Phactr4 regulates directional migration
of enteric neural crest through PP1,
integrin signaling, and cofilin activity
Ying Zhang,1Tae-Hee Kim,1,2and Lee Niswander3
Howard Hughes Medical Institute, Department of Pediatrics, Graduate Program in Cell Biology, Stem Cells, and Development,
Children’s Hospital Colorado, University of Colorado, Aurora, Colorado 80045, USA
Hirschsprung disease (HSCR) is caused by a reduction of enteric neural crest cells (ENCCs) in the gut and
gastrointestinal blockage. Knowledge of the genetics underlying HSCR is incomplete, particularly genes that
control cellular behaviors of ENCC migration. Here we report a novel regulator of ENCC migration in mice.
Disruption of the Phactr4 gene causes an embryonic gastrointestinal defect due to colon hypoganglionosis, which
resembles human HSCR. Time-lapse imaging of ENCCs within the embryonic gut demonstrates a collective cell
migration defect. Mutant ENCCs show undirected cellular protrusions and disrupted directional and chain
migration. Phactr4 acts cell-autonomously in ENCCs and colocalizes with integrin and cofilin at cell protrusions.
Mechanistically, we show that Phactr4 negatively regulates integrin signaling through the RHO/ROCK pathway
and coordinates protein phosphatase 1 (PP1) with cofilin activity to regulate cytoskeletal dynamics. Strikingly,
lamellipodia formation and in vivo ENCC chain migration defects are rescued by inhibition of ROCK or integrin
function. Our results demonstrate a previously unknown pathway in ENCC collective migration in vivo and
provide new candidate genes for human genetic studies of HSCR.
[Keywords: directional cell migration; enteric neural crest cell; Hirschsprung disease; Phactr4; PP1; b1 integrin; cofilin]
Supplemental material is available for this article.
Received September 18, 2011; revised version accepted November 16, 2011.
The enteric nervous system (ENS) has been called the
‘‘second brain,’’ as it is comprised of 100 million neurons
and plays an autonomous role in controlling many in-
testinal functions, including peristalsis, gastric and pan-
creatic secretion, and immune response (Heanue and
Pachnis 2007). The ENS is composed of neural crest-
derived neurons and glia that are organized into ganglia,
and these ganglia interconnect to form an enteric plexus.
Defects in ENS development in humans cause Hirsch-
sprung disease (HSCR), a common congenital disorder
occurring in 1:5000 live births. HSCR is characterized by
an absence of enteric neurons in terminal regions of the
gut due to an embryonic defect in ENS formation. The
receptor tyrosine kinase RET and the G protein-coupled
receptor endothelin receptor B (EDNRB) and their respec-
tive ligands, GDNF and EDN3, are critical in ENS de-
velopment (Heanue and Pachnis 2007; Amiel et al. 2008).
Mutations in c-Ret and EdnrB are responsible for ;50%
and 5% of HSCR cases, respectively (McCallion et al.
2003). However, the mechanisms responsible for many
of the remaining HSCR cases are still unclear. Moreover,
the complex inheritance pattern of HSCR indicates that
mutations at additional loci contribute to the disease.
Mice provide an excellent animal model to study the
genetics and mechanisms of ENS formation. During
mouse embryogenesis, ENS progenitors derive from vagal
and sacral neural crest cells (NCCs). At embryonic day
9.5 (E9.5), vagal NCCs emigrate from the neural tube and
invade the foregut, then migrate along the entire gastroin-
Sacral NCCs make a small contribution of neurons and
glial cells by colonizing the hindgut at E15.5 (Druckenbrod
andEpstein 2005).Different cellular processes such as neu-
ral crest specification, proliferation, differentiation, and
migration are important for complete innervation of the
gut (Asai et al. 2006; Simpson et al. 2007; Okamura and
Saga 2008; Wallace et al. 2009). The study of enteric NCC
(ENCC) migration has revealed complex cellular behaviors
at the migratory wave front. Close to the wave front, there
areafewsolitaryENCCs,and these helpdirect theforward
migration of ENCCs that follow as chains of cells, which
then spread out to form an interconnected network within
the gut (Young et al. 2004; Druckenbrod and Epstein 2005).
1These authors contributed equally to this work.
2Present address: Department of Medical Oncology, Dana-Farber Cancer
Institute, Harvard Medical School, D720 44 Binney Street, Boston, MA
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.179283.111.
GENES & DEVELOPMENT 26:69–81 ? 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org 69
In terms of ENCC migration, only a few genes are known
to be important, and these include L1 cell adhesion
molecule, b1-integrin, Ret, and matrix metalloprotease-2
(MMP-2) (Anderson et al. 2006; Asai et al. 2006; Breau et al.
2009; Anderson 2010).
Directed cell migration is crucial for complete coloni-
zation of the gut by the ENS. To migrate toward a chemo-
attractant, a cell must become polarized and form a single
dominant lamellipodial protrusion at its leading edge in
the direction of migration. Directional migration is a re-
sult of spatially restricted stable and persistent lamelli-
podium. Lamellipodial protrusionrequires reorganization
of the actin cytoskeleton, and this process is mediated by
multiple signals, including external cues, the intracellu-
lar polarity machinery, and adhesion receptors. Integrins
are cell surface transmembrane receptors clustered in
the leading edge and at cell–matrix adhesions, where they
connect the extracellular matrix (ECM) to the actin cy-
toskeleton network. When activated, integrins undergo
conformational change torecruit signaling moleculessuch
as focal adhesion kinase (FAK), SRC, and ERK to the cell–
matrix adhesions. This serves to control the temporal and
spatial activities of downstream small RhoGTPases to
direct local cell membrane dynamics (Geiger et al. 2001).
The actin-severing protein cofilin is a downstream target
of RhoGTPases, and the action of cofilin on actin poly-
merization helps generate propulsive force and directional
protrusions at the leading edge (Bernstein and Bamburg
2010). Small RhoGTPase can activate LIM kinase, which
phosphorylates cofilin at Ser3 to inhibit its activity (Arber
et al. 1998; Maekawa et al. 1999), whereas general serine/
threonine phosphatases such as protein phosphatase 1
(PP1) can activate cofilin by dephosphorylation (Ambach
et al. 2000; Larsen et al. 2003; Oleinik et al. 2010). Cofilin
does not regulate lamellipodia formation, but is required
for directional migration by controlling actin reorganiza-
tion to generate polarized lamellipodia at the leading edge
(Dawe et al. 2003; Ghoshet al. 2004).When cofilinactivity
is decreased in fibroblasts by b1 integrin-triggered phos-
phorylation of cofilin on Ser3 through the RhoA–ROCK1
pathway, cells undergo random intrinsic migration (Danen
et al. 2005). Thus, lamellipodial dynamics controlled by
cofilin and integrin signaling are crucial factors in dictat-
ing directional cell migration.
Genetic mutants can provide significant new insights
into the molecular mechanisms of cell migration. Starting
from a forward genetic screen in mice, we describe here
a novel function of the Phactr4 gene in ENCC migration.
Phactr4belongs toa small groupofproteinswithpredicted
PP1- and actin-interacting regulatory domains (Allen et al.
