Research Article Expression of the Wnt Receptor Frizzled-4 ...downloads.hindawi.com/journals/sci/2016/9076823.pdf · e Wnt signalling pathway plays a crucial role in the developm

Research ArticleExpression of the Wnt Receptor Frizzled-4 in the Human EntericNervous System of Infants

Katharina Nothelfer1 Florian Obermayr2 Nadine Belz1 Ellen Reinartz1 Petra M Bareiss1

Hans-Joumlrg Buumlhring3 Rudi Beschorner4 and Lothar Just1

1 Institute of Clinical Anatomy and Cell Analysis University of Tubingen Tubingen Germany2Department of Pediatric Surgery and Pediatric Urology University Childrenrsquos Hospital Tubingen Tubingen Germany3Division of Hematology Immunology Oncology Rheumatology and Pulmonology Department of Internal Medicine IIUniversity of Tubingen Tubingen Germany4Institute for Pathology and Neuropathology Department of Neuropathology University of Tubingen 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 Katharina Nothelfer 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

The Wnt signalling pathway plays a crucial role in the development of the nervous system This signalling cascade is initiatedupon binding of the secreted Wnt ligand to a member of the family of frizzled receptors In the present study we analysed thepresence of frizzled-4 in the enteric nervous system of human infants Frizzled-4 could be identified by immunohistochemistry ina subpopulation of enteric neuronal and glial cells in the small and large intestine Detection of frizzled-4 in the tunica muscularisby RT-PCR confirmed this receptorrsquos expression on the mRNA level Interestingly we observed distinct cell populations that co-expressed frizzled-4 with the intermediate filament protein nestin and the neurotrophin receptor p75NTR which have been reportedto be expressed in neural progenitor cells Flow cytometry analysis revealed that 60of p75NTR positive cells of the tunicamusculariswere positive for frizzled-4 Additionally in pathological samples of Hirschsprungrsquos disease the expression of this Wnt receptorcorrelated with the number of myenteric ganglion cells and decreased from normoganglionic to aganglionic areas of large intestineThe expression pattern of frizzled-4 indicates that this Wnt receptor could be involved in postnatal development andor functionof the enteric nervous system

1 Introduction

Neurons and glial cells of the peripheral nervous systemderive from neural crest stem cells (NCSCs) [1] Followinga distinct and well defined spatiotemporal pattern NCSCsmigrate out of the neural tube into embryonic tissue includ-ing the developing gut They enter the primitive foregut byembryonic day (E) 95 in mice and migrate in a rostral-to-caudal direction in order to colonize the entire gut by E145[2 3] In humans migration of enteric neural crest cells takesplace between gestational week 4 and gestational week 7 [4]During their journey along the gut a proportion of cellscontinues to proliferate while others begin to differentiate[5] into neurons and glial cells in order to generate acomplex neural network that coordinates bowel motility and

is involved in regulation of its secretory activity blood flowand modulation of the immune system [6 7] Impairmentof ENS development leads to incomplete colonization of thegut byNCSCs resulting in peristaltic dysregulation intestinalobstruction and enterocolitis as observed in Hirschsprungrsquosdisease (HSCR) [8ndash10] Much effort has been put intoresearch investigating the genetic regulation of neural crestdevelopment and its derivatives during the last decadesThis resulted in the identification of several major pathwaysregulating NCSC induction migration differentiation andinterconnection of developing neurons and glial cells [3 11]Wnt signalling has been reported to play a central role in thesedevelopmental processes in many model organisms In con-cert with members of the bone morphogenic protein (BMP)family and fibroblast growth factors Wnts regulate neural

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 9076823 12 pageshttpdxdoiorg10115520169076823

2 Stem Cells International

crest induction and specification In addition migration ofneural crest cells is dependent on theWnt signalling pathway[12 13]

TheWnt signalling cascade is initiated by binding of Wntto members of the frizzled transmembrane receptor familyand results in activation of pathways like the 120573-catenin-dependent pathway or the planar cell polarity- (PCP-)dependent pathway both of which have been shown to beinvolved in early neural crest development Recently thePCP pathway has been demonstrated to be additionallyinvolved in regulating neural interconnection during ENSdevelopment [14] However while the importance of Wnt-frizzled interaction is well established for early neural crestdevelopment its role in regulating postnatal neural crestderivatives is largely unknown

Interestingly there is evidence that neurogenesis con-tinues in postnatal mice in vivo upon stimulation withserotonin or following injury [15ndash17] A subpopulation ofcells of the postnatal ENS has been attributed with stem celllike properties although this niche is not well characterizedyet Nestin the low-affinity nerve growth factor receptorp75NTR and CD49b (alpha2-integrin) have been suggested torepresent markers for this population of enteric neural stemand progenitor cells [18]

Of the frizzled receptor family members frizzled-4 haspreviously been shown to be of particular interest withrespect to its role in the regulation of stem cell maintenanceneuroprotection and cell migration in the CNS [19ndash21] Inthe present study we examined the expression of this Wntreceptor in the enteric nervous system of infants by immuno-histochemistry RT-PCR and flow cytometry analysis Inorder to characterize the frizzled-4 positive cell populationwe performed colabelling experiments with various neuralmarkers that are expressed in neural and putative progenitorcells of the ENS

2 Materials and Methods

21 Human Gut Samples Human gut samples from smalland large intestine were obtained from infant male andfemale patients at the age of 4 weeks to 10 months whounderwent surgical enterostoma removal after treatmentfor necrotizing enterocolitis or anorectal malformations Inaddition tissue was collected from Hirschsprungrsquos diseasepatients who underwent a transanal endorectal pull-throughprocedure (Table 1) All samples were collected accordingto the guidelines and after approval of the Local EthicalCommittee at the University of Tubingen and with theconsent of the patientrsquos parents

22 Immunohistochemistry Immunohistochemistry wasperformed on 14 120583m thick cryosections Tissue slides werefixed in 4 (wv) paraformaldehyde in phosphate-bufferedsaline (PBS pH 74) for 20min at room temperature andsubsequently rinsed three times in PBS Slides whichwere stained with the ABC-detection system (VectorLabs Peterborough UK) were additionally pretreated in3 (vv) hydrogen peroxide PBS solution for 10min to

