Comparative Microarray Analysis of Proliferating and ... · ResearchArticle Comparative Microarray...

Research ArticleComparative Microarray Analysis of Proliferating andDifferentiating Murine ENS Progenitor Cells

Peter Helmut Neckel Roland Mohr Ying Zhang Bernhard Hirt and Lothar Just

Institute of Clinical Anatomy and Cell Analysis University of Tubingen Osterbergstrasse 3 72074 Tubingen Germany

Correspondence should be addressed to Lothar Just ljustanatomuni-tuebingende

Received 15 May 2015 Accepted 12 July 2015

Academic Editor Kodandaramireddy Nalapareddy

Copyright copy 2016 Peter Helmut Neckel et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Postnatal neural progenitor cells of the enteric nervous system are a potential source for future cell replacement therapies ofdevelopmental dysplasia like Hirschsprungrsquos disease However little is known about the molecular mechanisms driving thehomeostasis and differentiation of this cell pool In this work we conducted Affymetrix GeneChip experiments to identifydifferences in gene regulation between proliferation and early differentiation of enteric neural progenitors from neonatal miceWe detected a total of 1333 regulated genes that were linked to different groups of cellular mechanisms involved in cell cycleapoptosis neural proliferation and differentiation As expected we found an augmented inhibition in the gene expression of cellcycle progression aswell as an enhancedmRNAexpression of neuronal and glial differentiationmarkersWe further found amarkedinactivation of the canonicalWnt pathway after the induction of cellular differentiation Taken together these data demonstrate thevarious molecular mechanisms taking place during the proliferation and early differentiation of enteric neural progenitor cells

1 Introduction

The enteric nervous system (ENS) is a largely autonomousand highly complex neuronal network found in the gas-trointestinal tract Its two major plexuses are integrated intothe layered anatomy of the gut wall and together with cen-tral modulating influences exert control over gastrointesti-nal motility secretion ion-homeostasis and immunologicalmechanisms [1] In order to achieve this variety of functionsthe ENS is composed of a multitude of different neuronal andglial cell types and closely interacts with smooth muscle cellsandmyogenic pacemaker cells called interstitial cells of CajalFurthermore a population of neural stem or progenitor cellsin the ENS has been identified in rodents [2 3] and humansthat retain their proliferative capacity throughout adult lifeeven into old age [4 5] It is therefore not surprising that thecorrect functioning of the ENS as well as the regulation onenteric neural progenitor cells is subjected to the influence ofa myriad of transmitters neurotrophic and growth factorssignalling molecules and extracellular matrix componentswhich are not exclusively expressed by neural cell types [6]

Likewise the control of the development of the ENS is equallycomplex andmutations in its genetic programcan lead to fataldysplasia like Hirschsprungrsquos disease (HCSR) [7 8]

HSCR is hallmarked by an aganglionic distal bowelleading to life-threatening disturbances in intestinal motilityTodayrsquos therapeutic gold standard the surgical resection ofthe affected gut segments is nevertheless associated withproblematic long-term outcomes with regard to continence[9] In order to improve the therapeutic success the use ofautologous enteric neural stem cells was proposed [10] Thisconcept relies on the in vitro expansion of enteric neural stemcells derived from small biopsy materials However we arejust beginning to understand the molecular mechanisms thatunderlie neural stem cell biology and how this knowledge canbe used for optimizing in vitro culture conditions [11 12]

Genome-wide gene-expression analyses are a useful toolto examine the genetic programs and cellular interactionsand have been widely used to identify potential markersor signalling mechanisms especially in CNS neurospheresor cancer tissues Further gene-expression assays have alsohelped to unravel genetic prepositions associated with HSCR

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 9695827 13 pageshttpdxdoiorg10115520169695827

2 Stem Cells International

[13 14] though little effort has so far been put into charac-terizing the genetic profile of enteric neural stem cells in vitro[15]

Here we used an Affymetrix microarray analysis toevaluate the genetic expression profile of proliferatingmurineenteric neural stem cells and its changes during the earlydifferentiation in vitro

2 Materials and Methods

21 Cell Culturing Cell culturingwas conducted as describedpreviously [15] The handling of animals was in accordanceto the institutional guidelines of the University of Tuebingenwhich conform to the international guidelines

Neonatal (P0) C57BL6 mice without regard to sex weredecapitated and the whole gut was removed After removalof adherent mesentery the longitudinal and circular musclelayers containing myenteric plexus could be stripped as awhole from the small intestine Tissue was chopped andincubated in collagenase type XI (750UmL Sigma-AldrichTaufkirchen Germany) and dispase II (250 120583gmL RocheDiagnostics Mannheim Germany) dissolved in Hanksrsquo bal-anced salt solution with Ca2+Mg2+ (HBSS PAA PaschingAustria) for 30min at 37∘C During enzymatic dissociationthe tissue was carefully triturated every 10min with a firepolished 1mL pipette tip Prior to the first trituration step cellsuspension was treated with 005 (wv) DNAse I (Sigma-Aldrich) After 30min tissue dissociation was stopped byadding fetal calf serum (FCS PAA) to a final concentrationof 10 (vv) to the medium Undigested larger tissue pieceswere removed with a 40 120583m cell strainer (BD BiosciencesFranklin Lakes NJ USA) Residual enzymes were removedduring twowashing steps inHBSS at 200 gAfter dissociationcells were resuspended in proliferation culturemedium (Dul-beccorsquos modified Eaglersquos medium with Hamrsquos F12 medium(DMEMF12 1 1 PAA)) containing N2 supplement (1 100Invitrogen Darmstadt Germany) penicillin (100UmLPAA) streptomycin (100 120583gmL PAA) L-glutamine (2mMPAA) epidermal growth factor (EGF 20 ngmL Sigma-Aldrich) and fibroblast growth factor (FGF 20 ngmLSigma-Aldrich) Cells were seeded into 6-well plates (BDBiosciences) in a concentration of 25 times 104 cellscm2 Onlyonce before seeding the medium was supplemented withB27 (1 50 Invitrogen) EGF and FGF were added daily andculture medium was exchanged every 3 days All cultivationsteps were conducted in a humidified incubator at 37∘Cand 5 CO

2 An overview of the following cell culture

protocol is shown in Figure 1 During proliferation phaseof the culture cells formed spheroid-like bodies termedenterospheres After 5 days of proliferation free-floatingenterospheres were picked and transferred to petri dishes (Oslash60mm Greiner Bio One Frickenhausen Germany) in 5mLfresh proliferation medium and proliferation was continuedfor further 4 days

Single free-floating enterospheres (50 enterospheresdish) were picked again washed 3 times in Tris buffer andtransferred into new petri dishes containing either prolif-eration medium or differentiation medium Differentiationmedium consists of DMEMF12 containing N2 supplement

9 div start of differentiation

1 div 5 div

0 div 5 div spheres picked

Differentiation for 2 divProliferation for 9 div

Figure 1 Time schedule of enterosphere culture The timelineillustrates the schedule of in vitro culture Cells were isolated at 0div (days in vitro) and proliferated for 5 days Spheres were thenpicked and again proliferated for 4 days At 9 div enterosphereswere picked washed transferred to differentiation medium andincubated for 2 days before gene expression analyses were carriedout The micrographs show proliferating enterospheres after 1 and 5div Scale bar 200 120583m

(1 100) penicillin (100UmL) streptomycin (100 120583gmL) L-glutamine (2mM) and ascorbic acid-2-phosphate (200 120583MSigma-Aldrich)

Enterospheres were proliferated or differentiated for 2more days thereby forming the two experimental groupsldquoproliferationrdquo and ldquodifferentiationrdquoThe difference in expres-sion between those two groups (differentiation versus prolif-eration) was successively compared by microarray analysis asdescribed below

22 Affymetrix Microarray Analysis Affymetrix microarrayanalysis was conducted similar to previously published datain three independent experiments each with cell culturesprepared from 2 pups from the same litter [15] In each exper-iment free-floating enterospheres were picked as describedabove in order to diminish the fraction of adhesive fibroblastsand smooth muscle cells