2004).Little isknown of the invivofunctions ofthe Phactr
family. Our previous studies identified a missense muta-
tion of Phactr4 (called Phactr4humdy) within the PP1-
binding domain that disrupts PP1, but not actin, binding
and causes a misregulation of PP1 activity. During murine
neural tube closure and eye development, Phactr4 through
PP1 controls Rb phosphorylation and proliferation (Kim
et al. 2007). Here we report a HSCR-like defect in ENS
development in Phactr4humdymutant mouse embryos due
to defective collective cell migration of ENCCs. This
results in greatly reduced numbers of ENCCs in the
caudal gut, with abnormal accumulation of material in
the gut. Time-lapse live imaging of ENCC migration from
the neural tube and within the gut indicates that both
Phactr4 and PP1 are required for directed ENCC migra-
tion. Mutant ENCCs display random protrusions and
undirected migration, and Phactr4 acts cell-autonomously
intheregulationofcytoskeletal dynamics.Phactr4 protein
colocalizes with b1 integrin and cofilin at the tips of
lamellipodia. Biochemical studies show that Phactr4 is
required to negatively regulate integrin signaling, and
disrupted integrin signaling through RHO/ROCK leads
to misregulation of cofilin phosphorylation. Lamellipo-
dia formation and ENCC chain migration defects can
be rescued in vivo by inhibition of integrin signaling or
by activation of cofilin. Thus, Phactr4 regulates actin
cytoskeleton dynamics through cofilin activity that is
controlled by PP1 and integrin signaling during ENCC
migration. These data suggest Phactr4 and PP1 be consid-
ered as candidate genes in the etiology of human HSCR.
Phactr4humdyembryos exhibit intestinal
Phactr4humdy/humdymutant embryos displayed an intes-
tinal blockage phenotype with an abnormal accumula-
tion of material in the gut. Normally, intestines of wild-
type embryos at E18.5 were white and/or yellow in color,
but the intestines of mutant embryos were green and/or
dark red, indicating a gastrointestinal tract problem (Fig.
1A,B). Histological sections showed retention of meco-
nium in E18.5 mutant intestine (Supplemental Fig.
S1A,B). To characterize ENCCs in the gut, we analyzed
the expression of nicotinamide adenine dinucleotide phos-
phate (NADPH) diaphorase, which highlights the major
neuronal population in the myenteric plexus at E18.5 (Fig.
1C–J). Wild-type enteric neurons were clustered in myen-
teric ganglia interconnected by neurites in the stomach,
foregut, and midgut (Fig. 1C,E,G). However, mutant neu-
rons were largely individually localized and the neurite
pattern was disorganized (Fig. 1D,F,H). The number of
NADPH diaphorase-positive cells was normal in the
stomach, foregut, and midgut but showed a statistically
significant decrease in the mutant hindgut compared
with wild-type (30.2% decrease in the mutant) (Fig. 1K).
Ganglia hypotrophy was clearly observed in the mutant
hindgut (Fig. 1I,J).
The defect in ENCC number and organization was
observed early in development. RetTGM/+(Enomoto et al.
2001) was used to visualize ENCCs that are marked by
GFP expression under the control of the Ret promoter. By
E12.5, wild-type and mutant ENCCs colonized the gut to
post-caecum hindgut level (Fig. 1L–S). However, the
number of mutant ENCCs was reduced, and these cells
were more randomly distributed and often appeared as
individualized cells relative to the organized network in
wild type (Fig. 1R,S). Phactr4humdy/humdymutants die at
or before birth, and hence it was not possible to examine
Zhang et al.
70GENES & DEVELOPMENT
whether megacolon is present in Phactr4 mutants after
birth, similar to that observed in HSCR patients, although
this would be expected based on the embryonic hypogan-
glionic phenotype and retention of meconium.
Neural crest specification and ENCC proliferation
and differentiation are normal in Phactr4humdymutants
ENCC colonization of the gut is dependent on proper
neural crest specification, proliferation, and differentia-
tion. Molecular analyses of these processes showed no
apparent alteration. Neural crest specification appeared
normal, as revealed by neural crest markers AP2a and
Sox10 (Supplemetnal Fig. S2A–D). Our previous studies
showed Phactr4 regulates the cell cycle through PP1, Rb,
and E2F1. Although in the neural tube and retina the
humdy mutation causes excess cell proliferation (Kim
et al. 2007), analysis of phospho-histone 3 staining of
E9.5, E10.5, and E12.5 Phactr4humdy/humdy;RetTGM/+em-
bryonic ENCC in the gut (Supplemental Fig. S2E) showed
no significant difference between wild type and mutant.
Importantly, although loss of E2F1 function can rescue
exencephaly, coloboma, and abnormal proliferation of
neural progenitors in humdy embryos (Kim et al. 2007),
it could not rescue the ENS defects (Supplemental Fig.S1),
indicating a role for Phactr4 in the ENS that is indepen-
dent of the Rb–E2F1-regulated cell cycle. Mutant ENCCs
also underwent differentiation, as revealed by neuron and
glia markers Tuj1, PGP9.5, and GFAP (Supplemental Fig.
S2G–L). TUNELstaining for apoptotic ENCCs showed no
significant difference at E9.5, E10.5, and E12.5 (Supple-
mental Fig. S2F). However, after ENCC migration and
colonization of the gut, increased apoptosis was detected
in mutant ENCCs at E14.5. By E18.5, no apoptosis was
observed in wild-type or mutant ENS. Taken together,
these results indicate that the decreased number and
incomplete innervation by mutant ENCCs is not due to
an alteration in cell specification, proliferation, or differ-
presenting as green intestines versus the normal white or orange intestines of wild-type embryos (A). (C–J) Whole-mount NADPH
diaphorase staining of E18.5 wild-type (top panels) and Phactr4humdy(bottom panels) gut regions. (K) Statistically significant 30.2%
decrease in NADPH diaphorase-stained cells in the mutant hindgut, compared with wild type (E18.5). Data are expressed as mean 6
standard deviation (SD) in three independent experiments. (**) P < 0.01, Student’s t-test. (L–S) Confocal images of whole-mount gut
preparations from E12.5 wild-type;RetTGM/+(top panels) and Phactr4humdy/humdy;RetTGM/+(bottom panels) embryos. (L,M) Stomach.
(N,O) Foregut. (P,Q) Midgut. (R,S) Hindgut.
Colonic hypoganglionosis in Phactr4humdy/humdyembryos. (A,B) Material is retained in the intestines of E18.5 mutants (B)
Phactr4 and PP1 control ENS directional migration
GENES & DEVELOPMENT71
entiation. The reduced number of ENCCs at E12.5 cannot
be explained by cell death; however, the cell death seen at
E14.5 may contribute to the overall phenotype of hypo-
Phactr4 is required for ENCC migration
Another parameter critical to ENCC colonization of the
gut is directed cell migration. To explore the in vivo
migratory behavior of ENCCs within the gut, we used
time-lapse live imaging. RetTGM/+cells in organ explants
of E12.5 hindgut from wild-type and Phactr4humdy/humdy
embryos were imaged for up to 8 h (Fig. 2A,B; Supple-
mental Movies S1, S2). At the migration wave front, wild-
type ENCC chain migration was readily observed and cell
trajectories were in a relatively straight line from rostral
to caudal (Fig. 2A [panel a], C; Supplemental Movie S1).
