Stem Cells International
Volume 2016 (2016), Article ID 9695827, 13 pages
http://dx.doi.org/10.1155/2016/9695827

Research Article

Comparative Microarray Analysis of Proliferating and Differentiating 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 Tübingen, Österbergstrasse 3, 72074 Tübingen, Germany

Received 15 May 2015; Accepted 12 July 2015

Academic Editor: Kodandaramireddy Nalapareddy

Copyright © 2016 Peter Helmut Neckel et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Postnatal neural progenitor cells of the enteric nervous system are a potential source for future cell replacement therapies of developmental dysplasia like Hirschsprung’s disease. However, little is known about the molecular mechanisms driving the homeostasis and differentiation of this cell pool. In this work, we conducted Affymetrix GeneChip experiments to identify differences in gene regulation between proliferation and early differentiation of enteric neural progenitors from neonatal mice. We detected a total of 1333 regulated genes that were linked to different groups of cellular mechanisms involved in cell cycle, apoptosis, neural proliferation, and differentiation. As expected, we found an augmented inhibition in the gene expression of cell cycle progression as well as an enhanced mRNA expression of neuronal and glial differentiation markers. We further found a marked inactivation of the canonical Wnt pathway after the induction of cellular differentiation. Taken together, these data demonstrate the various 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 autonomous and highly complex neuronal network found in the gastrointestinal tract. Its two major plexuses are integrated into the layered anatomy of the gut wall and, together with central modulating influences, exert control over gastrointestinal motility, secretion, ion-homeostasis, and immunological mechanisms [1]. In order to achieve this variety of functions, the ENS is composed of a multitude of different neuronal and glial cell types and closely interacts with smooth muscle cells and myogenic pacemaker cells called interstitial cells of Cajal. Furthermore, a population of neural stem or progenitor cells in the ENS has been identified in rodents [2, 3] and humans that retain their proliferative capacity throughout adult life even into old age [4, 5]. It is therefore not surprising that the correct functioning of the ENS as well as the regulation on enteric neural progenitor cells is subjected to the influence of a myriad of transmitters, neurotrophic and growth factors, signalling molecules, and extracellular matrix components, which are not exclusively expressed by neural cell types [6]. Likewise, the control of the development of the ENS is equally complex and mutations in its genetic program can lead to fatal dysplasia like Hirschsprung’s disease (HCSR) [7, 8].

HSCR is hallmarked by an aganglionic distal bowel leading to life-threatening disturbances in intestinal motility. Today’s therapeutic gold standard, the surgical resection of the affected gut segments, is nevertheless associated with problematic long-term outcomes with regard to continence [9]. In order to improve the therapeutic success, the use of autologous enteric neural stem cells was proposed [10]. This concept relies on the in vitro expansion of enteric neural stem cells derived from small biopsy materials. However, we are just beginning to understand the molecular mechanisms that underlie neural stem cell biology and how this knowledge can be used for optimizing in vitro culture conditions [11, 12].

Genome-wide gene-expression analyses are a useful tool to examine the genetic programs and cellular interactions and have been widely used to identify potential markers or signalling mechanisms especially in CNS neurospheres or cancer tissues. Further, gene-expression assays have also helped to unravel genetic prepositions associated with HSCR [13, 14], though little effort has so far been put into characterizing the genetic profile of enteric neural stem cells in vitro [15].

Here, we used an Affymetrix microarray analysis to evaluate the genetic expression profile of proliferating murine enteric neural stem cells and its changes during the early differentiation in vitro.

2. Materials and Methods

2.1. Cell Culturing

Cell culturing was conducted as described previously [15]. The handling of animals was in accordance to the institutional guidelines of the University of Tuebingen, which conform to the international guidelines.

Neonatal (P0) C57BL/6 mice without regard to sex were decapitated and the whole gut was removed. After removal of adherent mesentery the longitudinal and circular muscle layers containing myenteric plexus could be stripped as a whole from the small intestine. Tissue was chopped and incubated in collagenase type XI (750 U/mL; Sigma-Aldrich, Taufkirchen, Germany) and dispase II (250 μg/mL; Roche Diagnostics, Mannheim, Germany) dissolved in Hanks’ balanced salt solution with Ca²⁺/Mg²⁺ (HBSS; PAA, Pasching, Austria) for 30 min at 37°C. During enzymatic dissociation the tissue was carefully triturated every 10 min with a fire polished 1 mL pipette tip. Prior to the first trituration step, cell suspension was treated with 0.05% (w/v) DNAse I (Sigma-Aldrich). After 30 min, tissue dissociation was stopped by adding fetal calf serum (FCS; PAA) to a final concentration of 10% (v/v) to the medium. Undigested larger tissue pieces were removed with a 40 μm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). Residual enzymes were removed during two washing steps in HBSS at 200 g. After dissociation, cells were resuspended in proliferation culture medium (Dulbecco’s modified Eagle’s medium with Ham’s F12 medium (DMEM/F12; 1 : 1; PAA)) containing N2 supplement (1 : 100; Invitrogen, Darmstadt, Germany), penicillin (100 U/mL; PAA), streptomycin (100 μg/mL; PAA), L-glutamine (2 mM; PAA), epidermal growth factor (EGF; 20 ng/mL; Sigma-Aldrich), and fibroblast growth factor (FGF; 20 ng/mL; Sigma-Aldrich). Cells were seeded into 6-well plates (BD Biosciences) in a concentration of 2.5 × 10⁴ cells/cm². Only once before seeding, the medium was supplemented with B27 (1 : 50; Invitrogen). EGF and FGF were added daily and culture medium was exchanged every 3 days. All cultivation steps were conducted in a humidified incubator at 37°C and 5% CO₂. An overview of the following cell culture protocol is shown in Figure 1. During proliferation phase of the culture, cells formed spheroid-like bodies termed enterospheres. After 5 days of proliferation, free-floating enterospheres were picked and transferred to petri dishes (Ø 60 mm; Greiner Bio One, Frickenhausen, Germany) in 5 mL fresh proliferation medium and proliferation was continued for further 4 days.

Figure 1: Time schedule of enterosphere culture. The timeline illustrates the schedule of in vitro culture. Cells were isolated at 0 div (days in vitro) and proliferated for 5 days. Spheres were then picked and again proliferated for 4 days. At 9 div, enterospheres were picked, washed, transferred to differentiation medium, and incubated for 2 days before gene expression analyses were carried out. The micrographs show proliferating enterospheres after 1 and 5 div. Scale bar: 200 μm.