Table 1 Human gut samples

Samplenumber Gender Patient age Tissue Pathology

1 Male 5 months Colon Normoganglionic2 Female 2 months Ileum Normoganglionic3 Female 5 months Sigma Normoganglionic4 Male 2 months Ileum Normoganglionic5 Male 10 months Colon Normoganglionic6 Female 3 months Sigma Normoganglionic7 Male 6 months Sigma Normoganglionic8 Female 4 months Ileocaecal Normoganglionic9 Male 7 months Colon Normoganglionic10 Male 4 weeks Colon Hirschsprung11 Male 6 weeks Colon Hirschsprung12 Male 7 months Colon Hirschsprung

block endogenous peroxidases After incubation with serumcontaining blocking buffer (PBS containing 01 (wv)BSA 4 (vv) goat serum and 03 (vv) Triton X-100) for30min sections were incubated with primary antibodiesdiluted in PBS 01 (wv) BSA and 01 (vv) Triton X-100overnight at 4∘C Primary antibodies and concentrationsare listed in Table 2 Following three washes with PBSthe sections were incubated with secondary antibodies for30min at room temperature in the same buffer Detectionwas performed with either fluorochrome-conjugatedsecondary antibodies (1 400 goat anti-rabbit IgG-Cy3(Dianova Hamburg Germany) goat anti-mouse IgG-Cy3 (Dianova Hamburg Germany) goat anti-mouseIgG-Alexa 488 (Life Technologies Darmstadt Germany))or biotinylated secondary antibodies (1 400 swine anti-rabbit IgG-biotin and rabbit anti-mouse IgG-biotin (DakoHamburg Germany)) when using the ABC-detectionsystem Incubation of the AB reagent was performedaccording to the manufacturer and visualization of the ABCsystem was carried out with 3-31015840diaminobenzidine (DABSigma Taufkirchen Germany) and hydrogen peroxidesolution For fluorescence detection the sections and cellswere washed three times in PBS and additionally stainedwith DAPI (41015840-6-diamidino-2-phenylindole 02120583gmL) PBSsolution for 10min Stained sections were dried embeddedin Kaiserrsquos gelatin (Merck Darmstadt Germany) andphotographed on an inverted microscope (Axiovert ZeissJena Germany)

23 RNA Isolation and RT-PCR Human gut samples weremechanically dissected with the visual aid of a stereomi-croscope After disposure of residual fat and mesenterictissue the tunica muscularis was peeled off the submu-cosal and mucosal layers and thoroughly minced TotalRNA was isolated from human tunica muscularis usingthe RNeasy Mini Kit (Qiagen Hilden Germany) accordingto the manufacturerrsquos instructions Possible contaminatinggenomic DNA was removed by treatment with DNase I (LifeTechnologies Darmstadt Germany) Reverse transcriptase

Stem Cells International 3

Table 2 Primary antibodies used for cell analysis

Primary antibodies Species Dilution Manufacturer120572-smooth muscle actin Rabbit 1 100 Spring Bioscience Pleasanton USAc-kit Rabbit 1 500 Dako Hamburg GermanyFzd4 (clone CH3A4)lowast Mouse Undiluted In-house (HJB)Fzd4 PE (clone CH3A4)lowast Mouse 1 50 Biolegend San Diego USAGFAP Rabbit 1 400 Dako Hamburg GermanyNestin Rabbit 1 1000 Abcam Cambridge UKp75NTR Rabbit 1 500 Promega Madison USAp75NTR APC Rabbit 1 50 Miltenyi Bergisch Gladbach GermanyPeripherin Rabbit 1 200 Merck Millipore Darmstadt GermanylowastMonoclonal mouse anti-human antibody CH3A4 against frizzled-4 was raised by immunization with the retinoblastoma cell line WERI-RB-1 and specificityfor frizzled-4 was verified by the selective recognition of HEK-293 cells transfected with human frizzled-4Thismolecule was clustered to CD344 at the HCDMworkshop in Quebec Canada (httpwwwhcdmorg)

PCR (RT-PCR) reaction containing oligo-(dT)23

anchoredmRNA primers (Sigma-Aldrich Taufkirchen Germany)Superscript II Reverse Transcriptase enzyme and RNase Outwas performed according to the supplierrsquos protocol (LifeTechnologies Darmstadt Germany)

To amplify the frizzled-4 receptor and the housekeepinggene GAPDH the following primer pairs (Sigma-AldrichTaufkirchen Germany) were used

Frizzled-4 (product size 605 bp)

51015840-CTCGGCTACAACGTGACCAAGAT-3101584051015840-AATATGATGGGGCGCTCAGGGTA-31015840

GAPDH (product size 452 bp)

51015840-ACCACAGTCCATGCCATCAC-3101584051015840-TCCACCACCCTGTTGCTGTA-31015840

The PCR reaction mixture was incubated at 95∘C for5min followed by 35 cycles of 95∘C for 30 s annealingtemperatures (119879An) for 40 s and 72∘C for 40 s and a finalcycle with a prolonged elongation time of 10min at 72∘CThe primer-specific annealing temperatures were as follows119879An (frizzled-4) = 58∘C and119879An (GAPDH) = 60∘C AmplifiedPCR products were analyzed by electrophoresis on a 1 (wv)agarose gel (Roth Karlsruhe Germany) in 1x TBE buffer(Tris base boric acid and EDTA) at 100V The productswere visualized with ethidium bromide (2 120583gmL) on UVlight The size of each PCR product was estimated by using a100 bp DNA ladder standard (Life Technologies DarmstadtGermany) The RT step was omitted as a control for pos-sible contamination of DNase-treated samples with residualgenomic DNA (negative control)

24 Flow Cytometry Cells used for flow cytometry analy-sis were obtained from human tissue by digestion of thetunica muscularis with 750UmL Collagenase XI (SigmaTaufkirchen Germany) and 05wv Dispase II (RocheMannheim Germany) in HBSS with Ca2+Mg2+ The diges-tion was carried out for a maximum of 1 h at 37∘C in orderto avoid digestion of surface epitopes Tissue residues werediscarded and the supernatant was centrifuged for 7min at

