Research Article

213

Canonical Notch signaling in the developing lung isrequired for determination of arterial smooth muscle
cells and selection of Clara versus ciliated cell fateMitsuru Morimoto1, Zhenyi Liu1, Hui-Teng Cheng2, Niki Winters3, David Bader3 and Raphael Kopan1,*
1

Department of Developmental Biology and Division of Dermatology, Washington University School of Medicine, Box 8103, Saint Louis,
MO 63110-1095, USA
2
Department of Internal Medicine, Far Eastern Memorial Hospital and National Taiwan University Hospital, Taipei, Taiwan
3
Stahlman Cardiovascular Research Laboratories, Program for Developmental Biology, and Department of Medicine, Vanderbilt University
Medical Center, Nashville, TN 37232-6300, USA*Author for correspondence (kopan@wustl.edu)

Journal of Cell Science

Accepted 23 October 2009
Journal of Cell Science 123, 213-224 Published by The Company of Biologists 2010
doi:10.1242/jcs.058669

Summary
Lung development is the result of complex interactions between four tissues: epithelium, mesenchyme, mesothelium and endothelium.
We marked the lineages experiencing Notch1 activation in these four cellular compartments during lung development and complemented
this analysis by comparing the cell fate choices made in the absence of RBPj␬, the essential DNA binding partner of all Notch receptors.
In the mesenchyme, RBPj␬ was required for the recruitment and specification of arterial vascular smooth muscle cells (vSMC) andfor regulating mesothelial epithelial-mesenchymal transition (EMT), but no adverse affects were observed in mice lacking mesenchymal
RBPj␬. We provide indirect evidence that this is due to vSMC rescue by endothelial-mesenchymal transition (EnMT). In the epithelium,
we show that Notch1 activation was most probably induced by Foxj1-expressing cells, which suggests that Notch1-mediated lateral
inhibition regulates the selection of Clara cells at the expense of ciliated cells. Unexpectedly, and in contrast to Pofut1-null epithelium,
Hes1 expression was only marginally reduced in RBPj␬-null epithelium, with a corresponding minimal effect on pulmonary
neuroendocrine cell fate selection. Collectively, the primary roles for canonical Notch signaling in lung development are in selectionof Clara cell fate and in vSMC recruitment. These analyses suggest that the impact of ␥-secretase inhibitors on branching in vitro
reflect a non-cell autonomous contribution from endothelial or vSMC-derived signals.
Key words: Notch, Lung, Clara cells, Ciliated cells, Arterial vascular smooth muscle cells

Introduction
Lung development is orchestrated by complex mesenchymalepithelial interactions that coordinate the temporal and spatial
expression of multiple regulatory factors that are required for properorgan formation. Distinct populations of stem cells contribute to
the epithelial and mesenchymal compartments (Mailleux et al.,2005; Perl et al., 2002; Rawlins et al., 2007; Rock et al., 2009). In
the tracheal epithelium, the basal cell generates mucous cells, Clara(secretory) and ciliated cells (Hong et al., 2004; Rock et al., 2009).
Smaller bronchi contain the latter two cell types and pulmonary
neuroendocrine cells (PNECs). The distal-most airway, the alveolus,
is lined with thin layers of flat Type I cells and cuboidal Type II
cells (Kimura and Deutsch, 2007; Rawlins and Hogan, 2006). At
the pseudoglandular stage (E11.5-16.5), during which most of
airway branching morphogenesis takes place, it is thought that theterminal buds contain a population of multipotent epithelial
progenitors (Perl et al., 2005). As the bronchial tree extends,
descendants of these cells give rise to lineage-restricted progenitorsthat produce Clara and ciliated cells (and possibly other cell types)
in the conducting airways (Cardoso and Lu, 2006; Perl et al., 2002).
The lung mesenchyme is comprised of multiple cell types,
including connective tissue, endothelial cells, lymphatics, smoothmuscle cells surrounding airways and blood vessels, myofibroblasts
involved in septum formation, and cartilage-forming cells in the
trachea. In addition, pleura-derived mesothelial cells cover the outersurface of the lung. The developmental origin of these cells is amatter of some dispute (Hall et al., 2000), with most cells believed
to be derived from the splanchnic mesenchyme, whereas other cells(endothelial, smooth muscle) are believed to invade the lung as it
expands (Cardoso and Lu, 2006; Galambos and Demello, 2007;
Hall et al., 2000). Although bronchial smooth muscle cells (bSMCs)
are derived from a distal lung mesenchyme lineage expressing
fibroblast growth factor 10 (FGF10) (Mailleux et al., 2005), vSMCs
are thought to derive from an invading population (Hall et al., 2000).
Evidence demonstrating the importance of Notch signaling in
the developing respiratory system is rapidly growing. Mice that are
genetically deficient in Hes1, a target of Notch signaling in several
biological systems, show hyperplasia of PNECs and a decreased
number of Clara cells, suggesting the bi-potential precursors of Claraand ciliated cells are separated from PNEC precursors via a Notchmediated lateral inhibition feedback loop (Ito et al., 2000; Shan etal., 2007). As this manuscript was being prepared for publication,a role for Notch signaling as a suppressor of the ciliated cell fate
was reported (Guseh et al., 2009; Tsao et al., 2009). Induced
expression of a constitutively active Notch1 intracellular domain(N1ICD) in lung epithelial cells throughout development promoted
mucous metaplasia and remarkably decreased the number of ciliatedcells (Guseh et al., 2009). Conditional removal of Pofut1, a
glycosyltransferase required for Notch signaling and possibly othercellular functions (Kopan and Ilagan, 2009), promoted ciliated cell
expansion at the expense of Clara cells (Tsao et al., 2009).
Interestingly, airway branching morphology was not altered by loss

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Journal of Cell Science 123 (2)of Notch signaling in the epithelium, despite previous loss-offunction reports demonstrating enhanced branching when lunganlagen were cultured in the presence of ␥-secretase inhibitors (GSI)(Tsao et al., 2008) or antisense Notch1 oligonucleotides (Kong etal., 2004). The discrepancy between in vivo and in vitro loss offunction analyses might be explained by an unknown function for
Notch signaling in lung mesenchyme.
Although these observations strongly suggest a role for Notch
signaling in the developing lung, several caveats limit our ability
to identify the cells in which Notch receptors function, and which
specific receptor(s) contribute to lung organogenesis.
Overexpression of N1ICD (Guseh et al., 2009) exposed the tissue
to non-physiological levels of Notch pathway activation in both thelevel and duration of the signal. Moreover, given that Hes1 canrespond to other signaling pathways (Yoshiura et al., 2007), notably
FGF (Nakayama et al., 2008), its activation might not depend on
Notch in every cellular context (Lee et al., 2007). To look at which
specific cell types require Notch activity during lung morphogenesis,
and to begin to assign functions to specific receptors, we examined
the role of Notch signaling in different compartments throughout
lung development. Given the dominant role suggested for Notch1,
we wished to visualize the lineages derived from cells experiencing
Notch1 activation. To map these lineages, we modified
N1IP::CRELOW (Notch1 Intramembrane Proteolysis) (Vooijs et al.,2007) to generate the N1IP::CREHI knock-in mouse strain in which
Cre activity was improved, thus increasing detection sensitivity.
These experiments were followed by detection of N1ICD with
epitope-specific antibodies to observe sites of Notch1 activity.
Finally, genetic inactivation of the canonical Notch pathway inepithelia or jointly in the mesenchyme and mesothelium wasachieved by removal of RBPj␬, the DNA binding partner of all four
Notch receptors and a core component of canonical Notch signaling(Kopan and Ilagan, 2009); more specifically, RBPj␬ is essential for
Notch-mediated Hes1 activation. We uncovered a specific function
for Notch signaling in the specification of the pulmonary vSMCs
and in mesothelial epithelial-mesenchymal transition (EMT). We
confirmed the function of Notch in selection of Clara or ciliatedcell fate and extended these observations, demonstrating a lateral
inhibitory role for Notch1 in this process and during Clara cellregeneration.
Results
Notch1 activation in lung mesenchyme is restricted to
specific lineages

