Growth factor signaling in lung morphogenetic centers: automaticity, stereotypy and symmetry

Abstract

Lung morphogenesis is stereotypic, both for lobation and for the first several generations of airways, implying mechanistic control by a well conserved, genetically hardwired developmental program. This program is not only directed by transcriptional factors and peptide growth factor signaling, but also co-opts and is modulated by physical forces. Peptide growth factors signal within repeating epithelial-mesenchymal temporospatial patterns that constitute morphogenetic centers, automatically directing millions of repetitive events during both stereotypic branching and nonstereotypic branching as well as alveolar surface expansion phases of lung development. Transduction of peptide growth factor signaling within these centers is finely regulated at multiple levels. These may include ligand expression, proteolytic activation of latent ligand, ligand bioavailability, ligand binding proteins and receptor affinity and presentation, receptor complex assembly and kinase activation, phosphorylation and activation of adapter and messenger protein complexes as well as downstream events and cross-talk both inside and outside the nucleus. Herein we review the critical Sonic Hedgehog, Fibroblast Growth Factor, Bone Morphogenetic Protein, Vascular Endothelial Growth Factor and Transforming Growth Factorbeta signaling pathways and propose how they may be functionally coordinated within compound, highly regulated morphogenetic gradients that drive first stereotypic and then non-stereotypic, automatically repetitive, symmetrical as well as asymmetrical branching events in the lung.

Figure 1

Growth factor interactions during lung bud outgrowth and lung bud arrest. In the left hand panel, a bud is beginning to extend. Fibroblast Growth Factor 10 (FGF10) expression is shown as a clump of green mesenchymal cells that chemoattracts the epithelium, shown in brown, towards the pleura shown in white. Sonic hedgehog (SHH) is expressed at low levels, which facilitates the chemotactic activity of FGF10. Bone Morphogenetic Protein 4 (BMP4) also plays key roles in bud extension. In the right hand panel, the bud has extended and is undergoing bud arrest. FGF10 has induced Sprouty2 (SPRY2) expression in the epithelium to a high level, which inhibits further chemotaxis in response to FGF10 signaling. BMP4 is also induced at a higher level and inhibits cell proliferation and hence bud extension. SHH acts through Patched (PTC), to negatively regulate Fgf10 expression in the mesenchyme near the bud tip. The net result is inhibition of cell proliferation and chemoattraction, culminating in bud arrest.

Figure 2

Potential interactions between Fibroblast Growth Factor7 (FGF7) and Fibroblast Growth Factor10 (FGF10) and cognate FGF receptors (FGFR1b and FGFR2b). FGF10 can activate both FGFR1b and FGFR2b. On the other hand, FGF7 only activates FGFR2b. Activation of FGFR1b by FGF10 may be responsible for chemotaxis, while epithelial cell proliferation and differentiation is mediated by both FGF10 and FGF7 activation of FGFR2b. This is mediated downstream by activation of specific target genes.

Figure 3

Sprouty is a rapidly inducible negative regulator of fibroblast growth factor (FGF) pathway signaling. The figure shows a model describing the interaction of murine Sprouty2 (mSPRY2) with other key signaling proteins in the FGF signaling pathway. In the upper panel, the FGF pathway is shown signaling the activation of MAP kinase/ERK2 via the FGFR, FRS2, Shp2, Grb2, Sos, Ras and Raf pathway. In the lower panel Sprouty2 (SPRY2) is shown binding FRS2 and Grb2 and displacing Shp2 from FRS2 and Grb2, thereby preventing subsequent activation of the Sos, Ras-GAP, Raf pathway, resulting in net inhibition of MAP kinase/ERK2 activation.

Figure 4

Signal transduction in the Transforming Growth Factor β(TGFβ) family pathway is finely regulated at many levels. Outside the cell latent Transforming Growth Factor β (LTGFβ) is activated by plasmin (uPA) among other unknown extracellular proteases. Thrombospondin-1 and β6 integrin play key roles in assembly and activation of the proteolytic complex. Free TGF β ligand is bound extracellularly and may be sequestered by Decorin. Noggin and Gremlin play similar roles to Decorin, but for Bone Morphogenetic Protein ligands. The TGF β type III receptor (IIIR), also termed betaglycan, presents ligand to the preformed TGF β type I (IR) and type II (IIR) receptor tetrameric signaling complex. This is particularly important with TGFβ 2, where betaglycan substantially increases its binding affinity for the receptor signaling complex. Non Smad signaling pathways activated by ligand binding include Ras-ERK, Rho-JNK, RhoA-p160RCCK, TAK1-p38MAPK and PP2A-S6 kinase. Ligand binding also facilitates phosphorylation and activation of the TGFβ IR serine-threonine kinase domain by the TGFβ IIR serine-threonine kinase domain. TGFβ IR in turn phosphorylates receptor Smads 2/3. The interaction of Smads with the TGFβ IR is facilitated by SARA. BAMBI is a dominant negative, kinase deficient isoform of TGFβ receptor. Smad 7 is an inhibitory Smad that inhibits Smad 2/3 association with Smad4, the co-Smad. Smad7 is a rapidly inducible negative regulator of TGFβ signaling. Phosphorylated receptor Smads 2/3 then associate with the co-Smad4 and translocate to the nucleus, where they coactivate or corepress certain specific target genes by binding to their respective transcription complexes, with or without directly contacting DNA, depending on the promoter in question. Smurf mediate ubiquitination of preformed Smad complexes, thereby negatively regulating Smad signaling to the nucleus. C-Ski and Sno-N are transcriptional factors that negatively regulate Smad activity in the nucleus.

