Endothelial tightening augmented neuronal activity in the SFO of water restricted mice. Hence, regulating the SFO vessel barrier may influence neuronal function in the context of water homeostasis.The circumventricular organs (CVOs) in the central nervous system (CNS) lack a vascular blood-brain barrier (BBB), creating communication sites for sensory or secretory neurons, involved in body homeostasis. Wnt/β-catenin signaling is essential for BBB development and maintenance in endothelial cells (ECs) in most CNS vessels. Here we show that in mouse development, as well as in adult mouse and zebrafish, CVO ECs rendered Wnt-reporter negative, suggesting low level pathway activity. Characterization of the subfornical organ (SFO) vasculature revealed heterogenous claudin-5 (Cldn5) and Plvap/Meca32 expression indicative for tight and leaky vessels, respectively. Dominant, EC-specific β-catenin transcription in mice, converted phenotypically leaky into BBB-like vessels, by augmenting Cldn5 Infections and diseases in the brain and spine can be very damaging and debilitating. Indeed, the central nervous system also needs a carefully controlled biochemical environment to survive. As such, all animals with a backbone have barriers and defenses to protect and preserve this key system. One of these is the blood-brain barrier, a physical barrier between the brain and the outside world. Where most blood vessels allow relatively free exchange of chemicals between the blood and surrounding cells, the blood-brain barrier controls what can move between the bloodstream and the brain.Yet, there are gaps in the blood-brain barrier, specifically within structures in the brain called the circumventricular organs. These leaky vessels allow the brain cells in these regions to monitor the blood and respond to changes, for example, by triggering sensations such as hunger, thirst or nausea. It is not clear what stops the blood-brain barrier from forming in these regions and what effect the presence of a barrier would have on the brains activity, or the health and behavior of the animal.Benz et al. have now used mice and zebrafish to examine the development and structure of the blood-brain barrier. The investigation revealed that the signals that induce the blood-brain barrier throughout the brain are absent in the circumventricular organs of both species. Next, by artificially activating a protein involved in cell-cell interactions in mice, Benz et al. created blood-brain barrier-like structures in circumventricular organs by converting the leaky vessels into tight ones. This change meant that the brain cells in these regions did not respond properly to water deprivation, which potentially may have affected the regulation of thirst in these mice.Understanding the blood-brain barrier could have a variety of impacts on how we treat diseases in the central nervous system. This includes stroke, brain tumors and Alzheimers disease. These findings could particularly help scientists to better understand conditions that affect basic needs like thirst and hunger. In vertebrates, the endothelial blood-brain barrier (BBB) is crucial for providing a permissive microenvironment for neuronal function. During developmental brain vascularization, blood vessels undergo Wnt/β-catenin signaling, driven by Wnt7a/7b that is required for angiogenesis as well as for BBB formation . In the organum vasculosum of the lamina terminalis, OVLT), the subfornical organ (SFO) and the area postrema (AP) are considered as sensory organs  are a number of small midline structures found in all vertebrate brains, located around the third and fourth ventricle. CVOs have a rich capillary plexus, which physiologically lacks BBB properties . These cy organs .The leaky vessels of the CVOs evidently have different morphological and structural characteristics from those of typical BBB vessels, lacking a cellular organization as neuro-vascular unit (NVU), without distinctive astrocytic endfeet and ECs with numerous fenestrations and vesicles . InteresBeside the leaky vasculature of the CVOs, free diffusion of substances into the brain parenchyma is prohibited by tanycytes, specialized cells of the ependymal lining. Tanycytes contain long processes, unlike typical ependymal cells, which project to the parenchyma of the CVOs making contact to the fenestrated vascular wall of the CVOs .The detailed function of all CVOs has not intensely been explored in the past, but more recently, the SFO together with the OVLT, the median preoptic nucleus (MnPO) and the PP were identified as a functional circuit, regulating drinking behavior and water homeostasis of the organism . The SFOAlthough a cell type-specific expression analysis by single cell sequencing, as it has been performed for the brain parenchyma , has notGiven the fact that endothelial Wnt/β-catenin signaling is necessary for BBB development and maintenance, the question remains if the pathway is instrumental in the establishment of vascular heterogeneity in the CNS. Hence, we addressed the question if Wnt/β-catenin signaling is operational in CVO vessels during development and if local regulation of β-catenin signaling is involved in establishing a leaky vascular phenotype in CVOs. Finally, we asked if dominant activation of β-catenin in ECs can overwrite the leaky vessel fate thereby affecting CVO function.+//Cldn5- followed by a Plvap/Meca32-//Cldn5+ EC, suggesting a locally confined regulation of barrier properties. Dominant, genetic activation of β-catenin signaling  in ECs resulted in tightening of CVO blood vessels, evidenced by the switch from a Plvap/Meca32+//Cldn5- to a Plvap/Meca32-//Cldn5+ vascular phenotype. Vessel tightening was accompanied by a significant reduction in endothelial fenestrations, that likely contributed to the reduction of transcellular permeability evidenced by decreased tracer leakage. Interestingly, endothelial tightening did not coincide with the formation of astrocytic endfeet towards a BBB-like NVU. Finally, we observed augmented neuronal activity in the SFO under thirst conditions after sealing CVO vessels, supported by significantly increased neuronal c-fos staining in the SFO of GOF mice.Here we show by investigating CVOs during BAT-gal reporter mouse development that at any embryonic stage analyzed, starting from E13.5, when the first SFO primordium could be identified, to P21, no activation of β-catenin signaling in CVO vessels could be detected. Focusing on the SFO as a crucial CVO in the regulation of water homeostasis, we show that blood vessels in the caudal portion were mainly leaky evidenced by Plvap/Meca32 staining, whereas capillaries in the rostral portion of the organ were tighter. Interestingly, within the vessel continuity, individual ECs might be Plvap/Meca32+ cells at any developmental stage analyzed. Instead, adjacent, non-endothelial cells in the ependymal lining as well as cells in the stroma of the organ showed active Wnt/β-catenin signaling, evidenced by nuclear β-galactosidase staining  mice by confocal and light sheet microscopy.+, but also showed a considerable degree of intermingling with Cldn5+ ECs , we intended to analyze the distribution of leaky and tight vessels within the SFO by staining for Plvap/Meca32 and Cldn5, respectively . In ordedn5+ ECs .-//Cldn5+, suggesting that these vessels possess BBB properties. Instead, vessels of the caudal SFO region were mainly Plvap/Meca32+//Cldn5-, providing evidence for their leaky phenotype  double-transgenic mice were induced with tamoxifen (TAM) either directly after birth for three -CreERT2:Ctnnb1We initially determined recombination efficiency in the brain by analyzing Cdh5(PAC)-CreERT2:mTmG reporter mice , control vessels exhibited high levels of Plvap/Meca32 and low levels of Cldn5 immunolabeling . InteresWhen analyzing different timepoints after TAM induction of adult GOF mice for the expression of Plvap/Meca32 and Cldn5, we observed that activation of β-catenin signaling significantly suppressed Plvap/Meca32 and induced Cldn5 already by day 16 . MaximalTo understand if beside Cldn5 also other tight junction components are regulated upon β-catenin activation in SFO endothelial cells, we stained for occludin (Ocln) and zonula occludens 1 (ZO-1). Analyzing the junctional localization of Ocln normalized to the vessel length in the SFO, we observed only a punctuated staining of Ocln at endothelial cell-cell junctions of controls as previously described by Morita et al. . In vessAs the upregulation of the junctional proteins Cldn5 and Ocln support the interpretation of an SFO vessel tightening in GOF mice, it remained to be clarified if vessel permeability is indeed affected by dominant β-catenin activation in ECs. To this end, GOF and control mice were intravenously injected with FITC-bovine serum albumin (FITC-BSA) (~68 kDa), and examined after 1.5 hrs of circulation. Analysis of FITC-BSA leakage normalized to vessel area revealed a significant reduction of extravasation in SFO vessels of the GOF versus control mice . SpecifiTo strengthen the observation of SFO vessel tightening, we performed β-catenin GOF experiments also with the Pdgfb-iCreERT2 mouse driver line , resultiGiven the increase in Cldn5 expression in SFO vessels, we addressed if the endothelial tightening may also have an effect on the organization of the NVU within the core region of the SFO, in which no astrocytic endfeet are formed around vessels. Therefore, we stained GOF and control SFOs for the astrocytic endfeet markers aquaporin-4 (Aqp4), α-dystroglycan (αDag) and Kir4.1  and 2, aAs previously shown for astrocytic endfeet proteins , leaky SIn order to further characterize the blood vessels in the SFO of β-catenin GOF mice, we employed electron microscopy to visualize their subcellular phenotype. As expected, the vessels in control SFOs showed the typical large and lacuna-like structure with an extensive ECM circumference . MoreoveUpon the observation that dominant endothelial activation of Wnt/β-catenin signaling established barrier properties in SFO vessel, we wanted to elucidate if the tightening of SFO vessels affects neuronal function in this organ.+ cells . β-Catenin GOF and control mice were subjected to water restriction 26 days after induction by TAM  if the Wnt/β-catenin pathway is operational in ECs of CVOs during murine development and in the adult mouse and zebrafish, b) if endothelial-specific, dominant activation of β-catenin transcription could convert the leaky vascular phenotype in CVOs and c) if the latter may have an effect on CVO function.The principle findings of this study are: 1) Wnt/β-catenin signaling is undetectable in CVO vessels during BAT-gal reporter mouse development; 2) similarly, β-catenin-mediated transcription is strongly reduced in the adult zebrafish OVLT; 3) SFO vessels are heterogenous regarding the expression of Plvap/Meca32 and Cldn5; 4) upon genetic β-catenin GOF in ECs, leaky SFO vessels are partially converted into tight vessels; 5) functional conversion of SFO vessel towards a BBB-like identity affects neuronal activity in the SFO.Wnt/β-catenin is crucial for brain vascularization and BBB development, by regulating endothelial sprouting as well as by promoting a BBB expression profile in ECs, respectively . CVOs arAs the Wnt/β-catenin pathway is considered a master switch for barriergenesis, we hypothesized that β-catenin transcription is not operational during CVO vascularization. The data provided here support this interpretation, as in BAT-gal reporter mice, from the initial identification of the SFO primordium at E13.5, none of the investigated developmental stages revealed a single β-galactosidase-positive vessel within the CVOs  and 2. AStill, it has to be noted that vessels in the SFO, OVLT and PP are heterogenous regarding the expression of Plvap/Meca32 and Cldn5 . SimilarThe present findings may suggest that, at least to some degree, vascular phenotypes in the SFO are locally regulated, which would be in line with their role providing local access for neurons to the blood milieu. If vessel differentiation might also be dynamically regulated to control water homeostasis in a circadian rhythm , is currAlthough the regulation of Cldn5 on the promotor level and the role of β-catenin herein requires additional investigation, the tightening of SFO vessels by Cldn5 protein upregulation in β-catenin GOF mice is consistent with previous reports in other regions of the brain . InteresBeside the mere upregulation of Cldn5 and Ocln, endothelial β-catenin GOF resulted in the abolishment of fenestrations and strengthened inter-endothelial junctions. These findings are well in line with a reduction in VEGF signaling in glioma ECs upon Wnt/β-catenin activation via the downregulation of VEGF receptor 2  and upregulation of VEGFR1 . This suOne of the main questions investigated in the