research article expression profile of sonic hedgehog...

Research ArticleExpression Profile of Sonic Hedgehog Pathway Members in theDeveloping Human Fetal Brain

Julia Tichy123 Jenny Zinke3 Benedikt Bunz1 Richard Meyermann1

Patrick N Harter3 and Michel Mittelbronn3

1 Institute of Pathology and Neuropathology University of Tuebingen 72076 Tuebingen Germany2Senckenberg Institute for Neurooncology Goethe University Frankfurt 60528 Frankfurt am Main Germany3Edinger Institute Institute of Neurology Goethe University Frankfurt 60528 Frankfurt am Main Germany

Correspondence should be addressed to Michel Mittelbronn michelmittelbronnkgude

Received 10 January 2015 Revised 29 March 2015 Accepted 30 March 2015

Academic Editor Anton M Jetten

Copyright copy 2015 Julia Tichy et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The Sonic Hedgehog (SHH) pathway plays a central role in the developing mammalian CNS In our study we aimed to investigatethe spatiotemporal SHH pathway expression pattern in human fetal brains We analyzed 22 normal fetal brains for Shh PatchedSmoothened andGli1-3 expression by immunohistochemistry In the telencephalon strongest expression of Shh Smoothened andGli2 was found in the cortical plate (CP) and ventricular zone Patched was strongly upregulated in the ventricular zone and Gli1in the CP In the cerebellum SHH pathway members were strongly expressed in the external granular layer (EGL) SHH pathwaymembers significantly decreased over time in the ventricular and subventricular zone and in the cerebellar EGL while increasinglevels were found in more superficial telencephalic layers Our findings show that SHH pathway members are strongly expressedin areas important for proliferation and differentiation and indicate a temporal expression gradient in telencephalic and cerebellarlayers probably due to decreased proliferation of progenitor cells and increased differentiation Our data about the spatiotemporalexpression of SHH pathway members in the developing human brain serves as a base for the understanding of both normal andpathological CNS development

1 Introduction

The Hedgehog (HH) pathway plays a central role in theembryonic and fetal development of both invertebrates andvertebrates [1] This pathway essentially regulates cellularstemness proliferation apoptosis migration and differenti-ation [2ndash6] While the hedgehog gene was first described inDrosophila melanogaster its homologous genes in mammalsmeanwhile consist of the hedgehog family members Indian(IHH) Desert (DHH) and Sonic hedgehog (SHH) [7ndash9]In general the hedgehog proteins interact with the trans-membrane receptor Patched (PTCH) the latter inhibitingthe cascade-initiating receptor Smoothened (SMO) in theabsence of hedgehog ligands [10] The Patched-mediatedinhibition of Smoothened is abolished in case of HH ligandsbinding to the Patched receptor [11] The SMO activationelicits a nuclear translocation of the transcription factors Gli1Gli2 and Gli3 [12] Although the definite functions of the

Gli family members are unclear there is rising evidence for apredominantly transcription-activating function of Gli1 andGli2 [13 14] In contrast Gli3 is supposed to act more likely asan inhibitor of transcription [15]While the hedgehog proteinin Drosophila is a key player in the formation of segmentpolarity its best characterized mammalian homologue Sonichedgehog is involved in the development of the CNS limbsskeleton and internal organs [16ndash20] In the mammaliancentral nervous system (CNS) SHH is responsible for thepatterning of the spinal cord and brain as well as for theproliferation of the cerebellar granule precursor cells [21]Furthermore it induces the differentiation of Bergmann gliain the developing cerebellum [21]The importance of the SHHpathway for normal development and organ homeostasis isreflected by the fact that mutations in the SHH pathway leadto severe malformations and the development of malignantneoplasms [22 23] While pathogenic mutations of theSHH pathway among other diseases cause holoprosencephaly

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 494269 15 pageshttpdxdoiorg1011552015494269

2 BioMed Research International

[20 22] a deletion of the Gli3 gene may result in the Greigcephalopolysyndactyly syndrome [24 25] On the other handan increased activation of the SHH pathway may lead toneoplastic transformation of various tissues including breast[26] skin [27] and brain [21] Most interestingly neoplasmsassociated with mutations of the SHH pathway occur in areaswhere SHH is essential for the regulation of cellular stemnessand differentiation such as in the cerebellum the brainarea where medulloblastomas emerge [28 29] Although theexpression of the molecules of the SHH pathway in humansis very well studied under pathological conditions there is alack of knowledge concerning the spatiotemporal expressionpattern in the normal developing human CNS The aim ofour study was to determine the nonpathological expressionpattern of the SHH pathway members in a cohort of normalhuman fetal brains at different stages of gestation to gaininsights into the role of the SHH pathway in normal CNSdevelopment

2 Materials and Methods

21 Clinical Data and Tissue Specimens We investigated 22human fetal brains from the 12th to the 28thweek of gestation(Table 1) For the present study a cohort of fetuses with-out pathological CNS alterations was selected according toclinical data family history and neuropathological analysesperformed by two experienced neuropathologists (MM andRM) The samples belong to the human tissue brain bank ofthe Institute of Pathology and Neuropathology University ofTuebingenGermany [30] All tissue specimenswere paraffin-embedded The study protocol was endorsed by the ethicalcommittee of the University of Tuebingen Germany

22 Immunohistochemistry All samples were fixed in 4formalin (pH 74) and embedded in paraffin Paraffin blockswere cut and sections of 4 120583m thickness were deparaffinizedand rehydrated in chloroform (3 times 10min) and alcohol at adecreasing concentration (2 times 100 96 and 70 for 2mineach) ending in distilled water The slides were then heatedin a microwave oven in 001 citrate buffer (pH 60) for 15minfor antigen retrievalThe endogenous peroxidase was blockedby a methanolH

2O2treatment (60mL 3H

2O2and 190mL

methanol) for 15min Tominimize nonspecific binding of theantibodies porcine serum (1 10) was applied Tissue sampleswere incubated overnight at 4∘C with the following primaryantibodies polyclonal goat anti-Shh (Biozol Eching Ger-many dilution 1 240 in TBS) polyclonal rabbit anti-Patched(Biozol Eching Germany dilution 1 50 in TBS) polyclonalrabbit anti-Smoothened (Biozol Eching Germany dilution1 200 in TBS) polyclonal rabbit anti-Gli1 (Biozol EchingGermany dilution 1 300 in TBS) polyclonal rabbit anti-Gli2 (Abcam Cambridge UK Dilution 1 300 in TBS)and monoclonal mouse anti-Gli3 (Biozol Eching Germanyclone 2C9 dilution 1 200 in TBS) antibodies After specificbinding of the primary antibodies corresponding biotiny-lated anti-mouse anti-goat or anti-rabbit F(ab1015840)

2fragments

(DAKO Hamburg Germany) were applied at a dilutionof 1 400 The peroxidase-conjugated avidin-biotin complex

Table 1 Clinical and epidemiological data

Case Week ofgestation Clinical data

1 20 Induced abortion cardiac malformation2 16 Intrauterine death collapsed amniotic sac3 14 Induced abortion preterm amniorrhexis4 12 Intrauterine death ruptured gravidity5 22 Postpartal death preterm birth6 13 Ejection7 14 Intrauterine death preterm amniorrhexis8 18 Intrauterine death preterm amniorrhexis9 22 Intrauterine death preterm amniorrhexis10 21 Intrauterine death anhydramnion

11 28 Intrauterine death twin-to-twin transfusionsyndrome

12 16 Induced abortion suspected nephronagenesis

13 14 Induced abortion on parents demand

14 13 Intrauterine death preterm abruption ofplacenta


16 19 Induced abortion anhydramnios geminigravidity


18 21 Intrauterine death chorioamnionitisplacentitis




22 26 Postpartal death preterm abruption ofplacenta

(ABC) technique (DAKO Hamburg Germany) with 331015840-diaminobenzidine (DAB Sigma Deisenhofen Germany) assubstrate was used to visualize the specific antigen binding(see Supplementary Figure 1 in the Supplementary Mate-rial available online at httpdxdoiorg1011552015494269)Finally the samples were counterstained with hematoxylinand mounted As negative controls for each antibody slideswere incubated in TBS without primary antibody (Sup-plementary Figure 1) Colon carcinoma samples served aspositive control for all antibodies (Supplementary Figure 1)

