1989 — 1998 |
Soriano, Philippe M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Retroviruses as Probes For Development @ Fred Hutchinson Cancer Research Center
Mutations affecting early development of the mouse embryo will be generated using gene traps in embryonic stem (ES) cells. In this approach, a reporter gene is introduced into ES cells by a retrovirus, but expression of the reporter can only originate from the promoter of a gene into which it has inserted. Since the reporter gene used in these experiments encodes beta-galactosidase activity, expression of the mutated gene can be conveniently followed in heterozygous mice. The consequences of gene disruption can be assessed by breeding the mice to homozygosity. In previous work, 42 lines of mouse mutants have been generated, which display a diversity of beta-galactosidase expression pattern during development. A high proportion of overt phenotypes was observed among these strains, since provirus insertion leads to embryonic lethality in 18 strains and to male sterility in two others. In one lethal strain, the provirus has disrupted the gene which encodes the transcription factor TEF1, leading to cardiac and central nervous system defects at midgestation. We will continue to analyze these strains by histology to understand the defects in mutant embryos, and by cloning the mutated gene. We will also use gene traps to identify genes induced during development in response to two types of factors thought to play critical roles in early development: retinoic acid, a factor believed to have the properties of a morphogen; and growth factors, whose receptors are protein tyrosine kinases implicated in several classical mouse mutations affecting early mouse development. It is anticipated that these studies will lead to the identification of genes playing key roles in early mouse development, and help identify, using genetics, critical components in specific signaling pathways.
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0.958 |
1995 — 1999 |
Soriano, Philippe M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Insertional Mutagenesis and Mouse Development @ Fred Hutchinson Cancer Research Center
The major aims of this proposal are to determine the phenotypic consequences of activating Src family kinases during development, and to understand the mechanisms by which these phenotypes arise. Src family kinases are negatively regulated by phosphorylation of a C- terminal tyrosine by two kinases, Csk and Ctk. Targeted disruption of Csk results in neural tube defects and embryonic lethality. In addition, a number of kinase substrates localized to focal adhesions, such as paxillin, and to the cytoskeleton, such as cortactin, become hyperphosphorylated in Csk- cells due to increased Src family kinase activity. To understand the consequences of activating multiple or individual kinases in development, mice will be derived carrying targeted mutations in the ctk gene, conditional mutations in the csk or ctk genes, and activating mutations in Src PTK genes. To test if alterations in cell adhesion might explain the phenotypes due to activation of Src family kinases, mice mutant for cortactin and paxillin will be generated. These mutations will be used to investigate the role of different cell adhesion mechanisms in the phenotype due to Src family kinase activation. This research should be useful to understand the role of kinase regulatory- and adhesion- pathways in normal embryonic development and in neoplastic transformation.
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0.958 |
1999 — 2010 |
Soriano, Philippe M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gene Expression, Function and Mutagenesis @ Fred Hutchinson Cancer Research Center
Growth factors induce a variety of cellular responses including proliferation, survival and migration but their final target genes remain for the most part unknown. In this proposal, we will focus on identifying targets for platelet derived growth factors (PDGFs) which are involved in various aspects of muscle and cardiovascular development, as well as upstream factors that control their expression. We will use a gene trap approach in ES cells to identify and clone genes whose expression is induced or repressed by PDGF, to study their expression pattern, and to assess the consequences of loss of function in mutant mice. We will concentrate our efforts on genes that are expressed or result in mutant phenotypes in cell types which are deficient in PDGF or PDGF receptor mutant mice. We will study PDGF-dependent gene transcription in cells isolated from somites and presomitic mesoderm, in which PDGF has been implicated for proper patterning, using subtraction-suppression PCR and hybridization to "DNA chip" microarrays. To identify factors that are genetically upstream of PDGF, we will use new gene trap vectors that activate a gene constitutively. The PDGF and PDGF receptor genes will be targeted with a promoterless reporter gene, allowing identification of PDGF upstream factors by increased expression of the reporter. Expression of the trapped genes will be disrupted following Cre recombination, resulting in loss of function mutations. This methodology will allow us to identify PDGF upstream factors as well as their normal physiological role. These studies should help us understand growth factor regulatory mechanisms, and provide information on the specificity and interplay of growth factor signaling pathways in physiological processes.