present work is how the vasculature in the sensory CVOs like the SFO functionally cooperates with the neurons and other stromal cells to achieve proper physiological regulation of fundamental body parameters like water homeostasis. So far, the vasculature has drawn little attention in this respect, even though considerable progress has recently been made to unravel the regulation of drinking behavior by the SFO, OVLT and the PP . SpecifiAlthough own preliminary experiments aiming to pharmacologically tighten CVO vessels with a systemically administered Wnt/β-catenin activator did not result in SFO vessel tightening (data not shown), this might be a potential way to therapeutically modulate water intake. Interestingly, patients that chronically receive LiCl, an FDA-approved drug for bipolar disorders and a potent Wnt/β-catenin activator, frequently develop polyuria that is linked to altered anti-diuretic hormone  function, which is released by the PP. Moreover, many patients develop polydipsia and urinate more frequently . Hence iad libitum if not declared otherwise. All experimental protocols, handling and use of mice were approved by the Regional Council Darmstadt, Germany (V54-19c20/15-FK/1052 and V54-19c20/15-FK/1108). Wildtype (WT) C57BL6/J as well as transgenic animals were used. The following mouse strains were included Cdh5(PAC)-CreERT2  were maintained under standard conditions at 28°C and a 14 hr light/10 hr dark cycle, in accordance with European and national animal welfare and ethical guidelines . Transgenic lines used in this study were Tg(kdrl:Hsa.HRAS-mCherry)s896 (ia4 (Zebrafish (rry)s896  and Tg(7896 (ia4 . After e+/wt) were bred with C57BL6/J mice to generate either heterozygous positive pups for β-galactosidase or homozygous negative control littermates. At different developmental stages  embryos were harvested. For postnatal day 0 (P0) pups were sacrificed by decapitation, for postnatal day 21 (P21) pups were sacrificed by cervical dislocation. Brain preparation was performed in ice cold PBS and followed by overnight fixation in 4% PFA in PBS. For cryo-sectioning the whole brain was embedded in Tissue-Tek O.C.T. after incubation in 12/15/18% sucrose.To investigate β-catenin activity in ECs, Wnt/β-catenin reporter mice . After 1.5 hr mice were sacrificed by cervical dislocation. The embedded brain was cryo sectioned .To induce thirst, animals were water restricted for 72 hr. Therefore, the animals had no free access to water and got only a restricted amount of water every 24 hr according to their initial body weight . During Mice were i.p. injected with either 3 M or an isotonic (0.15 M) NaCl solution (150 µl/20 g mouse) as described in Zimmerman et al. . After aTo label blood vessels, 80 µl tomato lectin Alexa 649  were injected i.v. in adult mice. After 4 min of circulation time animals were sacrificed by cervical dislocation. After overnight fixation (4% PFA in PBS) whole mount tissue samples were blocked and permeabilized with 0.2% gelatin from bovine skin, Type B, 0.5% Triton in PBS from 24 hr up to one week according to their size. Antibodies (Key resource table) were applied in blocking buffer supplemented with 0.1% saponin. To fix the staining samples were incubated for one hour in 4% PFA.At first the tissue was embedded in low melt agarose. The following dehydration and delipidation protocol was adapted from Orlich et al. and Renier et al. . In brieEither native frozen tissue or sucrose embedded samples were cryo-sectioned coronal or sagittal in 10 µm thickness and then fixed with 4% PFA for 10 min at room temperature or with ice cold MetOH for 3 min. To block/permeabilize tissue slides were incubated for 1 hr (overnight for vibratome section) . Primary antibodies (Key resource table) were incubated for 2 hr (24 hr for vibratome section) and secondary for 1 hr (4 hr for vibratome section) in antibody incubation buffer . If required, sections of PFA-fixed samples were subjected to antigen-retrieval (*) by boiling slides for 45 min in AR6 Buffer . After cooling them down and an additional washing step, slides were stained as described above.Images were acquired using either a Nikon 80i wide field fluorescent microscope, or a Nikon C1si Confocal Laser Scanning Microscope, together with NIS-Elements Microscope Imaging Software for image analysis . SFO vessels were defined as regions of interest (ROI) for area measurements. Staining was evaluated as a ratio of Cldn5 or Plvap/Meca32 to vessel area, evidenced by Podxl or Cdh5 labeling.+ neurons as well as the total number of nuclei within the SFO, defined as ROI, were counted and the ratio of c-fos + to total nuclei was calculated.The number of c-fosAnimals were anesthetized and transcardially perfused with PBS/heparin for 1 min followed by 4 min with 4% PFA in cacodylate buffer . The SFO was whole mount prepared in ice cold PBS directly after brain isolation. Afterwards the tiny SFO whole mount tissue pieces were post-fixed with 4% PFA and 2% glutaraldehyde/CB overnight at 4°C.Prior to embedding the tissue was incubated in 1% Os for 2 hr at RT followed by dehydration in graded acetone including contrast enhancement with uranyl acetate solution at 4°C o/n. Samples were embedded in Epon finally polymerized at 60°C for 24 hr. Ultra-thin sections (50 nm) were cut with Leica Ultracut UCT and analyzed using a Tecnai Spirit BioTWIN FEI electron microscope at 120kV. Images were taken with an Eagle 4K CCD bottom-mount camera. For the quantification of fenestrations, 5 SFO vessels were analyzed for each animal (n = 4 per genotype).+) and control (Cre-) mice.RNA isolation was done using the RNeasy plus Microkit (Qiagen) according to the manufacturer recommendations with DNase on-column digestion (Qiagen) like suggested in the RNeasy Minikit (Quiagen). For cDNA synthesis  57 ng RNA were used from SFO tissue of β-catenin GOF  was performed in technical triplicates for each sample using the Absolute qPCR SYBR Green Fluorescein Mix  according to the manufacturer's protocol. Rplp0 was used as a housekeeping gene for normalization. Expression data were analyzed with ∆∆ct method. Primer sequences used for cDNA amplification by qRT-PCR are listed in No statistical tests were used to predetermine sample size. Several independent experiments were performed to ensure reproducibility. The investigators were blinded by the experimental design during the analysis of the experiments shown in The number of biological replicates is provided as ‘n’ in the legend of each figure. Technical replicates, such as the number of sections analyzed or replicates for qRT-PCR analyses are indicated in the figure legend and the respective material and methods section, respectively. Results are shown as mean ±SEM. Statistical significance was assessed by an unpaired t-test using GraphPad Prism version 6.0 . p-Values were considered significant at p<0.05 and individual p-values are provided in each figure. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, including Elisabetta Dejana as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by K VijayRaghavan as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Dritan Agalliu (Reviewer #2); Christer Betsholtz (Reviewer #3).Thank you for submitting your article "Low Wnt/β-catenin signaling determines leaky vessels in the subfornical organ and affects water homeostasis in mice." for consideration by eLife. However, all the three referees raise important points to be considered before acceptance.The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. The reviewers have found your paper of interest and potentially acceptable for publication in Summary:The paper from Liebner laboratory investigates how CVO vessels, known to lack BBB and be permeable to water, solutes, and hormones, are regulated in relation to Wnt/β-catenin signaling – a central inducer of the BBB. The authors find (1) using Wnt reporter technologies, that Wnt signaling is undetectable in CVOs throughout development in mice and in adult zebrafish. (2) They describe a cellular phenotypic heterogeneity in the CVO vessels regarding Plvap and Cldn5 expression and they suggest that the latter might be regulated in a circadian fashion. (3) Using an inducible genetic β-catenin GOF approach, they show conversion of the CVO vessel phenotype into a BBB-like phenotype. (4) They show results that may indicate that this conversion has consequences for neuronal activity in the CVO.eLife. The manuscript is well-written and well-illustrated in general.This manuscript is an interesting and valuable contribution to the field that deserves publication in While going through the comments, please note in particular:Additional controls for the role of canonical Wnt/β-catenin signaling in the observed control of permeability are asked for. If experimentally feasible, an earlier control of β-catenin signaling in SFO would also be important. Please see if these are speedily do-able and if not pleased adequately discuss these points. It will be useful to have this experimentally addressed, though.A more accurate analysis of the permeability properties of SFO vessels and of tight junction organization and more accurate analysis of astrocyte polarization and matrix protein organization are also asked. More specifically, the authors should make better images for their c-fos data and show that it is indeed neurons that express c-fos.The authors could remove the circadian Cldn5 data (premature), the Aqp4 and CD13 data (confusing).Essential revisions:The gain-of-function mutation of β-catenin is induced in endothelial cell specifically. It is known that endothelial cells can produce soluble neural mediators. An important question, therefore, is whether the observed neural hyperactivation in vivo, induced by β-catenin gain of function in endothelial cells, is due to reduced vascular permeability or to a change of endothelial cell angiocrine activity. This is a crucial aspect for a correct interpretation of the results. Would the authors be able to see the same c-fos upregulation in neural cells by limiting permeability with different tools? For instance, mice Meca32- PLVAP knock out? Src inhibitors? VEGF inhibitors? other more specific tools? If this these experiments are not speedily addressable, they should be carefully and frontally discussed.A point that should be clarified is whether β-catenin does not play any role in the early angiogenesis of the SFO. The authors could not see, using reporter mice, a significant increase in β-catenin activity of the SFO-endothelial cells starting from 13.5E. However, β-catenin may be important in the very first steps of angiogenesis of SFO and then decline, as it occurs in other regions of the brain microcirculation. It could be, therefore, that it is already undetectable at E13.5 while active at earlier times. This is not irrelevant to understand SFO vascularization mechanisms and function. Here too, if experiments to address this are not speedily feasible, please discuss this point adequately.A major functional feature of the BBB is to exclude various circulating molecules from entering the CNS. However, a functional assessment of barrier properties in SFO blood vessels after β-catenin stabilization is missing. The authors need to inject intravenously small or large MW tracers and assess their permeability into the SFOs under conditions of Wnt/β-catenin activation in endothelial cells.Does overexpression of a stabilized form of β-catenin in the adult mice induce expression of Claudin-5 and suppresses MEca32 at the SFO?Do the blood vessels in SFO acquire Occludin protein expression at cell junctions with β-catenin overexpression? The authors need to show some staining with Occludin antibodies.The TEM data, clearly show that in GFO mice, pericytes and astrocytes are in close contact with endothelial cells. However, astrocytes do not appear to be polarized . Are other astrocyte polarity markers (e.g. Dystroglycan or Kir 4.1) absent in this region?One potential explanation for the observation that astrocytes do not form proper endfeet in the SFO region could be due to the absence of basement membrane proteins. The authors need to examine the expression of some of the key basement membrane proteins such as Collagen IV, Laminin α2 etc.The authors nicely demonstrate that β-catenin GOF mice have higher c-Fos levels with water restriction than wild-type mice. Underwater restrictions, mice may show abnormal behaviors under specific behavioral tasks . It may be useful to test if there are any potential behaviors (e.g. rewarding behaviors) where GOF mice may perform worse than wild-type mice due to higher c-Fos activation.The evidence for circadian regulation of Cldn5 expression weak. While the quantifications in Figure 4E may appear convincing, the immunostainings shown in Figure 4A-D are not convincing. The amount of vasculature captured