Article

Eya1 controls cell polarity, spindle orientation, cell fate and Notch signaling in distal embryonic lung epithelium

Developmental Biology and Regenerative Medicine Program, Saban Research Institute, Childrens Hospital Los Angeles, Keck School of Medicine of University of Southern California, 4661 Sunset Boulevard, Los Angeles, CA 90027, USA.
Development (Impact Factor: 6.46). 04/2011; 138(7):1395-407. DOI: 10.1242/dev.058479
Source: PubMed
ABSTRACT
Cell polarity, mitotic spindle orientation and asymmetric division play a crucial role in the self-renewal/differentiation of epithelial cells, yet little is known about these processes and the molecular programs that control them in embryonic lung distal epithelium. Herein, we provide the first evidence that embryonic lung distal epithelium is polarized with characteristic perpendicular cell divisions. Consistent with these findings, spindle orientation-regulatory proteins Insc, LGN (Gpsm2) and NuMA, and the cell fate determinant Numb are asymmetrically localized in embryonic lung distal epithelium. Interfering with the function of these proteins in vitro randomizes spindle orientation and changes cell fate. We further show that Eya1 protein regulates cell polarity, spindle orientation and the localization of Numb, which inhibits Notch signaling. Hence, Eya1 promotes both perpendicular division as well as Numb asymmetric segregation to one daughter in mitotic distal lung epithelium, probably by controlling aPKCζ phosphorylation. Thus, epithelial cell polarity and mitotic spindle orientation are defective after interfering with Eya1 function in vivo or in vitro. In addition, in Eya1(-/-) lungs, perpendicular division is not maintained and Numb is segregated to both daughter cells in mitotic epithelial cells, leading to inactivation of Notch signaling. As Notch signaling promotes progenitor cell identity at the expense of differentiated cell phenotypes, we test whether genetic activation of Notch could rescue the Eya1(-/-) lung phenotype, which is characterized by loss of epithelial progenitors, increased epithelial differentiation but reduced branching. Indeed, genetic activation of Notch partially rescues Eya1(-/-) lung epithelial defects. These findings uncover novel functions for Eya1 as a crucial regulator of the complex behavior of distal embryonic lung epithelium.

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Available from: Saverio Bellusci
1395RESEARCH ARTICLE
INTRODUCTION
The correct functioning of lung epithelium is essential to life.
Mammalian lung development begins when two primary buds
consisting of an inner epithelial layer surrounded by mesenchyme,
arise from the laryngotracheal groove in the ventral foregut. These
buds undergo stereotypic rounds of branching and outgrowth to
give rise to a tree-like respiratory organ, which contains different
specialized epithelial cell types organized along the proximodistal
axis (Cardoso, 2000; Warburton et al., 2000; Warburton, 2008;
Metzger et al., 2008). In order to function effectively, the alveolar
surface must form a selectively permeable monolayer where cell-
cell contact provides important spatial cues that are required to
generate cell polarity/communications (Nelson, 2003a; Nelson,
2003b; Boitano et al., 2004).
Cell polarity, the asymmetry in distribution of cellular
constituents within a single cell, is fundamental to cellular
functions and essential for generating cell diversity. Epithelial cells
have a characteristic apicobasal polarity, which is necessary for
their function as barriers between different extracellular
environments (Drubin and Nelson, 1996; Mostov et al., 2000). In
epithelial cells, the axis of polarity that will determine the
orientation of the apical-basal cell division plane is defined by the
cell fate determinants (CFDs), e.g. Numb and Par proteins. Intrinsic
CFDs are asymmetrically localized in dividing cells, and
preferentially segregate into one of two sibling daughters in order
to mediate asymmetric divisions (Betschinger and Knoblich, 2004).
The regulation of spindle orientation is often associated with cell
polarity regulation in polarized cells in model organisms. The
orientation and positioning of mitotic spindles, which determine the
plane of cell division, are tightly regulated in polarized cells such
as epithelial cells by intrinsic and extrinsic cues, e.g. cell
polarity/geometry. Orientation of mitotic spindle and cell division
axis can impact normal physiological processes, including
epithelial tissue branching and differentiation (Betschinger and
Knoblich, 2004). Despite their likely importance for lung
branching, little is known about cell polarity and spindle
orientation, and factors/mechanisms that regulate these processes
are not well understood in the embryonic lung epithelium.
The Eyes Absent (Eya) proteins possess dual functions as both
protein tyrosine phosphatases and transcriptional co-activators, and
are involved in cell-fate determination and organ development
(Jemc and Rebay, 2007). In mammals, Eya1-4 and sine oculis (Six)
family genes exhibit synergistic genetic interactions to regulate the
development of many organs (Xu et al., 1997a; Xu et al., 1997b;
Ford et al., 1998; Coletta et al., 2004). Eya1
–/–
and Six1
–/–
mouse
embryos have defects in the proliferation/survival of the precursor
cells of multiple organs, and die at birth (Xu et al., 1999; Xu et al.,
2002; Li et al., 2003; Zou et al., 2004). The phosphatase function
of Eya1 switches Six1 function from repression to activation in the
nucleus, causing transcriptional activation through recruitment of
co-activators, which provides a mechanism for activation of
specific gene targets, including those regulating precursor cell
Development 138, 1395-1407 (2011) doi:10.1242/dev.058479
© 2011. Published by The Company of Biologists Ltd
1
Developmental Biology and Regenerative Medicine Program, Saban Research
Institute, Childrens Hospital Los Angeles, Keck School of Medicine of University of
Southern California, 4661 Sunset Boulevard, Los Angeles, CA 90027, USA.
2
Faculty
of Medicine, Kagawa University, Kita-gun, Japan.
*Authors for correspondence (aelhashash@chla.usc.edu; dwarburton@chla.usc.edu)
Accepted 9 January 2011
SUMMARY
Cell polarity, mitotic spindle orientation and asymmetric division play a crucial role in the self-renewal/differentiation of epithelial
cells, yet little is known about these processes and the molecular programs that control them in embryonic lung distal epithelium.
Herein, we provide the first evidence that embryonic lung distal epithelium is polarized with characteristic perpendicular cell
divisions. Consistent with these findings, spindle orientation-regulatory proteins Insc, LGN (Gpsm2) and NuMA, and the cell fate
determinant Numb are asymmetrically localized in embryonic lung distal epithelium. Interfering with the function of these
proteins in vitro randomizes spindle orientation and changes cell fate. We further show that Eya1 protein regulates cell polarity,
spindle orientation and the localization of Numb, which inhibits Notch signaling. Hence, Eya1 promotes both perpendicular
division as well as Numb asymmetric segregation to one daughter in mitotic distal lung epithelium, probably by controlling aPKCz
phosphorylation. Thus, epithelial cell polarity and mitotic spindle orientation are defective after interfering with Eya1 function in
vivo or in vitro. In addition, in Eya1
–/–
lungs, perpendicular division is not maintained and Numb is segregated to both daughter
cells in mitotic epithelial cells, leading to inactivation of Notch signaling. As Notch signaling promotes progenitor cell identity at
the expense of differentiated cell phenotypes, we test whether genetic activation of Notch could rescue the Eya1
–/–
lung
phenotype, which is characterized by loss of epithelial progenitors, increased epithelial differentiation but reduced branching.
Indeed, genetic activation of Notch partially rescues Eya1
–/–
lung epithelial defects. These findings uncover novel functions for
Eya1 as a crucial regulator of the complex behavior of distal embryonic lung epithelium.
