cells with the extracellular matrix and/or with each other. These interactions create architectural and structural entities that enable the tissues in the embryo to restrain the physical forces encountered during development. Regulated cell adhesion is also often the driving force of morphogenetic movements. This review goes beyond the adhesive aspect of cadherins, focusing on their roles as signaling molecules in development. We discuss how cadherins, through their effects on cell proliferation, cell death, cell polarization, and differentiation, play a role in the formation of tissues and organs in the developing embryo.

Beta-catenin is a multifunctional protein involved in cell adhesion and communication. In response to signaling by Wnt growth factors, beta-catenin associates with nuclear TCF factors to activate target genes. A transactivation domain identified at the C-terminus of beta-catenin can stimulate expression of artificial reporter genes. However, the mechanism of target gene activation by TCF/beta-catenin complexes and the physiological relevance of the beta-catenin transactivation domain still remain unclear. Here we asked whether the beta-catenin transactivation domain can generate a Wnt-response in a complex biological system, namely axis formation during Xenopus laevis embryogenesis. We show that a chimeric transcription factor consisting of beta-catenin fused to the DNA-binding domain of LEF-1 induces a complete secondary dorsoanterior axis when expressed in Xenopus. A LEF-1-beta-catenin fusion lacking the C-terminal transactivation domain is impaired in signaling while fusion of just the beta-catenin transactivator to the DNA-binding domain of LEF-1 is sufficient for axis-induction. The latter fusion molecule is blocked by dominant negative LEF-1 but not by excess cadherin indicating that all events parallel or upstream of the transactivation step mediated by beta-catenin are dispensable for Wnt-signaling. Moreover, beta-catenin can be replaced by a heterologous transactivator. Apparently, the ultimate function of beta-catenin in Wnt signaling is to recruit the basal transcription machinery to promoter regions of specific target genes.

The interaction between beta-catenin and LEF-1/TCF transcription factors plays a pivotal role in the Wnt-1 signaling pathway. The level of beta-catenin is regulated by partner proteins, including glycogen synthase kinase-3beta (GSK-3beta) and the adenomatous polyposis coli (APC) tumor suppressor protein. Genetic defects in APC are responsible for a heritable predisposition to colon cancer. APC protein and GSK-3beta bind beta-catenin, retain it in the cytoplasm, and facilitate the proteolytic degradation of beta-catenin. Abrogation of this negative regulation allows beta-catenin to translocate to the nucleus and to form a transcriptional activator complex with the DNA-binding protein lymphoid-enhancing factor 1 (LEF-1). This complex is thought to be involved in tumorigenesis. Here we show that covalent linkage of LEF-1 to beta-catenin and to transcriptional activation domains derived from the estrogen receptor or the herpes simplex virus protein VP16 generates transcriptional regulators that induce oncogenic transformation of chicken embryo fibroblasts. The chimeras between LEF-1 and beta-catenin or VP16 are constitutively active, whereas fusions of LEF-1 to the estrogen receptor are regulatable by estrogen. These experiments document the oncogenicity of transactivating LEF-1 and show that the transactivation domain normally provided by beta-catenin can be replaced by heterologous activation domains. These results suggest that the transactivating function of the LEF-1/beta-catenin complex is critical for tumorigenesis and that this complex transforms cells by activating specific LEF-1 target genes.

The Armadillo family is formed by proteins which possess an Arm domain comprising multiple copies of a 42-amino-acid motif, the Arm repeat, initially described for the Drosophila segment polarity gene product Armadillo. The Arm domain serves in protein-protein interactions which are required for the family members Armadillo, beta-catenin and plakoglobin to mediate cell-cell adhesion and Wnt/Wingless signalling. Similarily, p120cas, the Arm domain containing src substrate, also binds to cadherins and becomes tyrosine phosphorylated in response to a variety of stimuli. However, a putative function of p120cas in adhesion or signalling has not yet been demonstrated. It has also not been shown until now that an Arm domain is a common signal transduction motif. Using Xenopus embryos we show by expression of murine p120cas1B (mp120cas1B) in ventral blastomeres that this catenin cannot replace beta-catenin function in dorsal axis formation. Thus, the presence of an Arm domain per se is not sufficient to activate the Wnt/Wg pathway. Indeed, injection of mp120cas1B into dorsal blastomeres led instead to delayed blastopore closure and posteriorized phenotypes with malformed head structures indicative of disturbed gastrulation movements. Because neither convergent extension behaviour nor adhesion to fibronectin was altered in the injected embryos we assume that mp120cas1B influences motility or orientation of migrating mesodermal cells.

The E-cadherin-catenin adhesion complex has been the subject of many structural and functional studies because of its importance in development, normal tissue function and carcinogenesis. It is well established that the cytoplasmic domain of E-cadherin binds either beta-catenin or plakoglobin, which both can assemble alpha-catenin into the complex. Recently we have identified an alpha-catenin binding site in beta-catenin and plakoglobin and postulated, based on sequence analysis, that these protein-protein interactions are mediated by a hydrophobic interaction mechanism. Here we have now identified the reciprocal complementary binding site in alpha-catenin which mediates its interaction with beta-catenin and plakoglobin. Using in vitro association assays with C-terminal truncations of alpha-catenin expressed as recombinant fusion proteins, we found that the N-terminal 146 amino acids are required for this interaction. We then identified a peptide of 27 amino acids within this sequence (amino acid positions 117–143) which is necessary and sufficient to bind beta-catenin or plakoglobin. As shown by mutational analysis, hydrophobic amino acids within this binding site are important for the interaction. The results described here, together with our previous work, give strong support for the idea that these proteins associate by hydrophobic interactions of two alpha-helices.

