RESEARCH ARTICLE 4365

Development 139, 4365-4373 (2012) doi:10.1242/dev.083840© 2012. Published by The Company of Biologists Ltd

Different assemblies of Notch receptors coordinate the
distribution of the major bronchial Clara, ciliated andneuroendocrine cells
Mitsuru Morimoto1,2,*,‡, Ryuichi Nishinakamura3, Yumiko Saga1,2 and Raphael Kopan4
SUMMARY
In the developing lung, it is thought that the terminal buds of elongating airways contain a population of multipotent epithelial
progenitors. As the bronchial tree extends, descendants of these cells give rise to lineage-restricted progenitors in the conducting
airways via Notch signaling, which is involved in the establishment of epithelial Clara, ciliated and pulmonary neuroendocrine (NE)cell populations. However, the precise molecular details of this selection process are still emerging. Our stepwise removal of thethree Notch receptors from the developing lung epithelium reveals that, whereas Notch2 mediates the Clara/ciliated cell fate decision
with negligible contributions from Notch1 and Notch3, all three Notch receptors contribute in an additive manner to regulate theabundance of NE cells and the size of the presumptive pulmonary neuroepithelial body (pNEB) as a result of mutual interactions
between NE cells and the Notch-dependent, SSEA-1+, CC10– cell population surrounding the pNEB (SPNC cells). Ectopic expressionof the Notch1 or Notch2 intracellular domain was sufficient to induce SSEA-1+ cells and to suppress pNEB formation without
expending Clara cells. We provide evidence that the additive functions of Notch receptors, together with other signaling pathways,
maintains the expression of Hes1, a key regulator of NE cell fate, and that maintenance of Hes1 expression in epithelial cells is key
to the regulation of pNEB size. These results suggest that two different assemblies of Notch receptors coordinate the numbers anddistribution of the major epithelial cell types in the conducting airway during lung organogenesis.

INTRODUCTION
Lung development relies on reciprocal mesenchymal-epithelialinteractions orchestrated by temporal and spatial expression waves
of multiple secreted factors and their downstream effectors(Morrisey and Hogan, 2010). Airway branching morphogenesis
takes place at E11.5-16.5 in the mouse (the pseudoglandular stage).
The terminal buds contain a population of multipotent epithelial
progenitors during this period, which give rise to lineage-restricted
descendants that produce at least seven major cell types in the‘stalk’ region (Perl et al., 2002b; Rawlins et al., 2009). Thus, theearly stalks form the proximal airway, then the distal airways andfinally the alveoli (Cardoso and Lü, 2006). The Clara, ciliated andpulmonary neuroendocrine (NE) cells are common throughout the
conducting airways, but their ratios vary along the proximodistal
axis. In the murine tracheal epithelium, the basal cell generates
goblet cells, a few Clara cells and many ciliated cells (Hong et al.,1

Division of Mammalian Development, National Institute of Genetics, 1111 Yata,
Mishima, Shizuoka 411-8540, Japan. 2Department of Genetics, The Graduate
University for Advanced Studies, Mishima, Shizuoka 411-8540, Japan. 3Departmentof Kidney Development, Institute of Molecular Embryology and Genetics (IMEG),
Kumamoto University, Kumamoto 860-061, Japan. 4Department of Developmental
Biology and Division of Dermatology, Washington University School of Medicine,Box 8103, 660 South Euclid Avenue, St Louis, MO 63110-1095, USA.
*Present address: Lung Development and Regeneration, RIKEN RIKEN Center for
Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047,
Japan‡
Author for correspondence (mmorimoto@cdb.riken.jp)
This is an Open Access article distributed under the terms of the Creative Commons Attribution
Non-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), which
permits unrestricted non-commercial use, distribution and reproduction in any medium provided
that the original work is properly cited and all further distributions of the work or adaptation are
subject to the same Creative Commons License terms.

Accepted 28 August 20122004; Rock et al., 2009). In the distal bronchioles, Clara cells are
more abundant than ciliated cells; a few NE cell clusters (calledneuroepithelial bodies, or NEBs) per unit area are also present. The
distal-most alveoli are lined with a single thin layer of flat type I
cells and cuboidal type II cells (Morrisey and Hogan, 2010).
The Notch signaling pathway plays an important role in thedeveloping respiratory system. Notch genes encode singletransmembrane receptors that mediate short-range communication
between cells in all animal species (Kopan and Ilagan, 2009). When
Notch binds to its ligand expressed on adjacent cells, a negative
control region unfolds that permits the shedding of its extracellulardomain by ADAM proteases. A subsequent cleavage within thetransmembrane domain executed by the enzyme -secretase (Mummand Kopan, 2000; Kopan and Ilagan, 2009) releases the Notch
intracellular domain (NICD). NICD translocates to the nucleus,
where it associates with Rbpj/Cbf-1, a DNA-binding protein. The
NICD/Rbpj complex recruits the adaptor mastermind and assembles
a transcription activation complex on target promoters (Lubman etal., 2004). In mammals, four Notch homologs (Notch1 to Notch4)
and at least five ligands (Jag1, Jag2, Dll1, Dll3 and Dll4) mediate
these signaling events. There are many auxiliary factors thatmodulate Notch signaling. One of these, protein O-fucosyltransferase
1 (Pofut1), conjugates O-fucose to specific extracellular EGF repeats,
modifications that are essential for productive Notch-ligand
interactions in mammals (Okamura and Saga, 2008; Stahl et al.,2008). Although Pofut1 may have additional substrates, Pofut1-nullmice display Notch signaling defects that are similar to those
associated with the loss of -secretase or Rbpj (Shi and Stanley,
2003; Okamura and Saga, 2008).
Conditional removal of Pofut1 or Rbpj from lung bud epithelia
promoted ciliated cell expansion at the expense of Clara cells (Tsaoet al., 2009; Morimoto et al., 2010). By contrast, constitutive

