Pair-Wise Regulation of Convergence and Extension Cell Movements by Four Phosphatases via RhoA

Various signaling pathways regulate shaping of the main body axis during early vertebrate development. Here, we focused on the role of protein-tyrosine phosphatase signaling in convergence and extension cell movements. We identified Ptpn20 as a structural paralogue of PTP-BL and both phosphatases were required for normal gastrulation cell movements. Interestingly, knockdowns of PTP-BL and Ptpn20 evoked similar developmental defects as knockdown of RPTPα and PTPε. Co-knockdown of RPTPα and PTP-BL, but not Ptpn20, had synergistic effects and conversely, PTPε and Ptpn20, but not PTP-BL, cooperated, demonstrating the specificity of our approach. RPTPα and PTPε knockdowns were rescued by constitutively active RhoA, whereas PTP-BL and Ptpn20 knockdowns were rescued by dominant negative RhoA. Consistently, RPTPα and PTP-BL had opposite effects on RhoA activation, both in a PTP-dependent manner. Downstream of the PTPs, we identified NGEF and Arhgap29, regulating RhoA activation and inactivation, respectively, in convergence and extension cell movements. We propose a model in which two phosphatases activate RhoA and two phosphatases inhibit RhoA, resulting in proper cell polarization and normal convergence and extension cell movements

Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice.

Intestinal mucosal cells have proved difficult to culture in vitro. Many attempts have been made to develop long-term cultures of these cells either by direct culturing or by attempting to immortalize these cells by using a range of transforming viral genes, but with little success. The recent development of a transgenic mouse bearing a temperature-sensitive mutation of the simian virus 40 large tumor antigen gene (tsA58) has enabled us to initiate conditionally immortalized cultures of epithelial cells from both small intestinal and colonic mucosa of adult mice. Crypts were isolated from either the small intestines or colons of young adult mice and cultured at the permissive temperature (33 degrees C) in medium containing conditioned medium from a human colon carcinoma cell line, LIM1863. Crypts from both tissues yielded cultures of epithelial cells that have now been in culture for more than 12 months with regular passaging. The epithelial nature of the cells has been confirmed by staining with anti-keratin antibodies. The intestinal origin of the cells was demonstrated by the ability of the cells to synthesize low levels of both brush border peptidases and a disaccharidase. The levels of expression of these enzymes were modulated by the addition of sodium butyrate or phorbol myristate acetate to the medium, which resulted in an increase in the synthesis of the peptidases and a decrease in the synthesis of the disaccharidase. The cells proliferate continuously at the permissive temperature (33 degrees C), but proliferation ceases at the nonpermissive temperature (39.5 degrees C). To our knowledge, this is the first description of the establishment of epithelial cell lines from both small intestine and colon of the same mouse strain. The success reported here indicates that this transgenic mouse will be a useful source of tissue for the study of the mechanisms that control the proliferation and eventual differentiation and senescence of the cells of the intestinal mucosa. These mice will also be a useful source of cells for attempts to culture cells from other tissues that have proved difficult to culture in vitro

Studies on precursor cells in the rodent pancreas

SIGLEAvailable from British Library Document Supply Centre- DSC:DX171911 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

DIRECT DERIVATION OF CONDITIONALLY IMMORTAL CELL-LINES FROM AN H-2KB-TSA58 TRANSGENIC MOUSE

Studies on cell lines have greatly improved our understanding of many important biological questions. Generation of cell lines is facilitated by the introduction of immortalizing oncogenes into cell types of interest. One gene known to immortalize many different cell types in vitro encodes the simian virus 40 (SV40) large tumor (T) antigen (TAg). To circumvent the need for gene insertion in vitro to generate cell lines, we created transgenic mice harboring the SV40 TAg gene. Since previous studies have shown that TAg expression in transgenic mice is associated with tumorigenesis and aberrant development, we utilized a thermolabile TAg [from a SV40 strain, tsA58, temperature sensitive (ts) for transformation] to reduce the levels of functional TAg present in vivo. To direct expression to a broad range of tissues, we used the mouse major histocompatibility complex H-2Kb promoter, which is both widely active and can be further induced by interferons. tsA58 TAg mRNA was expressed in tissues of all animals harboring the hybrid construct. Development of all tissues was macroscopically normal except for thymus, which consistently showed hyperplasia. Fibroblast and cytokeratin+ thymic epithelial cultures from these mice were readily established without undergoing crisis and were conditionally immortal in their growth; the degree of conditionality was correlated with the levels of tsA58 TAg detected. One strain of H-2Kb-tsA58 mice has been bred through several generations to homozygosity and transmits a functional copy of the transgene