210 g washed twice with HBSS and filtered through a 40120583mcell strainerThe resulting cell pellet was resuspended in 20120583Lblocking buffer (polyglobin (Talecris Biotherapeutics Frank-furt am Main Germany) diluted 1 10 in DPBS05 BSA(05 (wv) BSA in Dulbeccorsquos PBS without Ca2+Mg2+))and incubated on ice for 10min Subsequently cells werediluted with DPBS05 (wv) BSA to a concentration of2 times 105 cells per 50 120583L and antibodies were added for15min on ice P75NTR was detected with rabbit anti-p75NTR-APC (CD271 Miltenyi Bergisch Gladbach Germany 1 50)Staining of frizzled-4 was achieved either by incubation witha mouse anti-frizzled-4 antibody (25120583L104 cells undilutedhybridoma supernatant) followed by a rabbit anti-mouseIgG-FITC secondary antibody (Dako Hamburg Germany1 15) or by using a mouse anti-frizzled-4 PE-conjugatedantibody (Biolegend San Diego USA 1 50) All stainingswere compared according to isotype controls (rabbit IgG-APC (Miltenyi Bergisch Gladbach Germany 1 50) mouseIgG (Biozol Eching Germany 1 1000) and mouse IgG-PE(Biolegend San Diego USA 1 50)) and no differences inthe amount and intensity of frizzled-4 staining were observedbetween the two different staining procedures Followingincubation with the antibodies cells were washed withDPBS05 (vv) BSA and centrifuged for 7min and 210 g at4∘CThe resulting pellet was resuspended in DPBS05BSAand acquired on a FACSCanto II system (BD BioscienceHeidelberg Germany) Data was analysed using FlowJosoftware

3 Results

In the present study we analysed the expression of the Wntreceptor frizzled-4 in the enteric nervous system of humaninfants aged between 4 weeks and 10 months First immuno-histochemical staining processes for frizzled-4 glycoproteinwere performed on cryostat sections from small and largeintestine Positive cells could be observed in all layers of thegut wall (Figures 1 2 and 3) RT-PCR analysis of mRNAisolated from the tunica muscularis confirmed the presenceof frizzled-4 receptormRNA (Figure 4) To further character-ize the positive cell population coimmunostaining processes


Nestin Nestin Fzd4

Fzd4

TMTS

M

TMTS

M

TMTS

M

Fzd4

Fzd4

(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

Fzd4p75NTRp75NTR

Fzd4p75NTRp75NTR

Figure 1 The Wnt receptor frizzled-4 is expressed in neural cells in all layers of the gut wall Immunohistochemical analysis for frizzled-4(Fzd4) p75NTR and nestin expression on cryostat sections of human colon (sample number 1) ((a1)ndash(a3)) Overview of combined p75NTR

(green) and Fzd4 (red) immunostaining p75NTR (a1) Fzd4 (a2) merge (a3) DAPI (blue) tunica muscularis (TM) tela submucosa (TSM)scale bar 200 120583m ((b1)ndash(b3)) Higher magnification of combined p75NTR (green) and Fzd4 (red) immunostaining of myenteric gangliap75NTR (b1) Fzd4 (b2) merge (b3) DAPI (blue) scale bar 50 120583m ((c1)ndash(c3)) Combined nestin (green) and Fzd4 (red) immunostaining ofmyenteric ganglia nestin (c1) Fzd4 (c2) merge (c3) DAPI (blue) scale bar 50120583m

of frizzled-4 with peripherin and GFAP were performed Asshown in Figures 2 and 7 this Wnt receptor is expressedat various intensities in subpopulations of enteric neuronsand glial cells (Figures 2 and 7) Frizzled-4 immunopositiveneural extensions were observed in the tunica muscularistela submucosa and the lamina propria mucosae (Figures1 2 and 3) Notably the base of the epithelial crypts wasalso surrounded by frizzled-4 positive neural extensions(Figure 3) Moreover the Wnt receptor was costained in

cell populations expressing the intermediate filament nestinand the neurotrophin receptor p75NTR (Figures 1 2 and7) Interestingly the amount of p75NTR positive cells thatcoexpressed frizzled-4 per ganglia was highly variable evenin the same gut area Frizzled-4 was not found in smoothmuscle cells of tunica muscularis tunica mucosa or the wallof blood vessels and was also absent in interstitial cells ofCajal which were stained with c-kit (Figure 2)


Nestin Fzd4 Nestin Fzd4

GFAP

c-kit Fzd4

Fzd4 GFAP Fzd4

Peripherin Fzd4Peripherin Fzd4

Fzd4 Fzd4

c-kit Fzd4

(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

(d1) (d2) (d3)

(e1) (e2) (e3)

p75NTR p75NTR

Figure 2 Frizzled-4 is detected in a subpopulation of neural cells Immunofluorescence for frizzled-4 (Fzd4) p75NTR nestin peripherinGFAP and c-kit on cryostat sections of human ileum (sample number 2) ((a1)ndash(a3)) Combined p75NTR (green) and Fzd4 (red)immunostaining of myenteric ganglia p75NTR (a1) Fzd4 (a2) merge (a3) DAPI (blue) ((b1)ndash(b3)) Combined nestin (green) and Fzd4 (red)immunostaining of myenteric ganglia nestin (b1) Fzd4 (b2) merge (b3) DAPI (blue) ((c1)ndash(c3)) Combined peripherin (green) and Fzd4(red) immunostaining of myenteric ganglia peripherin (c1) Fzd4 (c2) merge (c3) DAPI (blue) (d1ndashd3) Combined GFAP (green) and Fzd4(red) immunostaining of myenteric ganglia GFAP (d1) Fzd4 (d2) merge (d3) DAPI (blue) ((e1)ndash(e3)) Combined c-kit (green) and Fzd4(red) immunostaining of myenteric ganglia c-kit (e1) Fzd4 (e2) merge (e3) DAPI (blue) scale bar (andashe) 100 120583m


LMM

TSM

(a1) (a2) (a3)

(b1) (b2) (b3)

Figure 3 Epithelial crypts of the mucosa are surrounded by frizzled-4 positive neural extensions Immunofluorescence of frizzled-4 (Fzd4)on cryostat sections of human ileum (sample number 2) ((a1)ndash(a3)) Sagittal section Combined Fzd4 (red) and DAPI (blue) fluorescencestaining (a1) combined Fzd4 fluorescence (red) and brightfield view (a2) merge (a3) The broken lines mark the border between epithelialcells of the crypt base and the lamina propria mucosaeThe white arrowheads indicate the borders of the lamina muscularis mucosae (LMM)to the lamina propria mucosae and the tela submucosa (TSM) scale bar 50 120583m ((b1)ndash(b3)) Horizontal section of the crypt region CombinedFzd4 (red) and DAPI (blue) fluorescence staining (b1) combined Fzd4 (red) fluorescence and brightfield view (b2) merge (b3) The brokenlines mark the border between epithelial cells of some crypts and the lamina propria mucosae scale bar (a b) 50 120583m

(bp)