KEY WORDS: Embryonic lung, Polarity, Eya1, Progenitor cells, Numb, Notch, Spindle orientation, Mouse
Eya1 controls cell polarity, spindle orientation, cell fate and
Notch signaling in distal embryonic lung epithelium
Ahmed HK El-Hashash
1,
*, Gianluca Turcatel
1
, Denise Al Alam
1
, Sue Buckley
1
, Hiroshi Tokumitsu
2
,
Saverio Bellusci
1
and David Warburton
1,
*
DEVELOPMENT
Page 1
1396
proliferation/survival during organogenesis (Li et al., 2003).
Although Eya1 transcriptional activity has been extensively
characterized, little is known about the targets and functions of its
phosphatase activity. Moreover, the physiological requirements for
Eya1 phosphatase activity in the lung epithelium remain obscure.
Herein, we show that Eya1 is located in the distal epithelium,
wherein it regulates cell polarity, spindle orientation, and both
aPKCz phosphorylation and Numb segregation. Interfering with
Eya1 function in vivo or in vitro results in defective cell polarity,
spindle disorientation and Numb segregation into both daughters,
as well as inactivation of Notch signaling in embryonic lung
epithelium. Furthermore, activation of Notch signaling in Eya1
–/–
distal epithelium partially rescues Eya1
–/–
embryonic lung epithelial
defects.
MATERIALS AND METHODS
Animals
Eya1
–/–
, Spc-rtTA
+/–
and Notch1 conditional transgenic (NICD) mice, and
their genotyping have been published (Xu et al., 1999; Xu et al., 2002; Perl
et al., 2002; Yang et al., 2004). Wild-type littermates were used as controls.
Conditional NICD;Eya1
+/–
female mice were generated by intercrossing
Eya1
+/–
mice with NICD mouse strain. Eya1
+/–
Spc-rtTA
+/–
tet(o) Cre
+/–
mice were generated by intercrossing Eya1
+/–
mice with Spc-rtta
+/–
tet(o)
Cre
+/+
mouse strain previously generated in our laboratory. The resulting
Eya1
+/–
Spc-rtTA
+/–
tet(o) Cre
+/–
mouse males were intercrossed with
NICD;Eya1
+/–
females to increase Notch1 activity in the distal epithelium
of mutant lungs by generating NICD-Eya1
–/–
; Spc-rtTA
+/–
tet(o) Cre
+/–
mutant mice for analysis. Pregnant NICD;Eya1
+/–
females were maintained
on doxycycline (DOX) containing food (Rodent diet with 0.0625%
Doxycycline, Harlan) from E6.5 till sacrifice. Ten compound mutant
embryos, which showed more increase of pulmonary Notch1 expression
than Eya1
–/–
littermates, were generated at expected Mendelian ratios and
examined at different stages.
Phenotype analyses, antibody staining, western blot and
immunoprecipitation
Antibody staining on paraffin sections or fixed MLE-15 cells, western blot
and immunoprecipitation were performed in triplicates using commercially
available antibodies following the manufacturers instructions and standard
protocols as described previously (Tefft et al., 2005; Tefft et al., 2002;
Buckley et al., 2005; del Moral et al., 2006a; del Moral et al., 2006b).
Briefly, for alveolar type-2 (AEC2) cells, cells were isolated from lavaged
lungs using the method of Dobbs et al. (Dobbs et al., 1986), and cultured
for 24 hours. The cells were lysed in RIPA buffer, centrifuged and the
supernatant containing ~1 mg protein was pre-cleared by incubation with
rabbit IgG and protein A/G agarose, then centrifuged. The cleared
supernatant was immunoprecipitated with 3 mg Eya1 antibody followed by
overnight incubation with protein A/G agarose, then washing before re-
suspension in electrophoresis sample buffer. The immunoprecipitate was
loaded onto Tris-glycine gel, with a lysates of AEC2 as a positive control,
and the non-specific proteins precipitated by rabbit IgG as a negative
control. The separated proteins were transferred to immobilon, and probed
overnight with a polarity protein antibody. Fluorescence intensity/protein
quantification were produced by densitometry analysis with the Image J
software as described (Carraro et al., 2009; Shigeoka et al., 2007).
Cell culture/transfection and in vitro phosphatase assay
Transfection of epithelial cells with siRNAs or Eya1 wild-type expression/
mutant (D323A) vectors and in vitro phosphatase assays were performed
following standard procedures as described previously (Carraro et al.,
2009; Cook et al., 2009; Dutil et al., 1994; Dutil et al., 1998). For siRNA
experiments, there is no change in cells of blank controls or lipofectamine
controls, and their data are not presented. The knockdown/ overexpression
efficiency was analyzed by western blot/ immunostaining of targeted
protein. In addition, we used an expression vector encoding a VP16 fusion
protein, and the transfection efficiency was further monitored by
fluorescence staining using anti-VP16 antibody. aPKCz inhibitor was used
at a concentration of 50 mmol/l, at which it is effective without displaying
cytotoxicity [as reported in different cell systems (Davies et al., 2000;
Buteau et al., 2001)].
Quantification of LGN/NuMA/Insc localization and spindle
orientation and statistical analysis
Mitotic cells and polarity orientation were identified by phospho-histone3/
pericentrin staining. Quantification of LGN/NuMA/Insc localization and
spindle orientation was performed as described previously (Lechler and
Fuchs, 2005). Statistical analysis was performed as described previously
(Carraro et al., 2009).
RESULTS
Eya1 is expressed in embryonic lung distal
epithelium and controls both cell polarity and
proper spindle orientation
Eya1 protein phosphatase is expressed in the nucleus and cytoplasm,
where it functions as a cytoplasmic protein phosphatase
(Fougerousse et al., 2002; Xiong et al., 2009). Two lines of reasoning
have led us to examine Eya1 functions in distal lung epithelial cell
polarity. First, Eya1 has a polarized (mostly apical) expression
pattern in the distal epithelial tips, particularly from E12.5-E13 (Fig.
1A,B,C), similar to polarity proteins Numb/LGN/Insc (Fig. 1E,F;
Fig. 2A,G). Second, other members of the protein phosphatase
family, e.g. protein phosphatase 2A, are crucial regulators of cell
polarity, spindle orientation and cell fate in Drosophila neural
epithelium (Ogawa et al., 2009; Wang C. et al., 2009). In this study,
E14-E14.5 was used as the developmental stage of choice to analyze
the behavior of distal epithelium because cell proliferation and
expression of progenitor cell markers Sox9, Id2 and N-myc (Mycn
– Mouse Genome Informatics) are relatively high. In addition,
Eya1
–/–
early lung development is normal and Eya1
–/–
epithelial lung
phenotype is evident at E14-E14.5, as discussed later.
The polarity proteins LGN (Gpsm2 – Mouse Genome
Informatics), NuMA (Numa1 – Mouse Genome Informatics) and
Insc regulate mitotic spindle orientation during epithelial
morphogenesis (Siller and Doe, 2009; Zheng et al., 2010).
Epithelial cells in interphase or undergoing lateral/planar divisions
have a diffuse or basolateral localization of LGN, whereas cells
undergoing perpendicular (i.e. apical-basal) divisions have LGN
only at the apical cell side (Lechler and Fuchs, 2005). In wild-type
lungs, an apical staining of anti-LGN labeling was seen at the
cortex of most mitotic cells of distal epithelial tips (Fig. 2A,A,J),
which are highly mitotic (Bishop, 2004).
