Lunatic Fringe-mediated Notch signaling is required for lung alveogenesis

Abstract

Distal lung development occurs through coordinated induction of myofibroblasts, epithelial cells, and capillaries. Lunatic Fringe (Lfng) is a beta(1-3) N-acetylglucosamine transferase that modifies Notch receptors to facilitate their activation by Delta-like (Dll1/4) ligands. Lfng is expressed in the distal lung during saccular development, and deletion of this gene impairs myofibroblast differentiation and alveogenesis in this context. A similar defect was observed in Notch2(beta-geo/+)Notch3(beta-geo/beta-geo) compound mutant mice but not in Notch2(beta-geo/+) or Notch3(beta-geo/beta-geo) single mutants. Finally, to directly test for the role of Notch signaling in myofibroblast differentiation in vivo, we used ROSA26-rtTA(/+);tetO-CRE(/+);RBPJkappa(flox/flox) inducible mutant mice to show that disruption of canonical Notch signaling during late embryonic development prevents induction of smooth muscle actin in mesenchymal cells of the distal lung. In sum, these results demonstrate that Lfng functions to enhance Notch signaling in myofibroblast precursor cells and thereby to coordinate differentiation and mobilization of myofibroblasts required for alveolar septation.

Fig. 1.

Generation of Lunatic Fringe (Lfng) mutant mice. A: generation of Lfng mutant alleles. The Lfng⁺ wild-type allele is shown at the top with numbered boxes representing exons. The Lfng^neo allele contains 3 loxP sites and 2 Flippase recognition target (FRT) sites, a Pgk-neo cassette flanked by loxP and FRT sites. The Lfng^flox allele, with exon 2 floxed, was generated by crossing Lfng^neo/+ with Rosa-Flp mice. The Lfng⁻ allele, with exon 2 deleted, was generated by crossing Lfng^flox/+ with EIIa-Cre mice. The bars represent 5′ and 3′ probes for Southern blot analysis. Small and large arrowheads indicate primers for PCR genotyping and RT-PCR, respectively. B: Southern blot analysis of targeted embryonic stem (ES) cell genomic DNA digested with MPh1103I (M) and ClaI (C) using 5′ probe. C: Southern blot analysis of targeted ES cell genomic DNA digested with HindIII (H) using 3′ probe. D: a representative gel of PCR genotyping for Lfng mutant mice. E: RT-PCR on total RNA from Lfng^−/− and wild-type lungs. Note the Lfng^−/− mutant lung expressed reduced level of a truncated transcript. CRE, Cre recombinase.

Fig. 2.

Abnormal lung development in Lfng mutants. A and B: representative microcomputed tomography images of pulmonary vasculature at 10 wk of age (w.o.). C: mean linear intercepts of alveolar septae measured at indicated ages. Values shown are means ± SD. *P < 0.003 and **P < 0.0005. D–O: representative hematoxylin and eosin (H&E)-stained lung sections from Lfng^+/+ (D, F, H, J, L, and N) and Lfng^−/− (E, G, I, K, M, and O) mice at indicated ages. Note the differences between wild-type and mutant lungs starting at day 18.5 of gestation (18.5 dpc), at which point Lfng^−/− lung shows less opened saccules. Postnatal Lfng^−/− lungs continue to show signs of defective alveolar development, including thickened alveolar walls, lack of alveolar septation, and large alveolar size. Scale bars, 200 μm. P, postnatal day.

Fig. 3.

Immunohistochemical analysis of major cell types in the distal lung at 17.5 dpc. A–L: representative photomicrographs of immunostained lung sections at 17.5 dpc with anti-β₆-integrin (A and B), anti-surfactant protein C (SP-C; C and D), anti-CD31 (E and F), anti-smooth muscle actin-α (sma; G and H), anti-Ki67 (I and J), and anti-cleaved caspase-3 antibodies (K and L). Scale bars, 50 μm. M: Western blot analysis of β₆-integrin in lung tissues from Lfng^+/− and Lfng^−/− mice at 17.5 dpc with β-actin as loading control. N: Western blot analysis of aquaporin 5 in lung tissues at 17.5 dpc and P5. Arrow indicates mature form of aquaporin 5. O: quantification of immunostaining from 17.5-dpc lung sections. Shown are percentages of SP-C⁺ cells, Ki67⁺ cells, and cleaved caspase-3⁺ cells normalized to the total number of distal lung cells within each image (excluding bronchiolar airways and blood vessels). Data are derived from 2 sections in each of 3 Lfng^+/− and 3 Lfng^−/− animals, respectively. Student's t-test was performed to assess significance.

Fig. 4.

Immunohistochemical analysis of major cell types in the distal lung during alveolarization. A–L: representative photomicrographs of immunostained lung sections at P5 with anti-T1α (A and B), anti-SP-C (C and D), anti-CD31 (E and F), anti-sma (G and H), anti-Ki67 (I and J), and anti-cleaved caspase-3 antibodies (K and L). Scale bars, 50 μm. Arrows point to capillary endothelial cells (inset in E), sma⁺ myofibroblast cells (G and H), and Ki67⁺ proliferating cells (I and J), respectively. Insets in E and F are higher magnification views from the same images. M: quantification of immunostaining from P5 lung sections. Shown are percentages of SP-C⁺ cells, Ki67⁺ cells, and cleaved caspase-3⁺ cells normalized to the total number of distal lung cells within each image (excluding bronchiolar airways and blood vessels). Data are derived from 2 sections in each of 3 Lfng^+/− and 3 Lfng^−/− animals, respectively. Student's t-test was performed and found no significance (P > 0.1).

