RESEARCH ARTICLE
631
Development 137, 631-640 (2010) doi:10.1242/dev.040550
© 2010. Published by The Company of Biologists Ltd
Vitamin A facilitates enteric nervous system precursor
migration by reducing Pten accumulation
Ming Fu1, Yoshiharu Sato1, Ariel Lyons-Warren1, Bin Zhang3, Maureen A. Kane4, Joseph L. Napoli4 and
Robert O. Heuckeroth1,2,5,*
SUMMARY
Hirschsprung disease is a serious disorder of enteric nervous system (ENS) development caused by the failure of ENS precursor
migration into the distal bowel. We now demonstrate that retinoic acid (RA) is crucial for GDNF-induced ENS precursor migration,
cell polarization and lamellipodia formation, and that vitamin A depletion causes distal bowel aganglionosis in serum retinolbinding-protein-deficient (Rbp4–/–) mice. Ret heterozygosity increases the incidence and severity of distal bowel aganglionosis
induced by vitamin A deficiency in Rbp4–/– animals. Furthermore, RA reduces phosphatase and tensin homolog (Pten) accumulation
in migrating cells, whereas Pten overexpression slows ENS precursor migration. Collectively, these data support the hypothesis that
vitamin A deficiency is a non-genetic risk factor that increases Hirschsprung disease penetrance and expressivity, suggesting that
some cases of Hirschsprung disease might be preventable by optimizing maternal nutrition.
INTRODUCTION
The enteric nervous system (ENS) is a complex network of
neurons and glia within the bowel that controls most aspects of
intestinal function (Furness, 2000). Hirschsprung disease
(HSCR), a partially penetrant life-threatening defect affecting
1:5000 children, is caused by the failure of ENS precursor
colonization of the distal bowel (Skinner, 1996). ENS precursors
originate in the vagal, sacral and upper thoracic neural tube before
migrating extensively, proliferating and then differentiating to
form the ENS (Heanue and Pachnis, 2007). Most ENS precursors
arise from the vagal neural tube, enter the foregut and colonize the
bowel in a rostral to caudal progression (Newgreen and Young,
2002a; Newgreen and Young, 2002b). Because molecular
mechanisms required for ENS precursor migration, proliferation
and differentiation are complex, many distinct mutations cause
HSCR. Furthermore, HSCR penetrance and the extent of
aganglionosis are governed by genetic interactions (Amiel et al.,
2008; Cantrell et al., 2004; McCallion et al., 2003; Owens et al.,
2005).
We now present data supporting the hypothesis that nongenetic factors, including vitamin A, crucially determine the
penetrance of HSCR-like distal bowel aganglionosis. Vitamin A
(retinol), an essential nutrient, is a precursor to retinoic acid (RA),
which is generated by short-chain dehydrogenases/reductases and
retinaldehyde dehydrogenases (Raldh) (Maden, 2007; Napoli,
2004). RA binds Rar and Rxr receptors that heterodimerize and
then bind RA response elements (RARE) to regulate target genes
and influence tissue morphogenesis. Recent data suggest that both
Departments of 1Pediatrics, 2Developmental Biology, 3Pathology and Immunology,
and the 5HOPE Center for Neurological Disorders, Washington University School of
Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA. 4Department of
Nutritional Science and Toxicology, University of California, Berkeley, 119 Morgan
Hall, MC#3104, Berkeley, CA 94720, USA.
*Author for correspondence ([email protected])
Accepted 7 December 2009
excess RA (Pitera et al., 2001) and reduced RA synthesis cause
abnormal ENS development, but the molecular and cellular
mechanisms that RA influences are not known. Indeed, although
Raldh2–/– mice develop HSCR-like aganglionosis (Niederreither
et al., 2003), these mice have severe RA deficiency starting at
conception, so it is unclear at what stage RA is required for ENS
morphogenesis. Our new data demonstrate that RA is essential for
efficient ENS precursor migration through the developing bowel
and for lamellipodia formation in migrating cells.
Cell migration is complex, requiring polarization and coordinated
activation of many proteins including RhoA-related GTPases that
regulate the actin cytoskeleton. In particular, Rac1 activation at the
leading edge of migrating cells is crucial for lamellipodia formation.
Recent work (Jaffe and Hall, 2005; Kawauchi and Hoshino, 2008;
Vohra et al., 2007a; Yoshimura et al., 2006) demonstrated that Rac1
activation occurs via a positive feedback loop initiated by
conversion of phosphatidylinositol-2-phosphate (PIP2) to
phosphatidylinositol-3-phosphate (PIP3) via phosphatidylinositol3 kinase (Pik3) (Wang et al., 2002; Wedlich-Soldner and Li, 2003).
This could be initiated by glial cell line-derived neurotrophic factor
(GDNF) binding to its receptor Ret, a kinase commonly mutated in
human HSCR and required for ENS precursor migration (Takahashi,
2001).
Because RA deficiency prevents efficient ENS precursor
migration, we hypothesized that RA is required to maintain signals
needed for cell migration. We show that, in response to GDNF, Pik3
and PIP3 accumulate at the leading edge of migrating enteric crestderived cells and Pten (phosphatase and tensin homolog), an enzyme
that reverses the Pik3 activity, is absent from most actively migrating
ENS precursors. When Rar signaling is blocked, however, polarized
distribution of Pik3, PIP3 and Pten is abolished and Pten levels
dramatically increase in actively migrating cells. This is
accompanied by loss of lamellipodia and reduced migration toward
GDNF. This suggests that RA is required in the developing ENS to
reduce Pten and enhance ENS precursor migration. Collectively,
these data suggest that some cases of Hirschsprung disease might be
preventable by ensuring adequate maternal vitamin A levels during
early gestation.
