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INTRODUCTIONThe enteric nervous system (ENS) is a complex network ofneurons and glia within the bowel that controls most aspects ofintestinal function (Furness, 2000). Hirschsprung disease(HSCR), a partially penetrant life-threatening defect affecting1:5000 children, is caused by the failure of ENS precursorcolonization of the distal bowel (Skinner, 1996). ENS precursorsoriginate in the vagal, sacral and upper thoracic neural tube beforemigrating extensively, proliferating and then differentiating toform the ENS (Heanue and Pachnis, 2007). Most ENS precursorsarise from the vagal neural tube, enter the foregut and colonize thebowel in a rostral to caudal progression (Newgreen and Young,2002a; Newgreen and Young, 2002b). Because molecularmechanisms required for ENS precursor migration, proliferationand differentiation are complex, many distinct mutations causeHSCR. Furthermore, HSCR penetrance and the extent ofaganglionosis 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 non-genetic factors, including vitamin A, crucially determine thepenetrance 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 andretinaldehyde dehydrogenases (Raldh) (Maden, 2007; Napoli,2004). RA binds Rar and Rxr receptors that heterodimerize andthen bind RA response elements (RARE) to regulate target genesand influence tissue morphogenesis. Recent data suggest that both

excess RA (Pitera et al., 2001) and reduced RA synthesis causeabnormal ENS development, but the molecular and cellularmechanisms that RA influences are not known. Indeed, althoughRaldh2–/– mice develop HSCR-like aganglionosis (Niederreitheret al., 2003), these mice have severe RA deficiency starting atconception, so it is unclear at what stage RA is required for ENSmorphogenesis. Our new data demonstrate that RA is essential forefficient ENS precursor migration through the developing boweland for lamellipodia formation in migrating cells.

Cell migration is complex, requiring polarization and coordinatedactivation of many proteins including RhoA-related GTPases thatregulate the actin cytoskeleton. In particular, Rac1 activation at theleading 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 Rac1activation occurs via a positive feedback loop initiated byconversion of phosphatidylinositol-2-phosphate (PIP2) tophosphatidylinositol-3-phosphate (PIP3) via phosphatidylinositol-3 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 inhuman HSCR and required for ENS precursor migration (Takahashi,2001).

Because RA deficiency prevents efficient ENS precursormigration, we hypothesized that RA is required to maintain signalsneeded for cell migration. We show that, in response to GDNF, Pik3and PIP3 accumulate at the leading edge of migrating enteric crest-derived cells and Pten (phosphatase and tensin homolog), an enzymethat reverses the Pik3 activity, is absent from most actively migratingENS precursors. When Rar signaling is blocked, however, polarizeddistribution of Pik3, PIP3 and Pten is abolished and Pten levelsdramatically increase in actively migrating cells. This isaccompanied by loss of lamellipodia and reduced migration towardGDNF. This suggests that RA is required in the developing ENS toreduce Pten and enhance ENS precursor migration. Collectively,these data suggest that some cases of Hirschsprung disease might bepreventable by ensuring adequate maternal vitamin A levels duringearly gestation.

Development 137, 631-640 (2010) doi:10.1242/dev.040550© 2010. Published by The Company of Biologists Ltd

Departments of 1Pediatrics, 2Developmental Biology, 3Pathology and Immunology,and the 5HOPE Center for Neurological Disorders, Washington University School ofMedicine, 660 South Euclid Avenue, St Louis, MO 63110, USA. 4Department ofNutritional Science and Toxicology, University of California, Berkeley, 119 MorganHall, MC#3104, Berkeley, CA 94720, USA.

*Author for correspondence (Heuckeroth@kids.wustl.edu)

Accepted 7 December 2009

SUMMARYHirschsprung disease is a serious disorder of enteric nervous system (ENS) development caused by the failure of ENS precursormigration 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 retinol-binding-protein-deficient (Rbp4–/–) mice. Ret heterozygosity increases the incidence and severity of distal bowel aganglionosisinduced by vitamin A deficiency in Rbp4–/– animals. Furthermore, RA reduces phosphatase and tensin homolog (Pten) accumulationin migrating cells, whereas Pten overexpression slows ENS precursor migration. Collectively, these data support the hypothesis thatvitamin A deficiency is a non-genetic risk factor that increases Hirschsprung disease penetrance and expressivity, suggesting thatsome cases of Hirschsprung disease might be preventable by optimizing maternal nutrition.

KEY WORDS: Enteric nervous system, Migration, Retinoic acid, Lamellipodia, Pten, Hirschsprung disease, Mouse

Vitamin A facilitates enteric nervous system precursormigration by reducing Pten accumulationMing Fu1, Yoshiharu Sato1, Ariel Lyons-Warren1, Bin Zhang3, Maureen A. Kane4, Joseph L. Napoli4 and Robert O. Heuckeroth1,2,5,*

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MATERIALS AND METHODSMiceRbp4–/– (Quadro et al., 2005) and Ret+/– mice (Enomoto et al., 2001) werebred >10 generations to C57BL/6. For timed breeding studies, the day of thevaginal plug was considered as embryonic day 0.5 (E0.5). Wild-type CF-1mice were from Charles River. Mice were maintained on Purina PICOirradiated mouse diet 5058 until E7.5. Food was changed to synthetic chow(El Mel, St Charles, MO, USA) containing sufficient vitamin A (TestDiet5755; 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 weremaintained on synthetic diets until analysis.

