Both homozygous mutant males and females produced normal gametes but were infertile because of abnormal seminal vesicle and vaginal morphogenesis. An ins-TOPGAL transgenic reporter spatiotemporally sustained Wnt/β-catenin signalling during the corresponding organogenesis. Therefore, C429Sβ-catenin should provide new insights into β-catenin as a universal component of Wnt/β-catenin signal transduction.Wnt/β-catenin signalling regulates numerous developmental and homeostatic processes. Ctnnb1  is the only protein that transmits signals from various Wnt ligands to downstream genes. In this study, we report that our newly established mouse strain, which harbours a Cys429 to Ser missense mutation in the  Upon Wnt ligand-mediated stimulation, β-catenin escapes from its default degradation, which is regulated by N-terminal phosphorylation and subsequent ubiquitination1The canonical Wnt/β-catenin signalling pathway is one of the pathways activated by Wnt11C429Sβ-catenin mouse strain, which harbours a cysteine 429 in the seventh armadillo repeat. The ins-TOPGAL transgenic reporter67896in vivo.In this study, we report the development of a C429Sβ-catenin congenic mouse strain  was caused by drastic changes in the sperm ejection route . The spein vivo during organogenesis of the seminal vesicle and vagina. As shown in the previous report131513We used ins-TOPGAL reporter transgenic micein situ hybridization with a seminal vesicle marker  homozygotes, ins-TOPGAL revealed not only the normal seminal vesicle bud (white bracket) but also an extra bud (black bracket) and an abnormal bend at the very caudal end of the Wolffian duct  for the wild-type Wolffian duct in vaginal formation. For instance, during normal development, the Wolffian duct may act as a guide for vaginal elongation. In the homozygotes, the abnormally elongated Wolffian duct may have indirectly prevented vaginal elongation, leading to vaginal aplasia.In the homozygous females, abnormally sustained ins-TOPGAL expression was also observed near the vagina rather than in the vagina itself. Ins-TOLGAL was abnormally expressed where the residual Wolffian duct verged caudally on the vagina . The mutAlthough the morphological defects were hyperplastic in the seminal vesicle and hypoplastic in the vagina, the caudal Wolffian ducts were commonly hyperplastic in both sexes. As morphologically distinctive differences in the Wolffian ducts appeared, specifically excessive branching in males and simple elongation in females, the hyperplastic defects in the Wolffian ducts may occur after sex determination.β-catenin cDNA with the canonical Wnt/β-catenin TOP-FLASH reporter plasmid24C429S and wild-type β-catenin proteins were equivalent  that may contribute to the fine-tuning of Wnt/β-catenin signalling.The structural prediction software programs Sift3839C429Sβ-catenin may serve as a new model of human infertility. Many studies have been reported particularly focusing on the N-terminus, which is a well known hot spot for tumorigenesisin vivo, the C429G  and C429Y  mutations have also been recorded in the cBioportal  and COSMIC (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/) human cancer databases, respectively. On the other hand, human β-catenin has hardly been anticipated as a risk factor for infertility; only one research group has attempted such a study but could not find any such mutationsWNT ligand genes have been found to be crucial for infertility, such as WNT4 (OMIM 603490) in the Serkal syndrome (OMIM 611812). Regarding the phenotypes in mice, vaginal aplasia . Prof. Yumiko Saga at the National Institute of Genetics, Japan kindly provided the ins-TOPGAL (RBRC02228) mouse strainC429S/C429Sβ-catenin males (n = 16) were mated with C57BL6/J females for >1 month.The The sperm transportation route was traced using the trypan blue dye. The dye was injected using a 27-gauge needle into the vas deferens of sacrificed 20-week-old mice following lower abdominal incision and exposure. The genital tracts were then removed by cutting the vas deferens, urethra and ureters. The seminal vesicles were further treated in the Hank's CMF solution containing 1% collagenase to remove the surrounding membranesVaginal plugs were collected with forceps. Each plug was suspended in 1× phosphate-buffered saline (PBS) and subsequently smeared on a glass slide. The samples were allowed to dry naturally and were stained with hematoxylin and eosin.C429Sβ-catenin was genotyped using the TaqMan® probe system . The following primers and probes were designed on the manufacturer's web site (https://www5.appliedbiosystems.com/tools/cadt/): F primer, 5′-CCTGTGCAGCTGGAATTCTCT-3′ and R primer, 5′-CTTGGCACACCATCATCTTGTTTT-3′; WT probe (VIC), 5′-CTAACCTCACTTGCAATAA-3′ and MT probe (FAM), 5′-CTAACCTCACTAGCAATAA-3′. PCR was performed under the conditions suggested by the manufacturer.In situ hybridization was performed according to standard protocols. The lower reproductive tracts (LRT) of foetal males were dissected and fixed overnight in PBS containing 4% PFA. LRTs were the dehydrated and stored in 100% methanol at −20°C until genotyping was completed. Prof. Nobuyuki Itoh of Kyoto University kindly provided the Fgf10 probe.3Fe(CN)3, 4 mM K4Fe(CN)6, 1 mM MgCl2 and 1 mg/ml X-gal in PBS), at 37°C on a rocking platform. The samples were subsequently fixed again in 15% neutral buffered formalin. Some specimens were cleared in SCALEVIEW-A2 regent  for 1 week. Other specimens were rinsed with PBS and used for paraffin sectioning.LRTs were fixed for 10 min in a fixative buffer . After rinsing twice with PBS, LRTs were incubated overnight in X-gal buffer . Histological sections were photographed on an Olympus AX80 microscope equipped with a DP50 digital camera and Studio Lite software . Some images were merged and trimmed using Photoshop CS6 .http://sift.jcvi.org/www/SIFT_enst_submit.html) assigned a score of 0.03 (‘Affected protein function'); a score nearer to 0 indicates more severe damage. PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) assigned a score of 0.953 (‘Possibly damaging'); a score nearer to 1 indicates more severe damage.We used the structural prediction software programs Sifthttp://thebiogrid.org/107880/summary/homo-sapiens/ctnnb1.html) and mouse studies (http://thebiogrid.org/198512/summary/mus-musculus/ctnnb1.html), respectively. The human and mouse β-catenin protein share 100% amino acid sequence identity. However, we should carefully consider proteins for which the interaction domains are uncharacterized or those also contain differences in the armadillo repeats.To date, in the BioGRID databaseC429Sβ-catenin strain and all β-catenin gene exons in the C429Sβ-catenin strain genome (Table S1). K.K. discovered the point mutations in the β-catenin gene . HiKa, HaKo and S.W. conducted in vitro fertilization and embryonic transfers (Table S3). H.T. and T.N. assisted with the histological analysis . Y.N. totalled the numbers of genotypes recorded in our mouse database . S.M. and R.F. made public database search to show how significant the C429S mutation. All authors reviewed the manuscript.T.M. and Y.G. wrote the main text. T.M. conducted most of the experiments in this study. Y.I. genotyped the Supplementary information


Triple Negative subset of (TN) Breast Cancers (BC), a close associate of the basal-like subtype (with limited discordance) is an aggressive form of the disease which convey unpredictable, and poor prognosis due to limited treatment options and lack of proven effective targeted therapies.We conducted an expression study of 240 formalin-fixed, paraffin-embedded (FFPE) primary biopsies from two cohorts, including 130 TN tumors, to identify molecular mechanisms of TN disease.The annotation of differentially expressed genes in TN tumors contained an overrepresentation of canonical Wnt signaling components in our cohort and others. These observations were supported by upregulation of experimentally induced oncogenic Wnt/β-catenin genes in TN tumors, recapitulated using targets induced by Wnt3A. A functional blockade of Wnt/β-catenin pathway by either a pharmacological Wnt-antagonist, WntC59, sulidac sulfide, or β-catenin  SiRNA mediated genetic manipulation demonstrated that a functional perturbation of the pathway is causal to the metastasis- associated phenotypes including fibronectin-directed migration, F-actin organization, and invasion in TNBC cells. A classifier, trained on microarray data from β-catenin transfected mammary cells, identified a disproportionate number of TNBC breast tumors as compared to other breast cancer subtypes in a meta-analysis of 11 studies and 1,878 breast cancer patients, including the two cohorts published here. Patients identified by the Wnt/β-catenin classifier had a greater risk of lung and brain, but not bone metastases.These data implicate transcriptional Wnt signaling as a hallmark of TNBC disease associated with specific metastatic pathways. Stratification of breast cancer (BC) into distinct histological or molecular subtypes has clinical utility for prognosis of outcome and prediction of treatment . Breast We analyzed mRNA expression from archival formalin-fixed paraffin-embedded (FFPE) tumor specimens from two breast cancer cohorts. Our data, together with meta-analysis of previous breast cancer microarray studies, indicate Wnt pathway activation in TNBC subtypes and provides evidence for an increased Wnt/β-catenin signaling associated with high grade, poor prognosis, and metastatic disease. In light of reports from Reis-Filho’s team , who estArchived FFPE tumor specimens were obtained from St. Mary’s Hospital, Montreal, Quebec, Canada (Quebec cohort) and Grady Memorial Hospital, Atlanta, GA (Georgia cohort) according to institutional guidelines (Emory University). Tumor content (> 50% inclusion criteria) was assessed by a board certified pathologist. Cohort sizes of 107 and 166 patients from St. Mary’s and Grady Hospitals respectively were analyzed at the Emory Biomarker Service Center . Both Georgia and Quebec samples (FFPE) were acquired following the acceptance of our protocols by the ethics committees  of the respective institutions. Archived FFPE samples were used for the study. All archived FFPE tumour specimens were obtained from St Mary’s Hospital  according to institutional guidelines.Data Deposition: Microarray data curetted under GEO series accession numbers [GSE17650 & GSE18539].FFPE samples were analyzed using DASL  expression chemistry , on the Illumina human cancer panel and a custom panel with breast cancer related genes. Depending upon tissue availability, specimens were obtained via either three 5 μm sections or 0.5 mm cores. Deparaffinization, RNA extraction and RNA purification were carried out using commercially available RNA High Pure Kit  modified as previously described . Prior thttp://www.cap.org/apps/docs/laboratory_accreditation/summary_of_recommendations.pdf]; HER2 testing [http://www.cap.org/apps/docs/committees/immunohistochemistry/summary_of_recommendations.pdf])”. Once standardized, the similar protocol was followed in both the cohorts. The comparison of IHC data with DASL data for ER, PR and HER2 had been carried out as mentioned in our earlier publication. Our data show that the concordance of DASL data with IHC data for all three receptors is very high, which is consistent with our previous published work relating mRNA and IHC protein levels [To determine whether the DASL assay yields comparable data to IHC data, the DASL assay gene intensity (expression) data were compared with the available IHC protein expression data for ER, PR and HER2 on the set of tumor samples. Standard clinical IHC testing was conducted for ER, PR and HER2 according to guidelines based on ASCO/CAP. This is now stated in the Methods section . “In total, we obtained 87 FFPE breast carcinomas that had previously been scored for the breast cancer markers, ER, PR and HER2 by immunohistochemistry (IHC) according to guidelines based on the ASCO/CAP recommendations for ER, PR and HER2 testing , and lysates were