The complexity of β-catenin action and conflicting results on the role of β-catenin in maintaining the pluripotent state have made it difficult to understand its precise cellular and molecular functions. To attempt this issue we have generated new genetically modified mouse embryonic stem cell lines allowing for the deletion of β-catenin in a controlled manner by taking advantage of the Cre-ER-T2 system and analyzed the effects in a narrow time window shortly after ablation. By using this approach, rather then taking long term cultured β-catenin null cell lines we demonstrate that β-catenin is dispensable for the maintenance of pluripotency associated genes. In addition we observed that the removal of β-catenin leads to a strong increase of cell death, the appearance of multiple clustered functional centrosomes most likely due to a mis-regulation of the polo-like-kinase 2 and furthermore, alterations in chromosome segregation. Our study demonstrates the importance of β-catenin in maintaining correct cellular functions and helps to understand its role in embryonic stem cells. Mouse embryonic stem cells (ES cells) are isolated from the inner cell mass of pre-implantation embryos at blastocyst stage and exhibit the two characteristics defining embryonic stem cells, which are prolonged self-renewal properties and the ability to differentiate into all three germ-layers – so called pluripotency del/floxβcat -line (hereafter referred to as SR1 line). Second, a Cre-ER-T2 expression cassette was introduce into this line and stable clones isolated . By introducing the Cre-ER-T2 system we were able to genetically ablate β-catenin at a defined time-point by adding tamoxifen into the culture medium Chemical inhibition of GSK3β or short-term treatment with soluble Wnt3a provided the initial evidence for an important role of Wnt/β-catenin signaling in maintaining pluripotency Our study provides insights into the early cellular events after ablation of β-catenin and helps to understand its function in embryonic stem cells.Experiments were performed in agreement with the German law on the use of laboratory animals as well as biosafety (S1) and institutional guidelines of the Max Planck society. The use of animals was approved by “Regierungspräsidium Freiburg ” and the animal welfare office of the Max Planck Institue of Immunobiology and Epigenetics, Freiburg, Germany (KE-2iTO-6).2 on irradiated mouse embryonic fibroblasts feeder cells or on 0.1% gelatin  in Dulbecco’s Modified Eagle Medium (DMEM) , supplemented with 15% fetal bovine serum (FBS) , 1× non-essential amino acids , 1x L-glutamine , 1× PenStrep , 1× Sodium pyruvate , 0.001% β-mercaptoethanol  and 1000 U/ml LIF (Gibco-BRL). 4-hydoxy-tamoxifen (4-OHT)  was reconstituted in 70% ethanol to a final concentration of 1 mg/ml. For induction of recombination a single dose of 4-OHT was added to the culturing medium with 0.1 µg/ml final concentration. Cells were subsequently analyzed and used for further experiments at day one, two or three. Recombinant Wnt3a (R&D systems) was reconstituted  with 40 µg/ml and stored at -20°C. Wnt3a was administrated to a final concentration of 100 ng/ml 4.5 hours before harvesting the cells for RNA extraction. siRNAs  were transfected using Lipofectamine RNAiMAX Reagent  according the manufacturers protocol.ES cells were cultured at 37°C with 5% COβcatdel/flox mouse ES cells  containing one null allele and one allele with exons 2–6 flanked by loxP sites βcatdel/flox embryos at E3.5 blastocyst stage using standard protocols and LIF containing medium βcatdel/flox ES cells transfected with the pCAGGS-Cre-ER-T2-IRES-Puro vector. A stable clonal line was isolated and expanded . Treatment of these cells with 4-OHT leads to the generation of βcatdel/del alleles.β-catenin sh-RNA sequence that allowed us upon doxycycline administration to down-regulate β-catenin. The following β-catenin–targeting sequence was used: 5′AAAGTATTCACCCACACTG 3′.Wild-type W4 ES cells Total RNA was extracted using TRI Reagent . Subsequently, 1 µg of RNA was reverse transcribed using First strand cDNA Synthesis Kit  and 5–10 ng cDNA used to perform quantitative RT-PCR using ABsolute QPCR ROX Mix  on a ABI PRISM 7300 real time instrument according the manufacturers protocols. Results were obtained from at least three experiments performed in triplicates using the ΔΔCT method.Ddit4l , Dffb , Perp , Plk1 , Plk2 , Plk3 , Plk4 . All other primer pairs can be found in The following primers were used: 3VO4 and 1 Complete Protease Inhibitor Cocktail (Roche). The cell lysate was incubated  and finally centrifuged . The supernatant was retained, quantified using BCA (Thermo Scientific) and subsequent analyzed by SDS-PAGE and Western-blotting according standard protocols.Cells were rinsed PBS , scraped, transferred into 1.5 ml reaction tubes and resuspended in ice-cold RIPA lysis buffer  containing 1 mM EDTA, 0.2 mM Dithiothreitol DTT, 50 mM NaF, 2 mM NaCells were plated on gelatin-coated glass cover slips. Cells were rinsed twice with PBS and fixed with ice-cold methanol for 5′ at -20°C. After washing  the cells were blocked for 1 h . Next, cells were incubated with primary antibodies for 1 h in blocking buffer. After washing  the cells were incubated with the appropriate secondary antibody in blocking buffer. Finally, cells were washed  and mounted . Fluorescent images were collected with a fluorescence microscope .Acetyl-p53 (1∶1000 (WB), 1∶200 (IF), Cell signaling, 2525), Centrin , E-Cadherin , GAP-DH , Nanog (1∶1000 (WB), 1∶200 (IF), Rolf Kemler, 2). Theafter, cells were washed by three quick rinses with PBS (RT) before addition of nocodazole-free medium (RT). Subsequent, every minute  one dish was fixed with cold methanol to allow for the analysis of a time series.Cells were first incubated for 1 h at 4°C in a humid chamber in normal culture medium followed by an incubation for 1 h in a cell culture incubator with nocodazole (50 ng/ml) containing medium  was performed using the In Situ Cell Death Detection Kit, TMR Red (Roche) according to manufacturer’s instruction. Quantification was performed by standard FACS protocols. The experiment was performed four times.βcatdel/flox ES cells (hereafter referred to as SR1 line) were transfected with a vector carrying a Cre-ER-T2 cassette under the control of a chicken beta-actin promoter. By taking advantage of the vector based pyromycin selection cassette a clonal ES cell line was established . ARβ1 cells do form compact colonies and are virtually indistinguishable from SR1 cells in respect to colony size, compactness, growth rate und cell shape. Administration of 100 ng/ml 4-hydroxy-tamoxifen (4-OHT) to ARβ1 cells was sufficient to achieve efficient recombination of the β-catenin locus resulting in ES cells exhibiting genetic ablation of β-catenin (βcatdel/del).β-catenin mRNA levels are already down 24 h after 4-OHT administration  as being strongly up-regulated. We performed real-time PCR analysis and could verify the strong and significant up-regulation of Plk2 after β-catenin ablation in ARβ1 ES cells. Plk3 appeared in the RT-PCR experiments only slightly up-regulated, however, not statistically significant (Plk1-4 by RT-PCR and obtained similar results (data not shown). Since polo-like kinases are known to localize to centrosomes, are involved in centrosome duplication and are necessary to ensure genomic stability we addressed these issues next on our system.We realized when culturing ARβ1 ES cells in the presence of 4-OHT that the growth rate of cells deficient for β-catenin was strongly impaired compared to SR1 or non-treated ARβ1 cells (data not shown). This observation together with our data pointing to a cell death phenotype led us hypothesize that chromosome stability or centrosome function might be impaired after ablation of β-catenin in mouse ES cells. Therefore, we re-examined our gene array data and identified the nificant . To confWe immunostained for centrosomes in β-catenin deficient ARβ1 ES cells by using an anti-γ-tubulin antibody and observed with high frequency cells with more than 2 centrosome-like structures . InteresPlk2 as being up-regulated after depletion of β-catenin. Since Plk2 is known to be involved in the regulation of centrosome duplication we wondered whether the appearance of multiple clustered centrosomes might be a consequence of this up-regulation. Therefore, we used a si-RNA approach to knock-down the mRNA levels of Plk2 after ablation of β-catenin in ARβ1 ES cells and observed a reduction of approximately 40% of the appearance of multiple clustered centromeres . Therefore, we have decided to generate new β-catenin mutant ES cell lines that would allow for Cre-mediated ablation of β-catenin by using the Cre-ER-T2 system del/flox:Cre-ER-T2(β-cat) to study its function βcatflox/flox or βcatdel/flox alleles were used and recombination had to be achieved by viral infection to drive Cre-recombinase expression or by transfection of Cre expression plasmids β-catenin mRNA is absent already 24 h after administration of 4-OHT. As expected, β-catenin protein decay is more delayed with protein levels being only