DEVELOPMENT

KEY WORDS: Lung, Notch signaling, Neuroendocrine, Mouse

Development 139 (23)4366 RESEARCH ARTICLE

MATERIALS AND METHODS
Mouse strains

We crossed SHH-Cre; N1flox/+, N2flox/+ or SHH-Cre; N1flox/+, N2flox/+, N3–/+
males to N1flox/ flox, N2 flox/ flox or N1flox/ flox, N2 flox/ flox, N3–/– females togenerate a Notch allelic series (see Fig. 1A) (Krebs et al., 2003; Harfe etal., 2004; Yang et al., 2004; McCright et al., 2006). SPC-rtTA, TetO-Cre;
Rosa-GFP-N1ICD and Ascl1 knockout embryos were provided aspreviously described (Guillemot et al., 1993; Perl et al., 2002a; Guseh etal., 2009). All mice were maintained on the C57BL/6 ⫻ CD1 mixed
background, with some transgenic lines also contributing FVB or 129
chromosome(s). Animal procedures were performed according to NIH
guidelines. The mice were maintained in the animal facility under the
Washington University animal care regulations or according to the Animal
Care and Use Committee of the NIG.

Immunohistochemistry

Fetal lungs were dissected and fixed in 4% paraformaldehyde for 1 hour toovernight at 4°C, embedded in paraffin and sectioned at 6-7 m. Sections
were rehydrated and treated with 0.3% hydrogen peroxide in methanol for
10 minutes before staining. The antibodies and conditions used forindividual immunohistochemical analyses are described in supplementarymaterial Table S1. We define only the main bronchus as the bronchus andthe terminal bronchiole as the bronchiole in this paper.
RNA isolation, cDNA synthesis and qRT-PCR analysis

E18.5 embryonic lung mRNA was isolated using the RNeasy kit (Qiagen)according to the manufacturer’s instructions. cDNA was synthesized using
the SuperScript III first-strand cDNA synthesis kit (Invitrogen).
Quantitative RT-PCR was performed on a TaKaRa Thermal Cycler Dice
Real Time System Single machine, using Power SYBR Green for Foxj1,
CC10, CGRP and Gapdh. Amplification and analysis 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. The PCR primer pairs used in thispaper were as follows: CC10-F, CACCAAAGCCTCCAACCTCTAC;
CC10-R, GGGATGCCACATAACCAGACTC; Foxj1-F, ATCGTCGTGCACATCTCGAAG; Foxj1-R, AGCAGAAGTTGTCCGTGATC;CGRP-F, GAGGGCTCTAGCTTGGACAG; CGRP-R, AAGGTGTGAAACTTGTTGAGGT; Gapdh-F, AATGTGTCCGTCGTGGATCTGA;
Gapdh-R, GATGCCTGCTTCACCACCTTCT.

RESULTS
Notch2 mediates Clara/ciliated cell selection
We and other groups have previously reported that deletion of Rbpj
or Pofut1 in developing lung epithelium leads to expansion ofciliated cells at the expense of the Clara cell population, and weshowed direct evidence for Notch1 receptor activation in Claracells (Tsao et al., 2009; Morimoto et al., 2010). Given that all four
mammalian Notch proteins bind to Rbpj with similar affinity(Kopan and Ilagan, 2009), we attempted to identify thecontributions of each Notch receptor during Clara/ciliated cell fateselection (Guseh et al., 2009; Tsao et al., 2009; Morimoto et al.,2010). We used SHH-Cre to genetically remove conditional Notch1
and Notch2 alleles from the developing endodermal epithelium inwild-type or Notch3-null animals, creating an allelic series
culminating in a triple knockout of all receptors (Shh-NTKO; Fig.
1A). Loss of Notch1 and Notch2 was confirmed by antibody
staining (supplementary material Fig. S1A-D). We correlated themagnitude of ciliated cell expansion with the number of remaining
Notch alleles (Fig. 1B,C) by measuring the levels of CC10 (a Claracell marker; Scgb1a1 – Mouse Genome Informatics) and Foxj1 (aciliated cell marker) mRNAs by qRT-PCR (Fig. 1). We observedthat CC10 expression was decreased by 50% when one allele of
Notch1 and one allele of Notch2 remained, which suggests a dosedependent additive function for Notch1 and Notch2 in maintaining
the Clara cell fate throughout lung epithelial development (Fig.
1B). However, whereas mice retaining only one allele of Notch2(Shh-Cre; N1f/f; N2+/f) retained 40.4% of CC10 expression, miceretaining one allele of Notch1 (Shh-Cre; N1+/f; N2f/f) lost CC10
expression (1.29% of wild-type levels). These data suggest that
despite the presence of activated Notch1 (N1ICD) in the nucleus
of lung epithelial cells (Morimoto et al., 2010), Notch2 makes adominant contribution in Clara cell fate determination. CC10
expression was reduced further in the Shh-Cre; N1f/f; N2f/f double
knockout (Shh-NDKO), and in the Shh-Cre; N1f/f; N2f/f; N3–/– triple
knockout (Shh-NTKO). These observations suggested that Notch1
and Notch3 make a modest contribution to Clara fate selection.
To examine the expansion of the ciliated cell fate, we quantified
the Foxj1 mRNA levels in the same samples. We saw a