DEVELOPMENT
KEY WORDS: Enteric nervous system, Migration, Retinoic acid, Lamellipodia, Pten, Hirschsprung disease, Mouse
RESEARCH ARTICLE
MATERIALS AND METHODS
Mice
Rbp4–/– (Quadro et al., 2005) and Ret+/– mice (Enomoto et al., 2001) were
bred >10 generations to C57BL/6. For timed breeding studies, the day of the
vaginal plug was considered as embryonic day 0.5 (E0.5). Wild-type CF-1
mice were from Charles River. Mice were maintained on Purina PICO
irradiated mouse diet 5058 until E7.5. Food was changed to synthetic chow
(El Mel, St Charles, MO, USA) containing sufficient vitamin A (TestDiet
5755; 22.1 IU vitamin A/g) or to vitamin A deficient food (TestDiet 5822;
<0.4 IU vitamin A/g) colored blue or yellow to avoid confusion. Mice were
maintained on synthetic diets until analysis.
Retinoid measurements
RA was quantified using an API-4000 (Applied Biosystems) LC/MS/MS
with atmospheric pressure chemical ionization in positive ion mode. Retinol
and retinyl esters were quantified by HPLC/UV (Kane et al., 2008a). Tissues
were harvested under yellow light, immediately frozen in liquid N2 and kept
at –80°C until assay. Samples were homogenized on ice, extracted and
analyzed as described (Kane et al., 2005; Kane et al., 2008b). Total protein
was determined by Bradford (Bio-Rad).
Slice culture
E12.5 CF-1 midgut sections (300-400 m length) from 400 m proximal to
the cecum were cultured on fibronectin-coated (250 g/ml) dishes in OptiMEM (Invitrogen), glutamine (2 mM), penicillin 100 IU/ml and
streptomycin 100 g/ml. Immediately after plating, cells were treated with
RA (10–7 M, Sigma, St Louis, MO, USA) or BMS493 (10–5 M, generously
provided by Dr Chris Zusi at Bristol-Myers Squibb). GDNF (100 ng/ml) was
added to cultures three hours later. Cultures were maintained for 16 hours
before fixation [4% paraformaldehyde (PFA), 10 minutes, 25°C].
Boyden chamber
Transwell supports (8.0 m pore size; 0.33 cm2 area; Corning 3422; Fisher
Scientific) were coated on both sides with 10% Matrigel (Fisher Scientific)
in PBS (4°C, 18 hours) and rinsed with PBS. Neural crest medium
[Dulbecco’s modified Eagle medium (DMEM), 10% chick embryo extract,
1% N2 supplement, 2% B27 supplement, penicillin 100 IU/ml, streptomycin
100 g/ml, -mercaptoethanol 50 M, all-trans-RA 35 ng/ml (~10–7 M),
bFGF 20 ng/ml, EGF 20 ng/ml] was added to the bottom and top chambers
with 50,000 dissociated E12.5 CF-1 gut cells/well in the top chamber,
prepared as previously described (Fu et al., 2006). Immediately after plating,
BMS493 (10–5 M) or vehicle (2 l ethanol) was added to the top well. Cells
were incubated 2 hours before adding GDNF (100 ng/ml) to the top or
bottom chambers and then 16 hours to allow migration. Cells on top of the
membrane were removed with Kimwipes. Cells on membrane bottoms were
fixed (4% PFA, 20 minutes, 25°C), blocked (1% BSA/PBS, 40 minutes,
25°C) and processed for immunohistochemistry.
Whole gut culture
The entire E11.5 CF-1 gastrointestinal tract was pinned to 2.5% agarose with
4-0 stainless steel filaments (Ethicon) and cultured in DMEM, 10% fetal calf
serum, penicillin 100 IU/ml and streptomycin 100 g/ml (Fu et al., 2006)
with or without all-trans-RA (10–7 M, Sigma) or BMS493 (10–5 M). Cultures
were maintained for 48 hours before fixing (4% PFA, 30 minutes, 25°C).
Dissociated cell culture
E12.5 CF-1 ENS precursor cells were maintained in culture as previously
described (Sato and Heuckeroth, 2008a). For Ret pixel intensity
measurements, ENS precursors from E13.5 Rbp4–/– mice deprived of
vitamin A starting at E7.5, or from wild-type C57BL/6 on a vitamin Acontaining diet, were cultured briefly (2 hours) before fixation.
Immunohistochemistry
After fixation, cells and organs were kept in TBST (100 mM Tris, 150 mM
NaCl, 0.2% Triton X-100) for 20 minutes at 25°C, blocked with 4% donkey
serum/TBST for 1 hour at 25°C and then incubated with primary antibody
for 18 hours at 4°C. The primary antibodies used were: TuJ1 (rabbit,
Covance, 1:100), Ret (goat; Neuromics, 1:100), P75NRT antibody (rabbit,
G323A Promega, 1:700), Alexa Fluor 488 phalloidin (mouse, Invitrogen,
Development 137 (4)
1:40), Pten (mouse, Covance, 1:40), PIP3 (mouse, Echelon Biosciences,
1:40), p85 (B-9) (mouse, Santa Cruz, 1:40) and Phox2b (rabbit, a generous
gift from Jean-François Brunet, 1:1000). Antibodies were visualized using
donkey anti-goat Alexa 594 (1:200) and donkey anti-rabbit Alexa 488
(1:100, Molecular Probes). Images were obtained with an Olympus BX60
microscope and Axiocam and AxioVision software (Zeiss). NIH Image J
was used for pixel intensity measurements of Ret immunohistochemistry.
Measurements were made in the cell body of p75NTR+ cells and we
subtracted background signal intensity.
Pten overexpression vector
Full-length Pten cDNA (Open Biosystems, Clone ID 5038272) obtained
using XbaI and XmaI was cloned into a modified version (FM) of the FUIV
[ubiquitin promoter – gene of interest-IRES-enhanced YFP (Venus)] (Araki
et al., 2004) using AgeI and NheI sites. Virus was produced by Washington
University Hope Center Viral Vectors Core using 293T cells, pCMV-G,
pMD-Lg, and RSV-REV (Li and Rossi, 2005). Viral titers were 1.4108
(TU/ml) for FM-Pten and 1108 (TU/ml) for FM.