slightly reduced after 24 h but almost completely absent after 48 h and 72 h. Importantly, we could show that recombined cells in contrast to non-recombined cells are not able to react on Wnt-signals by up-regulation of target genes. These data together with the expression of the pluripotency markers Oct4, Sox2, Nanog, Klf4, the presence of alkaline phosphatase and furthermore their cell morphology make us feel confident that our newly established cell line is a suitable tool to study β-catenin functions in ES cells. Importantly, the non-altered levels of expression of pluripotency markers 3 days after administration of 4-OHT are in agreement with results published by others that have used inducible recombination systems The molecular and cellular functions of β-catenin in ES cells are in the focus of interest for many years. Its dual function in maintaining adhesive cell-cell connections which also encloses direct and indirect regulation of signaling properties and its important role as key signaling molecule of the Wnt pathway turned out to be essential characteristics of ES cells and their biology. This dual function however, the enormous impact of the Wnt signaling pathway on many cellular processes and the additional cellular functions of β-catenin outside of the classical Wnt- and adhesion pathway e.g. in telomerase or centrosome biology Along with the ablation of β-catenin we observed that many ES cell colonies became smaller and cells started to detach. TUNEL analysis revealed that β-catenin ablated ES cells undergo apoptosis. It has been reported that neuronal stem cells in the developing embryonic forebrain undergo apoptosis after the loss of β-catenin as a consequence of the loss of N-cadherin mediated adhesion polo-like-kinase 2 a major regulator of centrosome duplication Plk2 might have accounted for the appearance of extra-centrosome-like structures and addressed this question by si-RNA knock-downs of Plk2 in β-catenin deficient ES cells and observed a reduction in the number of cells exhibiting extra centrosomes. This might hint to an indirect role of β-catenin in the formation of ectopic centrosomes by up-regulation of Plk2 due to the loss of β-catenin although also other mechanisms can be assumed. The presence of extra-centrosomes leads often to chromosome segregation defects and chromosomal instability a phenomenon often observed in cancer cells Our gene array analysis revealed also a strong up-regulation of the Klf4, Nanog, Oct4 and Sox2 two month after ablation of β-catenin. Nevertheless, our findings point to adaption and selection processes resulting in alteration of expression of pluripotency markers in long term cultured β-catenin null ES cells. However, our study also demonstrates the important impact of β-catenin on key processes such as centrosome function and chromosome segregation within ES cells. These data, together with previously published studies on the role of β-catenin in ES cells show that it is difficult to analyze straight the action of β-catenin in ES cells and demonstrates the pitfalls long term cultured mutant ES cells can exhibit due to the sensitivity of the system, their ability for compensation and the complexity of action of the β-catenin signaling pathways.We aimed to study the early events after ablation of β-catenin and could show that the loss of β-catenin does not lead to a loss of pluripotency markers within the first 72 hours. This is an important finding in respect to the discussions regarding the role of β-catenin in maintaining pluripotency by analyzing long term cultured mutant ES cells. Interestingly, we did not find statistical significant alterations in the expression of Figure S1Analysis of pluripotency markers in long-term cultured mutant ES cells. ARβ1 and SR1 ES cells were cultured for 2 month after 4-OHT induced ablation of β-catenin and analyzed by quantitative RT-PCR for the expression of pluripotency associated genes. The pluripotency genes Nanog, Oct 4, Sox 2 and Klf 4 reveal no statistically significant difference between SR1 and ARβ1 cells two month after treatment with 4-OHT. All PCRs were done in triplicates and repeated three times. All values are normalized to the corresponding SR1 cell values. Error bars = s.e.m.(TIF)Click here for additional data file.Figure S2Analysis of Cre expression levels in mutant and wt ES cells. (A) Comparison of the Cre mRNA expression levels in SR1 and stably transfected wild type W4 ES cells carrying a Cre-ER-T2 cassette. Quantitative RT-PCR analysis revealed that both cell lines exhibit similar levels of Cre mRNA (n = 3 for SR1 and n = 5 for W4 cells). (B) To analyze whether the presence of Cre-recombinase has an effect on the number of centrosomes we analyzed W4 Cre-ER-T2 cells after treatment with 4-OHT. No significant increase in centrosome number was observed after 4-OHT mediated induction of Cre expression. Error bars = s.e.m.(TIF)Click here for additional data file.


Our gene deletion analysis revealed that prostate epithelial β-Catenin is required for embryonic prostate growth and branching but is dispensable in the normal adult organ. During development, β-Catenin controls the number of progenitors in the epithelial buds and regulates a discrete network of genes, including c-Myc and Nkx3.1. Deletion of β-Catenin in a Pten deleted model of castration-resistant prostate cancer demonstrated it is dispensable for disease progression in this setting. Complementary overexpression experiments, through in vivo protein stabilization, showed that β-Catenin promotes the formation of squamous epithelia during prostate development, even in the absence of androgens. β-Catenin overexpression in combination with Pten loss was able to drive progression to invasive carcinoma together with squamous metaplasia. These studies demonstrate that β-Catenin is essential for prostate development and that an inherent property of high levels of this protein in prostate epithelia is to drive squamous fate differentiation. In addition, they show that β-Catenin overexpression can promote invasive prostate cancer in a clinically relevant model of this disease. These data provide novel information on cancer progression pathways that give rise to lethal prostate disease in humans.Prostate cancer is a major cause of male death in the Western world, but few frequent genetic alterations that drive prostate cancer initiation and progression have been identified. β-Catenin is essential for many developmental processes and has been implicated in tumorigenesis in many tissues, including prostate cancer. However, expression studies on human prostate cancer samples are unclear on the role this protein plays in this disease. We have used  in vivo analysis in the prostate of the β-Catenin protein, which has been shown to be important in many processes during embryogenesis and has been implicated in tumorigenesis. Our studies demonstrate that β-Catenin is essential for prostate development but is dispensable in the normal adult organ. Analysis of a mouse model of a frequently mutated human prostate tumour suppressor, Pten loss, revealed that β-Catenin is not required for neoplastic formation in this model, even in castrated conditions. However, increased β-Catenin levels can cooperate with Pten loss to promote the progression of aggressive invasive prostate cancer together with squamous metaplasia. These data uncover the role of β-Catenin in the prostate and provide new insights on how pathways interact to drive human prostate cancer.Prostate cancer is a major cause of male death in the Western world, but few genes involved in this disease have been identified. We have undertaken an in-depth  PTEN (phosphatase and tensin homolog deleted on chromosome 10), the gene rearrangement TMPRSS2-ERG, and the oncogene C-MYCSox9, Hoxb13 and Nkx3.1Prostate cancer is the most common male cancer in the developed world, and a leading cause of cancer-related death in men. To date, few common genes that promote prostate cancer progression have been identified, including the tumour suppressor The prostate develops from the endodermally derived urogenital sinus (UGS) in response to androgens that signal through the androgen receptor (AR) CTNNB1 gene) is crucial in many steps of embryogenisis and is involved in a number of cancers The cytoplasmic protein β-Catenin  in the embryonic and adult mouse. Loss of Ctnnb1 in epithelia during mouse prostate development results in failure of bud outgrowth and branching, while β-Catenin activation leads to squamous transdifferentiation through an androgen-independent mechanism. Surprisingly, deletion of Ctnnb1 in the adult prostate had no effect on normal prostate homeostasis. To study its function in prostate cancer we analysed the role of β-Catenin in the context of the frequently deleted PTEN gene. We demonstrate that β-Catenin expression is elevated after PTEN loss but plays no function in prostate cancer in intact or castrated animals. However, increasing the level of β-Catenin in combination with PTEN deletion leads to highly invasive prostate tumours with persistent squamous metaplasia.To better define the function