HOXA cluster. Most of the genes are epigenetically regulated independently of underlying mutations, although several are downstream targets of epigenetic modifier genes mutated in AML. The LSC epigenetic signature is associated with poor prognosis independent of known risk factors such as age and cytogenetics. Analysis of early haematopoietic progenitors from normal individuals reveals two distinct clusters of AML LSC resembling either lymphoid-primed multipotent progenitors or granulocyte/macrophage progenitors. These results provide evidence for DNA methylation variation between AML LSCs and their blast progeny, and identify epigenetically distinct subgroups of AML likely reflecting the cell of origin.Acute myeloid leukaemia (AML) is characterized by subpopulations of leukaemia stem cells (LSCs) that are defined by their ability to engraft in immunodeficient mice. Here we show an LSC DNA methylation signature, derived from xenografts and integration with gene expression that is comprised of 71 genes and identifies a key role for the  Leukaemic stem cells are prevalent in acute myeloid leukemia. Here, Jung and colleagues derive a signature of 71 methylated genes that characterise these stem cells and find multiple HOXA genes within the signature. Acute myeloid leukaemia (AML) is an aggressive malignancy of bone marrow precursors defective in their maturation and function345A number of both mouse and human studies have investigated the cell of origin in AML. Mouse studies have typically utilized retroviral oncogene transduction or knock-in models to explore this question and have generally led to the conclusion that committed progenitors, in particular common myeloid progenitors (CMP) and/or granulocyte/macrophage progenitors (GMP), serve as the cell of origin for most AML models. In one study of MN1-induced AML, retroviral transduction of single CMP, but not GMP or haematopoietic stem cells (HSC), resulted in the development of AML, indicating tight restriction of transformation by this oncogene1112IDH1/2, DNMT3A and TET2, and modulation of chromatin modifications such as ASXL1, EZH2 and others15IDH1 or IDH2 mutations are associated with globally increased DNA methylation15MLL fusions or mutations in NPM1, DNMT3A or FLT3 were associated with decreased DNA methylationDysregulation of the epigenome is a common feature in AML, as indicated by the recent discoveries that a number of epigenome-modifying genes are mutated in AML including some involved in the regulation of DNA methylation such as HOXA gene cluster. Finally, comparison of LSC with normal HSPC suggests that both L-MPP and GMP can give rise to LSC.Epigenetic signatures distinguishing normal haematopoietic stem and progenitor cells (HSPCs) are useful markers for survival analysis in AMLTo formally investigate epigenetic differences between LSC and blast progeny, we sought to identify differentially methylated regions (DMRs) between functionally defined AML LSC-enriched populations and their downstream non-engrafting blasts from a cohort of 15 primary patient samples. We obtained samples from 15 AML patients  and 2 an34in vivo (r=−0.12 (P=0.004) and −0.27 (P=0.003), respectively), whereas most hypomethylated DMRs in the engrafting populations were associated with transcriptional upregulation of associated genes  and exhibited >0.5 log2 ratio of differential expression between the LSC and Blast populations, with an inverse relationship between gene expression and DNA methylation within 2 kb of the transcriptional start site. We excluded gene body DMRs, as there was no statistically significant positive correlation in AML or normal haematopoiesis comparisons , there was 77% overlap . We observed statistically significant enrichment of these excluded DMRs in the DMRs without MLL cases . In addition, we performed an enrichment test to examine if the LSC epigenetic signature without MLL cases was enriched in the LSC epigenetic signature with all samples. We observed statistically significant enrichment between those two LSC epigenetic signatures . Thus, the presence of the MLL subtype was not a confounding variable in defining the LSC epigenetic signature with the inclusion of the HOXA cluster.Because the mixed-lineage leukaemia (MLL) subtype is itself associated with changes in expression of members of the  overlap . The LSCXA genes . Note thNPM1, ASXL1 and KAT6A as the most significant upstream regulators of the LSC epigenetic signature genes, primarily through regulation of HOXA genes including HOXA5, HOXA6, HOXA7, HOXA9 and HOXA10  cohort  cohort .DNMT3A, IDH1/2 and TET1/2, and upstream regulators of our LSC epigenetic signature, NPM1 and ASXL1 ; P=0.03, log-rank test; Next, we tested the association between expression of LSC epigenetic signature genes and clinical outcome using four different cohorts including TCGA293132DNMT3A, IDH1/2, TET2 and ASXL1 affected the prognostic impact of the LSC epigenetic signature in the TCGA cohort. As described previously, mutation in DNMT3A, but none of the other genes, was associated with patient overall survival  classification of the TCGA samples and found that the L-MPP-like cases mainly consisted of more immature M0, M1 and M2 types, while the more differentiated M4 and M5 types were enriched in GMP-like AML , and FLT3 and NPM1 mutations were enriched in the GMP-like group . DNMT3A and TET1 mutations were more enriched in the L-MPP group, but this was not statistically significant . Most oApart from its prognostic effect, the LSC epigenetic signature represents a molecular target that may improve patient survival and prevent relapse. Recently, epigenetic therapy with hypomethylating agents such as azacytidine and decitabine has been approved for the treatment of AML. Randomized trials demonstrated improved overall survival compared with chemotherapy, but also indicated limited effect on relapse rate in high-risk AMLHOXA cluster, suggesting this cluster is a key driver of LSC function. The HOXA cluster has been implicated as a key regulator of haematopoiesis and myeloid malignancyHOXA9 is known to be involved in increased proliferation of HSPC and leukaemogenesisHOXA9 has been found to be an adverse prognostic factor in AMLHOXA family members including posterior (HOXA7 and HOXA10) and anterior (HOXA6) members have been implicated in leukaemogenesis, as overexpression of these genes in normal mouse HSPC leads to increased self-renewal, transformation and development of myeloid malignanciesHOXA family is a key driver of AML LSC that may function in imparting aberrant self-renewal.More specifically, this LSC epigenetic signature was markedly enriched for the members of the As DNA methylation is a potential marker of cell identity, here we compared DNA methylomes of normal HSPC with LSC as a marker for the cell of origin in AML. Using this approach, we observed two subtypes in our cohort: L-MPP-like and GMP-like. These two subtypes were also identified in the TCGA cohort, suggesting that leukaemic transformation predominantly occurs at either the L-MPP or GMP stage of haematopoietic development. We found that other features of AML were associated with these two subtypes including FAB type, several mutations and cytogenetic abnormalities, suggesting that the cell of origin may drive key clinical features in AML.We newly identified a small subset of AML cells clustering with CMP and few samples clustering with HSC, MPP and MEP that could not be identified in smaller data sets including our own data and a previous study8 cells per donor, Catalogue#: ABM006) were purchased from ALLCELLS . A CD34+ cell-enrichment step was performed with the human progenitor cell-enrichment kit with CD61 depletion  on a RobSep machine from the same company. Human AML samples were collected from patient peripheral blood or bone marrow at Stanford Hospital, according to a Stanford-IRB-approved protocol (22264), and informed consent was obtained from all subjects. Peripheral blood mononuclear cells (PBMCs) or BMMCs were separated with Ficoll-Paque Plus , and cryopreserved in 1 × freezing medium +10% dimethylsulfoxide). All the AML experiments were conducted with cryopreserved PBMC or BMMC samples that were thawed and washed in IMDM medium containing 10% FBS.Fresh human bone marrow mononuclear cells (BMMCs) from healthy donors  were obtained from The Jackson Laboratory  and bred in a specific pathogen-free environment per Stanford Administrative Panel on Laboratory Animal Care Guidelines (Protocol 22264). Six- to eight-week-old female adult mice were exposed to 200 rad of gamma irradiation at least 2 h (up to 24 h) before transplantation. Up to 500 thousand fresh-sorted AML cell subpopulation were resuspended in 30 μl of Hank's balanced salt solution  containing 2% FBS, and injected intravenously via the tail vein using a 29-gauge needle. For each cell subpopulation, at least three technical replicates were performed by transplantation of three aliquot of cells into three mice. It was counted as ‘Yes' if at least one of the three mice was engrafted (NOD.Cg-ngrafted . Around 2 according to Stanford-IRB-approved protocol (22264). Bone marrows were isolated using scissors and needle flashing, then underwent hypotonic red cell lysis using ACK (ammonium–chloride–potassium) lysing buffer . BMMCs were stained with antibody combinations  according to the manufacturer's protocol. The genomic DNA (250–500 ng) was treated with sodium bisulfate using the Zymo EZ DNA Methylation Kit (ZYMO Research) as recommended by the manufacturer, with the alternative incubation conditions for the Illumina Infinium Methylation Assay. Converted DNA was eluted in 11 μl of elution buffer. DNA methylation level was measured using Illumina Infinium HD Methylation Assay (Illumina) according to the manufacturer's specifications. Methylation array data are deposited at the Gene Expression Omnibus (GEO) with accession number Raw intensity files were obtained using minfi packageP value cutoff used for downstream analysis was <0.01 that corresponded to Benjamini–Hochberg adjusted P value <0.1 (data not shown) unless different cutoff was designated in result part. Bumphunter 1.2.0 was used. Same method was applied to identify DMRs for the second DMR analysis of LSC versus Blast that we removed five LSC cases from two MLL patients (SU042 and SU046).Bump hunting method previously described was applied to identify DMRs in 450k array45A unit of 100 ng of genomic DNA from each sample was treated with sodium bisulfate using an EZ DNA Methylation Gold Kit (ZYMO Research) following the manufacturer's protocol. The bisulfate-treated DNA was PCR amplified using unbiased nested primers. Quantitative pyrosequencing was performed using a PSQ HS96 (Biotage) to validate DMR regions. The DNA methylation percentage at each CpG site was measured using the Q-CpG methylation software (Biotage). SssI-treated human genomic DNA was used as 100% methylated controls and human genomic DNA amplified by Repli-G mini kit (Qiagen) was used as the non-methylated (0%) DNA control. GSE63270).Total RNA was extracted from each FACS-sorted cell population using RNeasy Plus Mini  according to the manufacture's protocol. All RNA samples were quantified with 2100 Bioanalyzer , subjected to reverse transcription, two consecutive