Cell migration assay for virus-infected cells
E12.5 CF-1 whole gut was digested with collagenase (0.2 mg/ml) and
dispase (0.2 mg/ml) for 10 minutes at 37°C, triturated, centrifuged and
resuspended in the neural crest medium (see Boyden Chamber). Three
microlitres of virus was added to 106 cells in 50 l media. To prepare the
collagen gel, 0.25 volume of 5 DMEM was added to 1 volume of type I
rat tail collagen (4 mg/ml) in 0.02 N acetic acid (BD Biosciences). pH was
adjusted using 0.8 M NaHCO3 (final concentration 8 mM) and 200 mM
NaOH (final concentration 1.5 mM) to form gel and diluted with neural crest
medium to 2 mg/ml collagen. Fifty microliters of gel was added to 50 l
cells/virus mixture and the gel was cultured in 96-well U-bottom plates (BD
Biosciences) for 48 hours at 37°C. The cell-gel pellet was then transferred
to 4-well dishes and embedded in 1% collagen gel containing GDNF (200
ng/ml). After 24 hours, Venus+ cells that migrated from the cell-gel pellet
were counted after fixation in 4% PFA for 20 minutes at 25°C and
immunohistochemistry.
Statistical analyses
All experiments were performed at least three times and analyzed by t-test
or ANOVA analysis for multiple comparisons. P<0.05 was considered
significant. Data show mean ± s.e.m.
RESULTS
Vitamin A deficiency causes HSCR-like distal
bowel aganglionosis in vivo
To determine whether vitamin A (retinol) is required for efficient
bowel colonization by ENS precursors, we used serum retinolbinding-protein-deficient (Rbp4–/–) mice (Quadro et al., 1999;
Quadro et al., 2005). Rbp4 is required for transport of hepatic retinol
to peripheral tissues, so tissue retinoids in Rbp4–/– mice are primarily
derived from dietary retinyl ester incorporated into chylomicrons.
Thus, unlike wild-type mice that have large hepatic retinol stores
(Moore and Holmes, 1971) and are difficult to deplete (ClagettDame and DeLuca, 2002; Wolf, 1984), Rbp4–/– mice can be rapidly
depleted of peripheral tissue retinoids by removing vitamin A from
the diet. For these studies, Rbp4–/– mice were deprived of dietary
retinol starting at E7.5, 48 hours before crest-derived precursors
enter the bowel. Mice were analyzed at E14.5, shortly after ENS
precursors normally completely colonize the colon. The entire small
bowel and colon were stained using antibodies to Ret and neuronspecific beta-III tubulin (TuJ1, a neuronal lineage marker; Fig. 1).
These studies demonstrated striking distal bowel aganglionosis in
Rbp4–/– mice deprived of dietary retinol, but an apparently normal
ENS in Rbp4–/– mice maintained on retinol containing food (Fig.
1A). In fact, under vitamin A-sufficient conditions, only 2 out of 34
Rbp4–/– failed to completely colonize the colon with ENS precursors
at E14.5. By contrast, almost all Rbp4–/– mice fed vitamin A-
DEVELOPMENT
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RA enhances ENS precursor migration
RESEARCH ARTICLE
633
deficient food starting at E7.5 had distal bowel aganglionosis
(31/33). Some animals had total colonic aganglionosis (4/33). Thus,
simple diet-induced retinol deficiency appears to cause an HSCRlike phenotype.
Because retinol depletion causing loss of Ret and/or TuJ1
immunoreactivity in distal bowel ENS precursors is a potential
concern, we performed double-label immunohistochemistry with
antibodies to Ret and Phox2b in Rbp4–/– mice fed either retinoldeficient or -sufficient diets. Phox2b and Ret were expressed in the
same cells at the migration wavefront under both conditions (Fig.
1E). Furthermore, Ret immunoreactivity in ENS precursors
measured by pixel intensity demonstrated equal Ret abundance in
cells from wild-type C57BL/6 versus retinol-deprived Rbp4–/– mice
(WT: 26.61.3; retinol-deprived Rbp4–/–: 24.90.7, P0.21).
Therefore, immunohistochemistry for Ret, TuJ1 and Phox2b all
indicate that the distal bowel is not colonized in E14.5 Rbp4–/– mice
deprived of retinol starting at E7.5.
Rar signaling is required for ENS precursor
migration
To determine whether Rar signaling affects ENS precursor
migration, we used a modified Boyden chamber (Boyden, 1962) to
study ENS precursors at E12.5, the time that the distal colon is
colonized. Dissociated gut cells placed in the top chamber were
treated for 2 hours with control media containing RA or media
Table 1. E12.5 fetal retinoid levels
VAD (–)
VAS (+)
Retinol
Retinyl ester
Retinoic acid
4.500.44*
8.050.74
2.970.36*
5.150.49
0.0570.004*
0.120.008
Fetal retinoid levels in Rbp4–/– mice. Mice were changed at E7.5 to vitamin Acontaining [VAS (+)] or vitamin A-deficient diets [VAD (–)] and maintained on these
diets until analysis at E12.5. Total fetal retinol, retinyl ester and retinoic acid (RA)
levels were measured. Data are reported in nmol retinoid/gram protein s.e.m.
n22 VAD (–) mice; n16 VAS (+) mice. *, P<0.05 compared to VAS (+).
DEVELOPMENT
Fig. 1. Vitamin A deficiency causes distal bowel aganglionosis
in Rbp4–/– mice. (A)Rbp4–/– mice fed vitamin A-containing food
(top), had a dense network of neurons and fibers in the distal bowel
and almost all had ganglion cell bodies at the end of the colon.
Diagrams indicate positions of the most distal TuJ1+ ganglion cell
body or nerve fiber at E14.5 after feeding vitamin A-sufficient or
-deficient diets starting at E7.5. Each oval represents one mouse.
