E-cadherin is required for the proper activation of the Lifr/Gp130 signaling pathway in mouse embryonic stem cells.

The leukemia inhibitory factor (Lif) signaling pathway is a crucial determinant for mouse embryonic stem (mES) cell self-renewal and pluripotency. One of the hallmarks of mES cells, their compact growth morphology, results from tight cell adhesion mediated through E-cadherin, ß-catenin (Ctnnb1) and α-catenin with the actin cytoskeleton. ß-catenin is also involved in canonical Wnt signaling, which has also been suggested to control mES cell stemness. Here, we analyze Ctnnb1(-/-) mES cells in which cell adhesion is preserved by an E-cadherin-α-catenin (Eα) fusion protein (Ctnnb1(-/-)Eα mES cells), and show that mimicking only the adhesive function of ß-catenin is necessary and sufficient to maintain the mES cell state, making ß-catenin/Wnt signaling obsolete in this process. Furthermore, we propose a role for E-cadherin in promoting the Lif signaling cascade, showing an association of E-cadherin with the Lifr-Gp130 receptor complex, which is most likely facilitated by the extracellular domain of E-cadherin. Without Eα, and thus without maintained cell adhesion, Ctnnb1(-/-) mES cells downregulate components of the Lif signaling pathway, such as Lifr, Gp130 and activated Stat3, as well as pluripotency-associated markers. From these observations, we hypothesize that the changes in gene expression accompanying the loss of pluripotency are a direct consequence of dysfunctional cell adhesion. Supporting this view, we find that the requirement for intact adhesion can be circumvented by the forced expression of constitutively active Stat3. In summary, we put forward a model in which mES cells can be propagated in culture in the absence of Ctnnb1, as long as E-cadherin-mediated cell adhesion is preserved.

Differential requirements for ß-catenin during mouse development.

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

Wnt3a-dependent and -independent protein interaction networks of chromatin-bound ß-catenin in mouse embryonic stem cells.

Canonical Wnt signaling is repeatedly used during development to control cell fate, and it is often implicated in human cancer. ß-catenin, the effector of Wnt signaling, has a dual function in the cell and is involved in both cell adhesion and transcription. Nuclear ß-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 ß-catenin with and without Wnt3a stimulation. We uncovered previously unknown interactions of ß-catenin with transcription factors and chromatin-modifying complexes. Our proof-of-principle experiments show that ß-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 ß-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.

Biological and mathematical modeling of melanocyte development.

We aim to evaluate environmental and genetic effects on the expansion/proliferation of committed single cells during embryonic development, using melanoblasts as a paradigm to model this phenomenon. Melanoblasts are a specific type of cell that display extensive cellular proliferation during development. However, the events controlling melanoblast expansion are still poorly understood due to insufficient knowledge concerning their number and distribution in the various skin compartments. We show that melanoblast expansion is tightly controlled both spatially and temporally, with little variation between embryos. We established a mathematical model reflecting the main cellular mechanisms involved in melanoblast expansion, including proliferation and migration from the dermis to epidermis. In association with biological information, the model allows the calculation of doubling times for melanoblasts, revealing that dermal and epidermal melanoblasts have short but different doubling times. Moreover, the number of trunk founder melanoblasts at E8.5 was estimated to be 16, a population impossible to count by classical biological approaches. We also assessed the importance of the genetic background by studying gain- and loss-of-function ß-catenin mutants in the melanocyte lineage. We found that any alteration of ß-catenin activity, whether positive or negative, reduced both dermal and epidermal melanoblast proliferation. Finally, we determined that the pool of dermal melanoblasts remains constant in wild-type and mutant embryos during development, implying that specific control mechanisms associated with cell division ensure half of the cells at each cell division to migrate from the dermis to the epidermis. Modeling melanoblast expansion revealed novel links between cell division, cell localization within the embryo and appropriate feedback control through ß-catenin.

N-cadherin can structurally substitute for E-cadherin during intestinal development but leads to polyp formation.

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 beta-catenin (beta-cat; Ctnnb1) and enhanced expression of several beta-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.

Lineage specification of parietal epithelial cells requires ß-catenin/Wnt signaling.

ß-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 ß-catenin deficiency to renal epithelial cells at the late S-shaped body stage and the developing collecting ducts. The conditional ß-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 ß-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 ß-catenin/Wnt signaling is important during the late stages of nephrogenesis and for the lineage specification of parietal epithelial cells.

Mammalian cadherins and protocadherins: about cell death, synapses and processing.