Retinoid measurementsRA was quantified using an API-4000 (Applied Biosystems) LC/MS/MSwith atmospheric pressure chemical ionization in positive ion mode. Retinoland retinyl esters were quantified by HPLC/UV (Kane et al., 2008a). Tissueswere harvested under yellow light, immediately frozen in liquid N2 and keptat –80°C until assay. Samples were homogenized on ice, extracted andanalyzed as described (Kane et al., 2005; Kane et al., 2008b). Total proteinwas determined by Bradford (Bio-Rad).

Slice cultureE12.5 CF-1 midgut sections (300-400 m length) from 400 m proximal tothe cecum were cultured on fibronectin-coated (250 g/ml) dishes in Opti-MEM (Invitrogen), glutamine (2 mM), penicillin 100 IU/ml andstreptomycin 100 g/ml. Immediately after plating, cells were treated withRA (10–7 M, Sigma, St Louis, MO, USA) or BMS493 (10–5 M, generouslyprovided by Dr Chris Zusi at Bristol-Myers Squibb). GDNF (100 ng/ml) wasadded to cultures three hours later. Cultures were maintained for 16 hoursbefore fixation [4% paraformaldehyde (PFA), 10 minutes, 25°C].

Boyden chamberTranswell supports (8.0 m pore size; 0.33 cm2 area; Corning 3422; FisherScientific) 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, streptomycin100 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 chamberswith 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. Cellswere incubated 2 hours before adding GDNF (100 ng/ml) to the top orbottom chambers and then 16 hours to allow migration. Cells on top of themembrane were removed with Kimwipes. Cells on membrane bottoms werefixed (4% PFA, 20 minutes, 25°C), blocked (1% BSA/PBS, 40 minutes,25°C) and processed for immunohistochemistry.

Whole gut cultureThe entire E11.5 CF-1 gastrointestinal tract was pinned to 2.5% agarose with4-0 stainless steel filaments (Ethicon) and cultured in DMEM, 10% fetal calfserum, 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). Cultureswere maintained for 48 hours before fixing (4% PFA, 30 minutes, 25°C).

Dissociated cell cultureE12.5 CF-1 ENS precursor cells were maintained in culture as previouslydescribed (Sato and Heuckeroth, 2008a). For Ret pixel intensitymeasurements, ENS precursors from E13.5 Rbp4–/– mice deprived ofvitamin A starting at E7.5, or from wild-type C57BL/6 on a vitamin A-containing diet, were cultured briefly (2 hours) before fixation.

ImmunohistochemistryAfter fixation, cells and organs were kept in TBST (100 mM Tris, 150 mMNaCl, 0.2% Triton X-100) for 20 minutes at 25°C, blocked with 4% donkeyserum/TBST for 1 hour at 25°C and then incubated with primary antibodyfor 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,

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 generousgift from Jean-François Brunet, 1:1000). Antibodies were visualized usingdonkey anti-goat Alexa 594 (1:200) and donkey anti-rabbit Alexa 488(1:100, Molecular Probes). Images were obtained with an Olympus BX60microscope and Axiocam and AxioVision software (Zeiss). NIH Image Jwas used for pixel intensity measurements of Ret immunohistochemistry.Measurements were made in the cell body of p75NTR+ cells and wesubtracted background signal intensity.

Pten overexpression vectorFull-length Pten cDNA (Open Biosystems, Clone ID 5038272) obtainedusing XbaI and XmaI was cloned into a modified version (FM) of the FUIV[ubiquitin promoter – gene of interest-IRES-enhanced YFP (Venus)] (Arakiet al., 2004) using AgeI and NheI sites. Virus was produced by WashingtonUniversity Hope Center Viral Vectors Core using 293T cells, pCMV-G,pMD-Lg, and RSV-REV (Li and Rossi, 2005). Viral titers were 1.4�108

(TU/ml) for FM-Pten and 1�108 (TU/ml) for FM.

Cell migration assay for virus-infected cellsE12.5 CF-1 whole gut was digested with collagenase (0.2 mg/ml) anddispase (0.2 mg/ml) for 10 minutes at 37°C, triturated, centrifuged andresuspended in the neural crest medium (see Boyden Chamber). Threemicrolitres of virus was added to 106 cells in 50 l media. To prepare thecollagen gel, 0.25 volume of 5� DMEM was added to 1 volume of type Irat tail collagen (4 mg/ml) in 0.02 N acetic acid (BD Biosciences). pH wasadjusted using 0.8 M NaHCO3 (final concentration 8 mM) and 200 mMNaOH (final concentration 1.5 mM) to form gel and diluted with neural crestmedium to 2 mg/ml collagen. Fifty microliters of gel was added to 50 lcells/virus mixture and the gel was cultured in 96-well U-bottom plates (BDBiosciences) for 48 hours at 37°C. The cell-gel pellet was then transferredto 4-well dishes and embedded in 1% collagen gel containing GDNF (200ng/ml). After 24 hours, Venus+ cells that migrated from the cell-gel pelletwere counted after fixation in 4% PFA for 20 minutes at 25°C andimmunohistochemistry.

