Veraragavan P Eswarakumar
Yale University
Project start date: 2010-03-03
Project end date: 2015-02-28
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Veraragavan P Eswarakumar
MECHANISMS OF FGFR2 SIGNALING IN SALIVARY GLAND BRANCHING MORPHOGENESIS
Veraragavan P Eswarakumar, Assistant Professor
Yale University, 47 College Street, Ste 203, New Haven, Ct 06520-8047
Grant 1R01DE021088-01 from National Institute Of Dental & Craniofacial Research
Abstract: Fibroblast growth factors (FGF) and their receptors (FGFR) play a central role in salivary gland branching morphogenesis. In mice, targeted disruption of Fgf10 or its receptor, Fgfr2b, causes salivary gland agenesis, demonstrating the essential role of FGF10-FGFR2b signaling pathways in salivary gland development. However, the lack of salivary glands in Fgf10 or Fgfr2b knockout mice precludes further investigation of branching morphogenesis in these mouse models. Therefore, it is necessary to develop an alternative approach to gain insights about the role of Fgfr2b intracellular signaling pathways in salivary gland branching morphogenesis. We have developed a novel mouse model system in which specific signaling pathways downstream of Fgfr2 have been abrogated by a knock-in mutation, without compromising the tyrosine kinase activity of the receptor to activate other signaling pathways. Our hypothesis is that Fgfr2b signaling via Frs21 is essential for salivary gland branching morphogenesis. In Aim 1, we will (1) analyze the branching morphogenesis of submandibular glands (SMGs) from different mutant mouse strains by histology and ex vivo organ culture; (2) use mesenchyme-free SMG cultures to investigate epithelial morphogenesis; and (3) use cell lines expressing the mutant and wild type receptors to evaluate the mechanism of epithelial transphosphorylation of Frs21 by Fgfr1b and Fgfr2b during branching morphogenesis. In Aim 2, we will determine the role of Frs21-mediated Grb2 and Shp2 signaling pathways in SMG branching morphogenesis. Our hypothesis is that Shp2 is the critical mediator of Fgfr2b signaling via Frs21 for SMG branching morphogenesis. We will test this hypothesis using two strains of genetically engineered mutant mice. In the first mutant mouse model, the four Grb2 binding sites are mutated to phenylalanine (4F), and thus cannot recruit Grb2. In the second mutant mouse model, the two Shp2 binding sites are mutated to phenylalanine (2F), and thus cannot recruit Shp2. We will perform (1) histology and ex vivo SMG organ culture to determine the role of Grb2 and Shp2 in branching morphogenesis; and (2) recombinant SMG culture to determine whether the mutations are cell autonomous or non-cell autonomous by culturing wild type epithelium with Grb2 or Shp2 mutant mesenchyme and vice versa. In addition, we propose two approaches to identify novel genetic pathways a) genome-wide mRNA expression analysis in the epithelium and mesenchyme of the mutant SMGs, and b) expression-based pathway analysis of target genes in SMG. Collectively, this work will provide a detailed molecular picture of how FGF signaling, mediated by the Fgfr2b isoform and the docking protein Frs21, regulates salivary gland branching morphogenesis. The results of these studies may enable the design of novel methods for salivary gland regeneration using FGFR2b mediated pathways to regulate progenitor cell differentiation and morphogenesis. Salivary gland dysfunction caused by mutations, radiotherapy or chemotherapy affect the quality of life of patients by causing oral dryness, dental caries, hampered speech, and xerostomia. The overall goal of this proposal is to obtain a comprehensive molecular picture of the signaling pathways that are activated in response to Fgfr2b stimulation, specifically the Frs21-mediated pathways essential for the development of salivary glands. We anticipate that our findings will shed new light on fundamental intracellular signaling pathways essential for epithelial morphogenesis of salivary glands, and will enable the design of novel methods for salivary gland regeneration using FGFR2b-mediated pathways to regulate progenitor/stem cell differentiation and morphogenesis