DEVELOPMENT

expression of the Notch1 intracellular domain (N1ICD) in lungepithelial cells promoted mucous metaplasia and remarkably
decreased the number of ciliated cells (Guseh et al., 2009). Jag1
expression is detected exclusively in ciliated cells (Tsao et al., 2009),
and immunohistochemical analysis identified endogenous N1ICD in
their neighbors (Morimoto et al., 2010). In vivo fate-mapping
analysis of cells experiencing Notch1 activation provided evidence
that such cells generate Clara cells, but not ciliated cells or NE cells(Morimoto et al., 2010). These observations suggest that the primaryfunction of Notch1 in lung epithelial cells is to promote the Clara cellfate, either by suppressing the default ciliated cell fate via Notch1mediated lateral inhibition or by inducing Clara cells directly. In theadult lung, Notch1 is also activated in Clara cells that areregenerating after naphthalene-induced injury (Morimoto et al.,2010). Regeneration of the proximal airways from tracheal stem cells
relies on Notch activity to enable the cells to exit the stem cell nicheand differentiate into Clara and goblet cells (Rock et al., 2011).
In the absence of Hes1, hyperplasia of presumptive NEBs(pNEBs) occurs in embryonic lung at the expense of Clara cells(Ito et al., 2000; Shan et al., 2007). This phenotype was also seen
following conditional deletion of Pofut1 from lung epithelium(Tsao et al., 2009). Because Hes1 is a target of Notch signaling inseveral biological systems, this led to the conclusion that epithelial
Notch signaling was involved in NE cell fate selection. However,Rbpj-deficient mice on a mixed background retained Hes1
expression and displayed only a mild reduction in NE cellnumbers, resembling Hes1 heterozygotes. This may indicate derepression of Hes1 in Rbpj-null epithelia, involvement of Rbpjindependent Notch signaling in the regulation of pNEB size, or
regulation of Hes1 by additional pathways.
To clarify the role of specific Notch receptors in establishing thecellular identities in the lung epithelium, we generated an allelicseries for Notch1, Notch2 and Notch3 to eliminate Notch signalingfrom the lung epithelium while retaining Rbpj-mediated repression.
Quantitative and histological analyses identified Notch2 as theprimary receptor involved in Clara/ciliated cell fate selection, despite
the detection of N1ICD in presumptive Clara cells (Morimoto et al.,2010). By contrast, all three Notch receptors were involved inregulating NE fate selection in an additive manner. The triple Notch
knockout lung epithelium displays pNEB hyperplasia that resembles
the Hes1-null phenotype. Additional analyses revealed that the Dll1expressing NE cells are capped by SSEA-1+, peri-pNEB, N1ICD+,
CC10– (SPNC) cells. This novel epithelial cell population is involvedin the regulation of NE cell number during pNEB formation. Thus,
the numbers and distributions of the three major lung epithelial cell
types are established by two distinct Notch-dependent mechanisms:
Notch2 signaling controls Clara cell number, and three Notch
receptors in SPNC cells activate Hes1 to control pNEBs.

Notch proteins in lung epithelium

RESEARCH ARTICLE 4367

Fig. 1. Endodermal epithelium-specific stepwise removal of
Notch paralogs revealed the dominant contribution of Notch2 in
Clara/ciliated cell fate decision. (A)Two mating strategies were
employed to achieve endodermal epithelium-specific knockout of
Notch prologs. Male genotypes are noted on the left of the cross. (BD)For each genotype, total RNA was extracted from one or two wholelungs collected at E18.5. The relative gene expression levels of CC10 (B)and Foxj1 (C) in the lungs were measured by quantitative RT-PCR.
(B)CC10 expression is progressively diminished by reduction in the
Notch signal dose. Brackets highlight a comparison between SHH-Cre;
N1flox/flox, N2flox/+ and SHH-Cre; N1flox/+, N2flox/flox mice that unveiled aunique contribution of Notch2 in Clara cell fate determination.
(C)Accordingly, Foxj1 expression is upregulated in all mutants, reflecting
the expansion of ciliated cells (see Fig. 2A). Although elevation of Foxj1
expression correlates with a reduction of Clara cells, SHH-Cre; N1flox/flox;
N2flox/flox, SHH-Cre; N1flox/flox; N2flox/flox; N3–/+ and SHH-Cre; N1flox/flox,
N2flox/flox; N3–/– lungs show lower levels of Foxj1 expression relative to
Shh-RKO lungs (brackets). (D)Analysis of SHH-Cre; N2flox/flox lung also
demonstrated the reduction of CC10 and the elevation of Foxj1
expression in Notch2-null lung epithelium, while the Cgrp level was
unaltered. Error bars from technical triplicates indicate s.d. P values areshown above the bars.

