Author’s Accepted Manuscript

Ibuprofen slows migration and inhibits bowelcolonization by enteric nervous system precursorsin zebrafish, chick and mouse

Ellen Merrick Schill, Jonathan I. Lake, Olga A.Tusheva, Nandor Nagy, Saya K. Bery, LynneFoster, Marina Avetisyan, Stephen L. Johnson,William F. Stenson, Allan M. Goldstein, Robert O.Heuckeroth

PII: S0012-1606(15)30087-7DOI: http://dx.doi.org/10.1016/j.ydbio.2015.09.023Reference: YDBIO6922

To appear in: Developmental Biology

Received date: 28 July 2015Revised date: 31 August 2015Accepted date: 7 September 2015

Cite this article as: Ellen Merrick Schill, Jonathan I. Lake, Olga A. Tusheva,Nandor Nagy, Saya K. Bery, Lynne Foster, Marina Avetisyan, Stephen L.Johnson, William F. Stenson, Allan M. Goldstein and Robert O. Heuckeroth,Ibuprofen slows migration and inhibits bowel colonization by enteric nervoussystem precursors in zebrafish, chick and mouse, Developmental Biology,http://dx.doi.org/10.1016/j.ydbio.2015.09.023

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Title: Ibuprofen slows migration and inhibits bowel colonization by enteric nervous system

precursors in zebrafish, chick and mouse

Authors: 1Ellen Merrick Schill,

1Jonathan I. Lake,

1Olga A. Tusheva,

2,3Nandor Nagy,

4Saya K.

Bery, 5Lynne Foster,

1Marina Avetisyan,

6Stephen L. Johnson,

5William F. Stenson,

2Allan M.

Goldstein and 4Robert O. Heuckeroth

Affiliations: 1Department of Pediatrics,

5Department of Internal Medicine, and

6Department of

Genetics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO

63110, U.S.A., 2Department of Pediatric Surgery, Massachusetts General Hospital, Harvard

Medical School, 55 Fruit St., Boston, MA 02114, U.S.A., 3Department of Human Morphology

and Developmental Biology, Faculty of Medicine, Semmelweis University, Budapest, Hungary,

4Department of Pediatrics, The Children’s Hospital of Philadelphia Research Institute and the

Perelman School of Medicine at the University of Pennsylvania, Abramson Research Center,

3615 Civic Center Blvd, Philadelphia, PA 19104

Corresponding Author

Robert O. Heuckeroth M.D. Ph.D.

The Children’s Hospital of Philadelphia Research Institute

Perelman School of Medicine at the University of Pennsylvania

Abramson Research Center — Suite 1116I

3615 Civic Center Blvd.

Philadelphia, Pennsylvania 19104-4318, USA.

Phone: 215-590-1209; Fax: 215-590-3324; E-mail: Heuckerothr@email.chop.edu.

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Conflict of Interest: The authors have declared that no conflict of interest exists.

Key words: Enteric nervous system development, gene-environment interactions, migration

Abstract

Hirschsprung Disease (HSCR) is a potentially deadly birth defect characterized by the absence of

the enteric nervous system (ENS) in distal bowel. Although HSCR has clear genetic causes, no

HSCR-associated mutation is 100% penetrant, suggesting gene-gene and gene-environment

interactions determine HSCR occurrence. To test the hypothesis that certain medicines might

alter HSCR risk we treated zebrafish with medications commonly used during early human

pregnancy and discovered that ibuprofen caused HSCR-like absence of enteric neurons in distal

bowel. Using fetal CF-1 mouse gut slice cultures, we found that ibuprofen treated enteric neural

crest-derived cells (ENCDC) had reduced migration, fewer lamellipodia and lower levels of

active RAC1/CDC42. Additionally, inhibiting ROCK, a RHOA effector and known RAC1

antagonist, reversed ibuprofen effects on migrating mouse ENCDC in culture. Ibuprofen also

inhibited colonization of Ret+/- mouse bowel by ENCDC in vivo and dramatically reduced

bowel colonization by chick ENCDC in culture. Interestingly, ibuprofen did not affect ENCDC

migration until after at least three hours of exposure. Furthermore, mice deficient in Ptgs1 (COX

1) and Ptgs2 (COX 2) had normal bowel colonization by ENCDC and normal ENCDC migration

in vitro suggesting COX-independent effects. Consistent with selective and strain specific effects

on ENCDC, ibuprofen did not affect migration of gut mesenchymal cells, NIH3T3, or WT

C57BL/6 ENCDC, and did not affect dorsal root ganglion cell precursor migration in zebrafish.

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Thus, ibuprofen inhibits ENCDC migration in vitro and bowel colonization by ENCDC in vivo

in zebrafish, mouse and chick, but there are cell type and strain specific responses. These data

raise concern that ibuprofen may increase Hirschsprung disease risk in some genetically

susceptible children.

Introduction

The enteric nervous system (ENS) is an intrinsic neuronal and glial network in the bowel

that controls intestinal function (Furness, 2012; Sasselli et al., 2012). Most ENCDC that give rise

to the ENS begin to delaminate from vagal neural tube by E8.5 in mice and prior to week four in

humans (Fu et al., 2004; Kapur et al., 1992). Sacral ENCDC also contribute to distal bowel ENS

(Kapur, 2000; Wang et al., 2011). Vagal ENCDC migrate to the foregut and then begin

coordinated proliferation, rostro-caudal migration, and differentiation, a process that normally

results in interconnected neurons and glia throughout the bowel by E13.5 in mice and by week 7

in humans. This migratory route is one of the longest traversed by any cell during development.

In one in 5000 human infants, ENCDC do not colonize the bowel completely, causing

Hirschsprung disease (HSCR), a condition defined by the absence of distal bowel enteric neurons

(Heuckeroth, 2013; Hirschsprung, 1888; McKeown et al., 2013; Skinner, 1996).

HSCR is potentially fatal because aganglionic bowel (i.e., bowel without neurons)

tonically contracts, causing constipation, bilious vomiting, abdominal distension, enterocolitis,

and sepsis (Dasgupta and Langer, 2004; Heuckeroth, 2013; Skinner, 1996). Most children with

HSCR (80%) have only a short region of aganglionic bowel (Amiel et al., 2008; Suita et al.,

2005) and even modestly increased ENCDC bowel colonization (e.g. 5-10% increase) might

have prevented HSCR. For children with extensive aganglionosis, having only slightly greater

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bowel colonization by ENCDC could prevent the need for intravenous nutrition and associated

life-threatening infections. Therefore, it is valuable to identify non-genetic potentially avoidable

factors that influence ENCDC migration.

Because ENS development depends on many signaling pathways (Anderson et al., 2006;

Goldstein et al., 2013; Lake and Heuckeroth, 2013; Laranjeira and Pachnis, 2009), many

medicines may increase HSCR risk. One critical pathway includes RET receptor tyrosine kinase

(Pachnis et al., 1993; Schuchardt et al., 1994), the ligand glial cell line-derived neurotrophic

factor (GDNF), the co-receptor GFRα1 (Airaksinen and Saarma, 2002) and the small

RhoGTPases RAC1 and CDC42 that induce actin polymerization and reorganization at the

leading edge of migrating cells (de Curtis, 2008; Fukata et al., 2003; Goto et al., 2013; Ridley,

2006; Stewart et al., 2007; Vohra et al., 2007a). Among other roles, RAC1 induces lamellipodia,

promotes ENCDC migration in vitro and ex vivo and inhibits RHOA, a small GTPase (Stewart et

al., 2007; Wu et al., 2009). RHOA also inhibits RAC1 via its effector ROCK (Guilluy et al.,

2011; Nakayama et al., 2008). The complex signaling pathways needed for normal development

increase the vulnerability of ENS precursors to medicines that impact these and many other

signaling pathways.

By testing medicines commonly used during early human pregnancy, we discovered that

ibuprofen dramatically reduced bowel colonization by ENCDC in zebrafish and chick.

Furthermore, ibuprofen delayed Ret+/- mouse bowel colonization by ENCDC in vivo, but effects

were not observed in WT mice. Consistent with these observations, ibuprofen reduced migration

speed on 2-dimensional surfaces, reduced lamellipodia and reduced filamentous actin in murine

ENCDC. These changes correlated with reduced active RAC1/CDC42. Furthermore,

ibuprofen’s effects on migration could be prevented using a ROCK inhibitor. In contrast,

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ibuprofen did not slow migration or reduce lamellipodia in mouse gut mesenchymal or NIH3T3

cells. Surprisingly, mice with mutations in Ptgs (i.e., cyclooxygenase, COX), ibuprofen’s main

therapeutic target, had normal ENCDC bowel colonization and normal migration in vitro.