GA

PDH

Neg

ativ

eco

ntro

l

Fzd4

1000

500

800

200

100

Figure 4 Identification of frizzled-4 (Fzd4) mRNA by RT-PCR Total RNA was isolated from tunica muscularis Respective primer pairswere used for detection of Fzd4 (605 bp) and the housekeeping geneGAPDH (452 bp) Isolated RNAwithout subsequent reverse transcriptasestep served as negative control


Cou

nts

0

20

40

60

80

100

p75NTR-APC0 10

210

310

410

5

(a)C

ount

s

0

20

40

60

80

100

0 102

103

104

105

Fzd4-FITC

(b)

0

102

103

104

105

0 102

103

104

105

Fzd4-FITC

p75-

APC

(65)(173)

(03)

Fzd4+

Fzd4+

Fzd4minusp75NTR+

p75NTR+

p75NTRminus

(c)

0

20

40

60

80

100

Num

ber o

f cell

s (

)

Fzd4minus p75NTR+ Fzd4+ Fzd4+

p75NTRminus p75NTR+

(d)

Figure 5 A distinct frizzled-4 positive cell population is observed by flow cytometry Flow cytometry analysis of p75NTR and frizzled-4 (Fzd4)expression on cells isolated from tunicamuscularis (sample numbers 3 5ndash9) (a b)Histograms for p75NTR-APCandFzd4-FITC in comparisonto respective isotype controls The isotype control staining is shown as gray filled histogram (c) Representative dot plot of p75NTR-APC andFzd4-FITC costained cells Numbers indicate the percentage of stained cells in each gate (d) Quantification of the percentages of unstainedp75NTR Fzd4 positive and p75NTRFzd4 costained cells from six independent experiments Data are expressed as mean percentage plusmn SD ofthe total cell number

In summary we have shown by immunohistochemistrythat frizzled-4 is expressed in a subpopulation of entericnervous system cells which are positive for peripherin GFAPand p75NTR

In order to quantify the frizzled-4 positive cell pop-ulation enzymatically digested and immunolabeled cellsisolated from tunica muscularis were stained with frizzled-4

antibody and analysed by flow cytometry (Figure 5) Usingthis approach a distinct cell population could be detectedin six independent experiments 123plusmn 59 (mean plusmnSD) of the analysed cells stained positive for frizzled-4in comparison to 197plusmn 92 (mean plusmn SD) of cells thatwere positive for the neurotrophin receptor p75NTR Indeedcolabelling experiments demonstrated that 600plusmn 162


Mucosa

Submucosa

Myenteric plexus

Figure 6 Overview of immunostained section sliced from ldquoSwiss-rolledrdquo tissue of Hirschsprungrsquos disease Cryostat section from largeintestine diagnosed with Hirschsprungrsquos disease (sample number 10) was immunostained for peripherin scale bar 5mm The center of theldquoSwiss-rolledrdquo tissue marks the distal end of resected intestinal segment In the microscopic view at the upper left corner normoganglionichypoglanglionic and aganglionic areas are marked by an unbroken broken and dotted line respectively

(mean plusmn SD) of p75NTR positive cells expressed the Wntreceptor whereas almost all cells found positive for frizzled-4also expressed p75NTR (933plusmn 89 (mean plusmn SD)) Hencewe could identify a double-positive subpopulation of p75NTR+

frizzled-4+ cells which is clearly distinguishable from thefrizzled-4 negative population

Next we investigated the Wnt receptor expression pat-tern in gut samples of three patients presenting withHirschsprungrsquos disease In Figure 6 a representative overviewof a peripherin stained cryosection is shown which wassliced from a ldquoSwiss rolledrdquo tissue of Hirschsprungrsquos disease(Figure 6) In these sections the size and number of gangliatypically decreased from oral to anal The rudimentaryexpression of peripherin in the myenteric plexus at theanal (aganglionic) end represents peripherin expression inmyenteric nerve fibers [22] Immunohistochemical analysisof frizzled-4 in comparison to nestin p75NTR peripherinand GFAP revealed that in line with the other markers theexpression of frizzled-4 declined from normoganglionic toaganglionic areas of the large intestine (Figures 7 and 8)

4 Discussion

Wnt signalling plays an important role in the developmentof the peripheral nervous system It induces and specifies theneural crest and is involved in processes regulating neural

crest cell migration and formation of neuronal interconnec-tions in the developing gut [12ndash14]

Among the ten identified frizzled-Wnt receptors onlyfrizzled-4 is able to strongly bind Norrin Norrin is knownto regulate vascular development of the inner ear and retina[23] but has also been attributed to mediate neuroprotectiveeffects to retinal ganglion cells [20] In humans it is associatedwith Norrie disease a genetic disorder that primarily affectsthe eye and leads to blindness and to progressive hearingloss in a proportion of patients Although no gastrointestinalsymptoms were described in affected humans the Norrin-frizzled-4 interaction is suspected to play an important role incolonic mucosa regeneration and colonic tumorigenesis [24]

In addition frizzled-4 knockout mice suffer fromesophageal dysfunction apart from progressive cerebellarand auditory degeneration [25] While the authors ascribedchanges in esophageal motility rather to muscular malde-velopment and described the lower gastrointestinal ENS tobe unaffected in frizzled-4 knockout mice subtle changesin ENS architecture and the impact on the function of thegut beyond the stomach were not investigated in detail Thefact that the knockout of frizzled genes can lead to profoundneurogenic abnormalities of gastrointestinal tract motilitywhile ENS architecture appears to be almost normal hasbeen impressively demonstrated by Sasselli et al [14] Inthese experiments the loss of frizzled-3 led to developmentaldeficits in the ENS network and resulted in a gastrointestinal

Stem Cells International 9Fz

d4N

estin

Perip

herin

AganglionicHypoganglionicNormoganglionicp7

5N

TR

Figure 7The expression of frizzled-4 inHirschsprungrsquos disease decreases frombeing oral to anal Immunohistochemical analysis for frizzled-4 (Fzd4) in comparison to p75NTR nestin and peripherin on cryostat sections from large intestine diagnosed with Hirschsprungrsquos disease(sample number 10) Microscopic brightfield views were taken from normoganglionic hypoganglionic and aganglionic areas respectivelyscale bar 200 120583m

dysmotilityWhile the importance ofWnt signalling in neuralcrest and ENS development is well established the role of thispathway in the postnatal ENS is largely unknown Recentlypreliminary data indicate an anti-inflammatory activity ofWnt signalling in the postnatal enteric nervous system of rats[26]