In Eya1
–/–
distal epithelial tips, no apparent changes in LGN,
NuMA, Par3 and Insc expression levels were observed (Fig. 1G),
and most mitotic cells had a diffuse, basolateral or basal
localization of LGN (Fig. 2B,B,K). Closer inspection revealed that
cells with an apical localization of LGN accounted for about
86±5.0% of all mitoses in wild-type tip cells, but in Eya1
–/–
distal
epithelial tips, this number decreased markedly to about 5.0±4.0%
(Fig. 2C; P<0.05). These quantified data are further presented in
the diagrams in Fig. 2J,K, in which each dot represents the centre
of an LGN localization in a mitotic cell. Concomitantly, and as
shown in Fig. 2M, spindle orientations were overwhelmingly
lateral in Eya1
–/–
(i.e. parallel to the basement membrane), as
measured in mitotic cells at most distal epithelial tips in Eya1
–/–
compared with control lungs (Fig. 2L) and following methods
described by Lechler and Fuchs (Lechler and Fuchs, 2005).
Similarly, most Eya1
–/–
distal epithelial cells had a diffuse or
basolateral localization of NuMA and Insc, which were apically
localized in wild-type lungs (Fig. 2D-I; 87.0±6.0% versus 7±5.6%,
respectively; P<0.05), suggesting that Eya1 deletion changes cell
RESEARCH ARTICLE Development 138 (7)
DEVELOPMENT
Page 2
polarity/spindle orientation and induces lateral (i.e. planar) cell
divisions. Similarly, interfering with Eya1 functions disrupted
asymmetric localization of Par, myosin IIb (Myh10 – Mouse
Genome Informatics) and F-actin (Actg1 – Mouse Genome
Informatics) proteins (see Fig. S1 in the supplementary material).
To facilitate quantification of cells dividing perpendicularly
versus laterally, we stained E14 Eya1
–/–
distal epithelium for
centrosomes with anti-pericentrin antibody. Then, mitotic cells
were quantified based on centrosome orientation relative to the
basement membrane in order to distinguish parallel/lateral from
perpendicular spindle alignments in mitotic cells. Centrosomes that
were oriented at 0±30° to the basement membrane were classed as
parallel; those that were oriented at 90±30° were classed as
perpendicular. In Eya1
–/–
distal epithelium, most cell divisions
(87±3.0%) seemed to occur parallel/lateral to the basement
membrane, while about 12±5% mitotic cells had an alignment that
appeared perpendicular in contrast to wild-type cells where
perpendicular alignments were abundant (Fig. 2N-P).
LGN, Insc and NuMA control spindle orientation,
and Numb regulates the cell fate of lung
epithelial cells in vitro depending on Eya1
phosphatase activity
Next, we addressed whether murine LGN, Insc and NuMA functions
in the regulation of spindle orientation are conserved in the lung
epithelium, using gene-specific siRNA in the MLE-15 lung epithelial
cell line. MLE-15 cells were used in this study because of their
intense expression of different polarity proteins and progenitor/
differentiation cell markers. As in other epithelial cells (Lechler and
Fuchs, 2005), LGN, NuMA, Insc and Par3 had a mitosis-specific
polarized distribution in MLE15 cells, often localizing
asymmetrically to the cell cortex with one of the spindle poles
positioned directly below it, which indicates a perpendicular
alignment of the spindle (see Fig. S2A,B,J,K,L in the supplementary
material). Knock-down of Insc, Gpsm2 or Numa1 function caused
obvious mitotic defects, as judged by the misoriented and disrupted
morphology of mitotic spindles in transfected cells, compared with
control-siRNA-transfected cells (Fig. 3A-D and data not shown).
Conversely, Eya1 expression did not apparently change after
interfering with the function of different polarity proteins in vitro (see
Fig. S2Q-U in the supplementary material).
We next test Eya1 functions in controlling spindle-orientation-
regulatory proteins in culture. Although polarization of LGN,
NuMA and Insc in culture was more variable, it was observed in at
least 60±7% and sometimes as many as 73±6% of mitotic MLE-
15 cells (see Fig. S2I,M in the supplementary material). Upon Eya1
knockdown, LGN/Insc/NuMA/Par3 were seen at both apical and
basal cell sides or were diffuse (see Fig. S2C,D,N-P in the
supplementary material). Thus, the percent of cells with a polarized
localization of LGN/NuMA/Insc greatly decreased upon Eya1
knockdown to about 6-8%. Rescuing Eya1 function by expressing
wild-type murine Eya1 construct, not targeted by the siRNAs, into
these siRNA-transfected cells rescued the polarized distribution of
LGN/NuMA/Insc proteins (see Fig. S2I,M in the supplementary
material), while a phosphatase-dead mutant Eya1 failed to rescue
(examples are shown for LGN in Fig. S2A,C-H in the
supplementary material). This suggests that the polarized
localization of LGN/Insc/NuMA/Par, and hence proper spindle
orientations are dependent on Eya1 phosphatase activity.
The polarity protein Numb is essential in maintaining vertebrate
epithelial progenitors by allowing cells to choose progenitor over
differentiation fates, and specifies cell fate by repressing Notch
signaling (Petersen et al., 2004; Betschinger and Knoblich, 2004;
Hutterer and Knoblich, 2005). We therefore investigated Numb
functions in epithelial cell differentiation versus proliferation by
staining MLE15 cells for SP-B (Sftpb – Mouse Genome Informatics)
and Sox9, which are markers for epithelial differentiation and
progenitor cells, respectively. As shown in Fig. 3E-I, the number of
Sox9-positive cells increased fivefold (9.0±2.0% versus 50.3±5.0%,
respectively; P<0.05), while SP-B-positive differentiated cells greatly
decreased upon knockdown of Numb (60.0±4.0% versus 12.5±5.0%,
respectively; P<0.05). Moreover, Notch signaling was activated upon
1397RESEARCH ARTICLEEya1-mediated control of embryonic lung epithelium
Fig. 1. Eya1 and polarity proteins are
expressed in the lung. (A-C)
Antibody
staining shows widespread expression of
Eya1 in both lung epithelium and
mesenchyme at E11.5-E12.0 (arrowheads),
and strong polarized Eya1 signals in the
distal epithelium at E12.5-E14.0 (B,C;
arrowheads). (D-F)
Immunofluorescence
shows very week Numb expression at E11.5-
E12.0 distal epithelium (D), and strong
polarized Numb signals in the distal (inset a
in E and F; double arrowheads) rather than
proximal epithelium (inset b in E;
arrowhead) from E13-E13.5. (G)
Western
blot shows no apparent changes in the
expression of polarity proteins in E14 Eya1
–/–
lungs. Scale bars: 50
mm.
DEVELOPMENT
Page 3
1398
Numb knockdown, as indicated by increased signal/fluorescence
intensity for the Notch target genes Hes1/Hes5, and increased
number of Hes1-positive cells (Fig. 3J-P). This suggests a conserved
function for Numb in controlling cell fates and Notch signaling in
the lung epithelium.
Eya1 deletion enhances Numb expression and
phosphorylation, but inhibits its asymmetric
localization
Numb regulates cell polarity, and its phosphorylation/localization
is controlled by apically localized Par proteins during the
establishment of apical-basal polarity in mammalian epithelial
cells, which is necessary to maintain Numb asymmetric segregation
into one of the daughter cells and its function as a cell fate
determinant (Smith et al., 2007; Wang Z. et al., 2009).
The disrupted cell polarity, mislocalized Par3/6 and increased
lateral (planar) divisions in Eya1
–/–
mitotic distal epithelium (Fig.