Statistical analysesAll experiments were performed at least three times and analyzed by t-testor ANOVA analysis for multiple comparisons. P<0.05 was consideredsignificant. Data show mean ± s.e.m.

RESULTSVitamin A deficiency causes HSCR-like distalbowel aganglionosis in vivoTo determine whether vitamin A (retinol) is required for efficientbowel colonization by ENS precursors, we used serum retinol-binding-protein-deficient (Rbp4–/–) mice (Quadro et al., 1999;Quadro et al., 2005). Rbp4 is required for transport of hepatic retinolto peripheral tissues, so tissue retinoids in Rbp4–/– mice are primarilyderived 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 (Clagett-Dame and DeLuca, 2002; Wolf, 1984), Rbp4–/– mice can be rapidlydepleted of peripheral tissue retinoids by removing vitamin A fromthe diet. For these studies, Rbp4–/– mice were deprived of dietaryretinol starting at E7.5, 48 hours before crest-derived precursorsenter the bowel. Mice were analyzed at E14.5, shortly after ENSprecursors normally completely colonize the colon. The entire smallbowel and colon were stained using antibodies to Ret and neuron-specific beta-III tubulin (TuJ1, a neuronal lineage marker; Fig. 1).These studies demonstrated striking distal bowel aganglionosis inRbp4–/– mice deprived of dietary retinol, but an apparently normalENS in Rbp4–/– mice maintained on retinol containing food (Fig.1A). In fact, under vitamin A-sufficient conditions, only 2 out of 34Rbp4–/– failed to completely colonize the colon with ENS precursorsat E14.5. By contrast, almost all Rbp4–/– mice fed vitamin A-

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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 HSCR-like phenotype.

Because retinol depletion causing loss of Ret and/or TuJ1immunoreactivity in distal bowel ENS precursors is a potentialconcern, we performed double-label immunohistochemistry withantibodies to Ret and Phox2b in Rbp4–/– mice fed either retinol-deficient or -sufficient diets. Phox2b and Ret were expressed in thesame cells at the migration wavefront under both conditions (Fig.1E). Furthermore, Ret immunoreactivity in ENS precursorsmeasured by pixel intensity demonstrated equal Ret abundance incells from wild-type C57BL/6 versus retinol-deprived Rbp4–/– mice(WT: 26.6�1.3; retinol-deprived Rbp4–/–: 24.9�0.7, P0.21).Therefore, immunohistochemistry for Ret, TuJ1 and Phox2b allindicate that the distal bowel is not colonized in E14.5 Rbp4–/– micedeprived of retinol starting at E7.5.

Mild vitamin A deficiency increases HSCR-likedisease in genetically predisposed miceTo test the hypothesis that retinol depletion could increase HSCRpenetrance in genetically predisposed mammals, we analyzed theENS in Rbp4–/–;Ret+/– double-mutant mice. Ret mutations are themost commonly identified cause of human HSCR and Rbp4–/–

animals have mild vitamin A deficiency even when maintained onretinol containing food (Wendler et al., 2003). Furthermore, Ret+/–

mice are predisposed to aganglionosis (McCallion et al., 2003), buthave 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 onvitamin A containing food, many Rbp4–/–;Ret+/– animals have distalbowel aganglionosis (10/20) at E14.5 (Fig. 1C,D). Furthermore,dietary vitamin A deprivation caused more extensive bowelaganglionosis in Rbp4–/–;Ret+/– than in Rbp4–/– mice. Collectively,these data strongly support the hypothesis that vitamin A deficiencyis a nutritional risk factor for HSCR. To evaluate the severity ofretinoid depletion needed to cause ENS defects, we measured fetalretinoids in Rbp4–/– mice at E12.5, when migrating ENS precursorshave reached the mid-colon and are entering distal bowel. Theseanalyses demonstrated that switching to vitamin A-deficient diets atE7.5 caused 42-53% reductions in fetal retinoids at E12.5 (Table 1).Based on these findings, we investigated molecular and cellularmechanisms to explain our observations.

Rar signaling is required for ENS precursormigrationTo determine whether Rar signaling affects ENS precursormigration, we used a modified Boyden chamber (Boyden, 1962) tostudy ENS precursors at E12.5, the time that the distal colon iscolonized. Dissociated gut cells placed in the top chamber weretreated for 2 hours with control media containing RA or media

633RESEARCH ARTICLERA enhances ENS precursor migration

Table 1. E12.5 fetal retinoid levelsRetinol Retinyl ester Retinoic acid

VAD (–) 4.50�0.44* 2.97�0.36* 0.057�0.004*VAS (+) 8.05�0.74 5.15�0.49 0.12�0.008

Fetal retinoid levels in Rbp4–/– mice. Mice were changed at E7.5 to vitamin A-containing [VAS (+)] or vitamin A-deficient diets [VAD (–)] and maintained on thesediets 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.n22 VAD (–) mice; n16 VAS (+) mice. *, P<0.05 compared to VAS (+).