rounds of linear amplification, and production and fragmentation of biotinylated cRNA. A unit of 15 μg of cRNA from each sample was hybridized to HG U133 Plus 2.0 microarrays. Hybridization and scanning were performed according to the manufacture's instruction (Affymetrix). This step was performed at the PAN Center of Stanford University. Data were normalized by GC robust multi-array average method and analysed on R/Bioconductor. SU042 CD34+38+, BM2770 GMP, BM2759 L-MPP, BM2761 CMP and BM2770 CMP were removed from further analysis due to low quality. SU001 was excluded, as the samples from this patient were not included on expression array  according to the manufacture's instruction. PCR primers were designed to cover exon 3–11 of TET2, exon 4 of IDH1/2 and exon 7–23 of DNMT3A . The PCRcoxph function in R , with significance assessed by log-rank test. For gene expression, the genes in the LSC epigenetic signature were identified in expression data sets for which survival outcomes were available. The first principal component of their expression levels was computed, and patients were stratified as ‘high' or ‘low' relative to its median value. Survival differences between the groups were assessed by log-rank test. In multivariate analyses, age was incorporated as a continuous variable, mutations were coded as present/absent (1/0) and assessment of cytogenetic risk was treated as individual groups and done for intermediate versus low risk and high versus low risk  in four different cohorts. For DNA methylation data set (TCGA), patients were separated into two groups; LSC like and Blast like based on the methylation profile of each individual. Survival was compared between the two groups using the low risk . AnalysiTo assign cell identity of LSC/Blast to TCGA samples, mean methylation value of each LSC epigenetic signature (84 DMRs) for LSC/Blast (methylation profile) was retrieved and s.d. of the mean value for each signature was calculated. Then scores  for each TCGA sample regarding LSC and Blast profile was calculated using dnorm function with the mean and s.d. calculated in previous step. Maximum value of scores between the ones regarding LSC and Blast methylation profile was chosen, and then cell identity assigned. Same strategy was applied using the 216 DMRs for normal haematopoiesis to assign normal progenitor identity to TCGA samples in For clustering analysis, hclust function with ward method in R was used to generate all the cluster dendrogram analysis.P<0.05) was a χ2-test based on the number of overlapping DMRs between the excluded DMRs after removing MLL cases and DMRs without MLL cases (P<0.05), then DMRs only in the excluded DMRs and DMRs only in DMRs without MLL cases (P<0.05), and finally random DMRs from array background that do not overlap with either the excluded DMRs or DMRs without MLL cases (P<0.05).The test to examine the enrichment of the excluded DMRs after removing MLL cases in DMRs without MLL cases using a less strict statistical cutoff (www.ingenuity.com) was performed for pathway analysis.QIAGEN's Ingenuity Pathway Analysis  under accession codes GSE63409 and GSE63270, respectively.How to cite this article: Jung, N. et al. An LSC epigenetic signature is largely mutation independent and implicates the HOXA cluster in AML pathogenesis. Nat. Commun. 6:8489 doi: 10.1038/ncomms9489 (2015).Supplementary Figures 1-8, Supplementary Tables 1-16 and Supplementary ReferencesDMR lists for the comparisons in Table1LSC epigenetic signatureIngenuity pathway analysisIngenuity upstream regulator analysisAssociation of LSC epigenetic signature with DMRs of genetic mutationsDMRs for normal hematopoiesisR script for multivariate survival analysis


To examine microbe-mineral interactions in subsurface oceanic crust, we evaluated microbial colonization on crustal minerals that were incubated in borehole fluids for 1 year at the seafloor wellhead of a crustal borehole observatory  as compared to an experiment that was not exposed to subsurface crustal fluids (at nearby IODP Hole U1301B). In comparison to previous studies at these same sites, this approach allowed assessment of the effects of temperature, fluid chemistry, and/or mineralogy on colonization patterns of different mineral substrates, and an opportunity to verify the approach of deploying colonization experiments at an observatory wellhead at the seafloor instead of within the borehole. The Hole U1301B deployment did not have biofilm growth, based on microscopy and DNA extraction, thereby confirming the integrity of the colonization design against bottom seawater intrusion. In contrast, the Hole U1301A deployment supported biofilms dominated by Epsilonproteobacteria (43.5% of 370 16S rRNA gene clone sequences) and Gammaproteobacteria (29.3%). Sequence analysis revealed overlap in microbial communities between different minerals incubated at the Hole U1301A wellhead, indicating that mineralogy did not separate biofilm structure within the 1-year colonization experiment. Differences in the Hole U1301A wellhead biofilm community composition relative to previous studies from within the borehole using similar mineral substrates suggest that temperature and the diffusion of dissolved oxygen through plastic components influenced the mineral colonization experiments positioned at the wellhead. This highlights the capacity of low abundance crustal fluid taxa to rapidly establish communities on diverse mineral substrates under changing environmental conditions such as from temperature and oxygen. The deep subsurface biosphere represents a vast and relatively unknown environment. While this deep biosphere is estimated to comprise a significant fraction of the Earth's total biomass  enable examination of microbe-mineral interactions by slowly and continuously pumping crustal fluids over mineral substrates housed in an Initial CORK-based investigations, focused on a CORK network on the eastern flank of the Juan de Fuca Ridge Figure , indicatThe first microbe-mineral interaction studies from the Juan de Fuca Ridge flank subsurface, from experiments conducted at depth within CORKs at Integrated Ocean Drilling Program (IODP) Holes 1026B and U1301A from 2004 to 2008  and of massive basalt previously collected from the Juan de Fuca Ridge flank subsurface at IODP Hole U1301B  and goethite [FeO(OH)] in Sleeves 5 and 1, respectively (1−S (x = 0–0.2)] was linked to the same stainless steel tubing and allowed to freely vent , allowing subsurface fluids to flow through the sleeve at an unknown rate due to overpressure of the subsurface fluids compared to bottom seawater  in summer 2010. The plate was connected to the right bay of the Hole U1301A CORK wellhead during dive J2-498.The overall design principal of the mineral incubation devices  is described in detail elsewhere  verifies the absence of inadvertent contamination during FLOCS assembly/deployment/collection, (2) provides confirmation of the sterility of filtered seawater used in the FLOCS sleeves during deployment, and (3) confirms the physical integrity of colonization design against background seawater intrusion. Thus, Hole U1301B results verify that observations on the Hole U1301A materials resulted from exposure to volcanic reservoir fluids.Jason II dive J2-566 on cruise AT18-07 (R/V Atlantis) in summer 2011. Immediately after recovery, FLOCS sleeves were disassembled with ethanol and flame-sterilized tools, and samples were stored for shore-based DNA and microscopy analysis at −80°C.The FLOCS experiments were recovered during ROV in situ with acid to dissolve metal-containing compounds that precipitate upon exposure to oxic conditions  according to the manufacturer's protocols. DNA was quantified using a Qubit 2.0 fluorometer with HS DNA reagent kit (Invitrogen). Due to low biomass yields , environmental DNA was amplified using the illustra GenomiPhi V2 DNA Amplification Kit  according to manufacturer's protocols. The amplified DNA was then used as a template for PCR amplification of the full-length 16S rRNA gene using the universal primers B27F and U1492R  from the experiment, following approaches published previously  are typically warm (64°C) and anoxic yet relatively high in sulfate and calcium  observed in both bottom seawater and borehole fluids , indicating that no fluids  had passed through this experiment. Thus, further chemical analyses were not performed.To evaluate microbial colonization patterns on the different mineral substrates, environmental DNA was extracted from the samples, and then 16S rRNA genes of Bacteria were amplified, cloned and sequenced. Original DNA concentrations were below detection limit, so DNA was amplified with Phi29 Polymerase, then the 16S rRNA gene was amplified with bacterial specific primers via PCR. Hole U1301A minerals produced visible bands on an agarose gel, and the product was used for further cloning, whereas the Hole U1301B minerals did not generate visibly amplified DNA and no further cloning attempts were made. In total, 370 nearly full-length 16S rRNA gene sequences were obtained from Hole U1301A minerals, ranging from 23 to 82 clones per substrate Table .Arcobacter genus , and these were abundant on the iron oxides  and iron sulfides  but less abundant on the basalt samples  being closely related to Community similarity based on sequence membership, as evaluated by several different indices, consistently clustered Hole U1301A wellhead FLOCS mineral microbial communities as branching between Hole U1301A borehole-incubated minerals and seafloor-exposed minerals , with Hole U1301A fluids and a mineral biofilm from Hole 896A grouping more distantly Figure .To examine whether microbial biofilms formed on the surface of minerals incubated in the Hole U1301B FLOCS, mineral coupons were examined by SEM. No alteration products or microbial biofilms were observed on Hole U1301B mineral coupons via SEM Figure . SEM rev−1), both of which exacerbate low biomass conditions.In the quest to study microbial life supported by the deep crustal biosphere—a major focus of the international ocean drilling program  with a relatively diverse community, the representational biases are assumed to be minimal  and thus do not necessarily reflect subseafloor microbial activities at this site; however, our results show an adaptive response to changing thermal and redox regimes, and suggest an effective adaptation capacity potential for the colonization of mineral substrates by subseafloor crustal fluid biota.As a tool for examining mineral-microbe interactions in the warm (64°C) and anoxic crustal subsurface, deployment of mineral colonization experiments at the wellhead of crustal borehole observatories must be configured to mimic conditions at depth. This can be conducted in multiple ways; however the easiest means is to place such experiments within the open borehole of overpressured observatories from which hydrothermal fluids vent freely (Fisher et al., All authors contributed to the design of the experiment. KE and SH deployed the experiment; KE, GR, BO, SH, GW recovered the experiment. JB, GR, and AH analyzed samples, and all authors contributed to data analysis. JB, GR, KE, AH, and BO wrote the paper with input from all authors.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.


