The signal transduction adapter protein Disabled-2 (Dab2) is one of the two mammalian orthologs of the Drosophila Disabled. The brain-specific Disabled-1 (Dab1) functions in positional organization of brain cells during development. Dab2 is widely distributed and is highly expressed in many epithelial cell types. The dab2 gene was interrupted by in-frame insertion of beta-galactosidase (LacZ) in embryonic stem cells and transgenic mice were produced. Dab2 expression was first observed in the primitive endoderm at E4.5, immediately following implantation. The homozygous Dab2-deficient mutant is embryonic lethal (earlier than E6.5) due to defective cell positioning and structure formation of the visceral endoderm. In E5.5 dab2 (-/-) conceptus, visceral endoderm-like cells are present in the deformed primitive egg cylinder; however, the visceral endoderm cells are not organized, the cells of the epiblast have not expanded, and the proamniotic cavity fails to form. Disorganization of the visceral endodermal layer is evident, as cells with positive visceral endoderm markers are scattered throughout the dab2 (-/-) conceptus. Only degenerated remains were observed at E6.5 for dab2 (-/-) embryos, and by E7.5, the defective embryos were completely reabsorbed. In blastocyst in vitro culture, initially cells with characteristics of endoderm, trophectoderm, and inner cell mass were observed in the outgrowth of the hatched dab2 (-/-) blastocysts. However, the dab2 (-/-) endodermal cells are much more dispersed and disorganized than those from wild-type blastocysts, the inner cell mass fails to expand, and the outgrowth degenerates by day 7. Thus, Dab2 is required for visceral endodermal cell organization during early mouse development. The absence of an organized visceral endoderm in Dab2-deficient conceptus leads to the growth failure of the inner cell mass. We suggest that Dab2 functions in a signal pathway to regulate endodermal cell organization using endocytosis of ligands from the blastocoel cavity as a positioning cue.

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Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of Kras LA1/+ ;P53 R172HΔG/+ (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial-mesenchymal transition, including the TGF-β receptor 1 (TGFβR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFβRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelialmesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFβR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness.on-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the world (1). Approximately 7% of individuals born in the United States in 2013 will ultimately be diagnosed with lung cancer, and 160,000 die from this disease each year (1). The 5-y survival rate for lung cancer is around 16% of diagnosed cases (2). Much of the lethality of lung cancer is due to frequent diagnosis of the malignancy at the metastatic stage, when fundamental changes in tumor biology cause the disease to be refractory to many treatments. A better understanding of the biological processes that promote NSCLC metastasis promises to further improve clinical care of the patients. Kras LA1/+ ;P53 R172HΔG/+ (KP) mice provide a useful and wellvalidated model for the study of NSCLC metastasis. These mice combine a mutant p53 allele (p53 R175HΔG) with an activating KrasG12D allele (Kras LA1 ) (3), leading to development of adenocarcinomas resembling human NSCLC, which are often characterized by mutation of KRAS (∼30%) (4) and loss of TP53 (∼60%) (5). Many of the KP tumors metastasize to sites commonly seen in NSCLC patients (3). These features make the KP murine model a useful tool with which to evaluate factors that underlie NSCLC metastasis. Among the pathways activated...

The formation of the primitive endoderm layer on the surface of the inner cell mass is one of the earliest epithelial morphogenesis in mammalian embryos. In mouse embryos deficient of Disabled-2 (Dab2), the primitive endoderm cells lose the ability to position on the surface, resulting in defective morphogenesis. Embryonic stem cells lacking Dab2 are also unable to position on the surface of cell aggregates and fail to form a primitive endoderm outer layer in the embryoid bodies. The cellular function of Dab2, a cargo-selective adaptor, in mediating endocytic trafficking of clathrin-coated vesicles is well established. We show here that Dab2 mediates directional trafficking and polarized distribution of cell surface proteins such as megalin and E-cadherin and propose that loss of polarity is the underlying mechanism for the loss of epithelial cell surface positioning in Dab2-deficient embryos and embryoid bodies. Thus, the findings indicate that Dab2 is a surface positioning gene and suggest a novel mechanism of epithelial cell surface targeting.Epithelial cells are positioned on the outer surface of organs or the inner surface of glandular structures and are involved in diverse physiological functions. Simple epithelia consist of monolayered cells that form a sheet through cell-cell adherens junctions and attach to a basement membrane that lies underneath (1). Epithelial cells are polarized with the apical surface exposed, or free from cell-cell contact and the basal side lying in contact with a basement membrane or stromal cells. The cellfree apical space and basal contact are unique hallmarks of an epithelium and are likely positioning cues for the surface localization of the epithelial cells (2), although the molecular details and genes critical for epithelial cell surface positioning are yet uncertain and undefined. The concept that cues are required for epithelial surface positioning is also reinforced by observations of the disorganized growth of carcinomas. Carcinoma cells can be viewed as epithelial cells that have lost their ability to perceive surface positioning cues, and the neoplastic cells no longer obey the constraint imposed by tissue organization (3).The early embryos of vertebrates, especially mammalian species, have great plasticity, and significant cell dispersal, movement, and migration occur before their final positioning and acquisition of cell fates (4). Shown in the classical cell sorting experiments by Townes and Holtfreter (5), early embryonic amphibian cells of epidermis, endoderm, mesoderm, and neural plate, if dispersed, are able to segregate spontaneously upon aggregation, indicating cell positioning is an autonomous property.The primitive endoderm of mammalian early embryos is the first typical epithelial cell type derived that is capable of producing a basement membrane (6). Recent understanding is that the primitive endoderm cells arise from the differentiation of the pluripotent cells of the inner cell mass and migrate out to the surface to form the primitive endoderm layer (7...

The derivation of the primitive endoderm layer from the pluripotent cells of the inner cell mass is one of the earliest differentiation and morphogenic events in embryonic development. GATA4 and GATA6 are the key transcription factors in the formation of extraembryonic endoderms, but their specific contribution to the derivation of each endoderm lineage needs clarification. We further analyzed the dynamic expression and mutant phenotypes of GATA6 in early mouse embryos. GATA6 and GATA4 are both expressed in primitive endoderm cells initially. At embryonic day (E) 5.0, parietal endoderm cells continue to express both GATA4 and GATA6; however, visceral endoderm cells express GATA4 but exhibit a reduced expression of GATA6. By and after E5.5, visceral endoderm cells no longer express GATA6. We also found that GATA6 null embryos did not form a morphologically recognizable primitive endoderm layer, and subsequently failed to form visceral and parietal endoderms. Thus, the current study establishes that GATA6 is essential for the formation of primitive endoderm, at a much earlier stage then previously recognized, and expression of GATA6 discriminates parietal endoderm from visceral endoderm lineages. Developmental Dynamics 237: 2820 -2829, 2008.