Keywords: AFGF; ATP[{..}]protein-tyrosine O-phosphotransferase; Affect; Asialia; Asialias; BEK fibroblast growth factor receptor; BEK protein tyrosine kinase; BMP4; Binding Sites; Biological Models; Cancer Radiotherapy; Caries; Cell Communication and Signaling; Cell Differentiation; Cell Differentiation process; Cell Line; Cell Lines, Strains; Cell Signaling; CellLine; Cells; Cold-Insoluble Globulins; Combining Site; Culturing, in vitro Organ; Culturing, in vitro Vertebrate, Organ; Cytosolic Protein Tyrosine Phosphastase; DNA Synthesis Factor; Dental Decay; Dental caries; Development; Docking; Dryness; Dysfunction; EC 2.7; ECGF; ECGF-alpha; ECGF-beta; ECGFA; ECGFB; EPH- and ELK-Related Tyrosine Kinase; EPH-and ELK-Related Kinase; EPHA8; Embryo Development; Embryogenesis; Embryonic Development; Endothelial Cell Growth Factor; Engineering; Engineerings; EphA8 Protein; Ephrin Type-A Receptor 8; Ephrin Type-A Receptor 8 Precursor; Epithelial; Epithelium; FGF; FGF-2 receptor; FGF1; FGF1 gene; FGFA; FGFR Signaling Adaptor; FGFR Substrate 2; FGFR-2; FGFR2; FGFR2b; FN1; FNZ; FRS2; FRS2 protein, human; FRS2-Alpha; FRS2A; FRS2A protein, human; FRS2alpha protein, human; Fibroblast Growth Factor; Fibroblast Growth Factor Receptor 2; Fibroblast Growth Factor Receptor Substrate 2; Fibroblast Growth Regulatory Factor; Fibronectin 1; Fibronectins; Functional disorder; Gene Expression; Gene Targeting; Genetic; Genetic Alteration; Genetic Change; Genetic Models; Genetic defect; Genetically Engineered Mouse; Glycoprotein GP-2; Goals; HBGF; HBGF1; HEK3; Head and Neck, Salivary Glands; Histology; Hyposalivation; Hyposalivations; Intracellular Communication and Signaling; Investigation; Isoforms; Kinases; Knock-in; Knock-in Mouse; Knockout Mice; L-Phenylalanine; L-Tyrosine; LETS Proteins; Laminin; Large External Transformation-Sensitive Protein; Light; Mammals, Mice; Mediating; Mediator; Mediator of Activation; Mediator of activation protein; Mesenchymas; Mesenchyme; Methods; Mice; Mice, Knock-out; Mice, Knockout; Mice, Mutant Strains; Model System; Models, Biologic; Molecular; Morphogenesis; Mother Cells; Mouth Dryness; Murine; Mus; Mutant Strains Mice; Mutate; Mutation; Natural regeneration; Network Analysis; Null Mouse; Opsonic Glycoprotein; Opsonic alpha(2)SB Glycoprotein; Oral; Organ Culture; Organ Culture Techniques; PTK; PTPase; Pathway Analysis; Pathway interactions; Patients; Pattern; Phenylalanine; Phenylalanine, L-Isomer; Phosphotransferases; Phosphotyrosine Phosphatase; Phosphotyrosyl Protein Phosphatase; Photoradiation; Physiopathology; Play; Progenitor Cells; Protein Isoforms; Protein Tyrosine Kinase; Protein Tyrosine Kinase EEK; Protein Tyrosine Phosphatase; Proteins; QOL; Quality of life; Radiation therapy; Radiotherapeutics; Radiotherapy; Reactive Site; Receptor Protein; Recombinants; Recruitment Activity; Regeneration; Role; SUC1-Associated Neurotrophic Factor Target; Salivary Glands; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Molecule; Speech; Stem cells; Structure; Submandibular gland; Submaxillary Gland; System; System, LOINC Axis 4; TYR; Targetings, Gene; Testing; Transphosphorylases; Tyrosine; Tyrosine Kinase; Tyrosine Phosphatase; Tyrosine, L-isomer; Tyrosine-Protein Kinase Receptor EEK; Tyrosine-Specific Protein Kinase; Tyrosyl Phosphoprotein Phosphatase; Tyrosylprotein