23 Scoring We analyzed fetal cerebellar and telencephaliccortical layers In the telencephalon themarginal zone (MZ)the cortical plate (CP) the subplate (SP) the intermediatezone (IZ) the subventricular zone (SVZ) and the ventric-ular zone (VZ) were investigated In the cerebellum theexternal granule layer (EGL) the molecular layer (ML) andthe internal granule layer (IGL) were assessed Intensity of

BioMed Research International 3

staining reactivity and frequency were documented by asemiquantitative score The frequency score ranged from 0to 4 meaning 0 0-1 1 1ndash10 2 10ndash25 3 25ndash50 and4 gt50 of all cells showing a positive staining Likewiseintensity was recorded in a similar semiquantitative approachas follows 0 no staining 1 weak staining 2 moderatestaining 3 strong staining (raw data of the expression scoresare given in Supplementary Tables 1 and 2) The two resultsfor intensity of reactivity and expression frequency weremultiplied so that the ldquoMulti-Scorerdquo reflects both [30] Theevaluation and photographic documentation of the immuno-histochemical staining were performed using an OlympusBX50 light microscope

24 Statistical Analyses Ordinal scaled response variable(Multi-Score) and nominal explanatory variable (brain area)were analyzed with a contingency analysis and subsequentlytested using the likelihood-ratio test Multi-Score data isgiven as box plots in the figuresThe upper and lower whiskerextend from the ends of the box to the outermost datapoint that falls within the distances computed as follows3rd quartile + 15 lowast (interquartile range) or 1st quartile minus15 lowast (interquartile range) The association of the fetal agewith the response variable (Multi-Score) was analyzed byordinal logistic regression The cumulative logistic prob-ability plot provides an overview about what the logisticmodel is fitting For each 119909 value (WOG) the probabilityscale (in the 119910 direction) is divided into probabilities foreach response category (Multi-Score) The probabilities aremeasured as the vertical distance between the blue curveswith the total across all Y category probabilities sum to 1(see httpwwwjmpcomsupportdownloadspdfjmp statgraph guidepdf) Subsequently the hypothesis that the weekof gestation has no effect on the response variables wasanalyzed using the likelihood-ratio Chi-square test A signifi-cance level of alpha= 005was chosen for all tests therefore allvalues lower than 005 were considered as being significantFor adjustment of the 119901-values due to multiple testing weused themethod of Bonferroni-Holm Statistical analysis wasperformed using JMP 801 software (SAS Cary NC USA)

3 Results

31 Strongest Expression Levels of the SHH Pathway AreDetected in the Cortical Plate and the Ventricular Zone inthe Human Fetal Telencephalon In the human fetal telen-cephalon SHH was most strongly expressed in the CP(median 8 range 2ndash12) and the VZ (median 7 range 3ndash12) while its expression in the MZ (median 4 range 2ndash6)and the IZ (median 4 range 2ndash6) was significantly lowerIntermediate Shh levels were detected in the SP (median6 range 4ndash9) showing significantly higher expression ascompared to the MZ and IZ however not considerablydiffering from the levels in the CP or VZ The SVZ (median6 range 2ndash9) only displayed lower Shh levels as comparedto the CP (Figures 1(a) 2(a) and 3(a)) In contrast Patchedexpression was homogeneously distributed over the differenttelencephalic layers only showing significantly higher levels

in the VZ (median 8 range 4ndash12) as compared to the MZ(median 6 range 2ndash12) the SVZ (median 6 range 3ndash8) andthe IZ (median 4 range 2ndash9) (Figures 1(b) 2(b) and 3(b))The expression pattern of Smoothened was similar to Shhshowing no significant difference between the CP (median6 range 2ndash12) and the SVZ (median 4 range 2ndash8) howeverlacking the significant difference between the SP (median 6range 1ndash9) and the IZ (median 4 range 1ndash6) The CP stillshowed higher levels of Smoothened as compared to the MZ(median 4 range 1ndash6) and IZ (Figures 1(c) 2(c) and 3(c))In contrast to the Shh expression pattern Gli1 was only verymoderately regulated in the human fetal cortex however theCP (median 4 range 1ndash12) still exhibited significantly higherlevels as compared to the IZ (median 15 range 1ndash6) (Figures1(d) 2(d) and 3(d))The highest expression ofGli2 was foundin the CP (median 6 range 4ndash12) and the SP (median8 range 4ndash12) The CP showed significantly stronger Gli2expression levels as compared to theMZ (median 6 range 4ndash9) IZ (median 6 range 3ndash9) and the SVZ (median 6 range2ndash6) In addition the expression of Gli2 was significantlylower in the SVZ as compared to the SP (median 8 range4ndash12) and the VZ (median 7 range 2ndash12) (Figures 1(e) 2(e)and 3(e)) Finally the analysis of Gli3 expression in the humanfetal telencephalon revealed no significant differences in thedistinct cortical layers (Figures 1(f) 2(f) and 3(f)) Shh wasthe only molecule showing a diffuse expression comprisingintra- and extracellular compartments while Patched andSmoothened exhibited a cellular localisationTheGli proteinsmostly but not exclusively displayed a nuclear staining pat-tern The expression profiles of the SHH pathway membersrather showed a gradual increase or decrease in the transitionzones between distinct cortical layers than a clear-cut changein staining intensity or frequency Since staining intensitywas only indirectly reflected by the multi-scoring system weadditionally analyzed if any specific areas showed consider-able differences in the staining intensity for the SHH pathwaymoleculesWhile the staining intensity ofmost SHH pathwaymembers remained unchanged over the telencephalic areasShh itself was most intensively expressed in the CP andthe VZ (Figures 1(a)ndash1(f) and 2(a)ndash2(f)) Detailed statisticalcomparisons are indicated in Supplementary Table 3

32 In the Human Fetal Cerebellum Strongest Expression ofthe SHH Pathway Members Is Detected in the EGL and theIGL The members of the SHH pathway showed a similarexpression pattern in human fetal cerebellar layers StrongestSHH expression was found in the EGL (median 105 range4ndash12) whereas its expression was significantly lower in theML (median 6 range 2ndash8) and in the IGL (median 6 range4ndash9) The IGL also showed significantly stronger Shh levelsas compared to the ML (Figures 4(a) and 5(a)) SimilarlyPatched expression in the EGL (median 8 range 4ndash12) wasalso significantly stronger than in the ML (median 6 range4ndash8) and the IGL (median 6 range 3ndash12) Additionallythe expression of Patched in the IGL was significantlystronger as compared to the ML (Figures 4(b) and 5(b)) TheSmoothened expression pattern was similar to Shh showingsignificantly stronger levels in the EGL (median 8 range4ndash12) than in the ML (median 4 range 2ndash6) or the IGL


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SP

IZShh

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(c)

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(d)

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(e)

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Figure 1 Expression pattern of SHH pathway members in the normal human fetal cortex Immunohistochemistry for (a) Shh (b) Patched(c) Smoothened (d) Gli1 (e) Gli2 and (f) Gli3 in superficial telencephalic cortical layers including MZ (marginal zone) CP (cortical plate)SP (subplate) and IZ (intermediate zone) (examples are depicted from a 14th week of gestation fetus original magnification 200x)

(median 5 range 1ndash9) (Figures 4(c) and 5(c)) Gli1 was moststrongly expressed in the EGL (median 6 range 1ndash12) andIGL (median 6 range 2ndash12) while its expression in the ML(median 2 range 2ndash12) was significantly lower as comparedto both of them (Figures 4(d) and 5(d)) Gli2 expression wassignificantly stronger in the EGL (median 12 range 4minus12)as compared to the ML (median 6 range 4ndash9) and theIGL (median 8 range 4ndash12) In addition the ML showedsignificantly lowerGli2 levels as compared to the IGL (Figures4(e) and 5(e)) Gli3 expression was significantly stronger inthe EGL (median 9 range 6ndash12) as compared to the ML(median 6 range 4ndash12) and the IGL (median 6 range 4ndash12) (Figures 4(f) and 5(f)) In contrast to the findings in thetelencephalic layers a less gradual but more abrupt change

in the expression of SHH pathway molecules was observed(Figures 4(a)ndash4(f)) Furthermore no considerable changes inthe staining intensity could be detected for the SHH pathwaymolecules over the fetal cerebellar layers (Figures 4(a)ndash4(f))Detailed statistical comparisons are given in SupplementaryTable 4

