Am J Physiol Cell Physiol 299: C7–C10, 2010;
doi:10.1152/ajpcell.00145.2010.
Editorial Focus
Branching morphogenesis: Rac signaling “PIX” tubulogenesis. Focus on
“Pak1 regulates branching morphogenesis in 3D MDCK cell culture by a PIX
and ␤1-integrin-dependent mechanism”
James A. Marrs
Department of Biology, Center for Regenerative Biology and Medicine, Indiana University-Purdue University Indianapolis,
Indianapolis, Indiana
Branching Morphogenesis
Many epithelial organs develop complex three-dimensional
architecture by an amazing and complex process called branching morphogenesis. A variety of developmental model systems
of branching morphogenesis are being actively studied, including kidney, lung, and pancreas development (2, 6). Tracheal
branching morphogenesis in Drosophila uses the powerful
molecular genetic experimental toolbox developed in this
model (1, 2). Fundamental cellular mechanisms of branching
morphogenesis are shared between different epithelial organs
and between vertebrates and invertebrates. Vascular endothelial branching morphogenesis also share many of these conserved features (1, 2). Therefore, understanding cellular mechanisms of branching morphogenesis will be widely applicable
to cellular events during development and in normal tissue
maintenance.
Kidney developmental biology has a rich tradition of studying inductive signals for epithelial differentiation and branching morphogenesis (5, 24). In early mouse embryos (E11), the
ureteric bud emerges from the Wolffian duct and grows into the
Address for reprint requests and other correspondence: J. A. Marrs, Dept. of
Biology, Center for Regenerative Biology and Medicine, Indiana Univ.-Purdue
Univ. Indianapolis, 723 West Michigan St., SL 328 Indianapolis, IN 46202
(e-mail: [email protected]).
http://www.ajpcell.org
metanephric mesenchyme (Fig. 1A). Through an extensive
series of experiments, Grobstein, Saxén, Ekblom, and other
investigators (25) showed that there are reciprocal inductive
interactions between the ureteric bud epithelium and the metanephric mesenchyme. The ureteric bud induces the metanephric mesenchyme to form distinct nephron cell types found from
the glomerulus to distal convoluted tubule. The ureteric bud
epithelium produces the collecting system. Metanephric mesenchyme cells induce the ureteric bud to branch. The newly
branched ureteric bud induces more metanephric mesenchyme
epithelial induction (Fig. 1A). Waves of these differentiation
cycles produce the millions of nephrons needed for vertebrates
to exist on dry land.
The importance of cell adhesion molecules in metanephric
kidney development were recognized in the earliest studies by
Grobstein, Saxén, Ekblom, and others (8). Mesenchyme-toepithelial transformation induced by signals from the ureteric
bud produce condensates of mesenchyme expressing new cellto-cell and cell-to-extracellular matrix adhesion molecules.
Branching morphogenesis events similar to those in the
metanephric kidney ureteric bud are seen in other developing
organs, including Caenorhabditis elegans and Drosophila
(17). Combinations of molecular genetic techniques and advanced imaging methods were used to dissect cellular events in
these models, which shows that tubulogenesis is highly conserved (1).
In the submandibular gland, lung, and metanephric kidney,
branching morphogenesis requires coordinated extracellular
matrix/integrin adhesion signaling with tyrosine kinase growth
factor receptor signaling (3, 13, 32, 36). Significantly, ureteric
bud branching morphogenesis was blocked in conditional
knockout mice lacking ␤1-integrin expression in the ureteric
bud (32, 36), and these ␤1-integrin knockout ureteric bud
epithelial cells do not respond to glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor (FGF) (36).
Growth factor and integrin signaling pathways activate a Rac1
GTPase and Pak1 kinase-dependent pathway (27). Like mammalian branching morphogenesis, Drosophila also uses adhesion and growth factor-mediated signaling mechanisms for
tracheal development branching decisions (1, 2), suggesting
that these mechanisms are conserved among metazoans.
Cell Culture Models of Branching Morphogenesis
Cell culture models are able to recapitulate numerous aspects of branching morphogenesis. These in vitro branching
morphogenesis models are also induced by tyrosine kinase
growth factor receptor (HGFR, EGFR, etc.) activation (4).