In our study we analyzed the expression pattern offrizzled-4 in normal infant human gut tissue as well as tissue

collected from patients suffering from Hirschsprungrsquos dis-ease Immunohistochemical analysis revealed that frizzled-4is expressed in neural cells within the gastrointestinal tractSubpopulations of peripherin expressing neurons and GFAPpositive glial cells were stained with various intensities Incontrast frizzled-4 was expressed neither in enteric smoothmuscle nor in interstitial cells of Cajal Interestingly frizzled-4 expression was also observed in cell populations that


Fzd4

NestinFzd4

PeripherinFzd4

GFAPFzd4 GFAPFzd4

PeripherinFzd4PeripherinFzd4

NestinFzd4NestinFzd4

Fzd4 Fzd4

AganglionicHypoganglionicNormoganglionic

GFAPFzd4

p75NTR p75NTRp75NTR

Figure 8 Coimmunostaining processes reveal the parallel decrease of frizzled-4 with other neural markers in Hirschsprungrsquos diseaseImmunofluorescence for frizzled-4 (Fzd4 red) in combination with p75NTR nestin peripherin and GFAP (green) on cryostat sections fromlarge intestine diagnosed with Hirschsprungrsquos disease (sample number 10) cell nuclei were visualized with DAPI (blue) Fluorescence viewswere taken from normoganglionic hypoglanglionic and aganglionic area respectively scale bar 100 120583m

stained positive for the intermediate filament nestin and thecell surface antigen p75NTR Together with CD49b these pro-teins have been proposed to be expressed in ENS progenitoror stem cells [18] Indeed some evidence supports that neu-rogenesis also takes place in the postnatal gut Regeneration

of the ENS has been demonstrated to take place in micein vivo upon 5-HT stimulation or following injury of thegut [15ndash17] In addition the ability to isolate and propagatecells of the postnatal ENS of rodents and men indicatesthe persistence of progenitor or stem cell like neural cell


types in the postnatal intestine [27ndash30] In this context theidentification of suitable markers for isolation of these neuralprogenitor cells is of crucial importance for the developmentof cell based therapies Intriguingly our flow cytometryexperiments showed that gt93 of frizzled-4 expressing cellswere also positive for p75NTR whereas frizzled-4 positive cellsrepresent only about 60 of the p75NTR cell pool Thus thefrizzled-4 antibody recognizes a subpopulation of p75NTR

positive cells and might represent an interesting markerfor the isolation and characterization of these distinct cellpopulations However further experiments will be necessaryto investigate and compare the cell biological properties ofFzd4+p75NTR+ and Fzd4minusp75NTR+ cells

Interestingly our immunohistological findings demon-strate that the epithelial crypts are surrounded by frizzled-4 positive neural extension The intestinal crypt harbors theepithelial stem compartment that is strongly regulated by theWnt signalling pathway [31]The close localization of ENS cellextensions to this compartment indicates that the epithelialstem cell nichemight influence the enteric nervous system bysecreted Wnt pathway agonists such as Wnt3a or R-spondin

Diagnostic procedures use specific antibodies to iden-tify diseases such as Hirschsprungrsquos disease However theidentification of enteric ganglion cells can be challengingin human tissue especially if immature ganglion cells arepresent in newborns where these cells can be easily con-fused with endothelial cells and cells of mesenchymal orimmunological origin [32] In addition in primary diag-nostic suction biopsies specimens usually only include thesubmucosal plexus and are often of small size making aprecise diagnosis difficult Therefore immunohistochemicalanalysis using several antibodies targeted at various neuronaland glial antigens has been proposed to increase sensitivityand specificity of the diagnostic procedure In this study weevaluated the frizzled-4 expression pattern in comparisonto other cell markers in the various segments of the colonof patients suffering from Hirschsprungrsquos disease Immuno-histochemistry was performed for p75NTR nestin GFAPperipherin and frizzled-4 in the normally ganglionated gutthe transition zone and the aganglionic segment of the colonIn the examined tissues the number of frizzled-4 positivecells declined from normoganglionic to aganglionic areas oflarge intestine in accordance with the other neural markersWhether frizzled-4 might be useful as a putative diagnosticmarker for the diagnosis of Hirschsprungrsquos disease has to beinvestigated in a larger cohort of patients

5 Conclusion

In the present study we analysed the expression of theWnt receptor frizzled-4 in the enteric nervous system ofhuman small and large intestine Frizzled-4 was identified ina distinct subpopulation of enteric neurons and glia as wellas nestin and p75NTR positive cells in all layers of gut wallIn pathological human samples of Hirschsprungrsquos disease theexpression of this Wnt receptor decreased from normogan-glionic to aganglionic areas of large intestine The expressionpattern of frizzled-4 indicates that theWnt signalling pathway

might be involved in the postnatal development andorfunction of the enteric nervous system Additional studiesare necessary to characterize the frizzled-4 positive cellpopulation in more detail and to elucidate the biological roleof this Wnt receptor in the postnatal human enteric system

Conflict of Interests

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

Authorsrsquo Contribution

Katharina Nothelfer and Florian Obermayr contributedequally to the work

Acknowledgments

The authors would like to thank Susanne Stachon for herexcellent technical assistance The project was supported bya grant from the German Federal Ministry for Education andResearch (01GN0967)

References

[1] NM LeDouarin and EDupin ldquoMultipotentiality of the neuralcrestrdquo Current Opinion in Genetics amp Development vol 13 no5 pp 529ndash536 2003

[2] C Nishiyama T Uesaka T Manabe et al ldquoTrans-mesentericneural crest cells are the principal source of the colonic entericnervous systemrdquo Nature Neuroscience vol 15 no 9 pp 1211ndash1218 2012

[3] F Obermayr R Hotta H Enomoto and H M Young ldquoDevel-opment and developmental disorders of the enteric nervoussystemrdquoNature Reviews GastroenterologyampHepatology vol 10no 1 pp 43ndash57 2012

[4] A S Wallace and A J Burns ldquoDevelopment of the entericnervous system smooth muscle and interstitial cells of Cajal inthe human gastrointestinal tractrdquo Cell and Tissue Research vol319 no 3 pp 367ndash382 2005

[5] H M Young A J Bergner M J Simpson et al ldquoColonizingwhile migrating how do individual enteric neural crest cellsbehaverdquo BMC Biology vol 12 no 1 p 23 2014