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0.958 |
2001 — 2010 |
Soriano, Philippe M |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Signal Transduction and Mouse Development @ Fred Hutchinson Cancer Research Center
Growth factors induce a variety of cellular responses including proliferation, survival and migration. A number of growth factors activate receptors with intrinsic tyrosine kinase activity. Extensive studies in cell culture systems have shown that receptor engagement leads to binding of intracellular effectors, and the activation of individual signaling pathways. However, the physiological relevance of utilizing one or another pathway remains mostly unknown. In this proposal, we will focus on identifying the roles of individual signaling pathways downstream of platelet derived growth factors (PDGFs), which are involved in developmental and physiological responses in many tissues, including the vasculature, the kidney, the skeleton and neural crest derivatives. We will achieve our goals by generating mutant mice that express PDGF receptors in which the individual docking sites for various effectors have been mutated. We will further concentrate our efforts on determining if signaling pathways between PDGF receptors and other growth factor receptors are redundant, using genetic substitution methods. To further investigate the role of PDGF signaling in development, we will make use of conditional gene ablation to test the role of PDGF signaling in specific tissues. These studies should help us understand growth factor regulatory mechanisms, and provide information on the specificity and interplay of growth factor signaling pathways in physiological processes.
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0.958 |
2004 — 2008 |
Soriano, Philippe M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Gene Expression Function and Mutagenesis @ Fred Hutchinson Cancer Research Center
Platelet derived growth factors (PDGFs) are required for vascular development, cranial and cardiac neural crest cells, somitic mesoderm, extraembryonic lineages and in the kidney. The mechanisms by which they control cell proliferation, survival and migration have been deciphered in cultured cells, but the identity of the target genes that mediate such pleiotropic functions in development and the adult remain unknown. This proposal focuses on identifying PDGF targets using a novel platform, the gene trap array, in which thousands of cDNAs derived from gene trap disrupted loci in ES cells are spotted on DNA arrays. This approach combines all of the power of DNA array technologies with the possibility of readily generating mutant mice from the frozen ES cell stocks. The physiological roles of a subset of regulated genes will be assessed by deriving mutant mice from the trapped ES cell clones. Target genes will be further characterized by establishing if they are specific for one or the other PDGFR and if they are subject to regulation by other signaling pathways. Conditional gene trap vectors will be generated that allow spatio-temporal elimination of gene activity and the generation of allelic series at the trapped loci. The Gene Trap Array will be expanded to 10,000 mutant ES cell clones and genes mutated by these vectors will be identified by sequencing and their identity will be listed on a web-based platform. Critical components of PDGF signaling will be identified in neural crest cells, using two complementary approaches, the gene trap array or gain-of-function gene trap mutagenesis in a PDGFalphaR sensitized background. Gain-of-function alleles that synergize with a PDGFalphaR mutation will be converted to loss of function alleles to study the normal function of the gene. These studies may shed insight on birth defects associated with abnormal neural crest development, such as cleft palate or DiGeorge Syndrome.
