Dlg5 maintains apical aPKC and regulates progenitor differentiation during
Tamilla Nechiporuka,n, Olga Klezovitcha, Liem Nguyena, Valeri Vasioukhina,b,c,nn
aDivision of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, C3-168, Seattle, WA 98109, USA
bDepartment of Pathology, University of Washington, Seattle, WA 98195, USA
cInstitute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA
a r t i c l e i n f o
Received 31 August 2012
Received in revised form
21 February 2013
Accepted 22 February 2013
Available online 4 March 2013
a b s t r a c t
Cell polarity plays an important role in tissue morphogenesis; however, the mechanisms of polarity and
their role in mammalian development are still poorly understood. We show here that membrane-
associated guanylate kinase protein Dlg5 is required for proper branching morphogenesis and
progenitor differentiation in mammalian lung. We found that during lung development Dlg5 functions
as an apical–basal polarity protein, which is necessary for the apical maintenance of atypical protein
kinase C (aPKC). These results identify Dlg5 as a regulator of apical polarity complexes and uncover the
critical function of Dlg5 in branching morphogenesis and differentiation of lung progenitor cells.
& 2013 Elsevier Inc. All rights reserved.
Cell polarity is important for proper morphogenesis of all
mammalian organisms; however, the mechanisms of cell polarity
and, even more importantly, the particular role and significance of
these mechanisms in the major developmental events are still
poorly understood. Significant knowledge about the proteins
involved in apical–basal cell polarity was generated using such
(McCaffrey and Macara, 2012; Wodarz and Nathke, 2007). These
studies identified atypical PKC (aPKC)/Par3/Par6 proteins as
critical members of the apical cell polarity machinery, which
localize to the apical membrane domain and are necessary for the
establishment and maintenance of the apical membrane domain
identity (McCaffrey and Macara, 2009b). In contrast, the Par1,
Par4, Dlg, Lgl and Scribble proteins localize to the basolateral
membrane domain and are required for basolateral domain
formation and maintenance (Yamanaka and Ohno, 2008). In
general, the function and the mechanisms of the apical membrane
polarity complexes aPKC/Par6/Par3 are understood much better
than the function and the mechanisms of the basolateral polarity
proteins. Par3 and Par6 are the PDZ (PSD95/Dlg/ZO1) domain-
containing molecular adapter and scaffold proteins, which bind to
aPKC, the only enzyme in the apical polarity complex (McCaffrey
and Macara, 2009b). aPKC phosphorylates and negatively regu-
lates the function of Par1 and Lgl basolateral polarity proteins
(Betschinger et al., 2003; Hurov et al., 2004). Reciprocally, Par1
phosphorylates and negatively regulates the membrane associa-
tion and cell polarity function of Par3 (Benton and St. Johnston,
2003). Dlg is an essential basolateral polarity gene, which geneti-
cally interacts with Lgl and Scribble in Drosophila (Bilder et al.,
2000; Woods and Bryant, 1991). Dlg is a member of the Mem-
brane Associated Guanylate Kinase (MAGUK) proteins. The func-
tional role of Dlg in the regulation of cell polarity remains
obscure; however, MAGUK proteins usually function as protein
scaffolds that help to cluster multiple transmembrane and acces-
sory proteins to hold together the elements of individual signaling
pathways, and it is likely that Dlg performs similar function at the
lateral membrane domain (Yamanaka and Ohno, 2008).
Dlg5 is conserved throughout the Metazoan evolution gene
that differs from the Drosophila dlg and mammalian Dlg1-4
because in addition to guanylate kinase and PDZ domains, it
contains N-terminal CARD and coiled coil domains (Nechiporuk
et al., 2007). Function of Dlg5 in Drosophila has not been
investigated. Polymorphism in human Dlg5 protein sequence is
associated with predisposition to the Crohn’s disease: however,
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nCorresponding author. Current address: OHSU, Vollum Institute, Portland,
OR 97239, USA.
nnCorresponding author at: Division of Human Biology, Fred Hutchinson Cancer
Research Center, 1100 Fairview Avenue North, C3-168, Seattle,
WA 98109, USA. Fax: þ1 206 667 6524.