assayed for total protein (Bio-Rad protein assay kit) using BSA as a standard. The normalized lysates (20–40 μg protein) were resolved by 12.5% SDS-PAGE and transferred to nitrocellulose membranes .Breast cancer cell line (MDA-MB231) was seeded onto 6-well tissue culture dishes, and allowed to attach in culture medium supplemented with 10% FBS. A cell density of 60% to 70% was used for the transient transfection (Lipofectamine 2000) of β-catenin-specific SiRNA  into MDA-MB231 cells according to the manufacturer’s instructions. Transfected cells were collected after 24, 48, and 72 hours for analyses .A luciferase-based reporter gene was used to measure promoter activity of the TCF/LEF transcription factor . For SiRIn vitro wound healing assays were performed as previously described [Fibronectin directed migration assay was performed on Wnt-antagonist, WntC59  treated or β-catenin SiRNA transfected MDA-MB231 cells by transwell assay and scratch assay. Invasion assay was performed by transwell assay. Haptotaxis assays were carried out using transwell migration chambers (Costar Corp.) as previously described . Cells wescribed . In brieTo study the cytoskeletal arrangement, HCC38 and MDA-MB231TNBC cells were fixed, and permeabilized with PHEMO buffer. Phalloidin 555 was used for staining the cytoskeleton filamentous-actin and DAPI as a counter stain. Cells were imaged using a Zeiss  LSM 510 Meta confocal microscope with a Plan-Apochromat oil objective. Images were acquired using Zeiss LSM 510 software and processed using Adobe Photoshop CS3. To study the involvement of Wnt-pathway in integrin-directed migration in real time, video microscopy was performed. A scratch-would healing assay was performed on the confluent layer of cells . Time-lapse images were acquired with a Perkin Elmer Ultraview ERS  disk-spinning confocal system, mounted on a Zeiss Axiovert 200 M inverted microscope equipped with a 37°C stage warmer, incubator, and humidified CO2 perfusion system. Bright-field images were acquired with a Hamamatsu Orca-ER camera with a Plan-Neoflour 10× objective  at 10 minutes intervals for each image set.A full description can be found in the supplementary methods. In brief, DASL transcript intensities were quantile normalized in GenomeStudio and replicates were mean combined. Differential transcripts were determined using permutation testing  with a fMessenger RNA expression from FFPE samples were characterized using cDNA-mediated, Annealing, Selection, Extension, and Ligation (DASL)  for two Differentially expressed transcripts in the TNBC subtype of the Quebec and Georgia cohorts were annotated for canonical signal transduction pathways using the KEGG database . After Bin vitro using TNBC cell lines. A functional blockade of Wnt/β-catenin pathway by either a pharmacological Wnt-antagonist, WntC59, or β-catenin SiRNA mediated genetic manipulation demonstrated that a functional perturbation of the pathway is causal to the metastasis- associated phenotypes including fibronectin-mediated and invasion in TNBC cells. Similarly, Wnt/β-catenin signaling attenuator, sulindac sulfide also inhibited migration of TNBC cells on fibronectin (data not shown). We observed that the treatment with a pharmacological Wnt-antagonist, WntC59 blocked fibronectin-mediated migration and invasion in MDA-MB231 cells   is a dirTo address the issue of whether or not the modulation of Wnt-beta-catenin pathway causes the attenuation of its downstream signals, we tested the expression of active beta-catenin following beta-catenin SiRNA or sulindac sulfide treatment in MDA-MB-231 cells  and Wnt/β- catenin negative (Wnt-) patients with respect to overall survival, recurrence-free survival, metastasis-free survival, lung metastasis-free survival, brain metastasis-free survival, and lung metastasis-free survival in TNBC patients Figure . To inveOur study demonstrates two major findings that are consistent with current knowledge and advance our understanding of subset specific breast cancer etiology. First, using novel technologies to characterize FFPE materials we ascertained data consistent with clinical pathological subtype that also identified canonical and oncogenic Wnt signaling as an activated pathway in TNBC. These findings were confirmed using multiple published studies in diverse cohorts of patients across different microarray platforms, as well as independent experiments that identified Wnt induced targets which were consistently upregulated in TNBC subtypes. In the context of reports indicating associations of Wnt signaling with the basal-like subtype ,39, thes. Wnt signaling in osteoblasts regulates expression of the receptor activator of NFkappaB ligand, and inhibits osteoclastogenesis in vitro[Khramtsov et al., and others reported the association of Wnt signaling in TNBC with higher metastasis and poor prognosis ,41. This in vitro  Wnt signaling is upregulated in TNBC, and (2) Wnt/β-catenin signaling is associated with metastatic disease. Recently, Craig et al., have reported genome and transcriptome sequencing in prospective metastatic triple negative breast cancer [in vitro models.Wnt-C59 is a potent and selective Wnt signaling modulator with IC50 <0.11 nM in Wnt-Luc reporter assay for Wnt pathway inhibition, and with chemical/physical properties, suitable for t cancer . To furtPPARD) [Furthermore, other observations in this analysis include upregulation of Myc regulated genes  , uPAR (PPPARD) , and snaPPARD)   ,69, FOXAPPARD) , and MYBPPARD) . These dIn addition to the observed upregulation of Wnt signaling in TNBC, we also found association of Wnt signaling with metastatic disease. The Wnt/β-catenin classifier trained to identify oncogenic β-catenin signaling identified a disproportionate number of TNBC patients, supporting earlier observations of preferential Wnt activation in this subtype. Importantly, this classifier identified patients that were more likely to experience lung and brain metastases. These two metastatic routes have been associated with the basal-like subtype where Wnt signaling was noted as upregulated , and morTargeting the Wnt pathway has traditionally been difficult, but emerging modalities provide potential opportunities. Examples of small molecule inhibitors include sulindac, XAV939, ICG-001, and thiazolidinediones (TZDs) Figure . SulindaOur study contributes not only by identifying clinical markers associated with Wnt signaling such as histological grade 3 tumors and TNBC pathological subtype, but also increased risk for brain and lung metastases, thus recognizing Wnt signaling as a rational target in TNBC. The results of the study have implications for therapeutic target identification and the design of future clinical trials for this aggressive group of breast cancer. More genomic studies like this, however, are needed to create a genetic landscape of TNBC which will be utilized to differentiate “driver mutations” from “carrier mutations” and will guide therapeutics development. Individualized treatment will be possible only once we fully appreciate the biology of these genetic abnormalities.Introduction to Additional file 3: Details of Figure S1: Assay reproducibility in the Quebec cohort measured by a distribution of Pearson R2 coefficients between RNA replicates on the A) human, and B) breast cancer (BC) DASL panels. C) Overlap of gene content on the human (dashed line) and BC (solid line) DASL panels, and D) replicated samples on both platforms. E) Distribution of inter-platform Pearson R2 correlations for samples run on both platforms, and F) a Venn diagram of differentially regulated genes found using the overlap of patients and genes on the human and BC DASL panels. Expression of ESR1, PGR, and ERBB2 by pathology determined subtype for HR + (white), HER2+ (grey), and TN (black) subtypes in the Quebec cohort on the G) human and I) BC DASL panels, as well as the K) Georgia cohort on the BC DASL panel correspond with expected clinical pathological subtype. Cohort sizes by pathology subtype for the Quebec cohort on the H) human and J) BC DASL panels, as well as the L) Georgia cohort on the BC DASL panel are depicted by pie charts. Details of Figure S2: Analysis of upregulated probes in TNBC as compared to other subtypes in context with KEGG [ith KEGG  signal tith KEGG , E) UNCCith KEGG , and F) ith KEGG  were meaith KEGG , E) UNCCith KEGG  and F) Sith KEGG . Detailsith KEGG  pathway ith KEGG , E) UNCCith KEGG , and F) ith KEGG  measuredith KEGG , E) UNCCith KEGG , and F) ith KEGG . Hierarcith KEGG . Pathwayith KEGG , E) UNCCith KEGG , and F) TN: Triple negative; HR: Hormone receptor; ER: Estrogen receptor; PR: Progesterone receptor; HER2: Human epidermal growth factor receptor 2; OS: Overall survival; RFS: Recurrence-free survival; MFS: Metastasis-free survival; DASL: cDNA mediated annealing, selection, and ligation.The authors declare that they have no competing interests.ND was involved in designing / conducting the cell-line based experiments, and actively contributed to writing the manuscript. BB is the key contributor in the data analyses, and one of the key authors on writing the manuscript together with ND. CM helped design the DASL component of the study, performed the DASL analysis, and contributed to writing the manuscript. MO prepared RNA. ZC performed statistical analysis. PD contributed in designing experiments and contributed to writing the manuscript. WT prepared RNA and performed some DASL experiments. CC conducted pathological analysis. KK was involved in coordinating the study. GS conceived of the study, and participated in its design. MA participated in the design of the study and helped to draft the manuscript. MB helped conceive and design the molecular component of the study and contributed to writing the manuscript. BLJ participated in the overall conception and the design of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/537/prepubIllumia Cancer Panel.Click here for fileCustom Cancer Panel.Click here for fileFigure S1-Table S3: Assay reproducibility in the Quebec cohort measured by a distribution of Pearson R2 coefficients between RNA replicates. Figure S2: Analysis of upregulated probes in TNBC as compared to other subtypes in context with KEGG [Figure S3: Pathway expression of canonical KEGG signal transduction pathways [Figure S4: Canonical Wnt expression of KEGG [Figure S5: Pathway expression of experimentally derived oncogenic signaling pathways from Bild et al. [Figure S6: Experimentally induced Wnt/β-catenin pathway expression from Bild et al. [Figure S7: Expression of Wnt/β-catenin components in patients classified as Wnt compared to Wnt + (Wnt classifier signature) in each of the 11 studies analyzed in the meta-analysis shows greater expression of tumors classified as Wnt + (Wnt classifier signature). Table S3: Table identifying the number of patients from each cohort in a meta-analysis of 11 studies and 1,878 patients with pathological or intrinsic determined subtype, grade, and lymph node status. Each category is broken down by total number of Wnt + (Wnt classifier signature) and Wnt- patients identified by the Wnt/β-catenin classifier and then analyzed for overrepresentation using Fisher’s exact test.ith KEGG  signal tpathways  were mea of KEGG  pathway d et al.  measuredd et al. . SignifiClick here for fileSupplementary Analysis and Statistical Methods.Click here for fileReal-time Video Microscopy: A scratch-would healing assay was performed on the confluent layer of cells . Time-lapse images are acquired with a Perkin Elmer Ultraview ERS  disk-spinning confocal system, mounted on a Zeiss Axiovert 200 M inverted microscope equipped with a 37°C stage warmer, incubator, and humidified CO2 perfusion system. Bright-field images are acquired with a Hamamatsu Orca-ER camera with a Plan-Neoflour 10x objective  at 10 minutes intervals for each image set. HCC38 cells were treated with sulindac sulfide, and their movement was compared with the vehicle treated cells.Click here for fileReal-time Video Microscopy.Click here for file