33 Shh Pathway Expression Increases in Superficial Telen-cephalic Layers but Decreases in Periventricular Zones with theWeek of Gestation While in the MZ no significant changeswere found for all molecules of the SHH pathway in the CPonlyGli2 expression significantly increased (119901 = 00489)withthe week of gestation (Figure 6(a)) A significantly increasingexpression was found for both Patched (119901 = 00003) and


VZSVZ

IZ

Shh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)

Figure 2 Expression pattern of SHH pathway members in the normal human fetal cortex Immunohistochemistry for (a) Shh (b) Patched(c) Smoothened (d) Gli1 (e) Gli2 and (f) Gli3 in the internal telencephalic layers including IZ (intermediate zone) SVZ (subventricularzone) and VZ (ventricular zone) (examples are depicted from a 14th week of gestation fetus original magnification 200x)

Gli1 (119901 = 00161) in the IZ (Figures 6(b) and 6(c)) Incontrast to the increasing expression profiles of the Shhpathway members in the superficial telencephalic layerstheir expression levels decreased over time in periventricularregions While in the SVZ only Patched (119901 = 00018)expression levels significantly decreased during pregnancy(Figure 6(d)) 4 out of the 6 investigated molecules of theShh pathway showed a significant decreased expression in theVZ Shh (119901 = 00025) Patched (119901 = 00035) Smoothened(119901 = 00321) and Gli2 (119901 = 00066) (Figures 6(e)ndash6(h))

34 In the Human Fetal Cerebellum SHH Pathway ActivationDecreases with the Week of Gestation In the EGL of thehuman fetal cerebellum a significant decrease in both Shh(119901 = 0044) and Gli1 (119901 = 00031) expression was seen

over time while the other molecules remained unchanged(Figures 7(a) and 7(b)) In the ML only Gli1 (119901 = 00013)was significantly downregulated over time whereas all otherfactors displayed a stable expression (Figure 7(c)) In the IGLno considerable expression changes were observed over timeIn later stages of the fetal cerebellar development especiallydifferentiating Purkinje cells exhibited an increased expres-sion of most SHH pathway molecules while in other layerssuch as the EGL Shh and Gli1 were decreased (Figure 8)

4 Discussion

41 The Potential Role of the SHH Pathway in the Controlof Progenitor Cell Survival and Neuronal Differentiation inthe DevelopingHuman Fetal Telencephalon TheHh signaling


MZ CP SCZ IZ SVZ VZ

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Figure 3 SHH pathway members are differentially expressed in the developing human telencephalon Box plots of Multi-Score expressionvalues in different telencephalic areas are depicted for (a) Shh (b) Patched (c) Smoothened (d) Gli1 (e) Gli2 and (f) Gli3 For each factor119899 = 22 samples were assessed and expression values compared by a contingency analysis followed by the likelihood-ratio test (lowast119901 lt 005lowastlowast119901 lt 001 lowastlowastlowast119901 lt 00001)


IGL

ML

EGL

Shh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)

Figure 4 Expression patterns of SHH pathway members in the normal human fetal cerebellum Immunohistochemistry for (a) Shh (b)Patched (c) Smoothened (d) Gli1 (e) Gli2 and (f) Gli3 in the cerebellum including EGL (external granular layer) ML (molecular layer) andIGL (internal granular layer) (examples are depicted from a 18th week of gestation fetus original magnification 200x)

pathway is conserved among different species and playsa major role in the development of various organs andcell types [1] In our study we provide evidence that SHHpathway members are expressed in a specific manner in thedifferent layers of the human telencephalon In particularShh Smoothened and Gli2 showed increasing levels fromMZ to CP and decreasing levels from SP to IZ Strongestexpression of these factors was seen in the CP and VZPatched also showed high expression levels in MZ CP andVZ but low expression in the IZ and SVZ Concerning thetemporal expression pattern Shh Smoothened Gli2 andPatched levels decreased in the VZwith the week of gestationIn contrast in the CP Gli2 is the only molecule of the SHHsignaling pathway which increased during pregnancy

One of the first steps in CNS development is the prolif-eration of precursor cells and the differentiation of neuronalprogenitors into subclasses of mature neurons [31] In thezebrafish Shh is essential for the development of the time-dependent formation of the optical tectum [32] In additionin higher vertebrates Shh regulates the proliferation of neuralprecursor cells especially in the developing VZSVZ [33 34]This goes along with our findings of high expression levelsof the SHH pathway members especially in the VZSVZThe findings that highest expression levels of Shh and itspathway members were found in the VZ and decreasedwith the week of gestation might be related to the highproliferative capacity of this brain area at very early stages ofCNS development Further evidence for the important role


lowast

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Figure 5 SHH pathway members are differentially expressed in the developing human cerebellum Box plots of Multi-Score expressionvalues in different cerebellar areas are depicted for (a) Shh (b) Patched (c) Smoothened (d) Gli1 (e) Gli2 and (f) Gli3 For each factor 119899 = 22samples were assessed and expression values compared by a contingency analysis followed by the likelihood-ratio test (lowast119901 lt 005 lowastlowast119901 lt 001lowastlowastlowast119901 lt 00001)


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Figure 6 Continued


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Figure 6 SHH pathway members are differentially regulated during gestation in the developing human telencephalon (andashh) Areas showinga significant association of fetal age (week of gestation WOG) with the response variable (Multi-Score) were analyzed by ordinal logisticregression followed by the likelihood-ratio Chi-square test 119901-values are indicated accordingly The cumulative logistic probability plotprovides an overview about what the logistic model is fitting For each 119909 value (WOG) the probability scale (in the 119910 direction) is partitionedinto probabilities for each response category (Multi-Score) The probabilities are measured as the vertical distance between the blue curveswith the total across all Y category probabilities sum to 1

of a fine-tuned SHH pathway activation in progenitor cellcycle regulation is provided by findings in transgenic Shh-overexpressing mice that develop hyperplasia of the spinalcord and the VZ during embryogenesis [34] On the otherhand Palma et al showed that a selective inhibition of Shhleads to a significant decrease in the size of the murineSVZ [35] In contrast a downregulation of the Shh signalingis important to slow down cell division and further allowsthe cells to exit the cell cycle which is necessary for thetransition to nondividing neurons migrating away from theproliferation zones [36] The hypothesis that murine radialglial cells (RGC) intermediate progenitor cells (IPC) andmature neurons in the SVZ need low Shh levels to regulatetheir generation proliferation and cellular migration is alsocorroborated by our locoregional distribution pattern in thehuman fetal brain showing strongest downregulation of theSHH pathway between the SVZ and the IZ [36]

42 Expression of Gli Family Members in the Fetal Telen-cephalon Is Associated with Areas Involved in Proliferationand Differentiation The pattern of Gli1 and Gli2 expressionis strikingly similar to other data concerning murine telen-cephalon development where Gli1 expression is induced byShh and therefore also detected in close proximity to theShh expression signal [13] However previous studies withGli1minusminus mouse mutants revealed no detectable deficits incortical development [37] This leads to the assumption thatthere is no isolated function of Gli1 regarding proliferation ormigration In contrast Gli2minusminusmousemutants demonstrateda decrease in proliferation mainly in otherwise highlyproliferative cortical layers [38] This data indicates that Gli2might be a more important activating factor in the SHH

pathway cascade which goes well along with our findingsof almost identical expression pattern for Shh and Gli2Although the expression profile of Gli3 moderately resemblestheGli1 andGli2 signature no significant differences betweenthe developing telencephalic layers were observed Howeverit remains to be determined if Gli3 exerts a more inhibitoryor activating function during human fetal brain development[39] Different studies showed a correlation of Gli3 activa-tion and cell proliferation resulting in significantly smallerneocortical layers in Gli3minusminus mice [38 40] InterestinglyGli3SHH double mutants displayed a rescue of the brainmalformation caused by SHH single mutants [24 41] Inaddition many severe malformations occurring in Shh singlemutants are restored in embryos simultaneously lacking Gli3and Shh function [22] Therefore Shh may modulate theactivating and inhibitory function of Gli3 so that in theabsence of Shh Gli3 will be processed to the repressor formThis indicates that Gli3 alone or the balances of both proteinsare required for the normal fetal brain development in thetelencephalon [42]