Fig. 1. Vitamin A deficiency causes distal bowel aganglionosisin Rbp4–/– mice. (A)Rbp4–/– mice fed vitamin A-containing food(top), had a dense network of neurons and fibers in the distal boweland almost all had ganglion cell bodies at the end of the colon.Diagrams indicate positions of the most distal TuJ1+ ganglion cellbody 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 A-deprived mice have distal colon aganglionosis and reduced ENSdensity in the colonized colon (bottom). (B)Ret/TuJ1 double-labelimmunohistochemistry at the wavefront of ENS precursors of anE13.5 Rbp4–/– mouse maintained on vitamin A-deficient food startingat E7.5. The Ret signal is less intense than TuJ1, but staining overlapssignificantly and the wavefront visualized by Ret or TuJ1 antibodies isat the same position (n4 vitamin A-sufficient mice, n8 vitamin A-depleted mice). (C)Even when maintained on vitamin A, manyRbp4–/–;Ret+/– mice have distal bowel aganglionosis. When deprivedof vitamin A starting at E7.5, there is more extensive aganglionosis.(D)Summary of bowel colonization in mutant mice. (E)Double-labelimmunohistochemistry for Ret and Phox2b at the migrationwavefront of E13.5 Rbp4–/– mice maintained on a vitamin A-containing or -deficient diet starting at E7.5 shows colocalization ofRet and Phox2b-expressing cells. In all images, distal bowel is to theright. Scale bars: 100m in A; 50m in B,E. D

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containing a well-characterized pan-Rar antagonist, BMS493.GDNF was then added to the bottom chamber to induce ENSprecursor migration through the porous membrane. Cells werecultured 16 hours before Ret immunohistochemistry (Fig. 2).BMS493 competitively blocks all Rar isotypes (, and ) and hasbeen 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 BMS493develop the same spectrum of defects seen in vitamin A deficiency.GDNF in the bottom chamber markedly increased Ret+ ENSprecursor migration through the membrane. BMS493 completelyblocked chemoattractive effects of GDNF (Fig. 2E). Controlexperiments with GDNF in the top chamber only did not increaseRet+ cell migration through the membrane, suggesting that GDNFacts as a chemoattractant rather than simply increasing motility orproliferation.

Rar antagonism slows ENS precursor migration inorgan cultureTo further evaluate Rar effects on ENS precursor migration, E12.5mid-small bowel slices were cultured for 16 hours in media withGDNF plus RA, or GDNF plus the Rar antagonist BMS493.Under these conditions, ENS precursors migrate from gutexplants onto culture dishes and were visualized by videomicroscopy before Ret immunohistochemistry. Treatment withBMS493 considerably reduces migration of Ret+ ENS precursorsfrom gut explants in response to GDNF (Fig. 3A-D). Adding RAhad little effect on ENS precursor migration, suggesting thatexplants make enough RA to support migration in this assay.

Time-lapse microscopy confirmed that BMS493 did not influencecell survival in these short-term studies. Double-labelimmunohistochemistry for Ret and TuJ1 demonstrated no effectof Rar signaling on neuronal differentiation or the level of Retexpression (see Fig. S2 in the supplementary material). Parallelstudies of the effects of BMS493 on ENS precursor migration inwhole gut culture confirmed that Rar antagonism reduces distalbowel colonization. In this case, the position of the most distalganglion cell is the same in the presence or absence of BMS493,but the density of ENS precursors in the distal bowel isdramatically reduced by Rar antagonism (Fig. 4). BMS493 also

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Fig. 2. RA is required for efficient GDNF-induced ENS precursormigration. (A-D)E12.5 dissociated gut cells were placed in the topwells of Boyden chambers in retinoic acid (RA)-containing media with(D) or without (A-C) BMS493. After 2 hours, media with (C,D) orwithout (A,B) GDNF (100 ng/ml) was added to the bottom chamber. Asa control (B), GDNF was also added to the top, but not the bottomchamber. After 16 hours, ENS precursors that migrated throughmembranes were visualized using DAPI (blue) and Retimmunohistochemistry (red). (E)Analysis of Ret+ cells that migratedthrough membranes (n9 for each condition). Scale bar: 100m. *,P<0.0001 compared with GDNF in the bottom chamber.

Fig. 3. BMS493 reduces ENS precursor migration and lamellipodiaformation in slice cultures. (A-C)E12.5 mouse mid-gut slices werecultured to allow crest-derived cells to migrate onto the dish inresponse to GDNF. Cultures were maintained for 16 hours withoutadded retinoic acid (RA) (A), with added RA (B), or with BMS493 (C)before Ret immunohistochemistry (red) and DAPI staining. (D)Distancefrom the edge of the explant to the most distal Ret+ cells [4measurements/explant (see Fig. S1 in the supplementary material);n40 explants for each condition]. (E-L)Ret antibody (red) andPhalloidin (green) double-labeling demonstrate lamellipodia at theleading edge of crest-derived cells migrating from E12.5 mid-gut slicesmaintained in GDNF (E,I) or GDNF plus RA (F,J). Culture with GDNF plusBMS493 (G,K) or GDNF plus Pik3 inhibitor LY294002 (H,L) dramaticallyreduces lamellipodia formation and causes Ret+ cells to adopt veryunusual shapes. The arrow in I shows a typical lamellipodium (n>320cells analyzed; 12 slices per condition). (I-L)Higher magnification imagesof E-H. (M)Percentage of Ret+ cells at the leading edge of themigration wavefront with lamellipodia. Scale bars: 100m in A-C,E-H;25m in E�-H�. *, P<0.0001 versus GDNF. D

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appears to affect organization of the distal bowel ENS, but thesechanges are difficult to quantify. Collectively, these data supportthe hypothesis that RA is required for efficient GDNF-inducedENS precursor migration.