FLPo recombinase under control of the noradrenergic-specific Dopamine-Beta-Hydroxylase (DBH) gene. We found that by co-electroporating a circular vector expressing Cas9 and a locus-specific sgRNA, we could target FLPo to the DBH locus in ES cells with shortened 1 kb homology arms. Two different sites in the DBH gene were targeted; the translational start codon with 6–8% targeting efficiency, and the translational stop codon with 75% targeting efficiency. Using this approach, we established two mouse lines with DBH-specific expression of FLPo in brainstem catecholaminergic populations that are publically available on MMRRC (MMRRC_041575-UCD and MMRRC_041577-UCD). Altogether, this study supports simplified, high-efficiency Cas9/CRISPR-mediated targeting in embryonic stem cells for production of knock-in mouse lines in a wider variety of contexts than zygote injection alone.CRISPR/Cas9 mediated DNA double strand cutting is emerging as a powerful approach to increase rates of homologous recombination of large targeting vectors, but the optimization of parameters, equipment and expertise required remain barriers to successful mouse generation by single-step zygote injection. Here, we sought to apply CRISPR/Cas9 methods to traditional embryonic stem (ES) cell targeting followed by blastocyst injection to overcome the common issues of difficult vector construction and low targeting efficiency. To facilitate the study of noradrenergic function, which is implicated in myriad behavioral and physiological processes, we generated two different mouse lines that express  Toward knock-in transgenic mouse line production, targeted double stranded breaks have emerged as a powerful tool to increase rates of homologous recombination through homology-directed repair (HDR). The CRISPR system offers simplified sequence targeting for HDR and is comprised of a Cas9 nuclease that is guided to specific target sites by a short sgRNA complementary to the target sequence followed by a PAM motif (NGG). CRISPR strategies have been used with high efficiency for knockout mutations, small deletions, and oligo mediated HDR in mouse zygotes in a potentially shorter time frame. However, successful zygotic knock-ins and germline transmission of large targeting cassettes –7 remainin vitro, and show low rates of targeting efficiency. Toward addressing these problems, we have applied Cas9/CRISPR mediated HDR methods to ES cell targeting for large construct knock-in mouse generation. Since Cas9 mediated double stranded breaks have been shown to stimulate HDR with smaller homology targeting arms than traditional gene targeting vectors in several cell types, targeting vector construction becomes much simpler using only PCR products joined by isothermic cloning [While more time consuming, embryonic stem (ES) cell targeting and morula aggregation or blastocyst injections remains a common and at times more forgiving path to mouse line generation, particularly for low frequency targeting events or vectors of high complexity where large numbers of ES cell colonies can be rapidly screened. However, traditional ES cell targeting can also benefit from continued optimization, particularly when working with large or complex targeting vectors that are difficult to construct and work with  cloning  and in oIn our initial application of CRISPR/Cas9 integrated ES cell targeting, we chose to generate two FLPo mouse lines specific to Dopamine-Beta-Hydroxylase (DBH) expression. DBH is a key noradrenaline synthesizing enzyme and marker of brainstem and peripheral noradrenergic neurons and noradrenaline producing cells which are critically involved in a variety of behavioral and physiological processes including learning and memory, fear, anxiety, cardiovascular and respiratory function, arousal, mood, attention, and appetite, among others . Within To genetically access noradrenergic populations and demonstrate the feasibility of using CRISPR/Cas9 in ES cell gene targeting while maintaining pluripotency and germline transmission capabilities, we aimed to produce two DBH targeted FLPo mouse lines, one that is a FLPo knock-in into the DBH start codon, resulting in a null allele, and a second that is a p2a-FLPo knock-in into the 3’ end of the DBH coding sequence, leaving the DBH locus otherwise unperturbed.For cardiac perfusion mice were euthanized with an overdose of anesthetic . The pedal reflex was checked to ensure the mouse was fully anesthetized.Studies were approved by Baylor College of Medicine Institutional Animal Care and Use Committee under protocol AN-6171.Targeting vectors were constructed using standard cloning procedures. For the start codon knock-in mouse line (hereon known as DBH-FLPo) targeting vector, the 5’ homology arm is a 1kb fragment going from -1000 to +0 relative to the ATG of the gene. The 3’ homology arm is a 1kb fragment extending from +106 to +1105 position , was cloned into the BbsI sites of the px330 vector [Sequences of the targeting vectors and px330 vectors have been uploaded to GenBank  and plasmids are available upon request.Embryonic stem (ES) cells (AB2.2) were electroporated with 15–20 ug of varying ratios of the px330_DBH_sgRNA vector . NeomyciWe maintained colonies of all of our mouse strains by backcrossing to C57BL/6J mice. For routine genotyping, we carried out PCR amplification of DNA from ear punch preparations using the boiling alkaline lysis procedure . FLPo-spTo achieve germline deletion of the Lox2722-flanked neomycin cassette, we crossed DBH-FLPo and DBH-p2a-FLPo males with congenic Cre-deleter females (B6N-FVB-Tg(ACTB-Cre)2Mrt). For fate-mapping of FLP expression, we crossed DBH-FLPo and DBH-p2a-FLPo heterozygotes with homozygous RC::FEE mice that conditionally express EGFP upon FLPo recombination . Rosa26 To determine if homozygous mice were viable, we interbred heterozygous DBH-FLPo and DBH-p2a-FLPo mice and used a multiplexing locus-specific genotyping reaction for the offspring. DBH-FLPo specific genotyping primers were: 5’- GCCACTCTGCTTCGATTCTC, 5’- ACAGCAGTGCCGTACATG, and 5’- GGGATGATGGTGAACTCCCA and yield a 297 bp band (wt) and/or a 551 bp band (targeted). DBH-p2a-FLPo specific genotyping primers were: 5’-CCTAAGATCACCTCCACGCT, 5’-TCTCCGTTCAGTTGGACCTC and 5’ GGGATGATGGTGAACTCCCA and yield a 306 bp band (wt) and/or a 517 bp band (targeted).Dbhtm1(DBH-FLPo)Rray/Mmucd, MMRRC: 041577-UCD) and DBH-p2a-FLPo (Dbhtm2(DBH-FLPo)Rray/Mmucd, MMRRC: 041575-UCD) mouse lines have been accepted to the Mutant Mouse Regional Resource Center (MMRRC) and are unconditionally available to the academic research community.All animal experiments were performed with the approval of IACUC. Both the DBH-FLPo (Off-target sequences were identified using the Optimized CRISPR Design tool (crispr.mit.edu). The top 5 sequences for each sgRNA were amplified from selected targeted ES cells and sequenced to determine if any mutations or changes occurred in off target sites.4–8 week old adult mice were transcardially perfused with 0.1M phosphate-buffered saline (PBS) then with 4% paraformaldehyde (PFA) in PBS. Brains were dissected out and drop fixed for two hours in 4% PFA before a PBS rinse and equilibration in 20% sucrose in PBS. Brains were then sectioned into 30 μm sections, mounted on slides, and labeled with immunofluorescent antibodies. We stained overnight with anti-tyrosine hydroxylase antibody to identify catecholaminergic neurons  followed by 2 hours with donkey anti-rabbit Cy3 secondary  in 0.1% Triton-X in PBS (PBST) with 5% donkey serum. Images were collected on a Zeiss upright epifluorescent microscope.Co-stain cell count analysis was performed by manually counting on microscopy images the number of cells that either expressed EGFP without TH, TH without EGFP, or co-expressed EGFP and TH from 10 randomly selected images of known brainstem catecholaminergic populations from 3 mice per mouse line.-/- knockout generated [For the DBH-FLPo mouse line, the FLPo sequence was inserted into the transcriptional start site of the DBH gene. This resulted in the deletion of 106 bps of exon 1 and expression of FLPo  followinenerated . For posIn parallel, we attempted to create founder lines through direct oocyte injection, by 1) co-injection of the circular targeting vector without the neomycin resistance cassette and px330_DBH-sgRNA1 vector into the pro-nucleus; and 2) co-injection of the circular targeting vector without the neomycin resistance cassette and a px335_DBH_sgRNA1 vector that expresses a modified Cas9 nickase, also into the pro-nucleus . While tThus, we pursued the traditional ES cell electroporation route while integrating CRISPR/Cas9 to see if targeted double stranded break could increase the targeting efficiency with our reduced 1kb homology arms. We co-electroporated the px330_DBH_sgRNA1 vector with the targeting vector at four different molar ratios . Under the 0:1 and 0.5:1 px330 vector:donor vector conditions, we did not see any targeted clones, while under the 1:1 conditions and 10:1 conditions, we had 8.3% and 6.25% targeting, respectively . Previou+/- mice have no reported phenotypes, embryos during development have lower levels of noradrenaline and homozygous DBH-/- mice are embryonically lethal unless rescued by administration of dihydroxyphenylserine (DOPS), a noradrenaline precursor [While heterozygous adult DBHrecursor . Additiorecursor ,34, alcorecursor , post-trrecursor , migrainrecursor ,38, and recursor . To miniBased on our previous results suggesting that higher levels of the px330 vector resulted in higher targeting efficiencies, we co-electroporated the px330_DBH-sgRNA2 vector and targeting vector into ES cells using the highest 10:1 ratio. In strong contrast to the translational start knock-in, targeting of p2a-FLPo at the 3’ terminus of DBH gene resulted in a 12-fold increase of targeting efficiency (75%) over targeting into the 5’ terminus with the same length homology arms and cassette size . Becausein vitro transcribed Cas9 mRNA, transcribed sgRNA, and circular targeting vector into the oocyte cytoplasm; and 2) injection of Cas9 protein, transcribed sgRNA, and circular targeting vector into the oocyte cytoplasm  injection of ytoplasm . AlthougGenomic sequences that are similar to the sgRNAs used for targeted double stranded break may cause unintended gene mutations or editing at off-target sites. To detect potential off-target events, we predicted the off-target sites for each sgRNA and selected the top 5 sites for follow-up. We amplified and sequenced these loci from three correctly targeted ES cell clones for each line. We did not detect any genetic modification in the DBH-FLPo sgRNA off-target sites  or the D+/- animals have no known phenotypes. We saw no gross morphological or phenotypic deficits in our heterozygous DBH-FLPo or DBH-p2a-FLPo animals. To confirm that the DBH-FLPo is a recessive loss-of-function mutation, we interbred heterozygotes and genotyped offspring at weaning. We saw no homozygotes among 24 surviving pups  reporter line to reveal the expression of FLPo recombinase. Unlike the TgDBH-Cre line , expression of EGFP was limited to TH expressing populations in the brainstem, including the A6 locus coeruleus, A5, A7, and A1/C1 and A2/C2 populations . Cas9 mRNA was transcribed using the mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies AM1345) and cleaned up with the MEGAclear Transcription Clear-Up kit (Life Technologies AM1908). sgRNA was transcribed using the MEGAshortscript T7 kit (Life Technologies AM1354). Cas9 protein used was purchased from PNA Bio (PNA CB01). Injections were performed through the Baylor College of Medicine Genetically Engineered Mouse (GEM) Core.(PDF)Click here for additional data file.S1 FigA) Genotyping results from 24 intercrossed heterozygous DBH-FLPo offspring. Wt band is 297 bps and targeted band is 551 bps. No homozygous mice were seen upon weaning. B) Genotyping results from 33 intercrossed DBH-p2a-FLPo offspring. Wt band is 306 bps and targeted band is 517 bps. Ten out of the 33 pups were homozygous.(EPS)Click here for additional data file.