43 The Temporal Expression Pattern of the SHH Pathwayin the Human Fetal Telencephalon Indicates a DichotomousRole in Proliferation and Migration of Progenitor Cells Inthe normal developing human fetal brain neuronal precursorcells migrate radially away from the proliferation zones(VZSVZ) to the surface of the brain leading to the formationof the typical six-layered neocortex [43]Therefore migratoryprocesses in the developing fetal telencephalon are closelyassociated with cell cycle exit of neuronal progenitor cells[44] In contrast to the decrease of the proliferative activityin the VZSVZ the proliferation in the IZ and the SP


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(c)

Figure 7 SHH pathway members are differentially regulated during gestation in the developing human cerebellum (andashc) Areas showinga significant association of fetal age (week of gestation WOG) with the response variable (Multi-Score) were analyzed by ordinal logisticregression followed by the likelihood-ratio Chi-square test 119901-values are indicated accordingly The cumulative logistic probability plotprovides an overview about what the logistic model is fitting For each 119909 value (WOG) the probability scale (in the 119910 direction) is partitionedinto probabilities for each response category (Multi-Score) The probabilities are measured as the vertical distance between the blue curveswith the total across all Y category probabilities sum to 1

increases until the end of the last pregnancy trimester [45]Accordingly we found increasing expression levels of Patchedand Gli1 in the IZ with the week of gestation While strongexpression levels of all SHH pathwaymembers were observedin the CP only Gli2 increased significantly with the week ofgestation One possible explanation for the constantly highexpression levels of the SHH pathway members in the CPcould be that the proliferation activity already starts in the8th week of gestation while our study could only investigatehuman fetal brains deriving from the 12th until the 28th weekof gestation It is well known that neocortical migration andlaminar organization not only depends on radial migrationAngot et al provided evidence that Shh is a chemoattractivemolecule acting on neuronal progenitors in the adult SVZ

which in turn change their migratory behaviour towards anectopic Shh expression [46] This might be related to ourfindings showing constantly high expression levels of theSHH pathway members in the CP but decreasing levels inVZSVZ over time Komada and colleagues showed that inwild-type mice the early-born neurons form the deeper layerand the late-born neurons are the source for the superficiallayers of the CP In contrast in SMO knockout mice lackingnormal Shh signalling the early-born neurons were diffuselyspread over the dorsal telencephalon and the late-born didnot even migrate to the CP [36] Again this illustrates theimportance of a coordinate temporal regulation of the cellcycle and a successful migrationlamination process bothdepending on a well-functioning SHH pathway


Shh

(a)

Shh

(b)

Purkinje cell layer

EGL

MLIGL

Shh

(c)

Patched

(d)

Patched

(e)

Patched

(f)

Smoothened

(g)

Smoothened

(h)

Smoothened

(i)

Gli1

(j)

Gli1

(k)

Gli1

(l)

Gli2

(m)

Gli2

(n)

Gli2

(o)

Figure 8 Continued


Gli3

(p)

Gli3

(q)

Gli3

(r)

Figure 8 Temporal expression patterns of SHH pathway members in the normal human fetal cerebellum Immunohistochemistry for (a bc) Shh (d e f) Patched (g h i) Smoothened (j k l) Gli1 (m n o) Gli2 and (p q r) Gli3 showing examples of the cerebellar layers includingEGL (external granular layer) ML (molecular layer) and IGL (internal granular layer) (a d g j m p) 12th week of gestation (b e h k nq) 19th week of gestation and (c f i l o r) 28th week of gestation original magnification 200x

44 Temporospatial Changes in the Sources of Shh Expressionin Human Cerebellar Development Are Related to Prolifer-ation Migration and Differentiation Processes The humancerebellum consists of a three-layered structure of whichthe outermost external granular layer (EGL) is formed bycerebellar precursor cells invading from the rhombic lip andthen leading to the formation of the proliferating granule cellsof the expanding EGL [47] After proliferation these cells exitthe cell cycle and migrate along the Bergman glia to deeperlayers where they form the inner granular layer (IGL) [21 4748] The formation of the IGL lasts until several months afterbirth in the human brain [49]The SHH pathway is supposedto play a major role not only in proliferation of the granulecells but also in other precursor cell types in the developingCNS [48] In mice the blocking of the Shh signaling resultsin a reduced proliferation of the granule precursors cells inthe EGL in vivo [48] and in vitro [21]These findings indicatethat Shh secreted by EGL or Purkinje neurons is necessaryfor the proliferation and migration of the granule precursorcells [50] The high Shh levels in the EGL might confirm thehypothesis that Shh plays a role as enhancer of proliferationof granule precursor cells indicating a similar activity of Shhin the human and murine fetal cerebellum [21 51] Besidethe expression of Shh in the human EGL we also detectedhigh Shh levels in the IGL while being considerably lowerin the ML To stop cell division and allow an exit of the cellcycle for example to migrate a lowering of the Shh signalingis required [36] With regard to the temporal distributionpattern Shh significantly decreased between the 12th and28th week of gestation in the EGL potentially reflecting areduction in cellular proliferation and migration to the IGLOur results confirm recently published data showing a strongShh expression in the EGL between the 10th and 20th weekof gestation [52] In our cohort Shh is still present in theIGL until the 28th week of gestation and is not considerablydownregulated if compared to the EGL This could be due tothe fact that Shh downregulation might take place at the timeof birth or soon after in humans [52] On the other hand itis known that Shh plays a role in cell differentiation which isnecessary in the IGL after the immigration of granule cells[53] Shh is further present in the human EGL until week

17 and decreases after the 20th week when Purkinje neuronsform a monolayer suggesting the Purkinje neurons as thesource of the Shh signaling [52]

Additionally our investigation of the other members ofthe SHH pathway in the developing cerebellum revealedstrongest expression levels in the EGL and IGL suggestinga SHH pathway activation in these respective cerebellarareas In particular the expression of Gli1 which can betranscriptionally activated by SHH [13] and therefore be usedas a surrogate parameter for Shh activity [53] decreases inthe EGL with the week of gestation indicating an inhibitionof the Shh pathway a finding which was also seen in mouseembryos [21] In contrast to Patched Smoothened Gli2 orGli3 Gli1 also showed lower expression levels in the MLover the time of pregnancy The ongoing expression of SHHpathway members in other layers compared to the EGLsuggested that there has to be another source for the Shhexpression in the developing cerebellum Purkinje cells seemto be a potential cell type because these cells require intact Shhsignaling for proper development and maturation a processthat lasts for several months after birth [21] We could alsoconfirm that especially differentiating Purkinje cells show anincreased expression of Shh also in later stages of the humanfetal cerebellar development while other layers such as theEGL showed decreased Shh levels Concerning CNS tumorsthe medulloblastoma represents the most frequent malignantbrain tumor of the childhood and is currently classified at amolecular level indicating Shh pathway disturbances as oneof the key players for tumor development [54] Additionallyit is predicted that the source of medulloblastoma subtypes isthe granule neural precursor cells in the EGL being unable toexit the cell cycle due to overactivated Shh signaling [55]

5 Conclusion

In our study we show that the SHH pathway is active duringdifferent stages of the normal development in the differentlayers of the human brain Thus we could provide evidencethat a fine-tuned temporal Shh signaling is necessary fornormal human CNS development


Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Julia Tichy Richard Meyermann Patrick N Harter andMichelMittelbronn conceived and designed the experimentsJulia Tichy Benedikt Bunz Patrick N Harter and MichelMittelbronn performed the experiments Julia Tichy JennyZinke Benedikt Bunz Patrick N Harter and Michel Mit-telbronn analysed the data Richard Meyermann and MichelMittelbronn contributed reagents materials and financialsupport Julia Tichy Jenny Zinke Patrick N Harter andMichel Mittelbronn wrote the paper Michel Mittelbronnis the supervisor of the study All authors corrected andapproved the final version of the paper

References

[1] P W Ingham and A P McMahon ldquoHedgehog signaling inanimal development paradigms and principlesrdquo Genes andDevelopment vol 15 no 23 pp 3059ndash3087 2001

[2] N C Bambakidis M Petrullis X Kui et al ldquoImprovement ofneurological recovery and stimulation of neural progenitor cellproliferation by intrathecal administration of Sonic hedgehogrdquoJournal of Neurosurgery vol 116 no 5 pp 1114ndash1120 2012

[3] F Balordi andG Fishell ldquoHedgehog signaling in the subventric-ular zone is required for both the maintenance of stem cells andthe migration of newborn neuronsrdquo Journal of Neurosciencevol 27 no 22 pp 5936ndash5947 2007

[4] M H Elamin Z Shinwari S F Hendrayani et al ldquoCurcumininhibits the sonic hedgehog signaling pathway and triggersapoptosis in medulloblastoma cellsrdquo Molecular Carcinogenesisvol 49 no 3 pp 302ndash314 2010

[5] L Hayes S Ralls H Wang and S Ahn ldquoDuration of Shhsignaling contributes to mDA neuron diversityrdquo DevelopmentalBiology vol 374 no 1 pp 115ndash126 2013

[6] A Po E Ferretti EMiele et al ldquoHedgehog controls neural stemcells through p53-independent regulation ofNanogrdquoTheEMBOJournal vol 29 no 15 pp 2646ndash2658 2010

[7] Y Echelard D J Epstein B St-Jacques et al ldquoSonic hedgehoga member of a family of putative signaling molecules isimplicated in the regulation of CNS polarityrdquo Cell vol 75 no7 pp 1417ndash1430 1993

[8] J Mohler ldquoRequirements for hedgehod a segmental polaritygene in patterning larval and adult cuticle of DrosophilardquoGenetics vol 120 no 4 pp 1061ndash1072 1988

[9] C Nusslein-Volhard and E Wieschaus ldquoMutations affectingsegment number and polarity in Drosophilardquo Nature vol 287no 5785 pp 795ndash801 1980

[10] J TaipaleMK Cooper TMaiti and PA Beachy ldquoPatched actscatalytically to suppress the activity of Smoothenedrdquo Naturevol 418 no 6900 pp 892ndash897 2002

[11] MMurone A Rosenthal and F J de Sauvage ldquoSonic hedgehogsignaling by the patched-smoothened receptor complexrdquo Cur-rent Biology vol 9 no 2 pp 76ndash84 1999

[12] M Wickstrom C Dyberg T Shimokawa et al ldquoTargetingthe hedgehog signal transduction pathway at the level ofGLI inhibits neuroblastoma cell growth in vitro and in vivordquo

International Journal of Cancer vol 132 no 7 pp 1516ndash15242013

[13] J Lee K A Platt P Censullo and A Ruiz I Altaba ldquoGli1 isa target of Sonic hedgehog that induces ventral neural tubedevelopmentrdquoDevelopment vol 124 no 13 pp 2537ndash2552 1997

[14] A Ruiz i Altaba ldquoGli proteins encode context-dependentpositive and negative functions implications for developmentand diseaserdquo Development vol 126 no 14 pp 3205ndash3216 1999

[15] PDaiHAkimaru Y Tanaka TMaekawaMNakafuku and SIshii ldquoSonic hedgehog-induced activation of the Gli1 promoteris mediated by GLI3rdquo The Journal of Biological Chemistry vol274 no 12 pp 8143ndash8152 1999

[16] A Aguilar A Meunier L Strehl et al ldquoAnalysis of humansamples reveals impaired SHH-dependent cerebellar develop-ment in Joubert syndromeMeckel syndromerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 109 no 42 pp 16951ndash16956 2012

[17] B Balczerski S Zakaria A S Tucker et al ldquoDistinct spatiotem-poral roles of hedgehog signalling during chick and mousecranial base and axial skeleton developmentrdquo DevelopmentalBiology vol 371 no 2 pp 203ndash214 2012

[18] Y H Belgacem and L N Borodinsky ldquoSonic hedgehog signal-ing is decoded by calcium spike activity in the developing spinalcordrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 108 no 11 pp 4482ndash4487 2011

[19] J Mao B-M Kim M Rajurkar R A Shivdasani and A PMcMahon ldquoHedgehog signaling controls mesenchymal growthin the developingmammalian digestive tractrdquoDevelopment vol137 no 10 pp 1721ndash1729 2010

[20] S Odent T Attie-Bitach M Blayau et al ldquoExpression of theSonic hedgehog (SHH) gene during early human developmentand phenotypic expression of newmutations causing holopros-encephalyrdquo Human Molecular Genetics vol 8 no 9 pp 1683ndash1689 1999

[21] N Dahmane and A Ruiz i Altaba ldquoSonic hedgehog regulatesthe growth and patterning of the cerebellumrdquoDevelopment vol126 no 14 pp 3089ndash3100 1999

[22] K Aoto Y Shikata H Imai et al ldquoMouse Shh is requiredfor prechordal plate maintenance during brain and craniofacialmorphogenesisrdquoDevelopmental Biology vol 327 no 1 pp 106ndash120 2009

[23] M Kool A Korshunov M Remke et al ldquoMolecular subgroupsof medulloblastoma an international meta-analysis of tran-scriptome genetic aberrations and clinical data of WNT SHHGroup 3 and Group 4 medulloblastomasrdquo Acta Neuropatholog-ica vol 123 no 4 pp 473ndash484 2012

[24] C-C Hui D Slusarski K A Platt R Holmgren and A LJoyner ldquoExpression of three mouse homologs of theDrosophilasegment polarity gene cubitus interruptus Gli Gli-2 and Gli-3in ectoderm- and mesoderm-derived tissues suggests multipleroles during postimplantation developmentrdquo DevelopmentalBiology vol 162 no 2 pp 402ndash413 1994

[25] A Jamsheer A Sowinska T Trzeciak M Jamsheer-Bratkow-ska A Geppert and A Latos-Bielenska ldquoExpandedmutationalspectrum of the GLI3 gene substantiates genotype-phenotypecorrelationsrdquo Journal of Applied Genetics vol 53 no 4 pp 415ndash422 2012

[26] M Kubo M Nakamura A Tasaki et al ldquoHedgehog signalingpathway is a new therapeutic target for patients with breastcancerrdquo Cancer Research vol 64 no 17 pp 6071ndash6074 2004

[27] J Reifenberger M Wolter C B Knobbe et al ldquoSomaticmutations in the PTCH SMOH SUFUH and TP53 genes in


sporadic basal cell carcinomasrdquo British Journal of Dermatologyvol 152 no 1 pp 43ndash51 2005

[28] PGibson Y TongG Robinson et al ldquoSubtypes ofmedulloblas-toma have distinct developmental originsrdquoNature vol 468 no7327 pp 1095ndash1099 2010

[29] S Matsuo M Takahashi K Inoue et al ldquoThickened areaof external granular layer and Ki-67 positive focus are earlyevents ofmedulloblastoma in Ptch1+minusmicerdquoExperimental andToxicologic Pathology vol 65 no 6 pp 863ndash873 2013

[30] P N Harter B Bunz K Dietz K Hoffmann R Meyermannand M Mittelbronn ldquoSpatio-temporal deleted in colorectalcancer (DCC) and netrin-1 expression in human foetal braindevelopmentrdquo Neuropathology and Applied Neurobiology vol36 no 7 pp 623ndash635 2010

[31] T M Jessell ldquoNeuronal specification in the spinal cord induc-tive signals and transcriptional codesrdquoNature Reviews Geneticsvol 1 no 1 pp 20ndash29 2000