E-mail addresses: email@example.com (T. Nechiporuk),
firstname.lastname@example.org (V. Vasioukhin).
Developmental Biology 377 (2013) 375–384
the mechanisms of Dlg5 in Crohn’s disease are not well under-
stood (Stoll et al., 2004). In renal and mammary epithelial cell
lines, knockdown of Dlg5 activates cell migration and promotes
TGF-b-mediated epithelial–mesenchymal transition (Sezaki et al.,
2012; Smolen et al., 2010). To determine the physiological
function of Dlg5 in mammalian organism, we have previously
generated and analyzed Dlg5?/?mice (Nechiporuk et al., 2007).
We found that Dlg5?/?mice develop brain hydrocephalus and
kidney cysts. Biochemical analysis revealed an important function
of Dlg5 in facilitating the delivery of N-cadherin to the plasma
membrane (Nechiporuk et al., 2007).
In this study we analyzed the role of Dlg5 in developing
mammalian lung. The mammalian lung is one of the best-
studied examples of a developing organ that undergoes the highly
coordinated process of branching morphogenesis coupled with
timely progenitor cell differentiation. Together, these events
result in the formation of an organ containing branched airways
that terminate in millions of functional alveolar sacs enabling
adequate lung function (Metzger et al., 2008). Failure of proper
lung development can result in neonatal death or chronic
pulmonary disease, which is often associated with the enlarge-
ment of peripheral airspaces (Bourbon et al., 2009; Snider, 1992).
We show here that Dlg5 is required for proper mammalian lung
morphogenesis as Dlg5?/?mice display abnormal branching
morphogenesis and differentiation of lung progenitor cells and
develop completely penetrant lung airspace enlargement and
emphysema-like phenotype. We demonstrate that Dlg5?/?lung
epithelial cells display prominent apical–basal polarity defects,
which may be responsible for the defects in branching and
Failure of normal lung morphogenesis and emphysema-like
phenotype in Dlg5?/?mice.
We previously reported that approximately half of the
Dlg5?/?mice die perinatally (Nechiporuk et al., 2007). The
analysis of the surviving Dlg5?/?adults revealed prominent and
completely penetrant lung abnormalities. Therefore, here we
specifically focused on the analysis of the role of Dlg5 in murine
lung morphogenesis. Histological examination of adult lungs
demonstrated an emphysema-like phenotype in Dlg5?/?mice
with prominent dilatation of the distal airspaces and an overall
decrease in number of alveolar septa (Fig. 1A–B’). To assess the
origin of these morphological defects, we performed macroscopic
and histological analyses of the lungs from Dlg5?/?and wild-type
mice at different times of postnatal development. Similar to adult
Dlg5?/?animals, newborn Dlg5?/?pups displayed enlarged
distal airspaces that contained few alveolar septa and presented
with areas of collapsed lung parenchyma (Fig. 1C–D’, arrows). The
macroscopic analyses of 1-day-old (P1) lungs also revealed the
prominent enlargement of distal airspaces in Dlg5?/?
Since lung defects were already present in newborn mutants,
we histologically examined the lungs of Dlg5?/?and wild-type
mice at different times during embryonic development. We found
that the lung branching pattern was indistinguishable between
wild-type and Dlg5?/?embryos at E12.5. However, starting from
E13.5 and throughout the subsequent embryogenesis, 100%
Dlg5?/?lungs showed a significant decrease in the number,
accompanied by a prominent increase in the size of terminal
tubules within the developing lung (Fig. 1G–K’). We conclude that
the initial onset of lung abnormalities in Dlg5?/?mice occurs
between E12.5 and E13.5 of development.