Stress is increasingly present in everyday life in our fast-paced society and involved in the pathogenesis of many psychiatric diseases. Corticotrophin-releasing-hormone (CRH) plays a pivotal role in regulating the stress responses. The tree shrews are highly vulnerable to stress which makes them the promising animal models for studying stress responses. However, the mechanisms underlying their high stress-susceptibility remained unknown. Here we confirmed that cortisol was the dominate corticosteroid in tree shrew and was significantly increased after acute stress. Our study showed that the function of tree shrew CRH - hypothalamic-pituitary-adrenal (HPA) axis was nearly identical to human that contributed little to their hyper-responsiveness to stress. Using CRH transcriptional regulation analysis we discovered a peculiar active glucocorticoid receptor response element (aGRE) site within the tree shrew CRH promoter, which continued to recruit co-activators including SRC-1 (steroid receptor co-activator-1) to promote CRH transcription under basal or forskolin/dexamethasone treatment conditions. Basal CRH mRNA increased when the aGRE was knocked into the CRH promoter in human HeLa cells using CAS9/CRISPR. The aGRE functioned critically to form the “Stress promoter” that contributed to the higher CRH expression and susceptibility to stress. These findings implicated novel molecular bases of the stress-related diseases in specific populations. Stress is a condition that disturbs the physiological and psychological homeostasis of living organisms1Corticotrophin-releasing hormone (CRH) is a principle regulator of the hypothalamic-pituitary-adrenal (HPA) axis, which is the canonical pathway that regulates the physiological response to stressThe tree shrew (Tupaia belangeri) has much closer circadian rhythm to human compared with rodents. It has been employed to study a variety of neurological diseases, - in particular, psychosocial stresses162021In the present study, we aimed to characterize the tree shrew CRH gene and compare its key regulators of HPA axis with those of human. Special attentions were paid to the molecular mechanisms underlying their susceptibility to stress, especially the transcription of CRH gene in regulating the HPA axis and the stress responses. In this study, the CRH gene sequence of tree shrew was annotated which shows high similarity with human CRH. Binding affinities of CRH to its receptor and CRH-CRHR1 signaling transduction assays demonstrated that the function of tree shrew CRH was almost identical to human CRH. An aGRE site within the tsCRH promoter was identified which functioned as a key element that contributed to the higher CRH expression levels in the tree shrew when compared with that of human under basal conditions and after treatment with forskolin and/or dexamethasone. Using Cas9/CRISPR technology, we further determined that when the tree shrew aGRE site was added to the CRH promoter in human HeLa cells, increased CRH mRNA expression was observed. The corresponding element identified in human CRH promoter region is polymorphic and a registered (-811A-G) SNP may affect its GR binding. Our results highlight the critical role of the aGRE site in regulating CRH mRNA expression through GR binding which results in increased promoter activity and susceptibility to stress in the tree shrew.−1 and 12.1 ng ml−1 (between 1000 h and 1100 h), respectively. After 15 min of acute restraint stress, the plasma levels of corticosteroids in the same tree shrew were significantly increased    right. OApproximately 5 kb of the tree shrew CRH gene sequence was resolved using the genome walking method . ThroughTo evaluate the downstream effects of the single amino acid change, murine pituitary AtT20 cells were treated with synthesized human or tree shrew CRH peptides. Phosphorylation levels of PKA and ERK kinases were detected by western blot to evaluate the CRH induced signal pathway activations. Our results showed that both 100 nM human CRH (hCRH) and tree shrew CRH (tsCRH or tCRH) transiently activated ERK and PKA as early as at 5 min after treatment  and reached peak activation at 15 min after treatment , indicating a similar pattern of signal activation between hCRH and tsCRH . As cAMPTo determine whether the I40M substitution in tree shrew CRH would affect its protein structure and binding affinity to the receptor tsCRHR1, the secondary structures of dissolved hCRH and tsCRH were characterized using circular dichroism (CD) spectroscopy, and bioinformatical pre-analysis was used to predict the crystal structure of the tree shrew CRH-CRHR1-ECD (CRH and the Extracellular domain of CRHR1) binding complex based on the published human CRH-CRHR1-ECD structure. Our results revealed a refined conformational change in the secondary structures between tree shrew and human CRH, where tree shrew CRH contained a little lower α-helix and higher β-sheet contents . FurtherWe further identified the explicit binding affinities (Kd) of the human and tree shrew CRH ligands to their CRHR1 receptors respectively using surface plasma resonance (SPR), of which the specific binding kinetics were presented by RU curves recorded at 5 concentration gradients of hCRH and tsCRH . The resWe simultaneously evaluated differences in transcriptional regulation of the CRH gene between tree shrew and human. To analyze CRH promoter activity, fragments encompassing the CRH promoter region (800 bp and 2000 bp in length) from the human and tree shrew genomes were sub-cloned into the pGL3-basic vector for a luciferase reporter assay. In both CHO and Neuro-2a cells, higher transcriptional activity for the 2-kb fragment but not the 800-bp fragment was observed in tree shrew CRH construct when compared with the human construct . These r25Low GR expression occurs in Neuro-2a cells and much lower GR expression occurs in CHO cells . In bothTo identify the key cis elements responsible for higher promoter activity in the tree shrew, a transcription factor binding site search was performed using the TESS database. This analysis revealed a novel predicted CRE binding site in the tree shrew CRH gene promoter in addition to a corresponding proximal CRE site, which is not present in the human CRH gene promoter . SurprisGAACA NNN TGTCCG in which the underlined G and C are the most essential bases2830Transcription factor binding site prediction analysis also revealed an active glucocorticoid receptor binding site (aGRE) located in the tsCRH promoter region (−802 to −787: AGAACG GGA AGTCTTCT). It is highly consistent with the canonical + GRE consensus sequence ATo function as a nuclear transcription factor, activated GR recruits various co-activators to remodel the chromatin structure and to form and maintain a transcriptional pre-initiation complex at the aGRE site of the CRH gene promoter regionThe taxonomic history of tree shrews are complicated and had raised long debates22333438Corticosteroids are important mediators of stress responses and are shaped by both genetics and external factors early in life4414311We determined the sequence of the tree shrew CRH gene and identified an amino acid substitution in the mature CRH peptide relative to human CRH, next we found that tree shrew and human CRH induced similar responses through their respective HPA axis activation. We simultaneously investigated the transcriptional regulation of the CRH gene. After confirming that tree shrew CRH gene activity is controlled by canonical CREB and GR signaling, we identified an aGRE site in the CRH promoter region functioning as a key cis element, which contributes to the increased CRH transcription activity that lead to the high vulnerability of the tree shrew to stress. These observations updated the previous view that CRH is negatively regulated by glucocorticoids through the function of GR as a trans-repressor that bind to the CRH promoter in the hypothalamus26Similar to our study on transcription regulation of CRH gene, A SNP (-248C-T) within the CRH gene that has been identified in rhesus macaques appears to contribute to increased CRH promoter activity, meanwhile a similar SNP (-201C-T) has been found in human although little is known regarding its functionin vivo studies to demonstrate the role of aGRE function such as how excess exogenous cortisol affects CRH mRNA expression in the hypothalamic PVN region under basal or stressed conditions remains to be investigated. Previous study of long-term chronic exogenous cortisol treatments on tree shrews showed that their urinary cortisol value was 6–9 times the basal level, while exogenous corticosterone administration on rats that raise plasma hormone to 4 mg/100 ml  induced significant reduction in the diurnal corticosterone secretionFurthermore, the In conclusion, our findings provide a molecular basis to explain the high stress susceptibility of the tree shrew, a promising animal model for the study of the stress response and stress-related diseases.Tree shrews were obtained from Kunming zoology institute, Chinese Academy of Science. All of the animal experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals with approval from the Institutional Animal Care and Use Committee for University of Science and Technology of China.Genome walking kit was purchased from Clontech Inc,  and performed according to the manufacturer’s instruction. Genomic DNA was extracted from the tree shrew kidney. The complete coding sequence of CRH gene was deposited into NCBI database (accession no.: JQ697839.1).The primers used to resolve full length CRH gene and used in the following ChIP assays were both listed in −1 into 50 μg ml−1 and the proteins were immobilized on the sensor chip (GE) with around 2000 resonance unit (RU) increase. For kinetic measurement of interactions between CRH and CRHR1-ECDs, the protein samples were diluted in PBS buffer for SPR . Serially diluted ligands were injected at a flow rate of 30 μl min−1 for 3 min respectively, followed by a 2 min dissociation process per reaction. The response was detected as a function of time (sensorgram) at 25 °C.Eukaryotic expressed hCRHR1-ECD and tsCRHR1-ECD proteins were provided by Cusabio . SPR assays were launched on the BIACORE3000  apparatus. The 10 mM NaAc buffer (pH 4.5) was chosen to dilute receptor-ECDs from 1 mg ml2. For CRH induced signal activation Assay, after treatment with hCRH and tsCRH peptides  for