of β-Catenin in the normal and neoplastic prostate we have used Ctnnb1 in prostate epithelia. We used the Nkx3.1:Cre mouse line to drive the expression of Cre recombinase in epithelial cells at E17.5 just after bud induction, which in combination with a loxP containing Ctnnb1 allele (β-Cat) results in the excision of exons 2 to 6 and the production of a non-functional protein Nkx3.1:Cre mouse line is a knock-in allele and, therefore, heterozygous for Nkx3.1, β-Cat;Nkx3.1:Cre heterozygous animals were used as controls. At E18.5, β-Catenin is expressed at the membrane and in the cytoplasm of epithelial cells throughout the buds of control prostates and is lost in most cells of β-Cat;Nkx3.1:Cre mutant buds, although a small number of cells retain expression  on prostates dissected at E18.5 and grown in organ culture. Two well-described targets of WNT/β-Catenin signalling are Axin2, a negative regulator of the pathway, and c-Myc, a transcription factor that regulates multiple processes including cell proliferation and differentiation. Axin2 is expressed throughout the buds of control prostates with higher levels in the tips, while c-Myc is expressed in the bud tips , with only a few small remaining patches of c-Myc expression, presumably a result of epithelial cells that have not deleted β-Catenin. Interestingly, expression analysis of Nkx3.1, a gene important in prostate duct morphology Sox9 (n = 5), another key regulator of prostate development , while Shh is expressed at similar levels to control prostates (n = 4)  . This deNkx3.1:Cre to express a stabilized form of the protein (Actβ-Cat) in the developing mouse prostate epithelium, Actβ-Cat;Nkx3.1:CreActβ-Cat allele has loxP sites flanking exon 3 and Cre-mediated deletion results in the loss of the GSK3β regulatory phosphorylation sites, leading to stable expression of β-Catenin. At E18.5, the epithelial buds of Actβ-Cat;Nkx3.1:Cre mutants are larger than control prostates  and the morphology of the bud tip is irregular   , while Fgfr2 continues to be expressed in the novel structures of these mutants (n = 4)  . Similard growth . β-Catend growth . The nord growth . Areas od growth . Furtherd growth . The abnne (DHT) . Nkx3.1  (n = 4) . Loss ofβ-Cat) and mice with the PBCre transgene, which drives expression of Cre recombinase in mouse prostate epithelia from 2 weeks of age . H&E stained sections of β-Cat;PBCre prostates 6 months, 9 months and 19 months of age show no observable phenotype compared to control littermates   . In addi fertile . These dPTEN gene. To do this we used the well-defined Pten murine prostate cancer model Pten deletion in mice results in the formation of hyperplasia, prostatic intraepithelial neoplasia (PIN) and carcinoma. Pten null prostates were generated using a loxP containing Pten allele and the PBCre transgene . We first analysed the expression of β-Catenin protein to determine if β-Catenin levels are altered in these animals, as studies in prostate cancer cell lines have shown that Pten loss leads to β-Catenin accumulation F/F;PBCrePten prostates have a dramatic increase in the level of β-Catenin compared to controls, which is confirmed by Western blot , Pten;Ctnnb1 double mutant  and control animals  aged 3 months demonstrates a similar progression to PIN in Pten null mutants and in F/F;PtenF/F;PBCreβ-Cat mutants  animals with mice with the stabilized form of β-Catenin (Actβ-Cat) and the PBCre transgene. This allowed the comparison of prostates that are Pten null , have stabilized β-Catenin , Pten null in combination with stabilized β-Catenin  and controls . F/F;PBCreActβ-Cat;Pten prostates have higher levels of active non-phosphorylated β-Catenin than Pten null prostates and show a clear induction of LEF1, indicating these tumours have an increase in β-Catenin mediated transcription   . Comparave cells . These dCtnnb1 in the adult prostate has no effect on normal prostate homeostasis or a Pten model of prostate cancer. However, the functional significance of β-Catenin in prostate cancer is highlighted as increased levels can cooperate with Pten loss to drive progression to invasive carcinoma together with squamous transdifferentiation. The ability of β-Catenin to differentiate prostate epithelia into squamous cells in the embryo and adult suggests that this protein is playing a similar role in both developmental stages. These studies further highlight the use of developmental systems to inform on the function of genes and pathways in the adult.The role of β-Catenin in prostate development is not known and its function in prostate cancer is not clearly defined. We have undertaken an in depth genetic study in the embryonic and adult mouse to identify the role β-Catenin plays in these processes. Our developmental genetic studies demonstrate that epithelial β-Catenin is essential for early growth of the prostatic buds but that high levels of β-Catenin lead to the formation of squamous epithelia. Deletion of c-Myc at the tip of the developing prostate bud. c-Myc is a well-known regulator of proliferation and it is therefore likely that β-Catenin controls cell number in prostate buds through this gene Nkx3.1:Cre mouse line is first expressed after this has begun at E17.5. A recent study using an inducible system to delete β-Catenin prior to prostate formation has demonstrated that this protein is required for bud initiation Our studies show that during prostate development β-Catenin controls the proliferation of cells at the tip of the epithelial buds and regulates the number of p63 positive cells in this region. This lack of proliferation leads to a defect in prostatic bud growth and branching in mutant animals, consistent with other studies that have shown that the tips of the buds have high numbers of proliferating cells and are important in the formation of branches Nkx3.1 and Sox9. These, together with FGF signalling, through Fgfr2, and the Hedgehog signalling pathway, through Shh, are required for normal prostate development Nkx3.1 and Fgfr2 but having no effect on Sox9 and Shh expression. This suggests a complex gene network during prostate development with different pathway branches regulating prostate identity in parallel, and β-Catenin regulating Nkx3.1 and Fgfr2 in one of these branches. FGF signalling has been implicated in the early formation of prostate buds and β-Catenin could be acting through this pathway Fgfr2 was deleted using the Nkx3.1:Cre strain was not the same as in β-Cat;Nkx3.1:Cre mutants, suggesting that Fgfr2 is not the only factor responsible for the defects in growth and branching in the latter animals Prostate development is regulated by a set of transcription factors and signalling networks that are poorly defined. Two of the earliest markers of prostate identity are the transcription factors Ctnnb1 deletion, including LEF1, c-Myc and Axin2. In addition, several members of the WNT family have been shown to be expressed in the epithelia and mesenchyme of the developing prostate Our studies indicate that β-Catenin acts through the canonical WNT pathway to regulate prostate epithelial cell proliferation, as we observe the loss of expression of classical downstream genes of this pathway after Our data demonstrate that β-Catenin regulates the number of p63 positive progenitor cells in the developing prostate. Interestingly, WNT/β-Catenin signalling has been shown to control self-renewal of stem/progenitor cells of primary adult prostate epithelial cells and prostate cancer cell lines in prostate sphere assays Pten loss. This suggests that an intrinsic property of high levels of β-Catenin in prostate epithelia is to drive squamous differentiation. Squamous metaplasia is also seen after exogenous administration of estrogen to male mice. Estrogen has been shown to act through estrogen receptor alpha (ERα) in the epithelium to promote proliferation of p63 expressing basal cells and the formation of squamous metaplasia Actβ-Cat;PBCre prostates, however, this was not the case for F/F;PBCreActβ-Cat;Pten mice, suggesting that high ERα is not a requisite for squamous cell formation driven by high levels of β-Catenin (data not shown). Ectopic high levels of p63 expression were able to induce transdifferentiation and squamous metaplasia of mouse lung epithelium We have shown that during prostate development β-Catenin overexpression results in squamous epithelia differentiation, even in the absence of androgens. Similarly, squamous cells form when stabilized β-Catenin is expressed in the adult prostate, even after Pten mutant mice show resistant properties with proliferation of epithelial cells in the prostates of castrated animals Pten loss, it is not essential in neoplastic growth of Pten null cancer after androgen withdrawal. This data suggests that, in this CRPC model, β-Catenin does not act as a co-factor with AR or in an AR-independent manner to regulate neoplastic proliferation. The increase in β-Catenin expression we observe after Pten deletion is predominantly localized at the membrane and in the cytoplasm, with little transcriptionally active protein accumulating. This low level of active β-Catenin may not induce