Because humans and animals encounter various situations, the ability to adaptively decide upon responses to any situation is essential. To date, however, decision processes and the underlying neural substrates have been investigated under specific conditions; thus, little is known about how various conditions influence one another in these processes. In this study, we designed a binary choice task with variable- and fixed-reward conditions and investigated neural activities of the prelimbic cortex and dorsomedial striatum in rats. Variable- and fixed-reward conditions induced flexible and inflexible behaviors, respectively; one of the two conditions was randomly assigned in each trial for testing the possibility of condition interference. Rats were successfully conditioned such that they could find the better reward holes of variable-reward-condition and fixed-reward-condition trials. A learning interference model, which updated expected rewards  used in variable-reward-condition trials on the basis of combined experiences of both conditions, better fit choice behaviors than conventional models which updated values in each condition independently. Thus, although rats distinguished the trial condition, they updated values in a condition-interference manner. Our electrophysiological study suggests that this interfering value-updating is mediated by the prelimbic cortex and dorsomedial striatum. First, some prelimbic cortical and striatal neurons represented the action-reward associations irrespective of trial conditions. Second, the striatal neurons kept tracking the values of variable-reward condition even in fixed-reward-condition trials, such that values were possibly interferingly updated even in the fixed-reward condition. The cortico-basal ganglia circuit is involved not only in movement control, but also in inference-, experience- and reward-based decision making  were electrophysiologically recorded to investigate neural substrates of condition interference. We used rats because their parallel cortico-basal ganglia circuits for decision making are well examined and established ; two rats performed both the behavioral and electrophysiological experiments, and the remaining three rats performed only the behavioral experiments. Food was provided after the task to maintain animal body weight at no less than 85% of the initial level. Water was supplied freely.All experiments were conducted in a 36 × 36 × 37 cm experimental chamber (O'Hara & Co. Ltd.) placed in a sound-attenuating box  to characterize the behaviors in variable- and fixed-reward conditions. The extinction phase was conducted after the reward probability of variable-reward-condition block was identical to that of fixed-reward condition. In the extinction phase, we investigated the sensitivity to this treatment from the choice preferences of rats. Flexible or inflexible behaviors should change or retain choices with the outcome extinction, respectively.After rats practiced the free choice task, they were anesthetized with sodium pentobarbital  and placed in a stereotaxic frame (Narishige). Atropine sulfate (0.1 mg/kg) was also administered at the beginning of the surgery to reduce the viscosity of bronchial secretions  and a Cerebus data acquisition system (Cyberkinetics Inc.) with an amplified gain of 1000, a band-pass filter of 0.3–7500 Hz, and a sampling frequency of 30 kHz. We then applied an offline digital high-pass filter of 200 Hz . When the signal became below or above its root mean square (RMS) times 5.5, the signal was defined as spike activity , and a positive current of 10 μ A was passed for 10–20 s through one or two electrodes of each bundle to mark the final recording positions  were removed, and the remaining sequences of successful trials (in which rats successfully made a left or right choice) were used.Figure 4Bi). If the choice of each condition was independently learned and the interleaved trial caused no interference, conditional probabilities in the two trial sequences became the same.We first analyzed choice preferences during the extinction phase to identify whether rats had flexible or inflexible behaviors in the variable- and fixed-reward conditions. We then assessed the interference of variable- and fixed-reward conditions in the choice behaviors. We compared conditional choice probabilities between two trial sequences: repeated sequences , in which probabilities were calculated based on the action-outcome experience in the last trial with a same condition; and interleaved sequences , in which probabilities were calculated based on the experience in the next-to-last trial with the same condition, so that the last different-condition trial was ignored (a ∈ , the reward as r ∈  and the condition as C ∈ . We assumed that rats predicted the expected reward of each choice  in each condition, Qa, C: rats had four action values in total. A choice probability was predicted with the following soft-max equation based on the action values:C(t) and C were trial and non-trial conditions, i.e., C ≠ C(t); for example, when the presented trial was a variable-reward condition, C was a fixed-reward condition. GC(t) was a free parameter depending on the trial condition. This parameter adjusted the contribution of action values of a non-trial condition in the choice prediction.We analyzed choice behaviors of rats with reinforcement learning models and a fixed-choice model to test (i) whether interference occurred in choice learning, and (ii) whether it occurred in the value updating or action selection phase. We denoted the action as Cq was a free parameter depending on the value condition. If the fixed-choice model fit a choice behavior, the behavior had no-learning and no condition-interference in value updating.A fixed-choice model had the action value as a free parameter, assuming a constant value in all trials:Qa, C, in accordance with Ito and Doya (a(t), r(t) and C(t) were the action, reward, and condition at trial t, respectively. Action values of both variable- and fixed-reward conditions were updated every trial, irrespective of the trial condition. α1, α2, k1, and k2 were free parameters. α1 showed the learning rate in the chosen option, and α2 showed the forgetting rate in the un-chosen option. k1 and k2 indicated the strengths of reinforcers in reward and non-reward outcomes, respectively. α1 and α2 depended on the trial condition, C(t), and the action-value condition, C, to capture differences in (i) learning of variable- and fixed-reward conditions, and (ii) learning by its own condition and by the other condition. Equation (3) had 10 parameters in total.Figure and Doya :(3)Qa,V2 = k2 = 0, the equation became a standard Q-learning (Q-learning) , and we referred to the full-parameter equation as a differential forgetting Q-learning (DFQ-learning)  could take a variety of updating rules by selecting utilized parameters, so that updating rules for the values of variable- and fixed-reward conditions  could be different. When we set αC, C1,  = αC, C2,  = 0 where C ≠ C in value updating (Equation 3) and CG = 0 in action selection (Equation 1), the equations deal with variable- and fixed-reward conditions independently; we referred to the model as an independent model. When we set αC, C1,  = αC, C2,  = 0 in Equation (3), the model independently updated action values of each condition, but interferingly predicted the choices; we referred to it as an action interference model. Also, when we set CG = 0 in Equation (1), the model interferingly updated action values of the variable- and fixed-reward conditions; we referred to it as a learning interference model.When we set αInitial action values for reinforcement learning models were 0.5 in the left and right choices of the variable-reward condition , and were 0.9 and 0.5 in the optimal and non-optimal choices of the fixed-reward condition.Z, was defined as follows:N and z(t) were the number of trials and the likelihood at trial t, respectively. The likelihood, z(t), was defined as follows, with the predicted left choice probability P(a(t) = L):We employed the normalized likelihood to test how well the models fit the choice behaviors of rats  was a spike count with a time window of 600 ms at trial t. C(t), a(t), and r(t) were the trial condition , action (1 or −1 for the right or left choice), and reward (1 or −1 for the reward or non-reward outcome) at trial t, respectively. These variables at trial t − 1 were also included in the regression analysis as C(t − 1), a(t − 1), and r(t − 1). X(t) showed their interactions  with a dummy variable of 1 or −1; each interaction had 4, 4, 4, and 8 combinations, and the total was 20 combinations. When a neuron represented at least one combination of each interaction, we defined the neuron as interaction- or association-coding neuron. For example, when a neuron represented a combination of action and reward, i.e., a(t) × r(t), we defined the neuron as action-reward association coding. MC = C(t) were the five model variables for the presented-trial condition, consisting of the action values , QR, C(t)), state value , chosen value (Qa(t), C(t)) and policy  − QR, C(t)) . In contrast, values for the fixed-reward condition did not appear in the regression analysis, because the values were turned out to be constant and were difficult to capture with the analysis (see Results). T(t) was the trial number for detecting a slow drift of firing rate. When Equation (6) had significant regression coefficients , the neuron was defined as encoding the corresponding variables. In the model variables  and MC = V), we could not get enough neurons encoding each individual variable, because of our sparse recording. Thus, we defined neurons as value coding when they encoded at least one of the five model variables. Model variables were derived from the proposed reinforcement-learning model in which free parameters were set to achieve the maximum likelihood in each session.To test how neural activities in the prelimbic cortex and dorsomedial striatum were modulated during the task, we employed a stepwise multiple regression analysis . Regression analysis was used to investigate neural correlates with actions, rewards, conditions, and associations. The analysis also detected neural correlates with the variables in a reinforcement learning model  became as follows:First, to investigate neural correlates of actions , rewards  and R-O associations, regression analysis was conducted only with neural activities during variable-reward-condition trials. By reducing the condition terms at trial t, Equation (6) became as follows:Second, to investigate neural correlates of conditions  and S-O associations, we extracted trials in which rats selected the optimal side of fixed-reward condition. By focusing on the optimal side, we excluded a potential bias caused by the choice asymmetry in the fixed-reward condition in which rats mainly selected the optimal side. By reducing the action terms at trial In Equation (8), model variables had 4 terms because the chosen value became identical to the action value in either a left or right choice. Third, to investigate value-coding neurons, the regression analysis of Equation (6) was applied to neural activities in all trials. Value-coding neurons were also investigated in fixed-reward-condition trials; in this case, Equation (7) was applied for fixed-reward-condition trials.U-test, p = 1.12E-8 and 1.06E-8 in the high and low reward probabilities). Thus, the speed to find the better rewarding choice in variable-reward condition depended on the optimal side of fixed-reward condition, suggesting that there was some behavioral interference between the two conditions.Figure U-test, p = 3.30E-11 − 1.96E-34). This result indicated that rats had flexible and inflexible behaviors in variable- and fixed-reward conditions, respectively.Figure U-test, p = 1.58E-31 and 9.47E-30). Although choices in the fixed-reward condition were also significantly affected by the previous variable-reward-condition trial , the condition interference in fixed-reward condition was weak as compared to that in the variable-reward condition. Taken together, these results indicate that flexible behaviors are more likely affected by events in another condition than inflexible behaviors.If rats had condition interference, choices in one condition were affected with action-outcome experiences in the other condition. Figure l Figure  , while the results were completely opposite in the fixed-reward condition (p = 6.63E-6 − 4.33E-8), indicating that the choice strategy depended on the condition. Based on the results, we employed the FQ-learning and fixed-choice models for the