Philippe Soriano

Src family kinases are required for integrin but not PDGFR signal transduction

Src family kinases (SFKs) have been implicated as important regulators of ligand‐induced cellular responses including proliferation, survival, adhesion and migration. Analysis of SFK function has been impeded by extensive redundancy between family members. We have generated mouse embryos harboring functional null mutations of the ubiquitously expressed SFKs Src, Yes and Fyn. This triple mutation leads to severe developmental defects and lethality by E9.5. To elucidate the molecular mechanisms underlying this phenotype, SYF cells (deficient for Src, Yes and Fyn) were derived and tested for their ability to respond to growth factors or plating on extracellular matrix. Our studies reveal that while Src, Yes and Fyn are largely dispensable for platelet‐derived growth factor (PDGF)‐induced signaling, they are absolutely required to mediate specific functions regulated by extracellular matrix proteins. Fibronectin‐induced tyrosine phosphorylation of focal adhesion proteins, including the focal adhesion kinase FAK, was nearly eliminated in the absence of Src, Yes and Fyn. Furthermore, consistent with previous reports demonstrating the importance of FAK for cell migration, SYF cells displayed reduced motility in vitro. These results demonstrate that SFK activity is essential during embryogenesis and suggest that defects observed in SYF triple mutant embryos may be linked to deficiencies in signaling by extracellular matrix‐coupled receptors.

Neuronal position in the developing brain is regulated by mouse disabled-1.

During mammalian brain development, immature neurons migrate radially from the neuroectoderm to defined locations, giving rise to characteristic cell layers,. Here we show that targeted disruption of the mouse disabled1 ( mdab1 ) gene disturbs neuronal layering in the cerebral cortex, hippocampus and cerebellum. The gene encodes a cytoplasmic protein, mDab1 p80, which is expressed and tyrosine-phosphorylated in the developing nervous system. It is likely to be an adaptor protein, docking to others through its phosphotyrosine residues and protein-interacting domain. The mdab1 mutant phenotype is very similar to that of the reeler mouse. The product of the reeler gene, Reelin, is a secreted protein that has been proposed to act as an extracellular signpost for migrating neurons. Because mDab1 is expressed in wild-type cortical neurons, and Reelin expression is normal in mdab1 mutants, mDab1 may be part of a Reelin-regulated or parallel pathway that controls the final positioning of neurons.

The helix-loop-helix gene E2A is required for B cell formation

Heterodimers between tissue-specific basic-helix-loop-helix proteins and the gene products of E2A play major roles in determining tissue-specific cell fate. To understand the broad role of E2A in development, we have generated E2A mutant mice following homologous recombination in embryonic stem cells. Homozygous mutant mice develop to full term without apparent abnormalities, but then display a high rate of postnatal death. The surviving mice show retarded postnatal growth. Detailed examination of hematopoiesis reveals that the homozygous mutant mice contain no B cells while other lineages including T cell, granulocyte, macrophage, and erythroid are intact. The block to B cell differentiation occurs prior to immunoglobulin gene DH-JH rearrangement and the expression of the B lineage-specific marker B220. Surprisingly, heterozygous embryos contain, on average, about half as many B cells as wild-type embryos, suggesting the existence of a counting mechanism that translates levels of E2A into numbers of B cells.

Mena, a Relative of VASP and Drosophila Enabled, Is Implicated in the Control of Microfilament Dynamics

Drosophila Enabled is required for proper formation of axonal structures and is genetically implicated in signaling pathways mediated by Drosophila AbI. We have identified two murine proteins, Mena and Evl, that are highly related to Enabled as well as VASP (Vasodilator-Stimulated Phosphoprotein). A conserved domain targets Mena to localized proteins containing a specific proline-rich motif. The association of Mena with the surface of the intracellular pathogen Listeria monocytogenes and the G-actin binding protein profilin suggests that this molecule may participate in bacterial movement by facilitating actin polymerization. Expression of neural-enriched isoforms of Mena in fibroblasts induces the formation of abnormal F-actin-rich outgrowths, supporting a role for this protein in microfilament assembly and cell motility.

PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor.

