The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in β-catenin activity along the axis of the nephron tubule. By modifying β-catenin activity, we force cells within nephrons to differentiate according to the imposed β-catenin activity level, thereby causing spatial shifts in nephron segments. The β-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises β-catenin activity and promotes segment identities associated with low β-catenin activity. β-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating β-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning.DOI:http://dx.doi.org/10.7554/eLife.04000.001 The main function of the kidney is to filter blood to remove waste and regulate the amount of water and salt in the body. Structures in the kidney—called nephrons—do much of this work and blood is filtered in a part of each nephron called the glomerulus. The substances filtered out of the blood move into a series of ‘tubules’, another part of the nephrons, from where water and soluble substances are reabsorbed or excreted as the body requires. If the nephrons do not work correctly, it can lead to a wide range of health problems—from abnormal water and salt loss to dangerously high blood pressure.For organs and tissues to develop in an embryo, signalling pathways help cells to communicate with each other. These pathways control what type of cells the embryonic cells become and also help neighbouring cells work together to form specialised structures with particular functions. Much is unknown about how the nephron develops, including how its different structures coordinate their development with each other so that they form in the right position in the nephron.A protein called beta-catenin was already known to play an important role in the signalling pathways that trigger the earliest stages of nephron formation. Lindström et al. further investigated how this protein helps the nephron to develop by using a wide range of techniques, including growing genetically altered mouse kidneys in culture and capturing images of the developing nephrons with time-lapse microscopy. The combined results reveal that the levels of beta-catenin activity coordinate the development of the different structures in the nephron. The beta-catenin protein is not equally active in all parts of the nephron; instead, it forms a gradient of different activity levels. The highest levels of beta-catenin activity occur in the tubules at the furthest end of the developing nephron; this activity gradually decreases along the length of the nephron, and the glomerulus itself lacks beta-catenin activity altogether. Experimentally manipulating the levels of beta-catenin at different points along the nephron caused those cells to take on the wrong identity, causing parts of the nephron to form in the wrong place.Lindström et al. were also able to establish that the signalling pathway controlled by beta-catenin activity interacts with three other well-known signalling pathways as part of a network that controls nephron development. More research is required to find out which signal activates beta-catenin in the first place and from where in the kidney this signal comes. It also remains to be discovered how a particular cell in the tubule interprets the exact activities of the different signals to give the cell its specific identity for that place in the nephron. A better understanding of these sorts of processes will eventually help build new kidneys for people with kidney failure.DOI:http://dx.doi.org/10.7554/eLife.04000.002 All adult vertebrates depend on renal nephrons accurately performing a diverse set of roles that protect and maintain blood homeostasis. The diversity of roles that the nephron performs is reflected in the range of symptoms of abnormal nephron function and the consequent diseases range from, for example, Bartter syndrome , acidosis, and rickets to essential hypertension . In spit+ mesenchymal nephron progenitor-cells    in the non (MET) , and duron (MET) ; a Wt1+ segments . These ssegments . How thesegments , but the pathway . It remalineages . Post-MElineages . Wnt7b alineages . Notch slineages , 2007. Nlomeruli . Howeverlomeruli . N1ICD e and MET . WhetherUsing in vivo and ex vivo techniques we demonstrate that a gradient of β-catenin activity, along the proximal–distal nephron axis, controls the differentiation of segment-specific cell fates. We further investigate how β-catenin activity is prevented in the proximal and medial segments and show that BMP/PTEN/PI3K signalling in the medial nephron actively promotes the medial segment identity whilst blocking β-catenin activity. In addition, we show that modulating β-catenin or PI3K activity partially rescues the nephron segment defect phenotypes associated with the loss of Notch function. Our findings provide a model where multiple signalling pathways are integrated to control nephron segment-identity specification.TCF/Lef::H2B-GFP; gudmap.org) for S-shaped body nephrons. Confocal z-projections  suggestive of a single source and first-order decay of the activating signal  (Lef1 and Ccnd1) were also expressed along the gradient  . Target gradient . The disTCF/Lef::H2B-GFP and qRT-PCR analyses and confirmed that the inhibitors acted as expected , but not upstream (IWR1), CHIR-induced ectopic nephron formation was inhibited  and Tankyrase (IWR1) to positively and negatively modify β-catenin signalling ex vivo in whole organ cultures. We extensively characterised these small molecule inhibitors to ensure that they had their expected effect on the β-catenin signalling pathway; these data can be found in expected . Second,expected , 2012, wexpected . Increasexpected . Third, 2-CreGFP  and a Rose model  , and Wt1+/GFP reporter kidneys  compared to controls or kidneys with decreased β-catenin activity . The medial segment showed no significant change in length . These data confirm that both the distal and proximal nephron segments respond to increased β-catenin signalling according to our hypothesis, but the medial remained unchanged in size. Decreasing β-catenin signalling, on the other hand, resulted in much elongated nephrons (−14), as we had previously noticed   did not increase in response to CHIR as distal genes did. At the highest β-catenin activity levels used [6 µM], nephron formation was reduced   thus mir reduced  and the  reduced . To test reduced , 1955, a reduced .10.7554/Apc and Ctnnb1 (the gene encoding β-catenin) mutants that only result in mild increases in β-catenin activity. The Ctnnb1E654 model (Ctnnb1) leading to an increased propensity to signal, whereas the Apc1572T and Apc1638N models  kidneys (data not shown). Kidneys from Apc+/1572TCtnnb1+/+ and Apc+/1572Ctnnb1+/E654 embryos had normal nephrons , 10.6 µm/hr (PI3K), and 8.4 µm/hr (control) (data not shown).+ cells, strongly reduced the Jag1+ cells, and formed nephrons made of distal regions .Given the observed increase of the β-catenin reporter in response to blocking BMPR, by inhibiting BMPR and simultaneously activating β-catenin with CHIR, this should synergistically expand β-catenin signalling in the medial nephron segment where BMP activity was detected. The ectopic and tree-bound nephrons that formed in these dual-inhibitor conditions displayed very high β-catenin activity throughout the nephron structures  leads to reduced β-catenin signalling, whereas simulated decreased BMP signalling (via inhibition of BMPR) results in the opposite effect.Dll1 and Jag1. Furthermore, we showed activity of the BMP pathway in the segment affected when Notch is absent. Because we have shown that decreased β-catenin activity enhances the formation of the glomerular segment and PI3K inhibition promotes the medial segment, we tested if inhibition of β-catenin or PI3K could rescue these respective identities when Notch activity is lost. We first inhibited Notch using the γ-secretase DAPT as previously published (Lotus tetragonolobus lectin (LTL), Notch2−/− animals  . Together, these data show that proximal and medial nephron cells require Notch signalling and simultaneously need β-catenin and PI3K signalling to be kept at low levels, respectively.To date, the only other major pathway known to control nephron patterning is the Notch-signalling pathway . Notch-sublished . This re animals . Removal animals . Co-inhi animals . The Wt1me-lapse  and antime-lapse  confirmel, Hey1) . Co-inhil, Hey1) . Wt1+ ce segment . We asseroughout . Co-inhiA major outstanding question in kidney development has been to understand how the nephrons are patterned. It has been shown that Wnt via canonical β-catenin signalling induces nephrons to form and through non-canonical pathways regulate the nephron mesenchymal-to-epithelial transition as well as subsequent tubule elongation . Our datOur data combined suggest a model where a gradient of β-catenin activity specifies positional identities along the nephron axis. Additional signal transduction pathways modulate local β-catenin activity levels. Its action has a differential effect on nephron segments by controlling differentiation, maturation, and their size. While cell proliferation and apoptosis are likely to be essential in other aspects of nephrogenesis, our data exclude a direct role in the initial patterning mechanism. With the current data, it is difficult to identify key downstream targets and that way describe the next level of mechanism of this process, as many targets are also widely used as segmentation markers.TCF/Lef::H2B-GFP activity reporter showed a clearly recognisable visual gradient which we confirmed by quantifying the reporter signal along the nephron axis. Reporter activity is high at the distal end of the nephron, where it connects to the ureteric bud, and gradually reduces in the proximal direction, with the developing glomerulus at the extreme proximal end being devoid of β-catenin activity , which is normally characterized by low or absent β-catenin activity, does not form when β-catenin activity is increased, whereas it forms quicker when the activity of the pathway is decreased (+ and Jag1+), cells also respond to the level of β-catenin activity that is forced on them  compared to controls and when β-catenin signalling was increased  it is that drives the gradient or how β-catenin activity is antagonised in the proximal cells. For the Wnts, none of the published elongate . As thise defect . Wnt9b ie defect . AlthougLgr5 as a potential modifier of Wnt signalling establishing the β-catenin gradient. The nephrons forming in Lgr5+/EGFP-IRES-CreERT2 homozygotes were morphologically indistinguishable to heterozygotes or control animals ; Columbia University, New York (USA); Maine Medical Center, Maine (USA); the Beatson Institute, Glasgow (UK); and the Erasmus Medical Centre, Rotterdam (The Netherlands). All animal experiments and animal use were carried out according to regulations specified by the Home Office (MRC/UoE), approved by the Animal Ethics Committee and carried out in accordance with Dutch and international legislation (Erasmus) and conducted under PHS guidelines and approved by the relevant Institutional Animal Care and Use Committees (Columbia/Maine). All animal experiments were performed under Project Licenses 60/3788 and 60/4473.TCF/Lef:H2B-EGFP (Tg(TCF/Lef1-HIST1H2BB/EGFP)61Hadj) (Wt1+/GFP (Wt1tm1Nhsn) (Pax8+/Cre (Pax8tm1(cre)Mbu) mice (Rosa26eYFP/eYFP (Gt(ROSA)26Sortm1(EYFP)Cos) animals (Six2+/GCiP (tdRFP (Gt(ROSA)26Sortm1Hjf) (Ctnnb1E654 (Ctnnb1tm1.2Wvv) (Apc+/1572T (Apctm2Rfo) (Apc+/1638N (Apctm1Rak) (Lgr5+/EGFP-IRES-CreERT2 (B6.129P2-Lgr5tm1(cre/ERT2)Cle/J) . Embryos were generated through timed mating, with noon of the day a vaginal plug was found considered E0.5. )61Hadj)  were crotm1Nhsn)  mice werbu) mice  were cro animals . Six2+/Gx2+/GCiP  mice werrtm1Hjf) . Ctnnb1Em1.2Wvv) , Apc+/15ctm2Rfo) , and Apcctm1Rak)  were int2)Cle/J)  were crod before .2 on 0.4 µm PET Transwell membranes . The culture medium consisted of MEM , 10% FCS, and 1% Pen/Strep. Isolated mesenchyme was induced with spinal cord as previously described , ICG001 , IWR1 (TOCRIS), salinomycin (SIGMA), BIO , Ly294002 (TOCRIS), LDN-193189 , methotrexate (SIGMA), were used as specified in the text.Kidneys were dissected from E12.5 embryos and cultured at 37°C with 5% COescribed . Media wanti-jagged1 , anti-E-cadherin , anti-wt1 , anti-LEF1 , anti-Pax8 , anti-Pax2 , anti-phospho-β-cat  , anti-phospho-β-cat (S45) , pan β-cat (SIGMA), anti-dephospho-β-cat , anti-podocalyxin, anti-laminin (SIGMA), LTL , anti-ZO1 , anti-pSMAD , anti-pAKT , 6-CF (SIGMA), PNA (Vector Labs), Annexin V , TUNEL , anti-phospho-β-cat (S552) .Fluorescent secondary antibodies were purchased from Invitrogen Molecular Probes: anti-mouse IgG 488, anti-rabbit IgG 594, anti-goat IgG 488, anti-goat IgG 594, anti-mouse IgG 647, anti-rabbit IgG 488, anti-goat IgG 350.Organ cultures were fixed in −20°C methanol or 4% PFA in 1× PBS. 4% PFA was used for all tissues with fluorescently labelled proteins. Fixed tissues were washed thoroughly in 1× PBS prior to addition of primary antibodies and incubation at 4°C O/N. Samples were again washed thoroughly in 1× PBS before incubation with secondary antibodies at 4°C O/N. Excess secondary antibodies were washed off thoroughly with 1× PBS before mounting in Vectashield (Vectorlabs).http://fiji.sc/), Volocity , and ImageJ (http://rsb.info.nih.gov/ij/) were used for image analysis and presentation. 3D videos were made in Fiji. Time-lapse videos were presented using ImageJ, Fiji, and IP-Lab. Brightness and contrast levels were adjusted in Adobe Photoshop CS5 and IP-Lab after quantification and for presentational purposes only.Live imaging microscopy was performed on a Nikon TiE (Perfect Focus System) with NIS-Elements 4.0. Imaging was performed using a 4× objective and a CoolSnap HQ2 CCD camera . Confocal microscopy was carried out on a Nikon A1R and an N-STORM/A1 Confocal and 10×–60× objectives were used and image stitching was carried out at 10× to capture whole kidneys. Nikon NIS-Elements 4.0, Fiji  was calculated and combined for all the nephrons analysed. Two-tailed Student's t tests were used to compare the medial with distal and medial with proximal segment.Kidneys were stained for phospho-β-cat (S33/S37/T41), Jag1, and Cdh1. Scans were made every 1 µm with a 63× objective through whole nephrons with an N-STORM/A1 confocal microscope. 3D volume reconstructions were generated in IMARIS. Very bright pixels from background noise were made into a volume and masked onto the original channel and removed. Volume measurements were made in 75 × 75 × 10 µm cubes. Three cubes were fitted per nephron segment, thus nine measurements were made per nephron. The Image intensity sum was calculated and the average found per segment and across five different nephrons. Two-tailed Student's Whole E12.5 kidneys were cultured in control, LDN-193189, or Ly294002 containing media. Time-lapse imaging was performed to capture GFP and brightfield. The GFP signal was captured far below saturation levels (saturated pixels in figures and videos are present due to brightness and contrast changes performed for publication purposes) as a 16-bit image every 20 min. To measure the GFP signal intensity, individual nephrons were selected from their point of formation and monitored for 22 hr. The point of formation was determined based on the brightfield view and the GFP signal, which was detectable at low levels in the pre-epithelial cells. The earliest signs of nephron formation were when cells aggregated prior to epithelialisation (pretubular aggregates). That was chosen as ‘time 0’. The 22 hr time-frame was chosen as this was the time taken for nephrons to develop to an S-shaped body stage in controls. To measure the intensity of nuclei along the nephron distal-to-proximal axis a 5-µm wide line was drawn down the length of the axis. The mean value across the line was measured along the whole length of the nephron. This gave readouts of the intensities of nuclei along the nephron and also provided the nephron lengths. Several nuclei were thus picked up within each nephron segment. The nephron axis was visible in the GFP and the brightfield channels and both were used to accurately reconstruct the nephron axis. The intensities were plotted against the axis length in 8-bit values vs µm position. Several nephrons from separate kidneys were measured in this fashion for each condition: 11 control nephrons, 12 LDN-193189 nephrons, 11 Ly294002 nephrons. The nephron lengths were used to calculate the changes in nephron lengths between each time-point and over the total 22 hr time-period. This allowed us to calculate the rate of nephron growth. To measure the ‘total intensity’ at the 22 hr time-point, the intensity of all pixels within the whole nephron at that time-point was measured. For this, 17 control nephrons, 18 LDN-193189 nephrons, and 18 Ly294002 nephrons were measured. The pixel values were plotted against their percentage frequency to obtain curves displaying the frequency of GFP signal intensities.Kidneys were treated with a range of CHIR concentrations  and immunostained for Lef1, Pax2, Pax8, Chd1. Using Fiji the mean secondary antibody signal-intensity was measured within 50-µm diameter circular sections in all ectopic nephrons using two kidneys per treatment. All comparisons between treatments were made using samples that had been simultaneously stained and imaged.H2B-GFP fluorescence was measured in all ureteric bud tips of four kidneys per treatment. Images for measurements were captured using the same settings throughout. Statistical analysis was performed using single factor ANOVAs (α 0.05) for single time-points .t tests of each group compared to controls. 5 Wt1+/GFP (Wt1tm1Nhsn) kidneys were tested per group.Kidneys were cultured and imaged as described above. Kidneys were scored for the development of crescent-shaped glomeruli and the time was recorded. Statistical analysis of timing of crescent-shaped glomerular formation was carried out using two-tailed Student's t tests were used to analyse statistical significance compared to controls.Confocal scans were carried out on kidneys cultured in specified conditions. Wt1, Jag1, Cdh1 immunofluorescent stains were used to measure the size of each nephron domain. Fiji software was used to measure the length of each domain. Where overlap of markers was seen, the mid-point of overlaps was taken as end of each domain. The mean domain-size was calculated and separate two-tailed Student's TCF/Lef:H2B-EGFP kidneys cultured in the specified conditions. Kidneys were stained for H3(S10P), Cdh1, and Hoechst. Positive nuclei were counted within nephrons. 21–22 nephrons were counted per condition.Confocal scans were performed on http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000); primer and probe details can be found in http://www.chibi.ubc.ca/matrix2png/bin/matrix2png.cgi).For TaqMan experiments, samples were placed in RNALater (Ambion). A minimum of three kidneys were used per replicate of each condition and all experiments were performed in triplicates. RNA was isolated from cultured kidneys using RNAeasy micro kits . cDNA was made using SuperScript II  and random primers . Assays were done with the Universal Probe Library (Roche) and were designed using the Universal Probe Library Assay Design Center  beta 1 (β-catenin); PTEN, phosphatase and tensin homolog; PI3K, phosphatidylinositide 3-kinase; AKT, thymoma viral proto-oncogene 1; BMP, bone morphogenetic protein; Apc, adenomatosis polyposis coli; Lgr5, leucine rich repeat containing G protein coupled receptor 5; Lhx1, LIM homeobox protein 1; Fgf8, fibroblast growth factor 8; Pax8, paired box gene 8; UB, ureteric bud; Pod., podocyte; Pt., parietal epithelium. 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 , a Reviewing editor, and 4 reviewers.Thank you for sending your work entitled “Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron” for consideration at The Reviewing editor and the 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.eLife, there are a few issues raised by the reviewers that merit your attention prior to further consideration.All four reviewers found your work to be comprehensive in scope and for the most part found the data to justify the conclusions drawn, and the manuscript well-written. In fact, one of the strengths cited by multiple reviewers was the comprehensiveness of the study relative to other existing work on the signaling pathways controlling kidney morphogenesis. That said, although your work is in principle suitable for publication in Below, I delineate the issues raised that if satisfactorily addressed would allow us to move forward. In the reviewers' view, these should not be insurmountable hurdles for you, and we'd expect that you should be able to address the issues expeditiously:Experimental issues:Nephron patterning is assessed using several parameters: maturation, segment length, protein expression and gene expression. The results of these distinct assessments are not explicitly compared and integrated into one conclusion.A changing set of segment specific markers is used for nephron segment identification e.g. .Although some of the pathway modulations are performed intermittently . Reviewers 1-3 provided some specific recommendations for recrafting the text to more coherently flow for the reader.Please pay attention to reviewer 3's comment that more is known regarding nephron development than the authors sometimes claim, as this is important to correct. Among them, the example cited is a good one, point out your claim that “The nephron β-catenin activity gradient is to our knowledge the first instance of a β-catenin activity gradient being described in any system”, when in fact this has been shown for multiple systems, including the hair follicle, hematopoietic and intestinal stem cell lineages.The review comments below should put this summary into context:Reviewer #1:This is a very interesting manuscript that defines an important role for canonical WNT signaling in the patterning of the nephron. Some evidence is provided suggesting that there is normally a WNT gradient going from distal (strong) to proximal (weak). Increasing WNT, chemically or genetically, results in increased distal and decreased proximal regions of the nephron. Decreasing WNT gives more complicated results, although it is argued that there is a more rapid maturation of the proximal nephron.The work is quite thorough in many respects, for example in using a multitude of chemical modifiers of WNT signaling, as well as genetic approaches. The characterization of resulting phenotypes is also generally excellent, with many outstanding images provided and a reasonable set of markers