in each section appears hugely variable and the Podxl staining for endothelium seems highly variable in strength. Podxl should be luminal and Cldn5 junctional (or is it?) and this is not at all apparent at the magnification shown. Also, if true, the circadian variation in Cldn5 immunoreactivity raises a number of important issues that should be addressed, including if it is junctional or non-junctional Cldn5 that varies, if the effect is transcriptional or not if other tight junction proteins such as occludin and ZO-1 are also regulated, and more. If it is true that TJs are regulated in a circadian fashion, it is obviously interesting, but the physiological consequences are unclear, given that Plvap expression is maintained, and therefore (presumably) fenestrations not regulated. Would TJ regulation matter for the permeability if fenestrations are kept? The authors speculate that the (possible) circadian regulation of Cldn5 could influence the circadian regulation of thirst and drinking behavior, but this is pure speculation, as far as I can judge. Again, how can Cldn5 matter on top of (presumably) open fenestrations? Given the many questions and the premature nature of this data, we suggest they be removed from the present manuscript, to allow a better focus on Wnt/β-catenin regulation of the CVO vessel phenotype. If Wnt signaling is involved in the circadian regulation of Cldn5 expression in CVO vessels, it would also make more sense to study short term effects of β-catenin GOF experiments; now the earliest time point studied in adults is 16 days following induction.Another area where conclusions are weak relates to the TEM analysis . The images shown do not appear to support the conclusions that vessels in the GOF mice are slimmer, less tortuous and have less ECM deposition. For the ECM, it does appear denser in GOF but this may reflect decreased water content of the ECM rather than a decreased amount of ECM per se.The data regards c-fos expression by neurons in the SFO are also an area where the conclusions do seem robust. Neuronal staining to show that this is neuronal and not glial localization is not there. Also, some of the staining patterns are puzzling. Should not c-fos should look similar at 0h and isotonic NaCl panels in Figure 7, but they look hugely different, casting doubt over the quantifications in Figure 7E and F.The authors find increased expression of Sox17 along with Cldn5 in GOF experiments and note in conjunction with the TEM data that this may signify arterial specification. However, this is unfounded speculation in my opinion. There are many markers for arterial EC in the brain  and Sox17 is not a particularly strong one. The authors should remove this statement or investigate possible arterialization more thoroughly.The authors investigate Aqp4 and CD13 staining patterns to check "other phenotypic BBB features", but although neither Aqp4 nor CD13 was found changed in the GOF situation, it is unclear why the experiments were done in the first place. In what way do the (negative) data display an absence of BBB features? The meaning of the homogenous-looking Aqp4 staining in the CVOs is interesting but may have nothing to do with the absence of a BBB. Does the staining reflect lack of end-foot restricted localization in astrocytes, or is it the tanycytes that express Aqp4 in the CVOs? Also, what does the CD13-staining mean? Is this mural cells or perivascular fibroblasts ? Essential revisions:The gain-of-function mutation of β-catenin is induced in endothelial cell specifically. It is known that endothelial cells can produce soluble neural mediators. An important question, therefore, is whether the observed neural hyperactivation in vivo, induced by β-catenin gain of function in endothelial cells, is due to reduced vascular permeability or to a change of endothelial cell angiocrine activity. This is a crucial aspect for a correct interpretation of the results. Would the authors be able to see the same c-fos upregulation in neural cells by limiting permeability with different tools? For instance, mice Meca 32- PLVAP knock out? src inhibitors? VEGF inhibitors? other more specific tools? If this these experiments are not speedily addressable, they should be carefully and frontally discussed.We thank the reviewer for getting into this discussion. Indeed, it is difficult, if not impossible to dissect the two effects downstream of β-catenin activation, namely the physical tightening of SFO vessels and the direct effects on endothelial cells, including their autocrine status.Because of the time constraints of the revision period, we could not establish genetic mouse models for Meca-32/Plvap deletion in endothelial cells.Although thoroughly addressing this question in vivo is a project on its own and would be beyond the scope of this manuscript, we have made an effort to repeat the water restriction experiments, using the Wnt pathway activator BIO-X  as well We systemically administered BIO-X by i.p. injections of 1mg/kg BIO-X/DMSO every third day for 21 days. For the final 72 hours, mice were water-deprived and subsequently sacrificed and analyzed for Meca32 as well as for Cldn5. Unfortunately, we cloud not detect significant down regulation of Meca32 and up regulation of Cldn5 in SFO vessels. Instead, the phenotype of physiologically leaky vessels persisted.Similar results were obtained, when mice were daily treated with 25mg/kg AZD0530 Scrinhibitor by oral gavage for 7 days. Scr inhibition did not result in any effects on Meca32 and Cldn5 expression evidenced by IF staining. Analysis of c-fos showed the expected increased staining in the SFO of water-restricted mice (data not shown).Currently, we are further analyzing the samples of the BIO-X- and AZD0530-treated mice for activation of β-catenin, by staining for nuclear β-catenin as well as for Lef-1, which will be informative for future experiments but will not be relevant for the revision of the present manuscript. Additional experiments with other compounds such as VEGF-inhibitors could not be performed due to time limitation.To understand if activation of the Wnt/β-catenin pathway in ECs may alter their angiocrine profile, we have analyzed as a first step primary mouse brain microvascular endothelial cells (pMBMECs) that were stimulated with either Wnt3a or PBS/BSA control. Given the importance of vascular endothelial growth factor A (VEGFA) for neuronal function , we analysed VEGFA expression in Wnt-activated ECs by qRT-PCR. Despite robust induction of Axin2 as a well-established Wnt/β-catenin target, we did not observe a concomitant VEGFA regulation .A point that should be clarified is whether β-catenin does not play any role in the early angiogenesis of the SFO. The authors could not see, using reporter mice, a significant increase in β-catenin activity of the SFO-endothelial cells starting from 13.5E. However, β-catenin may be important in the very first steps of angiogenesis of SFO and then decline, as it occurs in other regions of the brain microcirculation. It could be, therefore, that it is already undetectable at E13.5 while active at earlier times. This is not irrelevant to understand SFO vascularization mechanisms and function. Here too, if experiments to address this are not speedily feasible, please discuss this point adequately.We thank the reviewer for this comment. We completely agree and have also analyzed earlier stages of BAT-gal reporter mice however, E13.5 was the earliest timepoint at which we could identify and visualize the developing SFO. Indeed, at this timepoint angiogenesis of the SFO is ongoing. Still, we could not observe active Wnt/β-catenin signaling. Consequently, due to the fact that E13.5 was the earliest timepoint the SFO is identifiable, we could not go earlier in order to study Wnt/β-catenin activation. Maybe we haven’t made this point as clear as it should be. We have now introduced this explanation in the Results section. We hope that this clarifies the reviewer’s question.A major functional feature of the BBB is to exclude various circulating molecules from entering the CNS. However, a functional assessment of barrier properties in SFO blood vessels after β-catenin stabilization is missing. The authors need to inject intravenously small or large MW tracers and assess their permeability into the SFOs under conditions of Wnt/β-catenin activation in endothelial cells.We thank the reviewer for this important comment. The physiological assessment of barrier tightness is indeed one of the most relevant parameters to address changes in permeability. In order to tackle the reviewer’s comment, we have injected β-catenin GOF and controls with tamoxifen, waited for 26 days and injected the mice intravenously with FITC-BSA (~68 kDa) and 3 kDa TMR-dextran. The detailed procedure is incorporated in the Material and Methods section of the revised manuscript. We now describe that the permeability for FITC-BSA was significantly reduced in the GOF compared to control animals. This result is now shown in Figure 6. Unfortunately, we encountered technical problems with a new Lot of the 3kDa TMR-dextran tracer that, although injected the same way as the FITC-BSA tracer, did not reveal the vessels nor was it detectable in the parenchyma of the SFO and other CVOs. For the reviewers’ reference, we show the positive technical pilot with an older Lot, nicely demonstrating leakage of the small tracer . UnfortuWe hope that the reviewers feel that we have sufficiently shown the physiological tightening of SFO vessels by the reduced leakiness of the ~68 kDa tracer and it clarifies the reviewers’ question.Does overexpression of a stabilized form of β-catenin in the adult mice induce expression of Claudin-5 and suppresses MEca32 at the SFO?We thank the reviewer for this question. It might be that we were not clear enough in the original submission of our manuscript, but the data that we’ve presented in the original Figure 5  were derived from adult double transgenic mice. We have now clarified this in the figure legend as follows: “(A) Mouse model and (B) schedule of endothelial-specific β-catenin GOF induction by tamoxifen (TAM) in adult, 8-12 week old mice”.Additionally, we have added data on the RNA expression of occludin (Ocln), showing in Figure 5 of the revised manuscript similarly to Cldn5 no mRNA upregulation could be observed, although Meca32 was significantly decreased in the SFOs of β-catenin GOF mice. Although the lack of transcriptional regulation of the two junctional genes may suggest that they are no direct targets of βcatenin, we might also face problems of signal masking by tight vessels in the total RNA, isolated from whole mount preparations. We now discuss this in the Discussion section and suggest that future RNAseq analysis of FACS-sorted pools and/or single cells may help to answer this question.Do the blood vessels in SFO acquire Occludin protein expression at cell junctions with β-catenin overexpression? The authors need to show some staining with Occludin antibodies.We thank the reviewer for this comment. We have investigated occludin (Ocln) expression and localization in SFO vessels of β-catenin GOF and control mice. The staining procedure and the antibody is incorporated in the Material and methods section of the revised manuscript. We now show that beside Cldn5, also Ocln localization at cell-cell junctions is significantly increased in GOF mice . Additionally, we have analyzed the expression of Ocln on the mRNA level by qRT-PCR and now show that in line with the expression of Cldn5 also the RNA of Ocln was not significantly increased in the SFO of GOF mice. As mentioned above, this might be due to the masking of the specific endothelial RNA by the total RNA isolated from the whole mount preparations, which inevitably include the ependymal epithelium that also expresses Ocln. Analyzing FACS-sorted cells from the SFO unfortunately, was beyond the scope of the present work.In addition to Ocln, we have now also stained for the adherens and tight junction associated protein zonula occludens/tight junction protein 1 (ZO-/Tjp1), which we now show in Figure 5—figure supplement 1. As opposed to Cldn5 and Ocln, for ZO-1 we observed only a minor increase in junctional continuity of ZO-1 staining. This finding fits with a comparable distribution of VE-cadherin/Cdh5 in GOF and control vessels in the SFO , given that ZO-1 was shown to localize to adherens junctions if tight junctions are weak or absent . This is now discussed in the Discussion section.The TEM data, clearly show that in GFO mice, pericytes and astrocytes are in close contact with endothelial cells. However, astrocytes do not appear to be polarized . Are other astrocyte polarity markers (e.g. Dystroglycan or Kir 4.1) absent in this region?We thank the reviewer for bringing up this interesting issue. By showing the