Rar antagonism causes lamellipodia loss inmigrating ENS precursorsTo determine why Rar antagonism reduces ENS precursormigration, we examined Ret+ cells that migrated furthest fromE12.5 mid-small bowel slices. Cells maintained with RA had well-defined lamellipodia at the leading edge (Fig. 3F,J), but lamellipodiawere uncommon in BMS493-treated cultures (Fig. 3G,K). Analysisof Ret antibody and Phalloidin-stained cells confirmed that BMS493markedly reduces lamellipodia in rapidly migrating ENS precursors(Fig. 3M). Furthermore, BMS493-treated cells often had strikinglyabnormal morphology with filopodia-like projections, suggestingthat RA critically regulates the signaling machinery required forlamellipodia formation (Fig. 3K).

Antagonizing Rar increases Pten accumulation inmigrating ENS precursors and alters Pik3r1 andPIP3 distributionMany signaling components required for ENS precursor migrationand lamellipodia formation are known (Fig. 8) (Fukuda et al., 2002;Hall, 2005; van Weering and Bos, 1997; Vohra et al., 2007a). Thispathway begins with receptor-induced Pik3 activation. Pik3converts PIP2 to PIP3. PIP3 in membranes recruits proteins thatactivate Rac1, a key regulator of lamellipodia formation and part ofa positive feedback loop required for migration. Because previousstudies demonstrated that blocking Pik3 reduces distal bowelcolonization by ENS precursors (Natarajan et al., 2002), wehypothesized that RA deficiency might reduce Pik3 activity or alterlocalization. We therefore examined the cells that migrated furthestfrom gut slices with Pik3 and PIP3 immunohistochemistry.Migrating Ret+ cells maintained with GDNF and RA accumulatedPIP3 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 notseen 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. Wetherefore investigated other molecules that could affect lamellipodiaformation and cell migration.

As Pten reverses Pik3 activity by converting PIP3 to PIP2, wehypothesized that Rar antagonism might inhibit migration andlamellipodia formation by increasing Pten. To test this hypothesis,we cultured gut explants for 16 hours with GDNF, GDNF plus RAor GDNF plus BMS493. Remarkably, Pten was much moreabundant in migrating Ret+ cells after culture in BMS493 than afterculture with GDNF or GDNF plus RA (Fig. 5C-E). In the absenceof BMS493, most migrating Ret+ cells were Pten negative(91�1.3%). Even when Pten was present, it was largely excludedfrom the leading edge of cells (Fig. 5F,G). By contrast, Ret+ cellscultured with BMS493 had abundant Pten (61�5%) and noevidence of polarized Pten accumulation (Fig. 5E,H). These datasuggest that RA facilitates ENS precursor migration by reducingPten accumulation at least in the most actively migrating cells.Furthermore, increased Pten accumulation could account forabnormal cell shape, loss of lamellipodia and reduced migrationafter treatment with a Rar antagonist. If so, then blocking Pik3should similarly affect cell shape and migration. Consistent with thishypothesis, treatment of gut slices with Pik3 inhibitor LY294002significantly reduced ENS precursor migration from explants aspreviously reported (Natarajan et al., 2002) and reduced thepercentage of cells with lamellipodia (22�2%) (Fig. 3H,L,M).Furthermore, LY294002 induced abnormalities in cell shape closelyresembling BMS493-treated Ret+ cells. These data support thehypothesis that RA enhances ENS precursor migration by reducingPten accumulation.

Pten is not expressed in ENS precursors at themigration wavefront in vivoIf regulation of Pten by RA is crucial for normal ENS precursormigration, we might expect low Pten levels in the most activelymigrating ENS precursors within the bowel wall. Furthermore, Ptenaccumulation within ENS precursors should be regulated by Rar. Totest this hypothesis, we examined Pten in E11.5 whole gut segmentscultured for 48 hours in control media or with added RA orBMS493. These studies demonstrate that Pten protein levels are verylow in Ret+ cells at the leading edge of the migration wavefrontunder control conditions or with RA added, but that BMS493

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Fig. 4. Rar signaling is required for coloncolonization in organ culture. (A-C�) E11.5mouse gut cultured for 48 hours in control media(A), with added RA (B) or added BMS439 (C) wasstained with Ret (A-C) and PGP9.5 (A�-C�)antibodies. (A�-C�) Merged images. (D,F)Ret+ celldensity in the terminal 200m of colonized colonwas measured using a grid. (E)Position of the mostdistal Ret+ cell in colon relative to colon length.n15 control, 15 RA- and 27 BMS493-treated gutexplants. *, P<0.001 versus control. Scale bar:100m.

