FGF signaling is critical throughout all stages of lung development (56
). FGFR3 and FGFR4 signaling, in particular, appears to be critical for alveogenesis. We have confirmed that mice with combined deficiency of FGFR3 and FGFR4 develop postnatal airspace enlargement, here in a pure C57BL/6 background. We show that histological, morphometrical, biochemical, and molecular data are consistent with lung abnormalities initiating after birth in these mice. This conclusion is supported by quantitative assessment of lung structure and genome-wide expression profiling. We have also confirmed abnormalities in airspace elastic fiber production in compound mutant mice, and demonstrated that this involves coordinated overproduction of all elastic fiber components and accumulation of excessive elastic fibers lacking typical spatial restriction at alveolar entrance rings. We show that these abnormalities are not due to fibroblast-autonomous defects, but are associated with excessive production of AT2 paracrine factors capable of increasing alveolar fibroblast elastin expression. Finally, we demonstrate that the excessive, misplaced elastic fiber accumulation is associated with an apparent increase in the number of elastogenic alveolar mesenchymal cells expressing αSMA. Our data are consistent with the conclusion that defective AT2 paracrine signaling contributes to abnormal elastogenesis.
Compound FGFR3/4 mutant mice exhibit dwarfism and lung hypoplasia. Whether the dwarfism phenotype can exclusively account for the decrease in lung size is unknown. Interestingly, Klotho mutant mice (58
), Fgf23 mutant mice (59
), and Hoxa5 mutant mice (60
) were reported to exhibit growth retardation associated with the decreased lung size and postnatal airspace enlargement. However, FGFR3 mutant mice develop impaired growth, but no failure of alveolar septation (e.g., airspace enlargement) was reported (27
) nor observed in our studies. Therefore, we conclude that the dwarfism phenotype in compound mutant mice is not sufficient to explain alveogenesis defects. However, the data clearly suggest cooperative roles of FGFR4 with FGFR3 in coordinating body growth and lung development.
The role of FGFR signaling in airspace formation was also studied using transgenic mice expressing a soluble dominant-negative FGFR in the lung (61
). Embryonic inhibition of FGF resulted in abnormal airspace enlargement, whereas postnatal inhibition had no effect on alveogenesis, suggesting that FGFR function was critical during the prenatal period. These data are inconsistent with our current studies, which failed to identify any deficiencies at birth in compound mutant mice. Potential technical differences, including receptor specificity and expression patterns for the dominant-negative FGFR transgene, could explain the differences in these models. It is possible that the genomic, histological, biochemical, and molecular approaches that we used in the current study were not sensitive enough to identify specific prenatal changes that may exist in compound mutant mice. However, it is likely that failed alveogenesis in compound mutant mice results predominantly from postnatal effects.
Previous studies have described temporal and cell type–specific expression of FGFR3 and FGFR4 during lung development (19
). We sought to define the FGFR3/FGFR4–dependent mechanisms of alveolar formation during postnatal lung development in compound mutant mice using microarray analysis coincident with the histopathological observations. The genomic profiles revealed, among a small number of genes, the induction of multiple genes related to elastic fiber assembly in compound mutant mice. We validated these changes and observed coordinated induction in additional genes related to elastic fiber assembly, including tropoelastin, microfibril-related proteins (fibrillin-1, Mfap5 or microfibril-associated glycoprotein 2, fibulin 1, fibulin 5), and lysyl oxidases, Lox and Loxl1, all of which are essential for elastogenesis. Histochemical and biochemical analyses corroborated the findings at the mRNA level, confirming increased elastic fiber deposition in compound mutant lungs.
Decreased elastic fiber formation has been associated with airspace abnormalities in numerous gene-targeted animal models: elastin (12
); RAR/RXR (15
); fibrillin-1 (13
); loxl1 (64
); fibulin (65
). Excessive, disorganized elastic fiber accumulation can be a histopathological feature of impaired airspace formation in human bronchopulmonary dysplasia (66
) and in several animal models of this disease (17
). Reduced expression of FGFR3 and FGFR4 was observed in rodent models of BPD induced by neonatal hyperoxic exposure (17
), suggesting that impaired alveogenesis and excessive elastic fiber accumulation observed in these models may partly result from alterations in FGFR signaling. In total, these data indicate that appropriate quantitative and spatial deposition of elastic fibers is critical for normal airspace formation and lung function. We have not defined whether excessive elastogenesis observed in this model is causative for the failure of secondary crest elongation. Deficiencies in septation likely affect lung mechanics. Therefore, the overproduction of elastic fibers could be a compensatory response. Interestingly, our data are consistent with the conclusion that increased elastin accumulation in this model is a result of persistence of elastin gene expression in the compound mutant mouse at P28 when expression is repressed in the wild-type lung.