Total RNA of enterospheres of both groups was extractedusing the RNeasy Micro Kit (Qiagen) RNA quality wasevaluated on Agilent 2100 Bioanalyzer with RNA integritynumbers (RIN) of the samples in this study being in the rangefrom 8 to 10 RIN numbers higher than 8 are consideredoptimal for downstream application [16]

Double-stranded cDNA was synthesized from 100 ng oftotal RNA subsequently linearly amplified and biotinylatedusing the GeneChip WT cDNA Synthesis and AmplificationKit (Affymetrix Santa Clara CA USA) according to themanufacturerrsquos instructions 15 120583g of labeled and fragmentedcDNAwas hybridized toGeneChipMouseGene 10 ST arrays(Affymetrix) After hybridization the arrays were stainedand washed in a Fluidics Station 450 (Affymetrix) withthe recommended washing procedure Biotinylated cDNAbound to target molecules was detected with streptavidin-coupled phycoerythrin biotinylated anti-streptavidin IgGantibodies and again streptavidin-coupled phycoerythrinaccording to the protocol Arrays were scanned using theGCS3000 GeneChip Scanner (Affymetrix) and AGCC 30software Scanned images were subjected to visual inspection

Stem Cells International 3

to check for hybridization artifacts and proper grid alignmentand analyzed with Expression Console 10 (Affymetrix) togenerate report files for quality control

Normalization of raw data was performed by the PartekSoftware 66 applying an RMA (Robust Multichip Average)algorithm Significance was calculated using a t-test withoutmultiple testing correction (Partek) selecting all transcriptswith a minimum change in expression level of 15-foldtogether with a 119901 value less than 005

3 Results

In this study we investigated the changes of the geneticexpression profile that occur during the transition fromproliferating to differentiating enteric neural progenitor cellsin vitro Therefore we generated enterospheres by 9 dayin vitro cultures which then could be picked and eitherproliferated or differentiated for two more days (Figure 1)mRNA was subsequently extracted and gene expression ofthese two groups was analysed by Affymetrix microarrayanalysis

Analysis of mRNA expression was performed on aGeneChip Mouse Gene 10 ST array that determines theexpression profile of 28853 genes Each genewas interrogatedby a median of 27 probes that are spread along the full gene

In total the gene chip detected 1454 transcripts to beat least 15-fold differentially expressed between proliferatingand differentiating enterospheres 1333 of these transcriptscode for already identified proteins 541 genes were found tobe upregulated and 792 genes were found to be downregu-lated in comparison to proliferating enterospheres (see Sup-plementary Table 1 of the Supplementary Material availableonline at httpdxdoiorg10115520169695827)

We used the ingenuity pathway analysis software (IPA)and data mining with the science literature search enginehttpwwwncbinlmnihgovpubmed to divide the genesinto different groups according to their function duringcellular developmentThe largest functional group contained171 genes related to cell cycle and apoptosis (Table 1 Sup-plementary Table 2) Here we identified especially differentcyclin proteins and cell division cycle proteins that weremainly downregulated Further we found several genes thatare linked to neural development as well as genes regulatingneural stem cell proliferation and differentiation Further-more we also detected neuronal and glial differentiationmarkers and numerous genes involved in synapse formation(Table 2) It is noteworthy that we also identified a group ofgenes that are known to be involved in the differentiationof smooth muscle cells (Table 3) as well as in extracellularmatrix components (Table 4) Additionally we found reg-ulated genes related to canonical Wnt signalling indicatinga deactivation of this pathway during ENS progenitor celldifferentiation (Figure 2 Table 5)

4 Discussion

The proliferation and differentiation of enteric neural pro-genitor cells during embryonic and postnatal developmentare controlled by a complex interplay of various intrinsic

Table 1 Selected genes related to cell cycle

Gene Encoded protein Foldchange

Cell cycle

AURKA Aurora kinase A minus2712 STOPAURKB Aurora kinase B minus4146 STOPCCNA2 Cyclin A2 minus4652 STOP

CCNB1 Cyclin B1minus5752minus5820minus5857

STOP

CCNB2 Cyclin B2 minus3392 STOPCCND1 Cyclin D1 minus2476 STOPCCND3 Cyclin D3 minus1539 STOPCCNE1 Cyclin E1 minus1777 STOPCCNE2 Cyclin E2 minus2847 STOPCCNF Cyclin F minus3211 STOPCDC6 Cell division cycle 6 minus1936 STOPCDC20 Cell division cycle 20 minus3113 STOPCDC25B Cell division cycle 25B minus1636 STOPCDC25C Cell division cycle 25C minus2414 STOPCDC45 Cell division cycle 45 minus1769 STOPCDCA2 Cell division cycle associated 2 minus3461 STOPCDCA3 Cell division cycle associated 3 minus3003 STOPCDCA5 Cell division cycle associated 5 minus3053 STOP

CDCA7L Cell division cycle associated7-like minus4123 STOP

CDCA8 Cell division cycle associated 8 minus3467 STOPCDK1 Cyclin-dependent kinase 1 minus3227 STOPCDK15 Cyclin-dependent kinase 15 1618 GOCDK19 Cyclin-dependent kinase 19 1619 GO

CDK5R1 Cyclin-dependent kinase 5regulatory subunit 1 (p35) 1597 mdash

CENPA Centromere protein A minus1895 STOPCENPE Centromere protein E 312 kDa minus4140 STOP

CENPF Centromere protein F350400 kDa minus3927 STOP

CENPI Centromere protein I minus2899 STOPCENPK Centromere protein K minus2813 STOPCENPL Centromere protein L minus1864 STOPCENPM Centromere protein M minus3407 STOPCENPN Centromere protein N minus2465 STOPCENPU Centromere protein U minus1624 STOP

SKA1 Spindle and kinetochoreassociated complex subunit 1 minus1532 STOP



SKP2S-phase kinase-associatedprotein 2 E3 ubiquitin proteinligase

minus1845 STOP

SPC25 SPC25 NDC80 kinetochorecomplex component minus4148 STOP


Table 2 Neural differentiationdevelopment

Gene Encoded protein Fold changeNeural stem cells

ABCG2 ATP-binding cassette subfamily G (WHITE) member 2 (junior blood group) minus1526ASPM asp (abnormal spindle) homolog microcephaly associated (Drosophila) minus4911CDT1 Chromatin licensing and DNA replication factor 1 minus1528EGFL7 EGF-like-domain multiple 7 3132EPHA2 EPH receptor A2 minus1529ETV4 ets variant 4 minus1934ETV5 ets variant 5 minus2844

minus2651FABP7 Fatty acid binding protein 7 brain minus2095

Neural differentiationATOH8 Atonal homolog 8 (Drosophila) 1932AXL AXL receptor tyrosine kinase 2015CRIM1 Cysteine-rich transmembrane BMP regulator 1 (chordin-like) 1999CRLF1 Cytokine receptor-like factor 1 2382DAB1 Dab reelin signal transducer homolog 1 (Drosophila) minus2297ELK3 ELK3 ETS-domain protein (SRF accessory protein 2) minus1613ESCO2 Establishment of sister chromatid cohesion N-acetyltransferase 2 minus4767GAP43 Growth associated protein 43 1613GLDN Gliomedin 5809HMOX1 Heme oxygenase (decycling) 1 1884KLF9 Kruppel-like factor 9 1592Lmo3 LIM domain only 3 1542MAP6 Microtubule-associated protein 6 1874MYRF Myelin regulatory factor 2527NEUROD4 Neuronal differentiation 4 2036OLIG1 Oligodendrocyte transcription factor 1 2660Pvr Poliovirus receptor 1768RGS4 Regulator of G-protein signaling 4 1955S1PR1 Sphingosine-1-phosphate receptor 1 5073SOCS2 Suppressor of cytokine signaling 2 2052