Representative TuJ1-stained colon images are shown. Vitamin Adeprived mice have distal colon aganglionosis and reduced ENS
density in the colonized colon (bottom). (B)Ret/TuJ1 double-label
immunohistochemistry at the wavefront of ENS precursors of an
E13.5 Rbp4–/– mouse maintained on vitamin A-deficient food starting
at E7.5. The Ret signal is less intense than TuJ1, but staining overlaps
significantly and the wavefront visualized by Ret or TuJ1 antibodies is
at the same position (n4 vitamin A-sufficient mice, n8 vitamin Adepleted mice). (C)Even when maintained on vitamin A, many
Rbp4–/–;Ret+/– mice have distal bowel aganglionosis. When deprived
of vitamin A starting at E7.5, there is more extensive aganglionosis.
(D)Summary of bowel colonization in mutant mice. (E)Double-label
immunohistochemistry for Ret and Phox2b at the migration
wavefront of E13.5 Rbp4–/– mice maintained on a vitamin Acontaining or -deficient diet starting at E7.5 shows colocalization of
Ret and Phox2b-expressing cells. In all images, distal bowel is to the
right. Scale bars: 100m in A; 50m in B,E.
Mild vitamin A deficiency increases HSCR-like
disease in genetically predisposed mice
To test the hypothesis that retinol depletion could increase HSCR
penetrance in genetically predisposed mammals, we analyzed the
ENS in Rbp4–/–;Ret+/– double-mutant mice. Ret mutations are the
most commonly identified cause of human HSCR and Rbp4–/–
animals have mild vitamin A deficiency even when maintained on
retinol containing food (Wendler et al., 2003). Furthermore, Ret+/–
mice are predisposed to aganglionosis (McCallion et al., 2003), but
have almost normal ENS anatomy (Gianino et al., 2003).
Rbp4–/–;Ret+/– mice were switched to vitamin A-containing or
-deficient synthetic food starting at E7.5. Even when maintained on
vitamin A containing food, many Rbp4–/–;Ret+/– animals have distal
bowel aganglionosis (10/20) at E14.5 (Fig. 1C,D). Furthermore,
dietary vitamin A deprivation caused more extensive bowel
aganglionosis in Rbp4–/–;Ret+/– than in Rbp4–/– mice. Collectively,
these data strongly support the hypothesis that vitamin A deficiency
is a nutritional risk factor for HSCR. To evaluate the severity of
retinoid depletion needed to cause ENS defects, we measured fetal
retinoids in Rbp4–/– mice at E12.5, when migrating ENS precursors
have reached the mid-colon and are entering distal bowel. These
analyses demonstrated that switching to vitamin A-deficient diets at
E7.5 caused 42-53% reductions in fetal retinoids at E12.5 (Table 1).
Based on these findings, we investigated molecular and cellular
mechanisms to explain our observations.
634
RESEARCH ARTICLE
containing a well-characterized pan-Rar antagonist, BMS493.
GDNF was then added to the bottom chamber to induce ENS
precursor migration through the porous membrane. Cells were
cultured 16 hours before Ret immunohistochemistry (Fig. 2).
BMS493 competitively blocks all Rar isotypes (,  and ) and has
been used in embryo culture and in vivo to block retinoid action
(Chazaud et al., 1999; Chazaud et al., 2003; Hochgreb et al., 2003;
Mollard et al., 2000; Sato and Heuckeroth, 2008; Wendling et al.,
2000; Wendling et al., 2001). Animals treated with BMS493
develop the same spectrum of defects seen in vitamin A deficiency.
GDNF in the bottom chamber markedly increased Ret+ ENS
precursor migration through the membrane. BMS493 completely
blocked chemoattractive effects of GDNF (Fig. 2E). Control
experiments with GDNF in the top chamber only did not increase
Ret+ cell migration through the membrane, suggesting that GDNF
acts as a chemoattractant rather than simply increasing motility or
proliferation.
Development 137 (4)
Time-lapse microscopy confirmed that BMS493 did not influence
cell survival in these short-term studies. Double-label
immunohistochemistry for Ret and TuJ1 demonstrated no effect
of Rar signaling on neuronal differentiation or the level of Ret
expression (see Fig. S2 in the supplementary material). Parallel
studies of the effects of BMS493 on ENS precursor migration in
whole gut culture confirmed that Rar antagonism reduces distal
bowel colonization. In this case, the position of the most distal
ganglion cell is the same in the presence or absence of BMS493,
but the density of ENS precursors in the distal bowel is
dramatically reduced by Rar antagonism (Fig. 4). BMS493 also
Fig. 2. RA is required for efficient GDNF-induced ENS precursor
migration. (A-D)E12.5 dissociated gut cells were placed in the top
wells of Boyden chambers in retinoic acid (RA)-containing media with
(D) or without (A-C) BMS493. After 2 hours, media with (C,D) or
without (A,B) GDNF (100 ng/ml) was added to the bottom chamber. As
a control (B), GDNF was also added to the top, but not the bottom
chamber. After 16 hours, ENS precursors that migrated through
membranes were visualized using DAPI (blue) and Ret
immunohistochemistry (red). (E)Analysis of Ret+ cells that migrated
through membranes (n9 for each condition). Scale bar: 100m. *,
P<0.0001 compared with GDNF in the bottom chamber.
Fig. 3. BMS493 reduces ENS precursor migration and lamellipodia
formation in slice cultures. (A-C)E12.5 mouse mid-gut slices were
cultured to allow crest-derived cells to migrate onto the dish in
response to GDNF. Cultures were maintained for 16 hours without
added retinoic acid (RA) (A), with added RA (B), or with BMS493 (C)
before Ret immunohistochemistry (red) and DAPI staining. (D)Distance
from the edge of the explant to the most distal Ret+ cells [4
measurements/explant (see Fig. S1 in the supplementary material);
n40 explants for each condition]. (E-L)Ret antibody (red) and
Phalloidin (green) double-labeling demonstrate lamellipodia at the
leading edge of crest-derived cells migrating from E12.5 mid-gut slices
maintained in GDNF (E,I) or GDNF plus RA (F,J). Culture with GDNF plus
BMS493 (G,K) or GDNF plus Pik3 inhibitor LY294002 (H,L) dramatically
reduces lamellipodia formation and causes Ret+ cells to adopt very
unusual shapes. The arrow in I shows a typical lamellipodium (n>320
cells analyzed; 12 slices per condition). (I-L)Higher magnification images
of E-H. (M)Percentage of Ret+ cells at the leading edge of the
migration wavefront with lamellipodia. Scale bars: 100m in A-C,E-H;
25m in E-H. *, P<0.0001 versus GDNF.