All Notch receptors contribute to the regulationof pNEB abundance and size
We expected a similar expansion in Foxj1 in Shh-RKO, Shh-NDKO
and Shh-NTKO. However, we were surprised to see that Foxj1
expression in Shh-NDKO and Shh-NTKO was much lower than in
Shh-RKO, resembling instead Shh-Cre; N1f/f; N2+/f (230%) (Fig. 1C,
brackets). To address the cellular basis of this observation, we
assessedthe
Clara/ciliatedcell
distribution
using
immunohistochemistry (Fig. 2A). Foxj1-expressing ciliated cells
were distributed in a salt-and-pepper pattern in the distal conducting
airways, where Notch dose correlated with the magnitude of ciliatedcell expansion at the expense of CC10-postive cells (Fig. 2A, upper
panels). The distal conducting airways of Shh-Cre; N1+/f; N2f/f, ShhNDKO, Shh-NTKO and Shh-RKO mice were indistinguishable,
containing mostly ciliated cells. However, in bronchi, an expanded
Foxj1–, CC10– DAPI+ cell population was present in Shh-NDKO
and Shh-NTKO mice (Fig. 2A, lower panel, asterisks). Because theircell morphology suggested that they were pNEBs, we examined the
distribution of pNEBs in Shh-NTKO and Shh-RKO mice byimmunohistochemistry for the NE cell marker CGRP (Calca –Mouse Genome Informatics). Although pNEB abundance in ShhRKO mice remained similar to wild type and their size was only
minimally affected (Fig. 2B) (Morimoto et al., 2010), Shh-NTKO
bronchi contained an increased number of pNEBs with somedisplaying large increases in NE numbers (Fig. 2B, white asterisk).
To quantify the abundance of pNEBs in bronchi, we counted pNEBs
on histological sections and compared pNEB abundance among
genotypes using the Kolmogorov-Smirnov test (K-S test) (Fig. 2C;
Table 1). Interestingly, only Shh-NTKO lungs showed a significant
increase in the abundance of pNEBs (P<0.05). In addition, we
counted the number of NE cells in each pNEB and used the K-S test
to determine whether pNEB size was altered in different genotypes(Fig. 2D). A significant increase in NE cell numbers per pNEB was
detected in Shh-NTKO (P<0.001) and in Shh-NDKO (P<0.017),
indicating that pNEB size reflects the abundance of NE cells in apNEB. qRT-PCR for Cgrp mRNA further revealed that wheneverFoxj1 levels were lower than expected, a corresponding increase in
Cgrp levels (by up to 6.86-fold in Shh-NTKO lungs) was detected(Fig. 2E). These data indicate that all three Notch receptors
contribute in an additive manner to negatively regulate the pNEB
size (Fig. 2C-E). Furthermore, Notch1, Notch2 or Notch3 single
knockouts did not affect Cgrp expression, suggesting that thecontribution of any single Notch receptor is redundant in theregulation of pNEB size (Fig. 1D; supplementary material Fig. S2B,
Fig. S3).

DEVELOPMENT

complementary expansion of Foxj1 expression in Shh-Cre; N1f/f;
N2+/f and Shh-Cre; N1+/f; N2f/f cells: the former showed a twofold(230%) expansion in Foxj1 expression, while the latter, as well as
Shh-Cre; Rbpjf/f (Shh-RKO), displayed a greater than fourfold
increase in the expression of Foxj1 (Fig. 1C). Accordingly,
expanded ciliated cell populations were observed in the developing
lung epithelium of both genotypes (Fig. 2A). Individual conditionaldeletion of Notch1 or Notch2 confirmed the dominant contributionof Notch2 to Clara/ciliated cell selection (Fig. 1D; supplementarymaterial Fig. S2): Shh-Cre; N2f/f reproduced the phenotypes ofciliated cell expansion with a great reduction in Clara cell numbers,
whereas Shh-Cre; N1f/f was indistinguishable from wild type. This
unique role of Notch2 in Clara cell fate may be due to the greater
abundance of Notch2 (supplementary material Fig. S1A,C), to
activation of Notch2-specific targets or to an enhanced activation
profile of Notch2 in response to Jag1 (Hicks et al., 2000).

4368 RESEARCH ARTICLE

Development 139 (23)Fig. 2. Loss of Notch receptors affects NE cell numbers. For eachgenotype, two or three whole lungs were collected at E18.5 and at
least six paraffin wax-embedded sections were stained for eachindividual sample. (A)Immunofluorescence of E18.5 bronchioles (upper
panels) and bronchus (lower panels) stained with anti-Foxj1 (green)
revealed that the ciliated cell expansion progressed as the Notch dose
decreased. In addition, foci of Foxj1– cells are observed in the Notch
double knockout and triple knockout epithelia (asterisks). Dotted lines
demarcate the mesenchyme (left) and the epithelium (right).
(B)Immunohistochemistry for CGRP in E18.5 lungs shows a significant
increase and enlargement of pNEBs (brown) in the Notch triple
knockout but a moderate increase in Rbpj knockout epithelium.
(C)Frequency distribution of pNEB abundance. Eight or nine
photographs of proximal airways in 12 sections from two or three mice
were taken at ⫻20 magnification for individual genotypes: control(black), SHH-Cre; N1flox/+; N2flox/flox (green), Notch double knockout(blue), triple knockout (red) and Rbpj knockout (orange) embryos.
CGRP-positive clusters were determined as pNEBs on the monitor. Two
to 11 pNEBs were observed in each photograph. (D)Frequency
distribution of the numbers of NE cells per pNEB. The numbers of NE
cells in a pNEB were counted in 30 pNEBs randomly selected from antiCGRP-stained sections, for which at least two biological lung samples
were collected from E18.5 control (black), Notch double knockout(blue) and triple knockout (red) embryos. (E)Quantification of Cgrp
expression in E18.5 lungs estimates the magnitude of pNEB
enlargement in each genotype. Total RNA was extracted from one tothree whole lungs collected at E18.5 for each genotype. In comparison
with the control, the Notch triple knockout displayed the greatest
expansion, and Rbpj loss elevated the expression more than the loss ofany two Notch receptors. (F)Double immunofluorescence of E18.5
bronchus with anti-CGRP (green) and anti-N1ICD (red) determined
Notch activation in peri-pNEB cells (arrowheads). Scale bars: 10m in
A,F; 20m in B. Error bars from technical triplicates and multiple
samples indicate s.d. P values are shown above the bars.