Platelet-derived growth factors (PDGFs) are important in many types of mesenchymal cell. Here we identify a new PDGF, PDGF-C, which binds to and activates the PDGF α-receptor. PDGF-C is activated by proteolysis and induces proliferation of fibroblasts when overexpressed in transgenic mice. In situ hybridization analysis in the murine embryonic kidney shows preferential expression of PDGF-C messenger RNA in the metanephric mesenchyme during epithelial conversion. Analysis of kidneys lacking the PDGF α-receptor shows selective loss of mesenchymal cells adjacent to sites of expression of PDGF-C mRNA; this is not found in kidneys from animals lacking PDGF-A or both PDGF-A and PDGF-B, indicating that PDGF-C may have a unique function.

Requirement of pp60c-src expression for osteoclasts to form ruffled borders and resorb bone in mice.

Targeted disruption of the c-src proto-oncogene in mice has shown that src expression is required for normal bone resorption, since the src-deficient mutants develop osteopetrosis. To evaluate the mechanisms by which src-deficiency affects osteoclast function, we treated src-deficient mice with the stimulants of bone resorption, IL-1, parathyroid hormone, and parathyroid hormone-related protein, and analyzed the effects by quantitative bone histomorphometry and electron microscopy. Increased numbers of multinucleated cells with the morphological characteristics of osteoclasts appeared on bone surfaces, but these cells did not form ruffled borders or normal resorption lacunae. To confirm these in vivo findings, we cultured src-mutant bone marrow cells on dentine slices in the presence of 1,25 dihydroxyvitamin D3. Increased numbers of multinucleated cells were formed, but unlike normal murine bone marrow cells, they did not form resorption pits. These results indicate that osteoclasts appear in the absence of pp60c-src, but that pp60c-src expression is required for mature osteoclasts to form ruffled borders and resorb bone.

pp59fyn mutant mice display differential signaling in thymocytes and peripheral T cells

We have generated mutant mice that do not express pp59fyn, a nonreceptor protein tyrosine kinase related to pp60src, by homologous recombination in embryonic stem cells. fyn- mice did not display an overt phenotype. Because fyn is associated with the T cell receptor (TCR), thymocyte and T cell signaling was analyzed in the mutant background. Cross-linking of TCR-CD3 in thymocytes led to markedly reduced calcium fluxes and abrogated proliferation, whereas mature splenic T cells retained largely normal proliferation despite depressed calcium movements and IL-2 production. Similarly, proliferation induced by Thy-1 cross-linking was reduced in thymocytes but not in splenic T cells. fyn- thymocytes were impaired at a late stage of maturation and showed limited clonal deletion to the Mls-1a self-super-antigen but not to staphylococcal enterotoxin A. These results implicate fyn as a critical component in TCR signaling in thymocytes and, potentially, in the process that determines T cell repertoire in the adult mouse.

The Knockout Mouse Project

Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.

Widespread recombinase expression using FLPeR (flipper) mice.

Summary: As conditional genetic strategies advance, the need for multiple site‐specific recombinase systems has emerged. To meet this need in part, we have targeted the constitutive ROSA26 locus to create a mouse strain with generalized expression of the enhanced version of the site‐specific recombinase FLP (FLPe). This strain is designated FLPeR (“flipper”). Using this strain, extensive target gene recombination can be achieved in most tissue types, including cells of the developing germ line. FLPeR mice therefore serve two important functions: as a source of many different FLPe‐expressing primary cell lines and as a deleter strain. Moreover, because the FLPeR mouse is a 129‐derived strain, a 129 genetic background can be preserved when crossed to most ES cell‐derived mice. This enables conditional genetic alterations to be maintained on a standard background, a feature important for obtaining reproducible results and genetically defined controls. genesis 28:106–110, 2000.

Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons

We have used homologous recombination in embryonic stem cells to generate mice carrying a mutation in the gene encoding P0, an immunoglobulin-related recognition molecule and the major protein of peripheral nervous system myelin. These mice are deficient in normal motor coordination and exhibit tremors and occasional convulsions. Axons in their peripheral nerves are severely hypomyelinated and a subset of myelin-like figures and axons degenerate. The mutation leads to an abnormal regulation of some, but not all, molecules involved in myelination. These results demonstrate that P0 is essential for the normal spiraling, compaction, and maintenance of the peripheral myelin sheath and the continued integrity of associated axons. They further suggest that this protein conveys a signal that regulates Schwann cell gene expression.