Inhibition of Angiogenesis by a Mouse Sprouty Protein (original) (raw)
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Evidence that SPROUTY2 functions as an inhibitor of mouse embryonic lung growth and morphogenesis
Mechanisms of Development, 2001
Experimental evidence is rapidly emerging that the coupling of positive regulatory signals with the induction of negative feedback modulators is a mechanism of ®ne regulation in development. Studies in Drosophila and chick have shown that members of the SPROUTY family are inducible negative regulators of growth factors that act through tyrosine kinase receptors. We and others have shown that Fibroblast Growth Factor 10 (FGF10) is a key positive regulator of lung branching morphogenesis. Herein, we provide direct evidence that mSprouty2 is dynamically expressed in the peripheral endoderm in embryonic lung and is downregulated in the clefts between new branches at E12.5. We found that mSprouty2 was expressed in a domain restricted in time and space, adjacent to that of Fgf10 in the peripheral mesenchyme. By E14.5, Fgf10 expression was restricted to a narrow domain of mesenchyme along the extreme edges of the individual lung lobes, whereas mSprouty2 was most highly expressed in the subjacent epithelial terminal buds. FGF10 beads upregulated the expression of mSprouty2 in adjacent epithelium in embryonic lung explant culture. Lung cultures treated with exogenous FGF10 showed greater branching and higher levels of mSpry2 mRNA. Conversely, Fgf10 antisense oligonucleotides reduced branching and decreased mSpry2 mRNA levels. However, treatment with exogenous FGF10 or antisense Fgf10 did not change Shh and FgfR2 mRNA levels in the lungs. We investigated Sprouty2 function during lung development by two different but complementary approaches. The targeted overexpression of mSprouty2 in the peripheral lung epithelium in vivo, using the Surfactant Protein C promoter, resulted in a low level of branching, lung lobe edges abnormal in appearance and the inhibition of epithelial proliferation. Transient high-level overexpression of mSpry2 throughout the pulmonary epithelium by intra-tracheal adenovirus microinjection also resulted in a low level of branching. These results indicate for the ®rst time that mSPROUTY2 functions as a negative regulator of embryonic lung morphogenesis and growth. q
Mechanisms of Development, 2008
Tracheal occlusion during lung development accelerates growth in response to increased intraluminal pressure. In order to investigate the role of internal pressure on murine early lung development, we cauterized the tip of the trachea, to occlude it, and thus to increase internal pressure. This method allowed us to evaluate the effect of tracheal occlusion on the first few branch generations and on gene expression. We observed that the elevation of internal pressure induced more than a doubling in branching, associated with increased proliferation, while branch elongation speed increased 3fold. Analysis by RT-PCR showed that Fgf10, Vegf, Sprouty2 and Shh mRNA expressions were affected by the change of intraluminal pressure after 48h of culture, suggesting mechanotransduction via internal pressure of these key developmental genes. Tracheal occlusion did not increase the number of branches of Fgfr2b −/− mice lungs nor of wild type lungs cultured with Fgfr2b antisense RNA. Tracheal occlusion of Fgf10 LacZ/hypomorphic lungs led to the formation of fewer branches than in wild type. We conclude that internal pressure regulates the FGF10-FGFR2b-Sprouty2 pathway and thus the speed of the branching process. Therefore pressure levels, fixed both by epithelial secretion and boundary conditions, can control the branching process via FGF10-FGFR2b-Sprouty2.
Current Biology, 1999
In Drosophila embryos, the loss of sprouty gene function enhances branching of the respiratory system. Three human sprouty homologues (h-Spry1-3) have been cloned recently, but their function is as yet unknown [1]. Here, we show that a murine sprouty gene (mSpry-2), the product of which shares 97% homology with the respective human protein, is expressed in the embryonic murine lung. We used an antisense oligonucleotide strategy to reduce expression of mSpry-2 by 96%, as measured by competitive reverse transcriptase PCR, in E11.
Temporal effects of Sprouty on lung morphogenesis
Developmental Biology, 2003
Paracrine signaling mediated by FGF-10 and the FGF-R2IIIb receptor is required for formation of the lung. To determine the temporal requirements for FGF signaling during pulmonary morphogenesis, Sprouty-4 (Spry-4), an intracellular FGF receptor antagonist, was expressed in epithelial cells of the fetal lung under control of a doxycycline-inducible system. Severe defects in lobulation and severe lung hypoplasia were observed when Spry-4 was expressed throughout fetal lung development (E6.5-E18.5) or from E6.5 until E13.5. Effects of Spry-4 on branching were substantially reversed by removal of doxycycline from the dam at E12.5, but not at E13.5. In contrast, when initiated late in development (E12.5 to birth), Spry-4 caused less severe pulmonary hypoplasia. Expression of Spry-4 from E16.5 to E18.5 reduced lung growth and resulted in perinatal death due to respiratory failure. Expression of Spry-4 during the saccular and alveolar stages, from E18.5 to postnatal day 21, caused mild emphysema. These findings demonstrate that the embryonic-pseudoglandular stage is a critical time period during which Spry-sensitive pathways are required for branching morphogenesis, lobulation, and formation of the peripheral lung parenchyma.