2; see Fig. S1 in the supplementary material) raise the possibility
that Numb segregation/functions are disrupted in these cells, which
result in distribution of Numb equally to their two daughters at
cytokinesis after Eya1 deletion. To test this possibility, we first
examined Numb distribution in distal epithelial tips (Fig. 4). Numb
concentrates in the cell-cortex area overlying one of the two spindle
poles and is preferentially inherited by one of the two daughter
RESEARCH ARTICLE Development 138 (7)
Fig. 2. Eya1 deletion causes mislocalization of spindle-regulatory proteins, and increases parallel spindle alignments in mitotic distal
epithelium. (A,A,B,B,D,D,E,E,G,G,H,H) Immunofluorescence with specific antibodies shows that LGN, NuMA and Insc specifically localize to
apical cell sides of wild-type distal epithelial cells (A,A,D,D,G,G; arrowheads) and have a diffuse, basolateral or basal localization in Eya1
–/–
distal
epithelial cells (B,B,E,E,H,H; arrowheads). Broken line represents the collagen IV-stained basement membrane. A,B,D,E,G are electronic
magnifications from areas marked with asterisks in A,B,D,E,G, respectively. (C,F,I) Quantification of mitotic distal epithelial cells with apical
localization of LGN, NuMA or Insc for the experiments shown in A-H. This is expressed as a percentage of all mitotic distal epithelial cells.
*Significantly different from control (P<0.05; Student’s t-test). Error bars indicate s.e.m. (J,K)
Schematic representation of LGN localization in wild-
type (J) or Eya1
–/–
(K) distal epithelial cells. Each dot represents the centre of an LGN crescent in a single mitotic cell. (L,M)
Schematic representation
of spindle orientation in E14 wild-type (L) or Eya1
–/–
(M) distal epithelium. Each line represents the spindle axis of a single late mitotic cell.
(N,O)
Examples of distal epithelial mitotic cells that divide perpendicularly, as represented by the perpendicular orientation of pericentrin-stained
centrosomes (arrowheads/arrows) relative to the basement membrane (broken line; N), and others that have their centrosomes aligned parallel to
the basement membrane (O; arrowheads). (P)
Quantitation of the spindle orientations, which is expressed as a percentage of all divisions in the
distal epithelium, of the experiments shown in N,O for E14 wild-type/Eya1
–/–
lungs. Mitotic cells are quantified based on centrosome orientation
relative to the basement membrane in order to distinguish parallel from perpendicular spindle alignments.Bars carrying the same letter (a,b) are
significantly different from one another (*P<0.05; Student’s t-test). Data are mean±s.e.m. Scale bars: 50
mm.
DEVELOPMENT
Page 4
cells during asymmetric cell division (Knoblich et al., 1995). In
wild-type lungs, Numb was asymmetrically distributed and highly
concentrated at the apical side of distal epithelial cells with a little
or no staining at the basal pole (Fig. 4A,G). Conversely, Numb
staining markedly increased, but was diffused and localized at both
apical and basal cell poles in Eya1
–/–
distal epithelial cells (Fig.
4B,G).
Furthermore, closer inspection in mitotic cells revealed that
Numb staining is consistently concentrated as a crescent at the
apical pole of one (apical) daughter cell in 88±3% of wild-type
distal epithelial tip cells (Fig. 4C,F). Conversely, Numb seemed to
be inherited by both daughter cells in 84±6% of Eya1
–/–
mitotic
distal tip cells (Fig. 4D-F). This suggests that the more planar
(parallel) a cell division is (Fig. 2M,P), the more likely it is to
segregate Numb preferentially to both daughter cells in mitotic
Eya1
–/–
distal epithelial cells. This conclusion was further
confirmed in mitotic MLE15 cells in vitro (Fig. 4M,N). Numb
staining was cortical and started to be confined to one side of the
cell at prophase, then localized asymmetrically in metaphase/
anaphase, and was inherited by one daughter cell in anaphase/
telophase in most mitotic cells (Fig. 4M). Upon Eya1 knockdown,
Numb staining was diffuse in the cytoplasm at prophase and
became cortical later in metaphase (Fig. 4N). Numb failed to
localize asymmetrically in metaphase, and was inherited by both
daughters in anaphase/telophase in most mitotic cells (Fig. 4N).
In mammalian epithelium, phosphorylation of phosphotyrosine-
binding domain is essential for asymmetric localization of Numb
to the cortical membrane (Dho et al., 2006; Smith et al., 2007). We
therefore tested whether Numb phosphorylation changed in Eya1
–/–
lungs. Numb proteins were detected as two bands, with the higher
band representing the modified form of Numb (Rhyu et al., 1994).
If Numb phosphorylation changes, the modified form of Numb,
which is the putative phosphorylated form, will increase in
Eya1
–/–
lungs. Indeed, phosphorylated Numb increased in E14-
1399RESEARCH ARTICLEEya1-mediated control of embryonic lung epithelium
Fig. 3. Functions of polarity proteins in lung epithelium in vitro. (A,B)
Immunocytochemistry with a-tubulin antibody shows well-organized
and oriented spindle fibers (arrowheads) in MLE15 cells during early (A) and late (B) mitosis. (C,D)
Spindle fibers are disorganized/disoriented
(arrowheads) in mitotic MLE15 cells after Insc or Gpsm2 knockdown. (E-M)
Immunocytochemistry shows that MLE15-positive cells (arrowheads) for
Sox9 (F), Hes-1 (K) or Hes-5 (M) increase with strong nuclear staining, while SP-B-positive cells (H) decrease after Numb knockdown. (I)
Quantitation
of Sox9- or SP-B-positive cells, which is expressed as a percentage of all counted MLE15 cells, of the experiments shown in E-H. Bars carrying the
same letter (a,b) in I, O or P are significantly different from one another (*P<0.05; Student’s t-test). Data are mean±s.e.m. (N)
Western blot of the
experiments shown in J-M. (O)
Means fluorescence intensity of Hes-1 or Hes-5 staining for experiments showing in J-M. (P)
Quantitation of Hes-1-
positive cells, which is expressed as a percentage of all counted MLE15 cells, of the experiments shown in J-K. In O,P, Error bars indicate s.e.m. Scale
bars: 50
mm.
DEVELOPMENT
Page 5
1400
E14.5 Eya1
–/–
lungs (Fig. 4J). Moreover, phospho-Numb
immunoreactivity using phospho-Numb (Ser-295) antibody
increased in vivo (Fig. 4H-I,K) and was highest at the cell cortex
and in the nuclei of Eya1
–/–
distal epithelium (Fig. 4H-I).
Furthermore, Fig. 4L compares the mean fluorescence intensity of
phospho:total Numb of wild-type versus Eya1
–/–
distal epithelium,
showing that the phospho:total Numb was markedly altered
between wild-type and Eya1
–/–
epithelium.
Similarly, a polarized Numb signal localized to one side of the
cell was detected in MLE-15 cells in culture (Fig. 5A). Upon Eya1
knockdown, Numb was not polarized, was localized uniformly to
the cytoplasm/cell membrane as small puncta and exhibited
increased Ser295 phosphorylation (Fig. 5B,H,R). In the rescue
experiments, re-expression of wild-type Eya1, not targeted by the
siRNAs, rescued the polarized distribution and phosphorylation
level of Numb, whereas re-expression of the tyrosine-phosphatase-
dead mutant Eya1 did not (Fig. 5C,D,I,J,R). This suggests that
Numb phosphorylation is Eya1 dependent.
Recently, we reported that Eya1 controls the balance between
self-renewal and differentiation of distal epithelial cells, where
progenitor cells greatly decreased in number while differentiated
cell number increased in Eya1
–/–
embryonic lung epithelium. In
RESEARCH ARTICLE Development 138 (7)
Fig. 4. Eya1 is required for polarized apical localization and phosphorylation of Numb in distal epithelial cells. (A,B)
Immunofluorescence
for Numb shows preferential Numb localization to the apical side of distal epithelial cells in wild-type lungs (A; arrowheads). (B)
Increased Numb
expression but loss of its asymmetric localization in Eya1
–/–
distal epithelium (arrowheads). (C-E)
High magnification of wild-type/mutant mitotic
distal epithelial cells at anaphase/telophase shows that Numb localizes asymmetrically, and is inherited only by one daughter cell in wild-type lungs
(a in C). a is a daughter cell that does not inherit Numb. (D,E) Numb (arrowheads) fails to localize asymmetrically and is inherited by both daughter
cells (b,b,c,c in D and d,d,e,e in E) in Eya1
–/–
mitotic distal epithelium cells with planer/parallel (D) or perpendicular (E) divisions (E, which
represents the area marked with an asterisk in B), relative to the collagen IV-stained basement membrane (thick white broken lines in A-E).