staining of Aqp4 for astrocytic endfeet and by CD13 for pericytes, our intention was to visualize potential changes to the entire neuro-vascular unit (NVU) in SFO vessels of β-catenin GOF compared to control mice. That the leaky vessels of the mouse SFO do not exhibit an organization as an NVU was previously shown by Pócsai et al., (2015). Indeed, we could not observe Aqp4 polarisation at astrocytic endfeet in the control conditions, but equally not along with barrier tightening in GOF mice. As suggested by the reviewer we now investigated the polarisation markers α-dystroglycan (α-Dag) and Kir4.1 and obtained comparable results now shown in Figure 7—figure supplement 1 of the revised manuscript, suggesting that end feet polarization does not follow endothelial tightening. Specifically, dystroglycan shows weak and not strictly polarized staining around SFO vessel, whereas Kir4.1 seems to be mainly expressed by cells morphologically resembling tanycytes in the SFO. The co-localized distribution of dystroglycan and Kir4.1 around BBB vessels nicely confirmed the specific staining of these markers. This was not only true 26 days after TAM injection but also after 60 day, supporting the interpretation that the proper cellular arrangement of the NVU requires additional cues than endothelial Wnt/β-catenin signaling.One potential explanation for the observation that astrocytes do not form proper endfeet in the SFO region could be due to the absence of basement membrane proteins. The authors need to examine the expression of some of the key basement membrane proteins such as Collagen IV, Laminin α2 etc.We thank the reviewer for this thoughtful comment. We have investigated collagen IV, laminin α2 (Lama2) with specific antibody staining in SFO vessels of β-catenin GOF and control mice. The staining procedure and the antibodies are incorporated in the Material and methods section of the revised manuscript. We now show that collagen IV and Lama2 are indeed expressed around SFO vessels, and their distribution and localization follows the outline of the vessels. At least from these observations we concluded that the lack of proper astrocytic endfoot formation around SFO vessels has no obvious correlation to a lack of the most abundant basement membrane components. These findings are shown in the revised manuscript in Figure 7—figure supplement 2.The authors nicely demonstrate that β-catenin GOF mice have higher c-Fos levels with water restriction than wild-type mice. Underwater restrictions, mice may show abnormal behaviors under specific behavioral tasks . It may be useful to test if there are any potential behaviors (e.g. rewarding behaviors) where GOF mice may perform worse than wild-type mice due to higher c-Fos activation.We thank the reviewer for this mindful comment. In the manuscript by Goldstein et al. the authors strictly compare food versus water restricted animals in their behaviour and conclude that there are little to no dramatic changes in overall behaviour due to water restriction. Specifically, water-deprived mice perform better regarding rewarding behaviour than food-deprived mice.GLP1R neurons are activated purely by fluid consumption and not by reward-seeking behaviour or licking action per se. Consistent with the connection from MnPOGLP1R to SFOnNOS neurons, the activity of the SFOnNOS population mirrored precisely the calcium dynamics of MnPOGLP1R neurons”. The findings of Augustine et al. may therefore support our interpretation that, the increase in c-fos in the SFO of GOF water-deprived mice, relates to altered water homeostasis rather than altered overall behaviour. Moreover, we learned from our collaborators, that water deprivation as a widely used paradigm to augment the motivation of mice and other rodents, is not considered to lead to “abnormal” behaviour, given that water and food deprivation is the „normal “situation in wild mice, whereas water and food ad libitum could be rather considered as abnormal.In order to tackle the reviewer’s request, we contacted our collaborators Prof. Jochen Roeper and Dr. Natascha Diamantopoulou , with which we have already planned to investigate the impact of SFO vessel tightening on mouse drinking behaviour. It turned out that it was not feasible to establish and conduct a meaningful experiment within the two month revision timeframe, considering 26d of TAM induction, mouse adaptation and taming, as well as water deprivation and analysis. Although testing the behaviour of Ctrl and GOF mice is beyond the scope of the present study, we believe however, that there is good evidence, that the overall rewarding behaviour does not play a major role in our paradigm. This has been shown and discussed in Augustine et al., 2018, who stated that activation of neurons in the median preoptic nucleus (MnPO), as part of the terminal lamina “indicate that MnPONevertheless, we would like to emphasis that the question raised by the reviewer is very valid and that it was and is on our future agenda as it requires substantial expertise in behavioural sciences and adequate equipment (both will be provided by the group of Prof. Roeper). We hope the reviewers appreciate the limitations we faced regarding this question and leave the answer to future work.The evidence for circadian regulation of Cldn5 expression weak. While the quantifications in Figure 4E may appear convincing, the immunostainings shown in Figure 4A-D are not convincing. The amount of vasculature captured in each section appears hugely variable and the Podxl staining for endothelium seems highly variable in strength. Podxl should be luminal and Cldn5 junctional (or is it?) and this is not at all apparent at the magnification shown. Also, if true, the circadian variation in Cldn5 immunoreactivity raises a number of important issues that should be addressed, including if it is junctional or non-junctional Cldn5 that varies, if the effect is transcriptional or not if other tight junction proteins such as occludin and ZO-1 are also regulated, and more. If it is true that TJs are regulated in a circadian fashion, it is obviously interesting, but the physiological consequences are unclear, given that Plvap expression is maintained, and therefore (presumably) fenestrations not regulated. Would TJ regulation matter for the permeability if fenestrations are kept? The authors speculate that the (possible) circadian regulation of Cldn5 could influence the circadian regulation of thirst and drinking behavior, but this is pure speculation, as far as I can judge. Again, how can Cldn5 matter on top of (presumably) open fenestrations? Given the many questions and the premature nature of this data, we suggest they be removed from the present manuscript, to allow a better focus on Wnt/β-catenin regulation of the CVO vessel phenotype. If Wnt signaling is involved in the circadian regulation of Cldn5 expression in CVO vessels, it would also make more sense to study short term effects of β-catenin GOF experiments; now the earliest time point studied in adults is 16 days following induction.We thank the reviewers for this important comment. As suggested by the reviewers, we have removed the data on circadian regulation from the revised manuscript. Based on the other recommendations of the reviewers, we have now set Figure 5 of the original manuscript as Figure 4 and have included the novel data on occludin (Ocln) localisation and expression in SFO vessels of β-catenin GOF and control mice as Figure 5. We further thank the reviewer for the constructive suggestions on analyzing earlier time point after TAM induction, which we will consider in ongoing experimentsAnother area where conclusions are weak relates to the TEM analysis . The images shown do not appear to support the conclusions that vessels in the GOF mice are slimmer, less tortuous and have less ECM deposition. For the ECM, it does appear denser in GOF but this may reflect decreased water content of the ECM rather than a decreased amount of ECM per se.We thank the reviewer for this comment. As the description of vessels diameter and morphology in the TEM analysis was not based on any quantifications, we have analysed the vessel perimeter but could not identify a significant effect on vessel perimeter . ConsequThe data regards c-fos expression by neurons in the SFO are also an area where the conclusions do seem robust. Neuronal staining to show that this is neuronal and not glial localization is not there. Also, I some of the staining patterns are puzzling. Should not c-fos should look similar at 0h and isotonic NaCl panels in Figure 7, but they look hugely different, casting doubt over the quantifications in Figure 7E and F.+ cells . As opposed to the general nuclear staining by the fluorescent Nissl stain, the flounders are specific for neurons only, that we indicated by the dashed lines in Figure 8G. The images show a high magnification of a Nissl+/c-fos+ double positive neuron in Figure 8G and a Nissl+/c-fos- neuron in Figure 8H. We hope the pronounced differences in c-fos between the conditions are now comprehendible.We thank the reviewer for this important comment. We agree that the c-fos staining of the 0 hour water restricted mice and the NaCl controls should be comparable, given that we identified nearly no c-fos staining in these control conditions, which the images of the original submission did not support well, due to some background staining in the 0 h panel in Figure 7C. Moreover, the neuronal identity of cell that show nuclear c-fos staining was missing in the originally submitted manuscript. Therefore, we have performed novel staining of samples from the water-restricted mice, combining the vascular marker CD31 with the neuron-specific Nissl staining and c-fos. The novel images of the new Figure 8C, D now show a comparable staining pattern between water restriction at 0 hour and the isotonic NaCl injected mice. Additionally, the Nissl staining of the so-called Nissl flounders nicely documents the neuronal identity of the c-fosThe authors find increased expression of Sox17 along with Cldn5 in GOF experiments and note in conjunction with the TEM data that this may signify arterial specification. However, this is unfounded speculation in my opinion. There are many markers for arterial EC in the brain  and Sox17 is not a particularly strong one. The authors should remove this statement or investigate possible arterialization more thoroughly.We thank the reviewer for this comment. The intention to show Sox17 was exclusively to demonstrate the upregulation of a Wnt/β-catenin target, and it was only a note that Sox17 was previously shown as an arterial marker. As suggested by the reviewer, we have deleted the statement of arterialisation, but described Sox17 solely as a Wnt target (Results section).The authors investigate Aqp4 and CD13 staining patterns to check "other phenotypic BBB features", but although neither Aqp4 nor CD13 was found changed in the GOF situation, it is unclear why the experiments were done in the first place. In what way do the (negative) data display an absence of BBB features? The meaning of the homogenous-looking Aqp4 staining in the CVOs is interesting but may have nothing to do with the absence of a BBB. Does the staining reflect lack of end-foot restricted localization in astrocytes, or is it the tanycytes that express Aqp4 in the CVOs? Also, what does the CD13-staining mean? Is this mural cells or perivascular fibroblasts ?We thank the reviewer for making this comment. As mentioned above in context of the astrocytic end foot staining with α-dystroglycan (α-Dag), Kir4.1 and Lama2, we decided to keep and show also Aqp4. However, we clarified in the main text of the revised manuscript  that the intention to investigate Aqp4, and as suggested by the reviewers in addition α-Dag and Kir4.1, was to understand if the known lack of a BBB-like NVU organisation of astrocytes, pericytes and endothelial cells in the core of the SFO, was altered by β-catenin GOF in ECs. The combined findings from all markers stained suggest that dominant endothelial activation of β-catenin does not lead to a structural formation of an NVU in the core of the SFO. We believe that this is valuable information, opening new aspects of investigating cellular and molecular interactions at the NVU. Regarding the expression of Aqp4 in astrocytes or tanycytes, the novel data on Kir4.1 expression which likely resembles tanycytes and does not overlap with Aqp4, may suggest that Aqp4 is expressed in astrocytes. We agree with the reviewer that the data on pericytes were too preliminary and hence, we have removed these data from the revised manuscript. Just mentioning that we haven’t seen obvious effects on this cell type as “data not shown”, and that we haven’t investigated perivascular fibroblasts identified by Vanlandewick et al., 2018 (Discussion section).