In vivo fate mapping of cells that experienced Notch1 activation
with N1IP::CRELOW allows identification of lineages in which
Notch1 activity might be required (Vooijs et al., 2007). To enhance
our ability to image such lineages in the lung, we generated
N1IP::CREHI knock-in mice in which Cre recombinase [instead of
Cre–6-Myc-Tag (Cre-6MT)] replaced the Notch1 intracellulardomain. Ligand binding unfolds a negative regulatory domain,
triggers ectodomain shedding and enables ␥-secretase-mediatedproteolysis of the Notch transmembrane domain. This leads to therelease of Cre (Vooijs et al., 2007). When combined with a strain
carrying a conditional reporter, Cre-mediated excision of a loxPflanked ‘stop’ cassette constitutively activates reporter expression
and indelibly marks cells that experienced Notch activation and allof their progeny. In N1IP::CRELOW, the inefficient Cre-6MT marks
only a subset of cells (those experiencing moderate to high levelsof sustained Notch activity, such as endothelium) (Vooijs et al.,2007), whereas the new N1IP::CREHI, R26R strain marked cells

receiving moderate-to-low levels of Notch activity and therefore
increased coverage of Notch1 activation patterns [a full descriptionof this line will be provided elsewhere, but compare the lung image
shown here and in Vooijs et al. (Vooijs et al., 2007)]. We used
N1IP::CREHI, R26R mice to determine which lineages within thelung experienced Notch1 activation during development.
Scattered, ␤-galactosidase-labeled mesenchymal (Fig. 1A) andmesothelial cells (black arrowhead in Fig. 1A,B) were detected in
the lung mesenchyme at E13.5 in N1IP::CREHI, R26R mice. As thelung matured, the number of these cells increased (Fig. 1A,C,D). To
identify which mesenchymal cell types were derived from cells
experiencing Notch1 activation, we co-immunostained tissue sections
with anti-␤-galactosidase and cell-type-specific antibodies (SM22␣,
PECAM, SMA). The N1IP::CREHI reporter abundantly marks thevascular plexus (Fig. 1I-N) and both arterial endothelial cells andvSMCs (gray arrowheads in Fig. 1O-Q). By contrast, ␤-galactosidase
was not activated in bSMCs (Fig. 1G, white arrowheads in Fig. 1OQ) or myofibroblasts located at the tip of the alveolar septum (whitearrowheads in Fig. 1R-T). These data suggest that by E18.5, thedescendents of cells experiencing Notch1 activation contributed
extensively if not exclusively to endothelial and vSMC cells.
Notch signaling is required to commit mesenchymal cells
to the arterial smooth muscle cell fate

RBPj␬ is ubiquitously expressed in lung mesenchyme (Fig. 2A,
Fig. 3A). Dermo1-Cre (Yu et al., 2003) is expressed within the lungmesenchymal (supplementary material Fig. S1A-C) and mesothelial(see below) lineages; only a few endothelial cells are targeted bythis strain and no expression is detected in the epithelium (Yin etal., 2008). To test whether canonical Notch signaling is necessary
for mesenchymal lung development, we employed Dermo1-Cre (Yuet al., 2003) to delete floxed RBPj␬ alleles from the mesenchymal
and mesothelial lineages within the developing lung (Drm1-RKOmice). Drm1-RKO mice die within 24 hours due to a highly
penetrant ventricular septal defect (VSD; supplementary material
Fig. S2). Notably, Dermo1-Cre is expressed in the cardiac cushion
tissue that is generated by endothelial-mesenchymal transition(EnMT) (Lavine et al., 2008; Timmerman et al., 2004). This
indicates an unappreciated requirement for Notch signaling after
EnMT has occurred. Importantly, Drm1-RKO pups filled their lungs
with air and their breathing was not labored, consistent with normal
surfactant expression and lung function in Drm1-RKO mice.
By E10.5, Dermo1-Cre had efficiently deleted RBPj␬ frommesenchymal and mesothelial cells (Fig. 2B) (Yin et al., 2008), but
Drm1-RKO lungs were morphologically indistinguishable fromcontrols (Fig. 2C-H). Clara and Type II cells formed properly(supplementary material Fig. S3A-D), indicating that canonical
Notch signaling does not contribute to the complex mesenchymalepithelial feedback loops required for lung development (White etal., 2007; White et al., 2006; Yin et al., 2008). As expected, epithelial
cells (Fig. 3C) and vascular cells retain RBPj␬ protein (whitearrowheads in Fig. 3D-I) within Drm1-RKO lungs. Because
Dermo1-Cre is rarely active in endothelial lineages (supplementarymaterial Fig. S3E-G) (Yu et al., 2003), these observations argue
against conversion of lung mesenchyme, which is RBPj␬-depleted
at E10.5, to endothelium (Stenmark and Abman, 2005).
To quantify the contribution of the Dermo1-Cre lineage to thevascular and bronchial SMC lineages, we counted cells doublepositive for smooth muscle actin and ␤-galactosidase (SMA+, ␤gal+) in arteries and airways of Dermo1-Cre, R26R embryos. SMA
and SM22␣ are SMC markers, and ␤-galactosidase is a lineage

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Journal of Cell ScienceFig. 1. Mesenchymal cells experiencing Notch1 activation contribute
predominantly to the lung vasculature. X-gal staining of N1IP::CREHI,
R26R lung sections at E13.5 (A,B), E15.5 (C,D) and E17.5 (E,F).
N1IP::CREHI activity marked several mesenchymal and mesothelial cells(black arrowheads). X-gal and SM22␣ co-staining of lungs from
N1IP::CREHI, R26R (G) and Dermo1-Cre, R26R (H) mice at E15.5.
N1IP::CREHI activity labeled the vasculature but not peripheral bronchi (G),
whereas Dermo1-Cre-labeled cells commit to both arterial and bSMCs (H,black arrowheads). Co-immunostaining for ␤-galactosidase and PECAM (I-N)
or SMA (O-T) of N1IP::CREHI, R26R lung at E18.5. Distal endothelial (I-K)
and mesothelial cells (L-M, white arrowheads) express ␤-galactosidase. A
subset of vSMCs also expresses ␤-galactosidase (O-Q, gray arrowheads);
whereas bSMCs (O-Q, white arrowheads) or myofibroblasts in alveolar septa(R-T, white arrowheads) do not. Ar, artery; Br, bronchus. Scale bars: A, 0.5mm; B, 10␮m; C,E, 0.1 mm; D,F, 25␮m; G,H, 50␮m; I-T, 20␮m.