Kinase; Work; Xerostomia; Xerostomias; alpha 2-Surface Binding Glycoprotein; aptyalism; base; bek fgf receptor kinase; bek fibroblast growth factor receptor kinase; biological signal transduction; chemotherapy; cultured cell line; design; designing; dry mouth; experiment; experimental research; experimental study; fibroblast growth factor receptor 2b; fibroblast growth factor receptor substrate 2 protein, human; fibroblast growth factor receptor substrate 2, human; gene product; genome mutation; genome-wide; gland development; human FRS2 protein; hydroxyaryl protein kinase; insight; irradiation; mRNA Expression; mouse model; mouse mutant; mutant; mutant mouse model; novel; para-Tyrosine; pathophysiology; pathway; progenitor; protein tyrosine phosphate phosphohydrolase; public health relevance; receptor; receptor-1, bek-related fibroblast growth factor; recruit; regenerate; research study; response; social role; stem cell differentiation; tooth decay; tyrosyl protein kinase
Relevance: 7. Salivary gland dysfunction caused by mutations, radiotherapy or chemotherapy affect the quality of life of patients by causing oral dryness, dental caries, hampered speech, and xerostomia. The overall goal of this proposal is to obtain a comprehensive molecular picture of the signaling pathways that are activated in response to Fgfr2b stimulation, specifically the Frs2¿-mediated pathways essential for the development of salivary glands. We anticipate that our findings will shed new light on fundamental intracellular signaling pathways essential for epithelial morphogenesis of salivary glands, and will enable the design of novel methods for salivary gland regeneration using FGFR2b-mediated pathways to regulate progenitor/stem cell differentiation and morphogenesis
Project start date: 2010-07-01
Project end date: 2015-06-30
Budget start date: 1-JUL-2010
Budget end date: 30-JUN-2011
PFA/PA: PA-07-070
1R01DE021088-01 (2010): $401784
MECHANISMS OF FGFR2 SIGNALING IN CRANIOFACIAL DEVELOPMENT
Veraragavan P Eswarakumar, Assistant Professor
Yale University, 47 College Street, Ste 203, New Haven, Ct 06520-8047
Grant 1R01DE020823-01 from National Institute Of Dental & Craniofacial Research
Abstract: Craniofacial anomalies are the fourth most common congenital birth defects that occur in new born. Dominant gain-of-function mutations in the fibroblast growth factor receptor 2 (FGFR2) account for the majority of the human craniosynostosis syndromes including Crouzon, Pfeiffer, Jackson-Weiss, Seathre Chotzen and Apert syndrome which are characterized by the premature fusion of cranial sutures before the completion of brain growth. The cranial sutures are specialized joints which contain rapidly dividing osteoprogenitors and mesenchymal cells. The balance between proliferating and differentiating osteoprogenitors is finely regulated by quantitative signals from growth factor receptors including FGFR2, which is expressed by proliferating osteoprogenitors and down-regulated in differentiating osteoblasts. Our preliminary data show that mutations in FGFR2 preferentially affect cranial and facial bones of neural crest origin. Herein, we propose two Specific Aims to determine the mechanisms of FGFR2 signaling that govern craniofacial development and morphogenesis. In Aim 1.1, we propose to use the Cre/loxP system to investigate whether activation of a mutant receptor in neural crest cells alone is sufficient to cause craniosynostosis syndrome or whether it requires signals from paraxial mesodermal cells. We will conditionally activate the Crouzon mutation in neural crest cells or mesodermal cells using Wnt1-Cre or Mesp1-Cre mice, respectively. A universal dual reporter strain will be used to identify recombinant cells from non-recombinant