Requirement for beta-catenin in anterior-posterior axis formation in mice

The anterior-posterior axis of the mouse embryo is defined before formation of the primitive streak, and axis specification and subsequent anterior development involves signaling from both embryonic ectoderm and visceral endoderm. Tauhe Wnt signaling pathway is essential for various developmental processes, but a role in anterior-posterior axis formation in the mouse has not been previously established. Beta-catenin is a central player in the Wnt pathway and in cadherin-mediated cell adhesion. We generated beta-catenin-deficient mouse embryos and observed a defect in anterior-posterior axis formation at embryonic day 5.5, as visualized by the absence of Hex and Hesx1 and the mislocation of cerberus-like and Lim1 expression. Subsequently, no mesoderm and head structures are generated. Intercellular adhesion is maintained since plakoglobin substitutes for beta-catenin. Our data demonstrate that beta-catenin function is essential in anterior-posterior axis formation in the mouse, and experiments with chimeric embryos show that this function is required..

E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton

beta-Catenin is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E-cadherin and also with the tumor suppressor gene product APC, and the Drosophila homologue of beta-catenin, armadillo, mediates morphogenetic signals. We demonstrate here that E-cadherin and APC directly compete for binding to the internal, armadillo-like repeats of beta-catenin; the NH2-terminal domain of beta-catenin mediates the interaction of the alternative E-cadherin and APC complexes to the cytoskeleton by binding to alpha-catenin. Plakoglobin (gamma-catenin), which is structurally related to beta-catenin, mediates identical interactions. We thus show that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggest that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression

Neocortical dendritic complexity is controlled during development by NOMA-GAP-dependent inhibition of Cdc42 and activation of cofilin

Neocortical neurons have highly branched dendritic trees that are essential for their function. Indeed, defects in dendritic arborization are associated with human neurodevelopmental disorders. The molecular mechanisms regulating dendritic arbor complexity, however, are still poorly understood. Here, we uncover the molecular basis for the regulation of dendritic branching during cortical development. We show that during development, dendritic branching requires post-mitotic suppression of the RhoGTPase Cdc42. By generating genetically modified mice, we demonstrate that this is catalyzed in vivo by the novel Cdc42-GAP NOMA-GAP. Loss of NOMA-GAP leads to decreased neocortical volume, associated specifically with profound oversimplification of cortical dendritic arborization and hyperactivation of Cdc42. Remarkably, dendritic complexity and cortical thickness can be partially restored by genetic reduction of post-mitotic Cdc42 levels. Furthermore, we identify the actin regulator cofilin as a key regulator of dendritic complexity in vivo. Cofilin activation during late cortical development depends on NOMA-GAP expression and subsequent inhibition of Cdc42. Strikingly, in utero expression of active cofilin is sufficient to restore postnatal dendritic complexity in NOMA-GAP-deficient animals. Our findings define a novel cell-intrinsic mechanism to regulate dendritic branching and thus neuronal complexity in the cerebral cortex

Shp2/MAPK signaling controls goblet/paneth cell fate decisions in the intestine

In the development of the mammalian intestine, Notch and Wnt/{beta}-catenin signals control stem cell maintenance and their differentiation into absorptive and secretory cells. Mechanisms that regulate differentiation of progenitors into the three secretory lineages, goblet, paneth, or enteroendocrine cells, are not fully understood. Using conditional mutagenesis in mice, we observed that Shp2-mediated MAPK signaling determines the choice between paneth and goblet cell fates and also affects stem cells, which express the leucine-rich repeat-containing receptor 5 (Lgr5). Ablation of the tyrosine phosphatase Shp2 in the intestinal epithelium reduced MAPK signaling and led to a reduction of goblet cells while promoting paneth cell development. Conversely, conditional mitogen-activated protein kinase kinase 1 (Mek1) activation rescued the Shp2 phenotype, promoted goblet cell and inhibited paneth cell generation. The Shp2 mutation also expanded Lgr5+ stem cell niches, which could be restricted by activated Mek1 signaling. Changes of Lgr5+ stem cell quantities were accompanied by alterations of paneth cells, indicating that Shp2/MAPK signaling might affect stem cell niches directly or via paneth cells. Remarkably, inhibition of MAPK signaling in intestinal organoids and cultured cells changed the relative abundance of Tcf4 isoforms and by this, promoted Wnt/{beta}-catenin activity. The data thus show that Shp2-mediated MAPK signaling controls the choice between goblet and paneth cell fates by regulating Wnt/{beta}-catenin activity

Afadin requirement for cytokine expressions in keratinocytes during chemically induced inflammation in mice