employed.There are some significant unanswered questions that are raised by the results and the proposed model. As mentioned in the paper, which WNT is responsible for the proposed gradient? And why don't any Wnt knockouts show the predicted nephron patterning defect? Further, why does knockdown of WNT in the proximal region of the nephron, where WNT is already proposed to be extremely low, partially rescue the Notch mutant loss of proximal structures?Despite these questions there is a wealth of interesting data presented. There is a real lack of understanding of the forces that pattern the nephron and this paper begins to fill that important gap.Reviewer #2:The authors have investigated the role of canonical Wnt signaling in nephron patterning during murine renal development in ex vivo culture experiments. In addition, the effect of BMP and PTEN/PI3K/AKT signaling on nephron patterning as well as the interaction between these pathways was investigated.Extensive intervention studies have been undertaken with both pharmacological compounds as well as mutant mouse models, and the effect on patterning is assessed morphologically, immunohistochemically and by gene expression analysis. The results provide relevant and new insight into mechanisms involved in nephron patterning.However, I do have serious concerns regarding the way the data is presented and some concerns about the coherence of the experiments.Major comments:The number and organization of the figures and movies interferes with the readability of the manuscript. Reducing the number of movies, figures and panels will enhance the legibility of the manuscript.The presentation of the data and conclusion should be more structured:Conclusions are presented inconsistently in the manuscript body, the figure legends  and the Paragraphs in the Results section sometimes end with a conclusion and sometimes with just research results. The manuscript contains redundant information and conclusions: e.g.: “Since proliferation was not affected by either increasing or decreasing β-catenin signaling” and “we found that proliferation along the nephron axis does not change in response to stimulating or inhibiting β-catenin”, both in the Discussion, and “phospho- and dephospho-specific antibodies (indicative of protein targeted for break-down and active 400 β-catenin respectively)” and “antibodies for β-catenin that was already tagged for degradation”, in the Discussion and in the Results respectively. Improving the cohesion (in the presentation) of the different experiments would strengthen the manuscript.Nephron patterning is assessed using several parameters: maturation, segment length, protein expression and gene expression. The results of these distinct assessments are not explicitly compared and integrated into one conclusion.A changing set of segment specific markers is used for nephron segment identification e.g. .Although some of the pathway modulations are performed intermittently : “more efficient formation of the proximal domain”, “a strong positive effect on the development of the medial segment”, “increased rate of maturation”, “change in cell identity”.In the Discussion section, the sentence “Exploring the roles… of future studies”: In the Results, the authors mention that Lgr-5 is a β-catenin target, however Lgr5 is also a Wnt receptor component that can enhance canonical Wnt signaling . The LgrThe authors state that β-catenin staining in Reviewer #3:In this manuscript, the authors investigate pattern formation of the nephron. Although nephron development is a relatively neglected area of research, more is known regarding its development than the authors sometimes claim. Nonetheless, this manuscript provides a far more detailed and integrated view of nephron pattern formation than any other study to date. First, the authors conduct a detailed series of experiments showing that a beta-catenin activity gradient determines positional identity in a morphogenetic manner. This is shown primarily by using pharmacological inhibitors/activators of Wnt signaling along with a reporter of beta-catenin activity. The authors do a nice job verifying the specificity and fidelity of their system. Importantly, this pharmacological study is further supported genetically using various hypomorphic alleles of beta-catenin. The authors then show that, similar to the intestine, nephron patterning is further regulated through a BMP/PI3K network, which acts in modulating the more central-acting beta-catenin activity. Given the structural and functional similarities between the nephron and intestine, this is an attractive model. The authors also attempt to link their data with Notch signaling, which is also known to regulate nephron patterning. This section is somewhat confusing and is not well-integrated with the first two major findings. Overall, the authors have provided a more comprehensive study of nephron patterning than any previous study, and their study sheds some light on its subject that could not be gleaned from the current literature. However, some questions/concerns should be addressed prior to publication.Minor comments:The results showing no changes in proliferation or apoptosis should be better explained and integrated into the manuscript. Do the authors suggest this implies that differentiation is mainly responsible for the patterning effects? Actually, changes in proliferation and apoptosis are well-known mechanisms for regulating morphogenesis . At any rate, the authors should better clarify the purpose of these experiments and what they conclude from the results.The finding that the PI3K pathway is regulating beta-catenin is interesting. These pathways are known to interact, especially in the intestinal and hematopoietic systems, where Akt can activate beta-catenin by phosphorylation at Ser552. It would be interesting to see whether and where pS552-bcat positive cells were present during nephron patterning. Such data could further support the author's model.The authors sometimes discount previous findings. For instance, they claim that “The nephron β-catenin activity gradient is to our knowledge the first instance of a β-catenin activity gradient being described in any system”. This is surprising since such gradients have been described in multiple systems, including mammals.The authors should consider either better integrating and explaining the Notch data or removing this section from the manuscript. It would be appropriate to discuss what's known about Notch and how it might relate to their new findings in the Discussion section; however, the current presentation tends to leave the reader more confused than enlightened. Experimental issues:Nephron patterning is assessed using several parameters: maturation, segment length, protein expression and gene expression. The results of these distinct assessments are not explicitly compared and integrated into one conclusion.We have added a combining/concluding paragraph to the beginning of the Discussion. We have limited this to what are in our opinion the clearly proven facts. Other parts we consider more speculative are discussed elsewhere in the Discussion.A changing set of segment specific markers is used for nephron segment identification (e.g.).In the original manuscript we chose markers that best illustrated the phenotype described in the specific part of the manuscript. In particular, in cases where markers can be used in different ways, for instance genes that are known to be downstream targets of the pathways, we study as well as markers for spatial segments of the nephron, we thought this would help to illustrate our points. However, we appreciate that this has led to confusion for the reader. We now provide a consistent set of markers in the different experiments.In the original manuscript, we presented these data as overlapping line diagrams in different colours. However, the expression patterns of the additional genes we now show made these very confusing figures, even if different colours were combined with different line styles. Eventually we decided to present these data in the revised manuscripts in the form of heat maps. Although these have the disadvantage of that we cannot include statistical information in the form of error bars, this presentation is the clearest in showing the biological message coming from the experiments.Although some of the pathway modulations are performed intermittently , other pathway modulations are performed only continuously (e.g. BMP).We now provide the reversal experiments for the important inhibitors we use in the manuscript. In all but one of the inhibitors (the exception being IWR1) this resulted in at least partial reversal of the phenotype. Please note that a failure to reverse the phenotype does not necessarily imply the experiment is flawed. For instance, in some case inhibitors can bind irreversibly to their target making phenotypic rescue impossible. In other instances, the change that is made might be of such a nature that the patterning would simply not be able to reverse. IWR1, for example, causes the nephrons to mature quicker. Once segments have fully matured, they might no longer have the capacity to generate those segments that didn’t form due to the inhibitor’s effect. Therefore, withdrawal of the inhibitor would simply not allow for a reversal of the effects.Reviewer 2 points out that β-catenin staining inand Figure 1–figure supplement 2B, D only shows membrane bound β-catenin and not the nuclear β-catenin necessary for the transcription of Wnt target genes. In addition, the presence of phosphorylated β-catenin in the developing nephron in Figure F appears to be limited to one cell, which is insufficient to support the claim of a gradient of β-catenin phosphorylation. In addition, the supposed gradient of phosphorylated B catenin along the tubule is insufficiently demonstrated by Figure 1–figure supplement 2B. These figures do not sufficiently explain the β-catenin activity gradient.We appreciate the original data using the phospho- and dephospho-specific antibodies on its own was insufficient to prove the existence of a β-catenin activity gradient, and for this reason we interpreted it in the context of all other data (reporter and pathway modulation). Unfortunately, these antibodies are very difficult to use in the whole mount kidney organ culture system (we are not aware of other publications that show their use in this) and optimization options are limited. We did succeed in improving the data with the Ser33/37/Thr41 phospho-specific antibody and these data are now provided. To further support the activity gradient this signal is now quantified in different parts of the nephron which confirms the different signals in different parts of the nephron.We did not succeed in further improving the pSer45 and dephospho-specific data. As we do not want to present data that has, for technical reasons, limited relevance these have now been removed from the manuscript to reduce the complexity of the presented data. We stress the pSer33/37/Thr41 data should still be considered in the context of the complete manuscript, not as stand-alone data. This is now more clearly discussed in the Discussion section.Ctnnb1ex 3 mutation throughout the intestine, the biological effect as well as the intracellular localization of the mutant β-catenin varies in different parts