different time courses  or doses , cells were lysed in RIPA Buffer  containing phosphatase and protease inhibitor cocktails , samples were collected in loading buffer  and heated at 100 °C for 5 min. For transfection, 12 h after seeding, cells were co-transfected with reporter vector and a control plasmid phRL-TK  using X-treme 9 DNA transfection reagent  according to the producer’s manual.AtT-20, 293T, HeLa, Neuro-2a and CHO cells, purchased from ATCC, were grown in DMEM supplemented with 10% FBS and maintained in the 37 °C incubator containing 5% COSignal activations were detected by Western blot. Phosphorylation levels of p-PKA, p-Erk, PKA and Erk were immune-labeled by primary antibodies  and HRP conjugated secondary antibodies (Promega). Signals were developed by chemiluminescence (West PICO Pierce) followed by capturing and quantifying by Lumi-imaging system .Total RNA were isolated from AtT20 cells with 24 h of 100 nM hCRH and tsCRH incubation using Trizol Reagent (Sigma), and then were reverse transcribed and subjected to real time Q-PCR:Primers (5′to 3′): Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)TCAACGGATTTGGTCGTAT (F), ATGAGTCCTTCCACGATAC (R);Proopiomelanocortin (POMC):GTGCCAGGACCTCACCAC (F), CTTCCGGGGGTTTTCAGT (R);The reactions were performed in the ABI PRISM 7300 Detection System.2O 1:5) from AtT-20 cells incubated with hCRH and tsCRH for 8 h were measured with ACTH Elisa Kits .ACTH levels in supernatants (diluted by ddHKpnI and BglII or XhoI sites. Mutation was introduced by using Quickchange mutation kit (Stratagene) according to the instruction.800 or 2000 bp fragments corresponding to CRH promoter region were amplified using genomic DNA as the template and ligated into the luciferase reporter vector pGL3-basic (Promega) between Renilla luciferase activity was used to normalize the transfection efficiency.24 h after transfection, cells were collected and subjected to lysis in passive lysis buffer and luciferase activity was detected on Veritas Luminometer  using dual luciferase reporter assay (Promega), in which For ChIP and Co-IP assays, male tree shrew was decapitated, and the cerebral cortexes and hypothalamus were obtained. Solutions containing soluble chromatins from tree shrew tissues were immunoprecipitated with the anti-GRα antibody, anti-pCREB antibody (CST) as well as anti-SRC-1, anti-CRSP150, anti-Trap220 antibodies . Corresponding IgG proteins were used as negative controls. Subsequently DNA extraction, purification and amplification by PCR were performed with the primers listed in 293T cells were seeded 24 h before transfection. A Cas9+sgRNA (1:1 molar ratio) plasmid was transfected into cells using X-treme 9 DNA transfection reagent (Roche) at 50% confluency. 48–72 h later, cells were collected for genomic DNA extraction  and PCR analysis. The genomic region flanking the CRISPR target site for CRH gene was PCR amplified, and products were purified using Gel DNA extraction kit . The purified PCR products were mixed with T7 endonuclease buffer  and subjected to a re-annealing process. After re-annealing, products were treated with T7 endonuclease I and tested by DNA gel electrophoresis. Indel percentage was determined by the function:a stands for the integrated intensity of the undigested PCR band, and b and c are the integrated intensities of each cleavage band.−1) for 24–48 h, then were subcultured at very low density to enable cell clone formation. Each single clone was picked and cultured for 7 d, then subjected to genomic DNA and total RNA extraction and RT-qPCR analysis.For Cas9 nickase mediate genomic modification, donor plasmid DNA was co-transfected with plasmid in total of Cas9 nickase and two sgRNAs into Hela cells. 24 h later, cells were screened with puromycin .


Alterations in gut microbiome composition have an emerging role in health and disease including brain function and behavior. Short chain fatty acids (SCFA) like propionic (PPA), and butyric acid (BA), which are present in diet and are fermentation products of many gastrointestinal bacteria, are showing increasing importance in host health, but also may be environmental contributors in neurodevelopmental disorders including autism spectrum disorders (ASD). Further to this we have shown SCFA administration to rodents over a variety of routes  or developmental time periods can elicit behavioral, electrophysiological, neuropathological and biochemical effects consistent with findings in ASD patients. SCFA are capable of altering host gene expression, partly due to their histone deacetylase inhibitor activity. We have previously shown BA can regulate tyrosine hydroxylase (TH) mRNA levels in a PC12 cell model. Since monoamine concentration is known to be elevated in the brain and blood of ASD patients and in many ASD animal models, we hypothesized that SCFA may directly influence brain monoaminergic pathways. When PC12 cells were transiently transfected with plasmids having a luciferase reporter gene under the control of the TH promoter, PPA was found to induce reporter gene activity over a wide concentration range. CREB transcription factor(s) was necessary for the transcriptional activation of TH gene by PPA. At lower concentrations PPA also caused accumulation of TH mRNA and protein, indicative of increased cell capacity to produce catecholamines. PPA and BA induced broad alterations in gene expression including neurotransmitter systems, neuronal cell adhesion molecules, inflammation, oxidative stress, lipid metabolism and mitochondrial function, all of which have been implicated in ASD. In conclusion, our data are consistent with a molecular mechanism through which gut related environmental signals such as increased levels of SCFA's can epigenetically modulate cell function further supporting their role as environmental contributors to ASD. Thus, hepatic clearance was thought to impede achieving levels sufficiently high to affect systemic functions or metabolic and regulatory pathways. However, the distal colon bypasses the portal circulation enabling systemic access. Despite the difficulty in their measurement due to intracellular concentration and rapid metabolism, there is growing evidence that the potential systemic effects of SCFA  on physiology and pathology may have been underestimated rev. in .SCFA have a number of direct effects on gastrointestinal physiology. Along with acetate and butyrate, PPA is known to reduce gastric motility and increase the frequency of contractions, presumably via a reflex that involves direct contact of these short chain fatty acids with the terminal ileum Clostridia, Bacteriodetes, Desulfovibrio) Recent evidence suggests potential, but unproven, links between dietary, metabolic, immune, infective, and gastrointestinal factors and ASDs. Although inheritable factors, mostly implicated in synaptic transmission, have been traditionally studied in ASD Further to this, we One potential key mechanism where the metabolic products of an altered gut microbiome may contribute to ASD pathophysiology is via the alteration of gene expression associated with ASD mutations or ASD implicated genetic pathways in vitro cell system to examine molecular biological processes in neurobiology y activation of transcription factors via different signaling cascades (including Ca2+/cAMP mediated activation of CREB The rat pheochromocytoma (PC12) cell line is an extensively used in vitro system to extend our observations on the epigenetic effects of SCFA. Microarray technology was used to compare global changes in gene expression profiles following exposure to the structurally related SCFA PPA and BA. Furthermore, we sought to determine if the expression of these PPA dependent genes was related to canonical biological pathways implicated in ASD.Since most research on the effects of SCFA on gene expression is limited to BA and not PPA, in the present study we used rat PC12 cells as an 2 as described earlier 6–107 cells). Cells treated with SCFA continued to receive media supplemented with SCFA after electroporation. After incubation for an additional 24 hours, cells were harvested and crude cell lysates were prepared. Protein concentrations in total cell lysates were determined PC12 cells . All plasmid DNA constructs were purified by chromatography on Qiagen columns .To compare the effects of structurally related SCFA on endogenous TH gene expression, PC12 cells were treated with low concentrations of SCFA  or with vehicle for 48 hours. Total RNA was isolated from individual petri dishes using RNazol according to the manufacturer's protocol . Northern blot analysis was performed as described previously PC12 cells were treated with 1 mM PPA, 10 mM PPA, 1 mM BA, 6 mM BA or vehicle for 48 hrs. Total RNA samples from each experimental group were pooled together and after DNase treatment purified using RNeasy Mini Kit  as recommended by Affymetrix. The cDNA synthesis and microarray analyses were performed at Keck Affymetrix GeneChip Resource at Yale, New Haven, CT (NIH Neuroscience Microarray Consortium). The chip data was analyzed using Affymetrix GeneChip Operating Software (GCOS) version 1.2.1. Genes showing altered expression with fold change values of ≥2.0 were exported for functional annotation to MetaCore by GeneGO, Inc. The expression levels of selected genes  were validated through Northern blot analyses of the same RNA samples . Raw andTotal protein extracts were prepared from each petri dish of control (vehicle treated) and SCFA- treated PC12 cells for the indicated periods of time (see the figure legend). Proteins were separated on 10% SDS-PAGE, electroblotted onto a nitrocellulose membrane  and incubated with TH antibody  overnight. After incubation with secondary antibody  the immune reaction was visualized by enhanced chemiluminescent substrate from Pierce, utilizing a horseradish peroxidase label and Kodak XAR-5 film, as described by the manufacturer. To confirm equal loading, blots were re-probed with primary antibody for the house keeping protein β-actin . The blots were exposed to autoradiography and the x-ray films were scanned and quantified with Bio-Rad Quantity One software. The ratio TH/β-actin immunoreactivity were calculated for each sample and the results were presented as fold induction compared to the corresponding control group on the same Western blot.Data were expressed as mean ± SEM and normalized to the values in the