Figure 5

Morphogenetic "Turing" gradients and some of their major regulators in murine early airway branching. This conceptual figure shows some of the key morphogens and their major regulators diagramed as putative "Turing" gradients, within a branching early embryonic mouse lung lobe. In the bottom panel, both lateral (monopodial) and terminal (dipodial) epithelial branches are diagramed, within a coating of mesenchyme and pleura. In the 5 panels shown above this one, concentrations or activities of key morphogens and their respective regulator molecules are shown as arbitrary relative expression/activity "Turing" gradients. In the top panel Fibroblast Growth Factor10 (FGF10) is shown as a solid line. The FGF10 "Turing" gradient is highest near the pleura and its concentration/activity gradient decays through the peripheral mesenchyme and forms an asymmetrical gradient across the distal bipodial branch induction domain. FGF10 then remains low until it peaks once more within the proximal monopodial branch induction domain. The expression/activity of mSPRY2, shown as the dotted line, is induced by FGF10 within the epithelial branch tips. In contrast, the expression/activity of mSPRY4 peaks in the peripheral mesenchyme and in the mesenchyme between the branch tips. The net result is that FGF10 expression/activity is powerfully negatively regulated between branches, but is increased within branch tips. FGF10 expression/activity is symmetrical within monopodial branch tips, but within dipodial distal tips it is asymmetrical. We suggest that the relative symmetry of the FGF10 expression/activity "Turing" gradient may play a key role in determining whether a specific branch will be mono or dipodial. Also the relative activity of FGF10 and mSPRY2 may play a key role in determining interbranch length and setting up subsequent branch points. In the second panel, SHH is shown as the hatched line and HIP is shown as the solid line. The sharp induction of Hedghog Interacting Protein (HIP) within the branch tips serves to inhibit Sonic hedghog (SHH) expression/activity. As noted in the text, SHH expression/activity is highest in between branch tips, i.e. in places where branches are not supposed to occur. SHH likely plays a major role in negatively regulating FGF10 expression/activity at these inter-branch sites. Conversely, negative regulation of SHH expression/activity by HIP may facilitate FGF10 expression/activity at points where branches are genetically programmed to arise. In the third panel, Bone Morphogenetic Protein4 (BMP4) expression/activity is shown as the solid line. BMP4 expression/activity is relatively low between branches but is increased at branch tips. The activity/expression of Noggin, shown as the dotted line, is the inverse of BMP4. Noggin expression/activity is high between branches and low at branch tips. Gremlin expression/activity is shown as the hatched line. Gremlin follows the contour of BMP4. Thus, the net BMP4 concentration/activity "Turing" gradient peaks in branch tips and is relatively suppressed between them. BMP4 signaling elements however show a more complicated picture. In the fourth panel BMP Smads 1 and 5 concentration/activities are shown. Smad1 peaks within branch tips and is low between them. Smad 5 on the other hand is expressed within small clusters of cells out in the mesenchyme. In the fifth panel, Transforming Growth Factorβ 2 (TGFβ 2) is shown as the solid line, while its signaling Smads 2, 3 and 4 are shown together as the hatched line. TGFβ 2 expression/activity is quite widespread throughout both mesenchyme and epithelium, but peaks within branch tips. Smads 2, 3 and 4 peak within branch tips. Therefore it is likely that TGFβ 2 only signals to any significant extent within branch tips. We suggest that morphogenesis of the branching airway is determined by genes responding to the hard wired temporospatial net integration of the "Turing" gradient distribution of the above morphogens and probably others as well. This conceptual framework represents our latest model for considering this hypothesis.

Figure 6

Schematic diagram drawn after Mailleux et al, 2001, of Fibroblast growth factor 10 (Fgf10) and murine Sprouty2 (mSpry2) expression respectively adjacent to and within the epithlium of an epithelial branch tip in the periphery of an early embryonic mouse lung. In panel a., Fgf10 is beginning to be expressed in the mesenchyme at a point where a bud is about to arise. Note that there is a gap between the epithelium and the locus of Fgf10 expression. At that time mSpry2 is either not expressed or is expressed at low levels. In panel b., an epithelial bud has begun to arise and is moving towards the chemoattractive source of FGF10 located in the mesenchyme near the adjacent pleura. At this time mSpry2 expression is increasing within the distal epithelial tip. In panel c., the bud has extended to a point close to where it will begin to branch. The Fgf10 expression domain is beginning to spread out towards the sides of the tip and mSpry2 is expressed at a high level. In panel d., the bud is extending into the Fgf10 expression domain, which has by now thinned between the bud tip and the adjacent pleura and extends downwards on either side of the bud tip between it and the adjacent bud tips (not shown). The level of mSpry2 expression within the bud tip epithelium is now high and the bud has stopped extending and is about to split. In panel e., a tip-splitting event has occurred and the two daughter buds have just begun to diverge towards the lateral sources of FGF10 expression. The expression of mSpry2 continues within the daughter bud epithelial tips, but at a lower level. It should be noted that the expression ofmSpry2 is extinguished between the two daughter bud tips, implying that FGF10 signaling is no longer inducing mSpry2 at the latter location. This pattern of bud extension and gene expression is then repeated as the bud tips migrate towards the band of Fgf10 expression located along the edge of the primitive lobe, which we have termed herein the APR or apical pulmonary ridge.