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

[7] M Schemann ldquoControl of gastrointestinal motility by thelsquogut brainrsquomdashthe enteric nervous systemrdquo Journal of PediatricGastroenterology amp Nutrition vol 41 supplement 1 pp S4ndashS62005

[8] C E Gariepy ldquoDevelopmental disorders of the enteric nervoussystem genetic and molecular basesrdquo Journal of PediatricGastroenterology and Nutrition vol 39 no 1 pp 5ndash11 2004

[9] D Newgreen andHM Young ldquoEnteric nervous system devel-opment and developmental disturbancesmdashPart 2rdquoPediatric andDevelopmental Pathology vol 5 no 4 pp 329ndash349 2002

[10] D Newgreen andHM Young ldquoEnteric nervous system devel-opment and developmental disturbancesmdashpart 1rdquo Pediatric andDevelopmental Pathology vol 5 no 3 pp 224ndash247 2002


[11] 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

[12] R Mayor and E Theveneau ldquoThe role of the non-canonicalWnt-planar cell polarity pathway in neural crest migrationrdquoBiochemical Journal vol 457 no 1 pp 19ndash26 2014

[13] T Sauka-Spengler and M Bronner-Fraser ldquoA gene regulatorynetwork orchestrates neural crest formationrdquo Nature ReviewsMolecular Cell Biology vol 9 no 7 pp 557ndash568 2008

[14] V Sasselli W Boesmans P V Berghe F Tissir A M Goffinetand V Pachnis ldquoPlanar cell polarity genes control the connec-tivity of enteric neuronsrdquo The Journal of Clinical Investigationvol 123 no 4 pp 1763ndash1772 2013

[15] N M Joseph S He E Quintana Y Kim G Nunez andS J Morrison ldquoEnteric glia are multipotent in culture butprimarily form glia in the adult rodent gutrdquo Journal of ClinicalInvestigation vol 121 no 9 pp 3398ndash3411 2011

[16] C Laranjeira K Sandgren N Kessaris et al ldquoGlial cells inthe mouse enteric nervous system can undergo neurogenesis inresponse to injuryrdquoThe Journal of Clinical Investigation vol 121no 9 pp 3412ndash3424 2011

[17] M T Liu Y H Kuan J Wang R Hen and M D Gershonldquo5-HT4 receptor-mediated neuroprotection and neurogenesisin the enteric nervous system of adult micerdquo The Journal ofNeuroscience vol 29 no 31 pp 9683ndash9699 2009

[18] 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

[19] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 no 1 pp 781ndash810 2004

[20] R Seitz S Hackl T Seibuchner E R Tamm and A OhlmannldquoNorrin mediates neuroprotective effects on retinal ganglioncells via activation of the Wntbeta-catenin signaling pathwayand the induction of neuroprotective growth factors in Mullercellsrdquo Journal of Neuroscience vol 30 no 17 pp 5998ndash60102010

[21] G Abbruzzese A Gorny L T Kaufmann et al ldquoThe Wntreceptor Frizzled-4 modulates ADAM13 metalloprotease activ-ityrdquo Journal of Cell Science vol 128 no 6 pp 1139ndash1149 2015

[22] R Beschorner M Mittelbronn K Bekure and R MeyermannldquoProblems in fast intraoperative diagnosis in Hirschsprungrsquosdiseaserdquo FoliaNeuropathologica vol 42 no 4 pp 191ndash195 2004

[23] H L Rehm D S Zhang M C Brown et al ldquoVascular defectsand sensorineural deafness in amousemodel ofNorrie diseaserdquoThe Journal of Neuroscience vol 22 no 11 pp 4286ndash4292 2002

[24] K PlanutisM PlanutieneMMoyer A VNguyen C A Perezand R F Holcombe ldquoRegulation of norrin receptor frizzled-4by Wnt2 in colon-derived cellsrdquo BMC Cell Biology vol 8 no 1article 12 2007

[25] Y Wang D Huso H Cahill D Ryugo and J NathansldquoProgressive cerebellar auditory and esophageal dysfunctioncaused by targeted disruption of the frizzled-4 generdquo TheJournal of Neuroscience vol 21 no 13 pp 4761ndash4771 2001

[26] 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

[27] N Bondurand D Natarajan NThapar C Atkins and V Pach-nis ldquoNeuron and glia generating progenitors of themammalianenteric nervous system isolated from foetal and postnatal gutculturesrdquo Development vol 130 no 25 pp 6387ndash6400 2003

[28] 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

[29] 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

[30] 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 2214ndash2225e3 2009

[31] H Clevers ldquoThe intestinal crypt a prototype stem cell compart-mentrdquo Cell vol 154 no 2 pp 274ndash284 2013

[32] S K Holland R B Hessler M D Reid-Nicholson P Rama-lingam and J R Lee ldquoUtilization of peripherin and S-100immunohistochemistry in the diagnosis of Hirschsprung dis-easerdquoModern Pathology vol 23 no 9 pp 1173ndash1179 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


crest induction and specification In addition migration ofneural crest cells is dependent on theWnt signalling pathway[12 13]

TheWnt signalling cascade is initiated by binding of Wntto members of the frizzled transmembrane receptor familyand results in activation of pathways like the 120573-catenin-dependent pathway or the planar cell polarity- (PCP-)dependent pathway both of which have been shown to beinvolved in early neural crest development Recently thePCP pathway has been demonstrated to be additionallyinvolved in regulating neural interconnection during ENSdevelopment [14] However while the importance of Wnt-frizzled interaction is well established for early neural crestdevelopment its role in regulating postnatal neural crestderivatives is largely unknown

Interestingly there is evidence that neurogenesis con-tinues in postnatal mice in vivo upon stimulation withserotonin or following injury [15ndash17] A subpopulation ofcells of the postnatal ENS has been attributed with stem celllike properties although this niche is not well characterizedyet Nestin the low-affinity nerve growth factor receptorp75NTR and CD49b (alpha2-integrin) have been suggested torepresent markers for this population of enteric neural stemand progenitor cells [18]

Of the frizzled receptor family members frizzled-4 haspreviously been shown to be of particular interest withrespect to its role in the regulation of stem cell maintenanceneuroprotection and cell migration in the CNS [19ndash21] Inthe present study we examined the expression of this Wntreceptor in the enteric nervous system of infants by immuno-histochemistry RT-PCR and flow cytometry analysis Inorder to characterize the frizzled-4 positive cell populationwe performed colabelling experiments with various neuralmarkers that are expressed in neural and putative progenitorcells of the ENS