Additionally, live imaging experiments demonstrated that ENCDC required at least three hours

of exposure to ibuprofen before ENCDC migration speed was impacted, a much slower time

course than would be expected for a solely COX dependent effect. Collectively these studies

suggest ibuprofen use during early pregnancy could increase HSCR risk in some genetically

susceptible children. This may be important since 23.5 % of women in the United State take

ibuprofen during early pregnancy (Thorpe et al., 2013).

Materials and Methods

Zebrafish

Wild type AB zebrafish, fertilized in vitro, were exposed to drugs from 34 to 96 hours

post fertilization (hpf) in E3 media with 1% DMSO (Murphey and Zon, 2006). At 96 hpf,

zebrafish were stained with HuC/D antibody to visualize neurons. Uncolonized gut was

measured from most distal HuC/D+ cell to end of bowel. Drugs (from Sigma, St. Louis) tested,

number of fish evaluated and drug concentrations are in Table 1.

Avian intestine organ culture

Fertilized White Leghorn chicken eggs from commercial breeders were maintained in a

37°C humidified incubator. E6 midgut plus hindgut from umbilicus to cloaca was pinned to

Sylgard, cultured in DMEM, 100 U/mL penicillin-G, 0.1 mg/mL streptomycin (Sigma), DMSO

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(5 µM), +/- ibuprofen (250 µM) for 48 hours and then processed for immunocytochemistry. 5-

ethynyl-2’-deoxyuridine (EdU) (10 µM, Invitrogen) was added 3 hours before fixation.

Mice

Mice used include WT CF-1 (Charles River Laboratories), WT C57BL/6J (The Jackson

Laboratory), Ptgs1tm1Unc

(called Ptgs1 or Cox1, RRID:MGI_4366280), Ptgs2tm1Unc

(called Ptgs2

or Cox2, RRID:MGI_4366244) (C57BL/6J background) (Langenbach et al., 1995; Morham et

al., 1995), Sox10tm1Weg

(called Sox10+/- or Sox10 LacZ, RRID:MGI_4437131) (C3H

background) (Britsch et al., 2001) (from M. Wegner (Friedrich Alexander University Erlangen

Nuremberg, Erlangen, Germany) and M. Southard-Smith (Vanderbilt University, Nashville,

Tennessee, USA)), and Rettm1Jmi

(Ret (tau-EGFP-myc), Ret-TGM, RRID:MGI_3623107), called

Ret+/-, C57/B6J background)(Enomoto et al., 2001). The day of the vaginal plug was called

E0.5. Mice were genotyped by PCR as described in the references above.

Midgut Slice Explant Culture

E12.5 small bowel (midgut) was cut into 300-500 micron slices. These slices were

cultured on fibronectin-coated (250 µg/mL; Life Technologies) plastic or glass Lab-Tek

Permanox chamber slides (ThermoFisher) in OptiMem (Life Technologies), 2 mM L-glutamine

(Life Technologies), 100 IU/mL penicillin, 100 µg/mL streptomycin (Life Technologies).

Ibuprofen stock (1 g/L) was prepared fresh in media daily. Unless noted, ibuprofen was added

when slices were plated. Four hours after plating, GDNF (100 ng/mL final concentration) was

added to cultures, which were then maintained for an additional 16 hours (37oC, 5% CO2) after

GDNF addition. When needed, 10 µM bromodeoxyuridine (BrdU) was added four hours before

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fixation. Y-27632 (5 µM, Sigma, St. Louis MO) in DMSO was added four hours after plating

slices. 16, 16-dimethyl prostaglandin E2 and 16, 16-dimethyl prostaglandin F2 (Cayman

Chemicals, Ann Arbor, MI) dissolved in ethanol were added (final concentration 1 µM) at the

same time as GDNF (for 16, 16-dimethyl prostaglandin E2) or at plating (for 16, 16-dimethyl

prostaglandin F2). Control cultures received equivalent diluent volumes.

Immunoselected ENCDC Culture

E12.5 CF-1 small and large bowel were treated with collagenase (0.2 mg/mL) and

dispase (0.2 mg/mL), triturated, resuspended in media (Neurobasal® (Life Technologies), B27®

(50x, Life Technologies), 2 mM L-glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin),

and then exposed to rabbit anti-p75NTR (anti-nerve growth factor receptor, P75, EMD

Millipore, 1:1000, one hour, 4oC), followed by goat anti-rabbit coupled paramagnetic beads

(anti-rabbit IgG MicroBeads1:50, Miltenyi Biotec, GmbH) (30 min, 4oC). ENCDC (p75NTR

positive) were isolated with MACS columns (Miltenyi Biotec, GmbH), before culturing on poly-

d-lysine (100 µg/mL, Sigma, St. Louis) and laminin (20 µg/mL, BD Biosciences) coated glass

chamber slides (Sato and Heuckeroth, 2008). Cells were treated with GDNF (50 ng/mL) with or

without ibuprofen (250 µM) at plating and then grown 48 hours before fixation.

In Vivo Mouse Ibuprofen Treatment

Pregnant mice were fed Lab Diet 5053 (Test Diet, Richmond, IN) with or without 375

parts per million ibuprofen from E8.5 to until analysis.

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Embryo Sections

10 µm sagittal E12.5 CF-1 mouse sections were prepared from immersion fixed (4%

paraformaldehyde overnight, rotating, 4oC), sucrose treated (30% overnight), frozen (Optimal

Cutting Temperature Compound, Tissue-Tek) mice.

Immunohistochemistry

Fixed zebrafish (2% paraformaldehyde, two hours, 25oC) were washed in PBS, stained

with mouse anti-HuC/HuD (16A11) antibody (overnight, 4oC) and examined after Alexa Fluor®

anti-mouse 594 secondary (1:250, Life Technologies) (two hours, 25oC) treatment.

Mouse cells were fixed (4% paraformaldehyde, 25oC) for 20 minutes (slice and

dissociated culture) or 30 minutes (whole bowel)), washed (PBS or Tris-buffered saline plus

0.1% Triton-X (TBST)), and blocked (4% normal donkey serum/TBST, 1 hour, 25oC or

overnight 4oC) before primary antibodies (Table 2) were applied in TBST (4

oC, overnight). TuJ1

and Phalloidin were applied only two hours at 25oC. Samples were washed in PBS or TBST

before incubating with secondary antibodies (Alexa Fluor® Donkey anti-Rabbit, Donkey anti-

goat, Donkey anti-mouse 488, 594, or 647nm, Invitrogen, 1:400) and DAPI (100 ng/mL, 4’,6–

diamidine–2–phenylidole–dihydrochloride, Vector Labs) in TBST (1 hour, 25oC) and then

washing again in PBS or TBST before mounting (50% glycerol).

Avian bowel was fixed (4% paraformaldehyde in PBS, 1 hour), rinsed (PBS), incubated

in 15% sucrose/PBS (overnight, 4°C), then in 7.5% gelatin,15% sucrose/PBS (37°C, 1 hour),

before freezing (−50°C, 2-Methylbutane, Sigma). 10 µm frozen sections on Probe On Plus slides

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(Fisher Scientific) were stained with ENCDC specific anti-chicken N-cadherin antibody (Nagy et

al., 2012) Nuclei were DAPI stained. EdU was detected using Clik-iT EdU Imaging Kit

(Invitrogen).

Active RAC1/CDC42 and RHOA Measurements

NIH3T3 (ATCC) for scratch test were grown on glass 8-well chamber slides to 100%

confluence in DMEM (high glucose), 10% fetal calf serum (FCS), 100 IU/mL penicillin, and 100

µg/mL streptomycin. 250 µM ibuprofen was added two hours before scratching to remove some

cells using a 10 µL pipette tip. Media was changed after the scratch. For RAC1/CDC42 and

RHOA studies, NIH3T3 were starved (18 hours in 1 % FCS, then 24 hours in 0 % FCS), then

stimulated with 100 ng/mL RAC1/CDC42 Activator II (3 min, Cytoskeleton, Denver) or 100

µg/mL Rho Activator I (30 min, Cytoskeleton, Denver). During stimulation, RAC1/CDC42

control cells were kept in 0% FCS media while RHOA control cells were in 1 % FCS media.