(F) Quantification of late mitotic distal epithelial progenitors, with Numb inherited by one (1daug.) or both (2daug.) daughter cells in E14 wild-type
or Eya1
–/–
lungs. Bars carrying the same letter (a,b) in F, L are significantly different from one another (*P<0.05; Student’s t-test). Data are
mean±s.e.m. (G)
Morphometric analysis of Numb signal intensity/distribution of selected areas (thin yellow broken lines in A,B) shows loss of
polarized/asymmetric localization of Numb, which is distributed at both apical and basal sides of Eya1
–/–
distal epithelium. (H-I) Immunostaining
with specific Ser295 phospho-Numb antibody shows increased Numb phosphorylation in E14 Eya1
–/–
distal epithelium (I,I; arrowheads: staining in
the cytoplasmic side of cell membrane and in the nuclei) compared with control lungs (H,H; arrowheads). (H,I) High magnification of boxed areas
in panels H,I, respectively. (J,K)
Western blots of E14 lungs with anti-Numb (J), anti-Ser-295 phospho-Numb or anti-tyrosine phosphorylated aPKCz
antibody (K) show increased Numb/aPKCz phosphorylation in Eya1
–/–
lungs. Bars in J represent quantified western blot signals (mean±s.e.m.,
**P<0.001). Blue numbers in K represent relative band intensity. (L)
Mean fluorescence intensity of total Numb or phospho-Numb staining
compared between wild-type and Eya1
–/–
distal epithelium for experiments showing in A,B and H-I. Error bars indicate s.e.m. (M,N)
In control
mitotic MLE15 cells, Numb (arrowheads) segregated asymmetrically and was inherited by one daughter cell in anaphase/telophase. Upon Eya1
knockdown, Numb segregated to both daughters (N; arrowheads). Scale bars: 50
mm.
DEVELOPMENT
Page 6
addition, Eya1 overexpression in MLE15 cells increases Sox9-
positive progenitors cells, but decreases SP-B differentiated cells
(El-Hashash et al., 2011) (Fig. 7; see Fig. S3 in the supplementary
material), similar to Numb knockdown effects (Fig. 3E-I). We
therefore examined whether the magnitude of Eya1 effects in
balancing proliferation/differentiation of lung epithelial cells is
changed in a Numb knockdown background in vitro. As shown in
Fig. 5Q, the number of Sox9-positive progenitors increased
fivefold (9.0±5.0% versus 50±7.0%, P<0.05), while SP-B-positive
differentiated cells decreased (60.0±4.0% versus 12-14±3.0%,
respectively; P<0.05) following, respectively, Numb knockdown or
Eya1 overexpression in MLE-15 cells. Overexpression of Eya1,
together with the knockdown of Numb in MLE15 cells led to a
greater increase in the number of Sox9-positive cells (eightfold),
and a more severe decrease in the number of SP-B-positive cells
(45%; Fig. 5Q) compared with control cells.
1401RESEARCH ARTICLEEya1-mediated control of embryonic lung epithelium
Fig. 5. Eya1 regulates aPKC
z
and Numb phosphorylation. (A,B,G,H,M,N) Antibody staining of MLE15 cells shows changes of Numb
distribution/Ser295 phosphorylation (B,H; arrowheads) and aPKCz tyrosine phosphorylation (N; arrowheads) after Eya1 knockdown compared with
control cells. Arrowheads in A indicate polarized Numb staining. (C,D,I,J,O,P) Rescue of endogenous Eya1 function by co-transfection of murine siRNA
and murine wild-type or enzymatically inactive mutant Eya1 constructs (48 hours) in MLE15 cells reveals that Numb and aPKCz
distribution/phosphorylation (arrowheads) are dependent on Eya1 phosphatase activity. (E,K)
Inhibition of aPKCz in Eya1 siRNA-transfected MLE15 cells
rescues Numb distribution/Ser295 phosphorylation (arrowhead). (F,L)
Increased aPKCz tyrosine phosphorylation (arrowheads) in Eya1
–/–
distal lung
epithelium. (Q)
Quantitation of Sox9- or SP-B-positive cells, which is expressed as a percentage of all counted MLE15 cells, after interfering with the
function of Numb and/or Eya1. Bars carrying the same letter (a,b) are significantly different from the control of the same protein (*P<0.05; ANOVA-
Dunnett test). Data are mean±s.e.m. (R)
Western blot of Numb, phospho-Numb or phospho-aPKCz for experiments showing in A-E,G-K,M-P. Bar
graphs represent quantified western blot signals (mean±s.e.m.). Bars carrying the same letter (a,b,c) are significantly different from the control of the
same protein (**P<0.001; ANOVA-Dunnett test). (S)
Endogenous Eya1 was immunoprecipitated from AEC2 cells with a specific Eya1 antibody and
western blotting was performed with antibodies specific to different polarity proteins. Anti-Eya1IP of Eya1 siRNA-transfected cells was used as a control.
(T)
siRNA knockdown of endogenous aPKCz or different polarity proteins in epithelial cells (48 hours) and subsequent IP for Eya1 and western blot for
different polarity proteins. (U)
In vitro phosphatase assay using immunopurified wild-type Eya1 or enzymatically inactivated mutant protein (Eya1
D323A) and aPKCz protein. Graph represents quantified western blot signals normalized to input (n3; mean±s.e.m., **P<0.001). Scale bars: 50
mm.
DEVELOPMENT
Page 7
1402
Eya1 is essential for atypical protein kinase C
z
(aPKC
z
) phosphorylation
Eya1 has well-known tyrosine phosphatase activities (Li et al.,
2003). As Numb phosphorylation increased in Eya1
–/–
lungs on
Ser295 residue, which is phosphorylated by aPKCz leading to
Numb asymmetric localization (Smith et al., 2007), we therefore
tested whether tyrosine phosphorylated aPKCz is a direct substrate
for Eya1 phosphatase. aPKCz activity that is probed by a tyrosine
phosphorylated aPKCz antibody increased both in vivo at the cell
cortex of Eya1
–/–
distal epithelium, similar to Numb (Fig. 4K; Fig.
5F,L), and after Eya1 knockdown in MLE15 cells in vitro (Fig.
5N,R). Rescuing Eya1 function by expressing wild-type murine
Eya1 construct, not targeted by the siRNAs, into these Eya1siRNA-
transfected cells led to near-control level of phospho-aPKCz,
whereas re-expression of the tyrosine-phosphatase-dead mutant
Eya1 did not (Fig. 5O,P,R). This suggests that increased Numb
phosphorylation is likely to be due to the increased aPKCz
activity/phosphorylation in Eya1
–/–
lungs. This conclusion was
confirmed by inhibiting aPKCz activity in Eya1siRNA-transfected
MLE15 cells, which rescued the polarized distribution and
phosphorylation level of Numb (compare Fig. 5A,B,G,H with
5E,K,R).