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0.958 |
2012 — 2021 |
Soriano, Philippe M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Fgf Signaling Pathways and Craniofacial Development @ Icahn School of Medicine At Mount Sinai
The major aims of this proposal are to identify signaling mechanisms initiated by FGFs that underlie craniofacial morphogenesis. Loss of the FGFR1 receptor together with FGFR2 in neural crest cells leads to agenesis of multiple components of the midface and mandible, whereas hypomorphic mutations in Fgfr1/2 result in cleft palate. Elsewhere, FGFR2 rather than FGFR1 has a predominant role in salivary gland epithelia, and its activity is differentially regulated by various ligands. Engagement of the FGF signaling cascade leads to dimerization of the FGFRs, binding of multiple intracellular effectors and activation of cellular responses that converge on ERK1/2 and several other pathways. This application proposes: 1. To investigate how FGF signaling coordinates midface development. Combined loss of both Fgfr1 and Fgfr2 in neural crest cells leads to facial clefting that extends through the midline, agenesis of the midface, mandibular hypoplasia, and significant cell death in the lateral nasal and maxillary processes. To determine the origin of these midface defects, Fgfr1/2 mandibular and/or lateral nasal /maxillary process conditional mutants will be generated to investigate how loss of Fgfr1/2 in the mandible or lateral structures contributes to facial clefting. Furthermore, neural crest Fgfr1/2 mutants will be crossed to Bim mutants to disassociate alterations in patterning from BCL-2 family regulated cell death. 2. To identify signaling mechanisms promoted by Fgfr1 and Fgfr2 in craniofacial development. To identify signaling pathways that remain active in a previously generated Fgfr1 and Fgfr2 allelic series of point mutations that prevent the binding of single or multiple effector proteins and the initiation of specific signaling pathways, a proteomic screen will be performed in mouse embryonic palatal mesenchyme cells using endogenous epitope tagged FGFR1 and FGFR2 receptors. In a complementary approach, mice in which additional candidate tyrosine phosphorylation sites are disrupted on the receptors will be generated. 3. To characterize signaling pathways specified by ligand identity. An emerging theme in FGF signaling is that cellular responses in multiple physiological contexts can be encoded in the identity of the ligand and are not only specified at the level of the receptor. Signaling responses that are differentially encoded by FGF7 and FGF10 in submandibular salivary gland branching morphogenesis will be investigated. Critical downstream FGF pathways in this response will be further identified by transcriptional profiling following stimulation with each ligand, and morphogenetic responses that are sensitive to ERK1/2 and PI3K signaling and feedback inhibition pathways will be investigated. These proposed studies explore novel territories in the area of growth factor signaling in craniofacial biology and open new directions for the prevention of craniofacial birth defects.
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0.958 |
2012 — 2021 |
Soriano, Philippe M |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Growth Factor Signaling and Craniofacial Development @ Icahn School of Medicine At Mount Sinai
DESCRIPTION (provided by applicant): The major aims of this proposal are to identify mechanisms underlying midface development that are controlled by PDGF signaling. Loss of the PDGFRa or of its ligands PDGFA and PDGFC lead to facial clefting and improper development of the frontonasal process, whereas hypomorphic mutations in this pathway result in cleft palate. This application proposes: 1. To characterize the processes regulated by PDGFRa in cranial neural crest cells and craniofacial development. Loss of PDGFRa signaling affects two distinct processes, the development of the frontonasal masses and fusion at the midline. We will assess the ability of cranial neural crest cells to populate the frontonasal mass in PDGFRa conditional and hypomorphic mutants, using in vivo Cre lineage tracing and in vitro chemotaxis assays and cranial neural crest cell explants. We will also examine in detail the development of the frontonasal masses and midline fusion. 2. To investigate the pathways that operate downstream of PDGF engaged PI3K/AKT signaling in craniofacial development. Our previous genetic experiments have assigned a central role to the PI3K signaling pathway in mediating PDGF activity in craniofacial development, but further downstream pathways remain unknown. We will identify by mass spectroscopy the phosphorylation targets of PI3K/AKT in primary palatal mesenchyme cells. Genetic epistasis experiments between PDGFRa and phosphorylation target mutants will be conducted to identify their role in craniofacial development. 3. To establish a genetic pathway that instructs craniofacial patterning downstream of PDGF signaling. We have identified a number of transcriptional targets of PDGF in primary palatal mesenchymal cells, which are involved in cell migration and pattern specification in the frontonasal process and the palate. A selection of these PDGF target genes will be used to isolate mouse mutants. Phenotyping will be performed in null or conditional target gene mutants to determine changes in the frontonasal process and the palate. Genetic interactions with PDGFRa mutants will be performed to establish a genetic pathway operating downstream of PDGF in craniofacial development. Among growth factor signaling pathways, PDGF signaling has been exquisitely analyzed at a molecular and cellular level, and the proposed studies are anticipated to have significant impact in craniofacial biology because of the insight provided by this detailed knowledge. This proposal will thus open new directions for the prevention of craniofacial birth defects.
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0.958 |