of the gut (PMID 22287596). This is now also discussed in the manuscript. Therefore, although we agree this is an important observation, in our opinion it does not necessarily disprove the existence of the gradient.As for the lack of nuclear β-catenin, although we cannot exclude technical complications with the whole organ staining, the model that active signalling automatically leads to and requires nuclear localization is likely an oversimplification. For instance, in colorectal cancer  it has been shown that within the tumour there is a dynamic change in localization in different parts of the same tumour although all cells will carry the same cancer-inducing mutation (PMID 17306971). Likewise, in mice expressing the same activating Reviewer 1 points out an issue with the reliability of WNT/beta catenin reporters in detecting WNT signaling and that they should not be considered definitive .Axin2-lacZ knock-in line (Axin2 being an endogenous β-catenin target). Our preference for the mini-construct is based on the following: i) the relevance of endogenous target genes can differ in a cell type-dependent manner. For instance, c-Myc is a key β-catenin target in colorectal cancer (PMID 17377531) but Ccnd1 is not (PMID 15946945). If endogenous transcriptional responses are not uniform, reporters based on them will not be reliable in their responses; ii) endogenous reporters will only be fully reliable if β-catenin is the only pathway that controls its expression. This is unlikely to be the case. A reporter based on an endogenous β-catenin target gene can in that case truthfully report increased activity of the pathway, but reduction is not necessarily detected as other pathways could maintain its expression. In this respect, the simplicity of mini constructs as we used is a clear advantage, and we show that IWR1 treatment results in the expected decrease in signal intensity; iii) the use of lacZ in the Axin2 model gives an additional layer of arbitrary experimental conditions. Staining time can be chosen at will and the lacZ signal rapidly reaches a maximum. For subtle differences, as we show here, a fluorescent reporter and quantification methods as we employ them here is far superior.We fully agree with this reviewer that activity reporters need to be used with common sense. Whereas we have chosen to use an artificial mini-construct reporter, other labs prefer to use the We do emphasize that none of the experiments on the gradient should be seen in isolation (see the previous point), and this is now discussed in more detail.Moreover, the images don't completely agree with the quantifications shown.The detectors of the imaging systems, and therefore quantification of the signal, have a far better dynamic range than computer screens and printers. RGB images have a value range 0-255, with 255 being a fully saturated pixel. In the example of, for instance, the 39x difference between the proximal and distal end of the nephron (in the Results section), this would mean that an almost-but-just-not saturated pixel at the high end  would give a pixel with value 6.5 at the low end. On every printer and all but the best calibrated screens this would simply be black. For this reason we have chosen to quantify as much of our data as possible, it is the only way to see the nuances in the actual data. Visual recognition is due to the technical limitations of screens and paper simply not good enough. The fact that many structures are in close proximity of the ureteric bud (with very high reporter activity) makes it even more difficult to present proper imaging data that shows all the nuances that exist in the data. However, because this was an issue raised by several reviewers we chose to include additional data to more clearly display the different intensities. On this point, multiple reviewers have raised questions as to how a single WNT source might generate such a gradient and whether a gradient has been definitively shown.The details of the mode of Wnt secretion and spreading are hotly debated in the literature. We can only speculate that it is unlikely the  Wnt molecule would be traveling on the outside of the nephron, as this would imply movement through the basement membrane in the responding cell. This would indeed also be complicated, as implicated by the reviewers, that the convoluted 3D structure of the nephron would be difficult to imagine in a gradient, especially in the context of the growing organ. Intraluminal transport however would, by definition, follow the shape of the nephron irrespective of the shape and organ as a whole. It would allow the Wnt signal to be taken up by receiving cell via the apical side (i.e. no basement membrane), and would fit with the observation that cilia are present on that side of the cell. We could have included this speculation in the Discussion, but as at the moment our manuscript is written from the β-catenin angle and not the Wnt angle we have not yet done so. The second issue on the gradient is discussed above.A final issue relating to this point is what (where) is the source of the Wnt?Wnt7b and Wnt9b in the patterning of the nephron as we describe, and indicate that extensive additional analyses beyond the scope of this work are required to prove or disprove this. We also like to stress that because of this we refer consistently to a β-catenin activity gradient, not a Wnt gradient. We even mention that at present we cannot exclude the possibility this is a hypothetical Wnt-independent β-catenin function (though we think this is highly unlikely).We have extended the Discussion with some speculation of a potential role of Continuing along this line of ambiguity is the issue of why does both decrease and increase of WNT give increased maturation rate? . And why is the CTRL showing no crescent-shaped glomeruli until 3500 minutes ?We think there is a misunderstanding here. Only the decreased β-catenin signalling results in increased maturation rate . We agreSeveral of your reviewers also felt that your section on the link to Notch signalling is somewhat confusing and is not well-integrated with the first two major findings. This section needs bolstering with additional data to make a more compelling link.We thank the reviewers for this criticism. We have added data on the β-catenin activity reporter under Notch/β-catenin and Notch/PI3K modulated conditions. These data have made the link between Notch and the other pathways considerably stronger.Reviewer 3 points out that it would be straightforward to relate to other systems your finding that the PI3K pathway regulates beta-catenin. One way to explore these parallels is to evaluate whether Akt activates beta-catenin by phosphorylation at Ser552 in the nephron.We have added this experiment but our current data cannot support the reviewers’ hypothesis; we find staining for this antibody in the medial and distal segment  but thisWriting/Text issues:The number and organization of the figures, movies and supplementary figures interferes with the readability of the manuscript. Reducing the number of main and supplementary movies, figures and panels will enhance the legibility of the manuscript.We have tried to find a balance between reducing the figures and maintaining essential data. For instance, given the wide scepticism of specificity of pharmacological inhibitors we decided to keep all control experiments for this in figure supplements. Unfortunately, where in a biochemical paper this might be combined in a single western blot experiment, in our organ culture system this requires a lot more figures. On the other hand, in the old manuscript we showed the whole kidneys in figure supplements that our close-up data in the main figures was taken from to confirm we showed representative parts of the kidneys. To reduce complexity the majority of fields showing whole kidneys have now been removed and we have left the close-ups of nephrons.The presentation of the data and conclusions drawn needs to be more structured, and some of the statements, e.g. those of Reviewer 2, need to be more explicitly stated upfront . Reviewers 1-3 provided some specific recommendations for recrafting the text to more coherently flow for the reader.We have extended this; more reviewer-specific comments are addressed below.Please pay attention to Reviewer 3's comment that more is known regarding nephron development than the authors sometimes claim, as this is important to correct. Among them, the example cited is a good one, point out your claim that “The nephron β-catenin activity gradient is to our knowledge the first instance of a β-catenin activity gradient being described in any system”, when in fact this has been shown for multiple systems, including the hair follicle, hematopoietic and intestinal stem cell lineages.We thank the editor and reviewers’ for this comment and we have now toned down such statements or deleted them altogether where appropriate as this is not central to our work described here.The review comments below should put this summary into context:Reviewer #1:This is a very interesting manuscript that defines an important role for canonical WNT signaling in the patterning of the nephron. Some evidence is provided suggesting that there is normally a WNT gradient going from distal (strong) to proximal (weak). Increasing WNT, chemically or genetically, results in increased distal and decreased proximal regions of the nephron. Decreasing WNT gives more complicated results, although it is argued that there is a more rapid maturation of the proximal nephron.The work is quite thorough in many respects, for example in using a multitude of chemical modifiers of WNT signaling, as well as genetic approaches. The characterization of resulting phenotypes is also generally excellent, with many outstanding images provided and a reasonable set of markers employed.We thank this reviewer for these kind and positive remarks.There are some significant unanswered questions that are raised by the results and the proposed model. As mentioned in the paper, which WNT is responsible for the proposed gradient? And why don't any Wnt knockouts show the predicted nephron patterning defect?We address this point in the response to the joint review.Further, why does knockdown of WNT in the proximal region of the nephron, where WNT is already proposed to be extremely low, partially rescue the Notch mutant loss of proximal structures?We hypothesize that under normal conditions proximal cells try to maintain a sufficiently low β-catenin activity level. By inhibiting this further using IWR1 we would simply be giving these cells a helping hand, and reaching the proximal fate becomes easier and is reached quicker. Our new Notch inhibition data shows more homogenous medium to high β-catenin activity levels throughout the nephron. By combining this with IWR1 or PI3K inhibition, which on their own decreases β-catenin activity throughout the nephron or in the medial segment , we woulReviewer #2:The authors have investigated the role of canonical Wnt signaling in nephron patterning during murine renal development in ex vivo culture experiments. In addition, the effect of BMP and PTEN/PI3K/AKT signaling on nephron patterning as well as the interaction between these pathways was investigated.Extensive intervention studies have been undertaken with both pharmacological compounds as