We next assessed Eya1 phosphatase activity on aPKCz by co-
immunoprecipitation. The endogenous aPKCz forms a complex
with Par3/Par6/Numb, which binds to LGN/Insc/NuMA in
epithelial cells (Lechler and Fuchs, 2005; Suzuki and Ohno,
2006; Nishimura and Kaibuchi, 2007). Expectedly, Eya1 co-
immunoprecipitated aPKCz and other polarity proteins in AEC2
cell lysate (Fig. 5S). To determine whether Eya1 binds to aPKCz-
Par-Numb/polarity protein complex by binding to aPKCz, we
performed Eya1/aPKCz co-immunoprecipitation studies and
analyzed other polarity proteins in cells treated with aPKCzsiRNA.
Indeed, co-immunoprecipitation of Eya1, Numb, Par/polarity
proteins was not observed after aPKCz knockdown, but was
observed after knocking down Numb or other polarity proteins
(Fig. 5T).
RESEARCH ARTICLE Development 138 (7)
Fig. 6. Inhibition of Notch signaling in Eya1
–/–
lung distal epithelium. (A-F)
Immunohistochemistry with specific antibodies shows reduced
staining of activated-Notch1 (B), Hes1 (D) and Hes5 (F) in E14 Eya1
–/–
distal epithelium (arrowheads) compared with control lungs (A,C,E;
arrowheads). (G)
Western blots show reduction of activated-Notch1 and Hes-5 in E14 Eya1
–/–
lungs. (H)
Western blot of activated-Notch1 for
experiments shown in I-M. Blue numbers in G,H,S represent relative band intensity. (I,J)
Immunocytochemistry shows reduced activated-Notch1
expression in MLE-15 after Eya1 knockdown. (K,L)
Rescue of endogenous Eya1 function by co-transfection of murine siRNA and murine wild-type
or enzymatically inactive mutant Eya1 constructs for 48 hours in MLE15 cells reveals that Notch1 signaling/activity is dependent on Eya1
phosphatase activity. (M)
Inhibition of aPKCz in Eya1 siRNA-transfected MLE15 cells rescues activated-Notch1 expression. (N)
Mean fluorescence
intensity of Hes1 staining for experiments showing in O-R. *Significantly different from control (P<0.05; ANOVA-Dunnett test). Error bars indicate
s.e.m. (O-Q)
Immunocytochemistry of MLE-15 cells shows decreased Hes1 expression after Eya1 knockdown (O,P), but increased Hes-1 expression
upon Eya1 overexpression (Q; arrowheads). (R)
Hes1-positive cells with strong nuclear staining further increase after co-transfection of Numb siRNA
and wild-type Eya1 expression vector in MLE-15 cells (arrowheads). (S)
Western blot of Hes1 for experiments showing in O-R. (T)
Quantitation of
Hes1-positive cells, which is expressed as a percentage of all counted MLE15 cells, of the experiments shown in O-R. *Significantly different from
control (P<0.05; ANOVA-Dunnett test). Data are mean±s.e.m. Scale bars: 50
mm.
DEVELOPMENT
Page 8
To further determine whether aPKCz tyrosine phosphorylation
might be a target of Eya1 phosphatase activity, we performed an
in vitro phosphatase assay, mixing aPKCz protein with
immunopurified HA-tagged Eya1. As shown in Fig. 5U, wild-type
Eya1 significantly inhibited aPKCz phosphotyrosine, while the
phosphatase-inactive mutant protein had no significant effect.
Inhibition of Notch signaling in Eya1
–/–
lung distal
epithelium
Numb functions as a negative regulator of Notch in mammals and
Drosophila (French et al., 2002; Cayouette and Raff, 2002), and
inactivated Notch1 signaling in MLE15 lung epithelial cells (Fig.
3J-P). As Eya1 controlled Numb segregation/function (Figs 4, 5),
we next investigated whether Eya1 also regulates Notch signaling
in distal lung epithelium.
Signals for activated (cleaved) Notch1 and for its downstream
transcriptional targets Hes1 and Hes5 were strong in wild-type
distal epithelium, but greatly decreased in Eya1
–/–
distal epithelium
(Fig. 6A-F). This was also shown by immunoblot analysis (Fig.
6G). Similarly, activated Notch1 expression decreased after Eya1
knock-down in MLE-15 cells (Fig. 6I,J,H). Rescuing Eya1 function
by expressing wild-type murine Eya1 construct, not targeted by the
siRNAs, into these siRNA-transfected cells rescued the expression
levels of activated-Notch1, while a phosphatase-dead mutant Eya1
1403RESEARCH ARTICLEEya1-mediated control of embryonic lung epithelium
Fig. 7. Genetic activation of Notch signaling in Eya1
–/–
lungs partially rescues epithelial progenitor defects and branching phenotype.
(A,B,D-F) External appearance (A,B) and histological analysis (D-F) of control versus Eya1
–/–
lungs show reduced epithelial branching and size of
Eya1
–/–
lungs, which are restored in NICD; Spc-rtTA
+/–
-tet(O) Cre
+/–
Eya1
–/–
compound mutant lungs (A,B,F). The last panel in B is high magnification
of the yellow boxed area. (C)
Quantitation of Sox9- or SP-B-positive cells, which is expressed as a percentage of all counted MLE15 cells, after
interfering with the function of Notch1 and/or Eya1. *Bars carrying the same letter (a,b) are significantly different from the control of the same
protein (P<0.05; ANOVA-Dunnett test). Data are mean±s.e.m. (G,H,L,M,Q,R,U,V) Specific antibody staining shows similar polarized localization of
polarity proteins (arrowheads) between compound mutant and wild-type lung distal epithelium. Broken lines represent the collagen IV-stained
basement membranes. (I-K,N-P) Immunohistochemistry on serial sections shows reduced expression of progenitor markers Sox9 and Id2 in E14.5
Eya1
–/–
distal epithelium compared with control lungs (arrowheads). (K,P)
Sox9/Id2 expression is substantially rescued in NICD; Spc-rtTA
+/–
-tet(O)
Cre
+/–
Eya1
–/–
lungs (arrowheads). (S,T)
Western blots show changes of the expression of Sox9, Id2 and SP-C between wild-type, Eya1
–/–
and
compound mutant lungs. Blue numbers represent relative band intensity. Scale bars: 50
mm.
DEVELOPMENT
Page 9
1404
failed to rescue (Fig. 6K,L,H). Interestingly, inhibition of aPKC
activity in Eya1siRNA-transfected cells led to near-Eya1 wild-type
transfected level of activated Notch1 (Fig. 6K,M,H).
To determine whether Numb is involved in Eya1 control of
Notch signaling in the lung epithelium, we tested whether the
magnitude of Eya1 effects on Notch activity in MLE15 cells
changes in a Numb knockdown background. As shown in Fig.
6N,R,S,T, Hes1 nuclear signal levels and Hes1-positive cells
greatly increased, and were higher than Hes1 signals/number after
either Eya1 overexpression (Fig. 6Q,N,S,T) or Numb knockdown
(Fig. 3J,K,N-P), carried out separately in MLE15 cells.
Genetic activation of Notch signaling in Eya1
–/–
lungs partially rescues epithelial progenitor
defects and branching phenotype
Notch signaling promotes progenitor cell identity at the expense of
differentiated cell phenotypes (Jadhav et al., 2006; Mizutani et al.,
2007). It also controls cell fates in developing airways (Tsao et al.,
2009), while Notch activation inhibits the differentiation of distal
lung progenitors into alveolar cells (Guseh et al., 2009). Loss
of epithelial progenitors from E14-E14.5, reduced epithelial
branching/ lung size and increased epithelial differentiation are
major Eya1
–/–
lung phenotypes (Fig. 7A-E,I,J,N,O,S,T; see Fig. S3
in the supplementary material) (El-Hashash et al., 2011). We
therefore tested the hypothesis that inactivation of Notch signaling
causes the epithelial defects in Eya1
–/–
embryos by conditional
genetic increase of Notch1 levels in Eya1
–/–
lung epithelium, using
NICD; Spc-rtTA
+/–
tet(O) Cre
+/–
Eya1
–/–
compound mutant mice. No
changes in lung phenotype or gene/protein expression were evident
in controls: DOX-fed Spc-rtTa and Spc-rtTa-tet(O) Cre mice (data
not shown).