After culture ENCDC and NIH3T3 were fixed (4% paraformaldehyde, 20 min, 25oC),

permeabilized in TBST (55oC, one hour) and incubated with 20 µg GST-tagged PAK-PBD

protein (PBD-GST) (Cytoskeleton, Denver) or 100 µg GST-tagged Rhotekin-RBD protein

(RBD-GST) (Cytoskeleton, Denver) (1 hour, 4oC) in 100 µl TBST. Cells were PBS washed,

post-fixed (4% paraformaldehyde, 15 min, 25oC), and washed again (PBS) before anti-GST

immunohistochemistry.

Microscopy and Analysis

Micrographs were obtained with 1) Olympus BX60 or IX71, Axiocam CCD, Axiovision

software, 2) Olympus FV1000 confocal with Fluoview software, 3) Zeiss Axio Imager.A2,

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AxioCam MRm Rev.3, ZEN software, 4) Zeiss LSM 710 confocal, Zen software, 5) Nikon

Eclipse 80i, Spot camera, software version 3.3.1 (Diagnostic Instruments). FIJI (NIH ImageJ)

and Photoshop CS6 (Adobe) were used to process images (Schindelin et al., 2012) including

only cropping, stitching (Preibisch et al., 2009), rotating, centering, and uniform adjustments of

brightness, contrast and saturation. Confocal images show flattened Z–stacks. Time-lapse

imaging used an AxioObserver.Z1 microscope (Zeiss) with motorized stage and incubator with

temperature and CO2 controls. Live-imaging was at 37oC and 5% CO2.

ENCDC colonization of mouse bowel was measured from cecal tip to most distal TuJ1+

cell or neurite. For midgut slices, migration was measured in octants from slice edges to the

most distal ENCDC or mesenchymal cell (Supplemental Figure 1). Octants adjacent to other

slices were not evaluated. ENCDC were RET+ by immunohistochemistry. Mesenchymal cells

were identified by absent RET immunostaining and an actin-rich cytoskeleton after Alexa488-

phalloidin staining. Each slice was considered a single replicate.

Lamellipodia were defined as regions of obvious F-actin ruffling wider than the cell body

and were analyzed in phalloidin-stained ENCDC furthest from gut slices (Supplemental Figure

1). The longest neurite in RET+ cells were measured using FIJI (NIH ImageJ). Cell speed was

measured in time-lapse images using MTrackJ manually (Meijering et al., 2012).

Immunofluorescent pixel intensity was measured using FIJI on images taken at fixed exposure

times. Only RET+ cells that migrated furthest from slices were analyzed for fluorescence

intensity. All samples for which fluorescence intensity was compared were stained on the same

chamber slide.

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Statistical Analysis

SigmaPlot 11 (Systat Software), student’s t-test or one-way ANOVA was used for

comparisons. All studies include at least three biological replicates. Non-parametric data were

analyzed by ranks. Post-hoc Holm-Sidak or Dunn’s tests were used for multiple comparisons.

Data are plotted as mean +/- SEM unless noted. P<0.05 was considered significant.

Approvals

All studies were approved by Animal Studies Committee at Washington University

School of Medicine, Institutional Animal Care and Use Committee at The Children’s Hospital of

Philadelphia, and by Massachusetts General Hospital’s Institutional Subcommittee on Research

Animal Care.

Results

Ibuprofen inhibited enteric nervous system development in zebrafish

Zebrafish embryos were exposed to selected medicines that are used by > 0.5 % of

women during early pregnancy (Thorpe et al., 2013) (Table 1). Fish were treated during the

period that ENCDC migrate through bowel (34 to 96 hours post fertilization (hpf)) and then

stained with Elavl3/4 (HuC/D) antibody to show enteric neurons (Kuhlman and Eisen, 2007;

Lake et al., 2013). Ibuprofen and acetylsalicylic acid specifically reduced bowel colonization by

ENCDC at concentrations found in human blood with therapeutic dosing (Table 1). We focused

on ibuprofen because it inhibited bowel colonization by ENCDC in a dose dependent manner

(Figure 1 A-E) and because acetylsalicylic acid caused death at 660 µM (i.e., twice the dose that

caused ENS defects) suggesting more generalized toxicity. In contrast, at an ibuprofen dose that

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reduced bowel colonization by ENCDC (25 µM), fish looked grossly normal (Figure 1A - D).

Body length was slightly reduced (Figure 1A, C, F), but dorsal root ganglia (DRG), another

neural crest-derivative, were in a normal position after ibuprofen treatment (Figure 1A, C, G)..

Furthermore fish treated with 25 µM ibuprofen had a normal number of DRG neurons per

ganglion (DMSO 2.2 +/- 0.09, ibuprofen 2.1 +/- 0.1, p = 0.3, 2 tailed t-test). These data suggest

that ibuprofen inhibits zebrafish bowel colonization by ENCDC at concentrations that do not

globally disrupt development or block migration of other neural crest derivatives.

Ibuprofen inhibits ENCDC colonization of chick bowel in organ culture

To determine if ibuprofen affects ENS development in other species we cultured E6

chick hindgut for 48 hours with or without ibuprofen (250µM) and then stained with N-cadherin

antibody (Figure 2A-B). This ibuprofen concentration was selected based on work in mouse

culture (Figure 3) where higher ibuprofen doses are needed to affect ENCDC migration. This

ibuprofen level is comparable to human serum levels after consuming three over-the-counter

ibuprofen tablets (i.e., 600 mg, a commonly used dose) (Ritschel and Kerns, 2009). Remarkably,

ibuprofen-treated hindgut was almost devoid of ENCDC, whereas control bowel was almost

fully colonized by ENCDC (n=6 per treatment group). Proliferation of ENCDC was also

reduced by ibuprofen based on EdU incorporation into N-Cadherin expressing cells at the

leading edge of the migration wavefront (N-Cadherin+ EdU+/N-Cadherin x 100: Control 58 +/-

0.06%, ibuprofen 38 +/- 0.05%, P = 0.03, n = 6/group, t-test) as was proliferation in gut

mesenchymal cells (DAPI+ N-Cadherin- EdU+/DAPI+ N-Cadherin- x 100: Controls 20 +/-

1.5%, ibuprofen 13 +/- 0.9%, P = 0.03, n = 6/group, t-test). Thus in chick and in fish, ibuprofen

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dramatically reduced colon colonization by ENCDC. In chick, this was accompanied by reduced

proliferation of ENCDC and surrounding mesenchymal cells.

Ibuprofen slows ENCDC colonization of mouse bowel in vivo

To determine if ibuprofen affects mammalian ENCDC development, we fed ibuprofen to

two mouse lines with genetic defects that predispose to HSCR-like disease. Pregnant mice with

Ret or Sox10 mutations received ibuprofen containing or control chow from E8.5 to E12.5. We

selected these dates because E8.5 is one day before ENCDC enter fetal bowel and by E12.5

ENCDC have normally migrated to mid-colon. We then stained the bowel with an antibody to

neuron specific β3 tubulin (TuJ1) (Figure 2C-F). Ibuprofen-exposed Ret+/- mice had a small,

but significant reduction in bowel colonization by ENCDC (% colon colonization: untreated

Ret +/− 73.6 ± 3.9%, n=4; ibuprofen treated Ret+/- 65.2 ± 5.2% n=9, P<0.042, t-test). In

contrast, colon colonization by ENCDC was normal in ibuprofen treated E12.5 Sox10+/- mice

and in E12.5 WT littermates of Sox10+/- and Ret+/- animals (% colon colonization: untreated

Sox10+/- 59.4 ± 2.4% n=19; ibuprofen Sox10+/- 60.3 ± 2.3% n=13, p=0.79, t-test and data not

shown) (Figure 2F). These data show that in Ret+/- mice, ibuprofen can delay ENCDC

colonization of fetal bowel in vivo, although the effect is more subtle than in chick and fish.