learning rules of variable- and fixed-reward conditions, respectively. We then compared normalized likelihoods among the independent model, the action interference model, and the learning interference model , and this significant trend was the same when DFQ-learning and Q-learning were employed as the learning rules of variable- and fixed-reward conditions, respectively (p = 2.84E-8 − 1.94E-28). In the learning interference model, FQ-learning for the variable-reward condition updated the values with the events in both the variable-reward-condition and fixed-reward-condition trials, while the fixed-choice model for fixed-reward condition had a fixed-choice probability in all trials . Thus, these results indicate that (i) condition interference occurred in the value updating phase, (ii) the choices in variable-reward condition were affected adversely by events in the fixed-reward condition, and (iii) choices in the fixed-reward condition were not affected by events in either the variable- or fixed-reward conditions.First, we separately fit reinforcement learning models to the choices in variable- and fixed-reward conditions and analyzed normalized likelihoods in 2-fold cross validation Figure . In the l Figure , to testV, V1, ) was 0.734 ± 0.0192, while the learning rate with events in the fixed-reward condition  was 0.432 ± 0.0206. This indicates that interference from the fixed-reward condition was weaker than learning from the variable-reward condition .In the learning interference model, the degree of condition interference was captured by the free parameters, i.e., learning rates. We set the free parameters to achieve the maximum likelihood in each session; in variable-reward condition, the learning rate for updating values with events in the variable-reward condition  in the prelimbic cortex and 26 neurons  in the dorsomedial striatum. Some neurons were recorded from a slightly central part of the dorsal striatum, but we analyzed them as the part of dorsomedial striatum Figure  Stalnak.z-test, p < 0.05). In action coding, both prelimbic and striatal neurons participated significantly    Figure . A repre) Figure .n = 26)   and associations between conditions-rewards , we conducted regression analysis on the trials in which rats selected the optimal side of the fixed-reward condition (Equation 8). There was no significant difference in the number of prelimbic cortical and dorsomedial striatal neurons that functioned as reward-coding neurons Figure , consist) Figure . The num) Figure . A repre) Figure . Overall) Figure .In addition to elucidating neural coding of basic task features , investigations of the coding of decision variables  are important for understanding learning algorithms of rats. Values were derived from the learning interference model which achieved the highest normalized likelihood among the models Figure . Free paz-test, p < 0.05). Especially after a reward or no-reward cue, a larger proportion of neurons in the striatum encoded the values than in the prelimbic cortex    association Figure . The R-OIn addition, a stimulus-outcome (S-O) association was observed in the dorsomedial striatum Figure . This asSuch associative representations were mainly found after choices and reward cues in our study; at this time, value updating is required for deciding future actions. Value updating requires calculating a temporal difference error, which is known to be represented in midbrain dopaminergic neurons (Schultz et al., Neural potentiation in R-O associations Figure  or rewarThe prelimbic cortex and dorsomedial striatum mainly encoded values of the variable-reward condition, and not of the condition in the on-going trial Figures , 11. In Our behavioral analyses with reinforcement learning models indicate that rats had an interfering value-updating in the variable-reward condition. Our electrophysiological study suggests that this interfering value-updating is mediated by the prelimbic cortex and dorsomedial striatum. First, although some dorsomedial striatal neurons represented condition-reward associations, the prelimbic cortex and striatum associated actions with rewards irrespective of trial conditions. Second, striatal neurons kept tracking values of the variable-reward condition even during the fixed-reward condition, such that values were possibly interferingly updated even in the fixed-reward condition.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The rate of neurogenesis is determined by 1) the number of neural stem/progenitor cells (NSCs), 2) proliferation of NSCs, 3) neuron lineage specification, and 4) survival rate of the newborn neurons. Aging lowers the rate of hippocampal neurogenesis, while exercise (Ex) increases this rate. However, it remains unclear which of the determinants are affected by aging and Ex. We characterized the four determinants in different age groups  of mice that either received one month of Ex training or remained sedentary. Bromodeoxyuridine (BrdU) was injected two hours before sacrificing the mice to label the proliferating cells. The results showed that the number of newborn neurons massively decreased (>95%) by the time the mice reached nine months of age. The number of NSC was mildly reduced during aging, while Ex delayed such decline. The proliferation rates were greatly decreased by the time the mice were 9-month-old and Ex could not improve the rates. The rates of neuron specification were decreased during aging, while Ex increased the rates. The survival rate was not affected by age or Ex. Aging greatly reduced newborn neuron maturation, while Ex potently enhanced it. In conclusion, age-associated decline of hippocampal neurogenesis is mainly caused by reduction of NSC proliferation. Although Ex increases the NSC number and neuron specification rates, it doesn't restore the massive decline of NSC proliferation rate. Hence, the effect of Ex on the rate of hippocampal neurogenesis during aging is limited, but Ex does enhance the maturation of newborn neurons. Neural stem/precursor cells (NSCs) in the subventricular zone and subgranular zone of dentate gyrus in hippocampus produce new cells throughout adulthood ,2. In thHippocampal neurogenesis continues throughout life and is known to be affected by multiple factors. Among these factors, aging is a well-known negative regulator of hippocampal neurogenesis ,9–12. In+ cells by the number of nestin+ cells. As the newly proliferated cells differentiate into immature neurons, they stop expressing nestin and start to produce doublecortin (DCX) [+DCX+) cells was considered as the levels of neurogenesis. The neuron lineage specification rate was calculated by dividing the number of BrdU+DCX+ cells by the number of BrdU+ cells. The survival rate of newborn cells was determined by dividing the number of BrdU+ cells, at four weeks after BrdU injection, by the number of BrdU+ cells at two hours after BrdU injection. We further measured the number of dendrite branches and the dendritic lengths of the DCX+ immature neurons as indications of newborn neuron maturation.To answer this question, we characterized the four determinants of adult hippocampal neurogenesis in the exercise mouse (Ex) group and the sedentary mouse (Sed) control group, that are further categorized by age into subgroups. We used the stem cell marker, nestin, to label the NSCs and we used bromodeoxyuridine (BrdU), a thymidine analog, to label the proliferating cells . The numin (DCX) . DCX, exMale C57BL/6J mice obtained from the Laboratory Animal Center, National Cheng Kung University were used for all experiments. All experimental protocols were performed according to National Institutes of Health guidelines for animal research  and were approved by the National Cheng Kung University Institutional Animal Care and Use Committee. The four determinant elements of the adult hippocampal neurogenesis were analyzed at the ages of 3, 6, 9, 12 and 21 months. Six weeks before the due time, half of the animals were subjected to treadmill exercise as described below. Six mice were assigned to each group.The detailed protocol of Ex training has been described elsewhere . The 5-wBrdU  was intraperitoneally injected into mice one day after the completion of Ex training program. Two hours or four weeks after the BrdU injection, mice were anesthetized and perfused with ice-cold phosphate buffered saline as previously described . Brains The procedure for immunohistochemistry has been described previously . The par+DCX+ double immunohistochemical labeling, free-floating tissues were first immunostained with mouse anti-BrdU  and developed by reaction with 3,3′-diaminobenzidine using nickel-enhanced method (deep purple). The procedure was followed by second labeling with goat anti-DCX , then visualized by reaction with 3,3′-diaminobenzidine only (brown).For BrdU+ cells, that of BrdU+ cells and that of DCX+ cells were counted in every sixth section. To prevent observer bias, sections were coded to ensure that the observer was blind to their condition. Positive cells were counted through the 40X objective under a Ziess microscope. The total number of labeled cells per section was determined and divided by the slide selection ratio (i.e. 16/96), to obtain the total number of labeled cells in each dentate gyrus [A modified stereology protocol was used to quantify labeled cells . The entte gyrus .+ cells were selected to estimate the maturation rate of newborn neurons. Only those DCX+ cells showing minimal dendritic tree overlap with adjacent cells were included to avoid ambiguity [http://rsb.info.nih.gov/ij). The observer was blind to the experimental condition. Ten neurons in 10 brain slices obtained from each animal were measured. Five animals in each group were analyzed (n = 5 for each group).The dendritic branch number and length of DCXmbiguity . By usinp < 0.05) interactions were found.Data are presented as mean ± standard error. Two-way ANOVA was used to analyze the two main effects  and their possible interaction. Bonferroni post-hoc tests were performed if significant  and Ex  altered the number of BrdU+ cells, without interaction between these two factors .+ newborn neurons were mainly located adjacent to subgranular zone (+ newborn neurons projected into the granular cell layer or crossed the granular cell layer into the molecular layer (p < 0.001) and Ex  affected the number of DCX+ newborn neurons. However, a significant interaction between these two factors  was evident. Bonferroni post-hoc tests indicated that there were differences between the 3-month-old Ex and the 3-month-old Sed groups  and Ex  significantly influenced the number of BrdU+DCX+ cells. No interaction between these two factors was evident   . Compareold mice . These rTo further investigate the age effect on neurogenesis, we have added a group of 1-month-old mice .+ cell number in the hippocampal dentate gyrus of mice at the ages of 3, 6, 9, 12 and 21 months (p = 0.004) and Ex  altered the number of NSC, without significant interaction between these two factors (p = 0.276). Using 3-month-old mice group as a reference point, the decline of NSCs by the age of 9-month was insignificant, where the measured value was slightly more than 10% (The effects of aging and those of Ex on NSCs were determined by counting the nestin1 months . Two-way p > 0.5). Howeverthan 10% .We have also estimated the number of NSC of the 1-month-old mice , but not Ex , significantly changed the proliferation rate (p > 0.5). The proliferation rates of the 6-month-old and the 9-month-old mice were about 22% and 16% of the rate of the 3-month-old mice, respectively  post-BrdU injection interval, we failed to detect BrdU+ cells in some aged mice. These mice were excluded from the specification rate analysis. The results revealed that age  and Ex  had significant effects on the neuron lineage specification rate (p = 0.137). The lineage specification rate of the 9-month-old mice was about 14% of that of the 3-month-old mice . Therefore, we determined the neuron lineage specification rate by dividing the number of BrdUion rate . No inteold mice .+ cells in the mice, four weeks after receiving BrdU injection, by the number of BrdU+ cells in the mice two hours after BrdU injection. We only estimated the survival rates of mice in the 3, 9 and 21-month-old groups, which represent young, middle-age and old age mice, respectively. Two way ANOVA revealed that neither age  nor Ex  had any effect on survival rate. There was no interaction between the age and Ex factors   .