Drosophila larvae. β-Catenin is reduced at the lateral cell-cell junctions of wound-edge epidermal cells in the early healing stages. Destruction complex components, including Ck1α, GSK3β and β-TrCP, suppress β-catenin levels in the larval epidermis. Tissue-specific RNAi targeting these genes also caused severe WC defects. The RNAiCk1α-induced WC defect is related to adherens junctions because loss of either β-catenin or E-cadherin significantly rescued this WC defect. In contrast, RNAiTCF does not rescue the RNAiCk1α-induced WC defect, suggesting that Wnt signaling is not related to this defect. Direct overexpression of β-catenin recapitulates most of the features of Ck1α reduction during wounding. Finally, loss of Ck1α also blocked junctional E-cadherin reduction around the wound. Our results suggest that Ck1α and the destruction complex locally regulate cell adhesion to facilitate efficient wound repair.Skin wound repair is essential to restore barrier function and prevent infection after tissue damage. Wound-edge epidermal cells migrate as a sheet to close the wound. However, it is still unclear how cell-cell junctions are regulated during wound closure (WC). To study this, we examined adherens junctions during WC in  Summary: Reduction of the adherens junction component β-catenin is a crucial mechanism for regulating the cell-cell interactions that ensure rapid epidermal wound closure in Drosophila larvae. Drosophila studies in wound repair and regeneration have provided many insights at the molecular and cellular levels  are calcium-dependent adhesion complexes that are important for holding cells together within diverse epithelial tissues . Analysiy lethal . During y lethal . Specifiy lethal . E-cadhey lethal , which iy lethal . E-cadhey lethal . E-cadhey lethal . Whethery lethal , is not Drosophila imaginal discs (Drosophila), to activate downstream gene expression . This signal is specific because expression of an RNAi transgene targeting β-catenin significantly reduced it without reducing an independent membrane label . Epidermal expression of an E-cadherin RNAi transgene also decreased β-catenin levels at the junctions , and vice versa , indicating that junctional β-catenin depends upon E-cadherin expression as previously reported , a procedure that produces small and largely symmetrical wounds. Larval whole mounts were immunostained using anti-β-catenin antibodies  at varioreported . Ten minreported E-F, β-careported G revealereported H. These Ck1α, the key destruction complex kinase that promotes β-catenin degradation. β-Catenin was significantly increased when the larval epidermis expressed RNAiCk1α  and E-cadherin  were also increased in larval epidermis expressing the RNAiCk1α transgene via another epidermal driver  compared with the control .Because β-catenin levels are decreased near the wound C′,D′,H, Ck1αRNAi B,E compaCk1αRNAi A,E. IntrRNAiCk1α also caused a strong WC defect , arguing strongly against RNAi off-target effects. These results suggest that Ck1α negatively regulates β-catenin to facilitate WC. To test whether GSK3β and β-TrCP, other destruction complex components, also regulate β-catenin, we expressed RNAi transgenes targeting them in the larval epidermis. Larvae expressing RNAi transgenes targeting GSK3β or β-TrCP also exhibited a significant increased in β-catenin . Therefore, we used a temperature-sensitive allele of Gal80 . RNAiCk1α also increased β-catenin levels in the cytoplasm . Junctional E-cadherin was similarly upregulated in the epidermis expressing the RNAiCk1α transgene  compared with the control , although the trend of increased junctional β-catenin did not reach significance . Importantly, inducible expression of RNAiCk1α also caused a strong WC defect that was unaffected by expression of a control RNAi transgene . However, knocking down β-catenin significantly ameliorated the WC defect observed upon expression of RNAiCk1α . However, fz3-lacZ was not activated after wounding . Similarly, 6TH-LacZ was also expressed in the wing discs  but not activated in the epidermis after wounding . The JNK reporter, msn-LacZ, a positive control, was activated around the wound margin after wounding  as previously reported . However, 6TH-LacZ was activated in the epidermis expressing RNAiCk1α transgene , suggesting that the Wnt signaling is activated in this condition.As β-catenin is an important downstream transcriptional co-activator of Wnt signaling, we also tested whether Wnt signaling is activated upon wounding. Larvae bearing either eporters , were woreported . To testRNAiCk1α-induced WC defect is related to a role at AJs (as opposed to some other functions of β-catenin), we co-expressed an RNAi transgene targeting E-cadherin, the core component of AJs, to determine whether it could rescue the WC defect observed upon RNAiCk1α expression. We again used Gal80ts to temporally control the Gal4 driver activity  and β-catenin staining . The RNAiE-cadherin transgene also worked in this inducible paradigm . Importantly, knocking down E-cadherin significantly reduced RNAiCk1α-induced WC defects . Epidermal expression of the RNAiTCF transgene by itself did not block WC , consistent with interference with Wnt signaling. Epidermal expression of a RNAiTCF transgene did not ameliorate the WC defect induced by RNAiCk1α expression , was not activated upon β-catenin overexpression. However, another Wnt reporter, 6TH-LacZ, was activated . If β-catenin-induced Wnt activation is responsible for its WC defect, silencing TCF should rescue the observed WC defect. However, epidermal expression of the RNAiTCF transgene did not block the β-catenin-induced WC defect  as with the RNAiCk1α transgene . We also examined the sub-cellular locations of β-catenin in these animals and found that although β-catenin is highly expressed in the nuclear and cytoplasmic compartments , its levels on the membrane were modest.To test whether the WC defect caused by β-catenin overexpression is related to its function at AJs, we wounded larvae expressing both  defects G,H. To tαRNAi and β-catenin overexpression cause WC defects that are dependent on the levels of E-cadherin. However, these genetic manipulations differ in the levels and cellular distributions of both β-catenin and E-cadherin. Because β-catenin is a key protein that bridges E-cadherin at AJs with the actin cytoskeleton, we checked the distribution of F-actin in these two genotypes. In the control epidermis, the F-actin label LifeAct-mCherry is evenly distributed within epidermal cells and membrane levels are low , GL00021 (RNAi#2Ck1α), HMS02276 (RNAi#4Ck1α), JF02306 (RNAiTCF), JF01252 (RNAiβ-catenin), HMS00693 (RNAiE-cadherin), JF01504 (RNAiβ-TrCP) and JF01355 (RNAiLuciferase). UAS-RNAi lines from the Vienna Drosophila Research Center were: GD4256 (RNAi#3Ck1α) and KK108994 (RNAiGSK3β). To enhance RNAiGSK3β and RNAiβ-TrCP knockdown efficiency, larvae were raised at 29°C. Other transgenic lines from Bloomington Stock Center were: #8369, P{UAS-arm.Exel}2 (UAS-β-catenin); #60584, GFPTI{TI}shg; and #8529, P{UAS-lacZ.Exel}2. LacZ reporters used were: msn-LacZ  or lethal (RNAi#3UAS-Ck1α), larvae bearing tstubP-gal80 .Pinch wounding of the mid-third instar larvae was carried out according to our detailed protocol . Pinch wPoke wounding was carried out using a homemade mechanical probe similar to Von Frey filaments, which generate a constant force when bent. These probes were developed by Roger Lopez-Bellido, Patrick Huang and Thomas Wang in the Galko laboratory and are described elsewhere . To makemsn-LacZ, overnight for fz3-LacZ and 20 min for 6TH-LacZ.The third instar larval epidermis was dissected, fixed and immunostained as detailed previously . To highUAS-LifeAct-mCherry and different UAS transgenes via e22c-Gal4 were etherized and poke wounded before mounting. 15 min and 1 h after wounding, larvae were etherized, immobilized and immersed in 1:4 (v/v) diethyl ether to halocarbon oil  and covered with glass coverslips. An Olympus FV1000 Confocal microscope and Fluoview software were used to acquire images  signal was used to define the cell-cell junctions. The measured junctional β-catenin intensities were further subtracted by the mean intensity of cytoplasmic β-catenin of the same larva. Junctional β-catenin levels in poke-wounded third instar larvae at different time points were measured as follows: in ImageJ, junctional β-catenin intensities of four randomly selected lateral interfaces within the first row or the second row of wound-edge epidermal cells were measured and averaged. The ratios of averaged first row to second row wound-edge cells were compared between different time points. Unpaired two-tailed t-test (two groups) or one-way ANOVA  were used to test the significance of experiments: ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.Junctional β-catenin levels of unwounded third instar larvae of different genotypes were measured as follows: in ImageJ, a single line was randomly drawn along the anterior-posterior axis across six epidermal cells (cell tri-junctions were avoided). The peak signals at each junction were measured and averaged for each larva. The intensity of different genotypes was normalized to the


Musa acuminata L.) based on small RNA sequencing. The banana miR169 family clustered into two groups based on miRNA evolutionary analysis. Multiple sequence alignment indicated a high degree of sequence conservation in miRNA169 family members across 28 plant species. Computational target prediction algorithms were used to identify 25 targets of miR169 family members in banana. These targets were enriched in various metabolic pathways that include the following molecules: glycine, serine, threonine, pentose, glycerolipids, nucleotide sugars, starch, and sucrose. Through miRNA transcriptomic analysis, we found that ma-miR169a and ma-miR169b displayed high expression levels, whereas the other 16 ma-miR169 members exhibited low expression in the HG and Baxi banana cultivars. Further experiments indicate that there were negative relationships between ma-miR169a, ma-miR169b and their targets basing on their expression levels to Foc4  infection in resistant cultivars. But they were low expressed in susceptive cultivars. These results suggested that the expression levels of ma-miR169a and ma-miR169b were consistent with the resistance degree of the banana cultivars to Foc4. The analysis presented here constitutes a starting point to understand ma-miR169-mediated Fusarium wilt resistance at the transcriptional level in banana and predicts possible candidate targets for the genetic improvement of banana resistance to Foc4.MicroRNAs (miRNAs) play an important role in plant resistance to pathogen infections. However, little is known about the role of miRNAs in banana Fusarium wilt, which is the most economically devastating disease in banana production. In the present study, we identified and characterized a total of 18 miR169 family members in banana ( Arabidopsis thaliana miR172 was determined