[32] C G Feijoo M G Onate L A Milla and V A PalmaldquoSonic hedgehog (Shh)-Gli signaling controls neural progenitorcell division in the developing tectum in zebrafishrdquo EuropeanJournal of Neuroscience vol 33 no 4 pp 589ndash598 2011

[33] R Machold S Hayashi M Rutlin et al ldquoSonic hedgehog isrequired for progenitor cell maintenance in telencephalic stemcell nichesrdquo Neuron vol 39 pp 937ndash950 2003

[34] D H Rowitch B S-Jacques SM K Lee J D Flax E Y Snyderand A P McMahon ldquoSonic hedgehog regulates proliferationand inhibits differentiation of CNS precursor cellsrdquo Journal ofNeuroscience vol 19 no 20 pp 8954ndash8965 1999

[35] V Palma D A Lim N Dahmane et al ldquoSonic hedgehogcontrols stem cell behavior in the postnatal and adult brainrdquoDevelopment vol 132 no 2 pp 335ndash344 2005

[36] M Komada H Saitsu M Kinboshi T Miura K Shiota andM Ishibashi ldquoHedgehog signaling is involved in developmentof the neocortexrdquo Development vol 135 no 16 pp 2717ndash27272008

[37] H L Park C Bai K A Platt et al ldquoMouse Gli1 mutants areviable but have defects in SHH signaling in combination witha Gli2 mutationrdquo Development vol 127 no 8 pp 1593ndash16052000

[38] V Palma and A Ruiz I Altaba ldquoHedgehog-GLI signalingregulates the behavior of cells with stem cell properties in thedeveloping neocortexrdquoDevelopment vol 131 no 2 pp 337ndash3452004

[39] Y-S Kim G Nakanishi M Lewandoski and A M JettenldquoGLIS3 a novel member of the GLIS subfamily of Kruppel-likezinc finger proteins with repressor and activation functionsrdquoNucleic Acids Research vol 31 no 19 pp 5513ndash5525 2003

[40] T Franz ldquoExtra toes (Xt) homozygous mutant mice demon-strate a role for the Gli-3 gene in the development of theforebrainrdquo Acta Anatomica vol 150 no 1 pp 38ndash44 1994

[41] Y Litingtung and C Chiang ldquoSpecification of ventral neurontypes is mediated by an antagonistic interaction between ShhandGli3rdquoNature Neuroscience vol 3 no 10 pp 979ndash985 2000

[42] J Motoyama L Milenkovic M Iwama Y Shikata M P Scottand C-C Hui ldquoDifferential requirement for Gli2 and Gli3 inventral neural cell fate specificationrdquo Developmental Biologyvol 259 no 1 pp 150ndash161 2003

[43] O Marın and J L R Rubenstein ldquoCell migration in theforebrainrdquo Annual Review of Neuroscience vol 26 pp 441ndash4832003

[44] V S Caviness Jr R S Nowakowski and P G Bhide ldquoNeocorti-cal neurogenesis morphogenetic gradients and beyondrdquoTrendsin Neurosciences vol 32 no 8 pp 443ndash450 2009

[45] A Kendler and J A Golden ldquoProgenitor cell proliferationoutside the ventricular and subventricular zones during humanbrain developmentrdquo Journal of Neuropathology amp ExperimentalNeurology vol 55 no 12 pp 1253ndash1258 1996

[46] E Angot K Loulier K T Nguyen-Ba-Charvet A-P GadeauM Ruat and E Traiffort ldquoChemoattractive activity of SonicHedgehog in the adult subventricular zone modulates thenumber of neural precursors reaching the olfactory bulbrdquo StemCells vol 26 no 9 pp 2311ndash2320 2008

[47] H J ten Donkelaar M Lammens P Wesseling H O MThijssen and W O Renier ldquoDevelopment and developmentaldisorders of the human cerebellumrdquo Journal of Neurology vol250 no 9 pp 1025ndash1036 2003

[48] R J Wechsler-Reya and M P Scott ldquoControl of neuronalprecursor proliferation in the cerebellum by sonic hedgehogrdquoNeuron vol 22 no 1 pp 103ndash114 1999

[49] P Rakic and R L Sidman ldquoHistogenesis of cortical layers inhuman cerebellum particularly the lamina dissecansrdquo Journalof Comparative Neurology vol 139 no 4 pp 473ndash500 1970

[50] Y Choi P R Borghesani J A Chan andR A Segal ldquoMigrationfrom a mitogenic niche promotes cell-cycle exitrdquoThe Journal ofNeuroscience vol 25 no 45 pp 10437ndash10445 2005

[51] V A Wallace ldquoPurkinje-cell-derived Sonic hedgehog regulatesgranule neuron precursor cell proliferation in the developingmouse cerebellumrdquo Current Biology vol 9 no 8 pp 445ndash4481999

[52] P Haldipur U Bharti S Govindan et al ldquoExpression of sonichedgehog during cell proliferation in the human cerebellumrdquoStem Cells and Development vol 21 no 7 pp 1059ndash1068 2012

[53] J D Corrales G L Rocco S Blaess Q Guo and A L JoynerldquoSpatial pattern of sonic hedgehog signaling through Gli genesduring cerebellum developmentrdquo Development vol 131 no 22pp 5581ndash5590 2004

[54] P A Northcott A Korshunov HWitt et al ldquoMedulloblastomacomprises four distinct molecular variantsrdquo Journal of ClinicalOncology vol 29 no 11 pp 1408ndash1414 2011

[55] J Buhren A H A Christoph R Buslei S Albrecht OD Wiestler and T Pietsch ldquoExpression of the neurotrophinreceptor p75119873119879119877 inmedulloblastomas is correlated with distincthistological and clinical features evidence for a medulloblas-toma subtype derived from the external granule cell layerrdquoJournal of Neuropathology amp Experimental Neurology vol 59no 3 pp 229ndash240 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


[20 22] a deletion of the Gli3 gene may result in the Greigcephalopolysyndactyly syndrome [24 25] On the other handan increased activation of the SHH pathway may lead toneoplastic transformation of various tissues including breast[26] skin [27] and brain [21] Most interestingly neoplasmsassociated with mutations of the SHH pathway occur in areaswhere SHH is essential for the regulation of cellular stemnessand differentiation such as in the cerebellum the brainarea where medulloblastomas emerge [28 29] Although theexpression of the molecules of the SHH pathway in humansis very well studied under pathological conditions there is alack of knowledge concerning the spatiotemporal expressionpattern in the normal developing human CNS The aim ofour study was to determine the nonpathological expressionpattern of the SHH pathway members in a cohort of normalhuman fetal brains at different stages of gestation to gaininsights into the role of the SHH pathway in normal CNSdevelopment

2 Materials and Methods

21 Clinical Data and Tissue Specimens We investigated 22human fetal brains from the 12th to the 28thweek of gestation(Table 1) For the present study a cohort of fetuses with-out pathological CNS alterations was selected according toclinical data family history and neuropathological analysesperformed by two experienced neuropathologists (MM andRM) The samples belong to the human tissue brain bank ofthe Institute of Pathology and Neuropathology University ofTuebingenGermany [30] All tissue specimenswere paraffin-embedded The study protocol was endorsed by the ethicalcommittee of the University of Tuebingen Germany

22 Immunohistochemistry All samples were fixed in 4formalin (pH 74) and embedded in paraffin Paraffin blockswere cut and sections of 4 120583m thickness were deparaffinizedand rehydrated in chloroform (3 times 10min) and alcohol at adecreasing concentration (2 times 100 96 and 70 for 2mineach) ending in distilled water The slides were then heatedin a microwave oven in 001 citrate buffer (pH 60) for 15minfor antigen retrievalThe endogenous peroxidase was blockedby a methanolH

