Expression of Spred and Sprouty in developing rat lung

Sproutys and Sprouty-related proteins, Spred-1 and -2, are known inhibitors of fibroblast growth factor (FGF) signaling, which plays key role in lung branching morphogenesis and the development of other tissues. The present study demonstrates that Spreds are expressed in a variety of rat embryonic tissues (brain, intestine, heart, skin) including the lung. In the embryonic lung, Spreds and Sproutys are expressed during the early stages of branching morphogenesis, but their expression profiles are both distinct and overlapping. Spreds are predominantly expressed in mesenchymal cells in contrast to Sproutys, which are abundantly expressed in epithelial cells. Spred expression is especially strong in the regions of new bud formation both in the peripheral mesenchyme as well as in the epithelium. The peripheral region also expresses FGF-10 in the mesenchymal cells and FGF-9 in the mesothelial cells. The expression profiles suggest that Spreds, Sproutys and FGF-9/FGF-10 are part of epithelial-mesenchymal interactions, which are essential for the development and maintenance of normal lung branching pattern.     

Expression of Spred and Sprouty in developing rat lung

Sproutys and Sprouty-related proteins, Spred-1 and -2, are known inhibitors of fibroblast growth factor (FGF) signaling, which plays key role in lung branching morphogenesis and the development of other tissues. The present study demonstrates that Spreds are expressed in a variety of rat embryonic tissues (brain, intestine, heart, skin) including the lung. In the embryonic lung, Spreds and Sproutys are expressed during the early stages of branching morphogenesis, but their expression profiles are both distinct and overlapping. Spreds are predominantly expressed in mesenchymal cells in contrast to Sproutys, which are abundantly expressed in epithelial cells. Spred expression is especially strong in the regions of new bud formation both in the peripheral mesenchyme as well as in the epithelium. The peripheral region also expresses FGF-10 in the mesenchymal cells and FGF-9 in the mesothelial cells. The expression profiles suggest that Spreds, Sproutys and FGF-9/FGF-10 are part of epithelial-mesenchymal interactions, which are essential for the development and maintenance of normal lung branching pattern.     

Sprouty2 and Spred1-2 proteins inhibit the activation of the ERK pathway elicited by cyclopentenone prostanoids

Sprouty and Spred proteins have been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. In considering the functional role of these proteins, we explored their effects on ERK activation induced by cyclopentenone prostanoids, which bind to and activate Ras proteins. We therefore found that ectopic overexpression in HeLa cells of human Sprouty2, or human Spred1 or 2, inhibits ERK1/2 and Elk-1 activation triggered by the cyclopentenone prostanoids PGA(1) and 15d-PGJ(2). Furthermore, we found that in HT cells that do not express Sprouty2 due to hypermethylation of its gene-promoter, PGA(1)-provoked ERK activation was more intense and sustained compared to other hematopoietic cell lines with unaltered Sprouty2 expression. Cyclopentenone prostanoids did not induce Sprouty2 tyrosine phosphorylation, in agreement with its incapability to activate tyrosine-kinase receptors. However, Sprouty2 Y55F, which acts as a defective mutant upon tyrosine-kinase receptor stimulation, did not inhibit cyclopentenone prostanoids-elicited ERK pathway activation. In addition, Sprouty2 did not affect the Ras-GTP levels promoted by cyclopentenone prostanoids. These results unveil both common and differential features in the activation of Ras-dependent pathways by cyclopentenone prostanoids and growth factors. Moreover, they provide the first evidence that Sprouty and Spred proteins are negative regulators of the ERK/Elk-1 pathway activation induced not only by growth-factors, but also by reactive lipidic mediators.     

FGF8 spliceforms mediate early mesoderm and posterior neural tissue formation in Xenopus

The relative contributions of different FGF ligands and spliceforms to mesodermal and neural patterning in Xenopus have not been determined, and alternative splicing, though common, is a relatively unexplored area in development. We present evidence that FGF8 performs a dual role in X. laevis and X. tropicalis early development. There are two FGF8 spliceforms, FGF8a and FGF8b, which have very different activities. FGF8b is a potent mesoderm inducer, while FGF8a has little effect on the development of mesoderm. When mammalian FGF8 spliceforms are analyzed in X. laevis, the contrast in activity is conserved. Using a loss-of-function approach, we demonstrate that FGF8 is necessary for proper gastrulation and formation of mesoderm and that FGF8b is the predominant FGF8 spliceform involved in early mesoderm development in Xenopus. Furthermore, FGF8 signaling is necessary for proper posterior neural formation; loss of either FGF8a or a reduction in both FGF8a and FGF8b causes a reduction in the hindbrain and spinal cord domains.     