tracer used to quantify the contribution of Drm1-RKO cells to theselineages. To quantify the contribution of RBPj␬-deficient (RBPj␬–/–)cells to SMC, we stained for SM22␣ and RBPj␬ proteins (Fig. 3JL). SM22␣+, ␤-gal+ cells and SM22␣+, RBPj␬–/– cells contributed
equally to bSMC (Fig. 3M; 90% for both ␤-gal+ (gray arrowheads
in supplementary material Fig. S1A-C) and RBPj␬–/– cells (grayarrowheads in Fig. 3J-L). The absence of RBPj␬ protein from mostbSMCs indicated strongly that Notch signaling was dispensable forthe execution of the bSMC differentiation program.
In contrast to bSMC, a striking requirement for RBPj␬ wasobserved in vSMC. Whereas most vSMCs (81%) labeled with
SM22␣ and ␤-galactosidase, indicating a robust contribution fromthe Dermo1-Cre lineage (Fig. 1H and Fig. 3M; white arrowheads
in supplementary material Fig. S1A-C), only 15% of vSMC cells
were SM22␣+, RBPj␬–/– (Fig. 3J-M); the rest contained RBPj␬
protein and therefore must have arisen from outside the Dermo1Cre lineage. This indicates that although canonical Notch signaling
was not required for the execution of the bSMC differentiation
program, Notch signaling promoted the selection of the vSMC fate.
Finally, although the majority of endothelial cells appear to havebeen derived from outside the Dermo1-Cre lineage at E16.5, 21%of the endothelial cells (VEGFR2+) were ␤-galactosidase-positive
and, thus, Dermo1-Cre derived (Fig. 3M; gray arrowheads insupplementary material Fig. S1D-F). Interestingly, the fraction of
Dermo1-Cre-derived endothelial cells was reduced in Drm1-RKO
lungs (Fig. 3M).
N1IP::CRE mice detect a population of cells, only some of which
are engaged in Notch signaling. To look at which cells activatedNotch1 within the lung mesenchyme, we used anti-N1ICD antibody
to detect Notch1 activation. Double staining for N1ICD and SMA
at E14.5 revealed that Notch1 receptor is activated in endothelial
cells (Fig. 4A-C, asterisks), vSMCs and in peripheral mesenchymal
cells (Fig. 4A-C, white arrowheads) but never in bSMCs (Fig. 4AC, gray arrowheads). These observations complement the results
obtained with the N1IP::CREHI (Fig. 1), indicating that Notch1
signaling is required for the arterial vSMC fate in developing
mesenchyme and not in a general early precursor.
Platelet-derived growth factor receptor (PDGFR)-␤ is expressedin pericytes, the progenitor for vSMCs (Andrae et al., 2008), where
it regulates migration, proliferation and differentiation into vSMC
(Jin et al., 2008). We therefore measured the expression level of
PDGFR-␤ in Drm1-RKO mesenchyme at E14.5. PDGFR-␤ was
expressed in the wild-type mesenchyme (Fig. 4D-F) and in RBPj␬positive cells located within Drm1-RKO mesenchyme (whitearrowheads in Fig. 4G-I), but was absent from RBPj␬-negative cells,
as judged by immunohistochemistry (gray arrowheads in Fig. 4GI). Reduced PDGFR-␤ mRNA levels were confirmed by quantitative
RT-PCR; expression of endothelial controls (VEGFR1 and 2) was
not significantly altered in the same sample at E13.5 (Fig. 4J).
Because Notch signaling might also be required for cell proliferation(Sakata et al., 2004; Wang et al., 2003), we assessed cell proliferation
by immunohistochemical detection of phospho-histone H3 and216

Journal of Cell Science 123 (2)observed no differences in the number of cells positive for phosphohistone H3 between Drm1-RKO and wild-type E14.5 lungs (Fig.
4K-M, white arrow points to a proliferating RBPj␬-negative cell).
Endothelial-mesenchymal transition, but not epithelialmesenchymal transition of mesothelial cells, might rescue
vSMCs in RBPjk-deficient mesenchyme

Journal of Cell Science

During this analysis, we noticed that the overall numbers of vSMC
in Drm1-RKO lungs did not change, perhaps explaining the normal

Fig. 2. Ablation of RBPj␬ in the Dermo1 lineage does not disrupt lungdevelopment. Lung sections from E10.5 RBPj␬flox/flox (A) and Dermo1-Cre,
RBPj␬flox/flox (B) stained for RBPj␬. Note that Dermo1-Cre deleted RBPj␬
throughout the lung mesenchyme and mesothelium by E10.5 but not theepithelium or endothelium. At E18.5, lung morphology was examined using
hematoxylin and eosin staining in RBPj␬flox/flox control (C,E) or Dermo1-Cre,
RBPj␬flox/flox mutant (D,F) mice. The morphology of lungs from control andmutant mice is indistinguishable. PECAM staining (brown) revealed normal
vascular plexus in both controls (G) and mutants (H). Scale bars: 100 mm(A,B); 0.5 mm (C,D); 0.1 mm (E-H).

Fig. 3. RBPj␬-deleted mesenchymal cells are excluded from the vSMC
fate. Double staining for RBPj␬ and proSP-C in RBPj␬flox/flox (A), Dermo1Cre, RBPj␬+/flox (B), Dermo1-Cre, RBPj␬flox/flox (C) lungs at E16.5. Dashedlines indicate proSP-C-positive epithelial cells. In Dermo1-Cre, RBPj␬flox/floxlung mesenchyme, only a few cells were RBPj␬-positive, whereas
RBPj␬flox/flox and Dermo1-Cre, RBPj␬+/flox showed ubiquitous nuclear RBPj␬
staining. RBPj␬-positive cells in the mutant lung derived from outside theDermo1-Cre lineage. (D-L)Double staining for RBPj␬ and VEGFR2 (D-I) or
SM22␣ (J-L) in the Dermo1-Cre, RBPj␬flox/flox lung at E16.5 revealed thatmesenchymal RBPj␬ staining in the mutant lung colocalized with endothelial
cell markers (D-I, white arrowheads) and vascular (J-L, white arrowheads),
but not bronchial (J-L, gray arrowheads) SMCs. RBPj␬-positive mesothelial
cells were frequently observed in the mutant lung mesothelium (G-I, grayarrowheads). (M)Percentage of Dermo1-Cre lineage cells that contributed to
SMCs or vascular endothelium. The percentages of RBPj␬-positive (blue) or
RBPj␬-negative (orange) cells in distinct Dermo1-Cre, R26R lung populations
were determined at E16.5 by immunohistochemistry. Six to eight images fromeach group were taken at 400⫻ magnification, and the number of cells in each
population was counted. Whereas statistically identical fractions of RBPj␬positive and RBPj␬-negative cells contributed to bSMCs, RBPj␬-negative
cells contributed significantly less to the vSMCs and vascular endothelium
fates in Dermo1-Cre, RBPj␬flox/flox lungs. Error bars indicate s.d. *P<0.0001,**P<0.041. Scale bars: 20 mm.

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lung morphology and function in Drm1-RKO mice. We therefore
investigated the origin of the compensating RBPj␬-positive vSMC
population. One possible source is the mesothelium, a specialized
epithelium that covers the lung (Herrick and Mutsaers, 2004) inwhich some RBPj␬-positive cells are detected at E13.5 (grayarrowheads in Fig. 3G-I; supplementary material Fig. S4). The
mesothelium can undergo EMT and contribute vSMC to the gut(Wilm et al., 2005) and the heart (Cai et al., 2008; Zhou et al.,2008). To assess mesothelial contribution to lung vSMC, we
utilized Wt1-Cre RosaEYFP mice to fate map cells arising from thispopulation (Wilm et al., 2005). Wilm’s tumor protein 1 (Wt1) is amesothelium marker. As seen in the gut, Wt1 expression wasrestricted to lung mesothelium throughout development(supplementary material Fig. S5A-C), but yellow fluorescent protein(YFP) expression was detected within the lung parenchyma and inendothelial cells three months after birth. YFP-positive cells werealso observed in the mural wall, which consists of smooth muscle
and pericytes. Importantly, YFP-positive vSMC cells were rarely
seen in the lung (supplementary material Fig. S5D-I). Thus, weconclude that the mesothelial lineage does not constitute a major
population of vSMC progenitors (see also Que et al., 2008).
Several investigators have reported that endothelial cells cantransition into mesenchymal cells, a process called endothelialmesenchymal transition (EnMT) (Arciniegas et al., 2007).
Therefore, we tested whether endothelial cells contribute topulmonary vSMCs using Tie1-Cre, R26R mice (supplementarymaterial Fig. S6). Tie1-Cre is exclusively expressed in theendothelium, yet most vSMCs in the proximal pulmonary arteries
were double-positive for ␤-galactosidase and SMA in Tie1-Cre
mice, confirming that EnMT is responsible for forming most of theearly (proximal) vSMCs. The contribution of the Tie1-Cre lineage
to vSMC gradually declined; at the most distal positions, only anoccasional vSMC was ␤-galactosidase-positive. Distal contribution
from EnMT might have continued in Drm1-RKO mice, explaining
the presence of RBPj␬-positive vSMC cells in these mice. However,
we could not test this hypothesis directly because Tie1-Cre,
Notch1flox/flox embryos die at E9.5 due to the essential role of Notch1
in vascular development (Conlon et al., 1995; Huppert et al., 2000;
Krebs et al., 2000), prior to lung bud formation (supplementarymaterial Fig. S7) (Cheng, 2006).
Lung mesothelial cells contribute to mesenchyme via
Notch-triggered EMT