Oxygen Regulation of Airway Branching in Drosophila Is Mediated by Branchless FGF
Cell, 1999
network through the spiracular openings, and passes Stanford University School of Medicine through primary, secondary, and terminal branches to Stanford, California 94305-5307 reach the tissues. The network develops in the embryo by sequential sprouting of branches from 20 epithelial Summary sacs, each composed of 08ف cells (Samakovlis et al., 1996). The entire branching sequence occurs exclu-The Drosophila tracheal (respiratory) system is a tubusively by cell migration and changes in cell shape. Inilar epithelial network that delivers oxygen to internal tially, six small groups of cells migrate out from each tissues. Sprouting of the major tracheal branches is sac and form primary branches. This process is constereotyped and controlled by hard-wired developtrolled by Branchless (Bnl), a homolog of mammalian mental cues. Here we show that ramification of the fibroblast growth factors (Sutherland et al., 1996; Shilo fine terminal branches is variable and regulated by et al., 1997; Metzger and Krasnow, 1999). The gene is oxygen, and that this process is controlled by a local expressed in six clusters of cells arrayed around each signal or signals produced by oxygen-starved cells. sac. The secreted growth factor activates the Breathless We provide evidence that the critical signal is Branch-(Btl) FGF receptor (FGFR), a receptor tyrosine kinase less (Bnl) FGF, the same growth factor that patterns expressed on nearby tracheal cells, and guides their the major branches during embryogenesis. During larmigrations as they grow out and assemble into primary val life, oxygen deprivation stimulates expression of branches (Klambt et al., 1992; Reichman-Fried et al., Bnl, and the secreted growth factor functions as a 1994; Lee et al., 1996). The Bnl pathway also patterns chemoattractant that guides new terminal branches secondary branch sprouting, but by a different mechato the expressing cells. Thus, a single growth factor nism. High levels of Bnl induce expression of secondary is reiteratively used to pattern each level of airway branch genes, such as the pointed ETS domain tranbranching, and the change in branch patterning results scription factor in the cells at the tips of the outgrowing from a switch from developmental to physiological primary branches, stimulating these cells to form unicelcontrol of its expression. lular tubes called secondary branches (Lee et al., 1996; Sutherland et al., 1996). In this way, the Bnl pathway * To whom correspondence should be addressed (e-mail: krasnow@ planting a metabolically active tissue induced terminal cmgm.stanford.edu). † These authors made equal contributions. branches from neighboring segments to grow in and Cell 212
Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila
Development (Cambridge, England), 1999
Extracellular factors such as FGF and EGF control various aspects of morphogenesis, patterning and cellular proliferation in both invertebrates and vertebrates. In most systems, it is primarily the distribution of these factors that controls the differential behavior of the responding cells. Here we describe the role of Sprouty in eye development. Sprouty is an extracellular protein that has been shown to antagonize FGF signaling during tracheal branching in Drosophila. It is a novel type of protein with a highly conserved cysteine-rich region. In addition to the embryonic tracheal system, sprouty is also expressed in other tissues including the developing eye imaginal disc, embryonic chordotonal organ precursors and the midline glia. In each of these tissues, EGF receptor signaling is known to participate in the control of the correct number of neurons or glia. We show that, in all three tissues, the loss of sprouty results in supernumerary neurons or glia, respectively. Furthermor...
Genes & …, 1996
Receptor tyrosine kinases (RTKs) are members of a diverse class of signaling molecules well known for their roles in cell fate specification, cell differentiation, and oncogenic transformation. Recently several RTKs have been implicated in cell and axon motility, and RTKs are known to mediate chemotactic guidance of tissue culture cells. We have investigated whether the Drosophila FGF receptor homolog, Breathless (BTL), whose activity is necessary for each phase of branching morphogenesis in the embryonic tracheal system, might play a role in guiding the directed migration of tracheal cells. We found that expression of a constitutively active receptor during tracheal development interfered with directed tracheal cell migration and led to extra secondary and terminal branch-forming cells. Reduction in endogenous BTL signaling enhanced the cell migration defects while suppressing the ectopic branching defects. These results are consistent with a model for tracheal development in which spatially regulated BTL activity guides tracheal cell migration and quantitatively regulated BTL activity determines the patterns of secondary and terminal branching cell fates.
Molecular Mechanisms of Early Lung Specification and Branching Morphogenesis
Pediatric Research, 2005
The "hard wiring" encoded within the genome that determines the emergence of the laryngotracheal groove and subsequently early lung branching morphogenesis is mediated by finely regulated, interactive growth factor signaling mechanisms that determine the automaticity of branching, interbranch length, stereotypy of branching, left-right asymmetry, and finally gas diffusion surface area. The extracellular matrix is an important regulator as well as a target for growth factor signaling in lung branching morphogenesis and alveolarization. Coordination not only of epithelial but also endothelial branching morphogenesis determines bronchial branching and the eventual alveolar-capillary interface. Improved prospects for lung protection, repair, regeneration, and engineering will depend on more detailed understanding of these processes. Herein, we concisely review the functionally integrated morphogenetic signaling network comprising the critical bone morphogenetic protein, fibroblast growth factor, Sonic hedgehog, transforming growth factor-, vascular endothelial growth factor, and Wnt signaling pathways that specify and drive early embryonic lung morphogenesis. (Pediatr Res 57: 1-12, 2005) Abbreviations BMP, bone morphogenetic protein DKK, Dickkopf EGF (R), epidermal growth factor (receptor) ERK, extracellular regulated kinase FGF (R), fibroblast growth factor (receptor) FN, fibronectin LRP, lipoprotein receptor-related proteins MAP, membrane-associated protein PDGF, platelet-derived growth factor RAR, retinoic acid receptor sFRP, secreted Frizzled-related protein SHH, Sonic hedgehog Sp-C, surfactant protein C TGF-␣ (), transforming growth factor alpha (beta) VEGF (R), vascular endothelial growth factor (receptor)