Dlg5 is required for lung branching morphogenesis
Between days E9.5 and E16.5 of lung development (pseudo-
glandular stage) primary buds undergo the highly coordinated
process of branching morphogenesis that results in the formation
of tree-like structures that end with multiple terminal tubules
(Morrisey and Hogan, 2010). The histological abnormalities
observed in the Dlg5?/?lungs were consistent with a potential
defect in branching morphogenesis. To study branching, we
analyzed fixed lungs using macroscopic examination and whole-
organ immunostaining with anti-E-cadherin antibodies, which
facilitates 3-dimensional visualization of the bronchial tree
(Metzger et al., 2008). While the comparison of lungs from the
Dlg5?/?and wildtype littermate embryos revealed little differ-
ence at E12 and E12.5, prominent defects in branching morpho-
genesis were observed in Dlg5?/?lungs at E13.5 (Fig. 2A–D’).
E13.5 Dlg5?/?lungs display delay in branching, with an overall
decrease in the number of branches and a prominent dilation of
distal tubules (Fig. 2C–D’). Lung branching morphogenesis in the
developing mouse embryo is a highly stereotypical process that is
comprised of 3 branching modes: domain branching and planar
and orthogonal bifurcation of terminal buds (Metzger et al.,
2008). Comparison of left lung lobes in E13.5 wildtype and
littermates show a similar number of L branches
(L1–L5); however, all terminal buds are bifurcated (a–p) in the
wildtype, but not in the Dlg5?/?lungs (Fig. 2D–E, n¼4). More-
over, while the domain branching mode results in the formation
of D branches in both wildtype and Dlg5?/?lungs, the d3 branch
is missing in the high order L1 and L2 branches of the Dlg5?/?
embryos (Fig. 2D, D’). The decrease in the number of branches and
the dilation of terminal buds are consistent with the histological
phenotypes observed in E13.5–E15.5 Dlg5?/?lungs (Fig. 1H–I’).
We conclude that Dlg5 is required for proper lung branching
morphogenesis and that the absence of Dlg5 results in an overall
defect in branching with dilation of the distal tubules and
Epithelial–mesodermal interactions play an important role in
the regulation of lung development and defects in either epithe-
lial or mesenchymal tissues can result in an abnormal branching
morphogenesis (Morrisey and Hogan, 2010). To determine
whether Dlg5 is expressed in epithelial or mesodermal compart-
ments during lung development, we analyzed Dlg5 expression
using in situ hybridization. While Dlg5 was expressed throughout
the lung tissue, the highest levels of Dlg5 expression were present
in the epithelial tubes, the same structures that fail to branch and
instead dilate in Dlg5?/?embryos (Fig. 2F–G).
Several signal transduction pathways including Wnt, FGF,
PDGF and BMP signalings have been implicated in the regulation
of lung branching morphogenesis (Cardoso and Lu, 2006; De
Langhe and Reynolds, 2008). We therefore analyzed whether
Wnt, FGF, PDGF, or BMP signalings are perturbed in Dlg5?/?
lungs. Beta-catenin is pivotal for canonical Wnt signaling and
Dlg5 can physically interact with b-catenin (Wakabayashi et al.,
2003). To determine whether canonical Wnt signaling is affected
in Dlg5?/? ?lungs, we crossed our mutants with TOPGAL mice
carrying LacZ reporter for b-catenin (DasGupta and Fuchs, 1999).
Staining for LacZ activity did not reveal significant differences
between the control and Dlg5?/?lungs (Supplementary Fig. 1).
Similar to Wnt pathway, no significant differences in the levels of
phosphorylated FGFR2, PDGFa, or their downstream target, ERK1/
2, were found between wild-type and Dlg5?/?lungs (Supple-
mentary Fig. 2A–C). In addition, we did not find significant
differences in the phosphorylation of SMAD1/5, a downstream
target and transducer of BMP signaling (Supplementary Fig. 2D).
In situ hybridizations revealed overall similar levels of Fgf10
mRNA, although we noted that the Fgf10 expression pattern
T. Nechiporuk et al. / Developmental Biology 377 (2013) 375–384
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ydbio.2013.02.019.
Benton, R., St. Johnston, D., 2003. Drosophila PAR-1 and 14-3-3 inhibit Bazooka/
PAR-3 to establish complementary cortical domains in polarized cells. Cell
Betschinger, J., et al., 2003. The Par complex directs asymmetric cell division by
phosphorylating the cytoskeletal protein Lgl. Nature 422, 326–330.