Single free-floating enterospheres (50 enterospheres/dish) were picked again, washed 3 times in Tris buffer, and transferred into new petri dishes containing either proliferation medium or differentiation medium. Differentiation medium consists of DMEM/F12 containing N2 supplement (1 : 100), penicillin (100 U/mL), streptomycin (100 μg/mL), L-glutamine (2 mM), and ascorbic acid-2-phosphate (200 μM; Sigma-Aldrich).

Enterospheres were proliferated or differentiated for 2 more days, thereby forming the two experimental groups “proliferation” and “differentiation.” The difference in expression between those two groups (differentiation versus proliferation) was successively compared by microarray analysis as described below.

2.2. Affymetrix Microarray Analysis

Affymetrix microarray analysis was conducted similar to previously published data in three independent experiments, each with cell cultures prepared from 2 pups from the same litter [15]. In each experiment, free-floating enterospheres were picked as described above in order to diminish the fraction of adhesive fibroblasts and smooth muscle cells.

Total RNA of enterospheres of both groups was extracted using the RNeasy Micro Kit (Qiagen). RNA quality was evaluated on Agilent 2100 Bioanalyzer with RNA integrity numbers (RIN) of the samples in this study being in the range from 8 to 10. RIN numbers higher than 8 are considered optimal for downstream application [16].

Double-stranded cDNA was synthesized from 100 ng of total RNA, subsequently linearly amplified, and biotinylated using the GeneChip WT cDNA Synthesis and Amplification Kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. 15 μg of labeled and fragmented cDNA was hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix). After hybridization, the arrays were stained and washed in a Fluidics Station 450 (Affymetrix) with the recommended washing procedure. Biotinylated cDNA bound to target molecules was detected with streptavidin-coupled phycoerythrin, biotinylated anti-streptavidin IgG antibodies and again streptavidin-coupled phycoerythrin according to the protocol. Arrays were scanned using the GCS3000 GeneChip Scanner (Affymetrix) and AGCC 3.0 software. Scanned images were subjected to visual inspection to check for hybridization artifacts and proper grid alignment and analyzed with Expression Console 1.0 (Affymetrix) to generate report files for quality control.

Normalization of raw data was performed by the Partek Software 6.6, applying an RMA (Robust Multichip Average) algorithm. Significance was calculated using a -test without multiple testing correction (Partek), selecting all transcripts with a minimum change in expression level of 1.5-fold together with a value less than 0.05.

3. Results

In this study, we investigated the changes of the genetic expression profile that occur during the transition from proliferating to differentiating enteric neural progenitor cells in vitro. Therefore, we generated enterospheres by 9 day in vitro cultures, which then could be picked and either proliferated or differentiated for two more days (Figure 1). mRNA was subsequently extracted and gene expression of these two groups was analysed by Affymetrix microarray analysis.

Analysis of mRNA expression was performed on a GeneChip Mouse Gene 1.0 ST array that determines the expression profile of 28.853 genes. Each gene was interrogated by a median of 27 probes that are spread along the full gene.

In total, the gene chip detected 1454 transcripts to be at least 1.5-fold differentially expressed between proliferating and differentiating enterospheres. 1333 of these transcripts code for already identified proteins. 541 genes were found to be upregulated and 792 genes were found to be downregulated in comparison to proliferating enterospheres (see Supplementary Table of the Supplementary Material available online at http://dx.doi.org/10.1155/2016/9695827).

We used the ingenuity pathway analysis software (IPA) and data mining with the science literature search engine http://www.ncbi.nlm.nih.gov/pubmed/ to divide the genes into different groups according to their function during cellular development. The largest functional group contained 171 genes related to cell cycle and apoptosis (Table 1, Supplementary Table ). Here, we identified especially different cyclin proteins and cell division cycle proteins that were mainly downregulated. Further, we found several genes that are linked to neural development as well as genes regulating neural stem cell proliferation and differentiation. Furthermore, we also detected neuronal and glial differentiation markers and numerous genes involved in synapse formation (Table 2). It is noteworthy that we also identified a group of genes that are known to be involved in the differentiation of smooth muscle cells (Table 3) as well as in extracellular matrix components (Table 4). Additionally, we found regulated genes related to canonical Wnt signalling indicating a deactivation of this pathway during ENS progenitor cell differentiation (Figure 2, Table 5).

Table 1: Selected genes related to cell cycle.

Gene
Encoded protein
Fold change
Cell cycle

AURKA
Aurora kinase A
−2.712
STOP

AURKB
Aurora kinase B
−4.146
STOP

CCNA2
Cyclin A2
−4.652
STOP

CCNB1
Cyclin B1
−5.752 
−5.820 
−5.857
STOP

CCNB2
Cyclin B2
−3.392
STOP

CCND1
Cyclin D1
−2.476
STOP

CCND3
Cyclin D3
−1.539
STOP

CCNE1
Cyclin E1
−1.777
STOP

CCNE2
Cyclin E2
−2.847
STOP

CCNF
Cyclin F
−3.211
STOP

CDC6
Cell division cycle 6
−1.936
STOP

CDC20
Cell division cycle 20
−3.113
STOP

CDC25B
Cell division cycle 25B
−1.636
STOP

CDC25C
Cell division cycle 25C
−2.414
STOP

CDC45
Cell division cycle 45
−1.769
STOP

CDCA2
Cell division cycle associated 2
−3.461
STOP

CDCA3
Cell division cycle associated 3
−3.003
STOP

CDCA5
Cell division cycle associated 5
−3.053
STOP

CDCA7L
Cell division cycle associated 7-like
−4.123
STOP

CDCA8
Cell division cycle associated 8
−3.467
STOP

CDK1
Cyclin-dependent kinase 1
−3.227
STOP

CDK15
Cyclin-dependent kinase 15
1.618
GO

CDK19
Cyclin-dependent kinase 19
1.619
GO

CDK5R1
Cyclin-dependent kinase 5, regulatory subunit 1 (p35)
1.597
—

CENPA
Centromere protein A
−1.895
STOP

CENPE
Centromere protein E, 312 kDa
−4.140
STOP

CENPF
Centromere protein F, 350/400 kDa
−3.927
STOP

CENPI
Centromere protein I
−2.899
STOP

CENPK
Centromere protein K
−2.813
STOP

CENPL
Centromere protein L
−1.864
STOP

CENPM
Centromere protein M
−3.407
STOP

CENPN
Centromere protein N
−2.465
STOP

CENPU
Centromere protein U
−1.624
STOP

SKA1
Spindle and kinetochore associated complex subunit 1
−1.532
STOP

SKA2
Spindle and kinetochore associated complex subunit 2
−1.582
STOP

SKA3
Spindle and kinetochore associated complex subunit 3
−3.490
STOP

SKP2
S-phase kinase-associated protein 2, E3 ubiquitin protein ligase
−1.845
STOP

SPC25
SPC25, NDC80 kinetochore complex component
−4.148
STOP

Table 2: Neural differentiation/development.