cells. Sutures will be examined by histology, in situ hybridization and by microCT. In Aim 1.2, we propose to use a novel two color fluorescent system to study the mechanisms of FGFR2 signaling in early osteoprogenitors and mesenchymal cells. Gene expression studies will be performed on three defined homogeneous populations of cells isolated by Fluorescence Activated Cell Sorting based on the expression of osteogenic differentiation stage-specific fluorescent markers. Cell intrinsic versus extrinsic effects of the FGFR2 mutations will be studied by co-culture experiments. In Aim 2, we propose to determine the docking protein Frs21-dependent and independent signaling of FGFR2 that govern craniofacial development. We have shown that uncoupling of Frs21 from the mutant FGFR2 receptor rescues the craniosynostosis phenotype in mice. FGFR2 activation leads to phosphorylation of six tyrosine residues on Frs21, which in turn serve as docking sites for the recruitment of four Grb2 and two Shp2 signaling molecules. Our hypothesis is that Shp2 is the critical mediator of pathological signals of Frs21 from the activated FGFR2. We will test this hypothesis by using two genetically engineered Frs21 mutant mouse models that cannot recruit Grb2 or Shp2 in response to FGFR activation in the context of the Crouzon mutation. Collectively, this work will provide a detailed molecular picture of how FGF-signaling, mediated by the FGFR2 and the docking protein Frs21, regulates craniofacial patterning and development under normal and disease conditions. Dominant gain of function mutations in FGFR2 accounts for majority of the human craniosynostosis syndromes including Crouzon, Pfeiffer and Apert syndrome which are characterized by the premature fusion of cranial sutures before the completion of brain growth. The goal of this proposal is to obtain a comprehensive molecular picture of the signaling pathways that are activated in response to FGFR2 stimulation, which govern craniofacial development. It is anticipated that the findings will enable the design of novel pharmacological interventions for craniofacial disorders that are caused by dysfunction in FGFRs
Keywords: Accounting; Affect; Alanine; Alanine, L-Isomer; Animal Model; Animal Models and Related Studies; Animals; Apert syndrome; Articulation; BEK; BEK fibroblast growth factor receptor; BEK protein tyrosine kinase; BFR-1; Binding Sites; Biochemical; Biological Models; Birth; Birth Defects; Bone; Bone and Bones; Bone structure of cranium; Bone structure of face; Bones and Bone Tissue; Brain; Breeding; Cek-8 Kinase; Cell Body; Cell Communication and Signaling; Cell Signaling; Cells; Cephalic; Cleft Palate; Co-culture; Cocultivation; Coculture; Coculture Techniques; Color; Combining Site; Congenital Abnormality; Congenital Anatomic Abnormality; Congenital Anatomical Abnormality; Congenital Defects; Congenital Deformity; Congenital Malformation; Cranial; Cranial Sutures; Craniofacial Abnormalities; Craniosynostosis; Cranium; Cytosolic Protein Tyrosine Phosphastase; DNA Synthesis Factor; Data; Defect; Development; Disease; Disorder; Docking; Dysfunction; Dysplasia; EC 2.7; ECGF; Encephalon; Encephalons; Endothelial Cell Growth Factor; Engineering; Engineerings; Eph Receptor Ligands; Eph-A4 Receptor Tyrosine Kinase; EphA4 Protein; EphA4 Receptor; Ephrin Receptor A4; Ephrins; Equilibrium; FGF; FGF-2 receptor; FGF-R; FGFR; FGFR-2; FGFR2; FGFR2 gene; FGFR2c; Facial Bones; Fibroblast Growth Factor; Fibroblast Growth Factor Receptor 2; Fibroblast Growth Factor Receptor 2 Gene; Fibroblast Growth Factor Receptor Family; Fibroblast Growth Factor Receptors; Fibroblast Growth Regulatory Factor; Fluorescence-Activated Cell Sorting; Fractionation, Fluorescence Activated Cell Sorting; Functional