2335WIPF1 WASWASL interacting protein family member 1 1587

Neural differentiation markersCALB2 Calbindin 2 1616CNP 2101584031015840-Cyclic nucleotide 31015840-phosphodiesterase 1732GFAP Glial fibrillary acidic protein 2239MBP Myelin basic protein 1768Mturn Maturin neural progenitor differentiation regulator homolog (Xenopus) 1853OMG Oligodendrocyte myelin glycoprotein minus1822OPALIN Oligodendrocytic myelin paranodal and inner loop protein 39246PLP1 Proteolipid protein 1 1630S100B S100 calcium binding protein B minus1675TUBB2A Tubulin beta 2A class IIa 1608TUBB2B Tubulin beta 2B class IIb 1535TUBB3 Tubulin beta 3 class III 1976


Table 2 Continued

Gene Encoded protein Fold changeSynapse and neurotransmitters

ABAT 4-Aminobutyrate aminotransferase minus1512ADRA1D Adrenoceptor alpha 1D 1803ADRA2A Adrenoceptor alpha 2A 2900ADRA2B Adrenoceptor alpha 2B minus2093CHRM2 Cholinergic receptor muscarinic 2 1635CHRM3 Cholinergic receptor muscarinic 3 minus1715CHRNA7 Cholinergic receptor nicotinic alpha 7 (neuronal) 1772COMT Catechol-O-methyltransferase 1515DDC DOPA decarboxylase (aromatic L-amino acid decarboxylase) 1711DNM3 Dynamin 3 2643EPHA5 EPH receptor A5 2076GRIA3 Glutamate receptor ionotropic AMPA 3 minus1528GRIA4 Glutamate receptor ionotropic AMPA 4 minus1997GRIK2 Glutamate receptor ionotropic kainate 2 minus1565GRM5 Glutamate receptor metabotropic 5 minus1600HTR1B 5-Hydroxytryptamine (serotonin) receptor 1B G-protein-coupled minus2377HTR2B 5-Hydroxytryptamine (serotonin) receptor 2B G-protein-coupled 2205LRRTM2 Leucine-rich repeat transmembrane neuronal 2 3665LRRTM3 Leucine-rich repeat transmembrane neuronal 3 2210NTM Neurotrimin 1820PENK Proenkephalin 3478PRR7 Proline rich 7 (synaptic) 1788SLC10A4 Solute carrier family 10 member 4 1824

1867SLITRK2 SLIT and NTRK-like family member 2 minus2414SLITRK6 SLIT and NTRK-like family member 6 1672STON2 Stonin 2 4054STXBP3 Syntaxin-binding protein 3 1730Stxbp3b Syntaxin-binding protein 3B 1637SV2C Synaptic vesicle glycoprotein 2C 1929SYT6 Synaptotagmin VI 2571

Neurite outgrowthATF3 Activating transcription factor 3 2579DOK4 Docking protein 4 4937FEZ2 Fasciculation and elongation protein zeta 2 (zygin II) 1547NAV2 Neuron navigator 2 1647NRCAM Neuronal cell adhesion molecule 2496PLXNB3 Plexin B3 1739RGMA Repulsive guidance molecule family member a 1552RNF165 Ring finger protein 165 minus1548ROBO2 Roundabout axon guidance receptor homolog 2 (Drosophila) minus2211SEMA3B Sema domain immunoglobulin domain (Ig) short basic domain secreted (semaphorin) 3B 3692SEMA3E Sema domain immunoglobulin domain (Ig) short basic domain secreted (semaphorin) 3E 2877

SEMA4F Sema domain immunoglobulin domain (Ig) transmembrane domain (TM) and shortcytoplasmic domain (semaphorin) 4F 4891

SEMA6A Sema domain transmembrane domain (TM) and cytoplasmic domain (semaphorin) 6A minus1707SRGAP1 SLIT-ROBO Rho GTPase activating protein 1 1524UNC5B unc-5 homolog B (C elegans) minus1927


Table 2 Continued

Gene Encoded protein Fold changeGrowth factors

ARTN Artemin 2423FGF2 Fibroblast growth factor 2 (basic) 2264FGF5 Fibroblast growth factor 5 7704GDF10 Growth differentiation factor 10 minus2361GDF11 Growth differentiation factor 11 1604GDNF Glial cell derived neurotrophic factor 4325GFRA3 GDNF family receptor alpha 3 1707MET MET protooncogene receptor tyrosine kinase 6680NGFR Nerve growth factor receptor 1728NTRK3 Neurotrophic tyrosine kinase receptor type 3 minus1575SNX16 Sorting nexin 16 1641SPHK1 Sphingosine kinase 1 1704SPRY1 Sprouty homolog 1 antagonist of FGF signaling (Drosophila) minus1647

and extrinsic factors Their exact timing is crucial for propermigration and proliferation of neural crest cells and fortheir differentiation into the various neural cell types thatcompose the complex neural structures of the ENS Althoughresearch in recent years extended our understanding of ENSdevelopment and its pathologies [13] there are still manygenes and processes unknown Particularly factors regulatingneural progenitor proliferation and differentiation in thedeveloping and postnatal gut as well as cellular andmolecularinteraction systems remain largely elusive Here we used invitro cultures of enteric neural progenitor cells derived frommurine tunica muscularis to scan for molecular programsand signalling pathways acting on cell proliferation and earlydifferentiation

Our experiment aimed to elucidate gene regulations inenterospheres that occur while ENS progenitor cells leavetheir proliferative state and begin to differentiate into moredefined and specific cell types The results of the Affymetrixgene expression analysis showed the up- and downregu-lation of overall 1333 known genes that code for alreadyidentified proteins 171 of these genes could be linked tocell proliferation (Table 1 Supplementary Table 1) Amongstthem we detected genes coding for proteins related to thekinetochore complex (like NSL1 [17] NUF2 [18] SKA1-3 [19]and ZWILCH [20]) cyclin proteins [21] cyclin-dependentkinases (CDK) [22] and several types of centromere proteinsThe regulation of 145 of these genes strongly indicates aslowdown of cell cycle progression as it was intended bythe experimental deprivation of growth factor supplemen-tation by the end of the proliferation phase (see Section 3)Interestingly betacellulin (BTC) was upregulated nearly 6-fold although it was reported to promote cellular proliferationin the neural stem cell niche [23] Nonetheless the vastmajority of genes including all regulated cyclins cell divisioncycle proteins and kinetochore proteins were found to bedownregulated

We also checked the regulated genes for apoptosis mark-ers to see whether the stop in proliferation was related to cell

death (Supplementary Table 2) Since only 3 of 12 apoptoticgenes were regulated in the direction that indicates apoptosisit is unlikely that apoptosis played a leading role in theinterruption of proliferation Still the effect and regulationof apoptosis during enteric sphere cultures are an importantcornerstone of understanding enteric neural progenitorsin culture and in vivo and require further investigationTogether on a broad basis this dataset provides strong evi-dence that this cell culture design is applicable to decreasingthe proliferative rate of enteric neural progenitor cells withoutinducing cell death or apoptosis in an appreciable quantity

To further evaluate the proliferative conditions of celltypes present in enterospheres we focused on different cellspecific markers of neural progenitors as well as neuronsglial or smoothmuscle cells We consider this complex cellu-lar composition of the enterospheres an advantage comparedto more purified neural crest derived neurospheres as we areable to capture complex interactions and secretion mecha-nisms between cell types that might also play an importantrole in vivo Interestingly we found 8 genes involved in adultcentral or embryonic neural stem cells homeostasis (Table 2)The majority of genes like EPHA2 [24] are regulated in away that suggests that neural stem cells exit the proliferativecell cycle to enter differentiation programs This idea wassupported by the upregulation of numerous genes that driveneuronal and glial differentiation like NEUROD4 [25] orOLIG1 [26] In this context we identified several upregulatedgenes involved in proper myelination As enteric and centralglia cells are known to temporally express myelin-relatedproteins during development it is conceivable that thisregulation is part of the early glial differentiation program[27] Moreover also typical markers of differentiated neurons(class III beta-tubulin CALB2 [28]) and enteric glia (GFAP[29]) were found to be upregulated Intriguingly S100B acommon glia cell marker was downregulated contrasting therest of our data Again this might be due to the complexdifferentiation program of enteric glia in which S100B playsa role at later stages