Ibuprofen specifically inhibited murine ENCDC migration in vitro

To gain insight into why ibuprofen slowed bowel colonization by ENCDC, we cultured

E12.5 CF-1 mouse gut slices on fibronectin coated dishes in the presence or absence of ibuprofen

(Fu et al., 2013; Fu et al., 2010; Lake et al., 2013; Wang et al., 2010). Addition of GDNF to the

culture media enhanced ENCDC migration onto the dishes permitting drug effects on ENCDC

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migration, proliferation, cell death and differentiation to be evaluated. After 16 hours in culture,

ENCDC were identified by RET immunoreactivity. RET is expressed by all ENS precursors and

differentiated enteric neurons in vitro (Heuckeroth et al., 1998; Pachnis et al., 1993). ENCDC

migration was assessed by measuring the distance between gut slice edges and the most distant

ENCDC (Supplemental Figure 1). 250 µM ibuprofen reduced the distance ENCDC migrated

from gut slices, but migration distance was normal at lower ibuprofen concentrations (50 µM,

100 µM) (Figure 3A-B, I).

Since ENCDC migration might be affected by the number of migrating ENCDC and by

differentiation (Lake et al., 2013), we determined if ibuprofen altered ENCDC proliferation,

apoptosis, or differentiation. Double label immunohistochemistry for RET (expressed in

ENCDC and enteric neurons) plus BrdU, RET plus activated caspase-3, and RET plus TuJ1

(neuron specific β3 tubulin antibody that identifies early neurons) demonstrated ibuprofen had

no effect on murine ENCDC proliferation, apoptosis, or differentiation (Figure 3C-H and 3J-L).

These data suggest ibuprofen specifically affects murine ENCDC migration without altering

survival, proliferation or differentiation.

NIH 3T3 and gut mesenchymal cell migration was not affected by ibuprofen

To determine if ibuprofen affects migration of other cells, we performed “scratch test

migration assays” on confluent NIH3T3 fibroblasts. After two hours of ibuprofen treatment, we

created a wound to induce migration (Figure 4 A-D) and imaged for 24 hours. Ibuprofen did not

slow NIH3T3 migration (P = 0.75) (Figure 4 A-E). To determine if ibuprofen effects require

specialized media or factors produced by bowel, we examined CF-1 mesenchymal cells that

migrated from the gut along with ENCDC (Supplemental Figure 1). Like NIH3T3, gut

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mesenchymal cell migration was not affected by ibuprofen (Figure 4G). These data suggest that

ENCDC migration is especially sensitive to ibuprofen, compared to other cell types.

Ibuprofen-exposed ENCDC have reduced lamellipodia and filamentous actin

To assess if ibuprofen affected ENCDC morphology, we cultured CF-1 mouse fetal

bowel slices with or without ibuprofen for 16 hours and stained with phalloidin. Fewer

ibuprofen treated migrating ENCDC had lamellipodia than control ENCDC at all doses tested

(Figure 5A-F, I). In contrast, ibuprofen did not reduce well-formed lamellipodia in NIH 3T3

(Figure 4F) nor in gut mesenchymal cells (Figure 4H). This finding may underlie ibuprofen

effects on ENCDC migration because lamellipodia are thin membranous actin projections that

facilitate cell migration (Hall, 2005).

Lamellipodia formation is driven by actin polymerization and cross-linking. To assess

filamentous actin (F-actin), we quantified fluorescence intensity of Alexa488-phalloidin stained

ENCDC. Ibuprofen treated ENCDC had reduced Alexa488 staining suggesting that ibuprofen

affects ENCDC actin dynamics (Figure 5A-F, J, K). As actin polymerization is also important

for neurite growth and we assessed neurite length and found that ibuprofen reduced neurite

length by 36 % when dissociated ENCDC differentiate in vitro (Figure 5G-H, L). This contrasts

with studies showing ibuprofen enhanced neurite growth in corticospinal neurons, dorsal root

ganglion cells (Fu et al., 2007) and raphespinal neurons (Wang et al., 2009) via PPARγ and

reduced RHOA/ROCK signaling (Dill et al., 2010). Developing enteric neurons, however, have

much less PPARγ than dorsal root ganglion neurons (Figure 6) providing a possible explanation

for different ibuprofen effects. Collectively these data suggest ibuprofen slows ENCDC

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migration and reduces neurite length in ENCDC-derived neurons by altering actin cytoskeletal

dynamics.

Ibuprofen slows ENCDC migration but effects are delayed

To gain insight into the mechanism by which ibuprofen slows ENCDC migration, we

performed time-lapse microscopy on gut slice cultures with ibuprofen added, at the initiation of

imaging (Figure 7A-D, Supplemental Movie 1-2). Analysis of sequential images showed that

ENCDC persistence (net distance migrated / total distance migrated) was unaffected by

ibuprofen (data not shown), but ENCDC migration speed was reduced. Interestingly, the effect

on migration speed was only apparent after 3 hours of ibuprofen (i.e., during “imaging interval

4”, Figure 7A, E, Supplemental Movies 1-2). This is much longer than would be expected if

ibuprofen effects were mediated by COX inhibition, the primary therapeutic effect of ibuprofen

(Peppelenbosch et al., 1993; Vane, 1971). One possible explanation for delayed ibuprofen

responsiveness of ENCDC is that prolonged contact with tissue culture surfaces changes

ENCDC biology so that these cells become ibuprofen sensitive. To test this hypothesis, we

added ibuprofen just as gut slices were placed in culture (i.e. four hours before initiation of

imaging (Supplemental Movie 3)) and observed that ibuprofen-mediated effects on ENCDC

migration were already apparent when imaging began. This suggests that the delayed response of

ENCDC to ibuprofen was not dependent on a change in ENCDC phenotype after migration onto

the culture dish, but instead is consistent with the hypothesis that ibuprofen effects on ENCDC

migration were COX-independent.

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Ptgs1 -/- Ptgs2 -/- mice have normal appearing ENCDC migration in vitro and in vivo

To directly test if reduced COX activity affected ENCDC, we cultured mid-small bowel

slices from E12.5 Ptgs1-/- Ptgs2-/- (i.e., Cox1-/- Cox2-/-) mice on fibronectin and found no

difference in ENCDC migration or lamellipodia compared to WT (Figure 8A-B, E-F). We also

stained WT and Ptgs1 -/- Ptgs2 -/- E12.5 bowel with TuJ1antibody and found equivalent colon

colonization by ENCDC (Figure 8C-D, G). Finally, we tried to rescue ibuprofen effects on CF-1

ENCDC using stable analogs of two prostaglandins (PGE2 and PGF2) reported to facilitate cell

migration (16,16-dimethyl PGE2 or 16, 16-dimethyl PGF2 respectively), but did not observe any

effect on migration or lamellipodia (Figure 8H-K). These data also suggest that ibuprofen

effects on ENCDC migration may be COX-independent.

One caveat is that Ptgs mutant animals were on a C57BL/6 genetic background. We

therefore cultured E12.5 small bowel slices from C57BL/6 mice on fibronectin in media with

GDNF +/- ibuprofen. Ibuprofen did not reduce ENCDC migration from WT C57BL/6 gut

(Distance of most distant ENCDC from gut slice: WT 335 +/- 7 µm; 250 µM ibuprofen 343 +/- 7

µm, 4 biological replicates, 128 control slices, 161 ibuprofen slices, P = 0.39, t-test), however,

when this experiment was performed using C57BL/6 Ret+/- gut explants, ENCDC migration

was reduced by ibuprofen (untreated: 349 +/- 7 µm, 250 µM ibuprofen 324 +/- 7 µm, 4

biological replicates 125 control slices, 147 ibuprofen slices, P = 0.015, t-test), consistent with

our in vivo results. These data suggest that the C57BL/6 genetic background makes ENCDC

more resistant to ibuprofen effects on migration compared to CF-1.

18

Ibuprofen reduces RAC1 activity in migrating murine ENCDC

Reduced F-actin, lamellipodia, and neurite growth in ibuprofen treated ENCDC

suggested reduced RAC1 or increased RHOA activity (Bryan et al., 2005; Hall, 2005; Koh,

2006; Sato and Heuckeroth, 2008; Wang et al., 2003). We attempted to measure active RAC1

and RHOA in migrating ENCDC using sensitive G-LISA kits (Ferri et al., 2014; Nini and

Dagnino, 2010), but could not obtain reproducible results. Furthermore, we wanted to measure

RAC1 and RHOA activity only in cells that migrated furthest from cultured gut slices (i.e., distal

ENCDC, Supplemental Figure 1) because these cells were examined for all other studies.

