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First published online 15 April 2009
doi: 10.1242/dev.029249

Development 136, 1751-1759 (2009)
Published by The Company of Biologists 2009
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Notch signaling promotes airway mucous metaplasia and inhibits alveolar development

J. Sawalla Guseh1,2,*, Sam A. Bores1,2,*, Ben Z. Stanger3, Qiao Zhou1, William J. Anderson1, Douglas A. Melton1 and Jayaraj Rajagopal1,2,{dagger}

1 Department of Stem Cell and Regenerative Biology, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA.
2 Department of Internal Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.
3 Division of Gastroenterology, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.


Figure 1
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  		Fig. 1. Constitutive Notch expression in embryonic lung results in distal cyst formation. (A) Strategy to express activated Notch intracellular domain (NotchIC) in developing lung epithelium. The triangles represent loxP sites. (B,B') Lungs from E18.5 NotchIC transgenic pups and control littermates (B). GFP transgene activation is evident in NotchIC transgenic lungs and absent in control littermates (B'). (C,C') H&E staining of E18.5 control littermate (C) and NotchIC transgenic (C') lungs reveals dilated cysts in place of alveolar saccules. Scale bars: 100 µm in C,C'.

 

Figure 2
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  		Fig. 2. Constitutive Notch expression inhibits differentiation of distal alveolar saccules. (A,A') E18.5 Hes1 immunohistochemistry in control (A) and NotchIC transgenic (A') lungs. The brackets indicate control saccules (A) and corresponding cysts in transgenic animals (A'). BADJ, bronchioalveolar duct junction. (B,B') E18.5 Nkx2.1 immunohistochemistry (red) in control (B) and NotchIC transgenic (B') lungs. (C,C') E18.5 surfactant protein C (SPC) immunohistochemistry (red) in control (C) and transgenic NotchIC lungs (C'). GFP-positive (green) cells do not express SPC (arrow). However, SPC-positive cells persist when GFP is absent (arrowheads). The bracket indicates a GFP-negative saccule. (D,D') E18.5 E-cadherin immunohistochemistry (red) in control (D) and NotchIC-gfp transgenic lungs (D'). E-cadherin expression stops at the BADJ in control lungs but persists throughout the cystic epithelium in transgenic lungs. (E,E') E18.5 smooth muscle-myosin (SMM) immunohistochemistry (red) in control (E) and NotchIC-gfp (green) transgenic lungs (E'). SMM expression is restricted to proximal airways (dashed line) in control lung but is ectopically expressed surrounding distal cysts in transgenic lungs. (F,F') E18.5 CC10 immunohistochemistry (red) demonstrates normal CC10 patterning in transgenic animals compared with controls (inset) (F). A, airway. (F') Scattered CC10-positive cells (arrowheads) are found in GFP+ transgenic cysts. (G,G') E18.5 BrdU immunohistochemistry (red) of control (G) and NotchIC-gfp (green) transgenic (G') lungs after a 2 hour BrdU pulse. BrdU incorporation (percentage shown) is reduced in cystic epithelial cells compared with control alveolar epithelial cells. (H,H') TUNEL staining (red, percentage shown) of E18.5 control (H) and NotchIC transgenic (H') lungs reveals an increase in apoptosis in transgenic lungs compared with control. Scale bars: 100 µm.

 

Figure 3
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  		Fig. 3. Constitutive Notch activation leads to more mucous cells and fewer ciliated cells in E18.5 airway epithelium. (A-D) E18.5 immunohistochemistry of NotchIC airways. Transgene expression (green, B) is correlated with increased numbers of Muc5AC-positive cells (red, C). (C) Inset demonstrates the rare presence of mucous cells in control airway epithelium. (D) Color merge reveals that mucous cells co-label with GFP (yellow). Arrowheads indicate mucin-positive cells. (E-H) E18.5 immunohistochemistry of transgenic Notch airway epithelium shows few ciliated cells. Notch-transgene-expressing cells that are GFP-positive (green, F) lack EphA7 (red, G). (G) The inset demonstrates normal ciliated cell numbers in control epithelium as marked by EphA7 (arrowheads point to ciliated cells in red). (H) Rare residual ciliated cells marked by EphA7 (red) lack GFP (green) transgene expression. (I-L) The yellow-boxed regions from E-H are shown at high magnification in I-L, respectively. Merge reveals that GFP-positive NotchIC transgenic cells (arrowheads, green) are distinct from EphA7-positive (arrows, red) cells. (M,N) Quantification of Muc5AC+ mucus cells and EphA7+ ciliated cells in E18.5 airway epithelium demonstrates that Notch activation is associated with increased mucous cell differentiation (M) (P=0.006) but fewer ciliated cells (N) (P=0.003). GFP-negative cells in transgenic lungs showed normal ciliated cell differentiation, whereas GFP+ cells showed virtually no ciliated cell differentiation. Scale bars: 100 µm.