+ cells as an indication of newborn neuron maturation  and Ex , with no interaction between these two factors  (p < 0.001) and Ex . However, a significant interaction was observed between the two factors . Bonferroni post-hoc tests indicated that the dendritic lengths of the 9-month-old Ex mice were significantly longer than those of the 9-month-old Sed mice (We used the dendritic branch number and length of the DCXturation . Because= 0.195) . SimilarSed mice .We hypothesized that the levels of neurogenesis in adulthood were determined by the number of NSC, proliferation of NSC, neuron lineage specification of newborn cells, and survival rate of newborn neuron. We used these four elements to evaluate the effects of aging on adult hippocampal neurogenesis. Our results showed that the numbers of adult hippocampal neurogenesis decreased remarkably before middle age. By the age of 9 months, the number of newborn neurons in the hippocampus was less than 2% of that of the 3-month-old group. The big drop of neurogenesis during aging could not be explained by loss of NSC, as the number of NSC only marginally declined format the age of 3 months to the age of 9 months. However, the rates of NSC proliferation were largely reduced by about 85% during this time frame. Although the neuron lineage specification rate was also heavily decreased during this time frame, this step occurs after the cell proliferation stage. Therefore, we conclude that age-associated decline of adult hippocampal neurogenesis is caused by decline of NSC proliferation.One month of running exercise increased the levels of adult hippocampal neurogenesis. The degree of Ex effect on adult hippocampal neurogenesis was similar across different age groups (i.e. no interaction between Ex and age). Ex slightly increased the number of NSC and the rates of neuron lineage specification, without changing the rates of NSC proliferation. Furthermore, Ex increased maturation of newborn neurons without affecting their survival rate. Thus, Ex-increased adult hippocampal neurogenesis is likely derived from sustaining the number of NSC and increasing the rates of neuron lineage specification. Interestingly, Ex showed a strong effect on promoting the maturation of newborn neurons. These results were in agreement with previous findings that suggests running exercise potently increases the neuroplasticity  of post-mitotic neurons in several brain regions –21.+ cells was influenced by the number of NSC and the proliferation of NSC. When we adjusted the proliferation rate by dividing the number of BrdU+ cells by the number of nestin+ NSCs, the effect of Ex became insignificant. That is, the Ex-induced increase in the number of BrdU+ cells was mainly derived from the increased number of NSC. Therefore, we suggest that maintaining the NSC number is, at least in part, contributing to the positive effect of Ex on hippocampal neurogenesis during the process of aging.There are numerous reports showing that running exercise increases NSC division and number of newborn neuronal progenitor cells in rodents ,13,14,22+ NSC in the hippocampi of Fischer F344 rats was found to have greatly reduced by the time the rats were in middle age (10-month-old vs. 3-month-old) [+ proliferating cells varied greatly among different species of rodents [Whether the number of hippocampal NSC decreases with age is a debated issue. The number of nestinnth-old) . Howevernth-old) . Using Fnth-old) . The aut rodents . Further rodents .Regardless of the species-specific phenomenon, all evidence agrees that the number of newborn neurons in the hippocampus declines with age, especially before middle age. Our findings further revealed that even if the number of hippocampal NSCs has dropped, the trend was modest and only contributed slightly to the reduction of hippocampal neurogenesis during the process of aging. Rather, the remarkable decline of hippocampal neurogenesis during the process of aging is dictated by reduced proliferation of NSCs, a phenomenon that is associated with increased quiescence and decreased self-renewal ability of NSCs . Our res+DCX+ cells by the number of BrdU+ cells. Because the animals were killed 2 h after BrdU injection, while the expression of DCX usually begins several days after the birth of a cell, such a short interval may only allow a fraction of the total BrdU+DCX+ cells to be detected. In other words, the neuron lineage specification rate might be underestimated in this setting. Although longer intervals would allow more accurate estimations of the number of BrdU+DCX+ cells, the number of newborn neurons might also be confounded by the survival rate.Neuron lineage specification rate decreased with age, but could be increased by Ex. In this study, the neuron lineage specification rate was calculated by dividing the number of BrdUAdult hippocampal neurogenesis decreased steeply at a young age in mice. When mice reached 9 months of age, their neurogenesis capability was less than 5% of that of 3-month-old mice. The age-associated decline of hippocampal neurogenesis was not caused by diminished NSC number, but primarily by the reduced proliferation of NSCs. Ex increased adult hippocampal neurogenesis via maintaining the number of NSC and increasing the rates of neuron lineage specification. However, as Ex did not prevent the sharp decline of the NSC proliferation rate, the ability of Ex on restoring adult hippocampal neurogenesis during aging is limited. However, Ex did show a more pronounced effect on enhancing newborn neuron maturation.S1 FigSed: sedentary group. Bonferroni post-hoc test: * (p < 0.05), ** (p < 0.01), *** (p < 0.001) vs. respective Sed group. The standard deviations are too small to be seen in the scale setting.(TIF)Click here for additional data file.


Surprisingly, we found ß-catenin level and localization are heterogeneous in ESC colony by immunostaining and time-laps imaging of ß-catenin-mEGFP signals. Moreover, activation of Wnt signaling in ESC promoted expression level and nuclear localization of ß-catenin, and mRNA levels of Wnt antagonists, axin2 and dkk1, leading to upregulating Wnt/ß-catenin reporter in ESC state and Rax expression at differentiation culture day 7. Together, our results suggest that activation of Wnt signaling in ESC promotes the differentiation efficacy of rostral forebrain cells. Wnt-priming culture method may provide a useful tool for applications in the areas of basic science and molecular therapeutics for regenerative medicine.Wnt/ß-catenin signaling is crucial for maintenance of pluripotent state of embryonic stem cell (ESC). However, it is unclear how Wnt/ß-catenin signaling affects the differentiation ability of ESC, especially with regard to rostral forebrain cells. Here, using Rax, rostral forebrain marker, and Wnt/ß-catenin reporter lines, we report ratio of RaxThe online version of this article (doi:10.1007/s11626-015-9975-y) contains supplementary material, which is available to authorized users. Forebrain is derived from neural plate, called neural ectoderm  culture system, Wataya et al. showed efficient induction of hypothalamic tissues expressing Rax gene  analysis was performed as previously described . Then, we linearized Rosa26 ß-catenin-mEGFP targeting vector by a restriction enzyme and purified it by phenol-chloroform extraction to eliminate enzyme proteins. Using electroporation grade of linearized vector (7.5 μg), we introduced it into 1.0×106 E14Tg2a line via A24 program of Amaxa Nucleofector  based on the manufacturer’s instruction (day 0). Next at day 2, we add 200 μg/ml G418 sulfate  to select pooled homologous recombinant ESCs. Finally, at day 10, we extracted genome of pooled-drug-resistant ESCs and performed genotyping of ß-catenin-mEGFP knock-in allele by primers . We used pooled knock-in ESCs for Figs. Time-laps imaging and analysis. Live-imaging was performed using an incubator-combined confocal optic system  as previously described  using the company-provided protocol. As previous report . Data sets were first checked for standard error of the mean. The appropriate test for comparison was performed as follows; t tests (two samples) and Dunnett’s test (control and others) were used to generate P values .+ rostral forebrain by activating Wnt signaling through ß-catenin stabilization in mouse ESCs.In this study, we developed a new method for efficient differentiation of Raxet al.To date, the level of Wnt signaling activity in individual stem cells correlates with differences in lineage-specific differentiation propensity  manner  ß-catenin signals in each cell, we then tested activation of Wnt signaling in ESC state by addition of 10 μM CHIR, which inhibits GSK3ß to upregulate Wnt signaling through inhibition of ß-catenin degradation  to stabilize ß-catenin and Wntnce Fig. a. Surpriion Fig. a, b. FACion Fig. c, d. By ion Fig. e.Figure et al.et al.Although, Wnt inhibition in ESC differentiation promote rostral neural fate ; right, ß-catenin and DAPI (magenta) were observed by confocal microscope. The movie was taken at 1 frame per 0.5 μm step and played at 15 fps. (MP4 4442 kb)Z-scanning of ß-catenin expression along the apical-basal of ESC colony, related to Fig. Video 2f. Left, ß-catenin-mEGFP; right, DIC were observed by time-lapse imaging. The movie was taken at 1 frame per 15 min and played at 15 fps. (MP4 7882 kb)Dynamics of ß-catenin-mEGFP expression in ESC colony, related to Fig. Below is the link to the electronic supplementary material.


Angptl7-deficient mice by TALEN-mediated gene targeting and found that HSC compartments in Angptl7-null mice were compromised. In addition, wild type (WT) HSPCs failed to repopulate in the BM of Angptl7-null mice after serial transplantations while the engraftment of Angptl7-deficient HSPCs in WT mice was not impaired. These results suggest that Angptl7 is required for HSPCs repopulation in a non-cell autonomous manner.Successful expansion of hematopoietic stem cells (HSCs) would benefit the use of HSC transplants in the clinic. Angiopoietin-like 7 promotes the expansion of hematopoietic stem and progenitor cells (HSPC) in vitro and ex vivo. However, the impact of loss of Angptl7 on HSPCs in vivo has not been characterized. Here, we generated The online version of this article (doi:10.1186/s13045-014-0102-4) contains supplementary material, which is available to authorized users. Agiop in vivo . Several[ex vivo -9. Angpt[ex vivo . Recentlin vivo, we generated Angptl7-null mice by TALEN-mediated gene targeting  by RT-PCR and Western blotting  than Angptl7+/− and Angptl7+/+ mice , but similar numbers of erythroid precursors (CFU-E/BFU-E) and hematopoietic progenitors (CFU-GEMM) than WT BM (Figure Angptl7-deficient BM had normal levels of terminally differentiated hematopoietic cells, but decreased myeloid progenitors. To investigate whether other angiopoietin-like proteins compensate the loss of Angptl7 in BM, we compared the expression levels of other angiopoietin-like proteins in Angptl7-deficient BM stromal cells to that in WT BM stromal cells and found that their expression levels did not significantly change due to loss of Angptl7 (Additional file As HSCs undergo dramatic expansion in fetal liver during embryo development -13, we eAngptl7−/−, Angptl7+/− and Angptl7+/+ recipient mice. After WT donor BM cells were injected into lethally irradiated Angptl7−/−Angptl7+/− or Angptl7+/+ recipients without competitors (Figure Angptl7−/−, Angptl7+/− and WT recipient mice (Figure Angptl7−/− recipients were significantly lower than that in Angptl7+/− and WT recipients, suggesting that Angptl7 supports the maintenance of the HSC pool in BM (Figure Angptl7−/−, Angptl7+/− and WT recipient mice were collected for secondary transplantation. We found that the repopulating activity of cells originating from the primary Angptl7−/− recipients was significantly decreased compared with those from the primary Angptl7+/− and WT recipients at different time points (Figure Angptl7−/−, Angptl7+/− or WT BM cells into lethally irradiated WT recipients and measured the frequencies of donor cells in recipient mice (Figure Angptl7−/−, Angptl7+/− and WT LT-HSCs were similar in WT recipients (Figure Angptl7−/−, Angptl7+/− and WT donors at indicated time points (Figure in vivo.Since Angptl7 is secreted by stromal cells and binds to HSPCs , we specs Figure a, we foue Figure b, but thM Figure c-d. Conts Figure e. To tese Figure f. We fous Figure g-i. We fs Figure j. OveralIn this study, we demonstrated that deficiency of Angptl7 in the BM niche as a lack of exogenous Angptl7 stimulation impaired the reconstitution of HSCs in lethally irradiated mice, whereas endogenous deletion of Angptl7 in HSCs did not affect repopulation of HSCs in lethally irradiated mice. Thus, Angptl7 was indispensable for BM microenvironment to support HSC repopulation.