NICD; Spc-rtTA
+/–
tet(O) Cre
+/–
Eya1
–/–
compound mutant lungs
were comparable with doxycycline-untreated control lungs, albeit
smaller in size (Fig. 7A). Following induction with DOX feeding,
they showed increased lung size and restoration of both epithelial
branching and expression of distal epithelial progenitor markers
compared with lungs of Eya1
–/–
littermates (Fig. 7A-F,I-K,N-P,S,T;
see Fig. S3A-C in the supplementary material). Moreover, the
polarized cortical localization of polarity proteins (Fig.
7G,H,L,M,Q,R,U,V) and the expression levels of distal epithelial
differentiation markers (Fig. 7S,T; Fig. S3D-I in the supplementary
material) were restored into the wild-type control range in
compound mutant lungs versus Eya1
–/–
lungs, suggesting partial
but substantial rescue of the Eya1
–/–
hypoplastic lung phenotype.
Finally, we examined whether the magnitude of Eya1 effects in
balancing proliferation/differentiation of lung epithelial cells is
blunted in a Notch1 knockdown background in MLE-15 cells. As
shown in Fig. 7C, Notch1 knockdown reduced Sox9-positive
progenitors (60%; 10±3.0% versus 4.2±3.0%), but increased SP-B-
positive differentiated cells (61.0±3.0% versus 72±4.0%,
respectively; P<0.05). By contrast, the number of Sox9-positive
progenitors increased fivefold, while SP-B-positive cells greatly
decreased (78%) upon Eya1 overexpression. These changes were
blunted and the percentage of cells that are positive for Sox9 or SP-
B was restored into the control range in cells co-transfected with
Numb siRNA and wild-type Eya1 expression vector versus Eya1-
overexpressing cells alone (Fig. 7C).
DISCUSSION
The function and growth of pulmonary epithelial cells lining the
distal tubes/air sacs depend on their polarity, which its loss is
involved in lung cancers, chronic obstructive pulmonary disease
and disruption of lung epithelial differentiation (Matsui et al., 1999;
Xu et al., 2006). Yet, cell polarity remains uncharacterized in lung
epithelium. Herein, we demonstrate that distal lung epithelium,
which represents the epithelial progenitor pool (Rawlins et al.,
2009), is polarized with characteristic perpendicular divisions that
are controlled by Eya1 phosphatase.
Eya1 controls cell polarity and mitotic spindle
orientation in embryonic distal lung epithelium
Mammalian Eya1 protein phosphatase has been implicated in cell
polarity, because progressive Eya1 depletion results in the loss of
polarity in hair cells during inner ear development (Zou et al.,
2008). Here, we have extended these observations to the lung to
demonstrate that Eya1 is crucial for the maintenance of cell polarity
and mitotic spindle orientation of distal epithelium. Eya1
–/–
distal
epithelial cells exhibited a severe perturbation in the asymmetric
localization and organization of several polarity proteins (Figs 2, 4
and 5; see Figs S1, S2 in the supplementary material). Similarly,
members of protein phosphatase family are crucial regulators of
cell polarity and spindle orientation in Drosophila epithelial cells
(Wang et al., 2009; Ogawa et al., 2009).
How does Eya1 protein function to maintain cell polarity and
control mitotic spindle orientation? From the present study, Eya1
appears to exert this effect by influencing multiple processes,
including the apical cell localization of Par, Insc, LGN and NuMA
proteins, which are evolutionarily conserved and essential for the
formation of cell polarity/spindle orientation, as well as aPKCz/
Numb phosphorylation (discussed below). In mammalian
epithelium, the Par3/6 proteins localize predominantly to apically
located tight junctions and bind to aPKCz, Insc and LGN. This
binding is crucial for the establishment of epithelial polarity and
for apical-basal/perpendicular spindle orientation (Macara, 2004;
Suzuki and Ohno, 2006; Siller and Doe, 2009). Thus, the proper
localization of aPKCz-Par3/6-LGN-Insc polarity complex is crucial
for cell polarization (Ohno, 2001). Our findings that Eya1 may
bind to aPKCz and that Eya1 deletion causes mislocalization of
Par/Insc/LGN, together with increased planar cell divisions at the
expense of perpendicular/apical-basal division (Figs 2, 5; see Figs
S1, S2 in the supplementary material), provide strong evidence that
Eya1 is indeed required for controlling cell polarity and spindle
orientation in the embryonic lung.
Eya1 regulates Numb segregation and Notch
signaling in distal lung epithelium
Notch signaling is used for cell fate determination throughout the
animal kingdom, and differences in Notch activity between two
daughter cells determine their future fates. Thus, Notch signaling
promotes progenitor cell identity at the expense of differentiated cell
phenotypes (Jadhav et al., 2006; Mizutani et al., 2007). Differences
in the Notch activities between two daughter cells can be specified
by the asymmetric localization and inheritance of Numb, a negative
regulator of the Notch pathway (Guo et al., 1996; Cayouette et al.,
2001; Petersen et al., 2002; Shen et al., 2002). In the embryonic lung,
Notch signaling controls cell fates in developing airways (Post et al.,
2000; Tsao et al., 2008; Tsao et al., 2009), and Notch activation
inhibits the differentiation of distal progenitors into alveolar cells
(Guseh et al., 2009). Yet the role of asymmetric segregation of cell
fate determinant/Notch inhibitor Numb during lung development,
and the way the process might be regulated are still unknown.
Herein, the failure of polarized Numb localization after Eya1
knockout/knockdown (Figs 4, 5) supports our conclusion that one
of the principal functions of Eya1 is the regulation of asymmetric
RESEARCH ARTICLE Development 138 (7)
DEVELOPMENT
Page 10
Numb localization/segregation in mitotic lung epithelium. This
is further confirmed by our finding that Eya1 phosphatase
controls aPKCz phosphorylation, which is essential for Numb
phosphorylation and asymmetric localization/segregation (Dho et
al., 2006; Smith et al., 2007), as reported for other phosphatases
(Nunbhakdi-Craig et al., 2002). Indeed, aPKCz-dependent
phosphorylation of Numb inhibits its cortical/polarized localization
(Casanova, 2007). Increased Numb expression in Eya1
–/–
epithelium provides further evidence, because Numb localization
is also inhibited upon overexpression of the protein, presumably as
a result of saturation of the localization machinery (Rhyu et al.,
1994). Upon overexpression, Numb is segregated into both
daughter cells that then adopt the fate of the daughter that normally
inherits Numb (Rhyu et al., 1994). Moreover, mislocalization and
perturbation of Par3/6 and myosin IIb, together with inactivation
of Notch signaling, in Eya1
–/–
lungs further support our hypothesis
of Eya1 control of Numb segregation/expression, because myosin
IIB and Par proteins regulate Numb asymmetric segregation/
localization (Barros et al., 2003; Betschinger and Knoblich, 2004).
Moreover, high levels of Notch activation cause a reduction in
Numb protein levels (Chapman et al., 2006).