transcription of targets, such as LEF1, and have no affect on Pten-loss driven PIN. This is in contrast to our F/F;PBCreActβ-Cat;Pten model that has high levels of nuclear β-Catenin, increased levels of LEF1 expression and progression to an aggressive phenotype. Alternatively, the growth of Pten null prostate lesions deficient in β-Catenin may be due to compensation by the multiple downstream effectors of the PI3K/Akt pathway that are affected by Pten loss β-Catenin has been implicated in CRPC and has been shown to act as an AR co-factor in low androgen conditions Pten deficient prostate epithelia, a frequent genetic lesion in human prostate cancer, to invasive carcinoma suggesting that these pathways could interact during human prostate tumorigenesis. Consistent with this, recent exome sequencing has shown that members of the WNT pathway, including APC, are frequently mutated in aggressive prostate tumours, and these cancers are enriched for mutations in a PTEN interacting network K-ras and SV40 large T-antigen to drive tumour progression to invasive carcinoma K-ras in prostate epithelial cells have a loss of p63 expressing cells and do not display squamous metaplasia, consistent with our hypothesis that p63 expression is involved in transdifferentiation to squamous cells Our study, and work by others, has shown that activating β-Catenin through a stabilized form of the protein in the prostate leads to PIN and squamous metaplasia, with lesions that are highly proliferative but not invasive Pten deletion, mutant K-ras and SV40 large T-antigen to drive tumour progression suggests that it may be a general drug target in prostate cancer Pten and Actβ-Cat undergo Cre-mediated recombination at the same time within luminal cells, although some basal cells are also targeted. Therefore genetic lesions may need to occur in a step-wise fashion in patients so that β-Catenin expression is altered at a later stage in tumour formation. Alternatively, β-Catenin may need to interact with other genetic lesions or be upregulated in a different cell type to give rise to tumours that do not exhibit squamous differentiation. These studies provide novel information on the necessary steps in tumour development that are required for progression to the invasive phenotype seen in human patients with lethal prostate cancer.The ability of β-Catenin to cooperate with other oncogenic events such as Ctnnb1 conditional allele (β-Cat) have been described previously Ctnnb1 conditional allele (Actβ-Cat) were kindly provided by M.M. Taketo ARR2PBCre transgenic mice, PBCre, have been described previously Nkx3.1:Cre allele was created by introducing the Cre gene by homologous recombination into the Nkx3.1 locus and was kindly provided by Michael Shen. Mice with the conditional allele of Pten were obtained from The Jackson Laboratory Mice containing the −8 M dihydrotestosterone.E18.5 urogenital sinuses were dissected from male embryos and grown on Biopore membranes (MilliPore) with culture media (DMEM/F-12 HAM 1/1 mixture) supplemented with ITS+1 (Sigma-Aldrich I-2521) at 20 µl/ml, gentamicin (0.05 mg/ml), benzylpenicillin sodium (0.12 mg/ml), streptomycin sulphate (0.2 mg/ml), ampicillin (0.1 mg/ml) and 10Sox9 and Nkx3.1 have been described previously Axin2, c-Myc, Fgfr2 and Shh DIG containing probes were synthesized from PCR fragments containing T7 RNA polymerase promoter sequences as described previously WISH was carried out on an in situ processor (Intavis In Situ Pro) according to standard protocols 5′ AGGATGCTGAAGGCTCAAAGC 3′ andAxin2: 5′ GTAATACGACTCACTATAGGGACTCTGGATGGTCCCCAAAG 3′5′ AAGCTGGTCTCGGAGAAGCTGG 3′ andc-Myc: 5′ TAATACGACTCACTATAGGGAGGTTCAGGGATCTGGTCACG 3′5′ CGCAGGATGGACCTCTCTAC 3′ andFgfr2: 5′ GTAATACGACTCACTATAGGGCGACCAACTGCTTGAATGTG 3′;5′ GACAGCTCACAAGTCCTCAG 3′ andShh: 5′ GTAATACGACTCACTATAGGGACGTAAGTCCTTCACCA 3′.For each WISH probe, at least four embryos of each genotype were processed.Histological phenotype of samples was assessed on H&E stained sections. Serial sections were then stained for immunohistochemical analysis. Histological assessment was based on published guidelines and assisted by the pathologists R. Cardiff, D. Berney and G. Stamp 2O2 to block endogenous peroxidase activity. Sections were incubated in PBS with 10% sheep serum and then incubated with primary and secondary antibodies in PBS with 1% sheep serum. Sections were counterstained with hematoxylin. The following antibodies were used for immunohistochemistry; β-Catenin , AR , p63 , CK10 , CK1 , LEF1 , pAKT (Ser473) , Ki-67 , smooth-muscle actin , E-Cadherin . For DAB chromogen staining the ABC vector kit was used with biotinlyated secondary antibodies (Vector Laboratories) according to manufacturer's instructions and the DAB substrate (Dako). Secondary fluorescent antibodies were obtained from Molecular Probes and were used at a 1∶500 dilution. Fluorescent images were visualized and collected on a Leica TCS-SP2 confocal microscope. For all antibody stains, sections were stained from at least four animals of each genotype. Western blotting was performed using standard protocols, and the following antibodies were used; β-Catenin 1∶500; active non-phosphorylated β-Catenin 1∶500 (Millipore), LEF1 1∶1000, HSC70 1∶15,000 (Santa Cruz Biotechnology), β-Actin 1∶500 (Santa Cruz Biotechnology).Antibody stains were performed on paraffin sections. Tissues were fixed overnight in 4% paraformaldehyde (PFA), dehydrated by washing through an ethanol gradient series, washed in histoclear and embedded in wax. 4 µm sections were cut, treated with histoclear and rehydrated through an ethanol gradient series. Antigen retrieval was obtained by boiling the sections in citrate buffer (0.1 M sodium citrate pH6 and 0.05% Tween) and sections were treated with 3% HTo quantify proliferating cells, Ki-67 and β-Catenin double-labelled fluorescent immunohistochemistry was performed on sections and stained with nuclear DAPI. For developmental and adult studies prostatic ducts were identified using DAPI. For control ducts and ducts expressing stabilized β-Catenin Ki-67 positive cells were counted in the β-Catenin positive epithelia. For loss-of-function β-Catenin mutant buds Ki-67 positive cells were counted in the β-Catenin negative prostate epithelia. To quantify basal cells, p63 expressing cells were counted and shown as a percentage of the total epithelial cells. For developmental studies, cells were counted from a section of five embryos of each genotype. For adult studies, cells from at least 4 high power fields were counted per animal, which totalled more than 900 cells per animal. At least three animals of each genotype were analysed. All values are significant with p<0.05 using Student t-test unless otherwise stated.Tissues were dissected and fixed in PBS with 2% PFA and 0.1% glutaraldehyde for 30 min at 37°C. After washing in PBS, tissues were stained at 37°C in staining buffer 6, 5 mM K4Fe(CN)6 and 0.02% NP-40 in PBS) in the dark.Figure S1Actβ-Cat;Nkx3.1Cre mutant and control (Actβ-Cat) prostate organ cultures grown for 3 days. Arrows indicate area of high β-Catenin that has become squamous stratified epithelium. (B) H&E and IHC for β-Catenin on sections of Actβ-Cat;Nkx3.1Cre prostate organ cultures grown for 5 days with no DHT.Embryonic squamous formation of prostate epithelium by stabilized β-Catenin. (A) high magnification of β-Catenin and p63 IHC on sections of (TIF)Click here for additional data file.Figure S2β-Cat;PBCre mutant and control prostates. Sections cut through the dorsal-lateral lobe.β-Catenin is not required for adult prostate homeostasis. H&E stain and IHC for AR, Probasin, c-Myc and LEF1 on sections of (TIF)Click here for additional data file.Figure S3Pten null  prostates compared to controls. (B) undetectable levels of active non-phosphorylated β-Catenin are present in control prostates, while in F/F;PBCrePten prostates there is an accumulation of wild type endogenous active non-phosphorylated β-Catenin, as indicated. F/F;PBCreActβ-Cat;Pten prostates have very high levels of the smaller exon 3 deleted mutant active β-Catenin, as indicated. The anti-active β-Catenin antibody also detects a smaller unknown band in control and F/F;PBCrePten samples. LEF1 is not detected in control or F/F;PBCrePten prostates and is upregulated F/F;PBCreActβ-Cat;Pten prostates.Western blot analysis of β-Catenin and LEF1 in adult mouse prostate tissue. (A) β-Catenin levels increase in (TIF)Click here for additional data file.Figure S4Pten homozygous loss prostate cancer. (A) H&E stain on sections of 2-month-old F/F;PBCreActβ-Cat;Pten prostates. Left panel shows areas of adenocarcinoma and squamous metaplasia. Right panel shows epithelial cells invading into the surrounding stroma. (B) H&E stain and IHC for β-Catenin and p63 on sections of 3-month-old F/F;PBCreActβ-Cat;Pten prostates showing detail of adenocarcinoma and squamous metaplasia. Black arrows indicate mitotic figures. White arrows indicate cords of β-Catenin positive epithelial cells that have invaded the stroma. Arrowheads indicate squamous metaplasia.Stabilized β-Catenin and (TIF)Click here for additional data file.