control, taken as 1.0. Differences between the experimental groups from at least three independent experiments were evaluated by performing ANOVA followed by the Fisher's least significant difference test for experiments with more than two groups. A level of p≤0.05 was accepted as statistically significant.2+/cAMP-mediated signaling pathways in the transcriptional effects of structurally related SCFA.We have shown before To test this possibility, we also evaluated the role of the general transcription factor CREB in PPA-induced activation of TH gene promoter. In these experiments, PC12 cells were transiently transfected with plasmids carrying the luciferase reporter gene under the control of rat TH promoter in combination with either wild type CREB (positive control); dominant negative CREB expression vector(s) or unrelated DNA (pCMV-CAT expression vector) as we described before Our previous data revealed that exposure of PC12 cells to low concentrations of butyrate resulted in accumulation of endogenous TH mRNA see . To compTo determine whether SCFA-induced alterations in steady state TH mRNA levels were reflected in changes in TH protein, we treated PC12 cells with 1 mM PPA or BA for 24, 48 and 72 hours. Controls received only vehicle. Exposure to SCFA resulted in gradual increases in the relative immunoreactive TH protein levels, suggesting that the capacity of the cells to produce catecholamines is elevated .To identify other potential cellular targets for SCFAs, we used microarray technology allowing genome-wide simultaneous measuring of changes in gene expression. PC12 cells were exposed to SCFA , or vehicle for 48 hours. Total RNA was isolated and subjected to microarray analysis using Affymetrix GeneChip Rat Genome 230 2.0 microarray as described in Only few affected genes (79) fit the criteria when cells were treated with low dose of 1 mM PPA. The expression of a significantly higher number of genes was altered following exposure to 10 mM PPA (1587). At both doses, BA was more effective than PPA as it was evident by the number of differentially expressed transcripts and correlated with the higher potency of BA to induce histone hyperacetylation and alter global gene expression. For example, a total of 421 genes were significantly altered following treatment with 1 mM BA, and 3148 for 6 mM BA group. We report here the results from MetaCore enrichment analysis performed for 10 mM PA and 6 mM BA groups.Comparison analysis revealed 1599 differentially expressed genes unique for the BA experimental group and 355 for the PPA group. A large number of genes (1010) were identified as common for both, BA and PPA groups , S2, S3.Differentially expressed common genes included but were not limited to the following: those involved in the biosynthesis and degradation of dopamine, norepinephrine and serotonin , mitochondrial/fatty acid metabolism , oxidative stress , gap junction proteins, signaling pathways  receptors , see The top five most relevant gene networks, identified using the Analyze Networks (AN) algorithm with default settings  classification using MetaCore . The mosEmerging evidence links SCFA from diet, cellular metabolism or pharmacological treatments with regulation of neuronal gene expression, attendant neuronal function and even behavior The short chain fatty acid BA is a well known HDACI shown to induce differentiation, growth arrest and chromatin remodeling in a variety of cell lines, associated with specific-gene transcription regulation properties Our data correlate well with the fact that SCFA may enter the cell by passive diffusion and/or active transport via specific monocarboxylic acid/ketone transporters PPA can also exert its effect by activating nuclear receptor PPAR gamma The microarray data reported here are also consistent with the activation of CREB pathway – i.e. increased expression of CREB3L1 was observed in PPA (2.33 fold) and BA (4.2 fold) group, accompanied by elevated MAP4K1 (2-fold and 3.6 fold resp.), CAMK2G (2-fold for both SCFA) and CAMKV (increased by approximately 3-fold by PPA and BA). Increased P-CREB immunoreactivity was observed in ASD- hippocampus and white matter regions following intracerebroventricular infusion of SCFA  results in accumulation of TH mRNA and TH protein  indicatibi-directional perturbation of the dopaminergic pathways by both PPA and BA  and GTP cyclohydrolase 1 ; During the past few years, studies have begun to suggest that cholinergic systems in the brain might also be implicated in ASD The brain is vulnerable to mitochondrial dysfunction, oxidative stress and excitotoxicity P≤0.05  can in principle result in similar “loss” or “gain” of function and disrupt similar pathways/networks as genetic alterations associated with ASD.We applied MetaCore Gene Ontology (GO) bioinformatics tool In summary, the gene expression analysis in this study provides valuable insight into the complexity of possibilities and specific molecular pathways or processes altered in PC12 cells upon stimulation with SCFA, many of which, directly or indirectly, have been implicated in ASD. The study also underscores a novel capacity of SCFA to regulate multiple pathways and networks, which are relevant to brain functions and behavior. We believe that our data represent a useful initial cell model resource for identifying and functionally testing SCFA-responsive neuronal genes.in vitro system, as the effects of PPA on non-neural CNS cells  normally produced in the gut by microbial fermentation of dietary carbohydrates, may:Represent a novel evolutionary link to neural plasticity associated with different behavioral responses to a changing environment.Affect brain and behavior by multiple molecular mechanisms, some of which involve Ca2+/cAMP/CREB signaling pathways.Modulate expression of key genes  many of which have been implicated in neurodevelopmental disorders, such as ASD.Table S1List of differentially expressed genes unique for PPA.(XLS)Click here for additional data file.Table S2List of differentially expressed genes unique for BA.(XLS)Click here for additional data file.Table S3List for differentially expressed genes, common for PPA and BA.(XLS)Click here for additional data file.


Caenorhabditis elegans and Drosophila melanogaster) are unsuitable to model CKDs. Systematic structure/function comparisons revealed that the combination of ultrafiltration and flow-associated filtrate modification that is central to CKD etiology is remarkably conserved between the planarian excretory system and the vertebrate nephron. Consistently, both RNA-mediated genetic interference (RNAi) of planarian orthologues of human CKD genes and inhibition of tubule flow led to tubular cystogenesis that share many features with vertebrate CKDs, suggesting deep mechanistic conservation. Our results demonstrate a common evolutionary origin of animal excretory systems and establish planarians as a novel and experimentally accessible invertebrate model for the study of human kidney pathologies.Cystic kidney diseases (CKDs) affect millions of people worldwide. The defining pathological features are fluid-filled cysts developing from nephric tubules due to defective flow sensing, cell proliferation and differentiation. The underlying molecular mechanisms, however, remain poorly understood, and the derived excretory systems of established invertebrate models  have sought to fill in the gaps, but each model has its own limitations.Now, Thi-Kim Vu et al. propose that a flat worm called a planarian could provide a new and experimentally accessible animal model to study cystic kidney diseases. These flat worms get rid of their waste products via an excretory system that consists of branched tubules that spread throughout the body. Thi-Kim Vu et al. found that, like the tubules in the kidney, these tubules filter and then reabsorb chemicals from body fluids. Moreover, these processes are performed in different parts of the tubules, exactly as they are in the tubules in kidneys.Schmidtea mediterranea contains many genes that cause cysts to form when their equivalents are mutated in humans. Reducing the expression of these genes (and others that are involved in cilia formation) also caused cysts to form in the flat worms.The genome of a flat worm called These findings indicate that it is likely that the excretory systems of different animals have a shared evolutionary history. If so, the findings support the idea that cilia in kidney tubules send signals in response to fluid flow that affect kidney-specific stem cells. They also suggest that problems with these signals could be at the core of some human cystic kidney diseases. One of the next challenges will be to identify these cilia-associated signals. Finally, given that studies involving thousands of flat worms can be carried out with minimal cost, the ultimate goal is to develop flat worms into a new model to discover and investigate genes linked to human kidney diseases.DOI:http://dx.doi.org/10.7554/eLife.07405.002 Xenopus pronephros and zebrafish pro- and mesonephric kidneys, therefore, are increasingly being explored as model systems for human kidney disease  the pressure-driven ultra-filtration of blood plasma across the glomerulus, whereby molecular sieves prevent the passage of large macromolecules ; and (2) the subsequent modification of the resulting filtrate during its passage through the epithelial nephron tube . The par disease . Compoun disease . Both Caelegans; ) or uncoogaster; ). Furthecdysozoa . One succdysozoa . Here, wDrosophila nephrocytes  distributed throughout the entire body plan  Rink et. The barhrocytes . We co-ihrocytes . BecauseSchmidtea mediterranea genome identified 318 slc genes. Reciprocal BLAST analysis and sequence alignments revealed that S. mediterranea slcs represent 43 slc families  transporters recovers essential molecules from the primary filtrate or secretes waste products into the tubule lumen . The knofamilies . Express tissues , with 49slc relative to two previously characterized markers  segment (CAVII-1 expression were not available at the beginning of this study. Fluorescent in situ hybridization (FISH) mapping of putative protonephridial slc genes against the two markers and general tubule anatomy  revealed a significantly greater complexity of protonephridial cell types than previously appreciated . Interestingly, CD marker expressing segments were exclusively