Cyto

plas

mEx

trac

ellu

lar s

pace

Nuc

leus

DNA

FZD LRP56minus1571

WNT

DKK21731LRP41979

FRZB1938

NOTUM2631

NOV2050

APOE1704

DAAM2minus1993

PSRC1minus2235

SMURF11629

TNIK1677

TRAF4minus1673

EDIL32258

HIC1minus1731

JADE1minus1656

NARFminus1699

NKD13220

PRICKLE11536

TLE31714 TRIB2

minus1637

NEDD4L1588

FZD7minus2271

DVL

120573-catenin

120573-catenin

TCF

APC

LEF

AXIN21744

LRRK21677

GSK-3

LEF1minus2680

WNT5Aminus2325

TCF19minus2217

WNT7A2942

TCF7L1minus1762

Figure 2 Detected regulatory influences on the canonical Wnt pathway Scheme of the canonical Wnt pathway Pointy arrowheads indicatean activating blunt arrowheads an inhibitory influenceThe fold-change in expression of genes is written under the respective gene acronymsand colours indicate a general upregulation (red) or downregulation (green) For detailed explanation of the signalling cascade and regulatedgenes see text

Furthermore the establishment of neuronal cell commu-nication was strongly regulated Here we found an increasedexpression of genes related to synaptogenesis (LRRTM2 and3 [30] neurotrimin [31]) and to SNARE or vesicle proteinfunction (STXBP3 SV2C [32] and SYT6 [33]) We also iden-tified a number of genes involved in transmitter metabolism(COMT DDC) as well as neurotransmitter receptor like5-HT glutamate and adrenergic receptors However theregulation of those genes was highly variable shedding lighton the intricacy of synapse formation in the developingenteric nervous systemThis complexity is carried on by genes

related to axon sprouting and guidance like semaphorins [34]or RGMa [35]

Additionally we found that regulated genes directlyinvolved in the differentiation of muscle cells andor entericpacemaker cells called interstitial cells of Cajal (Table 3)Particularly interesting is the upregulation of a number ofgenes known to drive smooth muscle differentiation likeARID5B [36] FOSL2 [36] and genes that are expressedin differentiated smooth muscle cells in the intestine likeAFAP1 [37] ENPP2 [38] and CNN1 [39] as well as var-ious myosin and actin isoforms These data confirm the


Table 3 Differentiation of smooth muscle cellsICCs

Gene Encoded protein Fold changeSmooth muscle cells

ACTA2 Actin alpha 2 smooth muscle aorta 1693

ACTG2 Actin gamma 2 smooth muscleenteric 2336

ACTN1 Actinin alpha 1 minus1724AEBP1 AE binding protein 1 2702AFAP1 Actin filament associated protein 1 1638

ARID5B AT-rich interactive domain 5B(MRF1-like) 1521

Cald1 Caldesmon 1 minus1535CNN1 Calponin 1 basic smooth muscle 1652ENG Endoglin minus1552

ENPP1 Ectonucleotidepyrophosphatasephosphodiesterase 1 minus1522

ENPP2 Ectonucleotidepyrophosphatasephosphodiesterase 2 2959

ENTPD1 Ectonucleoside triphosphatediphosphohydrolase 1 1636

FOSL2 FOS-like antigen 2 2566GAMT Guanidinoacetate N-methyltransferase 1725MYO1E Myosin IE 1569MYO5A Myosin VA (heavy chain 12 myoxin) 1680MYO7B Myosin VIIB 1710MYO18A Myosin XVIIIA 1994MYPN Myopalladin 1570NEB Nebulin 1569Nebl Nebulette 2378NUP210 Nucleoporin 210 kDa minus1838RBM24 RNA binding motif protein 24 1548SMTN Smoothelin minus1778SSPN Sarcospan 1603TAGLN Transgelin 2706

ICCGUCY1A3 Guanylate cyclase 1 soluble alpha 3 minus1876GUCY1B3 Guanylate cyclase 1 soluble beta 3 minus2008

KIT v-kit Hardy-Zuckerman 4 felinesarcoma viral oncogene homolog minus1798

KITLG KIT ligand minus1541

fact that cultured spheroids are composed of different celltypes present in the intestinal tunica muscularis and fur-ther indicate that deprivation of growth factors inducesdifferentiation of smooth muscle cells resembling molecularprocesses in the developing gut In fact we among otherswere previously able to confirm the presence of smoothmuscle cells derived from enterosphere culture by BrdU-immunolabeling costudies [4] However it is noteworthy thata few genes related tomuscular differentiation (endoglin [40]smoothelin [41] NUP210 [42] caldesmon 1 [43] and ACTN1[44]) were downregulated contrasting the expression patternobserved in the majority of regulated genes This hints to

complex regulatory mechanisms controlling the myogenicdifferentiation program inwhich these genes are not requiredat all or in a different temporal sequence not mapped byour experimental design It is further remarkable that fivemarkers expressed in interstitial cells of Cajal (ICC) includingKIT [45] were downregulated

Moreover the regulation of 43 extracellular matrix pro-teins like collagens integrins proteoglycans and matrixmetallopeptidases points to a reconstruction of extracellularenvironment that has been discussed to influence neuralstem cell behaviour [46] (Table 4) Taken together theseresults illustrate the ongoing genetic programs during earlydifferentiation of enterospheres

Within the dataset it was of special interest to find partic-ularly many regulated genes related to the canonical Wntpathway (Table 5) The involvement of canonical Wnt sig-nalling has frequently been shown in the regulation of variousstem cell niches like intestinal epithelium or CNS derivedneural stem cells However these studies exhibited differentand partly contradicting outcomes which strongly hint to thevariable functions of canonicalWnt signals in different tissuesduring embryonic and postnatal development In previouswork we found regulation of several Wnt-related genes inthe context of thyroid hormone dependent differentiation ofenteric neural progenitor cells indicating a potential role ofthe canonical Wnt pathway activation during the prolifera-tion of this progenitor cell pool [15] CanonicalWnt signallinghas frequently been reviewed in the literaturemdashjust recentlyby Ring et al [47] In brief secretedWnt proteins bind to friz-zled receptors (FZD) complexed with low density lipoproteinreceptor-related protein 56 (LRP56) coreceptorsThereafterthe scaffolding protein disheveled (DVL) is recruited to FZDand inhibits the 120573-catenin destruction complex (AXIN2APC and GSK-3120573) Therefore 120573-catenin accumulates in thecytoplasm and translocates to the nucleus where it binds toTCFLEF transcription factors to initiate Wnt target geneexpression Interestingly our current data strongly indicatethat the canonical Wnt pathway is switched off during thefirst two days of enteric progenitor differentiation on severallevels of the signalling cascade (Figure 2) On the one handwe identified a downregulation of activating parts of thesignalling cascade itself like the receptor proteins FZD7 andLRP5 or the transcription factors TCF19 TCF7L1 and LEF1On the other hand inactivating elements of the pathwaylike parts of the 120573-catenin destruction complex AXIN2 andLRRK2 [48] were upregulated We also found numerousmodulators of the signalling cascade It is of interest that themajority of those genes are reported to inhibit the signallingprocess extracellularly or on receptor level (Notum [49]FRZB [50] DKK2 [51] and LRP4 [52]) in the cytoplasm(NEDD4L [53] NKD1 [54] PRICKLE1 [55] NOV [56] andAPOE [57]) or in the nucleus (TLE3 [58] EDIL3 [59])Furthermore we identified target genes of the canonical Wntpathway that were either upregulated (eg AXIN2 that exertsa negative feedback on the pathway) or downregulated likethe cell cycle progression genes CCND1 and SPRY4 [60] Wealso found a lower expression of SPRY2 [61] a Wnt targetgene and known inhibitor of GDNF signalling [62] in thedifferentiation group Together with a strong upregulation of