to be completely complementary to the open reading frame region of the target gene but was found to inhibit protein translation rather than cleaving mRNA (MicroRNAs (miRNAs) are a class of 18- to 22-nucleotide-long noncoding RNAs that play an important role in gene regulation at the posttranscriptional level in plants . Extensiing mRNA .TaNAC21/22, which is used to improve resistance to stripe rust in wheat species  is the most traded fruit species in the world, and it is widely consumed as a food commodity worldwide (Fusarium oxysporum f. sp. cubense (Foc) and is considered to be the most limiting disease for banana production worldwide. It spreads mainly through the soil and attacks banana plants of all ages, resulting in wilting and yellowing of banana leaves. There are four physiological races of Foc. The history and impact of Fusarium wilt can be summarized with an emphasis on tropical race 4, a “Cavendish”-killing variant of the pathogen that has spread dramatically in banana-planting countries  family members from the banana genome and further investigated their evolutionary relationship, target genes, and expression patterns in various banana cultivars after Foc4 infection. This systematic study will increase our understanding of miRNA169-mediated immunity to http://www.mirbase.org/). The BLAST parameters were the default routine settings of the database. The Latin name of miR169 members of plant species were got from the miRbase though the function of online database “Search by miRNA name or keyword.”In our previous study, we identified miR169 family members in banana using small RNA sequencing . The mathttp://plantgrn.noble.org/psRNATarget/) with the default parameter settings. We selected the V2 Scoring Schema (2017 release), and applied Musa genomics . psRNATarget is a new web server designed to integrate and analyze reverse complementary matching between target transcripts and small RNAs. Another important function is the evaluation of target-site accessibility through calculation of the unpaired energy utilized to unfold the secondary structure around the miRNA target site in the mRNA. The following parameters were used: penalty for opening gap = 2, penalty for extending gap = 0.5, expectation = 10, penalty for GU pair = 1, penalty for other mismatches = 1, HSP size = 19 and seed region = 2–7 nucleotides. KEGG pathway analysis (http://www.genome.jp/kegg/).The putative target genes for ma-miR169 were predicted using the plant miRNA target prediction online software psRNATarget . The sequences were aligned using ClustalW. Based on the neighbor-joining method, the molecular evolutionary history was inferred using 1,000 bootstrap replicates to assess the robustness of the tree branches.Molecular evolutionary analyses were conducted using the MEGA 7.0 program. To evaluate the reliability of the different phylogenetic groups among miR169 family members, a phylogenetic tree was constructed using the maximum likelihood algorithm in the MEGA 7.0 program. Editing was performed in FigTree v1.4.2 , HongYan banana (HY), FenJiao banana (FJ), and BaoDaojiao banana (BDJ), were obtained. BX is a triploid (AAA) cultivar with high yield, high quality, and the capacity for long-term storage. FJ is also a triploid cultivar, but with a different genotype (AAB), and it is characterized by good flavor, rapid ripening, and tolerance to abiotic stresses. BX and FJ re Fusarium wilt-susceptible cultivars that withers and die after infection. Other cultivars exhibit some resistance to Fusarium wilt, and the infection rate can be controlled at 5–8%. Both are widely cultivated in banana-planting areas. The seedlings used for RT-PCR were tissue culture seedlings that were not exposed to the environment, and the seedling height was approximately six to eight cm. qRT-PCR seedlings needed to be transplanted, and the seedling height was approximately 25–30 cm.6 conidia/mL). The pseudostem and roots of the banana were sampled and stored at −70 °C, and part of the postinfection banana was transverse and longitudinally cut for microscopic examination. Macroelements and microelements were supplied throughout the growing stage to maintain growth. The seedlings were grown under a long photoperiod (16:8 h/light:dark).All banana species were cultured and propagated separately. Foc4 was confirmed to efficiently infect banana plants and induce disease symptoms. At the seedling stage, the hydroponic suspension was replaced with a Foc4 spore suspension  tests. The http://www.mirbase.org/cgi-bin/browse.pl). A total of 209 mature miR169 sequences were predicted in 28 plant species . Five target genes  were marked in five metabolic pathways. These pathways included glycine, serine and threonine metabolism; pentose and glucuronate interconversion; glycerolipid metabolism; amino sugar and nucleotide sugar metabolism; and starch and sucrose metabolism  but repressed in FJ and BX (two cultivars sensitive to Foc4) after Foc4 treatment. There was a negative relationship between ma-miR169a, ma-miR169b and their targets basing on their expression levels to Foc4 infection. Thus, the expression levels of ma-miR169a and ma-miR169b are positively correlated with the resistance of banana cultivars to Foc4, implying a positive role of these miR169 family members in immunity to Foc4 in banana.This study identified 18 miR169 family members in banana. Their evolutionary relationships, target genes, and expression patterns in various cultivars after Foc4 infection were systematically analyzed. The ma-miR169a and ma-miR169b can be induced to upregulate by Foc4 and have high expression level in banana resistant varieties. Our study will give a novel sight on miR169’s function which associated with Fusarium wilt.10.7717/peerj.6209/supp-1Supplemental Information 1Supplemental file 1: MiR169 family mature sequences of all crops.Supplemental file 3: The targets of ma-miR169 family.Supplemental file 4: KEGG Pathway of the targets.Supplemental file 6: The FPKM values expressed of HG and BX cultivars.Supplemental file 7: The primer pairs for miRNAs and targets comfirmation and quantitative detection.Supplemental file 8: Expression profiles of mature ma-miR169a, ma-miR169b and their targets in root in response to Fusarium wilt.Click here for additional data file.10.7717/peerj.6209/supp-2Supplemental Information 2Evolutionary analysis clustered the miR169 family.Click here for additional data file.10.7717/peerj.6209/supp-3Supplemental Information 31) Glycine, serine and threonine metabolism, 2) Pentose and glucuronate interconversions, 3) Glycerolipid metabolism, 4) Amino sugar and nucleotide sugar metabolism, 5) Starch and sucrose metabolism.Click here for additional data file.


Cartilage damage is a crucial feature involved in several pathological conditions characterized by joint disorders, such as osteoarthritis and rheumatoid arthritis. Accumulated evidences showed that Wnt/β-catenin pathway plays a role in the pathogenesis of cartilage damage. In addition, it is experimentally documented that lncRNA (long non-coding RNA) HOTAIR plays a key role in the regulation of Wnt/β-catenin pathway based on directly decreased WIF-1 expression. Further, it is reported that Wnt/β-catenin pathway is a potent pathway to regulate the expression of MMP-13, which is responsible for degradation of collagen type II in articular cartilage. It is increasingly recognized that systems modeling approach provides an opportunity to understand the complex relationships and direct quantitative analysis of dynamic network in various diseases.A dynamic network of lncRNA HOTAIR-mediated Wnt/β-catenin pathway regulating MMP-13 is developed to investigate the dynamic mechanism of the network involved in the pathogenesis of cartilage damage. Based on the network modeling, the potential therapeutic intervention point Axin is predicted and confirmed by the experimental validation.Our study provides a promising strategy for revealing potential dynamic mechanism and assessing potential targets which contribute to the prevention of the pathological conditions related to cartilage damage. Cartilage damage is a central feature in joint diseases that is common in the patients with osteoarthritis (OA) and rheumatoid arthritis (RA) . EvidencInvestigation of potential molecular mechanisms of cartilage development and degeneration will offer important references for the treatment of related joint diseases. Increasing evidences indicate several crucial molecular pathways in cartilage damage, one of which is Wnt/β-catenin signaling pathway . The WntIn recent years, increasing evidence suggests that long non-coding RNAs (lncRNAs) play important roles in the regulation of a variety of biological processes , 15. It Fortunately, it has become increasingly recognized that systems modeling method offers a chance to understand the potential dynamic mechanism and direct quantitative analysis of signaling pathway in various biological processes , 23. It The Wnt/β-catenin signaling pathway has crucial roles in essential cellular processes such as cell growth, proliferation and apoptosis. Dysfunction of Wnt/β-catenin signaling pathway is involved in a host of diseases including cartilage damage in RA , 29. In Model describing such systems is carried out presently by numerical integration of Eqs. –25), wh, wh25), At the same time, the blue curves of Fig. In the present study, dynamic sensitivity analysis is carried out on a model of the HOTAIR-mediated Wnt/β-catenin pathway to determine how “sensitive” a model is to changes in the parameters causing changes in the dynamic behavior of the whole system. A sensitivity analysis for all parameters Table  of our m24) has the largest effect on the whole system which can affect 18 out of the total 37 reactions. While k16 (dissociation of phosphorylated β-catenin from Axin*•GSK3•APC*•β-catenin*) significantly affects 15 reactions, k19 (the degradation rate of Axin) affects 14 reactions, and k11 (binding rate of GSK3 to the APC•Axin complex), k− 11 (dissociation rate of GSK3 from the GSK3•APC•Axin complex), k13 (binding rate of APC to Axin), k− 13 (dissociation of APC from the APC•Axin complex) affect 13 reactions respectively. In summary, the influences of these parameters on the whole system obey the following order: k24 > k16 > k19 > k11 > k− 11 > k13 > k− 13. Interestingly, from these results we find that most of these parameters are all related to Axin. In particular, the most sensitive parameters are k24 and k19, all of which are directly relevant to the synthesis and degradation of Axin respectively. These observations indicate that the parameters related to Axin exhibit a marked dynamic effect in the whole model. It means that the dynamic behavior of HOTAIR-mediated Wnt/β-catenin pathway is highly sensitive to Axin, which may play a significant role in understanding the potential mechanism of cartilage damage. This implies that Axin may be a potential therapeutic intervention pointed to block cartilage damage.The number of reactions