Gene
Encoded protein
Fold change

Neural stem cells

ABCG2
ATP-binding cassette, subfamily G (WHITE), member 2 (junior blood group)
−1.526

ASPM
asp (abnormal spindle) homolog, microcephaly associated (Drosophila)
−4.911

CDT1
Chromatin licensing and DNA replication factor 1
−1.528

EGFL7
EGF-like-domain, multiple 7
3.132

EPHA2
EPH receptor A2
−1.529

ETV4
ets variant 4
−1.934

ETV5
ets variant 5
−2.844

−2.651

FABP7
Fatty acid binding protein 7, brain
−2.095

Neural differentiation

ATOH8
Atonal homolog 8 (Drosophila)
1.932

AXL
AXL receptor tyrosine kinase
2.015

CRIM1
Cysteine-rich transmembrane BMP regulator 1 (chordin-like)
1.999

CRLF1
Cytokine receptor-like factor 1
2.382

DAB1
Dab, reelin signal transducer, homolog 1 (Drosophila)
−2.297

ELK3
ELK3, ETS-domain protein (SRF accessory protein 2)
−1.613

ESCO2
Establishment of sister chromatid cohesion N-acetyltransferase 2
−4.767

GAP43
Growth associated protein 43
1.613

GLDN
Gliomedin
5.809

HMOX1
Heme oxygenase (decycling) 1
1.884

KLF9
Kruppel-like factor 9
1.592

Lmo3
LIM domain only 3
1.542

MAP6
Microtubule-associated protein 6
1.874

MYRF
Myelin regulatory factor
2.527

NEUROD4
Neuronal differentiation 4
2.036

OLIG1
Oligodendrocyte transcription factor 1
2.660

Pvr
Poliovirus receptor
1.768

RGS4
Regulator of G-protein signaling 4
1.955

S1PR1
Sphingosine-1-phosphate receptor 1
5.073

SOCS2
Suppressor of cytokine signaling 2
2.052

2.335

WIPF1
WAS/WASL interacting protein family, member 1
1.587

Neural differentiation markers

CALB2
Calbindin 2
1.616

CNP
2′,3′-Cyclic nucleotide 3′-phosphodiesterase
1.732

GFAP
Glial fibrillary acidic protein
2.239

MBP
Myelin basic protein
1.768

Mturn
Maturin, neural progenitor differentiation regulator homolog (Xenopus)
1.853

OMG
Oligodendrocyte myelin glycoprotein
−1.822

OPALIN
Oligodendrocytic myelin paranodal and inner loop protein
39.246

PLP1
Proteolipid protein 1
1.630

S100B
S100 calcium binding protein B
−1.675

TUBB2A
Tubulin, beta 2A class IIa
1.608

TUBB2B
Tubulin, beta 2B class IIb
1.535

TUBB3
Tubulin, beta 3 class III
1.976

Synapse and neurotransmitters

ABAT
4-Aminobutyrate aminotransferase
−1.512

ADRA1D
Adrenoceptor alpha 1D
1.803

ADRA2A
Adrenoceptor alpha 2A
2.900

ADRA2B
Adrenoceptor alpha 2B
−2.093

CHRM2
Cholinergic receptor, muscarinic 2
1.635

CHRM3
Cholinergic receptor, muscarinic 3
−1.715

CHRNA7
Cholinergic receptor, nicotinic, alpha 7 (neuronal)
1.772

COMT
Catechol-O-methyltransferase
1.515

DDC
DOPA decarboxylase (aromatic L-amino acid decarboxylase)
1.711

DNM3
Dynamin 3
2.643

EPHA5
EPH receptor A5
2.076

GRIA3
Glutamate receptor, ionotropic, AMPA 3
−1.528

GRIA4
Glutamate receptor, ionotropic, AMPA 4
−1.997

GRIK2
Glutamate receptor, ionotropic, kainate 2
−1.565

GRM5
Glutamate receptor, metabotropic 5
−1.600

HTR1B
5-Hydroxytryptamine (serotonin) receptor 1B, G-protein-coupled
−2.377

HTR2B
5-Hydroxytryptamine (serotonin) receptor 2B, G-protein-coupled
2.205

LRRTM2
Leucine-rich repeat transmembrane neuronal 2
3.665

LRRTM3
Leucine-rich repeat transmembrane neuronal 3
2.210

NTM
Neurotrimin
1.820

PENK
Proenkephalin
3.478

PRR7
Proline rich 7 (synaptic)
1.788

SLC10A4
Solute carrier family 10, member 4
1.824

1.867

SLITRK2
SLIT and NTRK-like family, member 2
−2.414

SLITRK6
SLIT and NTRK-like family, member 6
1.672

STON2
Stonin 2
4.054

STXBP3
Syntaxin-binding protein 3
1.730

Stxbp3b
Syntaxin-binding protein 3B
1.637

SV2C
Synaptic vesicle glycoprotein 2C
1.929

SYT6
Synaptotagmin VI
2.571

Neurite outgrowth

ATF3
Activating transcription factor 3
2.579

DOK4
Docking protein 4
4.937

FEZ2
Fasciculation and elongation protein zeta 2 (zygin II)
1.547

NAV2
Neuron navigator 2
1.647

NRCAM
Neuronal cell adhesion molecule
2.496

PLXNB3
Plexin B3
1.739

RGMA
Repulsive guidance molecule family member a
1.552

RNF165
Ring finger protein 165
−1.548

ROBO2
Roundabout, axon guidance receptor, homolog 2 (Drosophila)
−2.211

SEMA3B
Sema domain, immunoglobulin domain (Ig), short basic domain, secreted (semaphorin) 3B
3.692

SEMA3E
Sema domain, immunoglobulin domain (Ig), short basic domain, secreted (semaphorin) 3E
2.877

SEMA4F
Sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4F
4.891

SEMA6A
Sema domain, transmembrane domain (TM), and cytoplasmic domain (semaphorin) 6A
−1.707

SRGAP1
SLIT-ROBO Rho GTPase activating protein 1
1.524

UNC5B
unc-5 homolog B (C. elegans)
−1.927

Growth factors

ARTN
Artemin
2.423

FGF2
Fibroblast growth factor 2 (basic)
2.264

FGF5
Fibroblast growth factor 5
7.704

GDF10
Growth differentiation factor 10
−2.361

GDF11
Growth differentiation factor 11
1.604

GDNF
Glial cell derived neurotrophic factor
4.325

GFRA3
GDNF family receptor alpha 3
1.707

MET
MET protooncogene, receptor tyrosine kinase
6.680

NGFR
Nerve growth factor receptor
1.728

NTRK3
Neurotrophic tyrosine kinase, receptor, type 3
−1.575

SNX16
Sorting nexin 16
1.641

SPHK1
Sphingosine kinase 1
1.704

SPRY1
Sprouty homolog 1, antagonist of FGF signaling (Drosophila)
−1.647

Table 3: Differentiation of smooth muscle cells/ICCs.