Cantharidin is an active constituent of mylabris, a traditional Chinese medicine, and presents strong anticancer activity in various cell lines. Cantharidin is a potent and selective inhibitor of serine/threonine protein phosphatase 2A (PP2A). Our previous studies revealed the prospect of application of cantharidin, as well as other PP2A inhibitors, in the treatment of pancreatic cancer. However, the mechanisms involved in the anticancer effect of PP2A inhibitors have not been fully explored. The Wnt/β-catenin pathway is involved in cell migration and proliferation and participates in the progression of pancreatic cancer. If β-catenin is phosphorylated and degraded, the Wnt/β-catenin pathway is blocked. PP2A dephosphorylates β-catenin and keeps the Wnt/β-catenin pathway active. In the present study, we found that PP2A inhibitor treatment induced phosphorylation and degradation of β-catenin. The suppression on the migration and growth of PANC-1 pancreatic cancer cells could be attenuated by pretreatment with FH535, a β-catenin pathway inhibitor. Microarray showed that PP2A inhibitor treatment induced expression changes in 13 of 138 genes downstream of the β-catenin pathway. Real-time PCR further confirmed that FH535 attenuated the expression changes induced by PP2A inhibitors in 6 of these 13 candidate genes. These 6 genes, VEGFB, Dkk3, KRT8, NRP1, Cacnalg and WISP2, have been confirmed to participate in the migration and/or growth regulation in previous studies. Thus, the phosphorylation- and degradation-mediated suppression on β-catenin participates in the cytotoxicity of PP2A inhibitors. Our findings may provide insight into the treatment of pancreatic cancer using a targeting PP2A strategy. Pancreatic cancer is one of the most fatal solid malignancies, with a 5-year survival rate of only ~5%. There is no method for early detection of the cancer and most patients with localized cancer have no recognizable symptoms. As a result, most patients are not diagnosed until the cancer metastasizes to other organs . Less thMylabris is the dried body of the Chinese blister beetle. The use of mylabris as a traditional Chinese medicine in the treatment of tumor can be traced back to >2,000 years ago and it is still used as a folk medicine today . The actThe conserved Wnt/β-catenin pathway regulates stem cell pluripotency and cell fate decisions during development ,8. PreviMechanistically, cantharidin has been shown to be a potent and selective inhibitor of serine/threonine protein phosphatase 2A (PP2A) . Our preTherefore, in the present study, we investigated whether cantharidin, as well as other PP2A inhibitors, could suppress the migration and growth of pancreatic cancer cells through phosphorylation and degradation of β-catenin-mediated inhibition on the Wnt/β-catenin pathway.2 incubator with humidified atmosphere. Cells were passaged every 2–3 days for exponential growth.The human pancreatic cancer cell line PANC-1 was purchased from the American Type Culture Collection . Cells were maintained in DMEM medium . Medium was supplemented with 10% fetal calf serum (Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in a 5% COCantharidin, OA and SP600125 were purchased from Enzo Life Sciences International . Norcantharidin (NCTD) was purchased from Sigma . FH535 was purchased from Millipore .4 cells/well and grown to confluence. The monolayer culture was then artificially scrape-wounded with a sterile micropipette tip to create a denuded zone (gap) of constant width. Each well was washed with phosphate-buffered saline (PBS) twice to remove the detached cells before treatment. Cells that had migrated to the wounded region were observed using an XDS-1B inverted microscope  and photographed . Images were captured at various time points to monitor the wound healing process. The wound areas were measured using ImageJ .The cells were seeded in 96-well plates at a density of 1×104 cells/well. After treatment, MTT (Sigma) was added to each well at a final concentration of 0.5 mg/ml, followed by incubation at 37°C for 4 h. The medium was then removed and 800 μl dimethyl sulfoxide was added to each well. The absorbance of the mixture was measured at 490 nm using a microplate ELISA reader . The relative cell viability was calculated as follows: relative cell viability =  ×100%.Cellular growth was evaluated by the 3--2,5-diphenyl tetrazolium bromide (MTT) assay . The celTotal protein was extracted using a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, supplemented with protease inhibitor cocktail kit  and phosphatase inhibitor cocktail kit (Roche). The cytosol and nuclear extracts were prepared by NE-PER nuclear and cytoplasmic extraction reagents , supplemented with protease inhibitor cocktail kit (Roche). The protein extract was loaded, size-fractionated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Bio-Rad Laboratories). After blocking, the membranes were incubated with primary antibodies at 4°C overnight. Rabbit anti-phospho-β-catenin (Thr41/Ser45) antibody was purchased from Santa Cruz Biotechnologies . Mouse anti-active-β-catenin  antibody was purchased from Millipore. Mouse anti-β-catenin was purchased from Cell Signaling Technology . Rabbit anti-RPL38 and rabbit anti-histone H1 antibodies were purchased from Proteintech Group . The protein expression was determined using horseradish peroxidase-conjugated antibodies followed by enhanced chemiluminescence  detection. The intensity of the bands was captured by JS-1035 image analysis scanning system . RPL38 and histone H1 were used as internal controls for the total and nuclear extracts, respectively.Sample preparation and processing procedure were performed as described in detail in the Agilent GeneChip Expression Analysis Manual . Differentially expressed genes were screened using Agilent 44K human whole-genome oligonucleotide microarrays. The selection criterion was defined as a >1.5-fold difference in the level of expression (difference in upregulated expression >1.5-fold and difference in downregulated expression <0.67-fold). Hierarchical clustering of samples was performed by average linkage algorithm using TIGR MultiExperiment Viewer .Total RNA was extracted using TRIzol reagent (Life Technologies) according to the manufacturer’s protocol. After spectrophotometric quantification, 1 μg total RNA was used for reverse transcription in a final volume of 20 μl with AMV reverse transcriptase (Promega) according to the manufacturer’s instructions. Aliquots cDNA of corresponding to equal amounts of RNA were used for the quantification of mRNA by real-time PCR using the LightCycler 96 real-time quantitative PCR detection system (Roche). The reaction system (25 μl) contained the corresponding cDNA, forward and reverse primers and SYBR-Green PCR Master Mix (Roche). All data were analyzed using RPL38 gene expression as an internal standard. The specific primers are presented in Each experiment was performed at least in triplicate. Results are expressed as the mean value ± standard deviation (SD). Statistical analysis was performed using an unpaired Student’s t-test. A P-value <0.05 was considered to indicate a statistically significant difference.Previously, we found that cantharidin suppressed the proliferation of pancreatic cancer cells ,5. HowevIn our previous studies, cantharidin-induced activation of JNK suppressed pancreatic cancer cell growth ,5. As thTreatment with PP2A inhibitors, cantharidin, NCTD and OA, increased the phosphorylation level and decreased the nonphosphorylated form (active form) of β-catenin, accompanied by the reduction of total protein level of β-catenin . BlottinTo investigate whether the suppression on the β-catenin pathway is involved in the anticancer effect of PP2A inhibitors, we used FH535, a classic Wnt/β-catenin inhibitor , to veriCantharidin, NCTD and OA suppressed the migration and growth of pancreatic cancer cells, respectively. When cells were pretreated with FH535, the anti-migration and anti-growth effects of PP2A inhibitors were partially attenuated, suggesting that the anticancer effects of PP2A inhibitors were partially β-catenin pathway-dependent.http://www.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes)  . Only tht_genes) .We used real-time PCR to confirm the changes of these genes in the microarray analysis. Spearman’s rank correlation analysis revealed that the mRNA expression values obtained by real-time PCR correlated significantly with those obtained by microarray analysis for these 13 genes (P<0.01). The correlation coefficient R ranged between 0.828 and 0.896 .To verify whether the expression changes of these 13 genes induced by PP2A inhibitors were executed through β-catenin pathway-dependent manner, the cells were pretreated with FH535, followed by treatment with PP2A inhibitors. As shown in The Wnt/β-catenin pathway has been positively confirmed to regulate cell proliferation, migration, apoptosis, differentiation and stem cell self-renewal ,17. As tin vitro and in vivo evidence that Wnt/β-catenin signaling is involved in pancreatic cancer tumorigenesis. Aberration in canonical Wnt/β-catenin signaling activity has been documented in pancreatic cancer  is expressed in various human tumors, including colon and pancreatic cancer and glioblastoma, as well as in acute myelogenous leukemia  family, which participates in both physiologic and pathologic angiogenesis . VEGFB hleukemia ,47. Knocleukemia , breast leukemia  and glioleukemia  cells. Wleukemia . Previouleukemia  and promleukemia . As VEGFPP2A is generally considered to be a cancer suppressor. Inhibition of PP2A has been thought to be cancer promoting by induction of phosphorylation and activation of several substrate kinases, including c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), p38, Akt and protein kinase C (PKC) amongst others, most of which can accelerate growth –55. HoweThe JNK pathway, over-activation of which suppresses the growth of pancreatic cancer cells , fulfillIn conclusion, we demonstrated that PP2A inhibitors suppressed pancreatic cancer cell migration and growth through the Wnt/β-catenin pathway by phosphorylation and further degradation of β-catenin. This may be attributed to the selective downregulation of genes downstream of the Wnt/β-catenin pathway. Our findings provide a promising strategy for treatment of pancreatic cancer by targeting PP2A using PP2A inhibitors.