Table 4 ECM

Gene Encoded protein Fold changeCHSY3 Chondroitin sulfate synthase 3 minus1645COL6A5 Collagen type VI alpha 5 1527COL12A1 Collagen type XII alpha 1 minus1973COL14A1 Collagen type XIV alpha 1 6135COL16A1 Collagen type XVI alpha 1 1666COL18A1 Collagen type XVIII alpha 1 1595COL27A1 Collagen type XXVII alpha 1 1522COLGALT2 Collagen beta(1-O)galactosyltransferase 2 minus1564CSPG4 Chondroitin sulfate proteoglycan 4 minus2952CSPG5 Chondroitin sulfate proteoglycan 5 (neuroglycan C) minus1585CYR61 Cysteine-rich angiogenic inducer 61 1748ECM1 Extracellular matrix protein 1 2580HSPG2 Heparan sulfate proteoglycan 2 1923ITGA1 Integrin alpha 1 minus1665ITGA4 Integrin alpha 4 (antigen CD49D alpha 4 subunit of VLA-4 receptor) minus2324ITGA7 Integrin alpha 7 4203ITGA8 Integrin alpha 8 minus2262ITGA11 Integrin alpha 11 1762ITGB3 Integrin beta 3 (platelet glycoprotein IIIa antigen CD61) minus5342ITGB4 Integrin beta 4 1567KRT80 Keratin 80 2833LAMA4 Laminin alpha 4 minus1537LAMA5 Laminin alpha 5 1684LOX Lysyl oxidase 3250LOXL4 Lysyl oxidase-like 4 2427

2417MATN2 Matrilin 2 2570MMP2 Matrix metallopeptidase 2 (gelatinase A 72 kDa gelatinase 72 kDa type IV collagenase) 1668MMP9 Matrix metallopeptidase 9 (gelatinase B 92 kDa gelatinase 92 kDa type IV collagenase) minus5557MMP15 Matrix metallopeptidase 15 (membrane-inserted) minus2017MMP16 Matrix metallopeptidase 16 (membrane-inserted) minus1634MMP17 Matrix metallopeptidase 17 (membrane-inserted) 1612MMP19 Matrix metallopeptidase 19 3236MMP28 Matrix metallopeptidase 28 1956NDST3 N-deacetylaseN-sulfotransferase (heparan glucosaminyl) 3 minus5557P4HA1 Prolyl 4-hydroxylase alpha polypeptide I minus1958PLOD3 Procollagen-lysine 2-oxoglutarate 5-dioxygenase 3 2250UGDH UDP-glucose 6-dehydrogenase 1529

GDNF itself by 4325-fold thismight drive enteric progenitorcells into neural differentiation [12]

Taken together it is conceivable that canonical Wntsignalling plays a role in the maintenance of the entericprogenitor pool during proliferation and is switched off at thebeginning of differentiation conditions Indeed our previousgene expression analyses [15] as well as recently publishedcell culture experiments [63] and yet unpublished in vitroanalyses strongly support this hypothesis

5 Conclusion

This study focused on the changes in gene expression ofenteric neural progenitor cells occurring within the first twodays of transition from a proliferative state to differentiationin vitro Using microarray analysis we found a markedinhibition of cell cycle progression in general as well as strongevidence for neural stem cells differentiation into entericneurons and glia cells These findings were substantiated


Table 5 Wnt

Gene Encoded protein Fold changeWnt signaling cascade

FZD7 Frizzled class receptor 7 minus2271LEF1 Lymphoid enhancer-binding factor 1 minus2680LRP5 Low density lipoprotein receptor-related protein 5 minus1571LRRK2 Leucine-rich repeat kinase 2 1677TCF19 Transcription factor 19 minus2217F7L1 Transcription factor 7-like 1 (T-cell specific HMG-box) minus1762WNT5A Wingless-type MMTV integration site family member 5A minus2325WNT7B Wingless-type MMTV integration site family member 7B 2942

Target geneARL4C ADP-ribosylation factor-like 4C 2179AXIN2 Axin 2 1744CCND1 Cyclin D1 minus2476CSRNP1 Cysteine-serine-rich nuclear protein 1 1822RACGAP1 Rac GTPase activating protein 1 minus3201SPRY2 Sprouty homolog 2 (Drosophila) minus1771SPRY4 Sprouty homolog 4 (Drosophila) minus2771WISP1 WNT1 inducible signaling pathway protein 1 2489

Wnt antagonistsinhibitorsAPOE Apolipoprotein E 1704DKK2 Dickkopf WNT signaling pathway inhibitor 2 1731EDIL3 EGF-like repeats and discoidin I-like domains 3 2258FRZB Frizzled-related protein 1938HIC1 Hypermethylated in cancer 1 minus1731JADE1 Jade family PHD finger 1 minus1656LRP4 Low density lipoprotein receptor-related protein 4 1979NARF Nuclear prelamin A recognition factor minus1699NEDD4L Neural precursor cell expressed developmentally downregulated 4-like E3 ubiquitin protein ligase 1588NKD1 Naked cuticle homolog 1 (Drosophila) 3220NOTUM Notum pectinacetylesterase homolog (Drosophila) 2631NOV Nephroblastoma overexpressed 2050PRICKLE1 Prickle homolog 1 (Drosophila) 1536TLE3 Transducin-like enhancer of split 3 1714TRIB2 Tribbles pseudokinase 2 minus1637

Wnt activatorsDAAM2 Dishevelled associated activator of morphogenesis 2 minus1993PSRC1 Prolineserine-rich coiled-coil 1 minus2235TNIK TRAF2 and NCK interacting kinase 1677TRAF4 TNF receptor-associated factor 4 minus1673

by the upregulation of genes related to synapse formationand neural connectivity Most interesting we found that thistransition from enteric neural progenitor proliferation todifferentiation was accompanied by a considerable inactiva-tion of the canonical Wnt signalling pathway This togetherwith previous work strongly indicates that canonical Wntactivation is one of the driving mechanisms of enteric neural

progenitor proliferation and thus might play a role in thehomeostasis of this cell pool in vivo and in vitro

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Authorsrsquo Contribution

Peter Helmut Neckel and Roland Mohr contributed equallyto this work

Acknowledgments

The project was supported by a grant from the German Fed-eral Ministry for Education and Research (01GN0967) Theauthors would like to thank Andrea Wizenmann AndreasMack and Sven Poths for their helpful advice

References

[1] J B Furness ldquoThe enteric nervous system and neurogastroen-terologyrdquoNature Reviews GastroenterologyampHepatology vol 9no 5 pp 286ndash294 2012

[2] D Natarajan M Grigoriou C V Marcos-Gutierrez C Atkinsand V Pachnis ldquoMultipotential progenitors of the mammalianenteric nervous system capable of colonising aganglionic bowelin organ culturerdquoDevelopment vol 126 no 1 pp 157ndash168 1999

[3] G M Kruger J T Mosher S Bixby N Joseph T Iwashitaand S J Morrison ldquoNeural crest stem cells persist in the adultgut but undergo changes in self-renewal neuronal subtypepotential and factor responsivenessrdquo Neuron vol 35 no 4 pp657ndash669 2002

[4] M Metzger P M Bareiss T Danker et al ldquoExpansion anddifferentiation of neural progenitors derived from the humanadult enteric nervous systemrdquo Gastroenterology vol 137 no 6pp 2063e4ndash2073e4 2009

[5] M Metzger C Caldwell A J Barlow A J Burns and NThapar ldquoEnteric nervous system stem cells derived fromhumangut mucosa for the treatment of aganglionic gut disordersrdquoGastroenterology vol 136 no 7 pp 2214e3ndash2225e3 2009

[6] M J Saffrey ldquoCellular changes in the enteric nervous systemduring ageingrdquo Developmental Biology vol 382 no 1 pp 344ndash355 2013

[7] M D Gershon ldquoDevelopmental determinants of the indepen-dence and complexity of the enteric nervous systemrdquo Trends inNeurosciences vol 33 no 10 pp 446ndash456 2010

[8] F Obermayr R Hotta H Enomoto and H M Young ldquoDevel-opment and developmental disorders of the enteric nervoussystemrdquo Nature Reviews Gastroenterology and Hepatology vol10 no 1 pp 43ndash57 2013

[9] R J Rintala and M P Pakarinen ldquoLong-term outcomes ofHirschsprungrsquos diseaserdquo Seminars in Pediatric Surgery vol 21no 4 pp 336ndash343 2012