N1IP::CREHI labeled mesothelial cells (Fig. 1A-F), which
prompted us to examine whether Notch signaling regulated EMTin this population. We used the organ culture explant system
previously described (Wilm et al., 2005) to address this question.
Briefly, we labeled surface mesothelial cells in cultured embryoniclungs at E14.5 with a fluorescent chemical (CCSFE; 5-(and-6)carboxy-2,7-dichlorofluorescein diacetate succinimidyl ester) for
2 hours (Fig. 5A). Following washout, the lungs were incubatedfor 48 hours to examine the location of CCSFE-labeled mesothelial
cells. Some CCSFE-labeled cells were detected in the mesenchymeat the end of the chase (Fig. 5B), indicating that lung mesothelial
cells can undergo EMT ex-vivo. Transforming growth factor ␤(TGF␤), a general inducer for EMT, increased the number of
CCSFE-labeled cells migrating into the mesenchyme, and the
TGFRII inhibitor SD208 blocked EMT (Fig. 5C-E). To determine
whether Notch signaling participates in EMT and/or mesothelial
migration, we repeated the organ culture with embryonic lungs in
the presence of the ␥-secretase inhibitor DAPT (N-[N-(3,5-Fig. 4. Notch signaling is functionally activated in developing lungmesenchyme to induce expression of PDGFR-␤. (A-C)Double staining for
N1ICD (red) and SMA (green) illustrates Notch1 activation in endothelial cells(asterisk), vSMCs (white arrowheads) and pericytes, but not bSMC cells (grayarrowheads). Double staining for RBPj␬ and PDFGR␤ in Dermo1-Cre,
RBPj␬+/flox (D-F) or Dermo1-Cre, RBPj␬flox/flox (G-I) lungs at E14.5. Cells
double-positive for RBPj␬ and PDGFR-␤ are observed in Dermo1-Cre,
RBPj␬+/flox. In Dermo1-Cre, RBPj␬flox/flox lung mesenchymal cells, only RBPj␬positive cells express PDGFR-␤ (white arrowheads), whereas RBPj␬-negative
cells failed (gray arrowheads). Expression levels of PDGFR-␤, VEGFR1 and
VEGFR 2 were measured with quantitative RT-PCR for E13.5 whole lungs (J).
Mutants (orange) show significant reduction in expression of PDGFR-␤, but notother receptors. Error bars indicate s.d. *P<0.008, **P<0.006. (K-M)Double
staining for RBPj␬ and phospho-histone H3 (pHH3) on E14.5 Dermo1-Cre,
RBPj␬flox/flox lungs. RBPj␬-negative mesenchymal cells (white arrowheads) wereable to enter mitosis, suggesting that Notch signaling is dispensable formaintaining proliferation in the developing lung mesenchyme. Scale bars: 20␮m.

difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). ␥secretase is required for Notch cleavage and therefore for pathway
activity. DAPT treatment reduced mesothelial migration in a dosedependent manner (Fig. 5F-I). Addition of TGF␤ restored

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Journal of Cell Science 123 (2)Fig. 5. Notch signaling contributes to mesothelial EMT, and Notch deficiency can be rescued by TGF␤ in this process. Lung mesothelium in explants fromE14.5 embryos was labeled with CCFSE and visualized on frozen sections using an EGFP filter as indicated. (A)At day 0 of culture, only surface cells werepositive for CCFSE. (B)After two days in culture, some CCFSE-positive cells were observed in the mesenchyme. (C,D)Culture in 5 ng/ml TGF␤ increased themigration of CCFSE-labeled cells (white arrowheads). (E)Migration was inhibited by 5␮M SD208, a TGF␤ inhibitor. (F-I)Culture in DAPT containing medium
decreased CCFSE-labeled cells that migrated from the surface in a dose dependent manner (G-H, white arrowheads). TGF␤ allowed some migration in thepresence of 5␮M DAPT (I). (J,K)TAT-Cre protein treatment for E14.5 RosaYFP (J) and RosaN1ICD-GFP (K) lungs activated the expression of N1ICD-GFP or YFP
reporter in mesothelial cells and was followed by in vitro culture for 3 days. Migrated mesothelial cells were detected by staining with anti-GFP antibody on frozen
sections. (L)The number of migrated mesothelial cells was counted and classified by distance from the surface for each genotype. The number of cells and theirpercentage in the total population are shown. Scale bars: 50␮m.

migration in the presence of DAPT, indicating that in the presenceof strong TGF␤ signals, Notch signaling was dispensable for EMT(Fig. 5I).
To confirm that mesothelial EMT could be enhanced by Notch
signaling, we locally activated Notch signaling within the lungmesothelial cells in organ culture using a recombinant Crerecombinase fused to the HIV-TAT peptide (TAT-Cre) (Shaw etal., 2008; Xu et al., 2008). To induce constitutive Notch activity
in labeled mesothelial cells, we cultured embryonic lungs fromE14.5 RosaN1ICD-GFP (Murtaugh et al., 2003) or RosaEYFP mice (inwhich Cre activity will delete a loxP-flanked stop segment,
resulting in expression of N1ICD::GFP or YFP, respectively) with
5 ␮M TAT-Cre for 5 hours. Following cellar uptake of TAT-Cre
on the surface of the lungs and subsequent washout, growth media
was replaced and the lung organ cultures were maintained for an
additional 3-day period. Mesothelial EMT and migration wereassessed by immunohistochemistry with anti-GFP antibody (Fig.
5J-L). Whereas 37% of mesothelial cells underwent EMT incontrol ROSA-YFP cultures, 77% of cells expressing N1ICD::GFP
underwent EMT. Furthermore, 5% of the N1ICD cells migrated
more than 50 ␮m inwards, whereas control YFP+ cells were neverdetected that deep (white arrowheads in Fig. 5K,L). These results
indicate that Notch activation is sufficient to induce EMT in themesothelium and that it accelerated the mobility of mesotheliumderived cells.
Clara cells experience Notch1 activation during lungepithelial development