Grand Naine and cv. Rasthali leaves. In addition, several cultivar specific microRNAs along with their putative target transcripts were also detected in our studies. To validate our findings regarding the small RNA profiles, we also undertook overexpression of a common microRNA, MusamiRNA156 in transgenic banana plants. The transgenic plants overexpressing the stem-loop sequence derived from MusamiRNA156 gene were stunted in their growth together with peculiar changes in leaf anatomy. These results provide a foundation for further investigations into important physiological and metabolic pathways operational in banana in general and cultivar specific traits in particular.Micro RNAs (miRNAs) are a class of non-coding, short RNAs having important roles in regulation of gene expression. Although plant miRNAs have been studied in detail in some model plants, less is known about these miRNAs in important fruit plants like banana. miRNAs have pivotal roles in plant growth and development, and in responses to diverse biotic and abiotic stress stimuli. Here, we have analyzed the small RNA expression profiles of two different economically significant banana cultivars by using high-throughput sequencing technology. We identified a total of 170 and 244 miRNAs in the two libraries respectively derived from cv.  A large number of small RNAs have been identified in plants which play significant roles in the genetic regulation at the post transcriptional level , 2. Theshttp://www.statista.com/statistics/264001/worldwide-production-of-fruit-by-variety/). Bananas are grown in more than 120 countries across the globe and are regarded as an important food security crop. There are more than 50 different cultivars grown in the Indian subcontinent each bearing unique fruit characteristics [Grand Naine (AAA subgroup Cavendish) and Rasthali (AAB subgroup Silk).Banana is the fourth most important crop after the major cereal crops with a world production of 1.1 billion metric tonnes per year . The recent unearthing of the banana genome [ bananas . The fru bananas . Cultivaa genome , 16 has Among the thoroughly studied miRNAs in model plants is the miRNA156 which is highly conserved and expressed mostly in young plant parts and play important role in plant growth and development . miRNA15Musa spp.) cultivars namely Grand Naine (subgroup: Cavendish) and Rasthali (subgroup: Silk) having triploid genome composition and belonging to AAA and AAB group respectively. The suckers were grown under green house conditions. Tissue from the fresh fully expanded leaves was collected and used for small RNA isolation.Suckers were obtained from the two banana (2 (10 mM) and 2.5 volumes of chilled absolute ethanol. The samples were incubated at -20°C for 30 minutes and centrifuged at 16000 g for 20 minutes at 4°C to pellet down the total RNA. Total RNA obtained was dissolved in 45 μl RNase free water at 65°C. Further, the small RNA fraction was isolated from the total RNA using mirVana miRNA Isolation Kit  following the manufacturers instructions. Small RNA bound to the column was eluted in 40 μl RNase free water). Small RNA was separated based on the size using denaturing gel. Later the 5’ and 3’ adapters were ligated to the small RNAs and reverse transcribed and PCR amplified. Quality and quantity of the RNA was determined by using Bioanalyzer. The library construction and sequencing was performed at Genotypic Technology (P) Ltd  on the Illumina GAIIX platform.Total RNA was extracted from the fully expanded fresh banana leaves using Concert Plant RNA Reagent . The samples were incubated for 5 min at room temperature before separation of the supernatant. The supernatant was treated with 5M sodium chloride and the aqueous phase was extracted using chloroform. The chloroform extract was precipitated by adding MgClhttp://banana-genome.cirad.fr/ was used to analyze the genomic origins of the remaining small RNAs and subsequently small RNAs unique to each cultivar and similar in both the cultivars were identified.After the adapter sequences were trimmed, sequences with low quality or length less than 15 nucleotides were removed. The small RNA sequences obtained were compared with the miRBase database  and the Musa acuminata mRNA sequences using the “strict” settings . This option prevents the detection of target sites which contain gaps or non-canonical base pairing in this region). miRNA hits having minimum free energy < = -40 are assumed to be most probable targets for reported miRNA.For prediction of miRNA targets of the identified conserved and predicted novel miRNAs, miRanda target prediction tool was used. miRNA sequences were fed as input along with Rasthali and Grand Naine have been deposited at the National Centre for Biotechnology Information Gene Expression Omnibus archive under the Bioproject PRJNA274803 (accession numbers GSE65728 and GSE65727 respectively).Small RNA sequence data derived from OsmiR156a (accession no.: MI0000653) from rice was used as template to identify similar pre-miR156 sequences in the banana genome database. Several partial hits were examined in silico. These sequences were analyzed for their capacity to fold into typical miRNA hairpins. Homolog of one of these sequences was found in the EST database of banana (accession no.: ES434836). This sequence was aligned with the genomic sequence derived from Musa acuminata subspecies malaccensis (genomic location: GSMUA_Achr7G05222_001) sourced from banana genome hub and primers were designed in the complementary regions. A 175 bp sequence encompassing the stem loop sequence of miRNA156 gene was subsequently amplified from genomic DNA derived from banana cv. Rasthali leaves using these primers. This sequence was first cloned into pTZ57R/T cloning vector and then subsequently subcloned in p1301-MusaDAD114 in place of MusaDAD1 coding sequence to generate p1301-MusamiRNA156 binary vector. This construct was electroporated into Agrobacterium tumefaciens strain EHA105 for use in banana genetic transformation.Precursor miRNA (pre-miRNA) sequences comprising the stem loop of Agrobacterium-mediated genetic transformation method as described previously [Agrobacterium culture harbouring p1301-MusamiRNA156 binary vector was cocultivated with embryogenic cell suspension cultures of banana cv. Rasthali. The cocultivated banana cells were aspirated onto the glass fibre filters and placed on the medium supplemented with acetosynringone (ACS). After 3 days the cells along with the filters were transferred onto medium containing cefotaxime (400 mg/L) and incubated for another 3 days. On the seventh day post cocultivation the cells were transferred onto medium supplemented with cefotaxime (400 mg/L) and hygromycin (5 mg/L). The embryos developed on the hygromycin based medium were subcultured for three rounds after every 3 weeks. The embryos were germinated in medium containing 0.5 mg/L 6-benzylaminopurine (BAP). The shoots were multiplied in medium containing BAP and rooted in medium added with 1-naphthalene acetic acid (NAA). The rooted plantlets were hardened in green house under controlled conditions.The transgenic banana plants were generated by eviously , 24. TheMusamiRNA156 was detected by histochemical staining with 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc). The leaf tissue sample from transgenic banana plant and untransformed control was incubated overnight at 37°C in staining buffer , 10 mM EDTA, 0.5 mM K4Fe(CN)6, 0.5 mM K3Fe(CN)6, and 0.1% (v/v) Triton X-100) as described previously [The GUS activity in the transgenic banana plants overexpressing eviously . Followi12–18 primer and AccuScript Reverse Transcriptase . The cDNA preparation was diluted appropriately before being used for PCR amplification. PCR was carried out using the following primers: miRNA156 Fw: AGGAGATCGGCGACGGATAmiRNA156 Rv: GGGGGTTGACAGAAGAGAGTG. Following amplification protocol was performed: 94°C for 5 min followed by 35 cycles each with 94°C for 1 min, 56°C for 1 min and 72°C for 45 sec with a final extension 72°C for 5 min. The amplified products were separated on 1.5% (w/v) TAE-Agarose gel and visualized using ethidium bromide.Total RNA was extracted from the leaf tissue of a selected transgenic banana plant and an untransformed control banana plant as described above. cDNA was synthesized using oligo (dT)Transgenic banana plants along with the untransformed control banana plants were hardened together in the green house under controlled conditions. The plants were observed for change in the height, leaf anatomy and root growth after 1 month. The plants were further maintained in green house and observed for morphology after 3 and 6 months period. The abaxial surface of the leaves was observed under microscope after 1 month to study the change in the leaf cellular arrangement.Grand Naine (GN1) and Rasthali (NR1), belonging to subgroups Cavendish (AAA) and Silk (AAB) respectively, small RNA sequencing was carried out using Illumina sequencing platform. A total of 4981328 and 8889596 reads were obtained from Grand Naine and Rasthali small RNA libraries respectively  database. Mireap identified novel microRNA based on alignment, secondary structure, free energy and location on the precursor arm. 55% of reads that did not map to mirbase were successfully mapped on to banana genome in Rasthali whereas this number was a close 56% in case of Grand Naine. We predicted 81 novel microRNAs in Rasthali and 43 in Grand Naine  from the p1301-MusamiRNA156 derived banana plant and untransformed control banana plant was reverse transcribed into cDNA and used as template in the PCR amplification reaction. A single amplified product of 175 bp was clearly visible in both the samples  in transgenic banana plants. The fact that small RNA profiling of the two cultivars indicated a 5 fold difference in levels of MusamiRNA156 in young leaves justified its overexpression in transgenic banana plants.MiRNAs have recently emerged as a novel class of gene regulators. Several studies over the last decade have indicated their importance in normal growth and development of plants . Banana  genomes , we haveGrand Naine has three A genomes (derived from Acuminata lineage) and cv. Rasthali possesses two A genomes and a B genome . We selected these two cultivars based on their elite fruit characteristics and the economic importance. Investigations into the relative profiles of major miRNAs in the two cultivars are expected to lead to better understanding of the major physiological processes in the two cultivars. Taking advantage of the newly sequenced Acuminata and Balbisiana genomes [Among the two cultivars that we chose , cv.  genomes , 16 we cRasthali conclusively proves the earlier observations in other plants that unique DICER proteins process RNA hairpins with different origins [Like in other such studies, the small RNA sequencing of these two cultivars showed that the 24 nt length miRNA class dominates in the dataset by a good majority , 9, 10.  origins , 30.MusamiRNA156 was oevrexpressed in cv. Rasthali using its stem-loop sequence amplified from cv. Rasthali, we observed significant growth alterations in the transgenic lines indicating the involvement and the importance of miRNA156 in banana growth and development. The altered appearance of the transgenic lines could be seen even after 6 months of growth in a contained greenhouse. In contrast to other plants where miRNA156 has been overexpressed before, the growth penalty was not so severe in banana. This was despite the fact that we noticed strong overexpression of the MusamiRNA156 transcript derived from the T-DNA in transgenic lines as compared to equivalent controls.To extend the findings of this study, we chose to overexpress one microRNA which showed significant difference in the abundance in the two libraries. When The findings of this study will help in better understanding of a myriad of physiological processes in banana. The importance of small RNA based regulation of gene functions in plants has been recognized in the last decade. This study describing the small RNA profile of two important cultivars of banana will facilitate investigations into the roles played by small RNAs in economically important banana traits like fruit ripening and disease resistance.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.