Activation of Yap-Directed Transcription by Knockdown of Conserved Cellular Functions.

The Yap-Hippo pathway has a significant role in regulating cell proliferation and growth, thus controlling organ size and regeneration. The Hippo pathway regulates two highly conserved, transcription coactivators, YAP and TAZ. The upstream regulators of the Yap-Hippo pathway have not been fully characterized. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that allow activation of the YAP transcription coactivator at high cell density. Activation of the YAP transcription coactivator was monitored using a high-content, image-based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known YAP target gene). The effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology, such as components of the nuclear pore, by which YAP cytoplasmic-nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays.

Identification of a novel beta-chemokine, MEC, down-regulated in primary breast tumors.

Chemokines represent a family of low molecular weight secreted proteins that primarily function in the activation and migration of leukocytes. A number of additional functions of chemokines have also been identified including growth of tumor cells, angiogenesis and development. An iterative search for new chemokines has identified a cDNA that encodes a new member of the CC(beta) chemokine family. The gene has been named MEC, for mammary enriched chemokine. MEC expression was found at high levels in many mammary gland samples and was also detected at lower levels in several other epithelial-enriched tissues, such as salivary gland, colon, and prostate. Northern blot analysis demonstrates that MEC expression was highly reduced or eliminated in a majority of human breast tumors as compared to normal adjacent tissue. In situ hybridization demonstrates that MEC was abundantly expressed in normal mammary ductal epithelium, but expression was absent or reduced in various mammary tumor types of epithelial origin. These observations suggest that MEC may be useful as a diagnostic tool in oncology, and may play a role in regulating mammary carcinogenesis. The absence of MEC may also contribute to the host's immune response to tumors.

The transcription factor NF-ATc is essential for cardiac valve formation.

Nuclear factor of activated T cells (NF-AT) is the name of a family of four related transcription factors that may be needed for cytokine gene expression in activated lymphocytes. Here we report that mice with a targeted disruption of the NF-ATc gene show an unexpected and dramatic defect in cardiac morphogenesis, with selective absence of the aortic and pulmonary valves, leading to death in utero from congestive heart failure at days 13.5-17.5 of gestation. In contrast, tricuspid and mitral valve morphogenesis is normal. NF-ATc is the first transcription factor known to be expressed only in the endothelial cells of the heart. As in T cells, nuclear translocation of NF-ATc in cardiac endothelial cells is controlled by the calcium-regulated phosphatase calcineurin: NF-ATc remains cytoplasmic in normal embryos cultured with cyclosporin A, an inhibitor of calcineurin. Abnormal development of the cardiac valves and septae is the most frequent form of birth defect, yet few molecular regulators of valve formation are known. Our results indicate that NF-ATc may play a critical role in signal-transduction processes required for normal cardiac valve formation.

A region of mouse chromosome 16 is syntenic to the DiGeorge, velocardiofacial syndrome minimal critical region.

DGS and VCFS, haploinsufficiencies characterized by multiple craniofacial and cardiac abnormalities, are associated with a microdeletion of chromosome 22q11.2. Here we document synteny between a 150-kb region on mouse chromosome 16 and the most commonly deleted portion of 22q11.2. Seven genes, all of which are transcribed in the early mouse embryo, have been identified. Of particular interest are two serine/threonine kinase genes and a novel goosecoid-like homeobox gene (Gscl). Comparative sequence analysis of a 38-kb segment reveals similarities in gene content, order, exon composition, and transcriptional direction. Therefore, if deletion of these genes results in DGS/VCFS in humans, then haploinsufficiencies involving this region of chromosome 16 should recapitulate the developmental field defects characteristic of this syndrome.

Adenovirus-mediated gene transfer during initial organogenesis in the mammalian embryo is promoter-dependent and tissue-specific.