Tbx1 regulation of myogenic differentiation in the limb and cranial mesoderm

The T-box transcription factor Tbx1 has been implicated in DiGeorge syndrome, the most frequent syndrome due to a chromosomal deletion. Gene inactivation of Tbx1 in mice results in craniofacial and branchial arch defects, including myogenic defects in the first and second branchial arches. A T-box binding site has been identified in the Xenopus Myf5 promoter, and in other species, T-box genes have been implicated in myogenic fate. Here we analyze Tbx1 expression in the developing chick embryo relating its expression to the onset of myogenic differentiation and cellular fate within the craniofacial mesoderm. We show that Tbx1 is expressed before capsulin, the first known marker of branchial arch 1 and 2 muscles. We also show that, as in the mouse, Tbx1 is expressed in endothelial cells, another mesodermal derivative, and, therefore, Tbx1 alone cannot specify the myogenic lineage. In addition, Tbx1 expression was identified in both chick and mouse limb myogenic cells, initially being restricted to the dorsal muscle mass, but in contrast, to the head, here Tbx1 is expressed after the onset of myogenic commitment. Functional studies revealed that loss of Tbx1 function reduces the number of myocytes in the head and limb, whereas increasing Tbx1 activity has the converse effect. Finally, analysis of the Tbx1-mesoderm-specific knockout mouse demonstrated the cell autonomous requirement for Tbx1 during myocyte development in the cranial mesoderm

A human YAC transgene rescues craniofacial and neural tube development in PDGFRalpha knockout mice and uncovers a role for PDGFRalpha in prenatal lung growth

The platelet-derived growth factor alpha-receptor (PDGFRalpha) plays a vital role in the development of vertebrate embryos, since mice lacking PDGFRalpha die in mid-gestation. PDGFRalpha is expressed in several types of migratory progenitor cells in the embryo including cranial neural crest cells, lung smooth muscle progenitors and oligodendrocyte progenitors. To study PDGFRalpha gene regulation and function during development, we generated transgenic mice by pronuclear injection of a 380 kb yeast artificial chromosome (YAC) containing the human PDGFRalpha gene. The YAC transgene was expressed in neural crest cells, rescued the profound craniofacial abnormalities and spina bifida observed in PDGFRalpha knockout mice and prolonged survival until birth. The ultimate cause of death was respiratory failure due to a defect in lung growth, stemming from failure of the transgene to be expressed correctly in lung smooth muscle progenitors. However, the YAC transgene was expressed faithfully in oligodendrocyte progenitors, which was not previously observed with plasmid-based transgenes containing only upstream PDGFRalpha control sequences. Our data illustrate the complexity of PDGFRalpha genetic control, provide clues to the location of critical regulatory elements and reveal a requirement for PDGF signalling in prenatal lung growth, which is distinct from the known requirement in postnatal alveogenesis. In addition, we found that the YAC transgene did not prolong survival of Patch mutant mice, indicating that genetic defects outside the PDGFRalpha locus contribute to the early embryonic lethality of Patch mic

Desmosomal glycoprotein DGI, a component of intercellular desmosome junctions, is related to the cadherin family of cell adhesion molecules.

Among the variety of specialized intercellular junctions, those of the adherens type have the most obvious association with cytoskeletal elements. This may be with the actin microfilament system as in the zonula adherens or with intermediate filaments as in the macula adherens, or desmosome. In the former case, it is clear that transmembrane glycoproteins of the cadherin family are important adhesive components of the molecular assembly. We now show for desmosomes that a major glycoprotein component (desmosomal glycoprotein DGI) has extensive homology with the cadherins, defining an extended family, but also has unique features in its cytoplasmic domain that are likely to be relevant to the association with intermediate rather than actin filaments. A novel 282-residue extension contains repeats of approximately 29 amino acid residues predicted to have an antiparallel beta-sheet structure, followed by a glycine-rich sequence. As in the cadherins, the extracellular domain contains possible Ca2(+)-binding sequences and a potential protease processing site. The cell adhesion recognition region (His-Ala-Val) of the cadherins is modified to Arg-Ala-Leu