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.

Nanog is required for primitive endoderm formation through a non-cell autonomous mechanism.

Early lineage segregation in mouse development results in two, either CDX2- or OCT4/NANOG-positive, cell populations. CDX2-positive cells form the trophectoderm (TE), OCT4/NANOG-positive cells the inner cell mass (ICM). In a second lineage decision ICM cells segregate into Epiblast (EPI) and primitive endoderm (PE). EPI and PE formation depend on the activity of the transcription factors Nanog and Gata4/6. A role for Nanog, a crucial pluripotency factor, in preventing PE differentiation has been proposed, as outgrowths of mutant ICMs result in PE, but not EPI derivatives. We established Nanog-mutant mouse lines and analyzed EPI and PE formation in vivo. Surprisingly, Gata4 expression in mutant ICM cells is absent or strongly decreased, thus loss of Nanog does not result in precocious endoderm differentiation. However, Nanog-deficient embryos retain the capacity to form PE in chimeric embryos and, in contrast to recent reports, in blastocyst outgrowths. Based on our findings we propose a non-cell autonomous requirement of Nanog for proper PE formation in addition to its essential role in EPI determination.

Replacement of E-cadherin by N-cadherin in the mammary gland leads to fibrocystic changes and tumor formation.

INTRODUCTION: E-cadherin (E-cad; cadherin 1) and N-cadherin (N-cad; cadherin 2) are the most prominent members of the cadherin family of cell adhesion molecules. Although they share many structural and functional features, they are expressed in an almost mutually exclusive manner in vivo. METHODS: To explore functional differences between the two cadherins in vivo, we recently generated a knock-in line in which N-cad is expressed from the E-cad locus. In combination with a conditional gene inactivation approach, we expressed N-cad in the absence of E-cad (referred to as Ncadk.i.) in alveolar epithelial cells of the mammary gland starting in late pregnancy. RESULTS: We found that the sole presence of N-cad induces constitutively active fibroblast growth factor (Fgf) signaling and a precocious involution resulting in massive apoptosis of alveolar cells. To block apoptosis, we conditionally deleted one allele of p53 in Ncadk.i. mice and observed a temporal rescue of alveolar morphology and function. However, an accumulation of fibrotic tissue and cysts with increasing age and lactation cycles was observed. This phenotype closely resembled fibrocystic mastopathy (FM), a common disorder in humans, which is thought to precede breast cancer. Concordantly, 55% of Ncadk.i. mice harboring a heterozygous p53 deletion developed malignant and invasive tumors. CONCLUSIONS: Our results demonstrate a possible role for N-cad in the formation of fibrosis and cysts in the mammary gland. Moreover, we show that these lesions precede the development of malignant tumors. Thus, we provide a new mouse model to investigate the molecular mechanisms of fibrocystic mastopathy and the transition from benign to malignant tumors.

Protocadherins.

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.

Beta-catenin-mediated signaling and cell adhesion in postgastrulation mouse embryos.

beta-Catenin plays two major roles during the development of multicellular organisms. It is the downstream effector of the canonical Wnt signaling cascade, which is involved in many developmental processes and in tumor formation. Additionally, it is linked to classic cadherins and is required for the correct assembly and function of adherens junctions. beta-Catenin loss of function mutants show early gastrulation lethality. To address the role of beta-catenin in postgastrulation stages and to overcome the early embryonic lethality, we performed conditional gene targeting, using Cdx1::Cre, a newly established mouse line. By this approach, beta-catenin was depleted in the entire posterior embryo after the gastrulation process at embryonic day 8.0, when the three germ layers were established. We observed defects in signaling and adhesion which are temporarily separated. At an early event, known targets of Wnt/beta-catenin are down-regulated in the paraxial mesoderm. Moreover, Fgf8 and Wnt3a, the key players of the segmentation process, are down-regulated in the neural ectoderm (NE). Wnt3a expression was rescued in mutant embryos by exogenous Fgf and inhibition of Fgf signaling in wild-type embryos resulted in Wnt3a down-regulation. Based on these results, we assume the existence of an autoregulatory feedback loop in the NE where Fgf8 regulates Wnt3a, which in turn, by means of beta-catenin, maintains Fgf8 expression. In later stages, the lack of beta-catenin caused a progressive posterior disintegration. We found that beta-catenin is required for the correct localization of N-cadherin at the membrane of neural ectodermal cells and that its absence causes a disintegration of the neural tube.