Therefore we used a validated “in situ” method to analyze active RAC1/CDC42 or active

RHOA based on binding to p21 PAK binding domain (PBD) or rhotekin binding domain (RBD)

respectively, fused to glutathione-S-transferase (GST) (Supplemental Figure 2) (Li et al., 2002;

Lindsley et al., 2011). Gut slices were cultured with or without ibuprofen, fixed and incubated

with PBD-GST or RBD-GST followed by GST immunohistochemistry. Analysis of mean

fluorescence intensity in ENCDC that migrated furthest from the gut edge demonstrated less

PDB-GST immunoreactive protein in ibuprofen-treated ENCDC, suggesting less active

RAC1/CDC42 than in control ENCDC (Figure 9A-H, Supplemental Figure 2A, C). In contrast,

active RHOA levels were similar in ibuprofen-treated and control ENCDC based on RBD-GST

binding (Figure 9I-P, Supplemental Figure 2 B, D). Collectively these data suggest ibuprofen

hinders ENCDC migration by reducing active RAC1 without affecting RHOA activity.

19

ROCK inhibition rescues ibuprofen effects on ENCDC migration in vitro

RAC1 and RHOA are regulated in complex ways as they control actin cytoskeleton to

alter cell morphology (Guilluy et al., 2011; Hall, 2005; Parri and Chiarugi, 2010). We

hypothesized that if ibuprofen slowed ENCDC migration and reduced lamellipodia via reduced

RAC1 activation, it might be possible to rescue ibuprofen’s effects by inhibiting ROCK, a

RHOA effector kinase that inhibits RAC1. To test this hypothesis, CF-1 midgut slices were

cultured with the ROCK inhibitor Y-27632 (Figure 10). This drug increased migration of

ibuprofen-treated ENCDC to the level seen in control cells, but did not affect ENCDC migration

in the absence of ibuprofen (Figure 10M). These data suggest that excess RHOA/ROCK activity

relative to RAC1/CDC42 may underlie ibuprofen’s effect on ENCDC migration (Figure 11).

20

Discussion

Despite surgical treatment available for Hirschsprung disease since 1948 (Swenson and

Bill, 1948), approximately 5% of children with HSCR die at an early age (Rescorla et al., 1992;

Suita et al., 2005) and > 40% have problems after surgery (El-Sawaf et al., 2013; Menezes et al.,

2008). It would therefore be ideal if HSCR could be prevented from occurring in the first place.

We now believe this may be possible in some cases by optimizing maternal nutrition, health, and

medication use before conception and during early pregnancy. If non-genetic risk factors were

identified, targeted advice could be provided to families at “high genetic risk” for HSCR. From

this standpoint it is particularly valuable to identify common and avoidable exposures.

Using zebrafish to test medicines commonly used by pregnant women we discovered that

ibuprofen causes HSCR-like absence of neurons in developing fish bowel in vivo. Studies in

mice also show ibuprofen can slow colonization of fetal bowel in vivo. Most dramatically,

ibuprofen treatment of fetal chick bowel led to almost complete absence of colon colonization by

ENCDC. Detailed analysis of actively migrating mouse ENCDC confirmed ibuprofen slowed

migration, reduced lamellipodia, reduced filamentous actin, and reduced RAC1 activation.

These in vitro and in vivo data suggest ibuprofen use during the period that ENCDC colonize

fetal bowel (e.g. week 3-8 of human gestation) might increase HSCR risk.

Human studies are now needed to extend this work. Although ibuprofen reduced

ENCDC colonization of fetal bowel in three species, the dose needed and severity of inhibitory

effects differed between species. The reason for interspecies and interstrain differences is not

known, but may reflect differences in drug metabolism, the presence of alternative migration

21

modes, target sensitivity, or intracellular signaling. One valuable approach will be human HSCR

epidemiologic studies examining maternal medication use in early pregnancy.

To put the mouse in vivo studies in context, Ret+/- mice fed 375 ppm ibuprofen in chow

had a 12% reduction in ENCDC colonization of colon at E12.5. This dose was selected because

it suppresses COX-dependent inflammation (Lim et al., 2000; Ritschel and Kerns, 2009) and

gives serum levels (60-70 µM) (Morihara et al., 2005) within the human therapeutic dosing

range (Ritschel and Kerns, 2009). Because ibuprofen use in early pregnancy is common (almost

1 in 4) (Thorpe et al., 2013) and HSCR relatively rare (about 1:5000) (Amiel et al., 2008),

ibuprofen alone is unlikely to cause HSCR in humans even at higher doses. Ibuprofen might,

however, increase HSCR occurrence in the context of underlying genetic risk. This is important

because known genetic causes for HSCR are partially penetrant suggesting gene-gene or gene-

environment interactions cause HSCR. This was demonstrated dramatically in mice by the

observation that Ret+/- mice never have distal bowel aganglionosis (McCallion et al., 2003), but

develop HSCR-like disease more readily than WT when other mutations or non-genetic risk

factors are present (Arnold et al., 2009; Carrasquillo et al., 2002; Fu et al., 2010; Gunadi et al.,

2014; Lake et al., 2013; McCallion et al., 2003; Phusantisampan et al., 2012; Wallace and

Anderson, 2011). In contrast to mice, humans with a single inactive RET allele (i.e., ~30% of

HSCR cases) have about a 50% chance of having HSCR (Amiel et al., 2008). The vast majority

of these children have only a small region of distal aganglionic bowel, suggesting that even small

changes in bowel colonization efficiency by ENCDC could have an important effect on HSCR

occurrence. For example, in mice, male sex only slightly reduces bowel colonization by

ENCDC similar to the effect of ibuprofen (Vohra et al., 2007b), but in humans the male sex

increases HSCR occurrence four-fold.

22

From the standpoint of human teratogenicity, it may be important that ibuprofen effects

on ENCDC appear likely to be COX-independent. COX (PTGS) enzymes that make

prostaglandins (Vane, 1971) are the primary therapeutic target of ibuprofen and other NSAIDs,

but Ptgs1-/- Ptgs2-/- mice had normal bowel colonization by ENCDC and normal ENCDC

migration in vitro. Furthermore, prolonged ibuprofen exposure and high doses were needed to

slow migration. NSAIDs should inhibit COX enzymes much faster (with effects apparent within

10 minutes) and at low micromolar doses (IC50 = 2.1 µM for PTGS1 and 1.6 µM for PTGS2)

(Peppelenbosch et al., 1993; Tegeder et al., 2001). One caveat is that our Ptgs mutant mice were

C57BL/6 genetic background, which appears more resistant to ibuprofen effects than CF-1.

The precise mechanisms through which ibuprofen inhibits ENCDC migration remain

uncertain, but our observations suggest ibuprofen treated ENCDC have abnormal regulation of

actin dynamics. Reduced neurite growth in ibuprofen treated ENCDC was not anticipated since

ibuprofen increased neurite growth in several other neuron types (Fu et al., 2007; Wang et al.,

2009). We hypothesize this cell-type specific difference occurs because unlike other neurons

tested, ENCDC have low levels of PPARγ that is required for ibuprofen to reduce RHOA

activity and enhance neurite growth (Dill et al., 2010). The lack of ibuprofen effect on NIH 3T3

or CF-1 gut mesenchymal cell migration and lamellipodia further highlights how cell-type

specific gene expression impacts biology and emphasizes that extrapolation from one cell type to

another may be misleading.

Our studies suggest ibuprofen reduces active RAC1/CDC42 in migrating ENCDC. This

fits with emerging literature on the role of small RhoGTPases in neural crest-derived cell

migration. Elegant in vivo FRET studies showed RAC1 promotes ENCDC chain migration

through fetal bowel (Goto et al., 2013). Reduced RAC1 activity in ibuprofen treated ENCDC

23

provides a reasonable explanation for reduced migration and fewer lamellipodia. The ability of

ROCK inhibition to prevent ibuprofen induced changes in migration and lamellipodia, without

any apparent alteration of RHOA activity, may occur because RAC1 and RHOA usually inhibit

each other and ROCK is a RHOA effector kinase (Figure 9) (Nakayama et al., 2008; Sordella

and Van Aelst, 2008).

Conclusions: These data demonstrate that ibuprofen, a medication commonly used in the first

trimester of pregnancy, inhibits ENCDC migration and might increase HSCR risk in genetically

susceptible individuals. This study extends our previous observations demonstrating

environmental risk factors like vitamin A deficiency (Fu et al., 2010) and mycophenolate (Lake

et al., 2013) can cause HSCR-like disease in mice, especially when combined with predisposing

genetic changes. Human epidemiological studies are necessary to determine the extent to which

ibuprofen contributes to HSCR risk.