Gene
Encoded protein
Fold change

Smooth muscle cells

ACTA2
Actin, alpha 2, smooth muscle, aorta
1.693

ACTG2
Actin, gamma 2, smooth muscle, enteric
2.336

ACTN1
Actinin, alpha 1
−1.724

AEBP1
AE binding protein 1
2.702

AFAP1
Actin filament associated protein 1
1.638

ARID5B
AT-rich interactive domain 5B (MRF1-like)
1.521

Cald1
Caldesmon 1
−1.535

CNN1
Calponin 1, basic, smooth muscle
1.652

ENG
Endoglin
−1.552

ENPP1
Ectonucleotide pyrophosphatase/phosphodiesterase 1
−1.522

ENPP2
Ectonucleotide pyrophosphatase/phosphodiesterase 2
2.959

ENTPD1
Ectonucleoside triphosphate diphosphohydrolase 1
1.636

FOSL2
FOS-like antigen 2
2.566

GAMT
Guanidinoacetate N-methyltransferase
1.725

MYO1E
Myosin IE
1.569

MYO5A
Myosin VA (heavy chain 12, myoxin)
1.680

MYO7B
Myosin VIIB
1.710

MYO18A
Myosin XVIIIA
1.994

MYPN
Myopalladin
1.570

NEB
Nebulin
1.569

Nebl
Nebulette
2.378

NUP210
Nucleoporin 210 kDa
−1.838

RBM24
RNA binding motif protein 24
1.548

SMTN
Smoothelin
−1.778

SSPN
Sarcospan
1.603

TAGLN
Transgelin
2.706

ICC

GUCY1A3
Guanylate cyclase 1, soluble, alpha 3
−1.876

GUCY1B3
Guanylate cyclase 1, soluble, beta 3
−2.008

KIT
v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
−1.798

KITLG
KIT ligand
−1.541

Table 4: ECM.

Gene
Encoded protein
Fold change

CHSY3
Chondroitin sulfate synthase 3
−1.645

COL6A5
Collagen, type VI, alpha 5
1.527

COL12A1
Collagen, type XII, alpha 1
−1.973

COL14A1
Collagen, type XIV, alpha 1
6.135

COL16A1
Collagen, type XVI, alpha 1
1.666

COL18A1
Collagen, type XVIII, alpha 1
1.595

COL27A1
Collagen, type XXVII, alpha 1
1.522

COLGALT2
Collagen beta(1-O)galactosyltransferase 2
−1.564

CSPG4
Chondroitin sulfate proteoglycan 4
−2.952

CSPG5
Chondroitin sulfate proteoglycan 5 (neuroglycan C)
−1.585

CYR61
Cysteine-rich, angiogenic inducer, 61
1.748

ECM1
Extracellular matrix protein 1
2.580

HSPG2
Heparan sulfate proteoglycan 2
1.923

ITGA1
Integrin, alpha 1
−1.665

ITGA4
Integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)
−2.324

ITGA7
Integrin, alpha 7
4.203

ITGA8
Integrin, alpha 8
−2.262

ITGA11
Integrin, alpha 11
1.762

ITGB3
Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)
−5.342

ITGB4
Integrin, beta 4
1.567

KRT80
Keratin 80
2.833

LAMA4
Laminin, alpha 4
−1.537

LAMA5
Laminin, alpha 5
1.684

LOX
Lysyl oxidase
3.250

LOXL4
Lysyl oxidase-like 4
2.427

2.417

MATN2
Matrilin 2
2.570

MMP2
Matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)
1.668

MMP9
Matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase)
−5.557

MMP15
Matrix metallopeptidase 15 (membrane-inserted)
−2.017

MMP16
Matrix metallopeptidase 16 (membrane-inserted)
−1.634

MMP17
Matrix metallopeptidase 17 (membrane-inserted)
1.612

MMP19
Matrix metallopeptidase 19
3.236

MMP28
Matrix metallopeptidase 28
1.956

NDST3
N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 3
−5.557

P4HA1
Prolyl 4-hydroxylase, alpha polypeptide I
−1.958

PLOD3
Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3
2.250

UGDH
UDP-glucose 6-dehydrogenase
1.529

Table 5: Wnt.

Gene
Encoded protein
Fold change

Wnt signaling cascade

FZD7
Frizzled class receptor 7
−2.271

LEF1
Lymphoid enhancer-binding factor 1
−2.680

LRP5
Low density lipoprotein receptor-related protein 5
−1.571

LRRK2
Leucine-rich repeat kinase 2
1.677

TCF19
Transcription factor 19
−2.217

F7L1
Transcription factor 7-like 1 (T-cell specific, HMG-box)
−1.762

WNT5A
Wingless-type MMTV integration site family, member 5A
−2.325

WNT7B
Wingless-type MMTV integration site family, member 7B
2.942

Target gene

ARL4C
ADP-ribosylation factor-like 4C
2.179

AXIN2
Axin 2
1.744

CCND1
Cyclin D1
−2.476

CSRNP1
Cysteine-serine-rich nuclear protein 1
1.822

RACGAP1
Rac GTPase activating protein 1
−3.201

SPRY2
Sprouty homolog 2 (Drosophila)
−1.771

SPRY4
Sprouty homolog 4 (Drosophila)
−2.771

WISP1
WNT1 inducible signaling pathway protein 1
2.489

Wnt antagonists/inhibitors

APOE
Apolipoprotein E
1.704

DKK2
Dickkopf WNT signaling pathway inhibitor 2
1.731

EDIL3
EGF-like repeats and discoidin I-like domains 3
2.258

FRZB
Frizzled-related protein
1.938

HIC1
Hypermethylated in cancer 1
−1.731

JADE1
Jade family PHD finger 1
−1.656

LRP4
Low density lipoprotein receptor-related protein 4
1.979

NARF
Nuclear prelamin A recognition factor
−1.699

NEDD4L
Neural precursor cell expressed, developmentally downregulated 4-like, E3 ubiquitin protein ligase
1.588