disorder; Gene Expression; Gene Targeting; Generalized Growth; Genetic; Genetic Alteration; Genetic Change; Genetic defect; Genetically Engineered Mouse; Genetics, in situ Hybridization; Germ Lines; Goals; Growth; Growth Factor Receptors; HBGF; Histology; Human; Human, General; In Situ Hybridization; In Vitro; Intervention; Intervention Strategies; Intracellular Communication and Signaling; Isoforms; Joint structure of suture of skull; Joints; KGFR Gene; KSAM-1; Keratinocyte Growth Factor Receptor Gene; Kinases; Knock-in; Knock-in Mouse; L-Alanine; L-Tyrosine; Life; Link; Mammals, Mice; Man (Taxonomy); Man, Modern; Mediating; Mediator; Mediator of Activation; Mediator of activation protein; Mesenchymal; Mesenchymas; Mesenchyme; Mesoderm Cell; Mesodermal Cell; Methods; Mice; Mice, Mutant Strains; Model System; Models, Biologic; Molecular; Molecular Analysis; Molecular Genetic Abnormality; Morphogenesis; Mother Cells; Murine; Mus; Mutant Strains Mice; Mutate; Mutation; Nervous System, Brain; Neural Crest; Neural Crest Cell; Osteoblasts; PTPase; Parturition; Pathway interactions; Pattern; Phenotype; Phosphorylation; Phosphotransferases; Phosphotyrosine Phosphatase; Phosphotyrosyl Protein Phosphatase; Physiopathology; Population; Prevalence; Progenitor Cells; Proliferating; Protein Isoforms; Protein Phosphorylation; Protein Tyrosine Phosphatase; Proteins; Proto-Oncogene, Growth Factor Receptor; Reactive Site; Receptor Protein; Receptor, EphA4; Receptors, FGF; Recombinants; Recruitment Activity; Reporter; Role; Sek-1 Receptor Tyrosine Kinase; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Molecule; Site; Skull; Sortings, Fluorescence-Activated Cell; Specificity; Staging; Staining method; Stainings; Stains; Stem cells; Surgical sutures; Sutures; Syndrome; System; System, LOINC Axis 4; TK14; TYR; Targetings, Gene; Testing; Tissue Growth; Transphosphorylases; Tyrosine; Tyrosine Phosphatase; Tyrosine, L-isomer; Tyrosyl Phosphoprotein Phosphatase; Work; ing; balance; balance function; base; bek fgf receptor kinase; bek fibroblast growth factor receptor kinase; biological signal transduction; bone; cell body (neuron); coronal suture; craniofacial; craniofacies; cranium; design; designing; disease/disorder; dyscrasia; experiment; experimental research; experimental study; face bone structure; fibroblast growth factor receptor 2c; gain of function; gain of function mutation; gene product; genome mutation; genome-wide; in situ Hybridization Staining Method; interventional strategy; loss of function; mRNA Expression; model organism; mouse mutant; mutant; mutant mouse model; neural cell body; neuronal cell body; novel; ontogeny; ossa faciei; osteogenic; osteoprogenitor cell; para-Tyrosine; pathophysiology; pathway; premature; protein tyrosine phosphate phosphohydrolase; receptor; receptor-1, bek-related fibroblast growth factor; recruit; research study; response; social role; soma; stem; suture fusion; synostosis (cranial)
Relevance: 7. Dominant gain of function mutations in FGFR2 accounts for majority of the human craniosynostosis syndromes including Crouzon, Pfeiffer and Apert syndrome which are characterized by the premature fusion of cranial sutures before the completion of brain growth. The goal of this proposal is to obtain a comprehensive molecular picture of the signaling pathways that are activated in response to FGFR2 stimulation, which govern craniofacial development. It is anticipated that the findings will enable the design of novel pharmacological interventions for craniofacial disorders that are caused by dysfunction in FGFRs
Project start date: 2010-03-03
Project end date: 2015-02-28
Budget start date: 3-MAR-2010
Budget end date: 28-FEB-2011
PFA/PA: PA-07-070
1R01DE020823-01 (2010): $421295