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dramatically increases Pten in Ret+ cells within 200 m of thewavefront (Fig. 6A-C,E). Interestingly, behind the migrationwavefront, Pten was expressed in a subset of Ret+ cells under allconditions, and these Pten expressing cells largely correspond todifferentiating TuJ1+ neurons (Fig. 6D). Analysis of Pten in wild-type mice at E11.5 also demonstrated that Pten is largely excludedfrom Ret+ cells at the migration wavefront (data not shown).Collectively, these data support the hypothesis that Pten abundanceis regulated by RA and that reduced Pten facilitates ENS precursormigration.

Pten overexpression reduces GDNF-inducedmigration of ENS precursorsIf RA is important for ENS precursor migration because it reducesPten, then increased Pten should slow migration. To test thishypothesis, E12.5 cells from murine bowel were infected withlentivirus (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 embeddedin a drop of collagen gel, cultured for 48 hours to allow Ptenproduction, and then surrounded with additional GDNF-containingcollagen gel. During an additional 24 hours, ENS precursorsmigrated into the GDNF-containing gel. Most migrating cells werevirus-infected (FM virus: 75�7%; FM-Pten virus: 76�5%) andalmost all migrating cells were Ret+. Cells infected with the FM-

Pten virus, however, did not migrate as far from the edge of the cell-rich pellet as cells infected with FM virus (Fig. 7E-H). These datasuggest that low Pten is required for efficient ENS precursormigration and support the hypothesis that Rar signaling is requiredfor migration of ENS precursors into the distal bowel because RAreduces Pten accumulation in the most rapidly migrating cells.

DISCUSSIONRA signaling is essential for normal ENSdevelopmentBoth RA excess (Pitera et al., 2001) and reduced RA synthesis(Niederreither et al., 2003) affect ENS development, but the cellularand molecular mechanisms underlying these observations areuncertain. Our studies suggest that RA is part of an essential networkof signaling molecules that facilitate ENS precursor migration into

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Fig. 5. BMS493 alters cell shape, PIP3 and Pik3r1 distribution andPten abundance. (A-H)E12.5 mid-gut slices were cultured for 16hours with GDNF (A,A�,C,F), GDNF plus RA (D,G) or GDNF plusBMS493 (B,B�,E,H). (A,A�) Immunohistochemistry for PIP3 or Pik3r1demonstrates polarization of PIP3 and Pik3r1 in lamellipodia (arrows) ofmigrating Ret+ cells. (B,B�) In BMS493-treated cells, both PIP3 andPik3r1 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 cellscultured with GDNF or GDNF plus RA, but elevated in cells culturedwith BMS493. (F,G)Even when Pten is detected, Pten is localized to thetrailing edge of migrating cells grown in control media or with addedRA. (H)By contrast, Pten is distributed throughout the cytoplasm afterculture with BMS493. (I)Analysis of Pten–Ret double-labeling in cells atthe outer edge of the migration wavefront (GDNF, n621 cells; GDNF +RA, n685; GDNF + BMS493, n1069; six independent experiments).Scale bars: 25m in A-B�,F-H; 100m in C-E. *, P<0.0001.

Fig. 6. Pten protein levels are low in Ret+ cells at the leading edgeof the migration wavefront in organ culture. (A-C�) E11.5 wholeguts 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 leadingedge of the migration wavefront in the colon. (A�-C�) Merged images.Arrows indicate the direction of precursor migration. Ret+ cells at theleading edge of the migration wavefront have very low Pten levels afterculture in control media or with RA added. By contrast, Pten is abundantin all Ret+ cells after culture with BMS493, even at the leading edge ofthe wavefront. (D)Freshly isolated E12.5 mouse hindgut was stained forTuJ1 (D) and Pten (D�). The distal-most immunoreactive bowel is shown.(D�)Merged image. Most TuJ1+ cells are Pten+. Some TuJ1+ cells withfaint Pten staining are also detected (arrows). (E)Percentage of Ret+ cells that express Pten within 200m of the migration wavefront (n8 explants and 400 cells under each condition; *, P<0.001). Scale bars: 200m in A-C; 50m in D.

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the distal bowel. Using Rbp4–/– mice (Quadro et al., 2005; Wendleret al., 2003), we demonstrate that vitamin A deprivation inducesdistal bowel aganglionosis, but that Rbp4–/– animals rarely developbowel aganglionosis if maintained on vitamin A-containing food.Ret and Rbp4 mutations also synergistically cause bowelaganglionosis, even when mice are fed retinol. Furthermore, vitaminA deprivation after E7.5 in Rbp–/–;Ret+/– mice causes more extensiveaganglionosis than in Rbp4–/–;Ret+/+ animals, demonstrating a newgene-environment interaction causing HSCR-like disease. Together,these data provide compelling evidence that vitamin A is essentialfor normal ENS development and suggest that retinoid deficiencycould increase HSCR penetrance and the extent of aganglionosis,crucial issues for a disease with incomplete penetrance and variableexpressivity.