Including an analysis of the general metabolic index and renal function, immunohistochemical staining and reverse transcription real-time PCR for β-catenin were performed in the renal cortex of the rat models every 4 weeks. After the treatments of DP and irbesartan, the albuminuria level, kidney weight/body weight, and thickness of the glomerular basement membrane were decreased, but the expression of β-catenin was not downregulated in the renal cortex. The effective drug target of DP to ameliorate albuminuria and renal hypertrophy should not inhibit the upregulated expression of β-catenin in rats with STZ-induced early-stage diabetic damage.Diabetic nephropathy (DN) is one of the most common causes of end-stage renal failure. This study was performed to determine the effect of Dantonic Pill (DP) treatment on  Wnt proteins interact with receptor proteins and stabilize the downstream transcription regulator β-catenin by inhibiting β-catenin phosphorylation, which reportedly affects tubule formation and epithelial differentiation  = [urinary Cr (μmol/L) × urinary volume (ml)/serum Cr (μmol/L)]/[1/1440 (min)] × [1000/body weight (g)]) [The serum creatinine (serum Cr) concentration was determined using a commercial assay kit . The urine creatinine concentration was measured using an assay based on Jaffe's reaction according to the manufacturer's protocol (Creatinine Assay Kit). The creatinine clearance rate (Ccr) was calculated using the following equation: Ccr[ml/(minht (g)]) . The uriThe renal glomerulus and renal tubule mesenchymal lesions were observed following HE and PAS staining. Twenty renal glomeruli were randomly selected from the cortical area under 400x magnification by an experienced pathologist to measure the index of mesangial expansion. The index of mesangial expansion was scored by a quantitative estimate of the mesangial zone width in each glomerulus, expressed as a function of the total glomerular area : 0, normThe electron microscopy morphometric evaluation was performed as described below. A portion of the renal tissues was cut into 1 mm cubes, fixed in 2.5% glutaraldehyde, and postfixed in 1% osmium tetroxide. The samples were dehydrated in a graded alcohol series and embedded in Epon 812. Four ultrathin sections (60 nm) were cut with a diamond knife continuously and stained with uranyl acetate and lead citrate. The thickness of glomerular basement membrane (GBM) was examined with a Hitachi H-7650 (80 kV) transmission electron microscope .2O2 and methyl alcohol. Then, a microwave was used to repair the antigen, and the tissues were blocked with normal caprine serum and dribbled with attenuated antibody I (antirabbit). The rat β-catenin antibody was diluted at the ratio of 1 : 50, stained with biotin-labeled caprine antirabbit antibody I and diaminobenzidine, restained with hematine, dehydrated, cleared, and sealed with glycerin gelatin. Stained renal glomeruli and tubules that were randomly selected from 20 sights were observed under 400x magnification by an experienced pathologist. The positive staining rate was evaluated as the following: ≤25% positive cells (+), 26–50% positive cells (++), 51–75% positive cells (+++), and >75% positive cells (++++); each scored as 1, 2, 3, and 4 points, respectively, for the statistical analysis [Paraffin sections were routinely dewaxed, hydrated, and dipped in a 0.03 volume fraction of Hanalysis .β-catenin mRNA was detected by quantitative real-time polymerase chain reaction (RT-PCR). β-actin was used as the internal reference. The total volume of the PCR was 20 μL, with 30 ng of cDNA as the template. After an initial denaturation step at 95°C for 3 min, 40 cycles of 95°C for 30 sec and 60°C for 30 sec for annealing and extension were run on a CFX384 Dice real-time PCR system . A melting curve analysis was performed after the amplification was completed. The mRNA levels were normalized to the β-actin levels of the respective control and presented as a ratio. The primers  and their sequences, product lengths, and reactive conditions are shown in Total RNA was extracted from the renal tissues with the Trizol method according to the manufacturer's protocol. cDNA was synthesized by reverse transcription using a kit . The expression of P values < 0.05 were considered significant. SPSS 16.0 software was used in this study.The measurement data were expressed as the mean ± SD. Significant differences among the groups were analyzed using a factorial-designed ANOVA. The method of simple linear correlation analysis was employed to measure the correlation between two variables. P < 0.05). The body weights were lower in the diabetic rats than in the normal rats. However, there were no significant differences in any of the metabolic indices among the diabetic rats treated with vehicle, DP, and irbesartan during the 12 weeks (P > 0.05).There were four groups of rats in the study, and the metabolic indices, such as body weight, food intake, water intake, urine output, and blood glucose levels, for each group are summarized in P < 0.05). After 12 weeks of treatment with DP and irbesartan, compared with vehicle alone, the urine protein levels, KW/BW, and GBM thickness were decreased in the diabetic rats, but Ccr and the mesangial expansion index were not . The effects of DP and irbesartan on the renal functional and pathological examination in the diabetic rats are shown in The renal pathological examination by HE and PAS staining indicated that the mesangial matrix was increased and the renal tubule was vacuolarly degenerated in the diabetic rats. The urine protein levels, Ccr, KW/BW, mesangial expansion index, and GBM thickness were significantly higher in the diabetic rats compared with the normal rats (β-catenin mRNA expression in the STZ-induced diabetic rats was significantly upregulated compared with the control group (P < 0.05). The β-catenin mRNA expression in the STZ + DP and STZ + irbesartan groups was reduced, but these changes were not significantly different compared with the levels in the STZ + vehicle group (P > 0.05). Immunohistochemical staining of β-catenin in the STZ + vehicle group was greater than that of the control group (P < 0.05), but the staining in the STZ + DP and STZ + irbesartan groups was not less compared with the STZ + vehicle group (P > 0.05). The effect of DP and irbesartan on the β-catenin mRNA and protein expression levels is shown in For 12 weeks, the Correlation Analysis. There was no significant correlation between the mRNA and protein expression levels of β-catenin and the urinary protein level or the KW/BW in the diabetic rats (P > 0.05).DN is characterized by definite renal morphological and functional alterations. The features of early diabetic renal changes are glomerular hyperfiltration and glomerular and renal hypertrophy. This disease is characterized by thickening of GBM and mesangial matrix expansion . The datβ-tumor growth factor [The principle of formulation in TCM has been adopted to guide the selection of herbs (herb matching) in the multicomponent herbal formulae prescribed for the treatment of diseases. The most important and effective pharmacological ingredients identified in DP are danshensu and Panax notoginseng . Dan Sheh factor , vasculah factor , and conh factor  and inhih factor  to proteβ-catenin-dependent or β-catenin-independent pathways. There are generally two pools of β-catenin in cells: one that is tightly associated with cadherins at cell-cell junctions, and the other that is “free” in the cytosol/nucleus. The latter pool is involved in gene transcription regulation. In the resting state, cytosolic/nuclear β-catenin must be maintained at a very low level through the rapid turnover of free β-catenin [β-catenin activity is also regulated by tumor necrosis factor [β-catenin to promote signaling [β-catenin are known to mediate inflammation, angiogenesis, and fibrosis through the upregulation of intercellular adhesion molecule [β-catenin. Previous studies revealed that the Wnt pathway was identified to be associated with DN. The binding of specific Wnt proteins to receptor/coreceptor complexes transduces intracellular signals through either -catenin . Includis factor , N-cadhes factor . Moreoveignaling . The tarmolecule , plasminmolecule , and CTGmolecule , which amolecule . Therefoβ-catenin in the renal cortex was upregulated in our untreated diabetic rats compared with the nondiabetic control rats during the 16 weeks of this study. A similar study also showed upregulated β-catenin levels in both the renal cytosol and nuclei of the streptozotocin-induced diabetic rats [β-catenin expression levels was too slight to be significantly different compared with the diabetic control rats. These results suggested that DP and irbesartan decrease urinary albumin and renal hypertrophy to prevent renal injury in the early stage of DN through a mechanism that does not involve the inhibition of β-catenin. The explanation is that in experimental models the kinetics concerning the development of fibrosis is faster (weeks or a few months at best) compared with that of humans (years). Therefore, the treatment in animals might have to be started relatively early, before the irreversible destruction of the renal structure . The molecular mechanisms that underlie the progression of DN to end-stage renal failure are not well defined, thereby limiting access to potential therapeutic targets. Because albuminuria caused by diabetic nephropathy is an independent risk factor for cardiovascular events and death [β-catenin.Immunohistochemistry and real-time PCR analyses showed that the expression of tic rats . After 1nd death , a stratβ-catenin is very limited in DN due to the lack of reliable approaches to selectively assess its function. Such studies are important not only for the quality control of herbal medicine but also for the development of new pharmaceutical products in the prevention and management of diabetic nephropathy. In summary, DP may contribute to the further understanding of the mechanisms of DN and to the development of novel strategies for both its prevention and management. However, our understanding of the specificities of