To examine which cells receive the Notch signal, we used theanti-N1ICD V1744 antibody (which detects -secretase-cleaved
Notch1) to identify signal-receiving cells in the vicinity of thepNEB (Xu et al., 2010) (supplementary material Fig. S4). Double
staining for N1ICD and CGRP at E18.5 clearly visualized Notch1
activation in cells adjacent to Dll1-expressing NE cells, but neverin the NE cells themselves (Fig. 2F, arrowheads). Our previous
study in N1IP::CRE reporter mice, which permit the mapping ofcell lineages experiencing Notch1 activation in vivo (Vooijs et al.,2007), illustrated that cells experiencing Notch1 activation never
become NE cells (Morimoto et al., 2010). These observations
suggest that Notch activation did not occur in NE precursors, but

Notch signaling is indispensable to the SPNC cell
population and to the regulation of pNEB size
These peri-pNEB cells containing N1ICD may be epithelial
progenitor cells for non-NE cell types, including Clara and ciliatedcells. We examined the distribution of the progenitor/stem cellmarker SSEA-1/CD15 (Yanagisawa, 2011) to see whether peripNEB cells express this marker. At E15.5, SSEA-1-positive
epithelial cells are abundant in the proximal airways (Xing et al.,2010). At E18.5, the distribution of SSEA-1-positive cells in theproximal airway became restricted in the vicinity of pNEBs (Fig.
3A,B, white arrowheads). Confocal imaging for SSEA-1 and
CGRP confirmed the restricted expression pattern of this marker(supplementary material Fig. S5A). SSEA-1-positive cells were
negative for CC10, distinguishing them from Clara cells (Fig. 3B,
white and gray arrowheads). By contrast, SSEA-1-positive, CC10positive cells were still abundant in the distal bronchioles at E18.5(Fig. 3C, white arrowhead). Therefore, the SSEA-1 epitope
distinguishes peri-pNEB cells at E18.5 as a distinct, CC10– cell
population.
To further characterize the peri-pNEB cells, we analyzed serial
sections for the presence of N1ICD (Fig. 3D,E, white arrowheadand supplementary material Fig. S5B), and for Notch2 and Notch3(supplementary material Fig. S1E,F). Co-staining for Notch and
SSEA-1 differentiated these cells from Clara progenitors

DEVELOPMENT

rather NE cells could activate Notch signaling in their neighbors.
Notch activity is probably necessary for the proper regulation ofpNEB size during lung development.

Notch proteins in lung epithelium

RESEARCH ARTICLE 4369

Table 1. P values determined by the Kolmogorov-Smirnov test for pNEB abundance in the Notch allelic mutant series
Control
SHH-Cre, N1f/+, N2f/f
SHH-Cre, N1f/f,N2f/f
SHH-Cre, N1f/f,N2f/f,N3–/–
SHH-Cre, Rbpjf/f

Control

SHH-Cre, N1f/+, N2f/f

SHH-Cre, N1f/f,N2f/f

SHH-Cre, N1f/f,N2f/f,N3-/-

1.00000.33640.0464*0.98500.69940.0143*0.78040.0143*0.07310.0044**Significant difference in frequency distribution between two genotypes (P<0.05).

and pNEBs, we examined the distributions of SPNC cells and NE
cells in E18.5 Achaete-scute complex homolog 1 (Ascl1/Mash1)deficient lungs (Guillemot et al., 1993; Borges et al., 1997) (Fig. 4).
Ascl1 is a basic-HLH type transcription factor that is indispensable
in NE cells, and a presumed transcriptional activator of Dll1 that isrepressed by Hes1 (Ito et al., 2000; Shan et al., 2007). Loss of Ascl1
results in loss of NE cells (Guillemot et al., 1993; Borges et al.,1997). Immunohistochemistry on multiple sections found instead that
only a few SSEA-1+ cells remained in the Ascl1-null bronchus (Fig.
4D). This is not because of the dependence of the SSEA1 markers
on Ascl1, because bronchiolar SSEA-1+ CC10+ cell populations
were unaltered in the Ascl1 knockout (Fig. 4C). These data suggestthat the existence of SPNC cells, but not of the SSEA-1 antigen,depends on Ascl1-expressing NE cells in the developing bronchus.
To investigate whether Notch activation is needed for SPNC cellmaintenance, we examined the distribution of SPNC cells in ShhCre; N2f/f and Shh-Cre; N1+/f; N2f/f lungs. These mutants have

Fig. 3. Peri-pNEB, Notch signal-receiving cells in the bronchus visualized by SSEA-1 immunoreactivity. (A-C)Immunofluorescence stainingof E18.5 lung with anti-CC10 (green), anti-CGRP (red) and anti-SSEA-1 (blue) shows localizations of Clara, NE and SSEA-1+ cells. At the bronchus,
SSEA-1+ cells are restricted to the peri-pNEB area (white arrowheads in A and B). These are SSEA-1+ CGRP – CC10– cells. The SSEA-1+/pNEB clusters
are interspaced between Clara cells (gray arrowheads in B). At the bronchioles, SSEA-1+ cells that are often CC10+ are broadly distributed (whitearrowheads in C). (D,E)Immunofluorescence staining with anti-SSEA-1 (green) and anti-CGRP (red) (D), or anti-SSEA-1 (green) and anti-N1ICD (red)(E) illustrates the SSEA-1 immunoreactivity of peri-pNEB, Notch signal-receiving cells at the E18.5 bronchus (white arrowheads). A gray arrowhead
identifies N1ICD in a Clara cell. Some N1ICD-positive vascular smooth muscle and endothelial cells are observed in mesenchyme (asterisk). These
distribution patterns were confirmed in at least eight sections from three biological samples. Scale bars: 40m in A; 20m in B,C, D (left), E (left);
10m in D (right), E (right).