DEVELOPMENT
Rar antagonism slows ENS precursor migration in
organ culture
To further evaluate Rar effects on ENS precursor migration, E12.5
mid-small bowel slices were cultured for 16 hours in media with
GDNF plus RA, or GDNF plus the Rar antagonist BMS493.
Under these conditions, ENS precursors migrate from gut
explants onto culture dishes and were visualized by video
microscopy before Ret immunohistochemistry. Treatment with
BMS493 considerably reduces migration of Ret+ ENS precursors
from gut explants in response to GDNF (Fig. 3A-D). Adding RA
had little effect on ENS precursor migration, suggesting that
explants make enough RA to support migration in this assay.
RA enhances ENS precursor migration
RESEARCH ARTICLE
635
Fig. 4. Rar signaling is required for colon
colonization in organ culture. (A-C) E11.5
mouse gut cultured for 48 hours in control media
(A), with added RA (B) or added BMS439 (C) was
stained with Ret (A-C) and PGP9.5 (A-C)
antibodies. (A-C) Merged images. (D,F)Ret+ cell
density in the terminal 200m of colonized colon
was measured using a grid. (E)Position of the most
distal Ret+ cell in colon relative to colon length.
n15 control, 15 RA- and 27 BMS493-treated gut
explants. *, P<0.001 versus control. Scale bar:
100m.
Rar antagonism causes lamellipodia loss in
migrating ENS precursors
To determine why Rar antagonism reduces ENS precursor
migration, we examined Ret+ cells that migrated furthest from
E12.5 mid-small bowel slices. Cells maintained with RA had welldefined lamellipodia at the leading edge (Fig. 3F,J), but lamellipodia
were uncommon in BMS493-treated cultures (Fig. 3G,K). Analysis
of Ret antibody and Phalloidin-stained cells confirmed that BMS493
markedly reduces lamellipodia in rapidly migrating ENS precursors
(Fig. 3M). Furthermore, BMS493-treated cells often had strikingly
abnormal morphology with filopodia-like projections, suggesting
that RA critically regulates the signaling machinery required for
lamellipodia formation (Fig. 3K).
Antagonizing Rar increases Pten accumulation in
migrating ENS precursors and alters Pik3r1 and
PIP3 distribution
Many signaling components required for ENS precursor migration
and lamellipodia formation are known (Fig. 8) (Fukuda et al., 2002;
Hall, 2005; van Weering and Bos, 1997; Vohra et al., 2007a). This
pathway begins with receptor-induced Pik3 activation. Pik3
converts PIP2 to PIP3. PIP3 in membranes recruits proteins that
activate Rac1, a key regulator of lamellipodia formation and part of
a positive feedback loop required for migration. Because previous
studies demonstrated that blocking Pik3 reduces distal bowel
colonization by ENS precursors (Natarajan et al., 2002), we
hypothesized that RA deficiency might reduce Pik3 activity or alter
localization. We therefore examined the cells that migrated furthest
from gut slices with Pik3 and PIP3 immunohistochemistry.
Migrating Ret+ cells maintained with GDNF and RA accumulated
PIP3 and phospho-Pik3r1, the activated regulatory subunit of Pik3,
in lamellipodia. Both PIP3 and pPik3r1 were also abundant in Ret+
cell bodies (Fig. 5A,A). By contrast, pPik3r1 and PIP3 were not
seen at the edge of migrating Ret+ cells cultured with BMS493 (Fig.
5B,B). Because BMS493 treatment dramatically alters cell shape,
however, it was difficult to know if altered pPik3r1 and PIP3
distribution caused, or resulted from, the cell shape changes. We
therefore investigated other molecules that could affect lamellipodia
formation and cell migration.
As Pten reverses Pik3 activity by converting PIP3 to PIP2, we
hypothesized that Rar antagonism might inhibit migration and
lamellipodia formation by increasing Pten. To test this hypothesis,
we cultured gut explants for 16 hours with GDNF, GDNF plus RA
or GDNF plus BMS493. Remarkably, Pten was much more
abundant in migrating Ret+ cells after culture in BMS493 than after
culture with GDNF or GDNF plus RA (Fig. 5C-E). In the absence
of BMS493, most migrating Ret+ cells were Pten negative
(911.3%). Even when Pten was present, it was largely excluded
from the leading edge of cells (Fig. 5F,G). By contrast, Ret+ cells
cultured with BMS493 had abundant Pten (615%) and no
evidence of polarized Pten accumulation (Fig. 5E,H). These data
suggest that RA facilitates ENS precursor migration by reducing
Pten accumulation at least in the most actively migrating cells.
Furthermore, increased Pten accumulation could account for
abnormal cell shape, loss of lamellipodia and reduced migration
after treatment with a Rar antagonist. If so, then blocking Pik3
should similarly affect cell shape and migration. Consistent with this
hypothesis, treatment of gut slices with Pik3 inhibitor LY294002
significantly reduced ENS precursor migration from explants as
previously reported (Natarajan et al., 2002) and reduced the
percentage of cells with lamellipodia (222%) (Fig. 3H,L,M).
Furthermore, LY294002 induced abnormalities in cell shape closely
resembling BMS493-treated Ret+ cells. These data support the
hypothesis that RA enhances ENS precursor migration by reducing
Pten accumulation.