By P21, when lung epithelial development is nearly complete, allof the lineages derived from cells experiencing Notch1 activation(as marked by N1IP::CREHI, R26R) have been marked. Histologicalanalysis of intact lungs identified ␤-galactosidase-positive cells as
airway epithelial cells (Fig. 6A,B). Immunohistochemistry for CC10
(Clara cells; Fig. 6C), acetylated tubulin (ciliated cells; Fig. 6D),
and calcitonin gene related peptide (CGRP) (PNECs; Fig. 6E)
determined that ␤-galactosidase-positive cells differentiatedpredominately into Clara cells. A few ciliated cells were also labeled(see below). To obtain a three-dimensional (3D) image of how theepithelial lineages were distributed in the bronchial tree, we
manually removed alveolar capillary cells that are robustly labeledwith ␤-galactosidase, obscuring the epithelium. ␤-galactosidasepositive cells appeared throughout the conducting airway (Fig. 6F),
reaching the highest density in the distal conducting airways (Fig.
6G-I).
To examine the role of Notch signaling in lung epithelia, we
generated SHH-Cre, RBPj␬flox/flox (Shh-RKO) mice (Harfe et al.,
2004; Harris et al., 2006). Staining for RBPj␬ in developing ShhRKO lungs confirmed that RBPj␬ was absent from lung epithelial
cells but its expression remained intact in all other pulmonary
lineages (supplementary material Fig. S8A-D). Unlike Pfout1deficient mice that survive to weaning (Tsao et al., 2009), ShhRKO mutant mice die at birth from an undetermined cause,
apparently unrelated to the lung because breathing appeared
normal, mice were not cyanotic and no morphological pulmonary
defects were observed. Shh-RKO reproduced the phenotypes seen
with loss of Pofut1 (Tsao et al., 2009), namely, expansion of Foxj1positive ciliated cells at the expense of Clara cells throughout theentire lung epithelium (supplementary material Fig. S8I-O). The
stem cell population still gave rise to normal alveolar epithelial
cell types (Type II and Type I cells) in Shh-RKO lungs, indicatingthat the defect was restricted to the Clara and ciliated lineages(supplementary material Fig. S8P-S). Collectively, these data

Journal of Cell Science

Functions of Notch in the lungFig. 6. Cells experiencing Notch1 activation become Clara and ciliatedcells, but not PNECs. The fate of cells experiencing Notch activation was
visualized using X-gal staining in N1IP::CREHI, R26R lungs at P21.
␤-galactosidase-positive epithelial cells (A-E) were identified by double
staining for ␤-galactosidase and CC10 (C), acetylated tubulin (D) or CGRP
(E). The Notch-experienced cells contributed to Clara and some ciliated cells
but not PNECs. (F)X-gal staining for whole lungs revealed gradual
distribution from low (proximal, B) to high (distal, A) density of ␤galactosidase-labeled cells in conducting airways. (G-I)Areas indicated bydashed squares in F are magnified in G-I. Scale bars: A,B, 20␮m; F, 1 mm;
G-I, 100␮m.

suggest that Notch signaling functions during lung development
in a bi-potential progenitor to either induce the Clara cell fate or
to permit Clara cell differentiation by blocking a default ciliatedfate. To differentiate between these possibilities, we examinedciliated cell production during the pseudoglandular stage, whenciliated cells and Clara cells are determined from an epithelial
progenitor cell population residing at the branch tip among proSPC-positive cells (Fig. 7A). At E16.5, Foxj1 (which marks ciliatedcells) was observed only within the proximal airway in a ‘salt andpepper’ pattern (Fig. 7A, asterisk), suggesting that determinationof ciliated cell fate occurs in a transitional zone located between
distal stem cells and proximal differentiated daughters. By contrast,
nearly all the proximal cells expressed Foxj1 in RBPj␬-deficientepithelium (Fig. 7B, asterisk). The salt and pepper distribution of
Clara and ciliated cells further suggests a lateral inhibition
mechanism involving activation of Notch signaling in neighborsof nascent ciliated cells at the transition zone (Fig. 7E). To test
whether lateral interaction among decedents led to Notch1
activation, we used an anti-N1ICD antibody to detect activatedNotch1. Double staining for N1ICD and Foxj1 at E16.5 revealed219

Fig. 7. Clara and ciliated cell fates are delineated from bi-potential
progenitors through a lateral inhibition mechanism related to Notch
signaling. (A,B)Immunofluorescence of E16.5 distal tip to proximal epithelial
cells stained with anti-proSP-C (distal bud marker, red) and anti-Foxj1 (aciliated cell marker, green) revealed that early ciliated cells distribute in a ‘saltand-pepper’ fashion in the proximal epithelium of control lungs (A, asterisk).
In RBPj␬-deficient epithelium, nearly all proximal epithelial cells were Foxj1positive (B, asterisk). (C,D) Double staining for N1ICD (red) and Foxj1(green) demonstrates Notch1 activation in cells adjacent to Foxj1-positive
cells. Dotted square in C indicates area magnified in D. (E)Model of the roleof Notch signaling in the determination of Clara or ciliated cell fate. At thepseudoglandular stage, the elongating distal tip includes epithelial stem cells(purple). A subset of progenitor cells initiate Foxj1 expression (green) as theydifferentiate into ciliated cells. Foxj1-positive cells might activate Notch
signaling in neighboring cells (as marked by N1ICD, red) to suppress theciliated fate and promote Clara cell differentiation. Finally, the conducting
airways generate Clara (orange), ciliated cells (green trapezoid) and PNECs(blue) in the proximal airways. Scale bars: A-C, 50␮m; D, 25␮m.

a mutually exclusive distribution of these markers (Fig. 7C,D).
Coupled with N1IP::Cre labeling (Fig. 6), these data indicate thatneighbors of Foxj1-positive cells, but not the distal progenitors,
activate Notch signaling. Cells experiencing Notch1 activation
become Clara cells. The few ␤-galactosidase-positive ciliated cells
we saw were either derived from a labeled Clara cell (Perl et al.,2002) or indicate that Notch signaling is activated again duringformation of a ciliated cell from a Clara cell.
To test whether ciliated cells required canonical Notch signaling
subsequent to their formation, we generated FOXJ1-Cre,
RBPj␬flox/flox mutant mice in which RBPj␬ was deleted after theciliated fate was determined. These mice were viable and fertileand displayed no change in the frequency of ciliated or Clara cells(supplementary material Fig. S9). Examination of SP-C-rtTA,(tetO)7-Cre, RosaN1ICD-GFP triple transgenic mice revealed a
reduction in ciliated cells, establishing that N1ICD inhibits ciliatedcell fate acquisition [data not shown and Guseh et al. (Guseh et al.,2009)]. These observations expand the data reported earlier (Tsaoet al., 2009) and confirm that Pofut1 deletion expanded the ciliatedepithelium by eliminating Notch1 signals.

Journal of Cell Science

220

Journal of Cell Science 123 (2)Fig. 8. Notch1 signaling is involved in Clara cellregeneration in the adult lung following injury.
The fate of cells that experienced Notch1
activation was visualized in N1IP::CRELOW, R26R
lungs at P14. X-gal staining for whole lungs (A,B)
and lung sections (C,D) revealed mosaic ␤galactosidase activity in the epithelium (B-D,
black arrowheads). CC10 (E,G) or CGRP (F,H)
staining on neighboring X-gal-stained sections
identified ␤-galactosidase-positive Clara cells but
not PNECs. To induce airway injury andregeneration, conducting airways of
N1IP::CRELOW, R26R mice were injected with asingle dose of naphthalene. At 3 days after
injection, all ␤-galactosidase-positive cells had
disappeared (I,J). Subsequently, labeled cells
increased in number, forming small clusters (K,L).
CC10 staining revealed that Notch1 activation
occurred in regenerating epithelia before
differentiation into Clara cells (M-P), suggestingthat Notch1 signaling is involved in the terminal
differentiation into Clara cells rather than themaintenance of the stem cell and/or progenitor
population for lung epithelial cells.