NKD1
Naked cuticle homolog 1 (Drosophila)
3.220

NOTUM
Notum pectinacetylesterase homolog (Drosophila)
2.631

NOV
Nephroblastoma overexpressed
2.050

PRICKLE1
Prickle homolog 1 (Drosophila)
1.536

TLE3
Transducin-like enhancer of split 3
1.714

TRIB2
Tribbles pseudokinase 2
−1.637

Wnt activators

DAAM2
Dishevelled associated activator of morphogenesis 2
−1.993

PSRC1
Proline/serine-rich coiled-coil 1
−2.235

TNIK
TRAF2 and NCK interacting kinase
1.677

TRAF4
TNF receptor-associated factor 4
−1.673

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

4. Discussion

The proliferation and differentiation of enteric neural progenitor cells during embryonic and postnatal development are controlled by a complex interplay of various intrinsic and extrinsic factors. Their exact timing is crucial for proper migration and proliferation of neural crest cells and for their differentiation into the various neural cell types that compose the complex neural structures of the ENS. Although research in recent years extended our understanding of ENS development and its pathologies [13], there are still many genes and processes unknown. Particularly, factors regulating neural progenitor proliferation and differentiation in the developing and postnatal gut as well as cellular and molecular interaction systems remain largely elusive. Here, we used in vitro cultures of enteric neural progenitor cells derived from murine tunica muscularis to scan for molecular programs and signalling pathways acting on cell proliferation and early differentiation.

Our experiment aimed to elucidate gene regulations in enterospheres that occur while ENS progenitor cells leave their proliferative state and begin to differentiate into more defined and specific cell types. The results of the Affymetrix gene expression analysis showed the up- and downregulation of overall 1333 known genes that code for already identified proteins. 171 of these genes could be linked to cell proliferation (Table 1, Supplementary Table ). Amongst them we detected genes coding for proteins related to the kinetochore complex (like NSL1 [17], NUF2 [18], SKA1-3 [19], and ZWILCH [20]), cyclin proteins [21], cyclin-dependent kinases (CDK) [22], and several types of centromere proteins. The regulation of 145 of these genes strongly indicates a slowdown of cell cycle progression as it was intended by the experimental deprivation of growth factor supplementation 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 proliferation in the neural stem cell niche [23]. Nonetheless, the vast majority of genes including all regulated cyclins, cell division cycle proteins, and kinetochore proteins were found to be downregulated.

We also checked the regulated genes for apoptosis markers to see whether the stop in proliferation was related to cell death (Supplementary Table ). Since only 3 of 12 apoptotic genes were regulated in the direction that indicates apoptosis, it is unlikely that apoptosis played a leading role in the interruption of proliferation. Still, the effect and regulation of apoptosis during enteric sphere cultures are an important cornerstone of understanding enteric neural progenitors in culture and in vivo and require further investigation. Together, on a broad basis, this dataset provides strong evidence that this cell culture design is applicable to decreasing the proliferative rate of enteric neural progenitor cells without inducing cell death or apoptosis in an appreciable quantity.

To further evaluate the proliferative conditions of cell types present in enterospheres, we focused on different cell specific markers of neural progenitors as well as neurons, glial, or smooth muscle cells. We consider this complex cellular composition of the enterospheres an advantage compared to more purified neural crest derived neurospheres as we are able to capture complex interactions and secretion mechanisms between cell types that might also play an important role in vivo. Interestingly, we found 8 genes involved in adult central or embryonic neural stem cells homeostasis (Table 2). The majority of genes like EPHA2 [24] are regulated in a way that suggests that neural stem cells exit the proliferative cell cycle to enter differentiation programs. This idea was supported by the upregulation of numerous genes that drive neuronal and glial differentiation like NEUROD4 [25] or OLIG1 [26]. In this context, we identified several upregulated genes involved in proper myelination. As enteric and central glia cells are known to temporally express myelin-related proteins during development, it is conceivable that this regulation 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, a common glia cell marker, was downregulated contrasting the rest of our data. Again, this might be due to the complex differentiation program of enteric glia, in which S100B plays a role at later stages.

Furthermore, the establishment of neuronal cell communication was strongly regulated. Here, we found an increased expression of genes related to synaptogenesis (LRRTM2 and 3 [30], neurotrimin [31]) and to SNARE or vesicle protein function (STXBP3, SV2C [32], and SYT6 [33]). We also identified a number of genes involved in transmitter metabolism (COMT, DDC) as well as neurotransmitter receptor like 5-HT, glutamate, and adrenergic receptors. However, the regulation of those genes was highly variable shedding light on the intricacy of synapse formation in the developing enteric nervous system. This complexity is carried on by genes related to axon sprouting and guidance like semaphorins [34] or RGMa [35].

Additionally, we found that regulated genes directly involved in the differentiation of muscle cells and/or enteric pacemaker cells called interstitial cells of Cajal (Table 3). Particularly interesting is the upregulation of a number of genes known to drive smooth muscle differentiation like ARID5B [36], FOSL2 [36] and genes that are expressed in differentiated smooth muscle cells in the intestine like AFAP1 [37], ENPP2 [38], and CNN1 [39] as well as various myosin and actin isoforms. These data confirm the fact that cultured spheroids are composed of different cell types present in the intestinal tunica muscularis and further indicate that deprivation of growth factors induces differentiation of smooth muscle cells resembling molecular processes in the developing gut. In fact, we among others were previously able to confirm the presence of smooth muscle cells derived from enterosphere culture by BrdU-immunolabeling costudies [4]. However, it is noteworthy that a few genes related to muscular differentiation (endoglin [40], smoothelin [41], NUP210 [42], caldesmon 1 [43], and ACTN1 [44]) were downregulated contrasting the expression pattern observed in the majority of regulated genes. This hints to complex regulatory mechanisms controlling the myogenic differentiation program in which these genes are not required at all or in a different temporal sequence not mapped by our experimental design. It is further remarkable that five markers expressed in interstitial cells of Cajal (ICC) including KIT [45] were downregulated.