Many epithelial cells (primary cultures and cell lines) will form
an epithelial cyst when suspended in extracellular matrix ma-
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BRANCHING MORPHOGENESIS mechanisms are regulated during
organogenesis, tissue maintenance, and repair (17). We are
also beginning to understand the broad influence of these
morphogenesis signaling mechanisms during physiological
processes and disease progression. Branching morphogenesis
mechanisms are conserved between vertebrates and invertebrates. Common features include regulation of cell-to-cell
adhesion, cell-to-extracellular matrix adhesion, growth factor
and paracrine signaling, cell migration, differentiation, cell
growth, and cell survival. Many developmental model systems,
from Drosophila tracheal branching to metanephric kidney
development, are actively being exploited to understand fundamental mechanisms of branching morphogenesis (17).
In addition to in vivo development models, cell culture
models were developed that show incredible utility and versatility for examining cellular events of branching morphogenesis (22). Monolayer and cyst culture models using MadinDarby canine kidney (MDCK) cells can recapitulate several
aspects of branching morphogenesis, including important adhesion and polarity regulatory processes (4). An article by
Hunter and Zegers (10) illustrates the power of the threedimensional MDCK cell cyst model in the analysis of branching morphogenesis mechanisms.
Editorial Focus
C8
terials like type I collagen. MDCK cysts are used extensively
to examine cyst and tubule morphogenesis (22). This cell
culture model has helped us gain a more detailed and complete
picture of branching morphogenesis.
Collagen suspension cysts from epithelial cell lines like
MDCK cells arise from a single cell, growing into a spherical,
fluid-filled cyst (Fig. 1B, top) with the basal cell surface facing
the surrounding extracellular matrix outside the sphere, with
the lateral cell-to-cell contacts forming junctional complexes,
and with the apical cell surface facing the fluid-filled lumen.
The ability of MDCK and other epithelial cells to self-organize
into multicellular, polarized, and transporting epithelium is a
part of their differentiated epithelial state. Stages of cyst
development are highly programmed, which has allowed numerous laboratories to use this model to examine cell polarity
and morphogenesis mechanisms (22).
Expanding the utilization of these cyst models and applying
new technologies to these experiments should continue to yield
new, fundamental knowledge of cellular mechanisms of morphogenesis. MDCK cells can be modified for genetic loss-offunction and gain-of-function experiments (4). Green fluorescent protein (GFP) fusion proteins are expressed and used to
analyze dynamic cellular processes when combined with timelapse imaging and other advanced imaging applications (9).
Advances in GFP imaging technology, for example, using
fluorescence resonance energy transfer (FRET) or biosensor
probes to measure intracellular signaling (28) will be increasAJP-Cell Physiol • VOL
ingly applied to the MDCK cyst system to dissect cell polarity
mechanisms in high spatial and temporal resolution.
Additional MDCK cell culture methods are used to experimentally manipulate cell polarity, in particular, showing the
importance of extracellular matrix adhesion. In addition to
growing MDCK cell cysts in a collagen suspension, MDCK
cells can be grown in a media suspension, and the cysts that
develop have apical domains facing the growth medium and
basal domains facing the interior lumen, where basement
membrane is laid down (30). These cysts that develop in media
suspension can be embedded in a collagen matrix, forcing cells
to reverse their polarity by degrading extracellular matrix
within the lumen and reorganizing the junctional complexes
and membrane proteins to have apical domains facing into the
lumen and basal domains facing the new collagen matrix (31).
Another alternative culture method that produces similar polarity reversal events in synchrony is achieved by growing
MDCK cells in monolayer culture, and then, a collagen matrix
can be polymerized on top of the monolayer (26, 37). In
response to the collagen overlay, apical proteins are internalized, and new apical domains assemble on the lateral cell
surface. The apical domains forming at lateral domains expand
and fuse with other apical domains forming in other cells.
Eventually, these collagen overlay cultures generate two complete monolayers that face one another and share a common
lumen. These MDCK cell models helped characterize adhesion
cues required during cyst formation.