2O2treatment (60mL 3H

2O2and 190mL

methanol) for 15min Tominimize nonspecific binding of theantibodies porcine serum (1 10) was applied Tissue sampleswere incubated overnight at 4∘C with the following primaryantibodies polyclonal goat anti-Shh (Biozol Eching Ger-many dilution 1 240 in TBS) polyclonal rabbit anti-Patched(Biozol Eching Germany dilution 1 50 in TBS) polyclonalrabbit anti-Smoothened (Biozol Eching Germany dilution1 200 in TBS) polyclonal rabbit anti-Gli1 (Biozol EchingGermany dilution 1 300 in TBS) polyclonal rabbit anti-Gli2 (Abcam Cambridge UK Dilution 1 300 in TBS)and monoclonal mouse anti-Gli3 (Biozol Eching Germanyclone 2C9 dilution 1 200 in TBS) antibodies After specificbinding of the primary antibodies corresponding biotiny-lated anti-mouse anti-goat or anti-rabbit F(ab1015840)

2fragments

(DAKO Hamburg Germany) were applied at a dilutionof 1 400 The peroxidase-conjugated avidin-biotin complex

Table 1 Clinical and epidemiological data

Case Week ofgestation Clinical data

1 20 Induced abortion cardiac malformation2 16 Intrauterine death collapsed amniotic sac3 14 Induced abortion preterm amniorrhexis4 12 Intrauterine death ruptured gravidity5 22 Postpartal death preterm birth6 13 Ejection7 14 Intrauterine death preterm amniorrhexis8 18 Intrauterine death preterm amniorrhexis9 22 Intrauterine death preterm amniorrhexis10 21 Intrauterine death anhydramnion

11 28 Intrauterine death twin-to-twin transfusionsyndrome

12 16 Induced abortion suspected nephronagenesis

13 14 Induced abortion on parents demand









22 26 Postpartal death preterm abruption ofplacenta

(ABC) technique (DAKO Hamburg Germany) with 331015840-diaminobenzidine (DAB Sigma Deisenhofen Germany) assubstrate was used to visualize the specific antigen binding(see Supplementary Figure 1 in the Supplementary Mate-rial available online at httpdxdoiorg1011552015494269)Finally the samples were counterstained with hematoxylinand mounted As negative controls for each antibody slideswere incubated in TBS without primary antibody (Sup-plementary Figure 1) Colon carcinoma samples served aspositive control for all antibodies (Supplementary Figure 1)

23 Scoring We analyzed fetal cerebellar and telencephaliccortical layers In the telencephalon themarginal zone (MZ)the cortical plate (CP) the subplate (SP) the intermediatezone (IZ) the subventricular zone (SVZ) and the ventric-ular zone (VZ) were investigated In the cerebellum theexternal granule layer (EGL) the molecular layer (ML) andthe internal granule layer (IGL) were assessed Intensity of




3 Results





MZ CP

SP

IZShh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)






VZSVZ

IZ

Shh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)





4 Discussion



MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

(b)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowastlowast

(d)

lowastlowast


lowastlowastlowast

lowastlowastlowast

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(e)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(f)



IGL

ML

EGL

Shh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)





lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(a)

lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(b)

lowastlowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(c)

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(d)

lowastlowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(e)

lowastlowast

lowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(f)



000

025

050

075

100

10 15 20 25 30

46

8

9

12

WOG

Mul

ti-Sc

ore

CP Gli2 p = 00489

(a)

4

3

6

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

46

3

2

1

IZ Gli1 p = 00161

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(c)

6

4

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(d)

34

6

8

9

12

VZ Shh p = 00025

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(e)

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(f)

Figure 6 Continued


2

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(g)

2

4

6

8

12

9


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

(h)







000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

4

6

8

9

12

EGL Shh p = 0044

(a)

1

2

3

4

6

9

12

EGL Gli1 p = 00031

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

1

2

4

6

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore


(c)





Shh

(a)

Shh

(b)

Purkinje cell layer

EGL

MLIGL

Shh

(c)

Patched

(d)

Patched

(e)

Patched

(f)

Smoothened

(g)

Smoothened

(h)

Smoothened

(i)

Gli1

(j)

Gli1

(k)

Gli1

(l)

Gli2

(m)

Gli2

(n)

Gli2

(o)

Figure 8 Continued


Gli3

(p)

Gli3

(q)

Gli3

(r)





5 Conclusion







References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















3 Results





MZ CP

SP

IZShh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)






VZSVZ

IZ

Shh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)





4 Discussion



MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

(b)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowastlowast

(d)

lowastlowast


lowastlowastlowast

lowastlowastlowast

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(e)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(f)



IGL

ML

EGL

Shh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)





lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(a)

lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(b)

lowastlowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(c)

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(d)

lowastlowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(e)

lowastlowast

lowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(f)



000

025

050

075

100

10 15 20 25 30

46

8

9

12

WOG

Mul

ti-Sc

ore

CP Gli2 p = 00489

(a)

4

3

6

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

46

3

2

1

IZ Gli1 p = 00161

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(c)

6

4

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(d)

34

6

8

9

12

VZ Shh p = 00025

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(e)

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(f)

Figure 6 Continued


2

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(g)

2

4

6

8

12

9


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

(h)







000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

4

6

8

9

12

EGL Shh p = 0044

(a)

1

2

3

4

6

9

12

EGL Gli1 p = 00031

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

1

2

4

6

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore


(c)





Shh

(a)

Shh

(b)

Purkinje cell layer

EGL

MLIGL

Shh

(c)

Patched

(d)

Patched

(e)

Patched

(f)

Smoothened

(g)

Smoothened

(h)

Smoothened

(i)

Gli1

(j)

Gli1

(k)

Gli1

(l)

Gli2

(m)

Gli2

(n)

Gli2

(o)

Figure 8 Continued


Gli3

(p)

Gli3

(q)

Gli3

(r)





5 Conclusion







References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MZ CP

SP

IZShh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)






VZSVZ

IZ

Shh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)





4 Discussion



MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

(b)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowastlowast

(d)

lowastlowast


lowastlowastlowast

lowastlowastlowast

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(e)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(f)



IGL

ML

EGL

Shh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)





lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(a)

lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(b)

lowastlowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(c)

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(d)

lowastlowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(e)

lowastlowast

lowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(f)



000

025

050

075

100

10 15 20 25 30

46

8

9

12

WOG

Mul

ti-Sc

ore

CP Gli2 p = 00489

(a)

4

3

6

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

46

3

2

1

IZ Gli1 p = 00161

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(c)

6

4

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(d)

34

6

8

9

12

VZ Shh p = 00025

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(e)

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(f)

Figure 6 Continued


2

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(g)

2

4

6

8

12

9


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

(h)







000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

4

6

8

9

12

EGL Shh p = 0044

(a)

1

2

3

4

6

9

12

EGL Gli1 p = 00031

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

1

2

4

6

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore


(c)





Shh

(a)

Shh

(b)

Purkinje cell layer

EGL

MLIGL

Shh

(c)

Patched

(d)

Patched

(e)

Patched

(f)

Smoothened

(g)

Smoothened

(h)

Smoothened

(i)

Gli1

(j)

Gli1

(k)

Gli1

(l)

Gli2

(m)

Gli2

(n)

Gli2

(o)

Figure 8 Continued


Gli3

(p)

Gli3

(q)

Gli3

(r)





5 Conclusion







References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















VZSVZ

IZ

Shh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)





4 Discussion



MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

(b)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowastlowast

(d)

lowastlowast


lowastlowastlowast

lowastlowastlowast

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(e)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(f)



IGL

ML

EGL

Shh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)





lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(a)

lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(b)

lowastlowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(c)

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(d)

lowastlowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(e)

lowastlowast

lowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(f)



000

025

050

075

100

10 15 20 25 30

46

8

9

12

WOG

Mul

ti-Sc

ore

CP Gli2 p = 00489

(a)

4

3

6

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

46

3

2

1

IZ Gli1 p = 00161

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(c)

6

4

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(d)

34

6

8

9

12

VZ Shh p = 00025

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(e)

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(f)

Figure 6 Continued


2

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(g)

2

4

6

8

12

9


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

(h)







000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

4

6

8

9

12

EGL Shh p = 0044

(a)

1

2

3

4

6

9

12

EGL Gli1 p = 00031

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

1

2

4

6

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore


(c)