There were very few solitary ENCCs (Fig. 2E), and indi-
vidual cells were quickly joined by more rostral ENCCs
to form small and dynamic groups of cells as either
aggregates or chains, which efficiently invaded the hind-
gut. This pattern of migration and net speed (27.67 6 2.33
mm/h) (Fig. 2F) is consistent with previous observations of
wild-type ENCCs (Young et al. 2004). In contrast, in the
mutant, directionality of ENCCs at the wave front was
much more erratic (cell tracking was performed on three
wild-type and three mutant gut explants) (Fig. 2B [panel
b], D; Supplemental Movie S2), indicating that directional
ENCC migration is disrupted by loss of Phactr4 function.
Moreover, there was a large number of solitary ENCCs
detached from the population and located away from the
wave front chains (Fig. 2B). Quantification of the number
of solitary cells at the wave front showed a 3.6-fold
increase in the mutant (Fig. 2E), suggesting that Phactr4
is required to retain cell–cell adhesion at the migratory
wave front. Some solitary ENCCs rejoined the rostral
chains, whereas some eventually rounded up and un-
derwent cell death. This latter subset was small and was
detected by live imaging but not in fixed TUNEL-stained
samples. The net speed and persistence of leading cell
movements were significantly reduced in the mutant,
although the speed of individual mutant cells was in-
creased (Fig. 2F–H). Taken together, these observations
indicate that Phactr4 is required to retain ENCCs in a
chain at the migratory wave front and provide directional
migration to allow complete innervation of the gut.
Phacrtr4 acts cell-autonomously to regulate directed
Phactr4 is expressed in ENCCs, as shown by PCR from
FACs-sorted ENCCs (Supplemental Fig. S3A). Therefore,
we next asked whether Phactr4 acts cell-autonomously
to regulate ENCC migration. To study this, we dissected
gut segments from E13.5 wild-type or Phactr4humdy/humdy
embryos and cultured them in 3D collagen gels with
GDNF to stimulate ENCC migration out of the explant
(ENCCs visualized by immunostaining with p75NTR)
(data not shown). Wild-type ENCCs showed extensive
migration out of the explant (Fig. 3A,A9). Mutant ENCCs
showed limited migration, wherein ;85% of explants
showed limited migration from the hindgut (Fig. 3B9) and
;15% displayed severe migration defects from both the
midgut and hindgut (Fig. 3C,C9). Higher magnification of
wild-type ENCCs showed chains of elongated and polar-
ized cells, whereas mutant ENCCs were largely individ-
ual and displayed an altered cell shape with multiple
random protrusions (Fig. 3A0,B0,C0).
The variable migration defect was also evident in vivo.
At E12.5, wild-type ENCCs have colonized the caecum
completely and reached the hindgut (Supplemental Fig.
S3B,D). In contrast, the mutant phenotype ranged from
the most severe but rare cases where almost no ENCCs
were detected throughout the gut (Supplemental Fig. S3C),
likely reflecting a vagal neural crest emigration defect, to
or hindgut, even at E13.5 (Supplemental Fig. S3E,G).
Live imaging of ENCC migration from E13.5 gut
explants showed properties similar to those seen in vivo.
Wild-type ENCCs migrated out of the explants in chains,
Supplemental Movie S3).In contrast, mutantENCCs were
more solitary and moved very rapidly (2.75-fold increase
in ENCC speed compared with wild type) (Supplemental
Fig. S4E) but without specific direction, as shown by the
meandering trajectories and decreased persistence (Sup-
plemental Fig. S4B,D,E; Supplemental Movie S4). The
directionality of cell protrusions was also much more
erratic in the mutant than wild type (Supplemental Fig.
S4F,G). Together, these data indicate that Phactr4 is re-
quired cell-autonomously to regulate ENCC migration.
Phactr4 is also required cell-autonomously for vagal
NCC emigration from explants of E9.0 neural tube (visu-
alized with anti-p75NTRantibody). Wild-type NCCs mi-
a 32% decrease in NCCs that migrated from the humdy
neural tube (Supplemental Fig. S5A–C). Actin cytoskele-
ton visualization by phalloidin staining showed that wild-
type NCCs were elongated and polarized, with a single
dominant lamellipodia at the leading edge (Fig. 3D). In
contrast, humdy NCCs were less elongated, with mul-
tiple lateral lamellipodium, and more solitary cells were
observed (Fig. 3E). In vivo migration to the foregut was
also defective in E9.5 Phactr4humdy/humdy; RetTGM/+em-
bryos, with reduced number of ENCCs in the foregut (37%
decrease relative to wild type) (Supplemental Fig. S5D–F).
Together, these data indicate that Phactr4 acts cell-
autonomously to direct ENCCs throughout their migra-
tory pathway from neural crest emigration to ENCC mi-
gration into the foregut and along the intestinal tract.
These data also suggest that Phactr4 regulates mainte-
nance of chain formation and cell polarization, perhaps
by affecting cytoskeletal dynamics.
PP1 is also required for ENCC migration along the gut
The Phactr4humdymutation disrupts PP1 binding, and
therefore we tested whether Phactr4 acts through PP1
in the regulation of ENCC migration. PP1 activity was in-
and the migratory wave front was visualized by dynamic
Zhang et al.
72 GENES & DEVELOPMENT
type;RetTGM/+(A) and Phactr4humdy/humdy;RetTGM/+mutant (B) whole-mount gut showing the caudal progression of ENCCs. (A, panel
a) Wild-type ENCCs migrated as streams of cells in interconnected chains with a strongly rostral-to-caudal trajectory (left to right,
respectively). (B, panel b) In contrast, Phactr4humdyENCCs were largely solitary at or close to the migratory wave front with a random
trajectory. (A, panel a; B, panel b) Numbers indicate four different cells tracked over 8 h at 5-min intervals, and the migration track is
color-coded to indicate the relative time point. (C,D) Polar histograms represent the trajectories of the most caudal cell at 15-min
intervals in three explants of E12.5 hindgut. The trajectories were determined by drawing a straight line from the position of the most
caudal cell with its position 15 min previously, with 0 degree being the rostrocaudal axis of the gut. The number shown on the inner
arcs represent the frequency with which the cell was detected at that angle. (E,F) Quantification of the number of solitary cells at the
migration wave front (E) and net speed at which the migratory wave front of ENCCs migrated caudally along the gut (F). The net speed
was determined by measuring the distance between the location of the wave front at the beginning of the sequence and its location at
the end of the sequence (a minimum of 8 h later). (G,H) Analysis of persistence (ratio of the direct length from start to end divided by the
total track length) (G) and speed (H) of migrating ENCCs. Data are expressed as mean 6 SD in three independent experiments. (***) P <
0.001; (**) P < 0.01; (*) P < 0.05, Student’s t-test.
Live imaging of ENCC migration along the gut. (A,B) Still images from time-lapse movies of the hindgut of E12.5 wild-
Phactr4 and PP1 control ENS directional migration
GENES & DEVELOPMENT73
imaging of wild-type E12.5 RetTGM/+hindgut organ cul-
tures. InDMSO control treatment,ENCCs at the migratory
wave front migrated in long chains and efficiently colonized
the gut (Fig. 3F; Supplemental Movie S5). In contrast, treat-
ment with 100 nM OA caused individualization of ENCCs
and they showed limited and undirected migration (Fig. 3G;
Supplemental Movies S6–S8). PP1 functions cell-autono-
GDNF stimulation of ENCC migration from wild-type
hindgut explants. OA addition resulted in chain dissocia-
tion, and the ENCCs were individualized, no longer polar-
control in H). These studies serve to tie together Phactr4
and PP1 function in the control of ENCC migration.