affected by the key parameters from sensitivity analysis are shown visually with a histogram in Fig. 24). Therefore, to examine how k24 exhibits the largest effects on our system, some explorations on variation of the kinetic parameter space were implemented presently. The kinetic parameter (k24) is 100 times increased or decreased compared to the ‘basal’ value of k24 = 0.02 nM•min− 1, when all other rate constants were kept fixed. The results are shown in Fig. 24 = 0.0002, 0.02 and 2 nM•min− 1, respectively.The sensitivity analysis shows that the system is the most sensitive to the variation of the synthesis rate of Axin  and 50 nM•min− 1, when keeping all other rate constants fixed. When k16 changes from 0.5 to 0.005 nM•min− 1 (blue curve) or 50 nM•min− 1 (black curve), the oscillatory behavior of MMP-13 appears in an extremely constant manner. In Fig. 19 was changed as 0.00167, 0.167 and 16.7 nM•min− 1, with the values of other parameters were kept fixed. As k19 increases to 16.7 nM•min− 1 (black curve) or decreases to 0.00167 nM•min− 1 (blue curve), MMP-13 evolves to a steady state and no oscillations occur compared to the ‘basal’ value of k19 = 0.167 nM•min− 1. As seen in Fig. 24 (the synthesis rate of Axin) was set to 0.0002, 0.02  and 2 nM•min− 1.When k24 increases from 0.02 to 2 nM•min− 1 (black curve) or decreases from 0.02 to 0.0002 nM•min− 1(blue curve), the oscillations of MMP-13 disappear and subsequently reach a steady state. Since k24 is the synthesis rate of Axin that is a suppressor of Wnt/β-catenin pathway, the concentration of MMP-13 decreases when k24 rises to 2 nM•min− 1.As illustrated in Fig. 16, k19 and k24 have significant effect on the oscillations of the deterministic system, which are well consistent with the findings obtained from the sensitivity analysis. More interestingly, compared with the variation in the amplitude of k16, the variation in the amplitude of MMP-13 changes significantly when k19 and k24 are whether enlarged or lessened by 100 times on the basis of the ‘basal’ values. This indicates that the parameters k19 and k24 with respect to the synthesis and degradation of Axin exert more marked effect on the dynamics of the model than k16. In other words, the mechanism of collagen type II degradation mediated by MMP-13 in articular cartilage is highly sensitive to Axin, which may a potential target for developing a therapeutic strategy toward blockade of cartilage damage and should be investigated further in an experimental setting. All the above results not only show the significant roles of parameter perturbation for the whole system, but also prove the reliability of dynamic sensitivity analysis, which might provide a promising strategy for the prediction of potential therapeutic intervention point for the prevention of cartilage damage.These results demonstrate that the parameterskPrevious studies indicate that Wnt signaling induces Axin2 expression in different cell types. To determine if Wnt 3a induce Axin2 expression, we researched Axin2 mRNA expression in Wnt 3a-treated chondrocytes. The expression of Axin2 gene was rapidly induced by Wnt3a, while IL-1β had no effect on Axin2 expression Fig. a. Then, In this paper, a system-theoretic approach by employing a chemical kinetic reactions approach based on ODEs is introduced to construct the model of HOTAIR-mediated Wnt/β-catenin pathway which is used not only to quantitatively describe the dynamic mechanism of key components in the model, but also to predict the possible interventions for preventing cartilage damage.Firstly, using the kinetic parameters with appropriate biological values, the modeling work was carried out by numerical integration of 25 ODEs. Our model reveals the existence of biological oscillatory behavior through modeling the time series of the dynamic behavior of key signaling molecules in the system. The reasonability of this model has demonstrated by clearly oscillations which were due to the fact that Axin is a likely component of the negative feedback loop, which is activated by β-catenin and then is degraded by a complex of Axin, APC and GSK3. In addition, we also point out the role of HOTAIR on the regulation of Wnt/β-catenin pathway. Although the effect of HOTAIR regulation on the oscillatory pattern formation in the system is weak, HOTAIR plays a significant role in maintaining the equilibrium state and activates Wnt/β-catenin pathway by inhibiting WIF-1 expression. Secondly, the dynamic sensitivity analysis was performed to investigate the oscillatory dynamic behavior of the model with changes in parameters to explore which parameters were the most crucial ones impacting the whole system. The obtained result suggested that the parameters with respect to the synthesis and degradation of Axin had the largest impact on our model which implied the system is highly sensitive to Axin. Thirdly, some explorations on variation of the parameter space were carried out for detailed analysis to examine how the effect of Axin causes changes in the dynamic behavior of our model. The result was well consistent with the finding obtained from the dynamic sensitivity analysis. Finally, the experiment is applied to validate the reasonability of our strategy, thus to provide a credible method to assess therapeutic intervention points for the prevention of cartilage damage. The experimental observations were in agreement with our prediction, confirming that our method is reliable for developing a tractable therapeutic target toward blockade of cartilage damage.In conclusion, it is urgent to improve our understanding of the molecular mechanisms involved in cartilage breakdown and to develop new therapeutic intervention points. Not surprisingly, systems modeling approaches have become increasingly recognized as a tool to explore these problems as the complement of experimental work. The successful development of our strategy will not only be helpful for deep understanding of the underlying dynamic molecular mechanism of HOTAIR-mediated Wnt/β-catenin signaling pathway, but also provide a promising idea to predict the potential intervention for further experimental verification, so as to contribute to the prevention of the various pathological conditions related to cartilage damage.In this work, all the programs were implemented on a Dell workstation and the CPU running time is obtained on Intel Xeon E5–1650 v3 6-Core 3.5GHz 15MB CPU and 8 GB RAM. An ODE solver in MATLAB was used to carry out our deterministic simulation. Accuracy is controlled by setting an absolute tolerance of 10–8 applied to all the variables. The challenge for future work will be a focus on the improvement of accuracy and comprehensiveness in the model, since lack of reliable biological experimental data and parameters available for model construction limits the restoration of the real biological system.In this work, a mathematical model for HOTAIR-mediated Wnt/β-catenin dynamic network regulating MMP-13 was developed by employing a chemical kinetic reactions approach based on ODEs. The HOTAIR-mediated Wnt/β-catenin pathway is defined in terms of a number of signaling molecules and reaction steps, which forms the basis for the mathematical model of the pathway. It is well know that β-catenin is the central and essential factor of Wnt/β-catenin pathway, which is regulated by the cytoplasmic destruction complex formed by Axin, GSK-3 and APC . Hence ti) to active form (Dsha). At this point, GSK3 is dissociated from a so called destruction complex (step 10), which is the center of this model that compose of the phosphorylated forms of the scaffolds APC, Axin and GSK3  [The model is initiated based on the fact that lncRNA HOTAIR activated the Wnt signal by directly repressing the expression of WIF-1  . In the  14, 15) . The fun 14, 15) . The pho 14, 15) .The reaction steps of HOTAIR-mediated Wnt/β-catenin pathway are modelled using standard reaction kinetics, leading to a set of ODEs based on the law of mass action and Hill-type function, which are the mathematical methods that explain and predict the behaviours of the molecules in dynamic equilibrium. The time evolution of the model is governed by a system of 25 kinetic equations. The ODEs of the dynamic model are presented as follows:The kinetic parameter values used in 25 ODEs were chosen within a reasonable physiological range where available. Semi-arbitrary choice of parameter values were explored in order to best reproduce the essential responses associated with experimental measurements. All parameter values were obtained and estimated according to their relevant references. The parameter values and their biological explanations are given in Table SA) is a useful tool to understand the behavior of dynamic systems, which characterizes the influence of sensitive the parameter changes on the outputs of the system.Dynamic mathematical models usually involve a large number of physicochemical parameters to describe biological models, such as gene regulation, signaling and metabolic networks. It is necessary to investigate how the model changes with perturbations in the parameter values, since any small changes in the value of the parameters may drastically affect the output of system . Sensitii can be calculated as:A dynamic sensitivity value characterizes the response of state variable (output) to a parameter (input) variation on model at any time. Thus, the sensitivity of the model to the output Y with respect to a single parameter XSA) is defined as the relative change in state variable Y (output) divided by the relative change in the parameter Xi. It examines the effect of each parameter independently corresponding to the variable at one specific moment. The results are normalized in order to obtain the dimensionless scaled sensitivity of an input factor for plotting the visual presentation drawing.The local sensitivity index  were purchased from ScienCell Research Laboratories  and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) . For the experiments, the Ha-c cells were plated into a 6-well culture plate at 2 × 10The MMP-13 level in cell supernatant was detected using MMP-13 ELISA kit  according to the manufacturer’s protocol.The total RNA of Ha-c cells was isolated using TRIzol Reagent  following the manufacturer’s instructions. After isolating the total RNA from Ha-c cells, 1 μg was reverse transcribed using QuantiTect Reverse Transcription Kit . The specific transcripts were optimized by quantitative real-time PCR with QuantiTect SYBR Green PCR Kit (QIAGEN K.K.) and ABI 7500 real-time PCR system  were used to analyze results. Gene-specific primers used were as follows: Axin2 (GCTTGGAGACAATGCTGTTG as reverse and GAGGGAGAATGCGTGG ATA as forward), GAPDH (AGGGGCCATCCACGTCTTC as reverse and AGAAGGCTGGGGCTCATTTG as forward). Real-time PCR performed as 40 cycles for 30 s at 95 °C, 5 s at 95 °C, 30 s at 60 °C, 15 s at 95 °C, 1 min at 60 °C and 15 s at 95 °C. The date were calculated using the ∆∆Ct algorithm and were normalized to GAPDH expression.