Sprouty2 and Sprouty4 are essential for embryonic morphogenesis and regulation of FGF signaling

Sprouty genes encode cytoplasmic membrane-associated proteins that inhibit receptor tyrosine kinase signaling. Four orthologs of Drosophila Sprouty (dSpry) (Sprouty1-4) have been identified in mammals. Physiological function of Sprouty1 and Sprouty2 has been investigated using gene targeting approaches, however to date detailed examination of Sprouty4 knockout (KO) mice has not been reported. In this study, Sprouty4 KO mice were generated and characterized. Although a significant fraction of Sprouty4 KO mice died shortly after birth due to mandible defects, the remainder were viable and fertile. Growth retardation was observed for most Sprouty4-deficient mice, with nearly all Sprouty4 KO mice having polysyndactyly. ERK activation was sustained in Sprouty4 KO mouse embryonic fibroblasts (MEFs) in response to FGF, but not to EGF. Sprouty2 and Sprouty4 double KO (DKO) mice were embryonic lethal and showed severe defects in craniofacial, limb, and lung morphogenesis. These findings suggest both redundant and non-redundant functions for Sprouty2 and Sprouty4 on embryonic development and FGF signaling.     

Fgf9 signalling stimulates Spred and Sprouty expression in embryonic mouse pancreas mesenchyme

Epithelial-mesenchymal interactions are critical for normal pancreas development. Fibroblast growth factor (Fgf)-10 is expressed in the pancreatic mesenchyme and its signalling is required for normal growth and regulation of gene expression in the pancreatic epithelium. However, little is known about putative Fgf signalling to the mesenchyme. Here we have examined the embryonic pancreas expression of differentially spliced Fgf receptor isoforms and their targets; the Sprouty (Spry) and Spred family genes which are induced by Fgf signalling. Using qPCR to quantify mRNA levels in microdissected pancreatic epithelium and mesenchyme as well as in FACS isolated Pdx1-GFP(+) and -GFP(-) cell populations we demonstrate that several members of the Spred and Sprouty families are expressed in embryonic mouse pancreas and find Spred1 and -2 as well as Spry2 and -4 to be predominantly expressed in pancreatic mesenchyme. Using embryonic pancreas explant cultures we demonstrate that Spred1/2 and Spry2/4 expression is regulated by Fgf receptor signalling and is increased by treatment with Fgf9, but not by Fgf7 or Fgf10. We extend previous work showing that Fgf9 is expressed in pancreatic mesenchyme, and since Fgf9 is known to activate the mesenchyme-specific "c"-splice forms of Fgf receptors, while Fgf7 and -10 both activate the epithelium-specific "b"-splice forms of Fgf receptors, these results suggest that Fgf signalling is active in the pancreatic mesenchyme, where expression of Spred1/2 and Spry2/4 appear downstream of Fgf9 signalling.     

Requirements for FGF3 and FGF10 during inner ear formation

Members of the fibroblast growth factor (FGF) gene family control formation of the body plan and organogenesis in vertebrates. FGF3 is expressed in the developing hindbrain and has been shown to be involved in inner ear development of different vertebrate species, including zebrafish, Xenopus, chick and mouse. In the mouse, insertion of a neomycin resistance gene into the Fgf3 gene via homologous recombination results in severe developmental defects during differentiation of the otic vesicle. We have addressed the precise roles of FGF3 and other FGF family members during formation of the murine inner ear using both loss- and gain-of-function experiments. We generated a new mutant allele lacking the entire FGF3-coding region but surprisingly found no evidence for severe defects either during inner ear development or in the mature sensory organ, suggesting the functional involvement of other FGF family members during its formation. Ectopic expression of FGF10 in the developing hindbrain of transgenic mice leads to the formation of ectopic vesicles, expressing some otic marker genes and thus indicating a role for FGF10 during otic vesicle formation. Expression analysis of FGF10 during mouse embryogenesis reveals a highly dynamic pattern of expression in the developing hindbrain, partially overlapping with FGF3 expression and coinciding with formation of the inner ear. However, FGF10 mutant mice have been reported to display only mild defects during inner ear differentiation. We thus created double mutant mice for FGF3 and FGF10, which form severely reduced otic vesicles, suggesting redundant roles of these FGFs, acting in combination as neural signals for otic vesicle formation.     