Pten is not expressed in ENS precursors at the
migration wavefront in vivo
If regulation of Pten by RA is crucial for normal ENS precursor
migration, we might expect low Pten levels in the most actively
migrating ENS precursors within the bowel wall. Furthermore, Pten
accumulation within ENS precursors should be regulated by Rar. To
test this hypothesis, we examined Pten in E11.5 whole gut segments
cultured for 48 hours in control media or with added RA or
BMS493. These studies demonstrate that Pten protein levels are very
low in Ret+ cells at the leading edge of the migration wavefront
under control conditions or with RA added, but that BMS493
DEVELOPMENT
appears to affect organization of the distal bowel ENS, but these
changes are difficult to quantify. Collectively, these data support
the hypothesis that RA is required for efficient GDNF-induced
ENS precursor migration.
RESEARCH ARTICLE
Fig. 5. BMS493 alters cell shape, PIP3 and Pik3r1 distribution and
Pten abundance. (A-H)E12.5 mid-gut slices were cultured for 16
hours with GDNF (A,A,C,F), GDNF plus RA (D,G) or GDNF plus
BMS493 (B,B,E,H). (A,A) Immunohistochemistry for PIP3 or Pik3r1
demonstrates polarization of PIP3 and Pik3r1 in lamellipodia (arrows) of
migrating Ret+ cells. (B,B) In BMS493-treated cells, both PIP3 and
Pik3r1 were present but the distribution was not polarized.
(C-E)Immunohistochemistry for Ret (green) and Pten (red)
demonstrates that Pten levels are low or absent in Ret+ migrating cells
cultured with GDNF or GDNF plus RA, but elevated in cells cultured
with BMS493. (F,G)Even when Pten is detected, Pten is localized to the
trailing edge of migrating cells grown in control media or with added
RA. (H)By contrast, Pten is distributed throughout the cytoplasm after
culture with BMS493. (I)Analysis of Pten–Ret double-labeling in cells at
the outer edge of the migration wavefront (GDNF, n621 cells; GDNF +
RA, n685; GDNF + BMS493, n1069; six independent experiments).
Scale bars: 25m in A-B,F-H; 100m in C-E. *, P<0.0001.
dramatically increases Pten in Ret+ cells within 200 m of the
wavefront (Fig. 6A-C,E). Interestingly, behind the migration
wavefront, Pten was expressed in a subset of Ret+ cells under all
conditions, and these Pten expressing cells largely correspond to
differentiating TuJ1+ neurons (Fig. 6D). Analysis of Pten in wildtype mice at E11.5 also demonstrated that Pten is largely excluded
from Ret+ cells at the migration wavefront (data not shown).
Collectively, these data support the hypothesis that Pten abundance
is regulated by RA and that reduced Pten facilitates ENS precursor
migration.
Pten overexpression reduces GDNF-induced
migration of ENS precursors
If RA is important for ENS precursor migration because it reduces
Pten, then increased Pten should slow migration. To test this
hypothesis, E12.5 cells from murine bowel were infected with
lentivirus (FM-Pten) that expresses Venus (a fluorescent protein)
and Pten or with control lentivirus (FM) that only produces Venus.
Pten production by virus was verified in 293T cells (Fig. 7).
Dissociated E12.5 gut cells were infected with virus and embedded
in a drop of collagen gel, cultured for 48 hours to allow Pten
production, and then surrounded with additional GDNF-containing
collagen gel. During an additional 24 hours, ENS precursors
migrated into the GDNF-containing gel. Most migrating cells were
virus-infected (FM virus: 757%; FM-Pten virus: 765%) and
almost all migrating cells were Ret+. Cells infected with the FM-
Development 137 (4)
Fig. 6. Pten protein levels are low in Ret+ cells at the leading edge
of the migration wavefront in organ culture. (A-C) E11.5 whole
guts were cultured for 48 hours in control medium (A), with added RA (B)
or with BMS493 (C) before whole-mount immunohistochemistry with Ret
(A-C) and Pten (A-C) antibodies. Confocal images show the leading
edge of the migration wavefront in the colon. (A-C) Merged images.
Arrows indicate the direction of precursor migration. Ret+ cells at the
leading edge of the migration wavefront have very low Pten levels after
culture in control media or with RA added. By contrast, Pten is abundant
in all Ret+ cells after culture with BMS493, even at the leading edge of
the wavefront. (D)Freshly isolated E12.5 mouse hindgut was stained for
TuJ1 (D) and Pten (D). The distal-most immunoreactive bowel is shown.
(D)Merged image. Most TuJ1+ cells are Pten+. Some TuJ1+ cells with
faint Pten staining are also detected (arrows). (E)Percentage of Ret+
cells that express Pten within 200m of the migration wavefront
(n8 explants and 400 cells under each condition; *, P<0.001).
Scale bars: 200m in A-C; 50m in D.
Pten virus, however, did not migrate as far from the edge of the cellrich pellet as cells infected with FM virus (Fig. 7E-H). These data
suggest that low Pten is required for efficient ENS precursor
migration and support the hypothesis that Rar signaling is required
for migration of ENS precursors into the distal bowel because RA
reduces Pten accumulation in the most rapidly migrating cells.