2 Materials and Methods

21 Human Gut Samples Human gut samples from smalland large intestine were obtained from infant male andfemale patients at the age of 4 weeks to 10 months whounderwent surgical enterostoma removal after treatmentfor necrotizing enterocolitis or anorectal malformations Inaddition tissue was collected from Hirschsprungrsquos diseasepatients who underwent a transanal endorectal pull-throughprocedure (Table 1) All samples were collected accordingto the guidelines and after approval of the Local EthicalCommittee at the University of Tubingen and with theconsent of the patientrsquos parents

22 Immunohistochemistry Immunohistochemistry wasperformed on 14 120583m thick cryosections Tissue slides werefixed in 4 (wv) paraformaldehyde in phosphate-bufferedsaline (PBS pH 74) for 20min at room temperature andsubsequently rinsed three times in PBS Slides whichwere stained with the ABC-detection system (VectorLabs Peterborough UK) were additionally pretreated in3 (vv) hydrogen peroxide PBS solution for 10min to

Table 1 Human gut samples

Samplenumber Gender Patient age Tissue Pathology

1 Male 5 months Colon Normoganglionic2 Female 2 months Ileum Normoganglionic3 Female 5 months Sigma Normoganglionic4 Male 2 months Ileum Normoganglionic5 Male 10 months Colon Normoganglionic6 Female 3 months Sigma Normoganglionic7 Male 6 months Sigma Normoganglionic8 Female 4 months Ileocaecal Normoganglionic9 Male 7 months Colon Normoganglionic10 Male 4 weeks Colon Hirschsprung11 Male 6 weeks Colon Hirschsprung12 Male 7 months Colon Hirschsprung

block endogenous peroxidases After incubation with serumcontaining blocking buffer (PBS containing 01 (wv)BSA 4 (vv) goat serum and 03 (vv) Triton X-100) for30min sections were incubated with primary antibodiesdiluted in PBS 01 (wv) BSA and 01 (vv) Triton X-100overnight at 4∘C Primary antibodies and concentrationsare listed in Table 2 Following three washes with PBSthe sections were incubated with secondary antibodies for30min at room temperature in the same buffer Detectionwas performed with either fluorochrome-conjugatedsecondary antibodies (1 400 goat anti-rabbit IgG-Cy3(Dianova Hamburg Germany) goat anti-mouse IgG-Cy3 (Dianova Hamburg Germany) goat anti-mouseIgG-Alexa 488 (Life Technologies Darmstadt Germany))or biotinylated secondary antibodies (1 400 swine anti-rabbit IgG-biotin and rabbit anti-mouse IgG-biotin (DakoHamburg Germany)) when using the ABC-detectionsystem Incubation of the AB reagent was performedaccording to the manufacturer and visualization of the ABCsystem was carried out with 3-31015840diaminobenzidine (DABSigma Taufkirchen Germany) and hydrogen peroxidesolution For fluorescence detection the sections and cellswere washed three times in PBS and additionally stainedwith DAPI (41015840-6-diamidino-2-phenylindole 02120583gmL) PBSsolution for 10min Stained sections were dried embeddedin Kaiserrsquos gelatin (Merck Darmstadt Germany) andphotographed on an inverted microscope (Axiovert ZeissJena Germany)

23 RNA Isolation and RT-PCR Human gut samples weremechanically dissected with the visual aid of a stereomi-croscope After disposure of residual fat and mesenterictissue the tunica muscularis was peeled off the submu-cosal and mucosal layers and thoroughly minced TotalRNA was isolated from human tunica muscularis usingthe RNeasy Mini Kit (Qiagen Hilden Germany) accordingto the manufacturerrsquos instructions Possible contaminatinggenomic DNA was removed by treatment with DNase I (LifeTechnologies Darmstadt Germany) Reverse transcriptase














3 Results



Nestin Nestin Fzd4

Fzd4

TMTS

M

TMTS

M

TMTS

M

Fzd4

Fzd4

(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

Fzd4p75NTRp75NTR

Fzd4p75NTRp75NTR







GFAP

c-kit Fzd4

Fzd4 GFAP Fzd4


Fzd4 Fzd4

c-kit Fzd4

(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

(d1) (d2) (d3)

(e1) (e2) (e3)

p75NTR p75NTR



LMM

TSM

(a1) (a2) (a3)

(b1) (b2) (b3)


(bp)

GA

PDH

Neg

ativ

eco

ntro

l

Fzd4

1000

500

800

200

100



Cou

nts

0

20

40

60

80

100

p75NTR-APC0 10

210

310

410

5

(a)C

ount

s

0

20

40

60

80

100

0 102

103

104

105

Fzd4-FITC

(b)

0

102

103

104

105

0 102

103

104

105

Fzd4-FITC

p75-

APC

(65)(173)

(03)

Fzd4+

Fzd4+

Fzd4minusp75NTR+

p75NTR+

p75NTRminus

(c)

0

20

40

60

80

100

Num

ber o

f cell

s (

)


p75NTRminus p75NTR+

(d)






Mucosa

Submucosa

Myenteric plexus





4 Discussion






d4N

estin

Perip

herin


5N

TR






Fzd4

NestinFzd4

PeripherinFzd4

GFAPFzd4 GFAPFzd4



Fzd4 Fzd4


GFAPFzd4

p75NTR p75NTRp75NTR









5 Conclusion







Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














3 Results



Nestin Nestin Fzd4

Fzd4

TMTS

M

TMTS

M

TMTS

M

Fzd4

Fzd4

(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

Fzd4p75NTRp75NTR

Fzd4p75NTRp75NTR







GFAP

c-kit Fzd4

Fzd4 GFAP Fzd4


Fzd4 Fzd4

c-kit Fzd4

(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

(d1) (d2) (d3)

(e1) (e2) (e3)

p75NTR p75NTR



LMM

TSM

(a1) (a2) (a3)

(b1) (b2) (b3)


(bp)

GA

PDH

Neg

ativ

eco

ntro

l

Fzd4

1000

500

800

200

100



Cou

nts

0

20

40

60

80

100

p75NTR-APC0 10

210

310

410

5

(a)C

ount

s

0

20

40

60

80

100

0 102

103

104

105

Fzd4-FITC

(b)

0

102

103

104

105

0 102

103

104

105

Fzd4-FITC

p75-

APC

(65)(173)

(03)

Fzd4+

Fzd4+

Fzd4minusp75NTR+

p75NTR+

p75NTRminus

(c)

