Heparan sulfate–FGF10 interactions during lung morphogenesis

Abstract

Signaling by fibroblast growth factor 10 (FGF10) through FGFR2b is essential for lung development. Heparan sulfates (HS) are major modulators of growth factor binding and signaling present on cell surfaces and extracellular matrices of all tissues. Although recent studies provide evidence that HS are required for FGF-directed tracheal morphogenesis in Drosophila, little is known about the HS role in FGF10-mediated bud formation in the vertebrate lung. Here, we mapped HS expression in the early lung and we investigated how HS interactions with FGF10-FGFR2b influence lung morphogenesis. Our data show that a specific set of HS low in O-sulfates is dynamically expressed in the lung mesenchyme at the sites of prospective budding near Fgf10-expressing areas. In turn, highly sulfated HS are present in basement membranes of branching epithelial tubules. We show that disrupting endogenous gradients of HS or altering HS sulfation in embryonic lung culture systems prevents FGF10 from inducing local responses and markedly alters lung pattern formation and gene expression. Experiments with selectively sulfated heparins indicate that O-sulfated groups in HS are critical for FGF10 signaling activation in the epithelium during lung bud formation, and that the effect of FGF10 in pattern is in part determined by regional distribution of O-sulfated HS. Moreover, we describe expression of a HS 6-O-sulfotransferase preferentially at the tips of branching tubules. Our data suggest that the ability of FGF10 to induce local budding is critically influenced by developmentally regulated regional patterns of HS sulfation.

Systemic inactivation of Hs6st1 in mice is associated with late postnatal mortality without major defects in organogenesis

“…Interestingly, the lack of 6-O sulfate can be compensated for by the presence of 2-O, but not N-sulfates in the mixture (Fig. 4f and Izvolsky et al, 2003a). In another assay, culturing intact E11.5 lungs in medium containing 6-O desulfated heparins or 2-O desulfated heparins results in essentially the same phenotype.…”

“…Therefore, we expanded our previous observations (Izvolsky et al, 2003a) using selectively sulfated heparins in lung explant cultures from wild type mice to compare the effect of 6-O and 2-O sulfation on epithelial patterning. E11.5 lung epithelial explants devoid of mesenchyme and cultured in the presence of recombinant FGF10 are unable to survive if HS sulfation is prevented by the addition of sodium chlorate.…”

“…E11.5 whole lung explants were cultured as described (Izvolsky et al, 2003a). Briefly, lungs isolated from CD1 mice were placed on 0.8 lm filters (Millipore) on top of metal grids and cultured in 60 mm organ culture dishes for 48 h in BGJb medium (Invitrogen) containing 1% fetal calf serum.…”

“…Mesenchyme-free epithelium was obtained from E11.5 CD1 mouse lungs after Dispase treatment, and cultured as described (Izvolsky et al, 2003a). Briefly, epithelium was covered with Matrigel (BD Biosciences) and cultured in serum-free DMEM medium containing human recombinant FGF10 (2,500 ng/ml; R&D Systems), sodium chlorate (25 mM), and selectively 2-Oand 6-O-desulfated heparins (125 lg/ml; Neoparin Inc.) for 48 h.…”

Mice Deficient in Heparan Sulfate 6-O-Sulfotransferase-1 Exhibit Defective Heparan Sulfate Biosynthesis, Abnormal Placentation, and Late Embryonic Lethality

“…Furthermore, HS6ST-1 is expressed strongly at the tips of branching tubules in the developing lung (43), and FGF10 is preferentially bound to 6-O-sulfate residues rather than 2-O-sulfate residue in HS (7). Thus, a deficiency in HS6ST-1 is potentially relevant to impaired lung development.…”