The location specific motor pattern generation properties of the spinal cord along its rostro-caudal axis have been demonstrated. However, it is still unclear that these differences are due to the different spinal interneuronal networks underlying locomotions or there are also segmental differences in motoneurons innervating different limbs. Frogs use their fore- and hindlimbs differently during jumping and swimming. Therefore we hypothesized that limb innervating motoneurons, located in the cervical and lumbar spinal cord, are different in their morphology and dendritic signal transfer properties. The test of this hypothesis what we report here.Discriminant analysis classified segmental origin of the intracellularly labeled and three-dimensionally reconstructed motoneurons 100% correctly based on twelve morphological variables. Somata of lumbar motoneurons were rounder; the dendrites had bigger total length, more branches with higher branching orders and different spatial distributions of branch points. The ventro-medial extent of cervical dendrites was bigger than in lumbar motoneurons. Computational models of the motoneurons showed that dendritic signal transfer properties were also different in the two groups of motoneurons. Whether log attenuations were higher or lower in cervical than in lumbar motoneurons depended on the proximity of dendritic input to the soma. To investigate dendritic voltage and current transfer properties imposed by dendritic architecture rather than by neuronal size we used standardized distributions of transfer variables. We introduced a novel combination of cluster analysis and homogeneity indexes to quantify segmental segregation tendencies of motoneurons based on their dendritic transfer properties. A segregation tendency of cervical and lumbar motoneurons was detected by the rates of steady-state and transient voltage-amplitude transfers from dendrites to soma at all levels of synaptic background activities, modeled by varying the specific dendritic membrane resistance. On the other hand no segregation was observed by the steady-state current transfer except under high background activity.We found size-dependent and size-independent differences in morphology and electrical structure of the limb moving motoneurons based on their spinal segmental location in frogs. Location specificity of locomotor networks is therefore partly due to segmental differences in motoneurons driving fore-, and hindlimbs. Correlative analysis of the same type of neuron in different parts of the CNS has shown location specific morphological and electrotonic differences in pyramidal neurons of the hippocampus in the rat /16 = 0.46 being the total number of neurons is 16. Another index used to measure last order cluster formations was the Peterson’s index  = 0.62. Both indexes may have values between 0 and 1. The indexes are closer to 1 if cervical and lumbar MNs are more similar and they are getting smaller with increasing differences between MNs of the two spinal segments. The significance of segmental cluster formation tendencies  was tested by comparing these homogeneity indexes with those of artificially generated cluster formations, where segmental origins of MNs were assigned randomly prior to the cluster analysis. For each actual dendrogram 100 other artificial dendrograms were generated reflecting grouping tendencies of the real set of MNs with their segmental origins artificially randomized. The mean value of indexes was calculated for the 100 artificial dendrograms and the mean was then compared with the actually found index by one sample t-test. If this test showed a significant difference between the real and artificial indexes, then we concluded that MNs tended to form homogeneous groups determined by their segmental location in the spinal cord.To check if MNs have a tendency to form last order clusters where cervical and lumbar MNs are segregated, homogeneity (or similarity) indexes were used to measure segmental homogeneity within clusters. Measuring segmental homogeneities is feasible since increasing segmental differences among MNs are reflected in their increasing segregation tendency to different clusters shown by the cluster analysis, and as a consequence of segregation, the clusters become more and more homogeneous in terms of segmental origins of MNs they contain. This way, increasing segmental segregation of MNs between clusters may be measured by segmental homogeneities (homogeneity indexes) within clusters. Two different homogeneity indexes were used. In addition to the Peterson’s index  we defins index  and was The purpose of this study was to investigate differences between alpha motoneurons (MNs) located in the cervical and lumbar enlargements of the frog that innervate the muscles of forelimbs and hindlimbs. These MNs had ellipsoid or fusiform perikarya in the lateral area of the ventral horn. The dendritic arborization of these MNs could be divided into a dorsomedial, dorsal and lateral dendritic arrays with many dendrites extending to the lateral funiculus of the white matter. The lateral dendrites extended to the border of spinal cord where they formed a subpial meshwork Figure , charactThe mean surface area of somata for cervical MNs was about 35% bigger than that of the lumbar MNs but the difference did not reach the significant level because of the high variances in both spinal segments Table  was applThe total number of branch points was more numerous in lumbar MNs than in MNs of the cervical part of the spinal cord . The distributions of branch points over different path distance domains were also different . This indicates that difference in branch point frequencies was due to longer total length of dendrites with the same average tendency of branching in the lumbar MNs close to somata as also found by Dityatev et al. .Early analyses of dendritic orientation of limb moving MNs in frog spinal cord described three dendritic arrays that extended in the dorso-medial, dorsal and lateral directions ,43. BaseHere we compared orientation of MN dendrites in the lumbar and cervical levels of the cord. Polar histograms were created Figure  to show ms = Rmd) and inhomogeneous (Rms < Rmd) soma-dendritic membranes (see Methods) at different neuron resistances. Since it is difficult to infer how MNs’ dendritic architecture affects electrical signal propagation, we used the graphical approach of the morphoelectrotonic transformation . By thimd) and the soma (Rms). Note that both Rmd and Rms are different in these two METs; Rmd is bigger, while Rms is smaller in Figure md makes the MET more compact, while the decreasing Rms has the opposite effect on compactness. When the geometry and the METs of dendritic arbors were compared, careful inspection found non-proportional changes in the MET size of dendritic branches relative to their morphological appearance  of the MET may be different Figure B–C indicms < Rmd), cervical and lumbar MNs also differed in their mean MET distances measured to branch points.To compare different METs of dendrites (representing electrotonic architectures in different membrane models) of the same MN, a set of MET descriptors Table  was compDiscriminant analysis using MET descriptors classified cervical and lumbar MNs 100% and 95% correctly in all membrane models . Similar results were obtained when the number of descriptors was reduced. With just four descriptors  MNs were still classified 87% segmentally correctly. These results were cross validated by the 16-fold and the repeated random subsampling techniques  (in averms < Rmd) membrane. These values yield 84/533 = 0.16 proximal to distal ratio for the geometrical size of branches, and 1.97/2.50 = 0.79 and 1.94/1.68 = 1.15 ratios in the METs with homogeneous and inhomogeneous membranes respectively. This observation suggests non-proportional changes in the size of dendrites during the MET, which depend on the distance of dendritic branch from the soma.In case of the two previously selected dendritic branches we calculated their geometrical and MET size ratios. The lengths of the proximal and distal branches were 84 and 533 μm respectively  membrane model with 1.4 MΩ neuron input resistance was assumed.To investigate these changes further, log attenuations of PSPs to soma were determined from different regions of the dendrites located within 100 μm path distance domains from the perikaryon. Then, the computed attenuations were divided by the mean attenuation of PSPs measured from dendritic sites within 0–100 μm from the perikaryon. These relative attenuations were not linearly related to the path distances of locations where PSPs were generated indicating again a non-proportional change in size of dendrites during the METs Figure . Relativ−6) from the same geometrical distance in lumbar and in cervical MNs both in the homogeneous (Rms = Rmd), Figure ms < Rmd, Figure The mean somatopetal log attenuations of PSPs were determined as a function of path distance from the soma in different membrane models of the cervical and lumbar MNs Figure . GeneralNerve cells with significantly different sizes are likely to be different electrotonically too. However, it remains an important question if neurons with different sizes keep their electrotonic difference if their comparison is based only on their topological structure and size-dependency is ignored. Here we studied this issue by using size-independent comparisons of dendritic signal transfer properties in cervical and lumbar MNs. This type of electrotonic comparison is feasible since electrotonic structure - in analogy to the geometrical structure - is not only defined by metric-related properties, but also by the branching structure (topology) of dendritic trees affecting the shape of distributions of voltage and current transfer properties over the length of dendrites.th, 25th, 50th, 75th and 90th percentiles .In this set of analysis a constant current was injected to generate steady depolarizations in the midpoints of all cylindrical compartments of the cable model and voltage and current transfers to soma were measured. These transfer values were then processed as summarized above.th percentiles of distributions for the lumbar MNs  and the generally shifted nature of percentiles relative to those of the cervical MN group  in their steady-state voltage transfer properties. These segmental differences between MNs were detected at all levels of background synaptic activities with some tendency of MNs to get more similar with the decrease of background activity.By using somatopetal voltage transfers, our first observation was the higher variabilities for all, except the 90t-test, p < 0.02). MNs were getting more and more similar with the decrease of overall synaptic activity  with a less obvious general shift in the standardized distributions Figure D. Cluste Figure A, B, as  Figure .While steady-state approach measures transfer properties when the generation of PSPs can be approximated by a constant current injection, many synaptic events are short in time and are better approximated by transient conductance changes. Propagation properties of these transient voltage signals are different than those under steady-state circumstances. Therefore, we extended our analysis and investigated transfers of voltage transients. In these simulations PSPs were generated by brief conductance changes in dendritic points. The changes in shape parameters  of transient PSPs were computed during their propagation to the soma and descriptors of these changes were created as described earlier. Box plots of these descriptors, once again, showed that lumbar MNs were more variable than the cervical ones in the way the amplitudes of voltage transients were reduced during their propagation to the soma Figure B–C.t-test, p < 0.0005) than those calculated for clusters when segmental origin of MNs was artificially randomized  in these cases. Graphs of both homogeneity indexes tended to remain in the territory  where they represent no significant segregation of the two groups of MNs.  