Moreover, the regulation of 43 extracellular matrix proteins like collagens, integrins, proteoglycans, and matrix metallopeptidases points to a reconstruction of extracellular environment that has been discussed to influence neural stem cell behaviour [46] (Table 4). Taken together, these results illustrate the ongoing genetic programs during early differentiation of enterospheres.

Within the dataset, it was of special interest to find particularly many regulated genes related to the canonical Wnt pathway (Table 5). The involvement of canonical Wnt signalling has frequently been shown in the regulation of various stem cell niches, like intestinal epithelium or CNS derived neural stem cells. However, these studies exhibited different and partly contradicting outcomes, which strongly hint to the variable functions of canonical Wnt signals in different tissues during embryonic and postnatal development. In previous work, we found regulation of several Wnt-related genes in the context of thyroid hormone dependent differentiation of enteric neural progenitor cells indicating a potential role of the canonical Wnt pathway activation during the proliferation of this progenitor cell pool [15]. Canonical Wnt signalling has frequently been reviewed in the literature—just recently by Ring et al. [47]. In brief, secreted Wnt proteins bind to frizzled receptors (FZD) complexed with low density lipoprotein receptor-related protein 5/6 (LRP5/6) coreceptors. Thereafter, the scaffolding protein disheveled (DVL) is recruited to FZD and inhibits the β-catenin destruction complex (AXIN2, APC, and GSK-3β). Therefore, β-catenin accumulates in the cytoplasm and translocates to the nucleus where it binds to TCF/LEF transcription factors to initiate Wnt target gene expression. Interestingly, our current data strongly indicate that the canonical Wnt pathway is switched off during the first two days of enteric progenitor differentiation on several levels of the signalling cascade (Figure 2). On the one hand we identified a downregulation of activating parts of the signalling cascade itself like the receptor proteins FZD7 and LRP5 or the transcription factors TCF19, TCF7L1, and LEF1. On the other hand, inactivating elements of the pathway like parts of the β-catenin destruction complex AXIN2 and LRRK2 [48] were upregulated. We also found numerous modulators of the signalling cascade. It is of interest that the majority of those genes are reported to inhibit the signalling process 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], and APOE [57]), or in the nucleus (TLE3 [58], EDIL3 [59]). Furthermore, we identified target genes of the canonical Wnt pathway that were either upregulated (e.g., AXIN2 that exerts a negative feedback on the pathway) or downregulated like the cell cycle progression genes CCND1 and SPRY4 [60]. We also found a lower expression of SPRY2 [61], a Wnt target gene and known inhibitor of GDNF signalling [62], in the differentiation group. Together with a strong upregulation of GDNF itself by 4.325-fold, this might drive enteric progenitor cells into neural differentiation [12].

Taken together, it is conceivable that canonical Wnt signalling plays a role in the maintenance of the enteric progenitor pool during proliferation and is switched off at the beginning of differentiation conditions. Indeed, our previous gene expression analyses [15] as well as recently published cell culture experiments [63] and yet unpublished in vitro analyses strongly support this hypothesis.

5. Conclusion

This study focused on the changes in gene expression of enteric neural progenitor cells occurring within the first two days of transition from a proliferative state to differentiation in vitro. Using microarray analysis, we found a marked inhibition of cell cycle progression in general as well as strong evidence for neural stem cells differentiation into enteric neurons and glia cells. These findings were substantiated by the upregulation of genes related to synapse formation and neural connectivity. Most interesting, we found that this transition from enteric neural progenitor proliferation to differentiation was accompanied by a considerable inactivation of the canonical Wnt signalling pathway. This, together with previous work, strongly indicates that canonical Wnt activation is one of the driving mechanisms of enteric neural progenitor proliferation and thus might play a role in the homeostasis of this cell pool in vivo and in vitro.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Authors’ Contribution

Peter Helmut Neckel and Roland Mohr contributed equally to this work.

Acknowledgments

The project was supported by a grant from the German Federal Ministry for Education and Research (01GN0967). The authors would like to thank Andrea Wizenmann, Andreas Mack, and Sven Poths for their helpful advice.

References

J. B. Furness, “The enteric nervous system and neurogastroenterology,” Nature Reviews Gastroenterology & Hepatology, vol. 9, no. 5, pp. 286–294, 2012.

D. Natarajan, M. Grigoriou, C. V. Marcos-Gutierrez, C. Atkins, and V. Pachnis, “Multipotential progenitors of the mammalian enteric nervous system capable of colonising aganglionic bowel in organ culture,” Development, vol. 126, no. 1, pp. 157–168, 1999.

G. M. Kruger, J. T. Mosher, S. Bixby, N. Joseph, T. Iwashita, and S. J. Morrison, “Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential, and factor responsiveness,” Neuron, vol. 35, no. 4, pp. 657–669, 2002.

M. Metzger, P. M. Bareiss, T. Danker et al., “Expansion and differentiation of neural progenitors derived from the human adult enteric nervous system,” Gastroenterology, vol. 137, no. 6, pp. 2063.e4–2073.e4, 2009.

M. Metzger, C. Caldwell, A. J. Barlow, A. J. Burns, and N. Thapar, “Enteric nervous system stem cells derived from human gut mucosa for the treatment of aganglionic gut disorders,” Gastroenterology, vol. 136, no. 7, pp. 2214.e3–2225.e3, 2009.

M. J. Saffrey, “Cellular changes in the enteric nervous system during ageing,” Developmental Biology, vol. 382, no. 1, pp. 344–355, 2013.

M. D. Gershon, “Developmental determinants of the independence and complexity of the enteric nervous system,” Trends in Neurosciences, vol. 33, no. 10, pp. 446–456, 2010.

F. Obermayr, R. Hotta, H. Enomoto, and H. M. Young, “Development and developmental disorders of the enteric nervous system,” Nature Reviews Gastroenterology and Hepatology, vol. 10, no. 1, pp. 43–57, 2013.

R. J. Rintala and M. P. Pakarinen, “Long-term outcomes of Hirschsprung's disease,” Seminars in Pediatric Surgery, vol. 21, no. 4, pp. 336–343, 2012.

T. A. Heanue and V. Pachnis, “Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies,” Nature Reviews Neuroscience, vol. 8, no. 6, pp. 466–479, 2007.

L. Becker, J. Peterson, S. Kulkarni, and P. J. Pasricha, “Ex vivo neurogenesis within enteric ganglia occurs in a PTEN dependent manner,” PLoS ONE, vol. 8, no. 3, Article ID e59452, 2013.