Phactr4 mutation disrupts the actin cytoskeleton
and lamellipodium formation through regulation
of cofilin activity
To explore further the molecular mechanism underlying
Phactr4regulationofdirectional migration, weestablished
an in vitro system of wild-type and Phactr4humdy/humdy
mouse embryonic fibroblasts (MEFs). In a wound healing
assay, wild-type MEFs moved in a persistent fashion
to close the wound, whereas the mutant MEFs moved
randomly with erratic trajectories, mimicking the in vivo
migration defects (Fig. 4A–D; Supplemental Movies S9,
S10). To examine actin dynamics, we transfected MEFs
with Lifeact-EGFP (Riedl et al. 2008) and followed their
mously to control directed migration. (A–C)
Segments of E13.5 proximal midgut (MG) (A–
C) and distal hindgut (HG) (A9–C9) were cul-
tured in 3D collagen matrix with GDNF for 3
d and stained with phalloidin (green) to detect
the cytoskeleton and with Hoechst (blue) for
nuclei. Explants of wild-type gut showed
extensive migration of ENCCs out of both
gut segments (A,A9). Phactr4humdyENCCs are
responsive to GDNF, but their migration out
of the explant is variable. (C,C9) In severe
cases, cells from the midgut and hindgut
displayed limited migration. (B,B9) In mild
cases, only cells from hindgut displayed a mi-
gration defect. (A0–C0) Higher magnification
of wild-type ENCCs showed elongated cells
that migrate together in chains (A0), while
Phactr4humdyENCCs had altered cell shape
with random protrusions (B0,C0). (D,E) Vagal
NCCs labeled with phalloidin 48 h after
migrating from vagal neural tube explant.
Wild-type NCCs are elongated and polarized
(D), while Phactr4humdyNCCs had aberrant
cell shape with random protrusions (E). (F,G)
Still images from time-lapse movies of E12.5
wild-type;RetTGM/+hindgut explants treated
with DMSO (F) or 100 nM OA (G) for 3 h, then
OA was removed and fresh medium was
added, followed by time-lapse imaging over
8 h. Time is noted in minutes. Following OA
treatment, cells displayed undirected cell pro-
trusions and random cell movements. Num-
bers indicate different cells tracked over 8 h at
5-min intervals, and the tracks shown in the
right panels are color-coded to indicate the
relative time point. (H,I) Explants of wild-type
gut cultured with GDNF for 2 d and then
treated with DMSO (H) or 20 nM OA (I) for 1
h. (H) In the control explant, ENCCs were
polarized and maintained long chains. (I) In-
hibition of PP1 activity with OA resulted in
altered cell shape with random protrusions.
Red is anti-p75NTRantibody detecting the
ENCC, green is phalloidin detecting the cy-
toskeleton, and blue is Hoechst detecting
Phactr4 and PP1 act cell-autono-
Zhang et al.
74GENES & DEVELOPMENT
migration during wound healing. Wild-type cells at
the wound front had one large fan-like lamellipodia,
whereas mutant cells displayed multiple small lamelli-
podium and retraction fibers (Fig. 4E,F; Supplemental
Movies S11, S12, a single cell is shown for wild type and
mutant). Quantitative analysis revealed smaller size but
increased number of lamellipodium in mutant MEFs
To determine the subcellular localization of Phactr4,
Myc-tagged Phactr4wtor Phactr4humdyconstructs were
transfected into wild-type or mutant MEFs. This showed
the concentration of Phactr4 at the tips of lamellipodium.
Moreover, the Phactr4humdymutation did not affect its
localization (Fig. 5A,B), consistent with the ability of both
wild-type and mutant protein to bind to actin (Kim et al.
2007). Endogenous Phactr4 also localized to the lamelli-
podia, as visualized with a Phactr4-specific antibody (Fig.
5C,E). However, the actin cytoskeletal network was dis-
rupted in mutant cells, as phalloidin staining showed
multiple small lamellipodium as well as multiple re-
traction fibers on all sides of the mutant cell (Fig. 4F,
5B). Normally, polarized lamellipodia are a result of
cytoskeletal remodeling directed by small RhoGTPases.
RhoGTPase, plays an important role in regulating actin
dynamics by severing filamentous actin. Cofilin activity is
important for directional migration by reorganizing actin
protrusions in response to external guidance cues (Ghosh
et al. 2004; Paavilainen et al. 2004; Bernstein and Bamburg
2010). PP1 can activate cofilin by dephosphorylation
(Ambach et al. 2000; Larsen et al. 2003; Oleinik et al.
2010). Immunostaining of wild-type and mutant cells
showed that Phactr4 colocalized with cofilin at the tip of
the lamellipodium (Fig. 5C–F). The distinct localization of
Phactr4, combined with the fact that the humdy mutation
specifically disrupts PP1 but not actin binding, led us to
hypothesize that Phactr4 serves as a novel scaffold protein
to bridge PP1 to cofilin to coordinate actin cytoskeletal
dynamics. The relative amount of cofilin phosphorylated
on Ser3 in wounded cells was significantly higher in
mutant MEFs by both Western blot and immunostaining
(Fig. 5G,H). Increased phospho-Ser3 (pSer3)-cofilin was
also observed when PP1 activity was inhibited in wild-
type MEFs (Fig. 5G). Together, these data provide evi-
dence that Phactr4 acts through cofilin to regulate actin
Phactr4 colocalizes with b1 integrin at the tips
of lamellipodia and regulates integrin signaling
Integrins play key roles incontrolling directional migration
by regulating the actin cytoskeleton (Etienne-Manneville
and Hall 2001; White et al. 2007; Legate et al. 2009). A
conditional null mutation of b1 integrin in mouse ENCCs
shows defects in cell–cell adhesion and directional migra-
tion (Breau et al. 2006, 2009). Moreover, b1 integrin tends
to promote random migration through the Rho–ROCK–
cofilin pathway (Danen et al. 2005). We therefore asked
whether Phactr4 may interact with b1 integrin to co-
ordinate the actin cytoskeleton. First, we examined the
localization of Phactr4 protein in relation to b1 integrin
(ITGB1) in the MEF wound healing assay. This showed
a strong correlation between endogenous Phactr4 and
ITGB1 at the tips of lamellipodium in both wild-type and
mutant cells (Fig. 6A–B). Moreover, Phactr4 localizes to
mature but not nascent focal adhesions (Fig. 6F), sites
where integrin signaling activates one of its downstream
targets, FAK (Legate et al. 2009).
We then tested whether Phactr4 function is required for
mutant MEFs, compared with wild type, showed an in-
crease of phospho-FAK (pFAK) (Figure 6C,D), suggesting
Phactr4 may serve as a negative regulator of integrin
signaling. We also examined whether alteration in integrin
signaling would modulate other signaling pathways such
as ERK and AKT. However, this effect was specific, as no
Phactr4humdyMEFs. (A,B) Trajectories of mi-
gratory wild-type MEFs (A) or mutant MEFs
(B) in a wound healing assay. Cells tracked
over 15 h at 3-min intervals. The track is
color-coded to indicate relative time point.
(C,D) Quantification of persistence (C) and
speed (D) of migrating cells in a wound heal-
ing assay. (E,F) MEFs transfected with Lifeact-
EGFP construct. Wild-type cells show one pre-
dominant lamellipodia (E), whereas mutant
cells display increased numbers of small ran-
dom lamellipodium and retraction fibers (F).