[10] T A Heanue and V Pachnis ldquoEnteric nervous system develop-ment and Hirschsprungrsquos disease advances in genetic and stemcell studiesrdquoNature Reviews Neuroscience vol 8 no 6 pp 466ndash479 2007

[11] L Becker J Peterson S Kulkarni and P J Pasricha ldquoExvivo neurogenesis within enteric ganglia occurs in a PTENdependent mannerrdquo PLoS ONE vol 8 no 3 Article ID e594522013

[12] TUesakaMNagashimada andH Enomoto ldquoGDNF signalinglevels control migration and neuronal differentiation of entericganglion precursorsrdquoThe Journal of Neuroscience vol 33 no 41pp 16372ndash16382 2013

[13] T A Heanue and V Pachnis ldquoExpression profiling the devel-oping mammalian enteric nervous system identifies marker

and candidate Hirschsprung disease genesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 18 pp 6919ndash6924 2006

[14] B P S Vohra K TsujiMNagashimada et al ldquoDifferential geneexpression and functional analysis implicate novel mechanismsin enteric nervous system precursor migration and neuritogen-esisrdquo Developmental Biology vol 298 no 1 pp 259ndash271 2006

[15] RMohr PNeckel Y Zhang et al ldquoMolecular and cell biologicaleffects of 3531015840-triiodothyronine on progenitor cells of theenteric nervous system in vitrordquo Stem Cell Research vol 11 no3 pp 1191ndash1205 2013

[16] S Fleige and M W Pfaffl ldquoRNA integrity and the effect onthe real-time qRT-PCR performancerdquo Molecular Aspects ofMedicine vol 27 no 2-3 pp 126ndash139 2006

[17] S L Kline I M Cheeseman T Hori T Fukagawa and ADesai ldquoThe human Mis12 complex is required for kinetochoreassembly and proper chromosome segregationrdquo The Journal ofCell Biology vol 173 no 1 pp 9ndash17 2006

[18] E D Salmon D Cimini L A Cameron and J G DeLucaldquoMerotelic kinetochores in mammalian tissue cellsrdquo Philosoph-ical Transactions of the Royal Society B Biological Sciences vol360 no 1455 pp 553ndash568 2005

[19] A A Ye and T JMaresca ldquoCell division kinetochores SKAdad-dlerdquo Current Biology vol 23 no 3 pp R122ndashR124 2013

[20] Y Lu Z Wang L Ge N Chen and H Liu ldquoThe RZZcomplex and the spindle assembly checkpointrdquo Cell Structureand Function vol 34 no 1 pp 31ndash45 2009

[21] D G Johnson and C L Walker ldquoCyclins and cell cyclecheckpointsrdquo Annual Review of Pharmacology and Toxicologyvol 39 pp 295ndash312 1999

[22] M Malumbres E Harlow T Hunt et al ldquoCyclin-dependentkinases a family portraitrdquoNature Cell Biology vol 11 no 11 pp1275ndash1276 2009

[23] M V Gomez-Gaviro C E Scott A K Sesay et al ldquoBetacellulinpromotes cell proliferation in the neural stem cell niche andstimulates neurogenesisrdquo Proceedings of the National Academyof Sciences of the United States of America vol 109 no 4 pp1317ndash1322 2012

[24] K Khodosevich Y Watanabe and H Monyer ldquoEphA4 pre-serves postnatal and adult neural stem cells in an undifferen-tiated state in vivordquo Journal of Cell Science vol 124 no 8 pp1268ndash1279 2011

[25] E Abranches M Silva L Pradier et al ldquoNeural differentiationof embryonic stem cells in vitro a road map to neurogenesis inthe embryordquo PLoS ONE vol 4 no 7 Article ID e6286 2009

[26] V Balasubramaniyan N Timmer B Kust E Boddeke and SCopray ldquoTransient expression of Olig1 initiates the differentia-tion of neural stem cells into oligodendrocyte progenitor cellsrdquoStem Cells vol 22 no 6 pp 878ndash882 2004

[27] M Kolatsi-Joannou X Z Li T Suda H T Yuan and A SWoolf ldquoIn early development of the rat mRNA for the majormyelin protein P

0is expressed in nonsensory areas of the

embryonic inner ear notochord enteric nervous system andolfactory ensheathing cellsrdquo Developmental Dynamics vol 222no 1 pp 40ndash51 2001

[28] M I Morris D B-D Soglio A Ouimet A Aspirot andN Patey ldquoA study of calretinin in Hirschsprung pathologyparticularly in total colonic aganglionosisrdquo Journal of PediatricSurgery vol 48 no 5 pp 1037ndash1043 2013

[29] K R Jessen and R Mirsky ldquoGlial cells in the enteric nervoussystem contain glial fibrillary acidic proteinrdquo Nature vol 286no 5774 pp 736ndash737 1980


[30] T J Siddiqui R Pancaroglu Y Kang A Rooyakkers and AM Craig ldquoLRRTMs and neuroligins bind neurexins with a dif-ferential code to cooperate in glutamate synapse developmentrdquoThe Journal of Neuroscience vol 30 no 22 pp 7495ndash7506 2010

[31] S Chen O Gil Y Q Ren G Zanazzi J L Salzer and D EHillman ldquoNeurotrimin expression during cerebellar develop-ment suggests roles in axon fasciculation and synaptogenesisrdquoJournal of Neurocytology vol 30 no 11 pp 927ndash937 2002

[32] R Janz and T C Sudhof ldquoSV2C is a synaptic vesicle proteinwith an unusually restricted localization anatomy of a synapticvesicle protein familyrdquo Neuroscience vol 94 no 4 pp 1279ndash1290 1999

[33] B Marqueze F Berton and M Seagar ldquoSynaptotagminsin membrane traffic which vesicles do the tagmins tagrdquoBiochimie vol 82 no 5 pp 409ndash420 2000

[34] B C Jongbloets and R J Pasterkamp ldquoSemaphorin signallingduring developmentrdquo Development vol 141 no 17 pp 3292ndash3297 2014

[35] M Metzger S Conrad T Skutella and L Just ldquoRGMa inhibitsneurite outgrowth of neuronal progenitors frommurine entericnervous system via the neogenin receptor in vitrordquo Journal ofNeurochemistry vol 103 no 6 pp 2665ndash2678 2007

[36] J M Spin S Nallamshetty R Tabibiazar et al ldquoTranscriptionalprofiling of in vitro smooth muscle cell differentiation identifiesspecific patterns of gene and pathway activationrdquo PhysiologicalGenomics vol 19 pp 292ndash302 2005

[37] J M Baisden Y Qian H M Zot and D C Flynn ldquoThe actinfilament-associated protein AFAP-110 is an adaptor protein thatmodulates changes in actin filament integrityrdquo Oncogene vol20 no 44 pp 6435ndash6447 2001

[38] L E Peri K M Sanders and V N Mutafova-YambolievaldquoDifferential expression of genes related to purinergic signalingin smooth muscle cells PDGFR120572-positive cells and interstitialcells of Cajal in the murine colonrdquo Neurogastroenterology ampMotility vol 25 no 9 pp e609ndashe620 2013

[39] S J Winder B G Allen O Clement-Chomienne and M PWalsh ldquoRegulation of smooth muscle actinmdashmyosin interac-tion and force by calponinrdquoActa Physiologica Scandinavica vol164 no 4 pp 415ndash426 1998

[40] M L Mancini J M Verdi B A Conley et al ldquoEndoglin isrequired for myogenic differentiation potential of neural creststem cellsrdquo Developmental Biology vol 308 no 2 pp 520ndash5332007

[41] F T L van der Loop G Schaart E D J Timmer F CS Ramaekers and G J J M van Eys ldquoSmoothelin a novelcytoskeletal protein specific for smooth muscle cellsrdquo TheJournal of Cell Biology vol 134 no 2 pp 401ndash411 1996

[42] M A DrsquoAngelo J S Gomez-Cavazos A Mei D H Lacknerand M W Hetzer ldquoA change in nuclear pore complex compo-sition regulates cell differentiationrdquoDevelopmental Cell vol 22no 2 pp 446ndash458 2012