well as mutant mouse models, and the effect on patterning is assessed morphologically, immunohistochemically and by gene expression analysis. The results provide relevant and new insight into mechanisms involved in nephron patterning.We thank the reviewer for these kind words.Major comments:The number and organization of the figures and movies interferes with the readability of the manuscript. Reducing the number of movies, figures and panels will enhance the legibility of the manuscript.As discussed above and described in more detail below, we have addressed this in the best possible way, at least in our opinion.The presentation of the data and conclusion should be more structured. Conclusions are presented inconsistently in the manuscript body, the figure legends  and the figure itself . Paragraphs in the Results section sometimes end with a conclusion and sometimes with just research results. The manuscript contains redundant information and conclusions: e.g.: “Since proliferation was not affected by either increasing or decreasing β-catenin signaling” and “we found that proliferation along the nephron axis does not change in response to stimulating or inhibiting β-catenin”, both in the Discussion, and “phospho- and dephospho-specific antibodies (indicative of protein targeted for break-down and active 400 β-catenin respectively)” and “antibodies for β-catenin that was already tagged for degradation”, in the Discussion and in the Results respectively. Improving the cohesion (in the presentation) of the different experiments would strengthen the manuscript.We have, with the help of the reviewers’ comments, hopefully addressed some of the inconsistencies within the manuscript. We believe that most of these came from not explaining clearly enough our conclusions. We have now added short conclusions to the different paragraph which should be helpful to the reader. Some of the examples that the reviewer considered being redundant information and conclusions have been modified, but others we chose to keep as they have been touched open by other reviewers as important.We have also taken into account all the reviewers’ and editor’s comments to improve the flow and cohesion of the manuscript.We have had a critical look at which figure panels are essential, and as discussed above, we have extended the range of markers to be consistent throughout the manuscript in parts where there was some inconsistent use.Nephron patterning is assessed using several parameters: maturation, segment length, protein expression and gene expression. The results of these distinct assessments are not explicitly compared and integrated into one conclusion.We have added a section to the start of the Discussion combining these different types of data in a single model.A changing set of segment specific markers is used for nephron segment identification (e.g.).Although some of the pathway modulations are performed intermittently , other pathway modulations are performed only continuously (e.g. BMP).As discussed above, we now use a consistent set of markers and we have included a more general interpretation of the data. We have also added the reversal experiments for the other pathways as discussed above.Vague remarks should be replaced by more clearly defined statements. Different terms are used interchangeably in the Discussion section, but remain devoid of proper definition. Please clarify how these terms relate to the parameters measured (see above): “more efficient formation of the proximal domain”, “a strong positive effect on the development of the medial segment”, “increased rate of maturation”, “change in cell identity”.gudmap.org) for S-Shaped body nephrons to refer to distal, medial, and proximal segments. This is also mentioned in the text.We now specifically state that we use the nomenclature used by the GenitorUrinary Development Molecular Anatomy Project  might therefore not be the result but the cause of the β-catenin gradient. The latter hypothesis is rejected by the experiments with Lgr-5 KO kidneys. This consideration should be mentioned more explicitly in the manuscript.This is a very helpful comment and it has been added to the manuscript.The authors state thatsuggest that β-catenin activity is induced by (factors secreted by (?)) a single source with a first order decay. This concept is difficult to visualize due to the spiral morphology of the immature nephron. Please clarify. As discussed in our response to reviewer 1, this is indeed something we cannot address within the scope of this manuscript. Moreover, tools for tracking Wnt molecules in the 3D context of a developing context would be as useful as they would be technically challenging (for multiple reasons). Transport within the lumen is indeed a possibility as we now discuss.β-catenin staining inand Figure 1–figure supplement 2B, D only shows membrane bound β-catenin and not the nuclear β-catenin necessary for the transcription of Wnt target genes. In addition, the presence of phosphorylated β-catenin in the developing nephron in Figure F appears to be limited to one cell, which is insufficient to support the claim of a gradient of β-catenin phosphorylation made in the Discussion section. In addition, the supposed gradient of phosphorylated β-catenin along the tubule is insufficiently demonstrated by Figure 1–figure supplement 2B. These figures do not sufficiently explain the β-catenin activity gradient.This is addressed in the response to the joint review.Reviewer #3:In this manuscript, the authors investigate pattern formation of the nephron. Although nephron development is a relatively neglected area of research, more is known regarding its development than the authors sometimes claim. Nonetheless, this manuscript provides a far more detailed and integrated view of nephron pattern formation than any other study to date. First, the authors conduct a detailed series of experiments showing that a beta-catenin activity gradient determines positional identity in a morphogenetic manner. This is shown primarily by using pharmacological inhibitors/activators of Wnt signaling along with a reporter of beta-catenin activity. The authors do a nice job verifying the specificity and fidelity of their system. Importantly, this pharmacological study is further supported genetically using various hypomorphic alleles of beta-catenin. The authors then show that, similar to the intestine, nephron patterning is further regulated through a BMP/PI3K network, which acts in modulating the more central-acting beta-catenin activity. Given the structural and functional similarities between the nephron and intestine, this is an attractive model. The authors also attempt to link their data with Notch signaling, which is also known to regulate nephron patterning. This section is somewhat confusing and is not well-integrated with the first two major findings. Overall, the authors have provided a more comprehensive study of nephron patterning than any previous study, and their study sheds some light on its subject that could not be gleaned from the current literature.We also thank this reviewer for the kind words.The results showing no changes in proliferation or apoptosis should be better explained and integrated into the manuscript. Do the authors suggest this implies that differentiation is mainly responsible for the patterning effects? Actually, changes in proliferation and apoptosis are well-known mechanisms for regulating morphogenesis . At any rate, the authors should better clarify the purpose of these experiments and what they conclude from the results.We now explain this in more detail in the Discussion.The finding that the PI3K pathway is regulating beta-catenin is interesting. These pathways are known to interact, especially in the intestinal and hematopoietic systems, where Akt can activate beta-catenin by phosphorylation at Ser552. It would be interesting to see whether and where pS552-bcat positive cells were present during nephron patterning. Such data could further support the author's model.As discussed above, this experiment has been added in the new manuscript and has indeed strengthened our model. We thank the reviewer for this suggestion.The authors sometimes discount previous findings. For instance, they claim that “The nephron β-catenin activity gradient is to our knowledge the first instance of a β-catenin activity gradient being described in any system”. This is surprising since such gradients have been described in multiple systems, including mammals.This is addressed in the response to the joint review.The authors should consider either better integrating and explaining the Notch data or removing this section from the manuscript. It would be appropriate to discuss what's known about Notch and how it might relate to their new findings in the Discussion section; however, the current presentation tends to leave the reader more confused than enlightened.As discussed in the response to the joint review, we have added data to strengthen the links between Notch, PI3K and β-catenin and this has greatly improved the robustness of our proposed model.We have also introduced new data showing 60x 1 µm scans through TCF/LEF nephrons treated with DAPT and in combination with Ly294002 and IWR1. These new data show how in DAPT treated nephrons the whole nephron is positive of GFP. It is not clear whether this is a cause or effect since DAPT treatment stops the proximal segments from forming. IWR1 treatment on top of DAPT treatment effectively reduces the signaling further and this might explain why some Wt1+ cells are now rescued. DAPT+ Ly29 does not clearly reduce GFP expression but it does rescue growth and expression of Jag1.