We have examined the murine embryonic expression pattern of the cell adhesion molecule R-cadherin in muscle, kidney, thymus, and lung. In developing muscle, R-cadherin was first seen at 10.5 ‐11.5 days postcoitum in the somitic myotome. Consistently, we found R-cadherin expressed at the highest levels in the myotome, early skeletal muscle, and smooth muscle (both vascular and visceral), while very low levels of R-cadherin were detected in the heart. The expression pattern and subcellular localization of R-cadherin in developing skeletal muscle indicate a possible role in myoblast cell ‐cell interactions during both primary and secondary myogenesis. In the developing kidney, R-cadherin was first detected at 10.5 days postcoitum in the mesonephric epithelial tubule cells. In the metanephric kidney, it was specifically expressed in the pretubular aggregates, comma- and S-shaped bodies, proximal tubules, and collecting ducts. Thus, in the kidney, R-cadherin was associated with the mesenchymal ‐e pithelial transition. R-cadherin was also found in other developing epithelia, for example in the thymic epithelial cells. In the lung, R-cadherin was expressed at the highest levels in the smooth muscle surrounding the lung epithelial tubules. To test whether Rcadherin can direct formation of tissues, we constitutively expressed R-cadherin in E-cadherin-/- ES cells and examined histogenesis in teratomas derived from these cells. R-cadherin exclusively rescued formation of striated muscle and epithelia in the teratomas. R-cadherin’s ability to form epithelia in vivo was substantiated by its ability to rescue formation of cystic embryoid bodies in vitro. By comparing our data with the previously reported embryonic expression patterns and histogenetic activities of E- and N-cadherin, we suggest that R-cadherin plays an important role in the formation of striated muscle and possibly also of epithelia. q 1997 Academic Press

M-cadherin is a member of the multigene family of calcium-dependent intercellular adhesion molecules, the cadherins, which are involved in morphogenetic processes. Amino acid comparisons between M-cadherin and E-, N-, and P-cadherin suggested that M-cadherin diverged phylogenetically very early from these classical cadherins. It has been shown that M-cadherin is expressed in prenatal and adult skeletal muscle. In the cerebellum, M-cadherin is present in an adherens-type junction which differs in its molecular composition from the E-cadherin-mediated adherens-type junctions. These and other findings raised the question of whether M-cadherin and the classical cadherins share basic biochemical properties, notably the calcium-dependent resistance to proteolysis, mediation of calcium-dependent intercellular adhesion, and the capability to form M-cadherin complexes with the catenins. Here we show that M-cadherin is resistant to trypsin digestion in the presence of calcium ions but at lower trypsin concentrations than E-cadherin. When ectopically expressed in LMTK- cells, M-cadherin mediated calcium-dependent cell aggregation. Finally, M-cadherin was capable of forming two distinct cytoplasmic complexes in myogenic cells, either with alpha-catenin/beta-catenin or with alpha-catenin/plakoglobin, as E-and N-cadherin, for example, have previously been shown to form. The relative amount of these complexes changed during differentiation from C2C12 myoblasts to myotubes, although the molecular composition of each complex was unaffected during differentiation. These results demonstrate that M-cadherin shares important features with the classical cadherins despite its phylogenetic divergence.

The Drosophila fat facets (faf) gene is a ubiquitin-specific protease necessary for the normal development of the eye and of the syncytial stage embryo in the fly. Using a gene trap approach in embryonic stem cells we have isolated a murine gene with extensive sequence similarity to the Drosophila faf gene and called it Fam (fat facets in mouse). The putative mouse protein shows colinearity and a high degree of sequence identity to the Drosophila protein over almost its entire length of 2554 amino acids. The two enzymatic sites characteristic of ubiquitin-specific proteases are very highly conserved between mice and Drosophila and this conservation extends to yeast. Fam is expressed in a complex pattern during postimplantation development. In situ hybridisation detected Fam transcripts in the rapidly expanding cell populations of gastrulating and neurulating embryos, in post-mitotic cells of the CNS as well as in the apoptotic regions between the digits, indicating that it is not associated with a single developmental or cellular event. The strong sequence similarity to faf and the developmentally regulated expression pattern suggest that Fam and the ubiquitin pathway may play a role in determining cell fate in mammals, as has been established for Drosophila.

Canonical Wnt signaling is repeatedly used during development to control cell fate, and it is often implicated in human cancer. β-catenin, the effector of Wnt signaling, has a dual function in the cell and is involved in both cell adhesion and transcription. Nuclear β-catenin controls transcription through association with transcription factors of the TCF family and the recruitment of epigenetic modifiers. In this study, we used a strategy combining the genetic manipulation of mouse embryonic stem cells with affinity purification and quantitative mass spectroscopy utilizing stable isotope labeling with amino acids in cell culture to study the interactome of chromatin-bound β-catenin with and without Wnt3a stimulation. We uncovered previously unknown interactions of β-catenin with transcription factors and chromatin-modifying complexes. Our proof-of-principle experiments show that β-catenin can recruit the H3K4me2/1 demethylase LSD1 to regulate the expression of the tumor suppressor Lefty1 in mouse embryonic stem cells. The mRNA levels of LSD1 and β-catenin are inversely correlated with the levels of Lefty1 in pancreas and breast tumors, implying that this mechanism is common to mouse embryonic stem cells and cancer cells.