Arrowheads show the lamellipodia. (G,H)
Quantification of size (G) and number (H) of
lamellipodium. Data are expressed as mean 6
SD in three independent experiments. (**) P <
0.01; (***) P < 0.001, Student’s t-test.
Phactr4 and PP1 control ENS directional migration
GENES & DEVELOPMENT 75
significant change in phospho-Erk (pERK) or phospho-Akt
(pAKT)wasobserved (Figure6C,D),nor wasthereachange
in phospho-Numb (data not shown), another target of
PP1(Nishimura and Kaibuchi 2007). The increase in integ-
rin signaling is not due to up-regulation of integrin expres-
sion, as ITGB1 protein or mRNA level is not changed in
either MEFs or sorted ENCCs (Fig. 6C,E). Furthermore, in
a 1-h wound healing assay in the presence of GRGDTP
peptide to block integrin activity or a ROCK inhibitor
(Y27632), pSer3-cofilin levels were markedly decreased,
indicating that the abnormal increase in phospho-cofilin
in Phactr4humdymutant cells is dependent on both b1
integrin and ROCK activity (Fig. 7A). RGD treatment,
but not ROCK inhibitor, also decreased pFAK, suggest-
ing that the increase in pFAK is due to increased integrin
activity in mutant cells (Fig. 7A). Together, these results
provide evidence that Phactr4 controls cofilin phosphor-
ylation by down-regulating the activity of the b1 integ-
Rescue of ENCC directional migration by inhibition
of integrin or ROCK activity
Given the mechanistic relationship defined above and
the abnormal regulation of b1 integrin signaling and
phospho-cofilin in Phactr4 mutant cells, we sought to
directly determine whether the random migration and
loss of persistent movement was a consequence of in-
creased b1 integrin signaling. Indeed, treatment of mutant
MEFs with GRGDTP peptide to block interaction of
integrin with its ECM ligands, b1 integrin function-
blocking antibody (clone Ha2/5), or the ROCK inhibitor
restored persistent migration and rescued the formation
of a single large lamellipodium (Fig. 7B,C; Supplemental
Movies S13–S17). Moreover, cell–cell adhesion was also
rescued with RGD treatment, as cells migrated collec-
tively to close the wound (Fig. 7B; Supplemental Movie
S16). We also tested whether ENCC migration in vivo
could be rescued. Treatment of mutant hindgut organ
cultures with ROCK inhibitor showed a normalization of
directed cell movement and partial rescue of chain migra-
tion (Fig. 7D,E; Supplemental Movies S18–S20). Even
more strikingly, GRGDTP peptide treatment strongly
rescued mutant ENCC migration, resulting in restoration
of chain migration and normalized persistence and net
speed (Fig. 7D,E; Supplemental Movie S20), reminiscent of
wild-type ENCC migration (Fig. 2A). Thus, the loss of
Phactr4-dependent directional migration is due to an up-
regulation of ITGB1 signaling, which promotes random
migration. Collectively, these data reveal a novel role for
Phactr4 in controlling directional migration and that
Phactr4 acts at the lamellipodia to mediate integrin signal-
ing through the ROCK–cofilin pathway.
The coordinated migration of enteric neurons is essential
for their correct positioning and proper integration to
form the functional neuronal network of the mature ENS.
However, relatively little is known about the molecular
regulation of this collective cell migration. Here we identify
Phactr4 as a novel regulator of ENCC migration. Moreover,
we discovered a mechanistic link between Phactr4-medi-
ated and integrin-dependent actin cytoskeleton dynamics
(A,B) Myc-tagged Phactr4 wild-type (A) or humdy (B) construct
was transfected into wild-type or mutant MEFs, respectively.
MEFs were fixed and stained with anti-Myc antibody, phalloi-
din, and Hoechst, showing Phactr4 localization to the lamelli-
podium. (C–F) Detection of endogenous Phactr4 protein, with an
anti-Phactr4 antibody (green) showing colocalization with cofi-
lin (red) at the leading edge of lamellipodium in wounded wild-
type (D) or mutant (E) MEFs. The arrow shows colocalization at
the lamellipodium. (G) MEFs were grown to confluency in
a laminin-coated dish for 36 h and then wounded extensively
(evenly spaced wounds, 500 mm apart). Cells were allowed to
migrate into the wound for 1 h. Where indicated, 0.1 mM OA or
DMSO was added to wild-type MEFs during the wound healing
period. Western blot analysis of total lysates with the indicated
antibodies is shown. Quantification of protein expression
showed 46% increase of pSer-cofilin in mutant and 26% in-
crease in OA-treated wild-type cells. n = 6; (*) P < 0.05. (H)
Immunostaining of pSer3-cofilin in wounded wild-type (top
panel) and mutant (bottom panel) MEFs.
Cofilin activity is disturbed in Phactr4humdyMEFs.
Zhang et al.
76GENES & DEVELOPMENT
(Supplemental Fig. S6). Phactr4humdy/humdymutant em-
bryos exhibit intestinal hypoganglionosis. This defect is
independent of ENCC specification, proliferation, or
differentiation, and instead is the result of a defect in
ENCC migration. One of our key findings is that the
Phactr4humdymutation does not decrease the velocity of
wounding, MEFs were fixed and stained with anti-Phactr4 (green) and anti-ITGB1 (red) antibodies and Hoechst (blue). Phactr4
colocalizes with ITGB1 at the leading edge of lamellipodium in wild type and mutant. (C) MEFs were grown on laminin and then
wounded for 1 h. Western blot analysis of total lysates with the indicated antibodies is shown. (D) Quantification of protein expression
based on experiments such as shown in C showed 30% increase of pFAK in mutant cells. n = 6; (*) P < 0.05. (E) ITGB1 mRNA level by
quantitative RT–PCR of mRNAs isolated from MEFs or FACS-sorted ENCCs shows no significant change in RNA levels. n = 3. (F)
MEFs were plated on a fibronectin-coated coverslip for 15 min and 90 min, and stained with antibody against Phactr4 (green), focal
adhesion marker Vinculin (red), and nuclei marker Hoechst (blue). Phactr4 is localized to mature, but not nascent, focal adhesions in
both wild-type and mutant cells.
Phactr4 colocalizes with b1 integrin at the tip of lamellipodia and regulates integrin signaling. (A,B) Four hours after
Phactr4 and PP1 control ENS directional migration
GENES & DEVELOPMENT77
an individual cell, but does significantly reduce the per-
sistence and directionality of migration, which results in
disrupted chain migration both in vivo and in vitro. The
Phactr4humdymutation does not inhibit formation or
retraction of cell protrusions, but the orientation and
direction of the cell protrusions are significantly disrupted.
Mechanistically, Phactr4 protein colocalizes with b1 integ-
rin and cofilin at the protrusions and is found at mature
indicated, 5 mM Y27632, 100 mg/mL GRGDTP, or vehicle control was included during the wound healing period. Cells were lysed, and
the cellular content of pSer3-cofilin, phospho-Y925-FAK, and b-tubulin relative to total cofilin and FAK was determined by Western
blotting. Quantification of protein expression based on experiments such as shown. n = 3; (*) P < 0.05; (**) P < 0.01. (B) Confluent MEF
monolayers were wounded, and the cells were allowed to migrate into the wound in the presence of 5 mM Y27632, 100 mg/mL
GRGDTP, 10 mg/mL b1 integrin blocking antibody (Ha2/5 clone, Supplemental Movie S17), or vehicle control (DMSO). The cells were
imaged every 3 min for 15 h, and then tracked by Imaris software. Representative trajectories of migrating cells (top panels) and selected
phase-contrast images showing lamellipodia morphology of migrating cells (bottom panels). (C) Quantification of cell persistence. n >
300 track plots; (**) P < 0.01; (***) P < 0.001. Data are expressed as mean 6 SD. (D) Still images from time-lapse movies of E12.5
Phactr4humdy/humdy;RetTGM/+hindgut explants treated with DMSO, 20 mM Y27632, or 1 mg/mL GRGDTP and then imaged every 3
min for 16 h. Time is noted in hours. (Top panels) Cell trajectories were color-coded to indicate the relative time point. (E)
Quantification of cell persistence and net speed. (*) P < 0.05. Data are expressed as mean 6 SD in three independent experiments.