MRAP2. Our results suggest that MRAP2 is an important modulator of the energy homeostasis machinery that operates through the regulation of multiple GPCRs throughout the hypothalamus.Ghrelin is the only known circulating orexigenic hormone. It is primarily secreted by the stomach and acts at its receptor, the growth hormone secretagogue receptor 1a (GHSR1a), in the hypothalamus to signal hunger and promote food intake. The melanocortin receptor accessory protein 2 (MRAP2) was previously shown to regulate energy homeostasis through the modulation of the activity of the melanocortin-4 receptor and prokineticin receptors. In this study we identify MRAP2 as a partner of ghrelin-GHSR1a signaling. We show that MRAP2 interacts with GHSR1a and potentiates ghrelin-stimulated signaling both in vitro and in vivo. We demonstrate that in the absence of MRAP2, fasting fails to activate agouti-related protein neurons. In addition, we show that the orexigenic effect of ghrelin is lost in mice lacking  Melanocortin receptor accessory protein 2 (MRAP2) is an adaptor protein that contributes to melanocortin-4 receptor and prokineticin receptor 1 signalling. Here the authors show that MRAP2 also regulates ghrelin receptor signalling in the hypothalamus and starvation sensing in mice. These molecules act on the hypothalamus, through their respective receptors, to inform the brain on the energy state of the organism. Ghrelin is a peptide hormone secreted by the stomach3 during fasting to signal hunger to the brain5. Ghrelin acts through its receptor, the growth hormone secretagogue receptor 1a (GHSR1a), a G-protein-coupled receptor (GPCR) expressed in the hypothalamus, in particular in orexigenic agouti-related protein (AGRP) neurons8. Ghrelin function has been shown to be largely mediated through and to require AGRP neurons9. Whereas several GPCRs have been shown to be regulated by or to require the presence of accessory proteins, the interaction of GHSR1a with accessory proteins has not been investigated.Food intake and energy expenditure are tightly regulated by numerous hormones and neuropeptides secreted either by peripheral organs or centrally10. Mutations in MRAP2 have also been identified in obese humans10. MRAP2 is highly expressed in the hypothalamus where it is known to regulate the activity of the melanocortin-4 receptor11 and the prokineticin receptors12, GPCRs, that signal satiety and are required for the proper control of food intake and energy expenditure16. In this study we investigated if MRAP2 modulates ghrelin signaling in vitro and in vivo, and conclude that MRAP2 interacts with GHSR1a and is an important component of the ghrelin receptor signaling complex, thus establishing that in addition to being a regulator of satiety sensing through the modulation of MC4R and PKR1 activity, MRAP2 promotes hunger sensing by potentiating ghrelin signaling.The melanocortin receptor accessory protein 2 (MRAP2) is a single transmembrane protein that plays an important role in the control of energy homeostasis since the deletion of MRAP2 in mice causes severe obesityMrap2 knockout (KO) mice develop obesity and their weight start diverging from wild-type (WT) animals between 9 and 11 weeks of age10. To identify the cause of the obesity phenotype, we investigated how deleting MRAP2 impacts energy homeostasis in 11- to 13-week-old mice. At this age, Mrap2 KO animals were slightly but significantly heavier than their WT littermates  are essential for sensing energy needs and signaling hungering Fig. . In the ice Fig. . A seconals Fig. . Interesice Fig. . Altoget20. For this reason we performed immunofluorescence detection of MRAP2, using a previously extensively validated anti-MRAP2 antibody12, in brain slices from mice expressing tdTomado in AGRP neurons (AGRPCRE/tdTomato mice). We show that MRAP2 is expressed throughout the hypothalamus, including AGRP neurons  or AGRPCRE/MRAP2fl/fl mice. Consistent with a specific deletion of MRAP2 in AGRP neurons, the expression of MRAP2 in microdissected ARC from AGRPCRE/MRAP2fl/fl mice is about half of that of AGRPCRE/MRAP2+/+ animals We tested the ability of MRAP2 to form a complex with the ghrelin receptor using HEK293T cells transfected with 3 hemaglutinin (HA)-tagged GHSR1a (3HA-GHSR1a) and 3xFlag-tagged MRAP2 (MRAP2-3F). Transfected cells were lysed and the GHSR1a or MRAP2 were pulled down using monoclonal anti-HA or anti-Flag antibody, respectively. Western blotting revealed reciprocal co-immunoprecipitation Fig. . SpecifiGiven that MRAP2 interacts with GHSR1a, we tested whether it could modulate the density of the receptor at the cell surface. To this end we transfected Chinese hamster ovary (CHO) cells with 3HA-GHSR1a and either MRAP2 or green fluorescent protein . We then fixed the cells and measured both cell-surface (in non-permeabilized cells) and total (in permeabilized cells) expression of 3HA-GHSR1a by fixed-cell enzyme-linked immunosorbent assay (ELISA). MRAP2 had no significant effect on either the cell surface density or total GHSR1a expression Fig. . Thus, Mq/11, we measured ghrelin-induced inositol triphosphate (IP3) production (reported by the concentration of accumulated inositol phosphates) in CHO cells expressing MRAP2, GHSR1a, or GHSR1a and MRAP2. As expected, ghrelin does not stimulate IP3 production in cells only expressing MRAP2. In cells expressing GHSR1a, we found that the efficacy of the ghrelin-induced IP3 response was greatly enhanced by the presence of MRAP2  injections of ghrelin or saline in mice cannulated in the lateral ventricle of the brain and harvested the brains for immunofluorescence staining of cFos. Injection with ghrelin caused a significant increase in the number of cFos-positive neurons in the ARC compared to saline regardless of genotype. However, the response to ghrelin in Mrap2 KO mice was about half of that in WT littermates  caused a significant increase in food intake compared to saline-injected animals , and food intake was monitored for 3 h. ACPA caused a similar and significant increase in food intake compared to saline in both WT and Mrap2 KO animals of both genders Lowl from the Jackson Laboratory) with R26-LSL-tdTomato reporter mice from the Jackson Laboratory. GHSR1a KO mice were kindly provided by Dr. Jeffrey Zigman . All animals were maintained in the University of Iowa temperature controlled animal facility with 12 h light/dark cycles (6 a.m./6 p.m.). Animals were fed with standard rodent diet (NIH-31) and allowed free access to water. All experiments using mice were approved by the Animal Care and Use Committee at the University of Iowa.C57BL/6N Seven-week-old animals were anesthetized with intraperitoneal injection of a combination of xylazine and ketamine at dosage 10:100 mg/kg before being placed on a stereotaxic apparatus . After standard disinfection on surgical site, an incision was made to expose the skull and a small hole was drilled. A 26-gauge stainless steel cannula (PlasticOne) was placed into the left lateral ventricle  and fixed in place with dental cement. Mice were allowed to recover for 7 days post surgery before experiments. At the end of the study, the localization of the injection site was confirmed by injecting 2 μl blue ink into the cannula.Animals were deeply anesthetized with saturated isoflurane in a closed chamber and perfused with ice cold 0.1 M phosphate buffer solution , followed by ice-cold 4% paraformaldehyde (PFA) in PBS. Whole brains were dissected out and post fixed in fresh 4% PFA for12 h at 4 °C. Brains were then immersed in 30% sucrose in PBS at 4 °C until they sunk. Samples were embedded in Tissue-Tek OCT compound  and sectioned at 35 µm thickness using a cryostat . Free-floating sections were washed in PBS two times and incubated with blocking buffer containing 5% normal goat serum in PBS with 0.5% Triton X-100 for 1 h followed by rabbit polyclonal anti-cFos antibody  No. sc 52, Santa Cruz Biotechnology, TX, USA) in blocking solution (1:1000) overnight at 4 °C. After washed with PBS containing 0.1% Tween-20 (PBST), the sections were incubated in 1:500 Alexa Fluor 594 goat anti-rabbit IgG  for 1 h at room temperature. The sections were washed in PBST five times and mounted onto superFrost slides , air-dried at room temperature in a dark room and coverslipped with Prolong diamond antifade mountant with 4,6-diamidino-2-phenylindole . For cell counting, three different sections of the ARC of hypothalamus in each mouse were examined using × 20 magnification. cFos-positive cells were manually counted from entire ARC from five mice per group using the Olympus IX3 microscope system and Olympus Cellsens Dimension software.2. Cells were transfected using LipoD293 in vitro transfection reagent . The total amount of DNA transfected for each condition within an experiment was kept identical.CHO-K1, HEK293T, and CHO-M1 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) /F-12 supplemented with 5% v/v fetal bovine serum and 1% penicillin–streptomycin solution. Cultures were incubated at 37 °C in a humidified atmosphere containing 5% COn-dodecyl-β-maltoside in PBS with proteinase inhibitor and incubated at 4 °C for 10 min on an orbital shaker, followed by centrifugation at 15,000 × g for 10 min. The protein lysates were transferred into new centrifuge tubes and following antibodies, Purified mouse anti-HA.11 epitope tag antibody  or M2 mouse anti-Flag , were added into the cell lysates at 1/5000 dilution. The protein lysates were then incubated on a shaker for overnight at 4 °C. 20 µl of protein G dynabeads (Life Technologies) were added into each sample and incubated at 4 °C for 1 h. The beads were washed four times with wash buffer and sample buffer containing LDS loading buffer with 5% β-mercaptoethanol was added on the beads to elude the target protein and antibodies and boiled 5 min. The same amount of protein from each sample was separated by SDS-polyacrylamide gel electrophoresis on 10% gel and transferred to polyvinylidene fluoride membranes. The proteins were detected by western blot using mouse anti-HA for GHSR1a co-immunoprecipitation experiments and mouse anti-Flag for detecting MRAP2. After 1 h in blocking solution containing 5% milk in PBS with 0.1 % Tween-20, the membranes were incubated with the primary antibody diluted 1/5000 in blocking solution overnight at 4 °C. The membranes were washed with PBST for 5 min three times and then incubated with secondary goat anti-mouse horseradish peroxidase (HRP) antibody  at 1/5000 dilution for 45 min at room temperature followed by four washes with PBST. The WesternSure ECL substrate was applied on the membrane to detect the HRP-labeled secondary antibodies on the membrane and imaged on an cDigit scanner .HEK293T cells were transfected with human MRAP2-3Flag and 3HA-GHSR1a using LipoD293 according to the manufacturer’s protocol. At 24 h after transfection, the cells were lysed with ice-cold non-denaturing lysis buffer containing 0.1% CHO cells were transfected with GFP, 3HA-GHSR1a-LgBit + FKBP-SmlBit, FRB-LgBit + MRAP2-SmlBit, or 3HA-GHSR1a-LgBit + MRAP2-SmlBit. FKBP and FRB do not interact with GHSR1a and MRAP2, respectively, and were used to measure nonspecific NanoLuc recombination (Promega). Transfected cells were plated in a white opaque 96-well plate and allowed to attach overnight. Live-cell NanoBit substrate was added to the cells following the manufacturer’s direction and luminescence was measured 5 min later using a Spectramax i3 plate reader (Molecular Devices).Ad libitum-fed or 24 h-fasted mice were deeply anesthetized with isoflurane before removing the brain. ARC was isolated by microdissection and solubilized in lysis buffer  with protease inhibitor on ice. Protein concentration was determined by bicinchoninic acid assay and the same amount of protein was loaded on polyacrylamide gels for SDS separation. Western blot was performed using either anti-V5  or anti-pAMPK .After 24 h fasting, 11-week-old animals were deeply euthanized with saturated isoflurane in a closed chamber. Fresh whole hypothalami were collected into an ice-chilled tube and immediately frozen in dry ice and kept at −80 °C. Well-fed animals were used as control groups. Total RNA was extracted from hypothalamus using RNeasy lipid tissue mini kit  according to the manufacturer’s instruction and treated with RQ1 RNase-free DNase enzyme . A unit of 1 µg cDNA was synthesized using iScript reverse transcription kit for reverse transcription (RT)-qPCR according to the manufacturer’s instruction . Gene expression was quantified using mouse-specific predesigned probe-based quantitative PCR (qPCR) assays  in 20 µl of PCR mixture containing 1 × KAPA Probe Fast qPCR master mix , 50 ng of cDNA (Bio-Rad), 200 nM of each primer, and FAM/BHQ-1 hydrolysis probe. Quantitative real-time PCR was done in optical 96-well plates on an iCycler (Bio-Rad) using a fast cycling protocol; 95 °C for 3 min followed by 40 cycles of 3 s at 95 °C and 20 s at 60 °C. The beta actin mRNA level was quantified as an endogenous control. The amount of