Saverio Bellusci - US grants

Our proposal focuses on the localization, migration and mechanism of differentiation of the smooth muscle progenitor cells during embryonic lung development. Fibroblast Growth factor 10 is one of the earliest genes expressed in a regionalized manner in the distal mesenchyme of the embryonic lung. Our preliminary data indicate that FGF10 plays a role in myogenesis. We therefore propose to test the following Hypothesis: progenitor cells are present in the distal mesenchyme in the embryonic lung and Fgf10 can be used as a marker to characterize them. In addition, FGF10 is involved, directly or indirectly, to maintain the smooth muscle progenitor cell population and/or to allow, via BMP4, proper differentiation of these progenitors into smooth muscle cells. Aim 1: To determine temporal-spatial expression of LacZ and Fgf10 in a novel Fgf10-enhancer trap mouse transgenic line. Aim 2: To determine the migration of distal mesenchymal cells toward the distal epithelium and the expression of smooth muscle cell differentiation markers in these cells. Aim 3: To determine the role of Fgf10 in the maintenance and/or differentiation of smooth muscle progenitor cells in the embryonic lung. Aim 4: To determine the role of Bone Morphogenetic Protein 4, a downstream epithelial target of FGF10, in controlling smooth muscle progenitor cell differentiation. Health relevance: Abnormal proliferation of the smooth muscle cells around the bronchi has been shown in several human chronic diseases such as asthma, broncho-pulmonary dysplasia and lymphangioleiomyomatosis. Our proposal may identify novel targets to control the proliferation of smooth muscle.

DESCRIPTION (provided by applicant): Wnt signaling plays a critical role in normal development and cancer. This competitive renewal will determine the role played by the Wnt signaling in the formation of mesenchymal-derived lineages in the lung with an emphasis on parabronchial smooth muscle cells (PSMC). During organogenesis, the peristaltic movements generated by smooth muscle cells are essential to build up the internal pressure in the developing lung, physical force required for lung growth. Our proposal focuses on survival, proliferation and differentiation of the PSMC progenitors located in the peripheral mesenchyme during lung development. In the previous funding cycle, we have shown that these cells express Fgf10 and are located in the sub-mesothelial mesenchyme. These cells progressively relocate around the bronchi and under the influence of Bone morphogenetic 4, a growth factor expressed in the epithelium at high level and partially controlled by FGF10 itself, undergo differentiation into smooth muscle cells (Mailleux et al., 2005). In addition, we have also shown that FGF9, produced initially by the mesothelium was capable of maintaining the PSMC progenitors into an undifferentiated state both in vitro (del Moral et al., 2006) and in vivo (De Langhe et al., 2006). The key mechanistic paradigm that we will test in this proposal is that (-catenin signaling in the mesenchyme requires the formation of an active (-catenin/PITX2 transcriptional complex that will regulate the expression of Pitx2, Fgfr2 and c-Myc. In turn Fgfr2 and c-Myc are instrumental to control survival, proliferation and differentiation of PSMC progenitors. Central Hypothesis: (-catenin/PITX2 signaling axis in the mesenchyme controls survival, proliferation and differentiation of parabronchial smooth muscle cell progenitors during lung development in vivo. Aim 1: To determine the role of (-catenin signaling in the formation of the PSMCs by a loss of function approach by inactivating (-catenin throughout the lung mesenchyme (using the Dermo1Cre driver line) or in PSMC progenitors (using a Fgf10rtTA driver line). The preliminary data generated for this aim allow us to propose three sub-hypotheses that will be tested. Sub-hypothesis 1: (-catenin signaling in the mesenchyme requires the formation of a privileged (-catenin/PITX transcriptional complex. Sub-hypothesis 2: (-catenin/PITX signaling controls the expression of Fgfr2. Sub-hypothesis 3: FGFR2 signaling in the mesenchyme via PITX2 controls the formation of PSMC. HEALTH RELEVANCE: During organogenesis, the peristaltic movements generated by smooth muscle cells are essential to build up the internal pressure in the developing lung, physical force required for lung growth. Our proposal focuses on the role of (-catenin signaling in survival, proliferation and differentiation of the parabronchial smooth muscle cells progenitors located in the peripheral mesenchyme during lung development. We propose that this work will be useful to better understand pathologies where mesenchymal cells in general (whether they are undifferentiated or not) are affected. For example, abnormal proliferation of the smooth muscle cells around the bronchi occurs in several human chronic diseases such as asthma, broncho-pulmonary dysplasia and lymphangioleiomyomatosis.