Rescue of random migration both in vitro and in vivo. (A) MEFs were grown on laminin and then wounded for 1 h. Where
Zhang et al.
78 GENES & DEVELOPMENT
focal adhesions, and loss of Phactr4 function results in
increased integrin signaling and increased phosphorylation
of FAK and cofilin. Thus, Phactr4 regulates integrin sig-
naling and cofilin activity, and the coordination of these
activities by Phactr4 controls polarized protrusion and
directional migration. Most strikingly, the Phactr4humdy
ENCC migration defects were rescued by inhibiting
integrin function with an RGD peptide or by inhibiting
ROCK activity, indicating that Phactr4 acts via integrin-
mediated cofilin signaling and this functional relation-
ship is essential for directed ENCC migration.
Our work provides in vivo evidence that Phactr4 regu-
polarized morphology, while Phactr4 mutant cell shape
is greatly altered, with an increased number of random
protrusions around the circumference of the cell. Studies
in cultured cells of other Phactr family members support
our in vivo results. Phactr3 (scapinin) enhances cell motil-
ity by interacting with the actin cytoskeleton (Sagara et al.
2009). Moreover, each Phactr family member, when over-
expressed, leads to a change in cell shape and cell pro-
trusions of variable length and direction (Favot et al. 2005).
Our in vivo loss of Phactr4 function results also show
dramatic changes in cell shape and cell protrusions, in-
dicating that the level and/or localization of the Phactr
proteins are critical in regulating the organization of the
actin cytoskeleton. Indeed, Phactr4 is specifically local-
ized to cell protrusions, where the actin cytoskeleton is
actively remodeled during directed cell migration. To-
gether, the data indicate that the Phactr family proteins
have a common feature of modifying cell morphology to
affect cell motility.
The yeast Phactr4 homolog is Afr1, and it also regulates
actin dynamics. There is a specific Afr1 mutation that
disrupts PP1 binding and results in abnormal budd-
ing versus polarized budding in wild-type budding yeast
(Bharucha et al. 2008). In yeast, Afr1 brings PP1 to the
septin cytoskeleton. Previously, we showed that mouse
Phactr4 binds to actin and PP1, and the humdy mutation
specifically disrupts interaction of Phactr4 with PP1 (Kim
et al. 2007). This unique allele has served to reveal the in
vivo functions of Phactr4 and PP1 in cell cycle regulation
and cell migration. During neural and eye develop-
ment, Phactr4 helps retain PP1 in the cytoplasm to
control the activity of PP1 toward one of its targets, the
retinoblastoma protein (Kim et al. 2007). During ENCC
migration, we postulate that Phactr4 bridges PP1 and actin
to regulate actin cytoskeletal dynamics during directional
as well as pharmacological loss of PP1 function, impairs
ENCCmigration and causes undirected cell protrusions. It
is intriguing to speculate that Phactr4, through its locali-
zation at lamellipodia and focal adhesions, serves to pro-
vide subcellular substrate specificity to PP1. We show that
Phactr4 colocalizes with cofilin and cofilin phosphoryla-
tion increases dramatically when Phactr4 cannot bind
PP1. Cofilin activity is important for directional cell migra-
tion by maintaining a polarized actin cytoskeleton (Dawe
et al. 2003; Ghosh et al. 2004; Mouneimne et al. 2004). Here
we show that the random migration of Phactr4humdycells is
associated with increased levels of inactive phosphorylated
cofilin. Rho, Rac, and Cdc42 can activate LIM kinase,
which phosphorylates cofilin at Ser3 to inhibit its activity
(Arber et al. 1998; Maekawa et al. 1999), whereas protein
phosphatases such as PP1 serve to dephosphorylate cofilin
to enhance its activity. In our in vivo studies, the Rho/Rho
kinase pathway is responsible for cofilin phosphorylation
downstream from Phactr4 as mutant cells are rescued by
ROCK inhibition, resulting in persistent migration with
increased cofilin activity. Furthermore, inhibition of PP1
activity by OA stimulates an increase in cofilin Ser3 phos-
phorylation and causes random migration. Together, our
findings provide a new pathway by which Phactr4 and
PP1 act to regulate cofilin activity, which is required for
directed collective cell migration in vivo.
The orientation of cell membrane protrusions deter-
mines the direction and behavior of a migrating cell.
Intracellular signaling pathways at the leading edge that
control actin cytoskeleton remodeling can therefore con-
tribute to directional migration (Petrie et al. 2009). Integ-
rins play a key role in sensing external cues, such as
chemoattractants or wounds. Integrin signaling activates
FAK as well as PI3K and MAPK pathways. In addition,
integrin and its coreceptors can mediate adhesion forma-
tion, and the formation of new adhesions at the leading
edge can contribute to directional migration, in part by
modulating RhoGTPase activity to control protrusion
formation (White et al. 2007). The specific localization
of Phactr4 to membrane protrusions, coupled with the
role of Phactr4 in regulating integrin signaling, allows the
polarized activation of integrin signaling and adhesion
formation at the leading edge of cells. Here we show that
Phactr4 is a novel negative regulator of integrin signaling
and propose that Phactr4 bridges external signals with the
regulation of actin dynamics to reshape the cell and direct
its movement (Supplemental Fig. S6). It is interesting that
loss of one of the integrin-interacting proteins, integrin-
linked kinase (ILK), also shows defects similar to Phactr4
at focal adhesion sites. The absence of a-parvin or muta-
tions in the a-parvin-binding domain of ILK causes abnor-
mal contraction and cells fail to extend a persistent leading
edge, leading to random migration in smooth muscle cells
and collecting duct epithelial cells. These defects are due
to increased RhoA–ROCK activity that results in elevated
myosin light chain phosphorylation (Lange et al. 2009;
Montanez et al. 2009). Furthermore, inhibition of ROCK
activity can also rescue the defects in smooth muscle cells.
Phactr4 is detected at mature but not nascent focal adhe-
ILK/a-parvin complex found at integrin adhesions (Zhang
et al. 2002). Therefore, it is possible that Phactr4 may
spatially control the function of ILK/a-parvin/b1 integrin
signaling. In vitro studies using podocytes have also shown
that the stability of the ILK/a-parvin complex depends on
possible that Phactr4 acts through PP1 to regulate ILK/
a-parvin/b1 integrin complex stability. The precise molec-
ular mechanism by which the Phactr4/PP1 complex regu-
lates integrin signaling remains open for future research.