Afadin is a filamentous actin-binding protein and a mediator of nectin signaling. Nectins are Ig-like cell adhesion molecules, and the nectin family is composed of four members, nectin-1 to nectin-4. Nectins show homophilic and heterophilic interactions with other nectins or proteins on adjacent cells. Nectin signaling induces formation of cell-cell junctions and is required for the development of epithelial tissues, including skin. This study investigated the role of afadin in epithelial tissue development and established epithelium-specific afadin-deficient (CKO) mice. Although showing no obvious abnormality in the skin development and homeostasis, the mice showed the reduced neutrophil infiltration into the epidermis during chemical-induced inflammation with 12-O-tetradecanoylphorbol 13-acetate (TPA). Immunohistochemical and quantitative real-time PCR analyses showed that the expression levels of cytokines including Cxcl2, Il-1{beta} and Tnf-{alpha} were reduced in CKO keratinocytes compared with control keratinocytes during TPA-induced inflammation. Primary-cultured skin keratinocytes from CKO mice also showed reduced expression of these cytokines and weak activation of Rap1 compared with those from control mice after the TPA treatment. These results suggested a remarkable function of afadin, which was able to enhance cytokine expression through Rap1 activation in keratinocytes during inflammation

Establishment of kidney cancer organoid cultures

This protocol describes an efficient novel method for cultivating kidney cancer organoids, derived directly from human clear cell renal cell carcinomas (ccRCC) or sorted cancer stem cells. The organoids recapitulate tumor-specific characteristics and display inter-tumor heterogeneity. Here we explain how to establish the organoid cultures, maintain them in defined, serum-free medium and use them for patient-specific drug testing. Within 10-14 days, a patient-derived organoid culture is established and can be serially passaged. Compared to other protocols focusing on organoid cultures derived from pediatric kidney malignancies or healthy kidneys, the protocol described here is optimized for adult ccRCC organoid cultures. Organoids established using this protocol can be used to further investigate tumor biology in a complex 3D in vitro system, complement current preclinical model-based drug studies, and advance the development of personalized therapy design

E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton.

beta-Catenin is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E-cadherin and also with the tumor suppressor gene product APC, and the Drosophila homologue of beta-catenin, armadillo, mediates morphogenetic signals. We demonstrate here that E-cadherin and APC directly compete for binding to the internal, armadillo-like repeats of beta-catenin; the NH2-terminal domain of beta-catenin mediates the interaction of the alternative E-cadherin and APC complexes to the cytoskeleton by binding to alpha-catenin. Plakoglobin (gamma-catenin), which is structurally related to beta-catenin, mediates identical interactions. We thus show that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggest that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression

E-cadherin is crucial for embryonic stem cell pluripotency and can replace OCT4 during somatic cell reprogramming

We report new functions of the cell-adhesion molecule E-cadherin in murine pluripotent cells. E-cadherin is highly expressed in mouse embryonic stem cells, and interference with E-cadherin causes differentiation. During cellular reprogramming of mouse fibroblasts by OCT4, SOX2, KLF4 and c-MYC, fully reprogrammed cells were exclusively observed in the E-cadherin-positive cell population and could not be obtained in the absence of E-cadherin. Moreover, reprogrammed cells could be established by viral E-cadherin in the absence of exogenous OCT4. Thus, reprogramming requires spatial cues that cross-talk with essential transcription factors. The cell-adhesion molecule E-cadherin has important functions in pluripotency and reprogramming

The BCL9-2 proto-oncogene governs estrogen receptor alpha expression in breast tumorigenesis

The majority of human breast cancers express estrogen receptor {Alpha} (ER), which is important for therapy with anti-estrogens. Here we describe the role of BCL9-2, a proto-oncogene previously characterized as co-activator of Wnt/{beta}-catenin signaling, for mammary tumorigenesis in mice and human. ER positive human breast cancers showed overexpression of BCL9-2 and tamoxifen treated patients with high BCL9-2 demonstrated a better survival. BCL9-2 was upregulated during puberty and pregnancy in normal mammary epithelia, but downregulated in the involuted gland. BCL9-2 overexpression in vivo delayed the mammary involution and induced alveolar hyperplasia. Moreover, aged BCL9-2 transgenic mice developed ductal-like mammary tumors with high nuclear ER expression. We found, that primary cell cultures of BCL9-2 breast tumors responded to tamoxifen treatment. Moreover, BCL9-2 regulated the expression of ER and the proliferation of human breast cancer cells independently of {beta}-catenin. Finally, we describe a novel mechanism, how BCL9-2 regulates ER transcription by interaction with Sp1 through the proximal ESR1 gene promoter. In summary, BCL9-2 induces ER positive breast cancers in vivo, regulates ER expression by a novel {beta}-catenin independent mechanism in breast cancer cells, and might predict the therapy response to tamoxifen treatment