It is important to note that elastogenesis in the lung vasculature and airways, and in other tissues, was normal in compound mutant mice. This reinforces the specificity of the defect, and indicates that distinct regulatory mechanisms contribute to airspace elastic fiber production. We investigated lung fibroblasts to determine the mechanism for aberrant airspace elastogenesis in compound mutant mice. No induction in baseline or regulated elastic fiber gene expression, or any other abnormalities, were observed in mutant fibroblasts. No change in the expression of TGF-β1, which is thought to function as an autocrine inducer of lung fibroblast elastin production (72
), was found in the microarray data or by qPCR. Additional experiments were performed to identify differences in isolated fibroblasts derived from both wild-type and mutant mice. Heparin treatment, likely through activation of endogenous FGF activity, or exogenous FGF treatment resulted in similar inhibitory responses in both wild-type and mutant fibroblasts. Our data suggest that dysregulation of elastic fiber genes in vivo
is not a primary defect in lung fibroblasts, and that another cell type provided paracrine signals involved in the regulation of elastogenesis.
Conversely, we found changes in the expression of multiple paracrine factors from AT2 cells, including the elastogenic molecule, Igf1 (53
), and its binding protein, Igfbp2. Interestingly, lung expression of IGF1/IGF1R has been demonstrated (49
), and deficiency in either IGF1 or IGF1R leads to abnormal lung development (50
). The cellular and molecular mechanisms contributing to this phenotype have not been defined. Conflicting data exist regarding the ability of IGF1 to promote lung fibroblast elastin expression. One prominent study reported tissue specificity for the inductive effects of IGF1 on elastin expression, with a lack of response in lung fibroblasts (52
). However, an earlier study suggested some potential elastogenic effects of IGF1 in lung fibroblasts (53
). We observed an inductive response that was greater than that observed for other physiological inducers of lung fibroblast elastin gene expression, such as TGFβ or retinoic acid (54
). These data implicate epithelial–mesenchymal interactions in the regulation of airspace elastogenesis and its dysregulaiton in compound mutant mice. Additional factors, including peptides (e.g., TGFβ), hormones (retinoic acid), and physical forces are also likely to contribute to the regulation of airspace elastin gene expression during development.
Airspace mesenchymal cell heterogeneity, including, but not limited to, myofibroblasts, lipid-laden fibroblasts, and pericytes, has been well documented. Physiologically, airspace elastin is primarily produced by αSMA-expressing fibroblasts located at alveolar septal tips (75
), resulting in elastic fiber enrichment at alveolar entrance rings. Recent reports have demonstrated that appropriate FGF signaling is essential for myofibroblast differentiation and induction of αSMA expression during alveogenesis (62
). In compound FGFR3/FGFR4 mutant mice, we demonstrate a spatially unrestricted deposition of elastic fibers throughout the primary saccular wall. This was accompanied by an increase in αSMA immunostaining. The increase in elastogenesis and αSMA expression was coincident with a significant reduction in airspace cell proliferation, suggesting either excessive recruitment of elastogenic cells or a failure of differentiation and/or proliferation of nonelastogenic fibroblasts, normally found at the base of secondary crests. Interestingly, compound mutant AT2 cells showed increased expression of SDF1α. This molecule is a regulator of progenitor cell recruitment, vascular cell differentiation, and angiogenesis (76
), and has previously been shown to be suppressed by FGF signaling (79
Our current studies have a number of limitations. We used standard morphometry of liquid-inflated, constant pressure–fixed lungs. The objectives of this analysis were to quantify the magnitude of changes in airspace size, and to track the relative sizes of the airspaces of compound mutant mice during maturation. We acknowledge that the structural and biochemical changes may have significant effects upon pressure-normalized inflation, and that alternate methods of morphometric analysis would likely result in quantitative differences. However, given the magnitude of the abnormal pathology, the relative differences and our conclusions are unlikely to be changed. Another limitation concerns our studies of isolated fibroblasts. How the phenotype of these cultured cells resembles the population responsible for elastin expression in vivo is not clear. For instance, it unavoidably removes factors such as mechanical loading. Although our data indicate that excessive elastic fiber production from these cells reflects a response to paracrine factors, it would be premature to conclude that compound mutant fibroblasts are not actively involved in the lung pathology observed in these mice.
In conclusion, we have demonstrated that FGFR3/FGFR4 signaling contributes to the spatiotemporal specification of lung elastic fiber production. Mice with compound mutation of FGFR3/FGFR4 display disrupted postnatal lung growth, with airspace enlargement and excessive elastic fiber deposition. In vitro studies demonstrate that induction of elastin expression is not a primary defect in fibroblast cells per se, but result from paracrine signaling from airspace epithelial cells. Current studies focus on defining the mechanisms of Igf1 and Sdf1α upon lung mesenchymal cell differentiation and specification, which may represent a novel, airspace-specific mechanism regulating elastogenesis.