Phactr4 and PP1 control ENS directional migration
GENES & DEVELOPMENT 79
Phactr4 not only affects directional migration but also
ENCC chain migration. Phactr4humdymutants show an
increased number of solitary cells both in vivo and in
vitro, and Phactr4 acts cell-autonomously to maintain
cohesive ENCC chain migration, suggesting that Phactr4
is involved in cell–cell interactions. Time-lapse micros-
copy has revealed the importance of intercellular contacts
between migrating ENCCs (Young et al. 2004; Anderson
et al. 2006). L1 is a cell adhesion molecule expressed by
the developing ENS, and L1 inhibition causes ENCCs to
separate from their chains and became solitary (Anderson
et al. 2006). Very little is known about the molecular
regulation of ENCC migration beyond the proteins L1,
b1-integrin, and MMP-2, which are involved in cell–cell/
cell–matrix interactions and GDNF/RETsignaling. Here
we connected the function of Phactr4 with integrin; future
studies may reveal an interconnection with L1 and/or
MMP-2. Together, our studies provide the first in vivo
evidence for a key intracellular regulator of cytoskeletal
rearrangementsneeded forENCCcollective cellmigration
to and along the gut. Our results demonstrate a previously
unknown pathway in ENCC collective migration in vivo
human genetic studies of HSCR.
Materials and methods
Mouse strains and genotyping
Phactr4humdymutantembryos were genotyped withSSLPmarkers
(Kim et al. 2007), specifically D4ski4010L, D2ski4010R, D4ski55-
50L, and D4ski55-50R (Supplemental Table S1). The Phactr4humdy
mutation was maintained on C3H/HeNCrl background for >10
generations. RetTGMwas crossed into C3H/HeN background for
five generations before crossing with Phactr4humdy. RetTGMgeno-
type was determined by PCR analysis (Enomoto et al. 2001).
Organ cultures and time-lapse imaging
For collagen gel gut explant cultures, E13.5 proximal hindgut
segments were placed on a glass-bottomed dish (MatTek) coated
with 1 mg/mL 3D collagen gel (R&D Systems) with 10 ng/mL
GDNF (US Biological) as described (Young et al. 2001). To inhibit
PP1, explants were cultured for 2 d, and then 20 nM OA (Sigma)
was added to the explants for 1 h and then the explants were
washed with PBS followed by addition of culture medium. For
imaging, explants were placed in a heat- and humidity-controlled
LSM510 Meta confocal microscope. All images were acquired
with a 103 lens c-Apochromat NA 1.2. Time intervals for live-
figure and movie legends.
For ex vivo time-lapse imaging, E12.5 gut segments contain-
ing RetTGMGFP+cells were prepared as suspended explants as
described (Hearn et al. 1999) and cultured in a MatTek glass-
bottomed dish in DMEM with 10% fetal bovine serum and
0.075% penicillin/streptomycin. The region to be imaged was
suspended across a ‘‘V’’ cut in a piece of black Millipore filter
paper and held in place by attaching the mesentery to filter
paper. Up to six gut explants were imaged by time lapse using
a Zeiss Axioskop motorized stage microscope equipped with
a heated stage (Zeiss). Images were captured as described in the
Supplemental Material inthe figure and movie legends. To inhibit
PP1, 100 nM OA was added to the culture medium for 1 h or 3 h,
and then the medium was removed and fresh medium was added.
GRGDTP peptide (1 mg/mL; Calbiochem) or 20 mM Y27632
(Calbiochem) were added to the culture medium throughout the
period of live imaging.
For NCC emigration analysis, the vagal region of neural tubes
from E9.0 embryos was dissected and digested in 2 mg/mL
Dispase II (Roche) as described (Newgreen and Minichiello
1995). Explants were placed on a fibronectin-coated (50 mg/mL;
Sigma) 14-mm coverslip and incubated in 150 mL of complete
culture medium for 24 h.
MEFs were isolated from +/+ and Phactr4humdy/humdyE13.5
embryos as described (Abbondanzo et al. 1993).
Additional Materials and Methods are included in the Supple-
We thank Rytis Prekeris for helpful discussions, Heather Young
for helpful suggestions, Helen McBride for advice on gut explant
culture, and members of our laboratory, especially Jianfu Chen
and Carsten Schnatwinkel, for suggestions throughout this work
and for helpful comments on the manuscript. We thank Lori
of the Phactr4humdy;E2f1 embryos. This work was supported by
the Department of Pediatrics, and L.N. is an investigator of the
Howard Hughes Medical Institute. T.-H.K initiated the project
and phenotypic characterization, Y.Z. performed phenotypic
characterization, developed the live imaging and mechanistic
pathway, and wrote the paper. L.N. oversaw the research design
and data analyses and wrote the paper.
Abbondanzo SJ, Gadi I, Stewart CL. 1993. Derivation of embry-
onic stem cell lines. Methods Enzymol 225: 803–823.
Allen PB, Greenfield AT, Svenningsson P, Haspeslagh DC,
Greengard P. 2004. Phactrs 1–4: A family of protein phos-
phatase 1 and actin regulatory proteins. Proc Natl Acad Sci
Ambach A, Saunus J, Konstandin M, Wesselborg S, Meuer SC,
Samstag Y. 2000. The serine phosphatases PP1 and PP2A
associate with and activate the actin-binding protein cofilin
in human T lymphocytes. Eur J Immunol 30: 3422–3431.
Amiel J, Sproat-Emison E, Garcia-Barcelo M, Lantieri F, Burzynski
G, Borrego S, Pelet A, Arnold S, Miao X, Griseri P, et al. 2008.
Hirschsprung disease, associated syndromes and genetics:
A review. J Med Genet 45: 1–14.
Anderson RB. 2010. Matrix metalloproteinase-2 is involved in
the migration and network formation of enteric neural crest-
derived cells. Int J Dev Biol 54: 63–69.
Anderson RB, Turner KN, Nikonenko AG, Hemperly J,
Schachner M, Young HM. 2006. The cell adhesion mole-
cule L1 is required for chain migration of neural crest cells
in the developing mouse gut. Gastroenterology 130: 1221–
Arber S, Barbayannis FA, Hanser H, Schneider C, Stanyon CA,
Bernard O, Caroni P. 1998. Regulation of actin dynamics
through phosphorylation of cofilin by LIM-kinase. Nature
Asai N, Fukuda T, Wu ZQ, Enomoto A, Pachnis V, Takahashi M,
Costantini F. 2006. Targeted mutation of serine 697 in the
Ret tyrosine kinase causes migration defect of enteric neural
crest cells. Development 133: 4507–4516.
Bernstein BW, Bamburg JR. 2010. ADF/cofilin: A functional
node in cell biology. Trends Cell Biol 20: 187–195.
Zhang et al.
80 GENES & DEVELOPMENT
Bharucha JP, Larson JR, Konopka JB, Tatchell K. 2008. Saccha-
romyces cerevisiae Afr1 protein is a protein phosphatase 1/
Glc7-targeting subunit that regulates the septin cytoskeleton
during mating. Eukaryot Cell 7: 1246–1255.
Breau MA, Pietri T, Eder O, Blanche M, Brakebusch C, Fassler R,
Thiery JP, Dufour S. 2006. Lack of b1 integrins in enteric
neural crest cells leads to a Hirschsprung-like phenotype.
Development 133: 1725–1734.
Breau MA, Dahmani A, Broders-Bondon F, Thiery JP, Dufour S.
2009. b1 integrins are required for the invasion of the
caecum and proximal hindgut by enteric neural crest cells.
Development 136: 2791–2801.