Bilder, D., et al., 2000. Cooperative regulation of cell polarity and growth by
Drosophila tumor suppressors. Science 289, 113–116.
Bourbon, J.R., et al., 2009. Bronchopulmonary dysplasia and emphysema: in search
of common therapeutic targets. Trends Mol. Med. 15, 169–179.
Cardoso, W.V., Lu, J., 2006. Regulation of early lung morphogenesis: questions,
facts and controversies. Development 133, 1611–1624.
Chang, Y., et al., 2010. Discs large 5 is required for polarization of citron kinase in
mitotic neural precursors. Cell Cycle 9, 1990–1997.
DasGupta, R., Fuchs, E., 1999. Multiple roles for activated LEF/TCF transcription
complexes during hair follicle development and differentiation. Development
De Langhe, S.P., Reynolds, S.D., 2008. Wnt signaling in lung organogenesis.
Organogenesis 4, 100–108.
El-Hashash, A.H., et al., 2011. Eya1 controls cell polarity, spindle orientation, cell
fate and Notch signaling in distal embryonic lung epithelium. Development
Evans, M.J., et al., 1975. Transformation of alveolar type 2 cells to type 1 cells
following exposure to NO2. Exp. Mol. Pathol. 22, 142–150.
Funke, L., et al., 2005. Membrane-associated guanylate kinases regulate adhesion
and plasticity at cell junctions. Annu. Rev. Biochem. 74, 219–245.
Harris, K.S., et al., 2006. Dicer function is essential for lung epithelium morpho-
genesis. Proc. Natl. Acad. Sci. USA 103, 2208–2213.
Harris, T.J., Peifer, M., 2005. The positioning and segregation of apical cues during
epithelial polarity establishment in Drosophila. J. Cell Biol. 170, 813–823.
Hurov, J.B., et al., 2004. Atypical PKC phosphorylates PAR-1 kinases to regulate
localization and activity. Curr. Biol. CB14, 736–741.
Johnston, C.A., et al., 2009. Identification of an Aurora-A/PinsLINKER/Dlg spindle
orientation pathway using induced cell polarity in S2 cells. Cell 138,
Klezovitch, O., et al., 2004. Loss of cell polarity causes severe brain dysplasia in
Lgl1 knockout mice. Genes Dev. 18, 559–571.
Li, S., et al., 2003. The role of laminin in embryonic cell polarization and tissue
organization. Dev. Cell 4, 613–624.
McCaffrey, L.M., Macara, I.G., 2009a. The Par3/aPKC interaction is essential for
end bud remodeling and progenitor differentiation during mammary gland
morphogenesis. Genes Dev. 23, 1450–1460.
McCaffrey, L.M., Macara, I.G., 2009b. Widely conserved signaling pathways in the
establishment of cell polarity. Cold Spring Harbor Perspect. Biol. 1, a001370.
McCaffrey, L.M., Macara, I.G., 2012. Signaling pathways in cell polarity. Cold Spring
Harb. Perspect. Biol. 4 (6), pii: a009654, 10.1101/cshperspect.a009654.
McElroy, M.C., Kasper, M., 2004. The use of alveolar epithelial type I cell-selective
markers to investigate lung injury and repair. Eur. Respir. J. 24, 664–673.
Metzger, R.J., et al., 2008. The branching programme of mouse lung development.
Nature 453, 745–750.
Millauer, B., et al., 1993. High affinity VEGF binding and developmental expression
suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 72,
Morrisey, E.E., Hogan, B.L., 2010. Preparing for the first breath: genetic and cellular
mechanisms in lung development. Dev. Cell 18, 8–23.
Nechiporuk, T., et al., 2007. Failure of epithelial tube maintenance causes
hydrocephalus and renal cysts in Dlg5?/?mice. Dev. Cell 13, 338–350.