24

Acknowledgements

We thank Dr. Tatyana Svitkina for insightful guidance about the actin cytoskeleton, Dr.

Allen Mitchell for sharing unpublished data about medicine use in early pregnancy, Ryo Hotta,

Ming Fu, Elizabeth Wright-Jin, Rajarshi Sengupta, and Alisha Jamil, and the Mouse Genetics

Core at Washington University School of Medicine for assistance and advice. This work was

supported by Irma and Norman Braman Endowment (ROH), Suzi and Scott Lustgarten Center

Endowment (ROH), The Children’s Hospital of Philadelphia Research Institute (ROH), The

Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital

(grant nos. CH-II-1008-123, CH-II-2010-390, MD-II-2013-269) (ROH), NIH grants RO1

DK087715 (ROH), R01 GM059688 (SLJ), R37 DK33165 (WFS), RO1 DK080914 (AMG),

Burroughs Wellcome Fund Clinical Scientist Award in Translational Research (grant no.

1008525) (ROH), NIH F30 DK100101 (EMS) and by the NIH Medical Scientist Training

Program Training Grant T32 GM07200.

Author Contributions

EMS, JIL, NN, WFS, SLJ, AMG, and ROH designed experiments. EMS, OAT, NN,

SKB, MA, and LF collected data. EMS, OAT, AMG, NN, SKB, and ROH analyzed data. EMS

and ROH wrote the manuscript. All authors reviewed and edited.

25

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30

Table 1. Medicines tested for an effect on zebrafish ENS development

Medicine

Human

therapeutic

blood

concentration

(µM)

Lowest

concentration

that affects

ENCDC*

(µM)

Concentration range tested

(µM)

[Actual concentrations

tested (number evaluated

at each concentration)] Reference

Acetaminophen 65-130 2,315 331-3,308

[331 (9), 662 (8), 1650 (8),

2315 (7) 3308 (7)]

(Ritschel and

Kerns, 2009)

Acetylsalicylic

Acid 110-1,700 333 56-666

[56 (21), 111(22), 222 (22),

333 (24), 666 (7)]

(Ritschel and

Kerns, 2009)

Caffeine 10-50 257 5-1,030

[5 (21), 51 (15), 257 (17), 514

(18), 1030 (16)]

(Baselt,

1982)

Chlorpheniramine 0.013-0.025 256# 13-1,279

[13 (6), 26 (9), 128 (10), 256

(5), 1279 (6)]

(Ritschel and

Kerns, 2009)

Clomiphene 0.050** 83# 17-836

[(17 (9) 83 (≥5), 167 (≥5) 836

(≥5)]

(Ghobadi et

al., 2009)

Dextromethorphan 0.74-1.3 368 18-1,840

[18 (9), 37 (9) 184 (7) 368 (7),

1840 (≥5)]

(Ritschel and

Kerns, 2009)

Diphenhydramine 0.034-0.34 103# 17-343

[17 (9) 34 (9) 103 (≥5), 172

(≥5), 343 (≥5)]

(Ritschel and

Kerns, 2009)

Doxylamine 0.3** 1,287# 129-1,287

[129 (8), 257 (8) 1287 (6)]

(Ritschel and

Kerns, 2009)

Erythromycin 0.68-3.4 >1,363 14-1363

[14 (7), 68 (9), 136 (9), 681 (8),

1363 (9)]

(Ritschel and

Kerns, 2009)

Guaifenesin 7.6** >3,31 252-3,531

[252 (5), 504 (9), 1009 (9),

(Aluri and

Stavchansky,

1993)

31

2522 (7), 3531 (8)]

Ibuprofen 25-240 25 2.5-50

[2.5 (21) 5 (24), 12.5 (24), 25

(24), 50 (20)]

(Ritschel and

Kerns, 2009)

Loratadine 0.13** >1,306 65-1,306

[65 (12), 131 (12), 261 (9) 652

(9) 1306 (10)]

(Hilbert et al.,

1988)

Sulfamethoxazole 200-790 1974 400-7,896

[395 (10), 987 (11), 1974 (12),

3948 (11), 7896 (8)]

(Ritschel and

Kerns, 2009)

Table 1. Ibuprofen and acetylsalicylic acid inhibit bowel colonization by ENCDC in

zebrafish at doses within the human therapeutic range. *Data in this column indicate the

lowest drug concentration that causes a statistically significant increase in the length of distal

zebrafish bowel that lacks ENCDC (compared to 1% DMSO vehicle) after treatment with drug

from 34-96 hpf. #Indicates medicines that caused severe malformation or death at

concentrations listed (i.e., defects were not ENS selective). **Based on the available literature

we indicate peak human therapeutic blood concentration instead of therapeutic blood ranges.

32

Table 2. Primary and secondary antibodies for immunohistochemistry or immunoselection

Antibody Concentration Catalog # Source

Rabbit anti-Tuj1 1:10,000 PRB-435P Covance*; RRID:AB_10063850

Goat anti-RET 1:100 GT15002 Neuromics; RRID:AB_2179886

Mouse anti-BRDU

(Conjugated to Alexa-594)

1:100 A21304 Invitrogen; RRID:AB_221472

Rabbit anti-cleaved

caspase-3

1:100 9661 Cell Signaling Technology;

RRID:AB_2314091

Mouse anti-PPARγ 1:100 sc-7273 Santa Cruz; RRID:AB_628115

Phalloidin (Conjugated to

Alexa-488)

1:50 A12379 Invitrogen; RRID:AB_2315147

Rabbit anti-GST 1:100 sc-35614 Santa Cruz; RRID:AB_647587

Mouse anti-vinculin 1:100 V9131 Sigma; RRID:AB_477629

Mouse anti-HuC/HuD

(Biotin Conjugate (16A11))

1:800 A21272 Invitrogen; RRID:AB_10375876

Mouse anti-chicken N-

cadherin antibody (clone

6B3)

1:5 6B3 Developmental Studies

Hybridoma Bank, Iowa;

RRID:AB_528118

Rabbit anti-nerve growth

factor (NGF-receptor), P75

1:1000 AB1554 EMD Millipore

RRID:AB_90760

Anti-rabbit IgG

MicroBeads

1:50 130-048-602 Miltenyi Biotec

RRID:AB_244362

Alexa Fluor® 594 Donkey

anti-mouse

1:400

A21203 Molecular Probes (Invitrogen);

RRID: AB_141633

Alexa Fluor® 594 Donkey

anti-rabbit

1:400 A21207 Life Technologies:

RRID:AB_10049744

Alexa Fluor® 488 Donkey

anti-rabbit

1:400 A21206 Life Technologies;

RRID:AB_10049650

Alexa Fluor® 594 Donkey

anti-goat

1:400 A11058 Life Technologies;

RRID:AB_10563390

Alexa Fluor® 647 Donkey

anti-rabbit

1:400 A31573 Life Technologies;

RRID:AB_10561706

* Covance was acquired by BioLegend in 2014 (new catalog # 802001; RRID not yet assigned)

RRID = Research Resource Identifiers (https://www.force11.org/node/4856)

33

Figure Legends

Figure 1:

Ibuprofen reduced ENCDC colonization of zebrafish bowel. (A-D) Zebrafish were treated

with vehicle (1%DMSO) or ibuprofen (Ibu) from 34-96 hpf and then stained with HuC/D

antibody. (A) White arrows indicate dorsal root ganglia. (C) White arrows highlight enteric

neurons. Scale bar=500µm. (B, D) Higher magnification of fish midsection. Scale bar=100µm.

(E) Length of bowel from most distal HuC/D+ cell (arrows in B, D) to bowel terminus

(arrowheads in B, D). *P<0.05 (ANOVA on Ranks) (F) Length of whole zebrafish (1% DMSO

n=4, 25 µM Ibuprofen n=6). *P=0.025 (t-test). (G) Distance between DRG and dorsal zebrafish

edge (1% DMSO n=3, 25 µM ibuprofen n=4). *P>0.05 (t-test).

Figure 2:

Ibuprofen inhibited hindgut colonization by chick ENCDC ex vivo and mouse ENCDC in

vivo. (A, B) Ibuprofen (250 µM) almost completely blocked E6 chick colon colonization by N-

cadherin+ (green) ENCDC during 48 hour culture. (Scale bar=200 µm, n=6 per group). Insets

show the migration wavefront with EdU (red) and N-cadherin (green) immunohistochemistry.