Beta-catenin is dispensable for hematopoiesis and lymphopoiesis.

Beta-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 beta-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 beta-catenin independent. In contrast to earlier reports, these data exclude an essential role for beta-catenin during hematopoiesis and lymphopoiesis.

Neural progenitors of the postnatal and adult mouse forebrain retain the ability to self-replicate, form neurospheres, and undergo multipotent differentiation in vivo.

Somatic stem cells are reservoirs to replace lost cells or damaged tissue. Cells with neural stem cell (NSC) characteristics can be isolated from the postnatal mammalian brain into adulthood and expanded as neurospheres. We addressed the ability of these in vitro expanded putative NSCs to retain progenitor characteristics in vivo, in analogy to hematopoietic stem cells. When transplanted in utero, both postnatal and adult neural progenitors colonize host brains and contribute neurons and glia. In stark contrast to what has been reported when transplanted in postnatal hosts, epidermal growth factor-expanded cells also remain self-replicating and multipotent in vivo over many months and can be serially transplanted into multiple hosts. Surprisingly, embryonically transplanted NSCs remain in the neurogenic regions in adult hosts, where they express progenitor cell markers and continue to proliferate even after 6 months without tumor formation. These data indicate that spherogenic cells of the postnatal and adult mammalian brain retain their potential in vitro and in vivo throughout the life of the organism and beyond transplantation, which has important implications for cell replacement strategies.

Abrogation of E-cadherin-mediated cell-cell contact in mouse embryonic stem cells results in reversible LIF-independent self-renewal.

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 beta-catenin binding domain of E-cadherin, we show that beta-catenin null (betacat(-/-)) 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 beta-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 beta 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.

E-cadherin inhibits cell surface localization of the pro-migratory 5T4 oncofetal antigen in mouse embryonic stem cells.

Epithelial-mesenchymal transition (EMT) events occur during embryonic development and are important for the metastatic spread of epithelial tumors. We show here that spontaneous differentiation of mouse embryonic stem (ES) cells is associated with an E- to N-cadherin switch, up-regulation of E-cadherin repressor molecules (Snail and Slug proteins), gelatinase activity (matrix metalloproteinase [MMP]-2 and -9), and increased cellular motility, all characteristic EMT events. The 5T4 oncofetal antigen, previously shown to be associated with very early ES cell differentiation and altered motility, is also a part of this coordinated process. E- and N-cadherin and 5T4 proteins are independently regulated during ES cell differentiation and are not required for induction of EMT-associated transcripts and proteins, as judged from the study of the respective knockout ES cells. Further, abrogation of E-cadherin-mediated cell-cell contact in undifferentiated ES cells using neutralizing antibody results in a reversible mesenchymal phenotype and actin cytoskeleton rearrangement that is concomitant with translocation of the 5T4 antigen from the cytoplasm to the cell surface in an energy-dependent manner. E-cadherin null ES cells are constitutively cell surface 5T4 positive, and although forced expression of E-cadherin cDNA in these cells is sufficient to restore cell-cell contact, cell surface expression of 5T4 antigen is unchanged. 5T4 and N-cadherin knockout ES cells exhibit significantly decreased motility during EMT, demonstrating a functional role for these proteins in this process. We conclude that E-cadherin protein stabilizes cortical actin cytoskeletal arrangement in ES cells, and this can prevent cell surface localization of the promigratory 5T4 antigen.

Cdx1::Cre allele for gene analysis in the extraembryonic ectoderm and the three germ layers of mice at mid-gastrulation.

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.

Formation of multiple hearts in mice following deletion of beta-catenin in the embryonic endoderm.

Using Cre/loxP, we conditionally inactivated the beta-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 beta-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 beta-catenin in embryonic endoderm changes cell fate from endoderm to precardiac mesoderm, consistent with the existence of bipotential mesendodermal progenitors in mouse embryos.

Wnt3a plays a major role in the segmentation clock controlling somitogenesis.

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/beta-catenin signaling and the segmentation clock. Axin2, a negative regulator of the Wnt pathway, is directly controlled by Wnt/beta-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/beta-catenin signaling via a negative-feedback mechanism and that Wnt3a controls the segmentation process in vertebrates.

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, beta-catenin, and the phosphatase DEP-1/CD148.

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 beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-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-beta-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 conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility.

Using the Cre/loxP system we conditionally inactivated beta-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 beta-catenin -/- endothelial cells showed a different organization of intercellular junctions with a decrease in alpha-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 beta-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.