Furthermore, the lack of polarized Numb localization, and
consequently loss of the difference in Numb levels between two
daughter cells (both inherit Numb) may be responsible for the
failure of Eya1
–/–
cells to upregulate Notch signaling pathway and
hence to execute the epithelial progenitor cell self-renewal program
at distal tips. This may explain enhanced epithelial differentiation
and the great reduction of both Notch activity and expression of
epithelial progenitor cell markers in the Eya1
–/–
lung (Figs 4-7; see
Fig. S3 in the supplementary material). Indeed, Numb inheritance
in daughter cells acts to inhibit Notch signaling (Chapman et al.,
2006). Consistent with our results, Eya1 abrogation inhibits Notch
signaling during sensory progenitor development in mammalian
inner ear (Zou et al., 2008), whereas high levels of Eya1 inhibit
neuronal differentiation, but expand the pool of proliferative
neuronal progenitors (Schlosser et al., 2008). Our findings that
genetically increasing Notch activity in Eya1
–/–
lungs substantially
rescues the abnormal lung epithelial phenotype in vivo (Fig. 7; see
Fig. S3 in the supplementary material) provide strong evidence that
Eya1 is indeed required for controlling Notch signaling activity to
ensure appropriate self-renewal/differentiation of lung distal
epithelium. Whether Eya1 directly or indirectly regulates Notch
signaling will be the subjects of future study.
In our future studies, we plan to use a conditional knockout
approach to delete Eya1 specifically from the epithelial
compartment to further investigate its specific functional roles in
epithelial cell development. Nonetheless, the Eya1 mutants
reported herein provide a new mouse model for congenital lung
hypoplasia/malformations and help us to understand the
mechanisms that control lung epithelial morphogenesis.
Does distal lung epithelium divide
asymmetrically? Does Eya1 phosphatase control
asymmetric division in the lung?
Recent reports suggest that undifferentiated lung epithelial
progenitors undergo multiple division-linked cell fate decisions
[symmetric and asymmetric cell division (ACD)] that lead to an
apparently homogeneous expansion of the progenitor cell
population (Rawlins, 2008; Lu et al., 2008). No reports about ACD
in the embryonic lung have appeared as yet to our knowledge, but
our study provides some evidence to suggest that distal lung
epithelial cell populations that contain progenitor cells (Rawlins et
al., 2009) divide asymmetrically. For example, most of the distal
epithelial cells had apically localized Insc, LGN, NuMA and Par
proteins, with mitotic spindles aligned perpendicular to the
basement membrane and a characteristic asymmetric segregation/
inheritance of Numb (Figs 1, 2; see Fig. S1 in the supplementary
material). Indeed, a strict correlation exists between ACD and the
apical localization of these polarity proteins, perpendicular
alignment of mitotic spindles and asymmetric Numb segregation
in Drosophila/mammalian epithelium (Cayouette and Raff, 2002;
Cayouette and Raff, 2003; Haydar et al., 2003; Noctor et al., 2004;
Lechler and Fuchs, 2005). In this regard, our data suggest a crucial
role for Eya1 in controlling ACD, similar to other phosphatases
(Wang et al., 2009; Ogawa et al., 2009), because Eya1 abrogation
perturbed the organization of polarity proteins and spindle
orientation, as well as Numb segregation in distal embryonic lung
epithelium, providing a conceptual framework for future
mechanistic studies in this area.
Acknowledgements
We thank Drs M. Rosenfeld, R. Hegde, P. Nare and K. Kiyosh for Eya1
constructs/proteins. This study was funded by NIH-NHLBI P01 HL 60231, RO1s
HL 44060, HL44977 and GM grants, and by a CIRM grant to D.W. and A.H.E.-
H. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.058479/-/DC1
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  • Source
    • "Eya1 is a multifunctional protein and a possible target of RTK signaling [35]. Furthermore, Eya1 has recently been shown to be involved in controlling cell polarity, cell fate, orientation of the mitotic spindle and notch signaling [36] . Hence, these mutations may compromise some other unknown or some or all of these recently identified functions of Eya1. "
    [Show abstract] [Hide abstract] ABSTRACT: Mutations in the human EYA1 gene have been associated with several human diseases including branchio-oto (BO) and branchio-oto-renal (BOR) syndrome, as well as congenital cataracts and ocular anterior segment anomalies. BOR patients suffer from severe malformations of the ears, branchial arches and kidneys. The phenotype of Eya1-heterozygous mice resembles the symptoms of human patients suffering from BOR syndrome. The Eya1 gene encodes a multifunctional protein that acts as a protein tyrosine phosphatase and a transcriptional coactivator. It has been shown that Eya1 interacts with Six transcription factors, which are also required for nuclear translocation of the Eya1 protein. We investigated the effects of seven disease-causing Eya1 missense mutations on Eya1 protein function, in particular cellular localization, ability to interact with Six proteins, and protein stability. We show here that the BOR-associated Eya1 missense mutations S454P, L472R, and L550P lead to enhanced proteasomal degradation of the Eya1 protein in mammalian cells. Moreover, Six proteins lead to a significant stabilization of Eya1, which is caused by Six-mediated protection from proteasomal degradation. In case of the mutant L550P, loss of interaction with Six proteins leads to rapid protein degradation. Our observations suggest that protein destabilization constitutes a novel disease causing mechanism for Eya1.
    Full-text · Article · Jan 2014 · PLoS ONE
  • Source
    • "In harmony with these results, we previously reported decreased cell proliferation, but increased differentiation of distal epithelial cells/primitive alveoli in Eya1 À / À or Six1 À / À lungs at E18.5 (ElHashash et al., 2011a, b). This is probably because of loss of the balance between self-renewal and differentiation (El-Hashash et al., 2011c). The increase of SMA-positive cells prompted us to analyze changes in mesenchymal/fibroblast cell differentiation in order to further characterize the abnormally thick parenchyma of mutant lungs. "
    [Show abstract] [Hide abstract] ABSTRACT: The Eya1 gene encodes a transcriptional co-activator that acts with Six1 to control the development of different organs. However, Six1-Eya1 interactions and functional roles in mesenchymal cell proliferation and differentiation as well as alveolarization during the saccular stage of lung development are still unknown. Herein, we provide the first evidence that Six1 and Eya1 act together to regulate mesenchymal development as well as alveolarization during the saccular phase of lung morphogenesis. Deletion of either or both Six1 and Eya1 genes results in a severe saccular phenotype, including defects of mesenchymal cell development and remodeling of the distal lung septae and arteries. Mutant lung histology at the saccular phase shows mesenchymal and saccular wall thickening, and abnormal proliferation of α-smooth muscle actin-positive cells, as well as increased mesenchymal/fibroblast cell differentiation, which become more sever when deleting both genes. Our study indicates that SHH but not TGF-β signaling pathway is a central mediator for the histologic alterations described in the saccular phenotype of Eya1(-/-) or Six1(-/-) lungs. Indeed, genetic reduction of SHH activity in vivo or inhibition of its activity in vitro substantially rescues lung mesenchymal and alveolar phenotype of mutant mice at the saccular phase. These findings uncover novel functions for Six1-Eya1-SHH pathway during the saccular phase of lung morphogenesis, providing a conceptual framework for future mechanistic and translational studies in this area.
    Full-text · Article · Jul 2013 · Developmental Biology
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    • "Thus, CDC42 is a critical regulator of cell polarity in the developing lung and is required for spatial coordination of nuclear and/or cell positioning during cell proliferation. Recently loss of the EYA1 protein disrupted epithelial cell polarity and mitotic spindle orientation, resulting in abnormal lung development (El-Hashash et al., 2011). Significantly, changes in mitotic spindle orientation/angle were also demonstrated in the CDC42 deficient lung, suggesting that loss of CDC42 alters directional cell division, resulting in abnormal branching of the developing lung. "
    Full-text · Article · Feb 2013 · Developmental Biology
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