[43] J J-C Lin Y Li R D Eppinga QWang and J-P Jin ldquoChapter1 roles of caldesmon in cell motility and actin cytoskeletonremodelingrdquo International Review of Cell andMolecular Biologyvol 274 pp 1ndash68 2009

[44] S B Marston and CW J Smith ldquoThe thin filaments of smoothmusclesrdquo Journal ofMuscle Research and Cell Motility vol 6 no6 pp 669ndash708 1985

[45] KM Sanders and SMWard ldquoKitmutants and gastrointestinalphysiologyrdquoThe Journal of Physiology vol 578 no 1 pp 33ndash422007

[46] L S Campos ldquoNeurospheres insights into neural stem cellbiologyrdquo Journal of Neuroscience Research vol 78 no 6 pp 761ndash769 2004

[47] A Ring Y-M Kim and M Kahn ldquoWntcatenin signaling inadult stem cell physiology and diseaserdquo Stem Cell Reviews andReports vol 10 no 4 pp 512ndash525 2014

[48] D C Berwick and K Harvey ldquoLRRK2 an eminence griseof Wnt-mediated neurogenesisrdquo Frontiers in Cellular Neuro-science vol 7 article 82 2013

[49] S Kakugawa P F LangtonM Zebisch et al ldquoNotumdeacylatesWnt proteins to suppress signalling activityrdquoNature vol 519 no7542 pp 187ndash192 2015

[50] Y Mii and M Taira ldquoSecreted Wnt lsquoinhibitorsrsquo are not justinhibitors regulation of extracellular Wnt by secreted Frizzled-related proteinsrdquoDevelopment GrowthampDifferentiation vol 53no 8 pp 911ndash923 2011

[51] Y Kawano and R Kypta ldquoSecreted antagonists of the Wntsignalling pathwayrdquo Journal of Cell Science vol 116 part 13 pp2627ndash2634 2003

[52] A Ohazama E B Johnson M S Ota et al ldquoLrp4 modulatesextracellular integration of cell signaling pathways in develop-mentrdquo PLoS ONE vol 3 no 12 Article ID e4092 2008

[53] Y Ding Y Zhang C Xu Q-H Tao and Y-G Chen ldquoHECTdomain-containing E

3ubiquitin ligase NEDD

4L negatively reg-

ulates Wnt signaling by targeting dishevelled for proteasomaldegradationrdquo The Journal of Biological Chemistry vol 288 no12 pp 8289ndash8298 2013

[54] A Ishikawa S Kitajima Y Takahashi et al ldquoMouse Nkd1a Wnt antagonist exhibits oscillatory gene expression in thePSM under the control of Notch signalingrdquo Mechanisms ofDevelopment vol 121 no 12 pp 1443ndash1453 2004

[55] D W Chan C-Y Chan J W P Yam Y-P Ching and I OL Ng ldquoPrickle-1 negatively regulates Wnt120573-catenin pathwayby promoting Dishevelled ubiquitinationdegradation in livercancerrdquo Gastroenterology vol 131 no 4 pp 1218ndash1227 2006

[56] K Sakamoto S Yamaguchi R Ando et al ldquoThe nephroblas-toma overexpressed gene (NOVccn3) protein associates withNotch1 extracellular domain and inhibits myoblast differenti-ation via Notch signaling pathwayrdquo The Journal of BiologicalChemistry vol 277 no 33 pp 29399ndash29405 2002

[57] A Caruso M Motolese L Iacovelli et al ldquoInhibition of thecanonical Wnt signaling pathway by apolipoprotein E

4in PC12

cellsrdquo Journal of Neurochemistry vol 98 no 2 pp 364ndash3712006

[58] A J Hanson H A Wallace T J Freeman R D Beauchamp LA Lee and E Lee ldquoXIAP monoubiquitylates GrouchoTLE topromote canonicalWnt signalingrdquoMolecular Cell vol 45 no 5pp 619ndash628 2012

[59] A Takai H Inomata A Arakawa R Yakura M Matsuo-Takasaki and Y Sasai ldquoAnterior neural development requiresDel1 a matrix-associated protein that attenuates canonical Wntsignaling via the Ror2 pathwayrdquo Development vol 137 no 19pp 3293ndash3302 2010

[60] Y Katoh and M Katoh ldquoFGF signaling inhibitor SPRY4 isevolutionarily conserved target of WNT signaling pathway inprogenitor cellsrdquo International Journal of Molecular Medicinevol 17 no 3 pp 529ndash532 2006

[61] P Ordonez-Moran A Irmisch A Barbachano et alldquoSPROUTY2 is a 120573-catenin and FOXO3a target gene indicativeof poor prognosis in colon cancerrdquo Oncogene vol 33 no 15pp 1975ndash1985 2014


[62] T Taketomi D Yoshiga K Taniguchi et al ldquoLoss of mam-malian Sprouty2 leads to enteric neuronal hyperplasia andesophageal achalasiardquoNatureNeuroscience vol 8 no 7 pp 855ndash857 2005

[63] R Di Liddo T Bertalot A Schuster et al ldquoAnti-inflammatoryactivity of Wnt signaling in enteric nervous system in vitropreliminary evidences in rat primary culturesrdquo Journal ofNeuroinflammation vol 12 no 1 p 23 2015


[13 14] though little effort has so far been put into charac-terizing the genetic profile of enteric neural stem cells in vitro[15]

Here we used an Affymetrix microarray analysis toevaluate the genetic expression profile of proliferatingmurineenteric neural stem cells and its changes during the earlydifferentiation in vitro

2 Materials and Methods

21 Cell Culturing Cell culturingwas conducted as describedpreviously [15] The handling of animals was in accordanceto the institutional guidelines of the University of Tuebingenwhich conform to the international guidelines

Neonatal (P0) C57BL6 mice without regard to sex weredecapitated and the whole gut was removed After removalof adherent mesentery the longitudinal and circular musclelayers containing myenteric plexus could be stripped as awhole from the small intestine Tissue was chopped andincubated in collagenase type XI (750UmL Sigma-AldrichTaufkirchen Germany) and dispase II (250 120583gmL RocheDiagnostics Mannheim Germany) dissolved in Hanksrsquo bal-anced salt solution with Ca2+Mg2+ (HBSS PAA PaschingAustria) for 30min at 37∘C During enzymatic dissociationthe tissue was carefully triturated every 10min with a firepolished 1mL pipette tip Prior to the first trituration step cellsuspension was treated with 005 (wv) DNAse I (Sigma-Aldrich) After 30min tissue dissociation was stopped byadding fetal calf serum (FCS PAA) to a final concentrationof 10 (vv) to the medium Undigested larger tissue pieceswere removed with a 40 120583m cell strainer (BD BiosciencesFranklin Lakes NJ USA) Residual enzymes were removedduring twowashing steps inHBSS at 200 gAfter dissociationcells were resuspended in proliferation culturemedium (Dul-beccorsquos modified Eaglersquos medium with Hamrsquos F12 medium(DMEMF12 1 1 PAA)) containing N2 supplement (1 100Invitrogen Darmstadt Germany) penicillin (100UmLPAA) streptomycin (100 120583gmL PAA) L-glutamine (2mMPAA) epidermal growth factor (EGF 20 ngmL Sigma-Aldrich) and fibroblast growth factor (FGF 20 ngmLSigma-Aldrich) Cells were seeded into 6-well plates (BDBiosciences) in a concentration of 25 times 104 cellscm2 Onlyonce before seeding the medium was supplemented withB27 (1 50 Invitrogen) EGF and FGF were added daily andculture medium was exchanged every 3 days All cultivationsteps were conducted in a humidified incubator at 37∘Cand 5 CO

2 An overview of the following cell culture

protocol is shown in Figure 1 During proliferation phaseof the culture cells formed spheroid-like bodies termedenterospheres After 5 days of proliferation free-floatingenterospheres were picked and transferred to petri dishes (Oslash60mm Greiner Bio One Frickenhausen Germany) in 5mLfresh proliferation medium and proliferation was continuedfor further 4 days