T. Uesaka, M. Nagashimada, and H. Enomoto, “GDNF signaling levels control migration and neuronal differentiation of enteric ganglion precursors,” The Journal of Neuroscience, vol. 33, no. 41, pp. 16372–16382, 2013.

T. A. Heanue and V. Pachnis, “Expression profiling the developing mammalian enteric nervous system identifies marker and candidate Hirschsprung disease genes,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 18, pp. 6919–6924, 2006.

B. P. S. Vohra, K. Tsuji, M. Nagashimada et al., “Differential gene expression and functional analysis implicate novel mechanisms in enteric nervous system precursor migration and neuritogenesis,” Developmental Biology, vol. 298, no. 1, pp. 259–271, 2006.

R. Mohr, P. Neckel, Y. Zhang et al., “Molecular and cell biological effects of 3,5,3′-triiodothyronine on progenitor cells of the enteric nervous system in vitro,” Stem Cell Research, vol. 11, no. 3, pp. 1191–1205, 2013.

S. Fleige and M. W. Pfaffl, “RNA integrity and the effect on the real-time qRT-PCR performance,” Molecular Aspects of Medicine, vol. 27, no. 2-3, pp. 126–139, 2006.

S. L. Kline, I. M. Cheeseman, T. Hori, T. Fukagawa, and A. Desai, “The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation,” The Journal of Cell Biology, vol. 173, no. 1, pp. 9–17, 2006.

E. D. Salmon, D. Cimini, L. A. Cameron, and J. G. DeLuca, “Merotelic kinetochores in mammalian tissue cells,” Philosophical Transactions of the Royal Society B: Biological Sciences, vol. 360, no. 1455, pp. 553–568, 2005.

A. A. Ye and T. J. Maresca, “Cell division: kinetochores SKAdaddle,” Current Biology, vol. 23, no. 3, pp. R122–R124, 2013.

Y. Lu, Z. Wang, L. Ge, N. Chen, and H. Liu, “The RZZ complex and the spindle assembly checkpoint,” Cell Structure and Function, vol. 34, no. 1, pp. 31–45, 2009.

D. G. Johnson and C. L. Walker, “Cyclins and cell cycle checkpoints,” Annual Review of Pharmacology and Toxicology, vol. 39, pp. 295–312, 1999.

M. Malumbres, E. Harlow, T. Hunt et al., “Cyclin-dependent kinases: a family portrait,” Nature Cell Biology, vol. 11, no. 11, pp. 1275–1276, 2009.

M. V. Gómez-Gaviro, C. E. Scott, A. K. Sesay et al., “Betacellulin promotes cell proliferation in the neural stem cell niche and stimulates neurogenesis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 4, pp. 1317–1322, 2012.

K. Khodosevich, Y. Watanabe, and H. Monyer, “EphA4 preserves postnatal and adult neural stem cells in an undifferentiated state in vivo,” Journal of Cell Science, vol. 124, no. 8, pp. 1268–1279, 2011.

E. Abranches, M. Silva, L. Pradier et al., “Neural differentiation of embryonic stem cells in vitro: a road map to neurogenesis in the embryo,” PLoS ONE, vol. 4, no. 7, Article ID e6286, 2009.

V. Balasubramaniyan, N. Timmer, B. Kust, E. Boddeke, and S. Copray, “Transient expression of Olig1 initiates the differentiation of neural stem cells into oligodendrocyte progenitor cells,” Stem Cells, vol. 22, no. 6, pp. 878–882, 2004.

M. Kolatsi-Joannou, X. Z. Li, T. Suda, H. T. Yuan, and A. S. Woolf, “In early development of the rat mRNA for the major myelin protein P₀ is expressed in nonsensory areas of the embryonic inner ear, notochord, enteric nervous system, and olfactory ensheathing cells,” Developmental Dynamics, vol. 222, no. 1, pp. 40–51, 2001.

M. I. Morris, D. B.-D. Soglio, A. Ouimet, A. Aspirot, and N. Patey, “A study of calretinin in Hirschsprung pathology, particularly in total colonic aganglionosis,” Journal of Pediatric Surgery, vol. 48, no. 5, pp. 1037–1043, 2013.

K. R. Jessen and R. Mirsky, “Glial cells in the enteric nervous system contain glial fibrillary acidic protein,” Nature, vol. 286, no. 5774, pp. 736–737, 1980.

T. J. Siddiqui, R. Pancaroglu, Y. Kang, A. Rooyakkers, and A. M. Craig, “LRRTMs and neuroligins bind neurexins with a differential code to cooperate in glutamate synapse development,” The Journal of Neuroscience, vol. 30, no. 22, pp. 7495–7506, 2010.

S. Chen, O. Gil, Y. Q. Ren, G. Zanazzi, J. L. Salzer, and D. E. Hillman, “Neurotrimin expression during cerebellar development suggests roles in axon fasciculation and synaptogenesis,” Journal of Neurocytology, vol. 30, no. 11, pp. 927–937, 2002.

R. Janz and T. C. Südhof, “SV2C is a synaptic vesicle protein with an unusually restricted localization: anatomy of a synaptic vesicle protein family,” Neuroscience, vol. 94, no. 4, pp. 1279–1290, 1999.

B. Marquèze, F. Berton, and M. Seagar, “Synaptotagmins in membrane traffic: which vesicles do the tagmins tag?” Biochimie, vol. 82, no. 5, pp. 409–420, 2000.

B. C. Jongbloets and R. J. Pasterkamp, “Semaphorin signalling during development,” Development, vol. 141, no. 17, pp. 3292–3297, 2014.

M. Metzger, S. Conrad, T. Skutella, and L. Just, “RGMa inhibits neurite outgrowth of neuronal progenitors from murine enteric nervous system via the neogenin receptor in vitro,” Journal of Neurochemistry, vol. 103, no. 6, pp. 2665–2678, 2007.

J. M. Spin, S. Nallamshetty, R. Tabibiazar et al., “Transcriptional profiling of in vitro smooth muscle cell differentiation identifies specific patterns of gene and pathway activation,” Physiological Genomics, vol. 19, pp. 292–302, 2005.

J. M. Baisden, Y. Qian, H. M. Zot, and D. C. Flynn, “The actin filament-associated protein AFAP-110 is an adaptor protein that modulates changes in actin filament integrity,” Oncogene, vol. 20, no. 44, pp. 6435–6447, 2001.