located close to the dorsal body surface, supporting early reports suggesting that the protonephridial terminus was located in the dorsal epithelium  ace segment . Markersreciated . slc expivisions . Similarithelium . Consistslc genes defined 6 molecularly distinct segments along the proximal-distal axis of protonephridia.For the first time, our results trace the complete course of protonephridial tubules from the ultrafiltrating flame cells as proximal entry point to their terminus in the dorsal epithelium. Further, our systematic mapping of expression domains of slc genes with known substrate specificity by substrate class and site of expression revealed a striking segregation of similar transport activities into similar regions of the protonephridial tubule, indicating the functional specialization of different segments . In terrestrial vertebrates, IT and CD have tightly linked functions in water conservation, whereby urea secretion by the IT establishes high extracellular solute concentrations that aid in water reabsorption from the CD . As fresNPHS1 and NEPH1 cause slit diaphragm loss by fusion of neighboring foot processes into a continuous cytoplasmic sheet (foot process effacement), resulting in proteinuria and edema  planarians. Wild type flame cells display slit-shaped 35–40 nm wide fenestrae that form between 90–150 nm wide foot processes  planarians, which displayed equal uptake of both small and large molecular tracer in the PT (NPHS1-6- and NEPH-3(RNAi) planarians, we could not observe ultrastructural evidence of flame cell detachment at the time that loss of ultrafiltration was observed (not shown). Evidence from a rat model of nephrotic syndrome indicates that the upregulation of transcytotic transport processes across the effaced podocyte envelope could maintain a basal level of non-size-selective fluid flow (NPHS1-6- and NEPH-3(RNAi) phenotypes in planarians and human nephrotic syndrome demonstrate that the functional homology between planarian flame cells and vertebrate podocytes extends to common pathologies.Because flame cell numbers appeared normal in both intact and regenerating animals , we examrocesses , 4D. Undh intact , 3 and rh intact . The dexn the PT . Why woun the PT . Howevern the PT . Consistuid flow , yet theS. mediterranea genome harbors homologs to human NPHP1-9, except for NPHP2 and NPHP3  animals, particularly of the proximal segment. RNAi animals presented striking clump-like accumulations of PT marker expressing cells (NPHP(RNAi) animals  animals, the number of BrdU/POU2/3 double-positive cells in the vicinity of cell accumulations was notably increased (NPHP(RNAi) animals (POU2/3+/smedwi-1+/H3P+), neuronal- (pax6A+/smedwi-1+/H3P+) (HNF4+/smedwi-1+/H3P+) (NPHP5(RNAi) and NPHP8(RNAi) animals, we found no change in the fraction of proliferating neuronal progenitors and even a slight decrease in intestinal progenitor proliferation   and inte1+/H3P+)  progenit1+/H3P+) . Whereasferation . The obsgenitors  further NPHP8(RNAi) animals was faster and more severe under the increased proliferation condition, yet significantly diminished or even abolished under reduced or no proliferation, respectively (slc6a-13 and CAVII-1) as a direct cell accumulation metric  planarians display severe defects in cilia-driven gliding motility (NPHP(RNAi) animals indeed confirmed structural cilia defects, which appeared shorter (NPHP5(RNAi)) or much reduced in density (NPHP6/8(RNAi)) (NPHP5/6/8(RNAi) ) . EM imag/8(RNAi) . Togetheia genes   animals lost their cilia-dependent gliding ability (IFT88(RNAi) animals also developed cystic protonephridia (IFT88(RNAi) animals was also associated with increased proliferation  animals also developed edema and formed protonephridial cysts  are not only expressed, but also required for planarian flame cell ultrafiltration function ; and second, the excretory systems of existing invertebrate model systems do not recapitulate this critical aspect. In fruitflies and other insects, ultrafiltration and filtrate modifications are carried out in separate cell types and organs  . C. eleggle cell . Both argle cell , 2011. Tgle cell , 2011. IAsplanchna  or the inhibition of ciliary beating by interfering with axonemal dyneins leads to protonephridial progenitor overproliferation. Moreover, in both cases, accumulating protonephridial progenitors form dense aggregates with disorganized lumens that quickly compromise the osmoregulatory functions of the organs, leading to edema formation. Hence the disruption of planarian cilia leads to phenotypically very similar alterations as PKD in vertebrates. We therefore conclude that cilia-mediated flow sensing constitutes an ancient mechanism for maintaining excretory organ homeostasis.The nephrons of the adult human kidney also bear cilia. However, in contrast to the multiciliated and flow generating cilia of the planarian PT, the cells of the adult mammalian nephron bear a single, immotile primary cilium protruding into the lumen . DespitePlanarians have lost central components of centrosome duplication, which should therefore preclude their ability to form primary cilia . Since pIn humans, polycystin-1 and polycystin-2 are key components of cilia-mediated flow sensing . As streGiven the extensive evolutionary homology between excretory systems, how can the exclusive reliance on motile cilia driven flow generation in invertebrates be reconciled with their absence in adult vertebrate kidneys? The loss of motile cilia in vertebrate kidneys has been observed in coincidence with the increase in blood pressure in birds and mammals . This raNPHP(RNAi) phenotypes phenocopy human NPHP. In human NPHP, cysts arise at the corticomedullary junction of the kidneys, meaning that they mostly develop from the distal convoluted and collecting tubules  . The mosleA2 containing 20% glycerol, 2.5% DABCO and 4 M urea  for 3–4 hr under direct light. After in situ development, specimens were treated with 2 N HCl for 45 min at room temperature, and washed four times with PBSTx (0.3% Triton) for 1 hr. BrdU was detected using rat anti-BrdU antibody . Primary antibody was detected with HRP-conjugated anti-rat antibody .BrdU was administered by soaking animals in 15 mg/ml BrdU and 3% DMSO  for 1 hr as previously described  and chasTo assay ultrafiltration capacity of planarian protonephridia, 10 kDa tetramethylrhodamine-dextran  and 500 kDa fluorescein-dextran  at the concentration of 1 mg/ml were co-injected into the mesenchyme of the animals. After 2 hr, the animals were rinsed with an excess of 1× Montjuic salts, fixed in cold 4% paraformaldehyde , mounted in modified ScaleA2 and photographed using a Zeiss LSM-510 VIS confocal microscope. Dextran uptake was quantified by measuring the average fluorescence intensity per unit area using a standard signal to noise thresholding in Fiji . For immIntracellular pH was measured using ratiometric pH dye SNARF-5F-AM  at 5 μM (in DMSO with 20% wt/vol Pluronic F-127) as previously described , 2013. Ahttp://research.stowers.org/imagejplugins).To visualize cilia beating along the lateral body edge of the planarian head region, live worms are immobilized on the glass bottom dish using a microfluidic device and imaged on a Zeiss Axiovert 200 microscope under DIC optics using 63× objective. Series of images were captured at 250 frames per second with pixel number of 800 × 800 (exposure time is 3.97 ms) using an ORCA-Flash4.0 V2 C11440-22CU camera from Hamamatsu. Spatiotemporal image correlation spectroscopy was used to determine the speed of the cilia for each animal. In each time-lapse, 100 consecutive frames were manually selected in which the animal was stationary so that no image registration was required. A region of interest was manually drawn around the cilia in each time-lapse. The area outside this region was uniformly filled with the average intensity inside the region. Spatiotemporal correlation was then carried out in 32 × 32 pixel regions with a 16 pixel overlap between the regions to allow for highly localized motions to be accurately represented using the fast Fourier transform method. The average cilia displacement within the correlation image is represented by the maximum of the spatial cross-correlation between two images separated in time. The time correlation shift was a single frame, and all velocities were converted to micrometers per minute. This method was adapted from a previous paper , where it-test.Statistical analysis of the data was carried out in Excel. p values were determined using Student's 2) for overnight; (2) wash in wash buffer  for 1 hr (3–4 exchanges); (3) fix in 1% Osmium tetroxide in 0.1 M sodium cacodylate buffer (+1 mM CaCl2) for 2 hr; (4) wash in wash buffer for 1 hr (3–4 exchanges) and in distill water for 30 min (3–4 exchanges); (5) fix in 0.5% aqueous Uranyl Acetate (in dark) overnight; (6) wash in distill water for 30 min (3–4 exchanges); (7) and dehydrate in acetone 30% (20 min), 50% (20 min), 70% (overnight), 90% , and 100% . Specimens were then embedded in epon-araldite or Spurr's resin as follows: 25% resin/acetone for 3 hr; 50% resin/acetone for 2.5 hr; 75% resin/acetone overnight; 100% resin without accelerator with microwave at 350 W for 3 min on/3 min off/3 min on for 1 day (2 exchanges); 100% resin with accelerator with microwave at 350 W for 3 min on/3 min off/3 min on for 1 day (2 exchanges) and placed in 60°C oven for polymerization for 2 days. Ultra-thin 50–100 nm sections were collected using a Leica UC6 Ultramicrotome. TEM specimens were stained with Sato's lead (3 min)/4% Uranyl Acetate in 70% methanol (4 min)/Sato's lead (6 min) prior to imaging on a FEI Technai BioTwin at 80 kV equipped with a Gatan UltraScan 1000 digital camera.Specimens were prepared as following at 4°C on orbital rotator: (1) fix in cold 2.5% glutaraldehyde in 0.05 M or 0.1 M sodium cacodylate (contained 1 mM CaClRNAi feedings were performed as described previously . FeedingIFT88- and LRRC50(RNAi): 3 dsRNA feedings (2 days in between); DNAHβ-1(RNAi): 8 dsRNA feedings (2 days in between). review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom, Yukiko Yamashita, is a member of our Board of Reviewing Editors. Another one of the reviewers, Friedhelm Hildebrandt, has also agreed to share his identity.Thank you for sending your work entitled “Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ” for consideration at