Shh

(a)

Shh

(b)

Purkinje cell layer

EGL

MLIGL

Shh

(c)

Patched

(d)

Patched

(e)

Patched

(f)

Smoothened

(g)

Smoothened

(h)

Smoothened

(i)

Gli1

(j)

Gli1

(k)

Gli1

(l)

Gli2

(m)

Gli2

(n)

Gli2

(o)

Figure 8 Continued


Gli3

(p)

Gli3

(q)

Gli3

(r)





5 Conclusion







References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

(b)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

lowastlowastlowast

(d)

lowastlowast


lowastlowastlowast

lowastlowastlowast

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(e)

MZ CP SCZ IZ SVZ VZ

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(f)



IGL

ML

EGL

Shh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)





lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(a)

lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(b)

lowastlowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(c)

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(d)

lowastlowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(e)

lowastlowast

lowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(f)



000

025

050

075

100

10 15 20 25 30

46

8

9

12

WOG

Mul

ti-Sc

ore

CP Gli2 p = 00489

(a)

4

3

6

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

46

3

2

1

IZ Gli1 p = 00161

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(c)

6

4

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(d)

34

6

8

9

12

VZ Shh p = 00025

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(e)

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(f)

Figure 6 Continued


2

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(g)

2

4

6

8

12

9


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

(h)







000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

4

6

8

9

12

EGL Shh p = 0044

(a)

1

2

3

4

6

9

12

EGL Gli1 p = 00031

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

1

2

4

6

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore


(c)





Shh

(a)

Shh

(b)

Purkinje cell layer

EGL

MLIGL

Shh

(c)

Patched

(d)

Patched

(e)

Patched

(f)

Smoothened

(g)

Smoothened

(h)

Smoothened

(i)

Gli1

(j)

Gli1

(k)

Gli1

(l)

Gli2

(m)

Gli2

(n)

Gli2

(o)

Figure 8 Continued


Gli3

(p)

Gli3

(q)

Gli3

(r)





5 Conclusion







References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IGL

ML

EGL

Shh

(a)

Patched

(b)

Smoothened

(c)

Gli1

(d)

Gli2

(e)

Gli3

(f)





lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(a)

lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(b)

lowastlowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(c)

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(d)

lowastlowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(e)

lowastlowast

lowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(f)



000

025

050

075

100

10 15 20 25 30

46

8

9

12

WOG

Mul

ti-Sc

ore

CP Gli2 p = 00489

(a)

4

3

6

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

46

3

2

1

IZ Gli1 p = 00161

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(c)

6

4

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(d)

34

6

8

9

12

VZ Shh p = 00025

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(e)

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(f)

Figure 6 Continued


2

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(g)

2

4

6

8

12

9


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

(h)







000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

4

6

8

9

12

EGL Shh p = 0044

(a)

1

2

3

4

6

9

12

EGL Gli1 p = 00031

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

1

2

4

6

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore


(c)





Shh

(a)

Shh

(b)

Purkinje cell layer

EGL

MLIGL

Shh

(c)

Patched

(d)

Patched

(e)

Patched

(f)

Smoothened

(g)

Smoothened

(h)

Smoothened

(i)

Gli1

(j)

Gli1

(k)

Gli1

(l)

Gli2

(m)

Gli2

(n)

Gli2

(o)

Figure 8 Continued


Gli3

(p)

Gli3

(q)

Gli3

(r)





5 Conclusion







References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(a)

lowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(b)

lowastlowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(c)

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(d)

lowastlowast

lowastlowast

lowastlowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(e)

lowastlowast

lowastlowast

EGL ML IGL

12

10

8

6

4

2

0

Mul

ti-Sc

ore

(f)



000

025

050

075

100

10 15 20 25 30

46

8

9

12

WOG

Mul

ti-Sc

ore

CP Gli2 p = 00489

(a)

4

3

6

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

46

3

2

1

IZ Gli1 p = 00161

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(c)

6

4

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(d)

34

6

8

9

12

VZ Shh p = 00025

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(e)

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(f)

Figure 6 Continued


2

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(g)

2

4

6

8

12

9


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

(h)







000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

4

6

8

9

12

EGL Shh p = 0044

(a)

1

2

3

4

6

9

12

EGL Gli1 p = 00031

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

1

2

4

6

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore


(c)





Shh

(a)

Shh

(b)

Purkinje cell layer

EGL

MLIGL

Shh

(c)

Patched

(d)

Patched

(e)

Patched

(f)

Smoothened

(g)

Smoothened

(h)

Smoothened

(i)

Gli1

(j)

Gli1

(k)

Gli1

(l)

Gli2

(m)

Gli2

(n)

Gli2

(o)

Figure 8 Continued


Gli3

(p)

Gli3

(q)

Gli3

(r)





5 Conclusion







References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















000

025

050

075

100

10 15 20 25 30

46

8

9

12

WOG

Mul

ti-Sc

ore

CP Gli2 p = 00489

(a)

4

3

6

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

46

3

2

1

IZ Gli1 p = 00161

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(c)

6

4

8


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(d)

34

6

8

9

12

VZ Shh p = 00025

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(e)

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(f)

Figure 6 Continued


2

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(g)

2

4

6

8

12

9


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

(h)







000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

4

6

8

9

12

EGL Shh p = 0044

(a)

1

2

3

4

6

9

12

EGL Gli1 p = 00031

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

1

2

4

6

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore


(c)





Shh

(a)

Shh

(b)

Purkinje cell layer

EGL

MLIGL

Shh

(c)

Patched

(d)

Patched

(e)

Patched

(f)

Smoothened

(g)

Smoothened

(h)

Smoothened

(i)

Gli1

(j)

Gli1

(k)

Gli1

(l)

Gli2

(m)

Gli2

(n)

Gli2

(o)

Figure 8 Continued


Gli3

(p)

Gli3

(q)

Gli3

(r)





5 Conclusion







References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















2

4

6

8

12


000

025

050

075

100

10 15 20 25 30WOG

Mul

ti-Sc

ore

(g)

2

4

6

8

12

9


000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

(h)







000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

4

6

8

9

12

EGL Shh p = 0044

(a)

1

2

3

4

6

9

12

EGL Gli1 p = 00031

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

1

2

4

6

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore


(c)





Shh

(a)

Shh

(b)

Purkinje cell layer

EGL

MLIGL

Shh

(c)

Patched

(d)

Patched

(e)

Patched

(f)

Smoothened

(g)

Smoothened

(h)

Smoothened

(i)

Gli1

(j)

Gli1

(k)

Gli1

(l)

Gli2

(m)

Gli2

(n)

Gli2

(o)

Figure 8 Continued


Gli3

(p)

Gli3

(q)

Gli3

(r)





5 Conclusion







References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

4

6

8

9

12

EGL Shh p = 0044

(a)

1

2

3

4

6

9

12

EGL Gli1 p = 00031

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore

WOG

(b)

1

2

4

6

000

025

050

075

100

10 15 20 25 30

Mul

ti-Sc

ore


(c)





Shh

(a)

Shh

(b)

Purkinje cell layer

EGL

MLIGL

Shh

(c)

Patched

(d)

Patched

(e)

Patched

(f)

Smoothened

(g)

Smoothened

(h)

Smoothened

(i)

Gli1

(j)

Gli1

(k)

Gli1

(l)

Gli2

(m)

Gli2

(n)

Gli2

(o)

Figure 8 Continued


Gli3

(p)

Gli3

(q)

Gli3

(r)





5 Conclusion







References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Shh

(a)

Shh

(b)

Purkinje cell layer

EGL

MLIGL

Shh

(c)

Patched

(d)

Patched

(e)

Patched

(f)

Smoothened

(g)

Smoothened

(h)

Smoothened

(i)

Gli1

(j)

Gli1

(k)

Gli1

(l)

Gli2

(m)

Gli2

(n)

Gli2

(o)

Figure 8 Continued


Gli3

(p)

Gli3

(q)

Gli3

(r)





5 Conclusion







References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Gli3

(p)

Gli3

(q)

Gli3

(r)





5 Conclusion







References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















References
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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