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 & DEVELOPMENT 71
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.
72GENES & 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