cDNA in unknown samples was determined from a standard curve method. The target cDNA was compared to the amount of beta actin.Mrap2: primer 1, AGTCTGACACAAAGCTGTTCA; primer 2, GATTGGATTCTGGGTTGGTCT; probe, /56-FAM/TGTCTTCGT/ZEN/CAGCAAAGTCAGCACA/3IABkFQ/.Primer sequences were as follows. Mouse Pomc: primer 1, CTGTTCATCTCCGTTGCCA; primer 2, CATAGATGTGTGGAGCTGGTG; probe, /56-FAM/AGCGAGAGG/ZEN/TCGAGTTTGCAAGC/3IABkFQ/.Mouse Agrp: primer 1, CAACAGCAGAACACAACTCAG; primer 2, GCACAAGAGACCAGGACATC; probe, /56-FAM/AGATCAGCA/ZEN/AGCAAAGGCCATGC/3IABkFQ/.Mouse Npy: primer 1, ACAAGTTTCATTTCCCATCACC; primer 2, ACATCAATCTCATCACCAGACAG; probe, /56-FAM/CCCAGAACA/ZEN/AGGCTTGAAGACCCT/3IABkFQ/.Mouse β-actin: primer 1, AGGTCTTTACGGATGTCAACG; primer 2, ATTGGCAACGAGCGGTT; probe, /56-FAM/ATTCCATAC/ZEN/CCAAGAAGGAAGGCTGG/3IABkFQ/.Mouse CRE mice  with R26-LSL-tdTomato reporter mice to study the expression of Mrap2 protein in the AgRP neuron in the ARC of the hypothalamus. AgRPCRE/tdTomato male mice were perfused with 4% PFA in 0.1 M phosphate buffer, pH 7.4, whole brains were dissected out and post-fixed in fresh 4% PFA overnight at 4 °C. The brains were cryopreserved by incubating in 30% sucrose in 0.1 M phosphate buffer. Cryosections were cut at 40 µm thickness and incubated in 10 mM sodium citrate at 80 °C for 30 min. The sections were then blocked in 5% normal goat serum/0.4% Triton X-100 in PBS for 1 h and subsequently incubated with rabbit anti-MRAP2 antibody  1:1000 in blocking buffer overnight at 4 °C. The sections were washed with 0.1% Tween-20 in PBS three times and incubated with goat anti-rabbit Alexa Fluor 488 antibody 1:300 for 1 h in blocking buffer and washed four times with 0.1% Tween-20 in PBS before mounting on a glass slide. Slides were visualized with an Olympus IX83 inverted fluorescence microscope.We bred AgRPTotal inositol 1 phosphate (IP1) level was measured 48 h after transfection in adherent cells plated in a 384-well white opaque plate at a density of 14,000 cells/well. CHO-M1 cells (CHO cells stably expressing the muscarinic receptor) were transfected with MRAP2 alone, GHSR1a and empty vector, or GHSR1a and MRAP2 at a 1:5 ratio and plated in a 384-well plate the next day. IP1 accumulation was measured using the IP-One Tb HTRF kit (Cisbio Bioassays) according to the manufacturer’s instructions. Signals at 665 and 620 nm were detected using Spectramax i3 (Molecular Devices) equipped with a Cisbio HTRF module. Results were normalized to the signal obtained in response to 10 µM Carbachol for each condition.For measurements of cumulative food intake, mice were individually housed in standard cages and, after 7 days of acclimation, food consumption was measured daily at 10.00 a.m. For energy expenditure, food intake, and activity measurements, 7-week-old male mice were individually housed in the home cage for 1 week before housed in CLAMS center feeder cages for 5 days. Data such as food intake, oxygen consumption, heat, and locomotion were collected automatically over a 5-day period and then analyzed with the Oxymax/CLAMS software. Daily food intake and activity data over the experimental period were collapsed to generate average daily food consumption and activity. Heat production was analyzed using analysis of covariance (ANCOVA).The CHO-K1 cells were cultured in a 24-well plate and transfected transiently with 3HA-GHSR1a and MRAP2, 3HA-GHSR1a and empty vector, or empty vector only. The fixed-cell ELISA was performed at 24 h after the transfection as described previously (eLife paper). After washing with PBS, the cells were fixed with 4% PFA in PBS for 10 min and washed with PBS twice, blocked with either 5% milk in PBS for surface-expression measurement or 5% milk in RIPA lysis buffer containing 150 mM NaCl, 50 mM Tris, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate, pH 8.0, for total receptor expression measurement for 45 min. Cells were subsequently incubated with 1/5000 mouse anti-HA monoclonal antibody in the blocking buffer for 2 h at room temperature and washed for 5 min three times with PBS followed by incubation with 1/5000 anti-mouse-HRP antibody for 1 h at room temperature before the cells were washed with PBS for 5 min three times. A volume of 200 µl of ELISA substrate  was added into each well and reaction was stopped with 10% sulfuric acid when blue color was visible. Absorbance of ELISA reaction was measured at 450 nm using a Spectramax I3 plate reader.Mice were anesthetized in accordance with University of Iowa Carver College of Medicine guidelines. Mice were placed into stereotaxic frame and injection of 5 µl AAV1-GCamp6s virus and 5 µl AAV1-flox-tdTomato (UPenn vector core) were injected unilaterally into the ARC of the hypothalamus  with a flow rate of 1 µl/5 min. At completion of injection, needle was kept in injection site for an additional 15 min. Mice recovered from surgery for 7 days and tissue was processed for analysis 7–14 days after injection.d-glucose) bubbled with 95% O2 and 5% CO2. Slices were then transferred to a holding chamber containing oxygenated aCSF  for 30 min at 34 °C and then for another 30 min at 22 °C for recovery, and subsequently transferred to a submersion recording chamber continually perfused with 32 °C oxygenated aCSF (rate: 2 ml/min). Slices were then placed in the Olympus FVMPE-RS multiphoton scanning microscope and equilibrated for at least 15 min before each recording. Baseline GCamp6 signal was recorded for the first 5 min and then slice was perfused with 10 nM ghrelin for 15 min. Peak fluorescence during the 15 min ghrelin perfusion was normalized to baseline measurements for each neuron recorded. At the end of the experiment, slices were perfused with high-potassium solution  to induce depolarization and ensure cell viability.Coronal slices (400 µm) from AAV1-GCamp6s/AAV1-flox-tdTomato-injected mice were prepared, in accordance with University of Iowa Carver College of Medicine guidelines. Slices were cut using a Vibratome 1000 Plus  in ice-cold slicing buffer , 1 μg ACPA, or 5 μg ACPA  over a 1 min period using a 25 μl Hamilton syringe coupled to an injection cannula by a polyethylene tubing. After injection the injector was kept in place for 3 min to ensure full diffusion from the injector tip. Food intake was monitored at 0, 1, and 3 h after injection.Ghrelin-induced spontaneous food intake was observed in ad libitum Mrap2 KO mice and WO littermates during the light phase from 10 a.m. to 1 p.m. WT and t-test or regular two-way ANOVA was used to determine statistically significant differences between the groups tested time points and different samples. Energy expenditure data were analyzed by ANCOVA. p < 0.05 was taken as the level of significance in all statistical analyses.Statistical difference was analyzed using GraphPad Prism 6 program . The data were shown as mean ± SEM. One-way analysis of variance (ANOVA) followed by post hoc Tukey test or unpaired All data included in this study are available upon request.Supplementary information Supplementary Figures


Exploring the molecular mechanisms underlying directed differentiation is helpful in the development of clinical applications of mesenchymal stem cells (MSCs). Our previous study on dental tissue-derived MSCs demonstrated that secreted frizzled-related protein 2 (SFRP2), a Wnt inhibitor, could enhance osteogenic differentiation in stem cells from the apical papilla (SCAPs). However, how SFRP2 promotes osteogenic differentiation of dental tissue-derived MSCs remains unclear. In this study, we used SCAPs to investigate the underlying mechanisms.SCAPs were isolated from the apical papilla of immature third molars. Western blot and real-time RT-PCR were applied to detect the expression of β-catenin and Wnt target genes. Alizarin Red staining, quantitative calcium analysis, transwell cultures and in vivo transplantation experiments were used to study the osteogenic differentiation potential of SCAPs.SFRP2 inhibited canonical Wnt signaling by enhancing phosphorylation and decreasing the expression of nuclear β-catenin in vitro and in vivo. In addition, the target genes of the Wnt signaling pathway, AXIN2 (axin-related protein 2) and MMP7 , were downregulated by SFRP2. WNT1 inhibited the osteogenic differentiation potential of SCAPs. SFRP2 could rescue this WNT1-impaired osteogenic differentiation potential.The results suggest that SFRP2 could bind to locally present Wnt ligands and alter the balance of intracellular Wnt signaling to antagonize the canonical Wnt pathway in SCAPs. This elucidates the molecular mechanism underlying the SFRP2-mediated directed differentiation of SCAPs and indicates potential target genes for improving dental tissue regeneration.The online version of this article (doi:10.1186/s11658-017-0044-2) contains supplementary material, which is available to authorized users. Mesenchymal stem cells (MSCs) are considered a good cell source for therapies focused on tissue regeneration . First iWnts are potent regulatory proteins in stem cells, modulating proliferation and differentiation via both canonical and non-canonical pathways. WNT1, WNT3a and WNT8 can activate the canonical Wnt signaling pathway, which has β-catenin as a key mediator . Some stThe family of secreted frizzled-related proteins (SFRPs), which includes five human SFRPs members, binds directly to Wnts to prevent receptor binding and activation of Wnt signaling , 10. It SFRP2 is upregulated during osteogenesis of MSCs  and signOur previous study demonstrated that SFRP2 could enhance the osteogenic differentiation ability of SCAPs , which cIn this study, we used SCAPs to investigate the underlying mechanisms for SFRP2 action in osteogenic differentiation. Here, we reveal that SFRP2 inhibits the canonical Wnt/β-catenin signaling by increasing the level of phosphorylated β-catenin, inhibiting the nuclear expression of β-catenin and downregulating the target genes of the Wnt signaling pathway. In addition, SFRP2 could restore the osteogenic differentiation capacity impaired by WNT1 in SCAPs. These findings provide novel insight into the mechanisms underlying SFRP2-mediated directed differentiation of dental tissue-derived MSCs and their potentially valuable clinical applications.Dental tissues were obtained under approved guidelines set by the Beijing Stomatological Hospital, Capital Medical University with informed consent from the patients. Wisdom teeth were first disinfected with 75% ethanol and then washed with phosphate buffered saline (PBS). SCAPs were gently separated from the apical papilla of the root and then digested in a solution of 3 mg/ml collagenase type I  and 4 mg/ml dispase (Roche Diagnostics Corp.) for 1 h at 37 °C.Single-cell suspensions were obtained by passing the cells through a 70 μm Falcon strainer (BD Biosciences). MSCs were grown in a humidified, 5% CO2 incubator at 37 °C in DMEM alpha (Invitrogen), supplemented with 15% fetal bovine serum , 2 mmol/l glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). The culture medium was changed every 3 days. The phenotype, lineage markers and lineage differentiation potentials of SCAPs were identified in a previous study .Human embryonic kidney 293 T cells were maintained in complete DMEM with 10% FBS (Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen).SFRP2 cDNA was generated using a standard gene synthesis method and sub-cloned into the