Quantitative analysis of retinoids in Rbp4–/– mice suggests thateven mild vitamin A deficiency might increase HSCR penetrance.Previous fetal retinoid measurements demonstrated that E14.5Rbp4–/– mice have reduced retinol (59% reduction) and retinyl ester(31% reduction) compared with wild-type mice when maintained onvitamin A-sufficient food during gestation (Kim et al., 2008). Ourdata suggest that, combined with Ret heterozygosity, this modestreduction in fetal retinoids might be enough to cause disease.Depriving Rbp4–/– mice of dietary retinoids at E7.5 led to anadditional ~50% drop in fetal retinoid levels at E12.5. Together,these data suggest that 50-75% reductions in fetal retinoidsprofoundly influence ENS development. If these findings apply tohuman populations, then vitamin A deficiency is likely to be acommon predisposing factor for HSCR in many regions of the world(West, 2002).

Rar antagonism prevents lamellipodia formationand efficient ENS precursor migrationENS development requires precursor migration from the vagal andsacral neural crest into and through the developing bowel. Vagalcrest-derived cells have a very long migratory route and might

therefore be more sensitive to factors that reduce migrationefficiency than other cells. Several in vitro models, including fetalgut culture, slice culture, Boyden chamber assays and videomicroscopy, all demonstrated that blocking RA signaling reducedENS precursor migration efficiency through the bowel or out of thebowel in response to GDNF. In this regard, Boyden chamber assaysprovide particularly compelling evidence that ENS precursorsmigrate toward GDNF and that migration efficiency is RA-dependent.

Rar antagonism also dramatically affects the shape of Ret+ cellsmigrating onto culture dishes. Normally, most migrating ENSprecursors in vitro are polarized with well-defined lamellipodia atthe leading edge. By contrast, blocking Rar causes dramaticlamellipodia loss in migrating Ret+ cells and many cells assumeunusual shapes consistent with loss of polarity and cytoskeletaldysregulation. We acknowledge that the precise mechanismsrequired for ENS precursor migration in the three-dimensionalenvironment of the gut wall might be distinct from those used on atwo-dimensional tissue culture dish (Doyle et al., 2009), but ourconsistent finding that reduced Rar signaling in many differentassays inhibits ENS precursor migration makes it more probable thatcommon molecular mechanisms underlie these observations.Furthermore, recent studies delineated a model for GDNF-inducedcontrol of cytoskeletal dynamics to support neurite growth andmigration (Vohra et al., 2007a), providing testable hypotheses toexplain our observations.

RA prevents Pten accumulation in migrating ENSprecursors to facilitate migrationPten reverses Pik3 activity by converting PIP3 to PIP2. Pik3activation at the leading edge of cells is important for efficientmigration because it promotes Rac1 activation and lamellipodiaformation (Fukuda et al., 2002; Natarajan et al., 2002; Vohra et al.,2007a). In ENS precursors, BMS493-treated cells still haveabundant PIP3 and Pik3 immunoreactivity, making it difficult to be

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Fig. 7. Pten overexpression reduces ENSprecursor migration in vitro. (A-B�) FM-Ptenefficiently produces Pten. 293T cells that lacksignificant Pten were infected with lentivirus-expressing Venus (FM virus) (A-A�) or Venus plus Pten(FM-Pten virus) (B-B�). Pten expression in Venus+ cellswas evaluated by immunohistochemistry 48 hoursafter infection (A�,B�). (C-D�) E12.5 dissociated gutcells were infected with FM or FM-Pten, embedded incollagen gel, were cultured for 48 hours and thenplaced into a larger volume of GDNF-containingcollagen gel for 24 hours to encourage migrationbefore Ret immunohistochemistry. Phase contrast (C�)and Venus imaging (C) demonstrate that most cells(~75%) migrating from the pellet into GDNF-containing gel were Venus+. (D,D�) Essentially allmigrating cells were Ret+. (E-F�) FM-Pten-infectedcells migrate less rapidly from the cell-gel pellet thanFM-infected cells. (G)Diagram shows how cellmigration was evaluated. Cells within 100m of thecell-rich pellet were too dense to count. Cells in theregions of 100-200m and 200-400m from theedge of the cell-rich gel pellet were countedseparately. (H)Very few FM-Pten-infected cellsmigrated more than 200m from the edge of thecell-rich gel pellet. *, P<0.0001 versus FM. Scale bars:100m.

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convinced that changes in the distribution of these molecules causethe defective cell shape or migration observed after BMS493treatment. By contrast, Pten levels were much higher in migratingcells maintained in BMS493 than in cells grown with RA.Furthermore, migrating ENS precursors maintained with RA had astriking polarization of Pten protein with exclusion of Pten from theleading edge and accumulation of Pten at the trailing edge of the cell.This polarization of Pten was lost in BMS493-treated cells. Thesechanges in Pten abundance in response to altered Rar signalingprovide a reasonable explanation for observed changes in cell shape,polarity and migration efficiency. Furthermore, Pik3 inhibitors causea similar loss of lamellipodia and altered cell shape. We also showthat Pten overexpression reduces ENS precursor migration. Finally,we demonstrate that cells at the migratory wavefront of ENSprecursors, a well-defined population of actively migrating cells,have very little Pten, especially when compared with ENSprecursors slightly behind the wavefront. Together, theseobservations suggest that RA is required for efficient ENS precursormigration because Rar signaling reduces Pten accumulation in themost actively migrating cells and that this reduction in Pten permitsGDNF-induced Pik3 activity to support lamellipodia formation and