DEVELOPMENT

elsewhere. As we have reported previously (Morimoto et al., 2010),
Clara (Fig. 3E, gray arrowhead), mesenchymal vascular smoothmuscle and endothelial cell populations (Fig. 3E, asterisk) are all
positive for N1ICD during lung development. Interestingly,
although N1ICD detection requires tyramide amplification and is
not quantitative, the signal intensity of N1ICD in peri-pNEB cells
appeared faster and appeared more intense than in Clara cells,
perhaps reflecting increased levels of Notch1 activation in thesecells (Fig. 3E, gray arrowhead). We named these SSEA-1+ peripNEB N1ICD+ CC10– cells, ‘SPNC’ cells.
NE cell establishment in the proximal airway requires Hes1,
Pofut1 or Rbpj (Ito et al., 2000; Tsao et al., 2009). This requirement
prompted several authors to hypothesize that Dll1-expressing NE
cells activate Notch signaling in their multipotent neighbors,
preventing them from assuming the NE cell fate (Ito et al., 2000;
Shan et al., 2007). If true, SPNC and NE cells may form anequivalence group. To clarify the relationship between SPNC cells

4370 RESEARCH ARTICLE

Development 139 (23)abnormal Clara/ciliated cell fate allocation but maintain normalpNEB distribution with no or only a moderate increase in NE geneproducts (Fig. 1D; Fig. 2C,E). We observed fewer SPNC cells inlungs with these genotypes (Fig. 4F,G). By contrast, loss of SPNC
cells and a dramatic increase in NE cells was seen when all three
receptors were deleted (Shh-NTKO; Fig. 4H). Thus, SPNC cells
require Notch signals and appear to be dependent on NE cells,
presumably via the Notch ligand Dll1 expressed by NE cells.
SPNC cell numbers show an inverse relationship with NE cellnumbers, which is consistent with an additive function for all
Notch receptors in NE cell regulation (Fig. 2).
Forced Notch activation increased the SPNC celllike population but did not increase Clara cellnumbers
We further attempted to express a constitutively active Notch1(N1ICD) in developing lung epithelial cells using SPC-rtTA, TetOCre; Rosa-GFP-N1ICD tri-transgenic mice (Perl et al., 2002a;
Murtaugh et al., 2003) in which GFP-N1ICD is expressed following
a doxycycline pulse. Although DOX/rtTA toxicity to Type II lungepithelial cells caused lethality at birth (Morimoto and Kopan, 2009)
and the lung epithelial structure was altered by N1ICD expression(Guseh et al., 2009), the bronchial region of the triple transgenic lungwas still distinguishable (supplementary material Fig. S6). SSEA-1+
cells were now detected throughout the bronchial epithelium at E18.5(Fig. 5A), implying that Notch1 activation is sufficient to expand a
SPNC cell-like population. Ciliated cells and pNEBs became
infrequent and their size was reduced (Fig. 5B,C). Ciliated cells thatremained were GFP negative (Fig. 5D) and (Guseh et al., 2009),

most probably reflecting incomplete targeting with the Doxinduction system. If Notch signaling was sufficient to promote Claracell fate acquisition, the tri-transgenic mice would contain more
Clara cells. However, N1ICD expression slightly decreased thefrequency of Clara cells (compare Fig. 5E with Fig. 3B and Fig. 5F).
This indicates that persistent Notch signaling blocks differentiationof both ciliated and NE cells, and may even interfere with Clara celldifferentiation while maintaining the SPNC cell-like population. The
observation of SSEA-1+, CC10+ cells suggests that some SPNC
cells may differentiate into Clara cells in the presence of N1ICD(Fig. 5E).
To determine whether the Notch2 intracellular domain could
promote Clara cell expansion, we examined the lung epithelium of
E18.5 Shh-Cre; Rosa-N2ICD mice (Fujimura et al., 2010).
Although constitutive Notch signaling altered the overallmorphology of the lung and guts, more severely than in the tritransgenic mice, many parts of the lung epithelium showed
extensive SSEA-1 immunoreactivity (Fig. 5G). Again, whereassome SSEA-1+ cells also expressed CC10, we did not see an
expansion in Clara cells. These data indicate that Notch signaling
allows other factors to specify the Clara cell fate, most likely byblocking ciliated cell fate, and further indicates that the importanceof Notch2 may be derived by its abundant expression in Clara cells
and the selective affinity between the extracellular domains of
Notch2 and Jag1 (see Discussion).
DISCUSSION
Ito et al. (Ito et al., 2000) reported that the Notch-Hes1 pathway
regulates NE cell selection during lung epithelial development, but