Sprouty1 and Sprouty2 limit both the size of the otic placode and hindbrain Wnt8a by antagonizing FGF signaling

Multiple signaling molecules, including Fibroblast Growth Factor (FGF) and Wnt, induce two patches of ectoderm on either side of the hindbrain to form the progenitor cell population for the inner ear, or otic placode. Here we report that in Spry1, Spry2 compound mutant embryos (Spry1^−/−; Spry2^−/− embryos), the otic placode is increased in size. We demonstrate that the otic placode is larger due to the recruitment of cells, normally destined to become cranial epidermis, into the otic domain. The enlargement of the otic placode observed in Spry1^−/−; Spry2^−/− embryos is preceded by an expansion of a Wnt8a expression domain in the adjacent hindbrain. We demonstrate that both the enlargement of the otic placode and the expansion of the Wnt8a expression domain can be rescued in Spry1^−/−; Spry2^−/− embryos by reducing the gene dosage of Fgf10. Our results define a FGF-responsive window during which cells can be continually recruited into the otic domain and uncover SPRY regulation of the size of a putative Wnt inductive center.     

Vertebrate Sprouty genes are induced by FGF signaling and can cause chondrodysplasia when overexpressed.

The Drosophila sprouty gene encodes an antagonist of FGF and EGF signaling whose expression is induced by the signaling pathways that it inhibits. Here we describe a family of vertebrate Sprouty homologs and demonstrate that the regulatory relationship with FGF pathways has been conserved. In both mouse and chick embryos, Sprouty genes are expressed in intimate association with FGF signaling centers. Gain- and loss-of-function experiments demonstrate that FGF signaling induces Sprouty gene expression in various tissues. Sprouty overexpression obtained by infecting the prospective wing territory of the chick embryo with a retrovirus containing a mouse Sprouty gene causes a reduction in limb bud outgrowth and other effects consistent with reduced FGF signaling from the apical ectodermal ridge. At later stages of development in the infected limbs there was a dramatic reduction in skeletal element length due to an inhibition of chondrocyte differentiation. The results provide evidence that vertebrate Sprouty proteins function as FGF-induced feedback inhibitors, and suggest a possible role for Sprouty genes in the pathogenesis of specific human chondrodysplasias caused by activating mutations in Fgfr3.     

Hydrolysis of phosphatidylcholine is stimulated by Ras proteins during mitogenic signal transduction.

We have used a dominant inhibitory ras mutant (Ha-ras Asn-17) to investigate the relationship of Ras proteins to hydrolysis of phosphatidylcholine (PC) in the transduction of mitogenic signals. Expression of Ha-Ras Asn-17 inhibited NIH 3T3 cell proliferation induced by polypeptide growth factors or phorbol esters. In contrast, the mitogenic activity of PC-specific phospholipase C (PC-PLC) was not inhibited by Ha-Ras Asn-17 expression. Similarly, cotransfection with a cloned PC-PLC gene bypassed the block to NIH 3T3 cell proliferation resulting from expression of the inhibitory ras mutant. Hydrolysis of PC can therefore induce cell proliferation in the absence of normal Ras activity, suggesting that PC-derived second messengers may act downstream of Ras in mitogenic signal transduction. This was substantiated by the finding that Ha-Ras Asn-17 expression inhibited growth factor-stimulated hydrolysis of PC. Taken together, these results indicate that PC hydrolysis is a target of Ras during the transduction of growth factor-initiated mitogenic signals.     