Danen EH, van Rheenen J, Franken W, Huveneers S, Sonneveld
P, Jalink K, Sonnenberg A. 2005. Integrins control motile
strategy through a Rho-cofilin pathway. J Cell Biol 169: 515–
Dawe HR, Minamide LS, Bamburg JR, Cramer LP. 2003. ADF/
cofilin controls cell polarity during fibroblast migration.
Curr Biol 13: 252–257.
Druckenbrod NR, Epstein ML. 2005. The pattern of neural crest
advance in the cecum and colon. Dev Biol 287: 125–133.
Enomoto H, Crawford PA, Gorodinsky A, Heuckeroth RO,
Johnson EM, Milbrandt J. 2001. RET signaling is essential
for migration, axonal growth and axon guidance of develop-
ing sympathetic neurons. Development 128: 3963–3974.
Etienne-Manneville S, Hall A. 2001. Integrin-mediated activa-
tion of Cdc42 controls cell polarity in migrating astrocytes
through PKCzeta. Cell 106: 489–498.
Favot L, Gillingwater M, Scott C, Kemp PR. 2005. Overexpres-
sion of a family of RPEL proteins modifies cell shape. FEBS
Lett 579: 100–104.
Geiger B, Bershadsky A, Pankov R, Yamada KM. 2001. Trans-
membrane crosstalk between the extracellular matrix–cyto-
skeleton crosstalk. Nat Rev Mol Cell Biol 2: 793–805.
Ghosh M, Song X, Mouneimne G, Sidani M, Lawrence DS,
Condeelis JS. 2004. Cofilin promotes actin polymerization
and defines the direction of cell motility. Science 304: 743–
Heanue TA, Pachnis V. 2007. Enteric nervous system develop-
ment and Hirschsprung’s disease: Advances in genetic and
stem cell studies. Nat Rev Neurosci 8: 466–479.
Hearn CJ, Young HM, Ciampoli D, Lomax AE, Newgreen D.
1999. Catenary cultures of embryonic gastrointestinal tract
support organ morphogenesis, motility, neural crest cell
migration, and cell differentiation. Dev Dyn 214: 239–247.
Kim TH, Goodman J, Anderson KV, Niswander L. 2007. Phactr4
regulates neural tube and optic fissure closure by controlling
PP1-, Rb-, and E2F1-regulated cell-cycle progression. Dev
Cell 13: 87–102.
Lange A, Wickstrom SA, Jakobson M, Zent R, Sainio K, Fassler
R. 2009. Integrin-linked kinase is an adaptor with essential
functions during mouse development. Nature 461: 1002–
Larsen M, Tremblay ML, Yamada KM. 2003. Phosphatases in
cell–matrix adhesion and migration. Nat Rev Mol Cell Biol
Legate KR, Wickstrom SA, Fassler R. 2009. Genetic and cell
biological analysis of integrin outside-in signaling. Genes
Dev 23: 397–418.
Maekawa M, Ishizaki T, Boku S, Watanabe N, Fujita A, Iwamatsu
A, Obinata T, Ohashi K, Mizuno K, Narumiya S. 1999.
Signaling from Rho to the actin cytoskeleton through protein
kinases ROCK and LIM-kinase. Science 285: 895–898.
McCallion AS, Emison ES, Kashuk CS, Bush RT, Kenton M,
Carrasquillo MM, Jones KW, Kennedy GC, Portnoy ME,
Green ED, et al. 2003. Genomic variation in multigenic
traits: Hirschsprung disease. Cold Spring Harb Symp Quant
Biol 68: 373–381.
Montanez E, Wickstrom SA, Altstatter J, Chu H, Fassler R.
2009. a-Parvin controls vascular mural cell recruitment to
vessel wall by regulating RhoA/ROCK signalling. EMBO J
Mouneimne G, Soon L, DesMarais V, Sidani M, Song X, Yip SC,
Ghosh M, Eddy R, Backer JM, Condeelis J. 2004. Phospholi-
pase C and cofilin are required for carcinoma cell direction-
ality in response to EGF stimulation. J Cell Biol 166: 697–
Newgreen DF, Minichiello J. 1995. Control of epitheliomesen-
chymal transformation. I. Events in the onset of neural crest
cell migration are separable and inducible by protein kinase
inhibitors. Dev Biol 170: 91–101.
Nishimura T, Kaibuchi K. 2007. Numb controls integrin endo-
cytosis for directional cell migration with aPKC and PAR-3.
Dev Cell 13: 15–28.
Okamura Y, Saga Y. 2008. Notch signaling is required for the
maintenance of enteric neural crest progenitors. Develop-
ment 135: 3555–3565.
Oleinik NV, Krupenko NI, Krupenko SA. 2010. ALDH1L1
inhibits cell motility via dephosphorylation of cofilin by
PP1 and PP2A. Oncogene 29: 6233–6244.
Paavilainen VO, Bertling E, Falck S, Lappalainen P. 2004.
Regulation of cytoskeletal dynamics by actin-monomer-
binding proteins. Trends Cell Biol 14: 386–394.
Petrie RJ, Doyle AD, Yamada KM. 2009. Random versus direc-
tionally persistent cell migration. Nat Rev Mol Cell Biol 10:
Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D,
Bista M, Bradke F, Jenne D, Holak TA, Werb Z, et al. 2008.
Lifeact: A versatile marker to visualize F-actin. Nat Methods
Sagara J, Arata T, Taniguchi S. 2009. Scapinin, the protein
phosphatase 1 binding protein, enhances cell spreading and
motility by interacting with the actin cytoskeleton. PLoS
ONE 4: e4247. doi: 10.1371/journal.pone.0004247.
Simpson MJ, Zhang DC, Mariani M, Landman KA, Newgreen
DF. 2007. Cell proliferation drives neural crest cell invasion
of the intestine. Dev Biol 302: 553–568.
Wallace AS, Barlow AJ, Navaratne L, Delalande JM, Tauszig-
Delamasure S, Corset V, Thapar N, Burns AJ. 2009. Inhibition
of cell death results in hyperganglionosis: Implications for
enteric nervous system development. Neurogastroenterol
Motil 21: 768–e49. doi: 10.1111/j.1365-2982.2009.01309.x.
White DP, Caswell PT, Norman JC. 2007. avb3 and a5b1
integrin recycling pathways dictate downstream Rho kinase
signaling to regulate persistent cell migration. J Cell Biol
Yang Y, Guo L, Blattner SM, Mundel P, Kretzler M, Wu C. 2005.
Formation and phosphorylation of the PINCH-1–integrin
linked kinase–a-parvin complex are important for regulation
of renal glomerular podocyte adhesion, architecture, and
survival. J Am Soc Nephrol 16: 1966–1976.
Young HM, Hearn CJ, Farlie PG, Canty AJ, Thomas PQ, New-
green DF. 2001. GDNF is a chemoattractant for enteric
neural cells. Dev Biol 229: 503–516.
Young HM, Bergner AJ, Anderson RB, Enomoto H, Milbrandt J,
Newgreen DF, Whitington PM. 2004. Dynamics of neural
crest-derived cell migration in the embryonic mouse gut.
Dev Biol 270: 455–473.
Zhang Y, Chen K, Tu Y, Velyvis A, Yang Y, Qin J, Wu C. 2002.
Assembly of the PINCH–ILK–CH–ILKBP complex precedes
and is essential for localization of each component to cell–
matrix adhesion sites. J Cell Sci 115: 4777–4786.
Phactr4 and PP1 control ENS directional migration
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