Fibroblast growth factor 18 gives growth and directional cues to airway cartilage.

OBJECTIVES/HYPOTHESIS: The majority of congenital airway anomalies arise from deficits in the respiratory tract cartilage, emphasizing the importance of this cartilage to the form and function of the upper airway. The primary objective of this study was to characterize molecular mechanisms that regulate rate and direction of chondrocyte growth in the larynx and trachea. Our hypothesis for this study was that fibroblast growth factor 18 (FGF18) provides proliferative and directional cues to the developing laryngeal and tracheal cartilage in the mouse by up-regulating the cartilage-specifying gene, Sox9. STUDY DESIGN: Molecular genetic and histological analyses of gene expression and cartilage growth in a mouse model. METHODS: Controlled mating of wild-type FVB/N (Friend Virus B-type/NIH mouse) mice and FGF18 overexpressing mice were carried out, and embryos ranging from embryonic (E) day 10.5 to E18.5 were obtained. The respiratory tract, including the larynx, trachea, and lung, was removed through meticulous dissection, and subjected to whole-mount in situ hybridization with RNA probes, or was sectioned and subjected to immunohistochemistry. Respiratory tracts from FVB/N mice were grown in culture in the presence of exogenous FGF18 or known inhibitors of the FGF pathway, and then subjected to quantitative reverse transcriptase polymerase chain reaction to measure the expression of cartilage-specific genes. RESULTS: The upper respiratory tract begins as a simple out-pouching from the ventral foregut endoderm at E10.5. The chondrocytes that form the cartilaginous structures of the upper respiratory tract are located at the junction of the respiratory tract out-pouching and the ventral foregut endoderm. This population of chondrocytes then undergoes directional proliferation to eventually assume the mature three-dimensional configuration of the upper respiratory tract cartilaginous framework. Immunohistochemical localization of extracellular signal-regulated kinases, a known modulator of FGF signaling, demonstrated the presence of this enzyme at the periphery of growing cartilage. Explants of larynx-trachea-lung grown in culture with exogenous FGF18 demonstrated hyperplastic growth and directed growth towards the FGF18 source. Finally, both FGF18 overexpressing tracheas and tracheas cultured with exogenous FGF18 demonstrated increased expression of the cartilage-specifying gene, Sox9. CONCLUSIONS: FGF18 provided both directional and proliferative cues to chondrocytes in the developing upper respiratory tract. FGF18 exerted this effect on developing chondrocytes by up-regulating Sox9 expression. Laryngoscope, 2009.

Slit and robo expression in the developing mouse lung.

Mammalian lung development is mediated through complex interactions between foregut endoderm and surrounding mesenchyme. As airway branching progresses, the mesenchyme undergoes dramatic remodeling and differentiation. Little is understood about the mechanisms that direct mesenchymal organization during lung development. A screen for candidate genes mediating this process identified Slit, a ligand for the Roundabout (Robo) receptor previously associated with guidance of axonal projections during central nervous system development. Here, we demonstrate by in situ hybridization that two Slit genes (Slit-2 and Slit-3) and two Robo genes (Robo-1 and Robo-2) are expressed in fetal lung mesenchyme. Slit-2 and Robo-1 expression is present throughout mesenchyme at midgestation and is not detectable by newborn day 1. Slit-3 and Robo-2 expression is restricted to specific, complementary subsets of mesenchyme. Robo-2 is expressed in mesenchymal cells immediately adjacent to large airways, whereas Slit-3 expression predominates in mesenchyme remote from airway epithelium. The temporal and spatial distribution of Slit and Robo mRNAs indicate that these genes may direct the functional organization and differentiation of fetal lung mesenchyme.

Temporal and spatial regulation of VEGF-A controls vascular patterning in the embryonic lung.

Vascular endothelial growth factor-A (VEGF-A) is required for vascular development throughout the embryo and has been proposed to play an important role in pulmonary vascular patterning. Expressed by the embryonic respiratory epithelium, VEGF-A signals endothelial cells within the splanchnic mesenchyme. To refine understanding of the spatial and temporal role of VEGF-A in lung morphogenesis, isoform VEGF164 was expressed under conditional control in distal and proximal airway epithelial cells. Unexpectedly, increased expression of VEGF164 in distal lung disrupted peripheral vascular net assembly and arrested branching of airways tubules without altering endothelial cell proliferation or apoptosis. Peripheral airway branching and vascular smooth muscle patterning were also altered. In contrast, expression of VEGF164 by epithelial cells of the conducting airways caused atypical evaginations of small capillary-like vessels into large airways but did not alter peripheral vascular net assembly or branching morphogenesis. These data demonstrate that the differential response of endothelial cells in distal vascular beds and large central blood vessels is established early in lung development. Precise temporal and spatial expression of VEGF-A is required for vascular patterning during lung morphogenesis. Disruption of pulmonary vascular assembly perturbs reciprocal interactions with epithelium leading to altered airway branching morphogenesis.

Mesenchymal expression of vascular endothelial growth factors D and A defines vascular patterning in developing lung.

The lung has specific vascular patterning requirements for effective gas exchange at birth, including alignment of airways and blood vessels and lymphatic vessels. Vascular endothelial growth factors (VEGF) are potent effectors of vascular development. We examined the temporal and spatial expression of VEGF-D and specific VEGF-A isoforms at each stage of lung development. VEGF-D, expressed only by cadherin-11-positive cells of the mesenchyme, is first detected at embryonic day (E) 13.5, a period of active vasculogenesis. VEGFR-3, its cognate receptor, is detected earlier on days E11.5 to E14.5, in both blood vessels and lymphatic vessels and later, on day E17.5, in only lymphatic vessels. VEGF-A is expressed in the mesenchyme throughout lung development and also by the epithelium midway through organogenesis. Before E14, the predominant forms of VEGF-A are the soluble isoforms, VEGF-A120 and 164. Not until E14.5 do epithelial cells at the tips of expanding airways express VEGF-A, including VEGF-A188, an isoform with high affinity for extracellular matrix. Our results demonstrate unique temporal and spatial expression of VEGF-D and specific VEGF-A isoforms during lung development and suggest these related factors have distinct functions in vascular and lymphatic patterning of the lung.