DEVELOPMENT

Fig. 4. Notch signals coordinate the NE/SPNC cell populations. (A-C)Immunofluorescence staining of SSEA-1 (green) and CGRP (red) in E18.5
Ascl1 heterozygous (A) or null mutant (B,C) lungs. Wild-type and Ascl1+/– lungs contain pNEBs and SPNC cells (white arrowhead), whereas NE cells
are not detected in Ascl1-null mutants. We also see a dramatic decrease in SSEA-1+ cells at the bronchus in Ascl1-null mutants, suggesting that theexistence of this marker depends on a signal from Ascl1-dependent NE cells. SSEA-1-positive cells remain in Ascl1-deficient bronchioles, suggestingthat the presence of SSEA1 antigen in this cell population is independent of Ascl1 and pNEBs. (D)Quantitative analysis of SSEA-1+ epithelial cells inthe bronchi of E18.5 wild-type, Ascl1 heterozygous and null mutant mice. At least 10 photos of the proximal airways at ⫻20 magnification fromthree biological samples for individual genotype were examined for this analysis. In the mutant, a significant decrease in the number of SSEA-1+
cells is observed. Error bars indicate s.d. P values are shown above the bars. (E-H)Immunofluorescence staining for SSEA-1 (green) and CGRP (red)in wild-type (E), SHH-Cre;N2flox/flox (F), SHH-Cre;N1flox/+;N2flox/flox (G) and SHH-Cre;N1flox/flox;;N2flox/flox;N3–/– (H) lungs. The relative expansion of the
SPNC population (green) or pNEBs (red) is represented below. Scale bars: 10m in A-C,E; 20m in F-H.

Notch proteins in lung epithelium

RESEARCH ARTICLE 4371

did not establish the cell type requiring Hes1. It has been shown thattransgenic expression of N1ICD under the control of the CGRP
promoter can inhibit Ascl1 activity and thus NE cell differentiation(Shan et al., 2007). Subsequent investigations revealed that Notch
signaling regulates the Clara/ciliated cell fate (Guseh et al., 2009;
Tsao et al., 2009; Morimoto et al., 2010) but created a controversy
regarding the role of Notch in NE cell fate selection (Tsao et al.,2009; Morimoto et al., 2010). In the present study, we analyzed aseries of Notch loss-of-function alleles, which disclosed interesting
differences between the NE and Clara/ciliated cells fate decisions.
Evidence for Notch1 activation during Clara/ciliated cell fateselection is provided by the accumulation of -secretase-generated
nuclear N1ICD polypeptides and by linage tracing (Morimoto et al.,2010), yet we show conclusively that the contribution from Notch1
is minimal. We find that Notch2 regulates the Clara/ciliated cell fatedecision, perhaps reflecting differences in abundance amplified by
the response of Fringe-modified Notch1 and Notch2 to Jag1 (Hicks
et al., 2000). Fringe inhibits Notch1 activation by Jag1, whereas
activation of Notch2 is not inhibited by Fringe. If this interpretationis correct, we predict that in L-fringe deficient mice, Notch1 will beable to rescue Clara cells in Notch2-deficient lung epithelia. Because
Hes1 null lungs show only a 50% reduction in Clara cell numbers(Ito et al., 2000), and because Clara cells disappear in Notch2-null
epithelia and Shh-RKO mice, we conclude that Notch2-mediatedsignaling acts through genes other than Hes1 in Clara cell fate
determination (Fig. 6F,G). Hes5 is not a candidate Clara cell
regulator because Hes5-null mutants have no obvious developmental
defects and show no significant differences in the profile of epithelial
differentiation or the timing of Clara cell differentiation relative tocontrols (Tsao et al., 2011).

By contrast, the size and abundance of pNEBs appear to be
regulated by an additive activity of all three Notch receptors in
SPNC cells, most likely triggered by Dll1. Shh-NTKO mice havemultiple large pNEBs that scarcely express Hes1 (Fig. 5D,E). By
contrast, Shh-Cre; N1+/f; N2f/f and Shh-RKO mice do not show asignificant reduction in Hes1 protein (P0.17 and 0.24 in Student’s
t-test) or pNEB size (Fig. 5B,C,E). Combined with the observationthat NE cell frequency is moderately increased in the Hes1
heterozygote (Ito et al., 2000), we concluded that pNEB size isdetermined by the dose of Hes1 in SPNC cells. Although Hes1 is
a well-characterized Notch target gene in some cells, it can be
regulated by other pathways (Yoshiura et al., 2007; Nakayama etal., 2008). Thus, Rbpj ablation could lead to de-repression of Hes1
by the Rbpj co-repressor complex. Alternatively, the regulation of
Hes1 by Alk5 (Xing et al., 2010) suggests that Notch proteins and
TGF signaling co-regulate Hes1 during NE cell selection in an
RBPj-independent manner. The observation that Smad4 caninteract with NICD on selected promoters (Li et al., 2011) may
explain how Rbpj-independent (non-canonical) Notch signaling
induces Hes1 expression in Shh-RKO.
What is the mechanism for controlling the abundance and size ofpNEBs? A classical Notch-mediated lateral inhibition model has
been proposed to explain the Hes1 knockout phenotype (Ito et al.,2000). In this model, a pNEB induction field is formed, which isdefined by low-level expression of Ascl1, Dll1 and Notch receptors.
Ascl1 is presumed to drive expression of Dll1, which would activate
Notch receptors on adjacent cells. Notch in turn activates Hes1,
which inhibits Ascl1 to prevent the cell from assuming the NE cellfate. We have previously shown that Notch is activated aroundprimitive ciliated cells, consistent with the expected activation pattern

DEVELOPMENT

Fig. 5. Artificial Notch1 or Notch2 activation promotes the
SPNC cell-like population but not Clara cells.
(A)Immunofluorescence staining for SSEA-1 (green) and CGRP
(red) in SPC-rtTA;TetO-Cre;Rosa-GFP-N1ICD lungs. (B,C,F) The
numbers of pNEBs (B), NE (C) and Clara cells (F) were
quantitatively evaluated in wild-type or triple-transgenic lung. Tenor more photographs of proximal airways in nine sections fromthree mice were taken at ⫻20 magnification. The numbers ofpNEBs and NE cells were determined by counting cells on the PC
display. P values are shown above the bars. (D)Anti-Foxj1 (red)and anti-GFP (green) identified a few ciliated cells in E18.5 SPCrtTA;TetO-Cre;Rosa-GFP-N1ICD mice that were most likely due toincomplete recombination induced by Dox feeding.
(E)Immunofluorescence staining for anti-CC10 (red) and antiSSEA1 (green) determined that forced Notch1 activation does
not result in Clara cell hyper-expansion. (G)Immunofluorescencestaining for SSEA-1 (green) and CC10 (red) in SHH-Cre;RosaN2ICD mice. Notch2 activation also failed to promote Clara cells.
Scale bars: 20m in A; 10m in D,E,G.