DISCUSSION
RA signaling is essential for normal ENS
development
Both RA excess (Pitera et al., 2001) and reduced RA synthesis
(Niederreither et al., 2003) affect ENS development, but the cellular
and molecular mechanisms underlying these observations are
uncertain. Our studies suggest that RA is part of an essential network
of signaling molecules that facilitate ENS precursor migration into
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636
RA enhances ENS precursor migration
RESEARCH ARTICLE
637
Fig. 7. Pten overexpression reduces ENS
precursor migration in vitro. (A-B) FM-Pten
efficiently produces Pten. 293T cells that lack
significant Pten were infected with lentivirusexpressing Venus (FM virus) (A-A) or Venus plus Pten
(FM-Pten virus) (B-B). Pten expression in Venus+ cells
was evaluated by immunohistochemistry 48 hours
after infection (A,B). (C-D) E12.5 dissociated gut
cells were infected with FM or FM-Pten, embedded in
collagen gel, were cultured for 48 hours and then
placed into a larger volume of GDNF-containing
collagen gel for 24 hours to encourage migration
before Ret immunohistochemistry. Phase contrast (C)
and Venus imaging (C) demonstrate that most cells
(~75%) migrating from the pellet into GDNFcontaining gel were Venus+. (D,D) Essentially all
migrating cells were Ret+. (E-F) FM-Pten-infected
cells migrate less rapidly from the cell-gel pellet than
FM-infected cells. (G)Diagram shows how cell
migration was evaluated. Cells within 100m of the
cell-rich pellet were too dense to count. Cells in the
regions of 100-200m and 200-400m from the
edge of the cell-rich gel pellet were counted
separately. (H)Very few FM-Pten-infected cells
migrated more than 200m from the edge of the
cell-rich gel pellet. *, P<0.0001 versus FM. Scale bars:
100m.
Rar antagonism prevents lamellipodia formation
and efficient ENS precursor migration
ENS development requires precursor migration from the vagal and
sacral neural crest into and through the developing bowel. Vagal
crest-derived cells have a very long migratory route and might
therefore be more sensitive to factors that reduce migration
efficiency than other cells. Several in vitro models, including fetal
gut culture, slice culture, Boyden chamber assays and video
microscopy, all demonstrated that blocking RA signaling reduced
ENS precursor migration efficiency through the bowel or out of the
bowel in response to GDNF. In this regard, Boyden chamber assays
provide particularly compelling evidence that ENS precursors
migrate toward GDNF and that migration efficiency is RAdependent.
Rar antagonism also dramatically affects the shape of Ret+ cells
migrating onto culture dishes. Normally, most migrating ENS
precursors in vitro are polarized with well-defined lamellipodia at
the leading edge. By contrast, blocking Rar causes dramatic
lamellipodia loss in migrating Ret+ cells and many cells assume
unusual shapes consistent with loss of polarity and cytoskeletal
dysregulation. We acknowledge that the precise mechanisms
required for ENS precursor migration in the three-dimensional
environment of the gut wall might be distinct from those used on a
two-dimensional tissue culture dish (Doyle et al., 2009), but our
consistent finding that reduced Rar signaling in many different
assays inhibits ENS precursor migration makes it more probable that
common molecular mechanisms underlie these observations.
Furthermore, recent studies delineated a model for GDNF-induced
control of cytoskeletal dynamics to support neurite growth and
migration (Vohra et al., 2007a), providing testable hypotheses to
explain our observations.
RA prevents Pten accumulation in migrating ENS
precursors to facilitate migration
Pten reverses Pik3 activity by converting PIP3 to PIP2. Pik3
activation at the leading edge of cells is important for efficient
migration because it promotes Rac1 activation and lamellipodia
formation (Fukuda et al., 2002; Natarajan et al., 2002; Vohra et al.,
2007a). In ENS precursors, BMS493-treated cells still have
abundant PIP3 and Pik3 immunoreactivity, making it difficult to be
DEVELOPMENT
the distal bowel. Using Rbp4–/– mice (Quadro et al., 2005; Wendler
et al., 2003), we demonstrate that vitamin A deprivation induces
distal bowel aganglionosis, but that Rbp4–/– animals rarely develop
bowel aganglionosis if maintained on vitamin A-containing food.
Ret and Rbp4 mutations also synergistically cause bowel
aganglionosis, even when mice are fed retinol. Furthermore, vitamin
A deprivation after E7.5 in Rbp–/–;Ret+/– mice causes more extensive
aganglionosis than in Rbp4–/–;Ret+/+ animals, demonstrating a new
gene-environment interaction causing HSCR-like disease. Together,
these data provide compelling evidence that vitamin A is essential
for normal ENS development and suggest that retinoid deficiency
could increase HSCR penetrance and the extent of aganglionosis,
crucial issues for a disease with incomplete penetrance and variable
expressivity.
Quantitative analysis of retinoids in Rbp4–/– mice suggests that
even mild vitamin A deficiency might increase HSCR penetrance.
Previous fetal retinoid measurements demonstrated that E14.5
Rbp4–/– mice have reduced retinol (59% reduction) and retinyl ester
(31% reduction) compared with wild-type mice when maintained on
vitamin A-sufficient food during gestation (Kim et al., 2008). Our
data suggest that, combined with Ret heterozygosity, this modest
reduction in fetal retinoids might be enough to cause disease.
Depriving Rbp4–/– mice of dietary retinoids at E7.5 led to an
additional ~50% drop in fetal retinoid levels at E12.5. Together,
these data suggest that 50-75% reductions in fetal retinoids
profoundly influence ENS development. If these findings apply to
human populations, then vitamin A deficiency is likely to be a
common predisposing factor for HSCR in many regions of the world
(West, 2002).
RESEARCH ARTICLE
convinced that changes in the distribution of these molecules cause
the defective cell shape or migration observed after BMS493
treatment. By contrast, Pten levels were much higher in migrating
cells maintained in BMS493 than in cells grown with RA.
Furthermore, migrating ENS precursors maintained with RA had a
striking polarization of Pten protein with exclusion of Pten from the
leading edge and accumulation of Pten at the trailing edge of the cell.