Neumuller, R.A., Knoblich, J.A., 2009. Dividing cellular asymmetry: asymmetric cell
division and its implications for stem cells and cancer. Genes Dev. 23,
Nguyen, N.M., et al., 2005. Epithelial laminin alpha5 is necessary for distal
epithelial cell maturation, VEGF production, and alveolization in the develop-
ing murine lung. Dev. Biol. 282, 111–125.
Ohshiro, T., et al., 2000. Role of cortical tumour-suppressor proteins in asymmetric
division of Drosophila neuroblast. Nature 408, 593–596.
Peng, C.Y., et al., 2000. The tumour-suppressor genes lgl and dlg regulate basal
protein targeting in Drosophila neuroblasts. Nature 408, 596–600.
Rajagopal, J., et al., 2008. Wnt7b stimulates embryonic lung growth by coordi-
nately increasing the replication of epithelium and mesenchyme. Develop-
ment 135, 1625–1634.
Sasaki, H., Hogan, B.L., 1993. Differential expression of multiple fork head related
genes during gastrulation and axial pattern formation in the mouse embryo.
Development 118, 47–59.
Scavo, L.M., et al., 1998. Apoptosis in the development of rat and human fetal
lungs. Am. J. Respir. Cell Mol. Biol. 18, 21–31.
Sezaki, T., et al., 2012. Role of Dlg5/lp–dlg, a membrane-associated guanylate
kinase family protein, in epithelial–mesenchymal transition in LLc-PK1 renal
epithelial cells. PLoS One 7, e35519.
Siegrist, S.E., Doe, C.Q., 2005. Microtubule-induced Pins/Galphai cortical polarity in
Drosophila neuroblasts. Cell 123, 1323–1335.
Smolen, G.A., et al., 2010. A genome-wide RNAi screen identifies multiple RSK-
dependent regulators of cell migration. Genes Dev. 24, 2654–2665.
Snider, G.L., 1992. Parker B. Francis lecture. Animal models of chronic airways
injury. Chest 101, 74S–79S.
Stoll, M., et al., 2004. Genetic variation in DLG5 is associated with inflammatory
bowel disease. Nat. Genet. 36, 476–480.
Tang, N., et al., 2011. Control of mitotic spindle angle by the RAS-regulated ERK1/2
pathway determines lung tube shape. Science 333, 342–345.
Taniuchi, K., et al., 2005. Down-regulation of RAB6KIFL/KIF20A, a kinesin involved
with membrane trafficking of discs large homologue 5, can attenuate growth
of pancreatic cancer cell. Cancer Res. 65, 105–112.
Wakabayashi, M., et al., 2003. Interaction of lp-dlg/KIAA0583, a membrane-
associated guanylate kinase family protein, with vinexin and beta-catenin at
sites of cell–cell contact. J. Biol. Chem. 278, 21709–21714.
Warburton, D., et al., 2008. Stem/progenitor cells in lung development, injury
repair, and regeneration. Proc. Am. Thorac. Soc. 5, 703–706.
Weaver, V.M., et al., 2002. Beta4 integrin-dependent formation of polarized three-
dimensional architecture confers resistance to apoptosis in normal and
malignant mammary epithelium. Cancer Cell 2, 205–216.
Wert, S.E., et al., 1993. Transcriptional elements from the human SP-C gene direct
expression in the primordial respiratory epithelium of transgenic mice. Dev.
Biol. 156, 426–443.
Wodarz, A., Nathke, I., 2007. Cell polarity in development and cancer. Nat. Cell Biol.
Woods, D.F., Bryant, P.J., 1991. The discs-large tumor suppressor gene of Droso-
phila encodes a guanylate kinase homolog localized at septate junctions. Cell
Yamanaka, T., Ohno, S., 2008. Role of Lgl/Dlg/Scribble in the regulation of epithelial
junction, polarity and growth. Front. Biosci. 13, 6693–6707.
Zou, D., et al., 2008. Eya1 gene dosage critically affects the development of sensory
epithelia in the mammalian inner ear. Hum. Mol. Genet. 17, 3340–3356.
T. Nechiporuk et al. / Developmental Biology 377 (2013) 375–384