Replication-defective adenoviruses have received increasing attention as vectors for exogenous gene administration in a variety of experimental and pathological conditions. However, little information exists about their utility for in utero gene therapy, and no information exists concerning their efficacy for gene delivery during initial organogenesis in the mammalian embryo. To evaluate the feasibility of using these vectors for exogenous gene transduction during the initial stages of organogenesis in the mammal, we injected an adenovirus vector carrying the bacterial beta-galactosidase (lacZ) gene under the control of either the cytomegalovirus (CMV) promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) into early, post-gastrulation, mouse embryos, and evaluated expression following 36-48 h in culture. These studies suggest that adenovirus-mediated gene delivery may provide an efficient method of gene transduction during critical developmental stages with no detectable adverse effects on normal development during early morphogenesis. In addition, the type of promoter used had a significant effect on the tissue distribution of gene expression.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): alternatively spliced, functionally distinct isoforms expressed during mammalian cardiovascular development.

The establishment of the cardiovascular system represents an early, critical event essential for normal embryonic development. An important component of vascular ontogeny is the differentiation and development of the endothelial and endocardial cell populations. This involves, at least in part, the expression and function of specific cell surface receptors required to mediate cell-cell and cell-matrix adhesion. Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) may well serve such a function. It is a member of the immunoglobulin superfamily expressed by the entire vascular endothelium in the adult. It is capable of mediating adhesion by a heterophilic mechanism requiring glycosaminoglycans, as well as by a homophilic, glycosaminoglycan independent, mechanism. It has been shown to regulate the expression of other adhesion molecules on naive T cells. This report documents by RT-PCR and immunohistochemical analysis the expression of PECAM-1 during early post implantation mouse embryo development. PECAM-1 was expressed by early endothelial precursors first within the yolk sac and subsequently within the embryo itself. Interestingly, embryonic PECAM-1 was expressed as multiple isoforms in which one or more clusters of polypeptides were missing from the cytoplasmic domain. The sequence and location of the deleted polypeptides corresponded to exons found in the human PECAM-1 gene. The alternatively spliced isoforms were capable of mediating cell-cell adhesion when transfected into L-cells. The isoforms differed, however, in their sensitivity to a panel of anti-PECAM-1 monoclonal antibodies. These data suggest that changes in the cytoplasmic domain of PECAM-1 may affect its function during cardiovascular development, and are consistent with our earlier report that systematic truncation of the cytoplasmic domain of human PECAM-1 resulted in changes in its ligand specificity, divalent cation and glycosaminoglycan dependence, as well as its susceptibility to adhesion blocking monoclonal antibodies. This is the first report of naturally occurring alternatively spliced forms of PECAM-1 having possible functional implications.

Cell adhesion receptors and early mammalian heart development: an overview.

Cardiovascular development is the end result of a complex genetic program subject to regulation by signals transmitted between a cell and its extracellular environment. As cells encounter new extracellular matrices or establish new cell-cell interactions, new genes must be activated to accommodate the altered developmental situation within which the cell finds itself. This is likely reflected in a program of adhesion receptor and counter receptor expression on the surface of cells engaged in the morphogenesis. To understand the molecular basis of development, it is necessary to first determine if such a program exists and then to establish the role of various receptors and counter receptors in the particular morphogenetic process under investigation. To this end, we have initiated an investigation into expression of specific adhesion receptors during cardiovascular development in the mouse. Here, we demonstrate that platelet endothelial cell adhesion molecule (PECAM)-1 is an excellent marker for following vascular formation in the mammalian embryo. It is expressed during development in several alternatively spliced forms involving the cytoplasmic domain of the molecule. These forms differ in their ligand binding properties. Thus, a change in the cytoplasmic domain affects the folding of the molecule in such a way as to structurally alter the extracellular domain. Further, several receptors including the laminin receptor, the fibronectin receptor and a hyaluronic acid receptor, display specific expression patterns during heart development. These include differential expression in the endocardium and myocardium, down regulation during endocardial and myocardium, down regulation during endocardial cushion formation and cessation of expression in particular regions of the heart upon maturation. Interference with the function of one of these receptors (the fibronectin receptor) results in aberrant heart formation. These observations strongly support the concept that morphogenesis requires specific cell adhesion molecules that are expressed in precisely choreographed programs.