This polarization of Pten was lost in BMS493-treated cells. These
changes in Pten abundance in response to altered Rar signaling
provide a reasonable explanation for observed changes in cell shape,
polarity and migration efficiency. Furthermore, Pik3 inhibitors cause
a similar loss of lamellipodia and altered cell shape. We also show
that Pten overexpression reduces ENS precursor migration. Finally,
we demonstrate that cells at the migratory wavefront of ENS
precursors, a well-defined population of actively migrating cells,
have very little Pten, especially when compared with ENS
precursors slightly behind the wavefront. Together, these
observations suggest that RA is required for efficient ENS precursor
migration because Rar signaling reduces Pten accumulation in the
most actively migrating cells and that this reduction in Pten permits
GDNF-induced Pik3 activity to support lamellipodia formation and
Fig. 8. Signaling pathways controlling ENS precursor migration
and lamellipodia formation. (A)This simplified pathway shows that
Ret activation by GDNF causes Pik3 activation, generating PIP3 and
activating Cdc42 and Rac1 as part of a positive-feedback loop at the
leading edge of migrating cells. Rac1 is crucial for lamellipodia
formation. Pten reverses Pik3 activity and stops the positive-feedback
loop. RA signaling reduces Pten protein abundance in the most actively
migrating ENS precursors in vitro and at the leading edge of the
wavefront of Ret+ cells organ culture. (B)A model of how cells polarize
in response to chemoattractants. In the absence of chemoattractant,
signaling proteins might be distributed throughout the cell. In actively
migrating cells, Cdc42, Rac1, PIP3 and Pik3 accumulate at the front
edge of the cell, inducing cytoskeletal changes and lamellipodia
formation. Pten, Rho and Rho kinase (Rock) become restricted to the
trailing edge of the cell. High levels of Pten in ENS precursors deprived
of Rar signaling disrupts the positive-feedback loop and causes loss of
the cell polarity needed for migration.
Development 137 (4)
cell migration (Fig. 8). Accumulation of Pten in cells behind the
migratory wavefront might then be one signal that encourages cells
to stop migrating and differentiate. Pten expression might therefore
be required for formation of a distributed plexus of neurons and glia
along the bowel. This hypothesis is supported by the observation
that Pten is expressed in TuJ1+ cells behind the migration wavefront.
These observations are consistent with known roles for Pten in the
migration of neuronal precursors in the central nervous system (Li
et al., 2003; van Diepen and Eickholt, 2008) and in other cells (Kim
and Dressler, 2007; Leslie et al., 2007; Liliental et al., 2000; Sanchez
et al., 2005; Tamura et al., 1998), supporting the hypothesis that Rarinduced changes in Pten are crucial for normal ENS development.
Several novel aspects of this work should be emphasized. First,
effects of RA on Pten are rarely reported. Furthermore, two of the
three studies linking RA to Pten show that RA increases Pten
accumulation in other cell types (Hisatake et al., 2001; Lee et al.,
2006; Lee et al., 2007). Even in the ENS, RA-induced reductions in
Pten occur in only subsets of actively migrating cells. Thus, although
there are several conserved RARE sequences within 2 kb of the start
site for Pten transcription, regulation of Pten protein abundance in
ENS precursors is probably complex. Finally, suppressing Pten
might be expected to adversely affect ENS precursor migration by
increasing PIP3 and enhancing neurite growth (Srinivasan et al.,
2005). Our recent work suggests that to counteract this effect, RA
also suppresses ENS precursor neurite growth via reduced Smurf1
(Sato and Heuckeroth, 2008). Thus, in the ENS, RA has complex
stage-dependent effects that facilitate normal development, but these
activities require activation of networks of signaling molecules,
controlled protein subcellular localization, and changes in RA
responses as cells differentiate. One intriguing possibility is that RA
responsiveness of ENS precursors during development might be
influenced by Edn3 and Shh, proteins also reported to alter ENS
precursor migration, but this hypothesis will require additional
investigation (Barlow et al., 2003; Fu et al., 2004; Kapur et al., 1995;
Kapur et al., 1993; Kruger et al., 2003; Nagy and Goldstein, 2006;
Shin et al., 1999; Sidebotham et al., 2002; Sukegawa et al., 2000;
Vohra et al., 2007b).
Conclusions
These studies suggest the intriguing possibility that vitamin A
deficiency is a preventable cause of Hirschsprung disease. We now
hypothesize that although severe vitamin A deficiency causes many
developmental defects, and might contribute to some common
anomalies found in children with HSCR (e.g. congenital heart
disease), less severe deficiency acts synergistically with genetic
mutations to cause penetrant HSCR in individuals who might
otherwise have complete colonization of the bowel by ENS
precursors if fetal vitamin A supply was optimal. This is similar to
the observation that subclinical maternal folate deficiency
dramatically increases the risk of neural tube defects (Pitkin, 2007).
Furthermore, because RA signaling influences differentiation and
proliferation of ENS precursors (Sato and Heuckeroth, 2008a),
vitamin A deficiency could contribute not only to HSCR, but also to
other intestinal motility disorders.
These observations raise many questions. For example, if vitamin
A deficiency is a common cause of HSCR, why has this association
not been previously appreciated? One explanation is that although
vitamin A deficiency is an important global problem (West, 2002),
the epidemiology of HSCR has not been systematically investigated
in regions with prevalent vitamin A deficiency. Furthermore, in areas
where malnutrition is common, the phenotype of HSCR (abdominal
distension, growth failure and early death) might be easily missed,
DEVELOPMENT
638
as the diagnosis requires specialized techniques and a high index of
suspicion. Given our data and other analyses (Niederreither et al.,
2003; Pitera et al., 2001), a systematic study of HSCR epidemiology
and maternal vitamin A status now seems appropriate. These data
provide new hope that some cases of Hirschsprung disease might be
preventable by optimizing maternal nutrition.
Acknowledgements
We thank William Blaner for generously sharing Rbp4–/– mice, Chris Zusi for
providing BMS493, Bhupinder Vohra, Russell Van Gelder, Louis Muglia and
Jonathan Lake for helpful advice, and Sanjay Jain for sharing equipment. This
work was supported by NIH/RO1 DK57038, DK6459201, (DDRCC) NIH/P30DK52574, DK56341, Burroughs Wellcome Fund 1008525, Children’s Discovery
Institute CH-11-2008-123, P30-DK079333, NCRR C06 RR015502P30
NS057105 and March of Dimes FY02-182. Deposited in PMC for release after
12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.040550/-/DC1
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