The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.In this manuscript, Alvarado and colleagues provide convincing evidence to show that the planarian excretory system, protonephridia, serves as a powerful model system to study mammalian kidney development and pathology such as cystic kidney disease. All reviewers agreed that this is an excellent paper, and raised no major concerns. They have several specific comments, which we suggest authors to address prior to publication. We decided to communicate all comments from reviewers to the authors, instead of summarizing points to be revised, as they contain encouraging comments and praises.Reviewer #1:slc expression patterns relative to known excretory system markers, the authors were able to define six different domains within this system. Strikingly, clustering slc genes based upon known substrate specificity and comparing their expression domains to those observed in vertebrate nephrons revealed a remarkable degree of similarity in the overall structural/functional organization of the planarian and rodent systems. These parallels extend to mutations associated with cystic kidney diseases in humans. RNAi knockdowns of planarian nephrins resulted in ultrastructural defects in the slit diaphragms in planarian flame cells and disruption of filtration. RNAi knockdowns of nephrocystin homologs generated cyst-like structures within the protonephridial tubules. Remarkably, the formation of these cysts and the resulting osmoregulatory defects are dependent upon proliferation. Finally, the authors show that knockdowns of genes required for proper ciliary beating  also produced cysts within the excretory system.This manuscript by Vu et al. provides a comprehensive characterization of the planarian excretory system. The authors conducted systematic phylogenetic and expression analyses of the ∼300 solute carrier (slc) transporters encoded by the planarian genome, identifying 49 that were expressed in the protonephridia. By mapping the eLife seeks to publish “authoritative” papers, and I can think of no better word to describe the truly impressive body of work that the authors present here. They convincingly show the many ways in which the planarian osmoregulatory system parallels such systems in vertebrates, providing an invertebrate model that can help us understand both the evolution of osmoregulatory systems and their pathologies. This beautiful paper sets the standard for how such work should be conducted in this organism and will help guide all future work on its excretory system.To help strengthen the impact of this paper, I have only a few minor suggestions/comments that the authors may wish to consider addressing before publication:slc expression (A-H), and another figure emphasizing the structural/functional conservation with the rodent (J-L). In its current form, with the panels reduced to accommodate all of the data, these important conclusions are underemphasized.1) The data presented in NPHS1 (RNAi) flame cell shows loss of the slit diaphragms, so the observed loss of ultrafiltration (500 kDa dextrans can now enter the system) seems counterintuitive. It may be helpful for the readers if the authors could comment on how loss of the slit diaphragms can lead to loss of size selectivity.2) Ultrastructural analysis of the Reviewer #2 (Friedhelm Hildebrandt):Schmidtea mediterranea (planaria). In the first part of the manuscript, authors extensively characterized the planarian excretory system by identifying solute carrier (slc) genes and mapping their expression, and revealed that the planarian protonephridium is structurally and functionally similar to the vertebrate nephron. In the second part of the manuscript, authors knocked down planarian orthologues of human genes which, if mutated, cause kidney diseases including nephrotic syndrome and nephronophthisis. Authors showed by knocking down planarian NPHS1 and NEPH3 that planarian flame cells are functional homologues of vertebrate podocytes. Knockdown of planarian nephrocystin orthologues  led to development of cyst-like structures in proximal tubule segments. In addition, authors found that the cyst formation in planaria depends on overproliferation of protonephridial progenitor cells and loss of cilia function as a fluid flow generator/sensor. These have been also suggested as disease mechanisms of human cystic kidney diseases. Based on these findings, authors concluded that the planarian protonephridium not only has structural and functional homologies, but also shares pathologies with human nephron. In conclusion, authors suggest the planarian protonephridum as a model system to study human kidney diseases.This manuscript describes the protonephridial excretory system of This work is interesting and the data are generally clear and of high quality. The amount of work to characterize the protonephridial excretory system of planarians is enormous, and extensive, and it is convincing and will have an impact in the renal community. The following points need to be addressed before publication.NPHP8. Have authors checked the increased proliferation in planaria with NPHP5, NPHP6, IFT88, DNAHβ-1 or LRRC6 knockdown?1) The authors showed that cyst formation depends on increased proliferation of protonephridia progenitors upon knockdown of 2) The authors claim that fluid flow, which may be generated by cilia, is important for tubular function based on knockdown of motile cilia genes. However, whether the cilia-driven flow exists in planarian excretory system is not clear in the current data. Another possible scenario is that the motile cilia in the protonephridia may be important in clearing waste materials in a similar way to the ‘mucociliary clearance’ in the respiratory systems. Therefore, this review recommends that authors should tone the ‘cilia-driven fluid flow’ down in the Results and Discussion section.3) It seems that knockdown of either NPNP genes or PCD genes in planaria resulted in protonephridial cysts, in other words, the same pathologies. However, in humans NPHP and PCD are totally different entities and almost mutually exclusive. In most cases, NPHP genes do not affect motile cilia and PCD genes do not affect immotile cilia. In this regard, it may be possible that the mechanism of cyst formation in planaria is different from that in human. In addition, in human NPHP, cysts occur in corticomedullary junctions and most develops from distal convoluted tubules. But protonephric cysts in planaria form in proximal tubules. Please discuss about these issues. Reviewer #1:1) The data presented inare so striking that they warrant two separate figures: one emphasizing the spatial domains defined by slc expression (A-H), and another figure emphasizing the structural/functional conservation with the rodent (J-L). In its current form, with the panels reduced to accommodate all of the data, these important conclusions are underemphasized.Thank you for this suggestion. We split the former 2) Ultrastructural analysis of the NPHS1 (RNAi) flame cell shows loss of the slit diaphragms, so the observed loss of ultrafiltration (500 kDa dextrans can now enter the system) seems counterintuitive. It may be helpful for the readers if the authors could comment on how loss of the slit diaphragms can lead to loss of size selectivity.The reviewer pointed out an important discrepancy between the EM data and the dextran assay, which has also been raised in studies of nephrotic syndrome in vertebrates. In fact, many nephrotic syndrome patients present severe proteinuria in conjunction with podocyte foot process effacement without observable podocyte detachment or loss . We inte“Why would the fusion of foot processes into a continuous sheet result in loss of filtration size selectively […], yet the exact mechanisms remain to be determined in both humans and planarians”.Reviewer #2 (Friedhelm Hildebrandt):1) The authors showed that cyst formation depends on increased proliferation of protonephridia progenitors upon knockdown of NPHP8. Have authors checked the increased proliferation in planaria with NPHP5, NPHP6, IFT88, DNAHβ-1 or LRRC6 knockdown?NPHP5(RNAi) (NPHP8(RNAi) treated animals. We further added the analysis of IFT88- and LRRC50(RNAi) animals (We did check the increased proliferation in planarians after P5(RNAi) , which,  animals  and foun2) The authors claim that fluid flow, which may be generated by cilia, is important for tubular function based on knockdown of motile cilia genes. However, whether the cilia-driven flow exists in planarian excretory system is not clear in the current data. Another possible scenario is that the motile cilia in the protonephridia may be important in clearing waste materials in a similar way to the ‘mucociliary clearance’ in the respiratory systems. Therefore, this review recommends that authors should tone the ‘cilia-driven fluid flow’ down in the Results and Discussion section.Asplanchna that within 30 minutes after exposure to NiCl2, an inhibitor of cilia mobility, beating of cilia stopped and the bladder then ceased its normal fill-contraction cycle significantly, while other muscle and body movements appeared normal  It seems that knockdown of either NPNP genes or PCD genes in planaria resulted in protonephridial cysts, in other words, the same pathologies. However, in humans NPHP and PCD are totally different entities and almost mutually exclusive. In most cases, NPHP genes do not affect motile cilia and PCD genes do not affect immotile cilia. In this regard, it may be possible that the mechanism of cyst formation in planaria is different from that in human. In addition, in human NPHP, cysts occur in corticomedullary junctions and most develops from distal convoluted tubules. But protonephric cysts in planaria form in proximal tubules. Please discuss about these issues.We thank the reviewer for pointing out this aspect of our phenotypes. We have included a discussion paragraph comparing/contrasting the planarian phenotypes with human pathologies. This section now reads:“Planarians have lost central components of centrosome duplication […] suggesting the likely existence of flow sensing role of motile cilia in planarian protonephridia .”“In NPHP knockdown planarians, cysts primarily arise in the PT at early stage […] why cysts predominantly affect the distal convoluted and collecting tubules only, but not other tubule parts in human NPHP is still unknown.”