cell migration (Fig. 8). Accumulation of Pten in cells behind themigratory wavefront might then be one signal that encourages cellsto stop migrating and differentiate. Pten expression might thereforebe required for formation of a distributed plexus of neurons and gliaalong the bowel. This hypothesis is supported by the observationthat Pten is expressed in TuJ1+ cells behind the migration wavefront.These observations are consistent with known roles for Pten in themigration of neuronal precursors in the central nervous system (Liet al., 2003; van Diepen and Eickholt, 2008) and in other cells (Kimand Dressler, 2007; Leslie et al., 2007; Liliental et al., 2000; Sanchezet al., 2005; Tamura et al., 1998), supporting the hypothesis that Rar-induced 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 thethree studies linking RA to Pten show that RA increases Ptenaccumulation in other cell types (Hisatake et al., 2001; Lee et al.,2006; Lee et al., 2007). Even in the ENS, RA-induced reductions inPten occur in only subsets of actively migrating cells. Thus, althoughthere are several conserved RARE sequences within 2 kb of the startsite for Pten transcription, regulation of Pten protein abundance inENS precursors is probably complex. Finally, suppressing Ptenmight be expected to adversely affect ENS precursor migration byincreasing PIP3 and enhancing neurite growth (Srinivasan et al.,2005). Our recent work suggests that to counteract this effect, RAalso suppresses ENS precursor neurite growth via reduced Smurf1(Sato and Heuckeroth, 2008). Thus, in the ENS, RA has complexstage-dependent effects that facilitate normal development, but theseactivities require activation of networks of signaling molecules,controlled protein subcellular localization, and changes in RAresponses as cells differentiate. One intriguing possibility is that RAresponsiveness of ENS precursors during development might beinfluenced by Edn3 and Shh, proteins also reported to alter ENSprecursor migration, but this hypothesis will require additionalinvestigation (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).

ConclusionsThese studies suggest the intriguing possibility that vitamin Adeficiency is a preventable cause of Hirschsprung disease. We nowhypothesize that although severe vitamin A deficiency causes manydevelopmental defects, and might contribute to some commonanomalies found in children with HSCR (e.g. congenital heartdisease), less severe deficiency acts synergistically with geneticmutations to cause penetrant HSCR in individuals who mightotherwise have complete colonization of the bowel by ENSprecursors if fetal vitamin A supply was optimal. This is similar tothe observation that subclinical maternal folate deficiencydramatically increases the risk of neural tube defects (Pitkin, 2007).Furthermore, because RA signaling influences differentiation andproliferation of ENS precursors (Sato and Heuckeroth, 2008a),vitamin A deficiency could contribute not only to HSCR, but also toother intestinal motility disorders.

These observations raise many questions. For example, if vitaminA deficiency is a common cause of HSCR, why has this associationnot been previously appreciated? One explanation is that althoughvitamin A deficiency is an important global problem (West, 2002),the epidemiology of HSCR has not been systematically investigatedin regions with prevalent vitamin A deficiency. Furthermore, in areaswhere malnutrition is common, the phenotype of HSCR (abdominaldistension, growth failure and early death) might be easily missed,

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Fig. 8. Signaling pathways controlling ENS precursor migrationand lamellipodia formation. (A)This simplified pathway shows thatRet activation by GDNF causes Pik3 activation, generating PIP3 andactivating Cdc42 and Rac1 as part of a positive-feedback loop at theleading edge of migrating cells. Rac1 is crucial for lamellipodiaformation. Pten reverses Pik3 activity and stops the positive-feedbackloop. RA signaling reduces Pten protein abundance in the most activelymigrating ENS precursors in vitro and at the leading edge of thewavefront of Ret+ cells organ culture. (B)A model of how cells polarizein response to chemoattractants. In the absence of chemoattractant,signaling proteins might be distributed throughout the cell. In activelymigrating cells, Cdc42, Rac1, PIP3 and Pik3 accumulate at the frontedge of the cell, inducing cytoskeletal changes and lamellipodiaformation. Pten, Rho and Rho kinase (Rock) become restricted to thetrailing edge of the cell. High levels of Pten in ENS precursors deprivedof Rar signaling disrupts the positive-feedback loop and causes loss ofthe cell polarity needed for migration. D

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as the diagnosis requires specialized techniques and a high index ofsuspicion. Given our data and other analyses (Niederreither et al.,2003; Pitera et al., 2001), a systematic study of HSCR epidemiologyand maternal vitamin A status now seems appropriate. These dataprovide new hope that some cases of Hirschsprung disease might bepreventable by optimizing maternal nutrition.

AcknowledgementsWe thank William Blaner for generously sharing Rbp4–/– mice, Chris Zusi forproviding BMS493, Bhupinder Vohra, Russell Van Gelder, Louis Muglia andJonathan Lake for helpful advice, and Sanjay Jain for sharing equipment. Thiswork was supported by NIH/RO1 DK57038, DK6459201, (DDRCC) NIH/P30-DK52574, DK56341, Burroughs Wellcome Fund 1008525, Children’s DiscoveryInstitute CH-11-2008-123, P30-DK079333, NCRR C06 RR015502P30NS057105 and March of Dimes FY02-182. Deposited in PMC for release after12 months.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.040550/-/DC1

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RESEARCH ARTICLE Development 137 (4)

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