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Fig. 1. Comparison between branching morphogenesis during mammalian kidney development and during tubulogenesis in vitro. A: coordinate growth factor
and integrin signaling regulates branching morphogenesis during metanephric kidney development. Interfering with either growth factor or integrin/extracellular
matrix signaling produces similar phenotypes in conditional knockout mice, failure in ureteric bud branching morphogenesis (see text). B: branching
morphogenesis is modeled using HGF induced tubulogenesis with the kidney epithelial cell line Madin-Darby canine kidney (MDCK) cells grown in collagen
suspension as cysts. MDCK cell epithelial cells form polarized monolayers when grown on solid supports, but when single cell suspensions of MDCK cells are
polymerized in type I collagen, then cells divide and differentiate to form a spherical, multicellular cyst that has a monolayer epithelium than secretes and binds
a basement membrane facing the collagen matrix, and a free apical cell surface facing a fluid-filled lumen. HGF induces cells within these MDCK cysts to send
out extensions that ultimately leads to tubulogenesis (see text). Hunter and Zegars (10) demonstrate that HGF and integrin-dependent tubule initiation and growth
pathways are initiated by Pak1-mediated PIX signals.
Editorial Focus
C9
nent of the Pak1-PIX-GIT complex. This protein complex
regulates focal contact turnover and cell migration behaviors in
several cell types (34). To prevent Pak1 recruitment to the
complex, Hunters and Zegers (10) expressed DN-Pak1 molecule with two-point mutations that prevented PIX binding,
which they called DN-Pak1-⌬PIX. Cysts expressing DN-Pak1⌬PIX do not induce precocious tubules (10), indicating that
tubulogenesis requires appropriately regulated Pak1 signaling
and Pak1/PIX/GIT complex assembly.
These important findings from the Zegers laboratory (10)
should also be considered within the context of their recent
findings that cadherin-mediated cell-to-cell adhesion and Pak1
signals from integrin-mediated cell-to-extracellular matrix adhesion coordinately regulate contact-mediated inhibition of cell
proliferation (15). Contact-mediated inhibition of cell proliferation activity also requires normal regulation of the Pak1-PIXGIT complex. Cadherin- and integrin-mediated adhesion signaling mechanisms also converge on phosphoinositide 3 kinase
(PI3 kinase) signaling mechanisms (15, 21). PI3 kinase signaling reciprocally can regulate cadherin-mediated cell-to-cell
adhesion and integrin-mediated cell-to-extracellular matrix adhesion mechanisms, suggesting that these mechanisms represent a cross-talk signaling pathway to coordinate cell adhesion,
proliferation, and migration.
There are very few toeholds into the puzzle of cross-talk
signaling mechanisms that permit coordinate regulation of
cell-to-cell matrix and cell-to-extracellular adhesion mechanisms. Thus findings from the Zegers laboratory (10, 15)
represent a very significant advance within this research avenue. These coordinate regulatory mechanisms are utilized during normal tissue development and tissue maintenance, but we
are learning that tipping the balance of cell differentiation is a
part of many disease processes. The epithelial-to-mesenchymal
transitions that occur during tumor progression and metastasis
represent a disease process that scrambles the normal balance
between cell-to-cell adhesion, cell-to-extracellular matrix adhesion, cell proliferation, and cell survival (14). In vitro models
like MDCK cystogenesis and tubulogenesis will continue to
provide important insight into difficult and complex cellular
processes. Similarly, application of new genetic, epigenetic,
imaging and other technologies to these models should generate more detailed information about branching morphogenesis
mechanisms in the near future.
Rac, Pak, and PIX: Branching Morphogenesis
DISCLOSURES
Branching morphogenesis in Drosophila trachea and mouse
kidney development require Rac signaling mechanisms, which
are recapitulated in the MDCK branching morphogenesis
model. An important effector for Rac signaling is Pak1, a
serine-threonine p21 (Rho family GTPase)-activated kinase
family member, and HGF and integrin signaling activate RacPak pathways (27). Hunter and Zegers (10) report that expressing a full-length kinase dead dominant-negative Pak1 mutant
(DN-Pak1) induces HGF-independent precocious tubulogenesis in MDCK cysts. DN-Pak1-induced tubulogenesis requires
integrin-mediated extracellular matrix adhesion, and DN-Pak1
induces myosin contractility. Hunter and Zegers (10) also
determined that the tubulogenesis phenotype induced by DNPak1 expression requires binding of the PIX protein (a Rac
guanine nucleotide exchange factor; Rac GEF) and a compoAJP-Cell Physiol • VOL
No conflict of interest, financial or otherwise, are declared by the author(s).
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