Cervical and lumbar limb-moving MNs of frogs have structurally different dendrites imposing different attenuations of voltage amplitudes during their propagation to the soma. 2) On the other hand, these structural differences in dendrites do not distinguish the two classes of MNs in the way how half-widths and rise times of transient potentials are changing during their dendritic propagation.We investigated morphological and electrical differences between cervical and lumbar spinal motoneurons (MNs) that innervate fore- and hindlimb muscles in adult frogs. We deliberately did not want to compare MNs according to the specific muscles they innervate ,45-48 raMNs undergo substantial developmental changes during embryological and postnatal life that affect the size of cell bodies, size and branching structure of dendrites and these changes are accompanied by physiological maturation of membrane properties like specific membrane resistance, neuron resistance, resting membrane potential, spike shapes and excitability . Some ofOverall, these findings suggest that MN maturation is highly dependent on rostro-caudal position of neurons and therefore intrinsic differences in morphology and electrical properties of MNs may be expected to occur along the rostro-caudal axis. These differences have been investigated in the present study.To investigate segmental differences between MNs, we used pair-wise comparisons of morophological and electrotonic properties, multivariate discriminant analysis and cluster analysis. These methods have been successfully applied in classifying spinal MNs in the turtle , tectal We used high-fidelity compartmental cable models with a set of different membrane properties to account for the variability in neuron resistances measured experimentally, to analyze the effects of the varying size of inhomogeneity in the soma-dendritic membrane and to mimic synaptic background activity. However, in our models we considered a passive membrane. This restriction is validated by a number of factors. Although there is an ever growing list of evidence that voltage-dependent ion channels are present in the dendritic membranes of different nerve cells  dendrites to total surface area and the low sensitivity of dendritic impulse propagation to the thickness of these dendrites we conclude, that the impact of 0.1–0.2 μm overestimation of diameters for the thinnest dendrites is unlikely to be significant in our study.We found segmental differences both in the numbers and distributions of branch points in dendrites of limb moving MNs of the cervical and lumbar segments. The bigger number of branch points in lumbar MNs does not merely mean more dendritic branches but also a topologically and electrotonically more complex dendritic architecture. Based on various morphological measures of dendritic complexity Dityatev et al.  found thLocations of branch points are related to signal propagation in the dendrites both in the presence and absence of voltage–dependent ion channels. In dendrites with passive membrane, the current transfer effectiveness generally changes abruptly at branch points altering the ‘cost’ of moving a synapse to a geometrically different location in terms of the change in the soma potential during synaptic activity ,78. In dComparison of the orientation of dendritic arborization also demonstrated differences between cervical and lumbar MNs. We found that the ventromedial extension of dendritic trees is more powerful in the cervical MNs. Experiments using retrograde cell degeneration technique showed that the ventromedial area in the gray matter of the cervical segments corresponds to the terminal fields of contralateral tectospinal pathways that do not extend to lumbar segments in frogs . Axon teThe morphology of MNs located in cervical, lumbar and sacral spinal segments have been investigated in several studies -48,84-87Comparison of our data on the morphology of lumbar MNs of frogs with MNs of cats showed several differences between the two species. The MNs in the frog presented smaller, elongated cell bodies emitting fewer stem dendrites. The size of the dendritic trees was about the half of that found in hindlimb innervating MNs of mammals ,88. LumbThe organization of dendritic arbor seemed to be also different in lumbar MNs of frogs and cats. While the dendritic trees of cat MNs emerge in almost all directions without any obvious preference, the dendritic trees of frog MNs are organized into dorsal, lateral and dorsomedial groups. The lateral dendrites dendrites form a dense subpial meshwork running parallel with the border of the spinal cord. This subpial dendritic plexus is well developed in lower vertebrates including anurans but it is reduced in mammals.ms) and dedndrites (Rmd). However, the detailed membrane properties (Rms and Rmd) of these large neurons are still not well known. In addition, the effective membrane resistance may be dependent on the activation state of synapses received by MNs in the functioning spinal networks. Therefore we used two approaches. First, we compared the METs of MNs by assigning different physiologically realistic somatic input resistances (1.4 or 5 MΩ) [ms = Rmd) and inhomogeneous (Rms < Rmd) soma-dendritic membrane with a canonical Rmd = 20000 Ωcm2 value for the dendrites  of the same neuron  was investigated on the similarities/dissimilarities of voltage and current transfer properties of cervical and lumbar MNs by taking Rmd = 20000 Ωcm2 as a control value. Higher and lower levels of synaptic activities were modelled by decreasing the Rmd to 5000 Ωcm2 and increasing it to 50000 Ωcm2, while the Rms was kept constant at 500 Ωcm2[Our major aim was to test if limb moving MNs are different electrically in the cervical and lumbar segments of the spinal cord. Factors shaping the electrical properties of neurons include the specific membrane resistance of the soma (Rr 5 MΩ) ,18,19 wi 500 Ωcm2,33,34  on MNs Comparison of the METs with the original geometry of dendrites showed increasingly disproportionate changes in MET sizes in the more distant locations. This was observed independently of the size of soma-dendritic membrane inhomogeneity and the resistance of the neurons. According to the cable theory, in an infinite cylinder with passive membrane the voltage is decaying exponentially with distance if a steady current is injected  Which distance domains of the dendrites do differentiate between cervical and lumbar MNs and 2) How these distance domains do change when properties of the soma-dendrite membrane are altered?Two distance domains, whose separation was at ~1500 μm distance, could be identified from where voltage attenuations to soma were characteristically related to the segmental origin of MNs. In this context we found the followings: i) PSPs propagating along the passive dendrites of cervical MNs attenuated less to soma than PSPs in lumbar MNs if the synapses were closer than ~1500 μm. The relationship is the opposite if PSPs were generated farther than ~1500 μm from the soma. ii) These findings are independent of the size of neuron resistivity and the inhomogeneity of the soma-dendritic membrane surface.ms < Rmd) nature of the soma-dendritic membrane would be kept, while the neuron input resistance becomes lower with the overall increase in synaptic activity. However, the impact of background synaptic activity on membrane resistance is presumably bigger in dendrites than in the soma since ~95–98% of the membrane surface is given by the dendrites and the majority of synapses are received there in frog spinal MNs [The observation that these relationships are independent of the size of neuron resistance suggests implications for the control of PSP attenuations by the uniform decrease or increase of background synaptic activity that changes the effective membrane resistance over the soma-dendritic membrane -31. In tnal MNs . Consequnal MNs ,26,27 anGlobal comparison of log attenuations (MET) of cervical and lumbar MNs resulted in a nearly 100% segmentally correct classification by discriminant analysis. Electrical structures of MNs were further investigated by the voltage and current transfers in dendrites at different intensities of synaptic background activity. In these analyses we used standardized distributions of the transfer values to isolate structure-related electrical differences from those related purely to size. Such structural differences were suggested by the different numbers and distributions of branch points in MNs of the two spinal segments and by the different distance-dependence of log attenuations in the two groups of MNs.This way, it was relevant to ask directly whether the electrotonic differences between fore- and hindlimb moving MNs are entirely due to their different metric (size-related) morphological properties or they are  consequences of the different dendritic structures.Voltage transfer properties showed differences in the two groups of neurons both in steady-state and in case of transient signals. This suggests that identical inputs propagate differently in the dendrites of cervical and lumbar MNs and the level of depolarization reaching the soma or the nearby axon hillock may be different. Threshold potentials were investigated experimentally in putative MNs along the spinal cord in young frogs and no significant tendency of changes was detected according to the rostro-caudal positions of neurons  suggestiHowever, it is disputed if the major determinant of firing is to exceed a certain voltage threshold or to deliver enough current (charge) to the soma since experimental evidence exists to support each view depending on the conditions of action potential initiation  direction . Our resUnder steady-state conditions we detected a general tendency of increasing segregation (differences) between the limb moving MNs as the background synaptic activity was increased. This tendency was more pronounced if current rather than voltage transfers were considered. A similar tendency of increasing differences in electrotonic properties during higher synaptic activities was reported in a comparative study on different classes of spinal neurons, including MNs, in the cat .Our findings on segmental segregation tendencies of limb moving MNs based on their morphological and dendritic signal transfer properties are strengthened by the consistency of the results obtained with the many different and validated statistical approaches, independently of the type of homogeneity index used, the choice of hierarchical cluster analysis and the weighting factors of descriptors.structural differences in motoneurons along the rostro-caudal axis of the spinal cord. The location-dependent differences are likely to affect input–output properties of these MNs.We showed location specificity of morphological and electrical transfer properties of the limb moving class of motoneurons in the frog spinal cord. Many of the location-specific differences were size-independent emphasizing the importance of The results present the first detailed systematic analysis of the location-dependent properties of spinal motoneurons and suggest that specificity of locomotor networks, which control fore- and hind limb movements, is partly due to differences in their motoneurons.These differences might reflect a basic initial segmental developmental pattern of MNs, which may then be refined according to the needs of specific muscles they innervate. This concept may obtain support from the experimental findings that the targets of these MNs are specified before the outgrowth of axon ,102. Furmax: Peak synaptic conductance; tmax: Time to peak of the synaptic conductance; Rmd: Specific dendritic membrane resistance; Rms: Specific somatic membrane resistance; RN: Neuron resistance.ANOVA: Analysis of variance; BRP: Branch point of a dendrite; CNS: Central nervous system; ENDP: End point of a dendrite; EPSP: Excitatory postsynaptic potential; Hox: Homeobox; MET: Morphoelectrotonic transformation; MN: Motoneuron; PAST: Palaeontological statistics; PSP: Postsynaptic potential; SEM: Standard error of means; SPSS: Statistical package for the social sciences; λ: Space constant; gThe authors declare that they have no competing interests.AS wrote the program codes, run simulations, carried out statistical tests and contributed to drafting the paper. JS participated in running simulations and in data analysis. IW and AB reconstructed the neurons. AB also contributed to the body text. EW designed the study, participated in analysis and interpretation of data, and finalized the text. All authors read and approved the final manuscript.


Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with limited therapeutic options. Activating β-catenin somatic mutations are found in ACC and have been associated with a poor clinical outcome. In fact, activation of the Wnt/β-catenin signaling pathway seems to play a major role in ACC aggressiveness, and might, thus, represent a promising therapeutic target.in vitro and in vivo effect of β-catenin inactivation using a doxycyclin (dox) inducible shRNA plasmid in H295R adrenocortical cancer cells line (clone named shβ).Similar to patient tumor specimen the H295 cell line derived from an ACC harbors a natural activating β-catenin mutation. We herein assess the β-catenin expression was detectable in shβ clones in comparison to control clones (Ctr). Accordingly, we observed a decrease in Wnt/βcatenin-dependent luciferase reporter activity as well as a decreased expression of AXIN2 representing an endogenous β-catenin target gene. Concomitantly, β-catenin silencing resulted in a decreased cell proliferation, cell cycle alterations with cell accumulation in the G1 phase and increased apoptosis in vitro. In vivo, on established tumor xenografts in athymic nude mice, 9 days of β-catenin silencing resulted in a significant reduction of CTNNB1 and AXIN2 expression. Moreover, continous β-catenin silencing, starting 3 days after tumor cell inoculation, was associated with a complete absence of tumor growth in the shβ group while tumors were present in all animals of the control group.Following dox treatment a profound reduction in In summary, these experiments provide evidences that Wnt/β-catenin pathway inhibition in ACC is a promising therapeutic target. Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine neoplasm, with a 5-year overall survival of around 40% CTNNB1, i.e. Catenin (cadherin-associated protein), beta 1) mutations CTNNB1 mutation has recently been shown Activation of the Wnt/β-catenin signaling pathway in adrenocortical tumorigenesis has recently been investigated in detail in vivo and in vitro effects of Wnt/β-catenin signaling pathway specific inactivation using short hairpin RNA (shRNA) in a tumor model for adrenocortical carcinoma (H295R).The aim of this study was to assess the CTNNB1  mRNA, and the control vector (pTer) were obtained from Dr. van de Wetering CTNNB1 expression was down-regulated at least 5 fold in a doxycyclin  dependent manner in comparison to three control clones (Ctr) transfected with pTer vector. All cell clones were investigated for their ability to express specific steroidogenic genes (StAR and CYP11B1) and their responses to the cAMP/PKA pathway which was found to be comparable to that of the parental cell line, H295R CTNNB1 (β-catenin) gene activating mutation, previously identified in the parental H295R cell line in vitro experiments were confirmed with equivalent results in 2–3 individual clones (Ctr and shβ).Adrenocortical carcinoma cell line H295R stably transfected with the Tet repressor (H295R/TR) was kindly provided by Dr. Lalli Total RNA or protein extractions and analysis from cell lines were performed as previously described As a Wnt/β-catenin pathway reporter construct driving expression of luciferase gene, the TopFlash plasmid (Top) was used which contains two copies of the β-catenin/T-cell factor TCF-binding sites whereas the FopFlash plasmid (Fop) contains two mutated copies of the β-catenin/TCF-binding sites Proliferation was measured by MTT assay (Promega). The cell cycle and apoptosis were analysed by flow cytometry as previously reported 6 cells of the individual clones were inoculated in a volume of 200 µl PBS subcutaneously into the neck of each mouse. For short-term therapeutic experiments dox treatment was initiated when the longest tumor diameters ranged between 0.2–0.9 cm in size (after 21–31 days). Doxycyline was added in a final concentration of 2 mg/ml to the drinking water in amber water bottles. After 9 days of dox treatment tumors were excised, fixed in formalin, embedded in paraffin, and 4 µm sections cut and stained with Hematoxylin-Eosin-Saffron. Immunohistochemistry for β-catenin was performed as previously described Female athymic NMRI nu/nu mice (6–8 weeks) were purchased from Harlan Winkelmann  and housed under pathogen-free conditions. All experiments were carried out following protocols approved by the Regierung von Oberbayern and in accordance with the german guidelines for animal studies. 15×10in vitro data with statistical analyses represent the quantification of at least three experiments. Control conditions were set as 100% and data were analyzed using Fisher's test. The statistical analysis for comparison of tumor weight after long-term therapeutic experiment in vivo was performed by Mann-Whitney test. Significance was set at P<0.05 (represented by * in figures); P<0.01 (**) and P<0.001 (***).All CTNNB1 gene mutation on the GSK3β phosphorylation site (S45P), exhibit a constitutive transcriptional activity of β-catenin-LEF/TCF CTNNB1 mRNA  and protein levels were significantly decreased   .The Wnt/β-catenin-LEF/TCF dependent transcription was studied by using the Top-Flash/Fop-Flash plasmids (Top and Fop). As expected, transfected H295R cells showed higher transcriptional activity of the β-catenin-LEF/TCF dependent luciferase reporter construct Top in comparison to the mutated Fop construct .Moreover, CTNNB1 silencing in H295R cell line demonstrated a significant decrease in proliferation  compared to shβ clone without CTNNB1 silencing (−dox) or the control clone . No such difference was observed in the control clone (Ctr). At this time point, we observed a reduction of two important proteins for G1/S transition, Cyclin A and CDK2 only in cells silenced for CTNNB1 , which was detectable already at an earlier time point  while no significant difference was observed in control clones.Similarly, no effect on apoptosis assayed by the flow cytometric analysis of annexine V incorporation in cells, was observed until 5 days while 10 days of dox induced  5 days, . LikewisTo evaluate the functional significance of β-catenin knock-down on tumor development we proceeded with investigation in a subcutaneous xenograft tumor model in athymic nude mice.CTNNB1 inactivation on established tumors (21 to 31 days after xenografting) for a duration of 9 days were performed to mirror the time course from our in vitro experiments  while control clones remained unaffected by dox treatment. Moreover, and in accordance with the cell culture experiments, immunohistochemical analysis , there was a significant impact of CTNNB1 silencing upon dox treatment in the shβ clone .In a first step, short term experiments with eriments . Similaranalysis  revealedhβ clone . Indeed,in vitro and in vivo experiments, demonstrate that direct and specific β-catenin inactivation lead to alterations of proliferation, cell cycle and apoptosis which lead to a dramatic decrease in tumor development in a tumor model for ACC. Further experiments are needed in order to know if β-catenin inactivation suppresses the growth or prevents engraftment of the cells. Nevertheless, these results confirm 1/the biological consequence of Wnt/β-catenin pathway activation in adrenocortical tumorigenesis, and 2/the major therapeutic interest to target this pathway.In many cancers, the Wnt/β-catenin signalling pathway plays an important role regulating cell growth, motility, and differentiation Inhibition of Wnt/β-catenin pathway in a subgroup of aggressive ACC seems to be a interesting therapeutic target and should be evaluated in more detail in the future.Table S1PCR conditions and antibodies used.(XLS)Click here for additional data file.