LV5 lentiviral vector (GenePharma Company). Full-length WNT1 cDNA fused to a hemagglutinin (HA) tag was generated using a standard gene synthesis method and sub-cloned into the pLNCX retroviral vector. Short hairpin RNAs (shRNAs) with complementary sequences of target genes were sub-cloned into the pLKO.1 lentiviral vector (Addgene). For viral infections, MSCs were plated overnight and then infected with retroviruses or lentiviruses in the presence of polybrene  for 6 h. After 48 h, the infected cells were selected with 2 μg/ml puromycin. Scrambled shRNAs (Scramsh) were purchased from Addgene. The target sequence for the shRNAs is: SFRP2 shRNA (SFRP2sh), 5′-ttgatgtaggttatctccttc-3′.The plasmids were constructed according to standard methods. All structures were verified by appropriate restriction digestion and/or sequencing. Human full-length Total RNA was isolated from MSCs with TRIzol reagent (Invitrogen). We synthesized cDNA from 2 μg aliquots of RNA using random hexamers or oligo(dT), and reverse transcriptase according to the manufacturer’s protocol (Invitrogen). Real-time PCR was performed with the QuantiTect SYBR Green PCR kit (Qiagen) and an iCycler iQ Multicolor Real-time PCR Detection System. The primers sequences are:GAPDH, forward primer, 5′-cgaacctctctgctcctcctgttcg −3′ and reverse primer, 5′-catggtgtctgagcgatgtgg-3′.AXIN2, forward primer, 5′-ctccccaccttgaatgaaga-3′ and reverse primer, 5′-gtttccgtggacctcacact-3′.MMP7, forward primer, 5′-aaactcccgcgtcatagaaa-3′ and reverse primer, 5′-ttctgcaacatctggcactc-3′.SCAPs were grown in mineralization-inducing media using the StemPro Osteogenesis Differentiation Kit (Invitrogen). Cells were induced for 5 days and then ALP activity was assayed with an ALP activity kit according to the manufacturer’s protocol (Sigma-Aldrich). Signals were normalized based on protein concentrations.To detect mineralization, cells were induced to mineralize for 2 weeks, fixed with 70% ethanol, and stained with 2% Alizarin Red (Sigma-Aldrich). To quantitatively determine calcium, Alizarin Red was de-stained with 10% cetylpyridinium chloride in 10 mM sodium phosphate for 30 min at room temperature. The concentration of calcium was determined by measuring the absorbance at 562 nm on a microplate reader and comparing the results to a standard calcium curve generated using calcium dilutions in the same solution. The final calcium level in each group was normalized to the total protein concentration detected in a duplicate plate.6 SCAPs were harvested by centrifugation. The pelleted cells were resuspended in 250 μl of cytosol extraction buffer A, incubated on ice for 10 min, mixed with 15 μl cytosol extraction buffer B, and incubated on ice for 1 min. The lysates were centrifuged, and the pellets were washed with cytosol extraction buffer A and then resuspended in 50 μl of cold nuclear extraction buffer. After incubation at 4 °C for 30 min with constant rotation, the suspension was spun at 12,000 g at 4 °C for 5 min. The nuclear extract was collected from the supernatant fraction.The nuclear extracts of SCAPs were prepared with a Nuclear Cytosol Extraction Kit (Applygen Technologies Inc.) according to the manufacturer’s instructions. Briefly, 5.0 × 10Cells were lysed in RIPA buffer consisting of 10 mM Tris–HCl, 1 mM EDTA, 1% sodium dodecyl sulfate (SDS), 1% NP-40, 1:100 proteinase inhibitor cocktail, 50 mM β-glycerophosphate and 50 mM sodium fluoride. The samples were separated on a 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes with a semi-dry transfer apparatus (Bio-Rad). The membranes were blotted with 5% dehydrated milk for 2 h and then incubated with primary antibodies overnight. The immune complexes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Promega) and visualized with SuperSignal reagents (Pierce). Primary antibodies against HA , phosphorylated β-catenin , and β-catenin . We also used a primary monoclonal antibody to detect the housekeeping protein beta-actin  or histone H3 .6 cells were mixed with 40 mg of HA/tricalcium phosphate ceramic particles , and then transplanted beneath the dorsal skin of 10-week old immunocompromised beige mice (nu/nu nude mice). These procedures were performed according to the approved animal protocol. Eight weeks later, the implants were harvested, fixed with 10% formalin, decalcified with 10% EDTA buffer (pH 8.0), and embedded in paraffin. To detect the expression of phosphorylated β-catenin, sections were stained immunohistochemically using antibody against phosphorylated β-catenin .This study was approved by the Animal Care and Use Committee of Beijing Stomatological Hospital, Capital Medical University. The animal experiments carried out in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals . Animals were purchased from the Institute of Animal Science of the Vital River Co., Ltd. No drugs or previous procedures were used. Approximately 4.0 × 105 SCAP-SFRP2 cells or SCAPs transfected with empty vector (SCAP-vector1) were seeded in the upper chamber, and 2 × 105 SCAP-HA-WNT1 cells or SCAPs transfected with empty vector (SCAP-vector2) were placed in the bottom chamber in the presence of DMEM alpha supplemented with 15% fetal bovine serum, 2 mmol/l glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. After 24 h, the culture medium was changed to mineralization-inducing medium using the StemPro Osteogenesis Differentiation Kit.Six Transwell plates were used in this study. Chambers with a 0.4 μm pore size membranes (Corning) were used to physically separate the SCAP-SFRP2 from the SCAP-HA-WNT1 cells. Approximately 2 × 10p ≤ 0.05 was considered significant.All statistical calculations were performed with SPSS 13. Student’s t test was performed to determine statistical significance. SFRP2-overexpressing constructs were transduced into SCAPs via lentiviral infection. Ectopic SFRP2 expression was confirmed using real-time RT-PCR . After selection, knockdown efficiency (90%) was verified using real-time RT-PCR  to target PCR Fig. . We discAPs Fig.  and f.SFRP2 decreased the expression of nuclear β-catenin in SCAPs , the levels of Wnt target genes, including C-myc, AXIN2, DKK1 and MMP7, were detected using real-time PCR. The results showed that silencing SFRP2 enhanced the expression of AXIN2 and MMP7 .After overexpression or knockdown of MP7 Fig.  and b, wMP7 Fig.  and d. HWNT1-expressing sequence into a retroviral vector. This construct overexpressing ectopic WNT1 was transduced into SCAPs via retroviral infection. Ectopic WNT1 expression was confirmed using western blot analysis , which is a co-receptor for Wnt, leads to a reduction in bone mass. However, a gain-of-function mutation in LRP5 causes an autosomal dominant disorder characterized by high bone density , 24. AnoSFRPs play a pivotal role in the Wnt pathway and mainly function as antagonists of Wnt signaling , 27. PreSFRP2 could enhance the phosphorylation of β-catenin and GSK-3β, and that it could inhibit the expression of nuclear β-catenin at the protein level. Accordingly, SFRP2 also affected the expression of Wnt target genes including AXIN2 and MMP7. Upregulation of SFRP2 could inhibit the expression of AXIN2 and MMP7 and vice versa. These results suggest that SFRP2 could act as an antagonist of the canonical Wnt signaling pathway in SCAPs. However, under certain circumstances, SFRP2 has been shown to display a synergistic effect on Wnt/β-catenin signaling, functioning as an active agonist of Wnt16b, facilitating cancer cell proliferation, migration and drug resistance [In this study, we found that sistance . In othesistance , 33.Besides, this has shown that SFRP2 had no effect on wnt5a expression at the mRNA level. It is speculated that SFRP2 might not affect the non-canonical wnt pathway. For other cell types and disease models, SFRP2 may exert different effects. SFRP2 has been shown to inhibit the proliferation of cardiac progenitor cells and prime them for cardiac differentiation by modulation of both canonical and non-canonical Wnt/Planar Cell Polarity (PCP) pathways through JNK . Thus, wSFRP2 was upregulated upon osteogenic differentiation of MSCs derived from dental tissue and bone marrow in a time-dependent manner. Moreover, gain-of-function studies showed that SFRP2 enhanced osteo/dentinogenic differentiation of SCAPs and the expression of the BSP, COL1A2, OPN, DSPP and DMP1 genes, which encode different extracellular matrix proteins of the bone and dentin. Moreover, SFRP2 was able to activate the expression of OSX independent of the transcription factors RUNX2. In addition, transplantation experiments demonstrated that SFRP2 considerably enhanced osteo/dentinogenesis in vivo [Our previous study demonstrated that  in vivo .SFRP2 cells during the process of osteogenesis and SCAP-SFRP2 cell transplanted samples, indicating that the Wnt signaling pathway was inhibited during osteogenesis process [We also found that low dose of SFRP2 recombinant protein (100 ng/ml) could effectively enhance the osteogenesis capacity of SCAPS, which may provide a simple and convenient means for clinical use. Consistently, this study showed that more phosphorylated β-catenin was detected in SCAP- process , 35.WNT1 decreased the osteogenic differentiation capacity of SCAPs. Furthermore, SFRP2 could rescue the WNT1-mediated impaired osteogenic differentiation in SCAPs in a competitive manner.Subsequently, we assessed whether the effect of SFRP2 in promoting osteogenesis was mediated by the Wnt pathway. Indeed, overexpression of SFRP2 has been implicated in the suppression of osteoblast activity during inflammation-induced bone loss, and it was shown to vary with the progression of inflammation [However, SFRP2 may also display an inverse effect on bone formation. SFRP2 protein has been reported to be widely expressed by multiple myeloma (MM) cells from patients. In MM, cell-derived SFRP2 plays an important role in the suppression of bone formation in advanced stages of the disease . In addiammation , 39. In ammation , 16. TheSFRP2 enhanced the osteogenic differentiation in SCAPs by inhibiting the canonical Wnt signaling pathway. Our work explored the molecular mechanisms underlying directed differentiation of MSCs mediated by SFRP2 and provided potential target genes for improving tissue regeneration mediated by dental tissue-derived MSCs.Our results showed that Additional file 1: Figure S1.SFRP2 did not affect the expression of WNT5a in SCAPs. (TIFF 1872 kb)Overexpression of Additional file 2: Figure S2.SFRP2 increased the expression of p-β-catenin during the process of osteogenesis. A – Expression of phosphorylated β-catenin increased in control SCAPs on days 7 and 10 during the osteogenic process. Overexpression of SFRP2 increased the expression of phosphorylated β-catenin. B – Quantitative analysis of p-β-catenin at different times based on western blot results. Student’s t test was performed to determine statistical significance. All error bars represent SD (n = 3). *p ≤ 0.05. (TIFF 1833 kb)Additional file 3: Figure S3.n = 3). *p ≤ 0.05. (sigma unit: unit of measurement of ALP activity). (TIFF 1174 kb)SFRP2 recombinant protein enhanced the osteogenic capacity of SCAPs. A – ALP activity and B – quantitative analysis of calcium concentration results showed that 100 ng/ml SFRP2 recombinant protein could enhance the osteogenic capacity of SCAPs. All error bars represent SD (