Neurotrophin receptor homolog (NRH1) proteins regulate mesoderm formation and apoptosis during early Xenopus development

Recent experiments suggest that Xenopus Neurotrophin Receptor Homolog 1 (NRH1) proteins act through the planar cell polarity pathway to regulate convergent extension movements during gastrulation and neurulation. We show in this paper that NRH1 proteins are also required for the proper expression of mesodermally expressed genes such as Xbra and Chordin, and to a lesser extent, of Xwnt11. Loss of NRH1 function is followed, during gastrula and neurula stages, by a dramatic increase in apoptosis. Apoptosis is delayed by injection of Xbra RNA, suggesting that cell death is a consequence, at least in part, of the down-regulation of this gene, and it is also delayed by expression of activated forms of Rho, Rac and Cdc42. These small GTPases have previously been implicated in the planar cell polarity pathway in Xenopus and, in other systems, in the regulation of apoptosis. We conclude that the effects of NRH1 proteins include the regulation of mesodermal gene expression and that the disruption of gastrulation that is caused by their loss of function is a consequence of the down-regulation of Xbra and other genes, in addition to direct interference with the planar cell polarity pathway. The apoptosis observed in embryos lacking NRH1 function is not an indirect consequence of the disruption of gastrulation, and indeed it may contribute to the observed morphological defects.     

Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals

At the border of the neural plate, the induction of the neural crest can be achieved by interactions with the epidermis, or with the underlying mesoderm. Wnt signals are required for the inducing activity of the epidermis in chick and amphibian embryos. Here, we analyze the molecular mechanisms of neural crest induction by the mesoderm in Xenopus embryos. Using a recombination assay, we show that prospective paraxial mesoderm induces a panel of neural crest markers (Slug, FoxD3, Zic5 and Sox9), whereas the future axial mesoderm only induces a subset of these genes. This induction is blocked by a dominant negative (dn) form of FGFR1. However, neither dnFGFR4a nor inhibition of Wnt signaling prevents neural crest induction in this system. Among the FGFs, FGF8 is strongly expressed by the paraxial mesoderm. FGF8 is sufficient to induce the neural crest markers FoxD3, Sox9 and Zic5 transiently in the animal cap assay. In vivo, FGF8 injections also expand the Slug expression domain. This suggests that FGF8 can initiate neural crest formation and cooperates with other DLMZ-derived factors to maintain and complete neural crest induction. In contrast to Wnts, eFGF or bFGF, FGF8 elicits neural crest induction in the absence of mesoderm induction and without a requirement for BMP antagonists. In vivo, it is difficult to dissociate the roles of FGF and WNT factors in mesoderm induction and neural patterning. We show that, in most cases, effects on neural crest formation were parallel to altered mesoderm or neural development. However, neural and neural crest patterning can be dissociated experimentally using different dominant-negative manipulations: while Nfz8 blocks both posterior neural plate formation and neural crest formation, dnFGFR4a blocks neural patterning without blocking neural crest formation. These results suggest that different signal transduction mechanisms may be used in neural crest induction, and anteroposterior neural patterning.     

Argonaute2 is essential for mammalian gastrulation and proper mesoderm formation

Mammalian Argonaute proteins (EIF2C1-4) play an essential role in RNA-induced silencing. Here, we show that the loss of eIF2C2 (Argonaute2 or Ago2) results in gastrulation arrest, ectopic expression of Brachyury (T), and mesoderm expansion. We identify a genetic interaction between Ago2 and T, as Ago2 haploinsufficiency partially rescues the classic T/+ short-tail phenotype. Finally, we demonstrate that the ectopic T expression and concomitant mesoderm expansion result from disrupted fibroblast growth factor signaling, likely due to aberrant expression of Eomesodermin. Together, these data indicate that a factor best known as a key component of the RNA-induced silencing complex is required for proper fibroblast growth factor signaling during gastrulation, suggesting a possible micro-RNA function in the formation of a mammalian germ layer.     

Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila

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. Furthermore, overexpression of sprouty in wing veins and ovarian follicle cells, two other tissues where EGF signaling is required for patterning, results in phenotypes that resemble the loss-of-function phenotypes of Egf receptor. These results suggest that Sprouty acts as an antagonist of EGF as well as FGF signaling pathways. These receptor tyrosine kinase-mediated pathways may share not only intracellular signaling components but also extracellular factors that modulate the strength of the signal.