(Scale bar=35 µm). mg=midgut, hg=hindgut. (C-E) Ibuprofen feeding from E8.5 to E12.5

reduced Ret +/- mouse colon colonization by ENCDC visualized with TuJ1 antibody (red). Scale

bars=400 µm. (E, F) Quantitative data show the proportion of the colon colonized by ENCDC

(i.e., length of colon containing ENCDC divided by total colon length) (Control n=4, ibuprofen

n=9). *P =0.042 (t-test). (F) Sox10 +/- colon was normally colonized by ENCDC when dams

were fed ibuprofen from E8.5 to E12.5 (Control n = 19, ibuprofen n=13). P=0.79 (t-test).

34

Figure 3:

Ibuprofen reduced murine ENCDC migration, but did not affect proliferation, caspase-3

activation, or neuronal differentiation. E12.5 CF-1 midgut slices cultured with or without

ibuprofen for 16 hours after GDNF addition were stained for (A, B) RET (red), phalloidin

(green), DAPI (blue) (scale bar=200µm), (C, D) RET (green), BrdU (red), DAPI (blue) (Scale

bar=100µm), (E, F) RET (red), cleaved-caspase 3 (green), DAPI (blue) (Scale bar=100µm), or

(G, H) RET (green), TuJ1 (red), DAPI (blue) (scale bar=100µm). (I) Ibuprofen reduced the

distance ENCDC migrated from gut explants at 250 µM and 500 µM. Control n=275 slices, 29

biological replicates; 50 µM ibuprofen n = 48 slices, 3 biological replicates; 100 µM ibuprofen n

= 55 slices, 3 biological replicates; 250 µM ibuprofen n=111 slices, 5 biological replicates; 500

µM ibuprofen n = 102 slices, 8 biological replicates) but not at lower ibuprofen concentrations.

**P<0.001 (ANOVA). (J) Mean percentage BrdU+ ENCDC (RET+ cells) (Control n=21 slices,

7 biological replicates; ibuprofen n=9 slices, 3 biological replicates). P=0.24 (t-test). (K) Mean

percentage cleaved-caspase 3+ ENCDC (RET+ cells) (Control n=15 slices, ibuprofen n=15

slices; both 3 biological replicates). P=0.36 (t-test). (L) Mean percentage TuJ1+ ENCDC (RET+

cells) (Control n=19 slices, ibuprofen n=23 slices; both 3 biological replicates). P=0.35 (t-test).

Figure 4:

Ibuprofen did not affect migration or lamellipodia of NIH 3T3 or mouse gut mesenchymal

cells. Confluent NIH 3T3 were pretreated with 250 µM ibuprofen (2 hours) before making a

scratch to remove some cells. (A, B) NIH3T3 immediately after the scratch was made. (C, D)

Two hours after the scratch. Cells were imaged every 5 minutes for 24 hours 250 µM

35

ibuprofen did not affect (E) average speed of gap closure (Control n=5 assays, ibuprofen; n=7

assays), P=0.76 (t-test) or (F) percentage of NIH 3T3 with lamellipodia (Control n=8 assays,

ibuprofen; n=9 assays). P=0.75 (t-test). (G, H) 250 µM ibuprofen did not affect distance CF-1

mesenchymal cells migrated from gut explants (Control n=19 slices, ibuprofen n=24 slices; 3

biological replicates). P=0.57 (t-test) or the percentage of mesenchymal cells with lamellipodia

after 16 hours (Control=9 slices, ibuprofen=9 slices; 3 biological replicates). P=0.52 (t-test).

Figure 5:

Ibuprofen reduced lamellipodia in migrating murine ENCDC and reduced neurite length

in differentiating murine enteric neurons. (A-F) E12.5 CF-1 midgut slices were cultured with

250 µM ibuprofen or control media for 16 hours after GDNF addition and then stained for (A, D)

RET, (B, E) Alexa488-phalloidin, and (C, F) DAPI (merged image). Arrows highlight well-

formed lamellipodia. (G, H) Dissociated immunoselected E12.5 CF-1 ENCDC cultured 48 hours

were stained for RET (red), TuJ1 (green) and DAPI (blue). Scale bars=50 µm. (I, J). (I) The

percentage of migrating ENCDC with lamellipodia was reduced by 50 µM or higher ibuprofen

concentrations. RET+ ENCDC most distant from the gut slices were analyzed. (Control n=61

slices, 13 biological replicates; 50 µM Ibuprofen n = 9 slices, 3 biological replicates; 100 µM

ibuprofen n=9 slices, 3 biological replicates, 250 µM ibuprofen n=27 slices, 9 biological

replicates; 500 µM ibuprofen n = 12 slices, 4 biological replicates). *P <0.05, **P <0.001,

(ANOVA, Holm-Sidak method). (J) Ibuprofen reduced mean Alexa488-phalloidin fluorescence

intensity for RET+ ENCDC (Control n=232 cells, ibuprofen n=236 cells; both 4 biological

replicates). *P<0.001 (Mann-Whitney Rank Sum). (K) Ibuprofen reduced longest neurite in

36

cultured differentiating ENCDC (Control n=50, ibuprofen n=76, both 3 biological replicates).

*P=0.009 (Mann-Whitney Rank Sum).

Figure 6:

E12.5 CF-1 mouse ENCDC had less immunoreactive PPARγγγγ than E12.5 dorsal root

ganglion (DRG) neurons. Sagittal sections were stained with antibodies to PPARγ (red) and

neuron specific beta 3 tubulin (green, TuJ1). (A-D) PPARγ is readily detectable in DRG

neurons. The boxed region in C contains DRG neurons that are immunoreactive for both PPARγ

and TuJ1 antibodies (yellow cells) and is enlarged in D to show DRG neurons immunoreactive

for PPARγ staining (red cells, white arrows). (E-H) Developing enteric neurons (TuJ1+ cells

highlighted with arrows in F) have much lower levels of immunoreactive PPARγ (i.e., no yellow

cells in the region of the ENS in G). The boxed region in G is enlarged in H to show the lack of

PPARγ staining (red) in the ENCDC. Arrows highlight the region of the developing ENS.

Images are from the same fetal mouse stained on the same slide. Scale bar = 100µm.

Figure 7:

Ibuprofen had delayed effects on murine ENCDC migration. (A) Experimental paradigms.

E12.5 CF-1 midgut slices were cultured on fibronectin. Ibuprofen was added four hours after

plating (ibuprofen late) or at plating (ibuprofen). Time lapse images were then obtained every

two minutes for four hours. (B-D) DIC images show final time points from time-lapse movies.

Tracks show trajectories for single cells that migrated from explants. Immunohistochemistry

after imaging confirmed tracked cells expressed RET. Scale bar = 100µm. (E) Mean ENCDC

migration speed during each one hour interval. Ibuprofen slowed ENCDC speed, but not until 3-

37

4 hours after ibuprofen exposure (Control n=130 cells, Ibuprofen n=180 cells, ibuprofen late

n=92 cells; all 3 biological replicates). *P<0.05 (ANOVA on ranks).

Figure 8:

Ibuprofen effects on murine ENCDC migration and lamellipodia do not appear to be

cyclooxygenase dependent. Ptgs1-/- Ptgs2-/- mice have normal ENCDC migration in vitro and

normal bowel colonization in vivo. (A, B) E12.5 WT C57BL/6 and Ptgs1-/- Ptgs2-/- midgut

slices were cultured 16 hours after GDNF addition and then stained with RET antibody (red),

phalloidin (green), and DAPI (blue). Scale bar=50 µm. (C, D) E12.5 bowel from WT and Ptgs1-

/- Ptgs2-/- mice was stained with Tuj1 antibody. Scale bar=500 µm. Ptgs1-/- Ptgs2-/- mutations

did not affect (E) the mean distance that ENCDC migrated from gut slices (WT n=57 slices, 5

embryos and Ptgs1-/- Ptgs2-/- n=48, slices, 7 embryos), P=0.55 (t-test), or (F) the mean

percentage of ENCDC with a well-formed lamellipodia (WT n=11 slices, 4 embryos and Ptgs1-/-

Ptgs2-/- n=10 slices, 5 embryos), P=0.38 (t-test), or (G) the extent of bowel colonization by

ENCDC in vivo (WT n=6 and Ptgs1-/- Ptgs2-/- n=5), P=0.23 (t-test). Only ENCDC most distant

from gut slices were evaluated for lamellipodia. (H-K) Stable Prostaglandin E2 (PGE2) and

Prostaglandin F2 (PGF2) analogs did not rescue ibuprofen-induced ENCDC migration defects.