At the same time, the modified epoxy resin curing product also has excellent heat and corrosion resistance. This work provides a new method for the study of epoxy resins with high performance.In this paper, a novel modified epoxy resin with an interpenetrating network structure for use as a grouting material with high toughness was prepared by a method of graft copolymerization between polyurethane prepolymer (PUP) trimer and epoxy resin (E-44). Polyurethane prepolymer was synthesized using poly(propylene glycol) (PPG) and 2,4-toluene diisocyanate trimer (TDIT) at 70 °C for 3 h. The graft copolymer was prepared by grafting polyurethane prepolymer onto the side chain of epoxy resin at 110 °C. The mechanical properties, fracture surface morphology, chemical structure, thermal properties, and corrosion resistance of the modified epoxy resin curing products were studied. Due to the beneficial flexible segments and the interpenetrating network structure, the results show that when the ratio of epoxy resin to polyurethane prepolymer is 10:2, the optimum mechanical properties are obtained; these include a compressive resistance of 184.8 MPa, impact property of 76.6 kJ/m Epoxy resin (EP) is a kind of polymer which contains two or more epoxy groups in its molecular chain and forms thermosetting products through various reactions between epoxy groups. It has the advantages of high mechanical strength , high bo2,4-toluene diisocyanate trimer (TDIT) is a kind of polymer formed by self-polymerization of 2,4-toluene diisocyanate ,25,26. TEther of bisphenol A type EP (E-44) and low-molecular-weight polyamide were supplied by Baling Petrochemical Co., Ltd. . TDIT and Allyl glycidyl ethers (AGE) were purchased from Jining Huakai Resin Co., Ltd. . Polypropylene glycol (PPG 1000) was purchased from Qingdao Chemical Science and Technology Co., Ltd. . 2,4,6-tris (dimethylaminomethyl) phenol (DMP-30) and 1,4-butanediol were provided by Chinese Medicine Group Chemical Reagent Co., Ltd. .TDIT was placed into a three-necked flask at 40 °C. Then, PPG 1000 was added into the flask, and the mixture was reacted for 3 h at 70 °C. The chain extender was then added and stirring was continued.E-44 and polyurethane prepolymer were mixed in a three-necked flask in a certain ratio given in , and uniAt room temperature, the curing agent (low-molecular-weight polyamide) was mixed with the modified epoxy resin in a stoichiometric molar ratio of 7:10. Subsequently, DMP-30  was added and stirred vigorously until a homogeneous system was obtained; the mixture was defoamed for 10 min in a vacuum box and then cured naturally.−1.The modified epoxy was analyzed by Fourier transform infrared spectroscopy (FT-IR)  using air as the background and scanning from 4000 to 500 cm−1 under an Ar atmosphere. The sample was heated from 25 to 600 °C.TG analysis was carried out using an STA 449C  equipped with a thermal analysis data station at a heating rate of 10 K·minSEM was conducted using a JEOL JSM 5900 LV scanning electronic microscope  with a tension of acceleration of 20 kV to characterize the fracture surfaces of specimens.The tensile properties and compressive strength of the cured materials were recorded using a microcomputer control 858 Mini Bionix universal testing machine (MTS Systems (China) Co., Ltd., Shenzhen, China). The flexural and compressive properties were tested according to Chinese Standard GB/T 2567-2008. The samples’ shapes and sizes were as shown in The cured epoxy resin was soaked in either water, sodium chloride solution (10 wt %), sodium hydroxide solution (10 wt %), or sulfuric acid solution (10 wt %) for 100 h. Then, the compressive strength and tensile strength were tested.Shown in 2, which is better than the 9.25 kJ/m2 in Park’s study and the 17.63 kJ/m2 in Hai-an Xie’s study [The effect of the amount of prepolymer on the compressive and impact properties of the specimens is shown in ’s study ,35. Comp’s study . The res’s study . When thThe SEM images of the impact fracture surfaces of the cured products are shown in −1, the isocyanate group at 2273.65 cm−1, and the stretching vibration peak of carbonyl at 1730.17 cm−1. The characteristic absorption peaks of E-44 (C) are at 3472.79 cm−1 and 914.51 cm−1, respectively corresponding to the hydroxyl group and the epoxy group. Comparing line B with line A, the infrared peak of EP-PUP disappears at 2273.65 cm−1, while the peak at 1730.17 cm−1 does not change. Compared with E-44, the modified epoxy resin has an obvious change: the absorption peak at 3472.79 cm−1 becomes weaker and wider. However, the characteristic absorption peak of epoxy resin at 914.51 cm−1 was retained. Therefore, it is clear that the change group is only the isocyanate group of the prepolymer and the hydroxyl group of E-44. This indicates that the synthesis of the modified epoxy resin was accomplished by reaction between the isocyanate group and the hydroxyl group. So, the mechanism of the modified epoxy resin is thus deduced from The structure of the modified epoxy was confirmed by FT-IR, as shown in Benefiting from the presence of PPG, as a flexible group embedded into the structure of the modified EP-PUP, and the structure of the interpenetrating network formed through graft copolymerization, the mechanical properties of toughness, impact and compression resistance, and elongation at break all showed excellent improvement. This is in agreement with the results presented in The corrosion resistance of the modified epoxy resin was studied by immersing the samples in either water, sodium chloride solution (10 wt %), sodium hydroxide solution (10 wt %), or sulfuric acid solution (10 wt %) for 100 h. As shown in 2 and the fracture elongation reached 32.3%. The compressive strength and tensile strength were increased to 184.8 and 40.6 Mpa, respectively. Further, the modified epoxy resin also has excellent heat resistance and corrosion resistance. The present work provides a simple and convenient pathway to prepare a new modified epoxy resin with high performance which may have potential applications in a wide range of fields.In summary, for the first time, a novel modified epoxy resin with high mechanical properties was prepared by a graft copolymerization method using TDIT. PPG, with its property of flexibility, was successfully embedded into the chains of PUP, and EP-PUP with an interpenetrating network structure was obtained. Due to the presence of the flexible PPG group and the complete structure of the interpenetrating network, the modified epoxy resins showed excellent mechanical properties including compressive strength, impact strength, tensile strength, and fracture elongation. With a mass ratio of PUP to EP close to 20%, the impact strength of the EP-PUP was significantly increased to 76.6 KJ/m


It has a critical role in cell-to-cell adhesion by linking cadherins to the actin cytoskeleton and has a central role in transcriptional regulation in the Wnt signaling pathway. We evaluated the diagnostic value of the Beta catenin immunohistochemistry marker in distinction of aggressive and non-aggressive Basal cell carcinoma.Basal cell carcinoma (BCC) is a common skin cancer arising from the basal layer of the epidermis and its appendages. They are locally invasive, aggressive, and destructive of skin and the surrounding struc- tures. β-Catenin in aggressiveness in BCC with higher rate of relapse.This cross sectional and descriptive-analytical study was done on archived formalin fixed, paraffin embed- ded tissue blocks in pathology library of Al-Zahra hospital in Isfahan city. We used immunochemistry to determinate the role of β-Catenin staining in aggressive group was more significant than the other group  and the mean percentage of membranous β-Catenin staining in non- aggressive group were significant more than the aggressive group. Intensity of membranous staining in both groups significant less than normal epithelium.A total of 76 samples were evaluated in two groups (aggressive &none aggressive). The mean percentage of cytoplasmic β-Catenin staining in aggressive BCC is more significant than non-aggressive subtypes, so this indicates that the use of β-Catenin IHC marker maybe helpful in the diagnosis of aggressive BCC.Cytoplasmic  Basal cell carcinoma (BCC) is a common skin cancer arising from the basal layer of the epidermis and its appendages. They are locally invasive, aggressive, and destructive of the skin and the surrounding structures including bone. Individuals with a history of BCC are at increased risk for subsequent lesions. Approximately 40 percent of patients who have had one BCC will develop another lesion within five years , 2.BCC is the most common cancer  in humans, especially in white- skinned people, although fortunately, the mortality is low. The incidence of BCC varies in different countries and it depends to sunlight exposure , 4. For β-Catenin is a multifunctional protein in the intracellular side of the cytoplasmic membrane coded by the CTNNB1 gene, which is located on chromosome 3p22.1. It has a critical role in cell-to-cell adhesion by linking cadherin to the actin cytoskeleton and has a central role in transcriptional regulation in the Wnt signaling path- way , casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3). CK1 and GSK3 sequentially phos- phorylate the amino-terminal region of β-catenin, resulting in β-catenin recognition by β-Trcp, an E3 ubiquitin ligase subunit, and subsequent β-catenin ubiquitination and proteasomal degradation . The aggressive group included: superficial–micronodular-morpheaform–metatypical and infiltrative. The non-aggressive group included: nodular-nodulocystic- fibroepithelial pinkus tumor. Exclusion criteria in this study were: 1. insufficient samples in blocks, 2. history of radiotherapy for treatment of the lesion, 3. incomplete admission data, and 4. specimens from relapse locations. Formalin-fixed and paraffin-embedded BCC blocks archived in pathology lab- oratory of al Zahra hospital, Isfahan, Iran, from 2014-2015 were used. Four micron sections were prepared from tissue blocks and stained by immunohistochemistry (IHC) for beta-catenin. The used antibody was monoclonal mouse antibody, subclass IgG1, kappa, clone CCH2 .Immunohistochemistry was performed using the streptavidin–biotin complex indirect immunoperoxidase method. Sections were dewaxed, rehydrated, and incubated with 0.3% hydrogen peroxide (H2O2) in water for 30 min to block endogenous peroxidase activity. Antigen retrieval was performed by microwaving for 20 min in 0.01 mol L citrate buffer (pH 6.0) at 750 W. After rinsing in phosphate-buffered saline (PBS), blocking theendogenous nonspecific bindings was performed by adding a blocking serum (for 30 min) and drying subsequently. The sections were then incubated with the primary antibody overnight at 40C , washed with PBS, and then incubated with peroxidase-streptavidin conjugate for 30 min. The sections were developed with diaminobenzidine solution (DAB) for 10 min and 0.1% H2O2 and counterstained with hematoxylin then Mounting .We used normal epithelium in specimens for the positive control for membranous staining. Tissue , samplesStatistical AnalysisAfter IHC staining and data collection, pattern (nuclear - cytoplasmic - membranous), percentage, cut off points, sensitivity, specificity, PPV and NPV of beta-catenin staining were determined and data analysis was performed in SPSS20 software and in independent T-test. Mann-Whitney, Wilcoxon rank sign Test, chi-square were used for statistical analysis.P=0.84). 47.4% of samples were female and 52.6% were male. Statistical analysis showed no statistical signifi- cant difference in the frequency of gender between groups (P=0.65). Also, there is no statistically significant dif- ference between the frequency of site of the tumor in groups (P=0.28).38 samples were studied in two groups with mean age 59.11 ± 9.65 in the aggressive group and 59.53 ± 8.37 in the non-aggressive group. Statistical analysis showed no statistical difference between mean age of two groups (P˂0.001) (P˂0.001). We used ROC curve for determining the diagnostic value in percentage for membranous and cytoplasmic beta- catenin staining. The cytoplasmic staining  . HoweverP˂0.001)    . Moreover, our results showed that there is no statistically significant difference in the distribution of frequency of Intensity of staining in internal control between groups (P=0.41).Also, the analysis, in both groups, showed significant intensity of membranous staining within groups was less than normal epithelium (P=0.25).In 2 years follow up of patients, one case in each group had a relapse (2.6%), where there was no statistically significant difference between the two groups . Also, we demonstrated that cytoplasmic beta-catenin staining in high risk subtypes of BCC was correlated with the aggressiveness of the tumor.Several studies demonstrated similar results to the current study. Auepemkiate et al. from Thailand , reporteIn South Korea, OH-St et al  reportedWe studied the diagnostic value of beta-catenin IHC marker to the distinction of aggressive from non-aggres- sive BCC. Our study showed that cytoplasmic beta-catenin staining in aggressive subtypes of BCC is more significant than in non-aggressive subtypes.