Notch signaling has a role in Clara cell regeneration but
not maintenance in the adult lungTo determine whether Notch1 was reactivated during Clara cellregeneration, we used N1IP::CRELOW, R26R mice (Vooijs et al.,2007) that label Clara cells infrequently compared to N1IP::CREHI(compare Fig. 8A,B and Fig. 6F,G). A few ␤-galactosidase-positive
cell clusters could be discerned within the conducting airway
epithelia at P14 (Fig. 8A,B). Histological analysis revealed that eachcluster included one to eight cells (Fig. 8C,D), and that these cells
were positive for CC10 (Clara cell marker) but negative for CGRP(PNEC marker). Next, we injured the airway epithelium in
N1IP::CRELOW, R26R mice with a single injection of naphthalene
and examined ␤-galactosidase staining patterns throughout theregeneration process. Naphthalene toxicity induced apoptotic deathof most Clara cells within conducting airways by 3 days. Following
this, lung epithelial stem cells initiate a regeneration program, which
is nearly complete 14 days after a single exposure to naphthalene(Plopper et al., 1992; Rawlins et al., 2007). Following injury, allepithelial ␤-galactosidase-positive cells disappeared (Fig. 8J), andregeneration was initiated from ␤-galactosidase-negative cells. ␤galactosidase-positive cells began to reappear 5 days post-injury(Fig. 8N) and increased in number within clusters as the epithelium
recovered (Fig. 8K,L). Furthermore, as judged by the timing of ␤galactosidase and CC10 expression at 5, 7 and 21 days post-injury,
Notch1 activation preceded Clara cell differentiation during
regeneration (Fig. 8M-P, Table 1).
To test whether Clara cells required Notch for their maintenance,
we generated triple-transgenic CCSP-rtTA, (tetO)7-Cre, RBPj␬flox/flox
mice. Although some lung toxicity and mosaicism was reported inthese mice (Sisson et al., 2006), mosaic deletion patterns of RBPj␬
would predict that no RBPj␬–/– Clara cells would be found 6 weeks
after doxycycline (DOX) administration if RBPj␬ played an
important role in Clara cell maintenance. When DOX was added

to the diet beginning at P31, many RBPj␬-null Clara cells weredetected by double staining (supplementary material Fig. S10)
indicating that they were not replaced by cells expressing RBPj␬.
Collectively, these results demonstrate that Notch1 activation did
not mark stem cells or their transient-amplifying daughters. Instead,
it was activated in cells during the final stages of differentiation,
where it might regulate mucous production [data not shown andGuseh et al. (Guseh et al., 2009)]. Notch signaling might not be
required for Clara cell differentiation or maintenance, but thisconclusion is confounded by the low turnover rates of the adultlung.
PNEC fate restriction by Hes1 is largely independent ofcanonical Notch signals

A role for Notch upstream of Hes1 in regulating PNEC fate selection
has been proposed (Ito et al., 2000). Pofut1-deficient mice appear
Table 1. Frequency of a b-galactosidase and CC10 doublepositive epithelial cell in regenerative N1IP::CRE, R26R lungafter naphthalene injection
b-gal+, CC10+
Total b-gal+
Section/sample

0 days

5 days7 days21 days

183
192
9/3
95.3%

6
81
6/2
7.4%

78
231
10/3
59.2%

260
326
10/3
79.8%

Data represent the number of b-galactosidase-positive (b-gal+) or bgalactosidase and CC10 double-positive cells in N1IP::CRE, R26R lungepithelium during the regenerative process. The total number of sections
examined and the percentage of double-positive cells of the total b-gal+ cell
population is shown. Although less than 8% of b-gal+ cells were doublepositive at 5 days after injury, the percentage increased to about 80% at 21days.

Functions of Notch in the lung221

tools, we report a function for Notch signaling in vSMC
specification and in regulating mesothelial EMT and migration rates,
and confirm recent observations describing Notch signaling as a
suppresser of ciliated cell fate that permits or induces secretory cell
formation (Guseh et al., 2009; Tsao et al., 2009). We extend theseobservations by providing evidence for direct Notch1 involvementin this process. Notch activation in regenerating Clara cells could
reflect regulation of mucous secretion (Guseh et al., 2009). Finally,
we report interesting differences between mice lacking RBPj␬ andPofut1 (longevity, role in Hes1 expression) and report a function
for Notch signaling after EMT occurred in the heart. This mightreflect an impact of Pofut1 on lung development via non-canonical
Notch activity or additional substrates. However, RBPj␬ is dedicatedto canonical Notch signaling in the lung, where RBPj␬-like protein(RBPL) will compensate for Notch-independent RBPj␬ activity
such as interaction with Pitf1a (Beres et al., 2006).

Journal of Cell Science

Vascular, but not bronchial, SMC development requires
Notch signaling

Fig. 9. Disruption of Notch signaling does not significantly change PNEC
specification or Hes1 expression. Immunostaining for CGRP (A,C) and Hes1
(B,D) in E18.5 SHH-Cre, RBPj␬+/flox control (A,B) and SHH-Cre, RBPj␬flox/flox
mutant (B,D) lungs. (E)RBPj␬ ablation in developing epithelium (orange)
minimally increased the number of CGRP-positive cells per focus, and of
CGRP-positive foci per section, compared to controls (blue), but thisdifference was not statistically significant. Counts represent average cellnumber per foci from six sections in each group. (F)Quantitative RT-PCR
analysis revealed no alteration in Hes1 mRNA expression in the RBPj␬deficient epithelium compared to controls. Error bars indicate s.d. *P<0.01.
Scale bars: A,C, 50␮m; B,D, 10␮m

to confirm this suggestion because they contain an increased
number of PNECs (Tsao et al., 2009). We thus examined the
distribution of PNECs in Shh-RKO mice within the lung epithelia
using immunohistochemistry. Although the average number and sizeof PNEC clusters was slightly increased in RBPj␬-deficient epithelia(Fig. 9A,C,E), this increase was similar to the increase reported in
Hes1 heterozygote lungs (Ito et al., 2000). Whereas Hes1 expression
was essentially abolished in the epithelia of Pofut1-deficient lungs(Tsao et al., 2009), Hes1 in RBPj␬-deficient epithelia was not
dramatically reduced at either the protein or mRNA levels as late
as E18.5 (Fig. 9B,D,F). Thus, Hes1 expression in lung epithelial
cells does not rely on canonical Notch signaling.
Discussion
In this study, we used genetic analyses to decipher where Notch
signaling acts during lung development. In addition, the new
N1IPHI::CRE, R26R mice permitted a higher resolution mappingof the lineages experiencing at least one round of Notch1 activation.
Conditional gene targeting was complemented by organ culture
experiments to confirm the role of activated Notch1. With these

Ablation of Notch signaling in lung mesenchyme and mesothelium
with the Dermo1-Cre transgene resulted in no obvious
morphological alterations. Published reports (Proweller et al., 2007)
established that canonical Notch signaling was not required for the
maintenance of the SMC cell fates; indeed, bSMC formed in theabsence of RBPj␬ as efficiently as they did in its presence. By
contrast, selection of the vSMC fate, or migration of SMCs arisingoutside of the arteries, was severely impaired. vSMC were strongly
labeled in N1IP::CREHI, R26R mice, implying that in addition toits established role in promoting endothelial cell differentiation intoa vascular network (Gridley, 2007), Notch1 activation was required
within vSMC precursors. A possible explanation for the involvementof Notch in arterial vSMC differentiation has been proposed basedon observations that three SMC marker genes (SM-MHC, SMA
and PDGFR-␤) responded to Notch activation (Doi et al., 2006;
Jin et al., 2008; Noseda et al., 2006). We find that expression of
PDGFR-␤ is significantly reduced in RBPj␬-deficient cells,
confirming that Notch signaling is required cell-autonomously for
PDGFR-␤ expression (Jin et al., 2008). Because PDGF signaling
plays a crucial role in the recruitment of pericytes and vSMC
progenitors during vasculogenesis (Andrae et al., 2008), loss of
RBPj␬ (and hence, PDGFR-␤) might impair vSMC recruitment.
Jagged1-expressing peripheral endothelial cells activate Notch
signaling on pericytes, promoting SMC differentiation (High et al.,2008; Liu et al., 2009). Therefore, Notch signaling is required bothfor recruitment and differentiation in vSMC. Accordingly, we
detected cells that experienced Notch1 activation in mesenchymal
cells surrounding the lung vasculature. Bronchial SMC
differentiation depends on FGF10 (Mailleux et al., 2005). Therefore,
Notch and FGF10 might separate vascular and bronchial SMC
progenitors, respectively, from mesenchymal stem cells.
Importantly, in the absence of RBPj␬ in vivo, vSMCs still formed
outside the Dermo1-Cre lineage, presumably to compensate for theloss of Notch signaling, obscuring a potential phenotype observedwith GSI or antisense oligonucleotides in organ culture.
Notch and TGFb signaling promote EMT in lungmesothelium