AIP) gene. A 50-year-old male presenting with headache was diagnosed with a large pituitary tumor by magnetic resonance imaging (MRI). His visual fields were intact and he exhibited no features of CD. Owing to an exuberant response to synacthen, an overnight dexamethasone suppression test was performed revealing inadequate suppression of plasma cortisol (419 nmol/l). Owing to tumor growth and visual field impairment, he underwent transsphenoidal surgery and developed hypocortisolemia. The pathology specimen revealed a sparsely granulated corticotrope adenoma. Postoperative MRI showed a large tumor remnant. The patient developed skin hyperpigmentation and a synacthen test demonstrated high basal and stimulated cortisol levels; an overnight dexamethasone suppression test showed no suppression (791 nmol/l) and elevated plasma ACTH levels (135 ng/l). A transcranial operation was performed followed by radiotherapy. Two months after radiotherapy, he developed secondary adrenocortical failure. Genetic testing revealed an AIP variant of unknown significance (p.R16H) without loss of the normal AIP allele in the tumor. A literature review showed ten CD patients with AIP gene variants, of whom five (including our case) were p.R16H. CD is occasionally dominated by pituitary tumor growth rather than symptoms of hypersecretion. The particular AIP gene variant identified in our patient is shared by four other reported cases of CD. Future studies are needed to assess whether the reported AIP gene variant is more than just coincidental.A patient of Cushing's disease (CD) characterized by a large tumor and only subtle symptoms of hormonal hypersecretion was examined. The patient had a germline variant in the aryl hydrocarbon receptor-interacting protein (CD is occasionally dominated by pituitary tumor growth rather than symptoms of hypersecretion.Resolution of both tumor remnant and hormonal hypersecretion may occur within 2 months after postoperative radiotherapy.The particular AIP gene variant identified in our patient is shared by four other reported cases of CD. Cushing's syndrome refers to the clinical manifestations induced by chronic exposure to excess glucocorticoids. It is a rare condition with an annual incidence of 3/million. It is, however, a frequently entertained differential diagnosis as it shares symptoms and signs with more prevalent conditions, such as type 2 diabetes and polycystic ovary syndrome. In addition, both the diagnosis and treatment of Cushing's syndrome are challenging, which have important prognostic implications. Cushing's disease (CD), defined as pituitary adrenocorticotropic hormone (ACTH) excess, is the most frequent cause of endogenous Cushing's syndrome with an incidence of 2/million per year. It is usually due to a microadenoma  and may be difficult to visualize; in contrast, macroadenomas  comprise only 4–10% of diagnosed patients AIP) gene have been shown to play a casual role in the development of pituitary adenomas, in particular large somatotropinomas occurring in young patients The etiology and pathogenesis of corticotrope adenomas remain unclear and the majority appears to be sporadic. In a minority of cases, Cushing's syndrome is associated with genetic syndromes such as McCune–Albright syndrome, MEN1, and Carney complex. More recently, germline mutations in the aryl hydrocarbon receptor-interacting protein  and exhibited no clinical features of anterior pituitary insufficiency, CD or acromegaly, but he did present symptoms of diabetes insipidus with pronounced thirst, polydipsia and polyuria (∼6 l/24 h), also during the night. Iodothyronine and thyroid-stimulating hormone levels were in the low normal range; a short synacthen test showed no evidence of ACTH insufficiency (cortisol levels (nmol/l): 654  and 981 (30 min)). The patient was started on oral desmopressin, and within 1 month, he was also substituted with levothyroxine and testosterone. A neuro-ophthalmological examination revealed intact visual fields. Owing to a moderately elevated serum insulin-like growth factor 1 (IGF1) level  and the exuberant response to ACTH stimulation, both a growth hormone (GH) profile during an oral glucose load and an overnight dexamethasone suppression test were performed. The former disclosed a near-normal nadir GH level of 0.32 μg/l (<0.30 μg/l), but the patient failed to suppress his morning plasma cortisol (nmol/l) to overnight dexamethasone (648 (afternoon) and 419 (morning)) combined with an unsuppressed ACTH level of 62 ng/l (7–64 ng/l). An MRI performed 7 months after the first one indicated tumor growth  performed due to a 1-year history of headaches. Twelve months before this, the patient underwent unilateral orchidectomy for a testicular seminoma and was in remission. In addition, the patient had a history of transitory cerebral ischemia and has been recently diagnosed with type 2 diabetes. The initial MRI revealed a pituitary macroadenoma with suprasellar extension a. The par growth b. Human 2). A postoperative GH profile with an oral glucose load showed a slightly elevated nadir GH level (0.37 μg/l) and serum IGF1 level of 248 μg/l (55–220 μg/l). An MRI performed 4 months after the operation demonstrated a large suprasellar tumor remnant; somatostatin analog treatment (Sandostatin LAR 10 mg/4 weeks) was attempted to allow for tumor shrinkage and normalization of GH. A repeat MRI after 6 months showed tumor growth but normal visual function, and treatment with a dopamine agonist (cabergoline 0.5 mg twice weekly) was added. The patient also appeared hyperpigmented and a synacthen test demonstrated high basal (797 nmol/l) and stimulated (1084 nmol/l) cortisol levels. Hydrocortisone treatment was discontinued and a subsequent overnight dexamethasone suppression test again showed lack of suppression (791 nmol/l) and elevated plasma ACTH levels (135 ng/l (reference range: 7–64 ng/l)). His glycemic control deteriorated and insulin treatment was started. The patient was underweight . The pathology specimen from the initial operation was re-evaluated and was shown to contain periodic acid-Schiff (PAS) positivity and ACTH immunoreactivity in a pattern consistent with a sparsely granulated corticotrope adenoma (AIP mutation and a variant of unknown significance was identified (p.R16H). The patient had no family history of pituitary disease. The seminoma and the pituitary adenoma tissue did not show any loss of the normal AIP allele  and ACTH levels (135 ng/l). Immunohistochemistry showed PAS positivity and ACTH immunoreactivity in a pattern consistent with a sparsely granulated corticotrope adenoma. Genetic testing indicated the MRI after the transcranial operation revealed a suprasellar tumor remnant and fractionated conventional radiotherapy was initiated 3 months postoperatively.A synacthen test was performed 2 months after the conventional radiotherapy and revealed secondary adrenocortical failure. The patient developed permanent panhypopituitarism and is currently treated with hydrocortisone, levothyroxine, testosterone, GH, and desmopressin.This case of CD is educational in several respects. The presenting symptom was headache, which led to an MRI revealing a pituitary tumor. It is important to recognize the spectrum of pituitary ACTH excess associated with corticotrope adenomas. The majority of patients with CD are diagnosed on the basis of classic symptoms and signs of hypercortisolism; the traditional challenge in the context of CD is rather the opposite than in our case: a patient presenting with persuasive symptoms and signs but without a distinct pituitary tumor. Most ACTH-producing pituitary adenomas are microadenomas (<10 mm in diameter); these small tumors are usually composed of basophilic, densely granulated corticotropes. In contrast, a less frequent scenario is that of a macroadenoma associated with only subtle features of glucocorticoid excess; these ‘whispering’ adenomas, distinct from the truly silent corticotrope adenomas, are almost always composed of chromophobic cells with faint immunoreactivity for ACTH that can be easily missed Our patient is unusual in that his pituitary adenoma caused central diabetes insipidus, which is usually attributed to other mass lesions involving the pituitary stalk or occurs following pituitary surgery.The initial endocrine evaluation included an elevated serum IGF1 measurement and a subsequent GH profile following an oral glucose load demonstrated a slightly elevated nadir GH level indicative of acromegaly despite the absence of classical symptoms and signs. Only few cases of pituitary tumors secreting both GH and ACTH are reported; in our case, the tumor tissue submitted for pathology examination was completely negative for Pit1 and GH and had no features of a somatotrope adenoma. We therefore find it unlikely that the tumor co-secreted GH, and it should be noted that elevated serum IGF1 levels have previously been reported in Cushing's syndrome (CS) Given the fact that the patient had an ACTH-producing adenoma and only a small proportion of the tumor was removed, it was also unexpected that hypocortisolemia developed after the first operation. The rapid resolution (4 months) of hypercortisolemia following the conventional radiation therapy targeting the suprasellar tumor remnant, and development of secondary adrenocortical failure are also unusual, as the effects of conventional radiation therapy on hypersecretion and tumor shrinkage usually set in after several years. We suggest that an infarction developed in the pituitary tumor remnant, which, to our knowledge, has been reported in only one previous case – also a patient with CD – following radiation therapy AIP gene c.47G>A, p.R16H. AIP mutations are associated with familial pituitary adenomas and more than 200 cases have been described displaying over 70 different mutations AIP mutations are predominantly GH-producing macroadenomas. Thus far, ten cases of CD have been reported AIP variant reported in our case is considered of unclear pathological significance. It has been identified in 0.3% of European Americans and 0.08% of African Americans in the general population. An in vitro functional study based on binding to one of the partners of AIP phosphodiesterase subtype 4A5 did not demonstrate a significant functional change attributable to this gene variant. Nevertheless, this gene variant has been reported in four additional patients with CD  has been linked to CD Finally, our case was diagnosed with a variant in the  with CD . In accoWritten informed consent was obtained from the patient.AIP mutation. S L Asa was responsible for pituitary tumor histology, the histological perspective of the case rapport. M Korbonits was responsible for the genetic perspective of the case rapport. J O L Jørgensen was in charge of the patient treatment and the CD perspective of case rapport.P T Dinesen and J Dal were involved in writing the case report and data collection. P Gabrovska, K Stals, and J Denes performed genetic examination of the pituitary adenoma tissue. M Gaustadnes and C H Gravholt performed genetic testing for