Fig. 5.

Defective myofibroblast cell differentiation in Lfng mutant lungs. A–H: representative immunostaining of lung sections with anti-sma antibody at 15.5 dpc (A and B), 18.5 dpc (C and D), P2 (E and F), and P9 (G and H). I: Western blot analysis of sma in lung tissues from Lfng^+/− and Lfng^−/− mice with β-actin as loading control. J–M: representative immunostaining of lung sections with anti-smooth muscle myosin heavy chain antibody at 17.5 dpc (J and K) and P5 (L and M). N–Q: representative immunostaining of lung sections with anti-PDGF receptor-α (PDGFRα) antibody at 15.5 dpc (N and O) and 18.5 dpc (P and Q). Arrows in C, E, G, J, and L indicate myofibroblast cells located at the growing septae. Arrows in N and O point at clusters of PDGFRα-positive cells surrounding the distal bronchiolar airways. Arrows in P and Q point at PDGFRα-positive cells migrated to alveolar walls. Scale bars, 50 μm.

Fig. 6.

Altered elastogenesis and collagen deposition in Lfng mutant lungs. A–F: representative photomicrographs showing Verhoeff staining of lung sections at P5 (A and B), P14 (C and D), and P42 (E and F). Arrows in C and E point at continuous elastic fibers (stained in dark brown) in wild-type lungs. Arrowheads in D and F indicate accumulation of disorganized elastic fibers in Lfng^−/− mutant lungs. G: Western blot analysis of elastin in lung tissues from Lfng^+/− and Lfng^−/− mice. H and I: representative photomicrographs of Masson trichrome staining in P42 lung sections showing increased deposition of collagen in Lfng^−/− mutant (stained in green, arrowhead in I). This increased deposition was observed in most areas of mutant lungs. Scale bars, 50 μm.

Fig. 7.

Expression of Lfng, Notch ligands, and receptors in developing lungs. Shown are representative photomicrographs of X-galactosidase (X-gal) staining in lung sections from Lfng^lacZ/lacZ (A and C–E), wild-type (B), Dll1^lacZ/+ (F and G), Dll4^lacZ/+ (H), Jagged1^β-geo/+ (I and J), Notch2^β-geo/+ (K), and Notch3^β-geo/+ (L) mice at indicated developmental stages. Double staining of X-gal (stained in blue) and anti-Mash1 (stained in brown) are shown in D, G, and J. Arrows point to neuroepithelial bodies (NEBs). Scale bars, 50 μm. Dll1/4, Delta-like ligands.

Fig. 8.

Genomic structure of Jagged1, Notch2, and Notch3 mutant alleles. A: Jagged1 was targeted in a secretory gene trap screen using a vector containing β-geo preceded by a transmembrane domain and flanked by a splicing acceptor and polyadenylation sites. The construct integrated into an intron between exons coding for EGF repeats #13 and #14, respectively, resulting in a truncated Jagged1 fused to β-geo. B and C: using the same vector, Notch2 and Notch3 were targeted to produce β-geo fusion proteins with 33 and 21 EGF repeats, respectively, of their extracellular domains. Gene trap ES cell lines KST248 (Jagged1), LST103 (Notch2), and PST033 (Notch3) were obtained from BayGenomics. En-2 SA, Engrailed-2 splice acceptor sequence; SVpA, polyA addition sequence from simian virus (SV40); DSL, Delta/Serrate/Lag-2 domain; VWCTM, Von Willebrand factor C domain, transmembrane domain; LNR, Lin-12 Notch repeat-containing region; PEST, Proline/Glutamic acid/Serine/Threonine rich sequences.

Fig. 9.

Defective myofibroblast cell differentiation in Notch2/3 (N2/N3) compound mutants and RBPJκ inducible knockout mice. A–C: representative photomicrographs of anti-sma immunofluorescence staining in lung sections from Notch2^β-geo/+, Notch3^{β-geo/β-geo}, and Notch2^β-geo/+Notch3^{β-geo/β-geo} embryos at 17.5 dpc. D–F: representative photomicrographs of H&E staining of lung sections from Notch2^β-geo/+, Notch3^{β-geo/β-geo}, and Notch2^β-geo/+Notch3^{β-geo/β-geo} mice at P21. G–J: representative photomicrographs of anti-sma staining (G and H) and anti-SP-C staining (I and J) in lung sections from doxycycline-exposed ROSA26-rtTA; tetO-Cre; RBPJκ^flox/+ and ROSA-rtTA; tetO-Cre; RBPJκ^flox/flox embryos at 18.5 dpc. Pregnant females containing these embryos were given doxycycline-containing drinking water from 14.5 to 18.5 dpc. K–P: representative photomicrographs of anti-sma staining (K and L), anti-SP-C staining (M and N), and anti-T1α staining (O and P) in lung sections from doxycycline-exposed ROSA26-rtTA; tetO-Cre; RBPJκ^flox/+ and ROSA-rtTA; tetO-Cre; RBPJκ^flox/flox pups at P0. Arrows in A, B, G, and K indicate sma⁺ myofibroblast cells. Scale bars represent 50 μm in A–C and G–P and 200 μm in D–F.