Because Dermo1-Cre is active in the mesenchyme and in themesothelium, and transdifferentiation of mesothelial cells via EMT
was described in the heart (Cai et al., 2008; Wada et al., 2003; Zhou
et al., 2008), the gut (Wilm et al., 2005) and the liver (Ijpenberg et

222

Journal of Cell Science 123 (2)al., 2007), we used Wt1-Cre, R26YFP mice to determine whether
vSMC were derived, in a Notch dependent manner, from themesothelium. Although YFP-positive cells were observed in
vascular endothelium and the mural wall, we concluded that themesothelial lineage (Wt1, Dermo1-positive) did not contributesignificantly to the vSMC population under normal conditions (seealso Que et al., 2008). Vital dye pulse-chase experiments,
constitutive Notch1 activation, and inhibitor studies in lung organ
cultures identified a role for Notch signaling alongside TGF␤ inmesothelial EMT. Thus, if mesothelial cells were involved in vSMC
rescue, in vitro inhibition of Notch signaling would greatly reduce
their ability to rescue. In addition, Notch activation enhanced
migration of mesothelium-derived cells; this finding might haveimportant implications for understanding the aggressive metastatic
nature of malignant mesothelioma because elevated Notch signaling
has been observed in malignant human mesothelial cells (Graziani
et al., 2008).

Journal of Cell Science

Notch signaling is essential for endothelial development

Another potential source for vSMCs in Dermo1-Cre, RBPj␬flox/flox
mice is endothelial cells, which remained positive for Notch
signaling and have been known to undergo EnMT in the lung
(Arciniegas et al., 2007). However, due to the early lethality
associated with endothelial-specific loss of Notch1, we could notdemonstrate a contribution of EnMT to vSMC in Dermo1-Cre, Tie1Cre, RBPj␬flox/flox animals. The origin of the rescuing cells, therefore,remains speculative, awaiting creation of an endothelial-specificFlp-recombinase-based reporter. Importantly, Notch signaling isnecessary to promote endocardial EnMT during formation ofcardiac valves (Timmerman et al., 2004), and activation of Notch
signaling is sufficient to induce EnMT in vitro (Noseda et al., 2004).
On the basis of our experiments, we can thus propose a
speculative model explaining how GSI and Notch1 antisense
oligonucleotides impact branching morphogenesis, yet two genetic
models of global Notch loss [this study and Tsao et al. (Tsao et al.,2009)] did not reproduce the branching phenotype. Endothelial cells
deficient in Notch signaling display branching and tube formation
defects (Gridley, 2007; Hellstrom et al., 2007). In our opinion, theprofound effects of GSI and antisense nucleotides on development
and branching of lung anlagen growing in organ culture can thus
be attributed to the disruption of vascular network formation, failed
recruitment of vSMC, failed compensation by EnMT, or somecombination of these. Indeed, DAPT-treated Tie2-GFP lungrudiment cultures show extensive migration and deficientvasculogenesis, whereas airway branching continues at an enhanced
rate (Robert Mecham, Washington University, St Louis, MO,personal communication). We conclude that the negative effects ofglobal Notch inhibition are most probably a reflection of losing thevascular endothelial network and/or its associated SMCs, whichmust therefore negatively regulate branching morphogenesis andpositively contribute to maintaining distal fates.
The primary function of Notch signaling in lung epithelial
cells is in permitting selection of Clara cell fate

Guseh and colleagues have reported that misexpression of aconstitutively active Notch1 fragment with a mosaic SPC-Cretransgene causes mucous metaplasia of the airway and decreases
the number of ciliated cells. In addition, this non-physiologicaland persistent activation generated alveolar cysts (Guseh et al.,2009). They interpreted this to suggest that Notch signaling
suppresses alveolar development. In contrast to these observations,loss-of-function analysis of RBPj␬, a core component of canonical
Notch signaling (this study), or Pofut1 (Tsao et al., 2009), whichmight be required for both canonical and non-canonical functions,did not support a role for physiological Notch signing within lungepithelial cells in regulating alveolar morphogenesis [(Morimotoand Kopan, 2009; Tsao et al., 2009) and data not shown]. RBPj␬or Pofut1-null epithelium did not display alterations in branchingmorphology and contained normal alveoli. We concur with theconclusion that loss of Notch signaling leads to loss of Clara cells
and provide evidence that Notch1 is involved. We describe a distalto-proximal transition zone in which ciliated cells induce Notch
activation in their neighbors, inhibiting them from selecting thesame fate and permitting development of Clara cells. Finally,
tracing the lineage of cells experiencing Notch1 activation indicatesthat these cells overwhelmingly assume the Clara cell fate (witha few ciliated cells labeled secondarily). Interestingly, Notch1 wasactivated again during epithelial regeneration following
pharmacological injury. Notably, Notch1 activation was not
involved in maintenance of the epithelial stem cells. Several
previous reports show that Wnt signaling promotes proliferationof the airway epithelial stem cells early during regeneration(Reynolds et al., 2008; Zhang et al., 2008). Induction of Notch
ligand by Wnt activity (Estrach et al., 2006) might trigger Notch
activation during Clara cell regeneration.
Notch signaling was reported previously to be involved in asimilar lateral inhibitory process, in which ciliated cells inhibit theirneighbors from assuming the same fate. In the zebrafish pronephros,transporting epithelia and multiciliated cells (MCCs) form in a salt
and pepper pattern (Liu et al., 2007). It has been shown that zebrafish
Jagged2 expression in presumptive MCCs induced activation ofzebrafish Notch3 in neighboring cells, blocking MCC fate anddriving the alternative transporting epithelial cell fate. In addition,a Hes1-related protein was involved (Ma et al., 2007). In Xenopus,
ciliated cells express Delta ligands to activate Notch signaling (andHes-related proteins), inhibiting the selection of ciliated cells byneighboring epidermal cells (Deblandre et al., 1999).
Deletion of Hes1 and Pofut impacts PNEC differently toloss of RBPj␬

Although Hes1 is a well-characterized Notch target gene in somecells, it can be regulated by other pathways (Nakayama et al., 2008;
Yoshiura et al., 2007). Accordingly, it has been reported that Hes1
and Pofut1 regulate PNEC foci number as well as size (Ito et al.,
2000; Tsao et al., 2009), yet RBPj␬-deficient mice retain Hes1
expression, and the number of PNECs was decreased only to theintermediate degree seen in Hes1 heterozygotes, not nulls. These
data suggests that Hes1 expression might be controlled by upstream
signals to which Pofut1 (but not RBPj␬) contributes. This could
either imply involvement of other pathways (Nakayama et al., 2008;
Yoshiura et al., 2007) or non-canonical Notch signaling.
Materials and Methods
Whole-mount X-gal staining

To visualize N1IP-CRE activity, the tracheas of 2- to 3-week-old N1IP-CRE, R26R
mice were filled with 0.2% GAD fixative (0.2% glutaraldehyde, 2 mM MgCl2 inPBS) before isolation. After removal of the lungs from the thorax, both were further
fixed in 0.2% GAD fixative for 1 hour at room temperature. The fixed lungs were
washed (in 2 mM MgCl2, 0.1% Tween-20, 0.05% dextrin in PBS) three times for 5
minutes. After washing, lungs were filled with X-gal solution (2 mM MgCl2, 35 mM
potassium ferrocyanide, 35 mM potassium ferricyanide, 1 mg/ml X-gal, 0.02% NP40, 0.01% Na deoxycholate in PBS). The X-gal-filled lungs were submerged in Xgal solution and incubated for 12-24 hours at 4°C in the dark, then post-fixed with
4% paraformaldehyde overnight at 4°C.