E12.5 CF-1 midgut slices were cultured in media with GDNF with or without ibuprofen for 16

hours and then stained for RET, F-actin (phalloidin) and with DAPI. (H, I) Distance from the gut

slice edge to the most distant RET+ ENCDC was measured. Neither 16, 16-dimethyl PGE2 (H)

nor 16, 16-dimethyl PGF2 (I) restored ENCDC migration to control levels in the presence of

ibuprofen, but 16,16-dimethyl PGE2 (H) did reduce ENCDC migration in the absence of

ibuprofen. (For PGE2 studies: Control n = 165 slices, ibuprofen n = 112 slices, 16, 16-dimethyl

38

PGE2 n = 137 slices, ibuprofen plus 16, 16-dimethyl PGE2 n = 53 slices; *P<0.05 compared to

Control. #P<0.05 compared to Prostaglandin E2 (ANOVA). For PGF2 studies: Control n =103

slices, ibuprofen n = 96 slices, 16, 16-dimethyl PGF2 n = 127 slices, ibuprofen plus PGF2 n =

107 slices; *P<0.05 compared to Control. #P<0.05 compared to Prostaglandin F2 (ANOVA on

Ranks)) (J, K) The percentage of ENCDC with well-formed lamellipodia was determined by

examining phalloidin stained ENCDC that had migrated furthest from the gut slice edge. Neither

16, 16-dimethyl PGE2 nor 16, 16-dimethyl PGF2 rescued lamellipodia in the presence of

ibuprofen (For PGE2 studies: Control n = 27 slices, ibuprofen n = 18 slices, PGE2 n = 27 slices,

ibuprofen plus PGE2 n =18 slices) *P<0.05 compared to Control. #P<0.05 compared to

Prostaglandin E2 (ANOVA on Ranks). For PGF2 studies: Control n = 105 slices, ibuprofen n =

83 slices, 16, 16-dimethyl PGF2 n = 105 slices, ibuprofen plus 16, 16-dimethyl PGF2 n =93

slices *P<0.05 compared to Control. #P<0.05 compared to Prostaglandin F2 (ANOVA)).

Figure 9:

Ibuprofen reduced active RAC1/CDC42, but did not affect active RHOA in migrating

murine ENCDC. E12.5 CF-1 midgut slices cultured 16 hours with GDNF were fixed,

permeabilized and incubated with (A-H) PBD-GST that binds active GTP-RAC1 and GTP-

CDC42, or (I-P) with RBD-GST that binds active GTP-RHOA. Cultures were then stained for

(A, D, I, L) RET (green), (B, E, J, M) GST (red), and (C, F, K, N) DAPI (blue; merged image).

Scale bar=50µm. (G, O) Mean fluorescence intensity for PBD-GST (G) and RBD-GST (O) for

ENCDC that migrated furthest from gut slices. (G) 250 µM Ibuprofen treated ENCDC had less

bound PBD-GST indicating reduced active RAC1/CDC42 (Control n=243 cells, ibuprofen

n=205 cells, both 4 biological replicates); *P<0.001 (Mann-Whitney Rank Sum), but did not

39

reduce (O) RBD-GST bound to active RHOA in actively migrating ENCDC (Control n=144

cells, ibuprofen n=136 cells, both 4 biological replicates). P=0.408 (Mann-Whitney Rank Sum).

(H, P) Because ENCDC shape is altered by ibuprofen and this may change mean fluorescence

intensity per pixel, total fluorescence intensity (mean fluorescence intensity/pixel x pixels/cell)

was determine for (H) active RAC1/CDC42 (via PGD-GST binding) (Control n=243 cells,

ibuprofen n=205 cells, both 4 biological replicates, and (P) active RHOA (via RBD-GST

binding) (Control n =144 cells, ibuprofen n=136 cells, both 4 biological replicates) per cell.

*P<0.001, Mann-Whitney Rank Sum. These data indicate that ibuprofen reduced total cellular

levels of F-actin and active RAC1/CDC42 without affecting active RHOA levels. Only ENCDC

furthest from the gut slice edge were analyzed.

Figure 10:

ROCK inhibition rescued ibuprofen effects on murine ENCDC migration. (A-L) E12.5 CF-

1 midgut slices were cultured with or without 250µM ibuprofen four hours before GDNF and 5

µM Y-27632 were added. Sixteen hours later cultures were fixed and stained for (A, D, G, J)

RET (red), (B, E, H, K) phalloidin (green), and (C, F, I, L) DAPI (blue; merged image). Scale

bar = 50 µm. (M) Distance from the gut slice edge to the most distant RET+ ENCDC was

measured. Y-27632 increased migration of ibuprofen treated ENCDC, but not control ENCDC

(Control n =43 slices, ibuprofen n=48 slices, Y-27632 n=54 slices, ibuprofen + Y-27632 n=59

slices, all from 7 biological replicates). *P<0.05 compared to control, #P<0.05 compared to

ibuprofen + Y-27632 (ANOVA on ranks).

Figure 11:

40

Model. RAC1 organizes the actin cytoskeleton to form lamellipodia and support migration.

Ibuprofen reduced ENCDC RAC1 activation. ROCK is a RHOA effector that inhibits RAC1. Y-

27632 inhibits ROCK to permit RAC1 activation, enhancing ENCDC migration.

Supplemental Figure Legends

Supplemental Figure 1: (Additional file 1: Schill Supplemental Figure 1.pdf)

Midgut slice explant culture and analysis. E12.5 CF-1 mouse midgut slices were cultured for

16 hours on fibronectin coated dishes in media with GDNF. ENCDC migrate from gut slices

onto the culture dish under these conditions. For analyses where we report the “Distance

migrated” by ENCDC from the slice, the slice area was divided into octants (dotted lines) and

the distance from the gut slice edge to the most distant ENCDC (RET immunoreactive cell, red)

was measured for each octant. Cell morphology was also assessed in “Distal ENCDC” (i.e., the

RET+ cells that had migrated furthest from the slice edge). Mesenchymal cells were identified

by their lack of RET immunoreactivity and their actin-rich cytoskeleton (stained green with

Alexa488-phalloidin). Phalloidin is a fungal bicyclic heptapeptide that binds filamentous, but

not G-actin (Govindan et al., 1972). The distance migrated by mesenchymal cells was

determined by measuring the distance from the gut slice edge to the most distant RET negative

cell with distinctive intense phalloidin staining.

Supplemental Figure 2: (Additional file 6: Schill Supplemental Figure 6.pdf)

41

PBD-GST and RBD-GST fusion proteins bind to cultured NIH 3T3 fibroblasts as predicted

using well-established methods to activate RAC1/CDC42 or RHOA. NIH 3T3 were serum

starved and then either (A, B) left unstimulated, (C) treated with RAC1/CDC42 Activator II to

activate RAC1 and CDC42, or (D) treated with RHO Activator I to activate RHOA. Cells were

fixed and treated with either PBD-GST (A, C) or RBD-GST (B, D) as in Figure 9. All cells were

then stained with antibodies to GST to visualize active RAC1/CDC42 or RHOA. Stimulated

cells had greater GST fluorescence compared to unstimulated cells for PBD-GST and RBD-GST

fusion proteins as expected after stimulation. Scale bar = 20 µm.

Supplemental Movies 1-3:

(Additional file 6: Movie 1 Control.avi)

(Additional file 7: Movie 2 Delayed ibuprofen.avi)

(Additional file 8: Movie 3 Ibuprofen.avi)

Time lapse images of migrating ENCDC. E12.5 CF-1 midgut slice explants were cultured for

4 hours before GDNF addition. Time lapse imaging began when GDNF was added and

continued for 4 hours. DIC images include a time stamp in “hours:minutes” in the upper left

hand corner.

Movie 1: No added ibuprofen.

Movie 2: Ibuprofen added just before imaging began.

Movie 3: Ibuprofen added 4 hours before imaging began.

42

Schill et. al. Highlights

1. Ibuprofen inhibits ENS precursor migration in zebrafish, chick and mouse.

2. Ibuprofen reduces lamellipodia and F-actin in cultured murine ENS precursors.

3. Ibuprofen reduces RAC1 activation in migrating ENS precursors.

4. Ibuprofen effects on ENS precursor migration appear to be COX independent.

5. Ibuprofen is commonly used in early pregnancy and might increase Hirschsprung risk.