 

Figure 4
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  		Fig. 4. Notch agonists and antagonists alter ciliated and mucous cell numbers in mouse tracheal explants. (A-C) E14.5 mouse tracheas (A) cultured in vitro for 10 days display normal mucous (B) and ciliated cell (C) differentiation. (D-F) Immunohistochemistry of tracheal explants: mucous cells (green) and ciliated cells (red) were present in control explants (D). Culture with the Notch ligand Dll4 results in increased mucous cell differentiation (green, E). Addition of a Notch signaling antagonist, DBZ, results in increased ciliated cell differentiation (red, F). (G) Incubation of tracheal explants in Dll4 increases mucous cells while decreasing ciliated cells. Addition of DBZ, the Notch antagonist, results in the near absence of Muc5AC-positive cells. Scale bars: 25 µm.

 

Figure 5
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  		Fig. 5. Notch antagonists decrease mucous cell differentiation in human airway epithelial cultures. (A) Human airway cultures incubated with Dll4 or IL13 (middle column) show substantial Muc5AC (green) immunostaining compared with control (left panel). DBZ addition to Dll4 or IL13 (right-hand column) decreased Muc5AC staining. (B) Increasing concentrations of DBZ decreased Muc5AC staining in control cultures (white) and in those cultures incubated with either Dll4 (gray) or IL13 (black). Scale bars: 100 µm.

 

Figure 6
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  		Fig. 6. Stat6 is not necessary for DLL4-induced mucous metaplasia. (A-C) Immunohistochemistry of control tracheas that were harvested at E14.5 and cultured for 7 days. Tracheas displayed normal, low levels of mucous differentiation, identified by Muc5AC staining (green) (A). Addition of IL13 (B) or DLL4 (C) induced mucous metaplasia. (A'-C') Immunohistochemistry of Stat6–/– tracheas that were harvested at E14.5 and cultured for 7 days. Tracheas displayed normal, low levels of mucous differentiation (A'). Addition of IL13 (B') failed to induce mucous metaplasia as predicted. Addition of DLL4 (C') induced mucous metaplasia. (D) Possible models for the interaction of Notch and the IL13/Stat6 signaling pathway, which both result in mucous metaplasia. Notch signaling cannot be upstream of Stat6 activation as Notch-induced metaplasia occurs in Stat6-null trachea. Notch signaling might be downstream of Stat6 signaling, although this is unlikely because Stat6 activation is not associated with Notch target induction. Therefore, Notch and Stat6 signaling may represent two parallel pathways for inducing mucous metaplasia. Scale bars: 100 µm.

 

Figure 7
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  		Fig. 7. Models of Notch action in mouse lung development. (A) Notch misexpression increases mucous cell differentiation and inhibits ciliated cell differentiation in proximal airway epithelium. (B) Notch downregulation is required for alveolar development. Constitutive Notch misexpression inhibits alveolar development and results in a dilated cystic epithelium. (C) Schematic representations of possible common and dual lineage progenitor models for lung development. In the common lineage model, a single progenitor lineage produces proximal and distal cell types. Notch signaling could block the transition from a proximal progenitor to a distal progenitor (a) or could block the differentiation of an already established distal progenitor (b) in this model. In the dual lineage model, in which there are distinct proximal and distal progenitors, Notch signaling may specifically block the differentiation of distal progenitors, whereas it would only modulate the specific cell-fate distribution of proximal progenitor progeny.

 

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© The Company of Biologists Ltd 2009