L. E. Peri, K. M. Sanders, and V. N. Mutafova-Yambolieva, “Differential expression of genes related to purinergic signaling in smooth muscle cells, PDGFRα-positive cells, and interstitial cells of Cajal in the murine colon,” Neurogastroenterology & Motility, vol. 25, no. 9, pp. e609–e620, 2013.

S. J. Winder, B. G. Allen, O. Clément-Chomienne, and M. P. Walsh, “Regulation of smooth muscle actin—myosin interaction and force by calponin,” Acta Physiologica Scandinavica, vol. 164, no. 4, pp. 415–426, 1998.

M. L. Mancini, J. M. Verdi, B. A. Conley et al., “Endoglin is required for myogenic differentiation potential of neural crest stem cells,” Developmental Biology, vol. 308, no. 2, pp. 520–533, 2007.

F. T. L. van der Loop, G. Schaart, E. D. J. Timmer, F. C. S. Ramaekers, and G. J. J. M. van Eys, “Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells,” The Journal of Cell Biology, vol. 134, no. 2, pp. 401–411, 1996.

M. A. D'Angelo, J. S. Gomez-Cavazos, A. Mei, D. H. Lackner, and M. W. Hetzer, “A change in nuclear pore complex composition regulates cell differentiation,” Developmental Cell, vol. 22, no. 2, pp. 446–458, 2012.

J. J.-C. Lin, Y. Li, R. D. Eppinga, Q. Wang, and J.-P. Jin, “Chapter 1: roles of caldesmon in cell motility and actin cytoskeleton remodeling,” International Review of Cell and Molecular Biology, vol. 274, pp. 1–68, 2009.

S. B. Marston and C. W. J. Smith, “The thin filaments of smooth muscles,” Journal of Muscle Research and Cell Motility, vol. 6, no. 6, pp. 669–708, 1985.

K. M. Sanders and S. M. Ward, “Kit mutants and gastrointestinal physiology,” The Journal of Physiology, vol. 578, no. 1, pp. 33–42, 2007.

L. S. Campos, “Neurospheres: insights into neural stem cell biology,” Journal of Neuroscience Research, vol. 78, no. 6, pp. 761–769, 2004.

A. Ring, Y.-M. Kim, and M. Kahn, “Wnt/catenin signaling in adult stem cell physiology and disease,” Stem Cell Reviews and Reports, vol. 10, no. 4, pp. 512–525, 2014.

D. C. Berwick and K. Harvey, “LRRK2: an éminence grise of Wnt-mediated neurogenesis?” Frontiers in Cellular Neuroscience, vol. 7, article 82, 2013.

S. Kakugawa, P. F. Langton, M. Zebisch et al., “Notum deacylates Wnt proteins to suppress signalling activity,” Nature, vol. 519, no. 7542, pp. 187–192, 2015.

Y. Mii and M. Taira, “Secreted Wnt ‘inhibitors’ are not just inhibitors: regulation of extracellular Wnt by secreted Frizzled-related proteins,” Development Growth & Differentiation, vol. 53, no. 8, pp. 911–923, 2011.

Y. Kawano and R. Kypta, “Secreted antagonists of the Wnt signalling pathway,” Journal of Cell Science, vol. 116, part 13, pp. 2627–2634, 2003.

A. Ohazama, E. B. Johnson, M. S. Ota et al., “Lrp4 modulates extracellular integration of cell signaling pathways in development,” PLoS ONE, vol. 3, no. 12, Article ID e4092, 2008.

Y. Ding, Y. Zhang, C. Xu, Q.-H. Tao, and Y.-G. Chen, “HECT domain-containing E₃ ubiquitin ligase NEDD₄L negatively regulates Wnt signaling by targeting dishevelled for proteasomal degradation,” The Journal of Biological Chemistry, vol. 288, no. 12, pp. 8289–8298, 2013.

A. Ishikawa, S. Kitajima, Y. Takahashi et al., “Mouse Nkd1, a Wnt antagonist, exhibits oscillatory gene expression in the PSM under the control of Notch signaling,” Mechanisms of Development, vol. 121, no. 12, pp. 1443–1453, 2004.

D. W. Chan, C.-Y. Chan, J. W. P. Yam, Y.-P. Ching, and I. O. L. Ng, “Prickle-1 negatively regulates Wnt/β-catenin pathway by promoting Dishevelled ubiquitination/degradation in liver cancer,” Gastroenterology, vol. 131, no. 4, pp. 1218–1227, 2006.

K. Sakamoto, S. Yamaguchi, R. Ando et al., “The nephroblastoma overexpressed gene (NOV/ccn3) protein associates with Notch1 extracellular domain and inhibits myoblast differentiation via Notch signaling pathway,” The Journal of Biological Chemistry, vol. 277, no. 33, pp. 29399–29405, 2002.

A. Caruso, M. Motolese, L. Iacovelli et al., “Inhibition of the canonical Wnt signaling pathway by apolipoprotein E₄ in PC12 cells,” Journal of Neurochemistry, vol. 98, no. 2, pp. 364–371, 2006.

A. J. Hanson, H. A. Wallace, T. J. Freeman, R. D. Beauchamp, L. A. Lee, and E. Lee, “XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling,” Molecular Cell, vol. 45, no. 5, pp. 619–628, 2012.

A. Takai, H. Inomata, A. Arakawa, R. Yakura, M. Matsuo-Takasaki, and Y. Sasai, “Anterior neural development requires Del1, a matrix-associated protein that attenuates canonical Wnt signaling via the Ror2 pathway,” Development, vol. 137, no. 19, pp. 3293–3302, 2010.

Y. Katoh and M. Katoh, “FGF signaling inhibitor, SPRY4, is evolutionarily conserved target of WNT signaling pathway in progenitor cells,” International Journal of Molecular Medicine, vol. 17, no. 3, pp. 529–532, 2006.

P. Ordóñez-Morán, A. Irmisch, A. Barbáchano et al., “SPROUTY2 is a β-catenin and FOXO3a target gene indicative of poor prognosis in colon cancer,” Oncogene, vol. 33, no. 15, pp. 1975–1985, 2014.

T. Taketomi, D. Yoshiga, K. Taniguchi et al., “Loss of mammalian Sprouty2 leads to enteric neuronal hyperplasia and esophageal achalasia,” Nature Neuroscience, vol. 8, no. 7, pp. 855–857, 2005.

R. Di Liddo, T. Bertalot, A. Schuster et al., “Anti-inflammatory activity of Wnt signaling in enteric nervous system: in vitro preliminary evidences in rat primary cultures,” Journal of Neuroinflammation, vol. 12, no. 1, p. 23, 2015.