Single free-floating enterospheres (50 enterospheresdish) were picked again washed 3 times in Tris buffer andtransferred into new petri dishes containing either prolif-eration medium or differentiation medium Differentiationmedium consists of DMEMF12 containing N2 supplement

9 div start of differentiation

1 div 5 div

0 div 5 div spheres picked

Differentiation for 2 divProliferation for 9 div

Figure 1 Time schedule of enterosphere culture The timelineillustrates the schedule of in vitro culture Cells were isolated at 0div (days in vitro) and proliferated for 5 days Spheres were thenpicked and again proliferated for 4 days At 9 div enterosphereswere picked washed transferred to differentiation medium andincubated for 2 days before gene expression analyses were carriedout The micrographs show proliferating enterospheres after 1 and 5div Scale bar 200 120583m

(1 100) penicillin (100UmL) streptomycin (100 120583gmL) L-glutamine (2mM) and ascorbic acid-2-phosphate (200 120583MSigma-Aldrich)

Enterospheres were proliferated or differentiated for 2more days thereby forming the two experimental groupsldquoproliferationrdquo and ldquodifferentiationrdquoThe difference in expres-sion between those two groups (differentiation versus prolif-eration) was successively compared by microarray analysis asdescribed below

22 Affymetrix Microarray Analysis Affymetrix microarrayanalysis was conducted similar to previously published datain three independent experiments each with cell culturesprepared from 2 pups from the same litter [15] In each exper-iment free-floating enterospheres were picked as describedabove in order to diminish the fraction of adhesive fibroblastsand smooth muscle cells

Total RNA of enterospheres of both groups was extractedusing the RNeasy Micro Kit (Qiagen) RNA quality wasevaluated on Agilent 2100 Bioanalyzer with RNA integritynumbers (RIN) of the samples in this study being in the rangefrom 8 to 10 RIN numbers higher than 8 are consideredoptimal for downstream application [16]

Double-stranded cDNA was synthesized from 100 ng oftotal RNA subsequently linearly amplified and biotinylatedusing the GeneChip WT cDNA Synthesis and AmplificationKit (Affymetrix Santa Clara CA USA) according to themanufacturerrsquos instructions 15 120583g of labeled and fragmentedcDNAwas hybridized toGeneChipMouseGene 10 ST arrays(Affymetrix) After hybridization the arrays were stainedand washed in a Fluidics Station 450 (Affymetrix) withthe recommended washing procedure Biotinylated cDNAbound to target molecules was detected with streptavidin-coupled phycoerythrin biotinylated anti-streptavidin IgGantibodies and again streptavidin-coupled phycoerythrinaccording to the protocol Arrays were scanned using theGCS3000 GeneChip Scanner (Affymetrix) and AGCC 30software Scanned images were subjected to visual inspection




3 Results





4 Discussion




Cell cycle



STOP












minus1845 STOP











Table 2 Continued








Table 2 Continued









Cyto

plas

mEx

trac

ellu

lar s

pace

Nuc

leus

DNA

FZD LRP56minus1571

WNT

DKK21731LRP41979

FRZB1938

NOTUM2631

NOV2050

APOE1704

DAAM2minus1993

PSRC1minus2235

SMURF11629

TNIK1677

TRAF4minus1673

EDIL32258

HIC1minus1731

JADE1minus1656

NARFminus1699

NKD13220

PRICKLE11536

TLE31714 TRIB2

minus1637

NEDD4L1588

FZD7minus2271

DVL

120573-catenin

120573-catenin

TCF

APC

LEF

AXIN21744

LRRK21677

GSK-3

LEF1minus2680

WNT5Aminus2325

TCF19minus2217

WNT7A2942

TCF7L1minus1762

























Table 4 ECM





5 Conclusion



Table 5 Wnt













Acknowledgments


References



























































4L negatively reg-






4in PC12












3 Results





4 Discussion




Cell cycle



STOP












minus1845 STOP











Table 2 Continued








Table 2 Continued









Cyto

plas

mEx

trac

ellu

lar s

pace

Nuc

leus

DNA

FZD LRP56minus1571

WNT

DKK21731LRP41979

FRZB1938

NOTUM2631

NOV2050

APOE1704

DAAM2minus1993

PSRC1minus2235

SMURF11629

TNIK1677

TRAF4minus1673

EDIL32258

HIC1minus1731

JADE1minus1656

NARFminus1699

NKD13220

PRICKLE11536

TLE31714 TRIB2

minus1637

NEDD4L1588

FZD7minus2271

DVL

120573-catenin

120573-catenin

TCF

APC

LEF

AXIN21744

LRRK21677

GSK-3

LEF1minus2680

WNT5Aminus2325

TCF19minus2217

WNT7A2942

TCF7L1minus1762

























Table 4 ECM





5 Conclusion



Table 5 Wnt













Acknowledgments


References



























































4L negatively reg-






4in PC12


















Table 2 Continued








Table 2 Continued









Cyto

plas

mEx

trac

ellu

lar s

pace

Nuc

leus

DNA

FZD LRP56minus1571

WNT

DKK21731LRP41979

FRZB1938

NOTUM2631

NOV2050

APOE1704

DAAM2minus1993

PSRC1minus2235

SMURF11629

TNIK1677

TRAF4minus1673

EDIL32258

HIC1minus1731

JADE1minus1656

NARFminus1699

NKD13220

PRICKLE11536

TLE31714 TRIB2

minus1637

NEDD4L1588

FZD7minus2271

DVL

120573-catenin

120573-catenin

TCF

APC

LEF

AXIN21744

LRRK21677

GSK-3

LEF1minus2680

WNT5Aminus2325

TCF19minus2217

WNT7A2942

TCF7L1minus1762

























Table 4 ECM





5 Conclusion



Table 5 Wnt













Acknowledgments


References



























































4L negatively reg-






4in PC12










Table 2 Continued









Cyto

plas

mEx

trac

ellu

lar s

pace

Nuc

leus

DNA

FZD LRP56minus1571

WNT

DKK21731LRP41979

FRZB1938

NOTUM2631

NOV2050

APOE1704

DAAM2minus1993

PSRC1minus2235

SMURF11629

TNIK1677

TRAF4minus1673

EDIL32258

HIC1minus1731

JADE1minus1656

NARFminus1699

NKD13220

PRICKLE11536

TLE31714 TRIB2

minus1637

NEDD4L1588

FZD7minus2271

DVL

120573-catenin

120573-catenin

TCF

APC

LEF

AXIN21744

LRRK21677

GSK-3

LEF1minus2680

WNT5Aminus2325

TCF19minus2217

WNT7A2942

TCF7L1minus1762

























Table 4 ECM





5 Conclusion



Table 5 Wnt













Acknowledgments


References



























































4L negatively reg-






4in PC12










Cyto

plas

mEx

trac

ellu

lar s

pace

Nuc

leus

DNA

FZD LRP56minus1571

WNT

DKK21731LRP41979

FRZB1938

NOTUM2631

NOV2050

APOE1704

DAAM2minus1993

PSRC1minus2235

SMURF11629

TNIK1677

TRAF4minus1673

EDIL32258

HIC1minus1731

JADE1minus1656

NARFminus1699

NKD13220

PRICKLE11536

TLE31714 TRIB2

minus1637

NEDD4L1588

FZD7minus2271

DVL

120573-catenin

120573-catenin

TCF

APC

LEF

AXIN21744

LRRK21677

GSK-3

LEF1minus2680

WNT5Aminus2325

TCF19minus2217

WNT7A2942

TCF7L1minus1762

























Table 4 ECM





5 Conclusion



Table 5 Wnt













Acknowledgments


References



























































4L negatively reg-






4in PC12





























Table 4 ECM





5 Conclusion



Table 5 Wnt













Acknowledgments


References



























































4L negatively reg-






4in PC12










Table 5 Wnt













Acknowledgments


References



























































4L negatively reg-






4in PC12












Acknowledgments


References



























































4L negatively reg-






4in PC12



































4L negatively reg-






4in PC12









Comparative Microarray Analysis of Proliferating and ... · ResearchArticle Comparative Microarray...

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