DESCRIPTION (provided by applicant): Surgical removal of part of the small intestine in newborn or adult leads to the adaptation of the remaining intestine. We propose that Fibroblast growth factor 10 plays a critical role in this adaptation process opening the way to new therapies to enhance gut function after resection. This proposal focuses on the role of the Fibroblast Growth Factor 10 (FGF10) signaling in gut adaptation after small bowel resection. Our preliminary data indicate that Fgf10 is ectopically expressed in the crypts of ileum after small bowel resection, suggesting a significant role for this growth factor in the adaptation process. Central Hypothesis: FGF10/FGFR2b signaling is a key regulator of intestinal epithelial progenitor cell survival, proliferation and differentiation in gut adaptation following small bowel resection in adult mice. Aim 1: To determine the functional role of FGFR2b signaling during gut adaptation following small bowel resection in adult mice using conditional knockdown of FGFR2b. Aim 2: To determine the regenerative role of FGF10 during gut adaptation following small bowel resection in adult mice by using a gain of function of Fgf10 in the adult intestinal epithelium. PUBLIC HEALTH RELEVANCE: The adaptability of the remaining intestine following small bowel resection is an important factor in the pathophysiological consequences resulting from such surgical procedure. Our proposal focuses on the role of the FGFR2b signaling pathway in the gut adaptation process. The elucidation of the mechanisms of action of FGF10 on the epithelium during the gut adaptation process will allow identifying mutations in this pathway in patients with small bowel syndrome as well as the development of innovative therapies to enhance gut adaptation after small bowel resection.Surgical removal of part of the small intestine in newborn or adult leads to the adaptation of the remaining intestine. We propose that Fibroblast growth factor 10 plays a critical role in this adaptation process opening the way to new therapies to enhance gut function after resection. This proposal focuses on the role of the Fibroblast Growth Factor 10 (FGF10) signaling in gut adaptation after small bowel resection. Our preliminary data indicate that Fgf10 is ectopically expressed in the crypts of ileum after small bowel resection, suggesting a significant role for this growth factor in the adaptation process.

Principal Investigator/Program Director (Last, first, middle): Bellusci, Saverio RESEARCH &RELATED Other Project Information 1. * Are Human Subjects Involved? m Yes l No 1.a. If YES to Human Subjects Is the IRB review Pending? m Yes m No IRB Approval Date: Exemption Number: 1 2 3 4 5 6 Human Subject Assurance Number 2. * Are Vertebrate Animals Used? l Yes m No 2.a. If YES to Vertebrate Animals Is the IACUC review Pending? m Yes l No IACUC Approval Date: 07-28-2005 Animal Welfare Assurance Number A3276-01 3. * Is proprietary/privileged information m Yes l No included in the application? 4.a.* Does this project have an actual or potential impact on m Yes l No the environment? 4.b. If yes, please explain: 4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or environmental impact statement (EIS) been performed? m Yes m No 4.d. If yes, please explain: 5.a.* Does this project involve activities outside the U.S. or m Yes l No partnership with International Collaborators? 5.b. If yes, identify countries: 5.c. Optional Explanation: 6. * Project Summary/Abstract 4119-summary.pdf Mime Type: application/pdf 7. * Project Narrative 4626-narrative.pdf Mime Type: application/pdf 8. Bibliography &References Cited 789-biblio.pdf Mime Type: application/pdf 9. Facilities &Other Resources 6084-SBRessources.pdf Mime Type: application/pdf 10. Equipment Tracking Number: Other Information Page 5 OMB Number: 4040-0001 Expiration Date: 04/30/2008 Principal Investigator/Program Director (Last, first, middle): Bellusci, Saverio FGF signaling in the epithelium and mesenchyme plays important functions during lung development. A member of the FGF family, FGF10 is a master player in the signaling network mediating epithelial-mesenchymal interactions in the lung. However, the specific mechanisms of action of FGF10 on the epithelium as well as the epithelial targets downstream of FGF10 in vivo are just emerging. In addition, how FGF10 impacts the formation of the smooth muscle cells, which are essential for alveolarization and lung function, is so far unknown. Our preliminary data indicate that we can use an Fgf10 hypomorphic mouse to decipher the role of FGF10 in lung development. Our preliminary results indicate that a) FGF10 signaling controls !-catenin signaling in the epithelium. b) FGF10 controls the amplification of the epithelial progenitors as well as maintains them in a "distal-like" phenotype and c) FGF10 signaling to the epithelium controls the formation of the alveolar smooth muscle cell progenitors. Furthermore, we recently reported that the pathological activation of an FGF10/FGFR2b autocrine feedback loop in the mesenchyme is responsible for the lung defects observed in a mouse model of Apert syndrome (De Langhe et al., 2006). Apert patients also exhibit many lung defects including fusion of tracheal cartilage, pulmonary aplasia, absent accessory lobe and defective interlobular septation. Our results also show that modulation of Fgf10 expression in this mouse model of Apert syndrome rescues the observed lung defects. This mouse Apert model will therefore allow us to determine how FGF signaling in the mesenchyme controls the differentiation and prevents the differentiation of the smooth muscle cell progenitors. Hypothesis: FGF10 is critical to control mesenchymal differentiation during normal and pathological lung development. Aim 1: To determine the role of epithelial FGF10 signaling during lung development using a newly described Fgf10 hypomorphic mouse. Aim 2: To determine the role of mesenchymal FGF signaling during lung development using a mouse model of Apert syndrome where Fgfr2b is ectopically expressed in the mesenchyme. Health relevance: FGF10 is a