Functions of Notch in the lungX-gal staining of tissue sections

␤-galactosidase-expressing fetal lungs were dissected in ice-cold PBS, then fixedwith 0.5% GAD fixative (0.5% glutaraldehyde, 2 mM MgCl2, 0.1% Tween-20 inPBS) for 1 hour at 4°C. The fixed lungs were washed in solution three times for 5
minutes each before equilibration with 30% sucrose in PBS. After embedding in
OCT compound (Sakura), frozen sections (5-6 mm) were generated and either stored
at –20°C or incubated in X-gal solution for 3-6 hours at 37°C. The stained sections
were counterstained with Nuclear Fast Red (Vector Laboratory).
Immunohistochemistry

Fetal lungs were dissected and fixed in 4% paraformaldehyde for 1 hour to overnightat 4°C, embedded in paraffin or OCT (for frozen sections) and sectioned at 6-7 ␮m.
4% formaldehyde was used for the detection of ␤-galactosidase. Sections wererehydrated and treated with 0.3% hydrogen peroxide in MeOH for 10 minutes before
staining. The antibodies and conditions used for individual immunohistochemical
analyses are described in supplementary material Table S1.

Journal of Cell Science

Lung organ culture

E14.5 lungs were collected from timed pregnant CD1 wild-type or N1IP::CRE, R26R
mice and labeled with 40 ␮M CCFSE (5-(and-6)-carboxy-2,7-dichlorofluoresceindiacetate, succinimidyl ester; Molecular Probes) in DMEM containing 10% FBS, 1mM L-glutamine and 1 mM penicillin-streptomycin for 2 hours at 37°C, 5% CO2.
The CCFSE was prepared as a 20 mM stock solution in DMSO. In some experiments,
samples were treated with 5 ␮M recombinant Tat-Cre protein for 5 hours at 37°C,
5% CO2. Subsequently, the labeled lungs were washed and cultured on a filter (cellculture insert, 1.0 ␮m pore size P.E.T. track-etched membrane; Falcon) in medium
without CCFSE or Tat-Cre for 48 hours at 37°C, 5% CO2. TGF␤ (2 ␮g/ml; R&D
Systems), SD208 (5 mM; Calbiochem) or DAPT (1 mM; Calbiochem) was added
to culture medium to appropriate final concentrations (described in each figure legend).
To prepare frozen blocks, at least three whole lungs at end time points were fixedusing fresh 4% formaldehyde for 1 hour at 4°C followed by equilibration with 30%
sucrose in PBS. Sections (6 ␮m) were cut and mounted with Vectamount with DAPI
(Vector Laboratories) or stained with anti-GFP-antibody (see supplementary material
Table S1) for Tat-Cre experiments. Using a Carl Zeiss Axio imager Z1 microscope,
the fluorescence of CCFSE was observed with the EGFP filter under 400⫻
magnification. To quantify mesothelial EMT, ten photographs of GFP-positive cells
were taken for each genotype under 400⫻ view.
Recombinant TAT-Cre protein

TAT-Cre recombinant protein was expressed and purified according to previously
described protocols (Peitz et al., 2002). Briefly, Escherichia coli TUNER(DE3)pLacI(Novagen) containing pTriEx(Novagen)-His-TAT-NLS-Cre was cultured with LB
medium containing 100 mg/ml ampicillin and 34 mg/ml chloramphenicol at 37°C
with shaking until the OD600 was 0.9-1.0. Expression was induced with 0.5 mM
isopropylthiogalactoside for 4 hours. Cells were harvested, resuspended in phosphatebuffer (50 mM Na2HPO4, 5 mM Tris pH 7.8, 500 mM NaCl) containing lysozyme(Sigma) and Benzonase (Novagen) for lysis. TAT-Cre protein was purified from thesupernatant with Ni-NTA matrix (Qiagen). The matrix was washed extensively withphosphate buffer containing 20 mM imidizole and eluted with phosphate buffer
containing 250 mM imidazole. Protein-containing fractions were pooled and dialyzed
against 600 mM NaCl, 20 mM HEPES, and subsequently against 600 mM NaCl, 20mM HEPES, 50% glycerol to concentrate.
Mouse strains

RBPj␬flox/flox mice were kindly provided by Tasuku Honjo, Kyoto University, Kyoto,
Japan (Tanigaki et al., 2002). Dermo1-Cre mice were generated by David Ornitz at
Washington University, St Louis, MO (Yu et al., 2003). FOXJ1-Cre mice were a kind
gift from Michael Holtzman, Washington University, St Louis, MO (Zhang et al.,2007). These mice were maintained on the CD1 background. Dermo1-Cre mice weremated to RBPj␬flox/flox mice to generate heterozygous Dermo1-Cre, RBPj␬+/flox mice,
which were then crossed to RBPj␬flox/flox mice to create Dermo1-Cre, RBPj␬flox/flox
conditional knockout animals. FOXJ1-Cre, RBPj␬flox/flox mice were generated in thesame way.
The N1IP::CRE (Vooijs et al., 2007), Wt1-Cre (Wilm et al., 2005) and CCSP-rtTA,(Tet)7O-Cre (Perl et al., 2002) mice were described previously. SHH-Cre iscommercially available at The Jackson Laboratory. All animal procedures were
performed according to NIH guidelines and maintained in the animal facility underWashington University animal care regulations.
RNA isolation, cDNA synthesis, and qRT-PCR analysis

Embryonic lung mRNA was isolated using the RNeasy kit (Qiagen) following the
manufacturer’s instructions. cDNA was synthesized using the SuperScript II firststrand cDNA synthesis kit (Invitrogen). Quantitative RT-PCR was performed on an
ABI 7500 machine using Power SYBR Green for Foxj1, CC10, Hes1 and Gapdh.
Amplification and analyses were performed according to the manufacturer’s
instructions. All reactions were normalized to Gapdh. Results were plotted as relative
expression compared with control, where control was scaled to 1.

223

The authors thank Adrian Shifren, David Ornitz, Rober Mecham andScott Boyle (from Washington University, St Louis, MO) for carefulreading of the manuscript. We also thank David Ornitz for the Dermo1Cre mice, Tasuku Honjo for the conditional RBPj␬flox/flox mice andSteven Brody for anti-FoxJ1 antibody. This work was supported by
Washington University and by NIH grants P50 CA094056 (David
Piwnica-Worms) and RO1 DK066408 (R.K.). M.M. was supported bya Toyobo Biotechnology Foundation Long-term Research Grant, theJapanese Society for the Promotion of Science and The Kanae
Foundation for the Promotion of Medical Science. We wish to thankPatricia Gonzalez-DeWhitt (Eli Lilly and Company, Indianapolis, IN)
for the generous gift of the rabbit anti-VLLS antibody. Deposited in
PMC for release after 12 months.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/123/2/213/DC1

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