0

20

40

60

80

100

Num

ber o

f cell

s (

)


p75NTRminus p75NTR+

(d)






Mucosa

Submucosa

Myenteric plexus





4 Discussion






d4N

estin

Perip

herin


5N

TR






Fzd4

NestinFzd4

PeripherinFzd4

GFAPFzd4 GFAPFzd4



Fzd4 Fzd4


GFAPFzd4

p75NTR p75NTRp75NTR









5 Conclusion







Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Nestin Nestin Fzd4

Fzd4

TMTS

M

TMTS

M

TMTS

M

Fzd4

Fzd4

(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

Fzd4p75NTRp75NTR

Fzd4p75NTRp75NTR







GFAP

c-kit Fzd4

Fzd4 GFAP Fzd4


Fzd4 Fzd4

c-kit Fzd4

(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

(d1) (d2) (d3)

(e1) (e2) (e3)

p75NTR p75NTR



LMM

TSM

(a1) (a2) (a3)

(b1) (b2) (b3)


(bp)

GA

PDH

Neg

ativ

eco

ntro

l

Fzd4

1000

500

800

200

100



Cou

nts

0

20

40

60

80

100

p75NTR-APC0 10

210

310

410

5

(a)C

ount

s

0

20

40

60

80

100

0 102

103

104

105

Fzd4-FITC

(b)

0

102

103

104

105

0 102

103

104

105

Fzd4-FITC

p75-

APC

(65)(173)

(03)

Fzd4+

Fzd4+

Fzd4minusp75NTR+

p75NTR+

p75NTRminus

(c)

0

20

40

60

80

100

Num

ber o

f cell

s (

)


p75NTRminus p75NTR+

(d)






Mucosa

Submucosa

Myenteric plexus





4 Discussion






d4N

estin

Perip

herin


5N

TR






Fzd4

NestinFzd4

PeripherinFzd4

GFAPFzd4 GFAPFzd4



Fzd4 Fzd4


GFAPFzd4

p75NTR p75NTRp75NTR









5 Conclusion







Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



GFAP

c-kit Fzd4

Fzd4 GFAP Fzd4


Fzd4 Fzd4

c-kit Fzd4

(a1) (a2) (a3)

(b1) (b2) (b3)

(c1) (c2) (c3)

(d1) (d2) (d3)

(e1) (e2) (e3)

p75NTR p75NTR



LMM

TSM

(a1) (a2) (a3)

(b1) (b2) (b3)


(bp)

GA

PDH

Neg

ativ

eco

ntro

l

Fzd4

1000

500

800

200

100



Cou

nts

0

20

40

60

80

100

p75NTR-APC0 10

210

310

410

5

(a)C

ount

s

0

20

40

60

80

100

0 102

103

104

105

Fzd4-FITC

(b)

0

102

103

104

105

0 102

103

104

105

Fzd4-FITC

p75-

APC

(65)(173)

(03)

Fzd4+

Fzd4+

Fzd4minusp75NTR+

p75NTR+

p75NTRminus

(c)

0

20

40

60

80

100

Num

ber o

f cell

s (

)


p75NTRminus p75NTR+

(d)






Mucosa

Submucosa

Myenteric plexus





4 Discussion






d4N

estin

Perip

herin


5N

TR






Fzd4

NestinFzd4

PeripherinFzd4

GFAPFzd4 GFAPFzd4



Fzd4 Fzd4


GFAPFzd4

p75NTR p75NTRp75NTR









5 Conclusion







Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


LMM

TSM

(a1) (a2) (a3)

(b1) (b2) (b3)


(bp)

GA

PDH

Neg

ativ

eco

ntro

l

Fzd4

1000

500

800

200

100



Cou

nts

0

20

40

60

80

100

p75NTR-APC0 10

210

310

410

5

(a)C

ount

s

0

20

40

60

80

100

0 102

103

104

105

Fzd4-FITC

(b)

0

102

103

104

105

0 102

103

104

105

Fzd4-FITC

p75-

APC

(65)(173)

(03)

Fzd4+

Fzd4+

Fzd4minusp75NTR+

p75NTR+

p75NTRminus

(c)

0

20

40

60

80

100

Num

ber o

f cell

s (

)


p75NTRminus p75NTR+

(d)






Mucosa

Submucosa

Myenteric plexus





4 Discussion






d4N

estin

Perip

herin


5N

TR






Fzd4

NestinFzd4

PeripherinFzd4

GFAPFzd4 GFAPFzd4



Fzd4 Fzd4


GFAPFzd4

p75NTR p75NTRp75NTR









5 Conclusion







Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Cou

nts

0

20

40

60

80

100

p75NTR-APC0 10

210

310

410

5

(a)C

ount

s

0

20

40

60

80

100

0 102

103

104

105

Fzd4-FITC

(b)

0

102

103

104

105

0 102

103

104

105

Fzd4-FITC

p75-

APC

(65)(173)

(03)

Fzd4+

Fzd4+

Fzd4minusp75NTR+

p75NTR+

p75NTRminus

(c)

0

20

40

60

80

100

Num

ber o

f cell

s (

)


p75NTRminus p75NTR+

(d)






Mucosa

Submucosa

Myenteric plexus





4 Discussion






d4N

estin

Perip

herin


5N

TR






Fzd4

NestinFzd4

PeripherinFzd4

GFAPFzd4 GFAPFzd4



Fzd4 Fzd4


GFAPFzd4

p75NTR p75NTRp75NTR









5 Conclusion







Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Mucosa

Submucosa

Myenteric plexus





4 Discussion






d4N

estin

Perip

herin


5N

TR






Fzd4

NestinFzd4

PeripherinFzd4

GFAPFzd4 GFAPFzd4



Fzd4 Fzd4


GFAPFzd4

p75NTR p75NTRp75NTR









5 Conclusion







Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


d4N

estin

Perip

herin


5N

TR






Fzd4

NestinFzd4

PeripherinFzd4

GFAPFzd4 GFAPFzd4



Fzd4 Fzd4


GFAPFzd4

p75NTR p75NTRp75NTR









5 Conclusion







Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Fzd4

NestinFzd4

PeripherinFzd4

GFAPFzd4 GFAPFzd4



Fzd4 Fzd4


GFAPFzd4

p75NTR p75NTRp75NTR









5 Conclusion







Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






5 Conclusion







Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Expression of the Wnt Receptor Frizzled-4 ...downloads.hindawi.com/journals/sci/2016/9076823.pdf · e Wnt signalling pathway plays a crucial role in the developm