4372 RESEARCH ARTICLE

Development 139 (23)of Notch-mediated lateral inhibition (Morimoto et al., 2010). We
examined the temporal sequence of pNEB formation relative to
NICD accumulation at an early pNEB developmental stage. Ascl1
protein and the N1ICD epitope were detected at E14.5, at the onsetof lung epithelial differentiation. If SPNC and NE cells are selectedfrom within an equivalence group of bi-potential progenitors, Notch
activation would be expected to occur in the cells surrounding each
NE cell. Endothelial and vascular smooth muscle cells, which wereused as controls for the staining reaction, contained N1ICD
throughout lung development (supplementary material Fig. S7,asterisk). By contrast, N1ICD was detected only next to NE cellclusters (pNEB) at E14.5 (supplementary material Fig. S7, grayarrowhead), but not next to individual NE cells (supplementarymaterial Fig. S7, white arrowhead) located more distally. Although
we have not ruled out the possibility that Notch inhibits NE celldifferentiation via lateral inhibition and thus determines the size of
the NE induction field (Fig. 6F), the data prompted us to hypothesizethat Dll1-Notch signaling might be not involved in repressing theneighbors of a nascent NE cell, but rather is deployed after NE celldifferentiation, most likely to specify SPNC cells. Supporting thisinterpretation is the observation that selective ablation of the variant
Clara cells surrounding the adult NEB caused hyperplasia (Hong etal., 2001). In this model, SPNC cells regulate the proliferation rates
of adjacent NE cells to control the size of pNEB. The molecularmechanism underlying pNEB size regulation by Notch signaling will
be clarified in future studies.
The adult lineage derived from SPNC cells is of interest.
However, because SSEA-1 is a carbohydrate antigen on the cellsurface, we cannot label the SPNC cells in vivo with a genetic tool.
Anti-SSEA-1 immunoreactive cells disappear from peri-pNEB
epithelial cells in the adult (data not shown). It is suggested that anequivalent population of SPNC cells expresses Uroplakin3A(Upk3a), and lineage labeling of Upk3A-expressing cells during
development shows that these cells contribute to Clara and ciliated

DEVELOPMENT

Fig. 6. Hes1 expression correlates with the severity of the pNEB defect in the Notch allelic series. (A-D)Hes1 protein levels (whitearrowheads) in E18.5 wild-type (A), SHH-Cre;N1flox/+;N2flox/flox (B), SHH-Cre;Rbpjflox/flox (C) and SHH-Cre;N1flox/flox;N2flox/flox;N3–/– (D) mice wereassessed by immunofluorescence. Scale bar: 10m. Asterisks indicate NE cells. (E)Mean Hes1 intensity within the epithelium was measured using
ImageJ. Normalization was performed by dividing the mean by the intensity from Hes1-negative NE cells within the same picture. At least four
pictures from two to three biological samples were measured for each genotype. Error bars indicate s.d. P values are shown above the bars.
(F-H)The proposed regulatoy mechanisms involved in Notch-dependent cell fate determination during bronchial epithelial development, which
integrate our findings with reports from other groups. In the pseudoglandular stage, epithelial progenitors (purple) located at the distal tip produce
descendants that differentiate into Clara (pink), ciliated (green), SPNC (red) and NE (blue) cells. See the text for details.

lineages in the adult lung (Guha et al., 2012). This observation isconsistent with our previous report that many Clara and a few
ciliated cells are labeled in N1IP::CRE reporter mice (Morimoto etal., 2010). We confirmed that SPNC cells express Upk3A (data notshown). Therefore, SPNC cells become Clara and/or ciliated cells
after the pNEB size has been determined.
In conclusion, this report demonstrates that although Notch
signaling is used reiteratively to organize the major cell types
within a tissue, each step uses different receptors. Selection is either
robust (pNEB regulation, where three Notch genes act with someredundancy in SPNC cells to negatively regulate NE cells) or
sensitive to perturbations (Clara cells, where it relays only the
Notch2 receptor signal). We speculate that these differences may
explain the constant spacing and size of pNEBs, and the diverse
patterns of Clara/ciliated cells throughout the conducting airway.
Acknowledgements
We thank Tasuku Honjo for the conditional Rbpjflox/flox mice, Thomas Gridley
for the conditional Notch2flox/flox, Francois Guillemot and Yukiko Goto for Ascl1
knockout mice, and Steven Brody for anti-FoxJ1 antibody. We also thank Drs
Guha and Cardoso for sharing unpublished information and for their criticalreading of this manuscript, and T. Shen and K. Yamamoto for their generaltechnical support.
Funding
This work was supported by Grant-in-Aid for Young Scientists (A) [22890233]
and (Start-up) [23689044] of the Ministry of Education, Culture, Sports,
Science and Technology, Japan, and by the National Institutes of Health [RO1
DK066408 to R.K.]. Deposited in PMC for immediate release.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.083840/-/DC1
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Notch proteins in lung epithelium