Vascular calcification (VC) is a pathological process underpinning major cardiovascular conditions and has attracted public attention due to its high morbidity and mortality. Chronic kidney disease (CKD) is a common disease related to VC. Ginsenoside Rb1 (Rb1) has been reported to protect the cardiovascular system against vascular diseases, yet its role in VC and the underlying mechanisms remain unclear. In this study, we established a CKD‐associated VC rat model and a β‐glycerophosphate (β‐GP)‐induced vascular smooth muscle cell (VSMC) calcification model to investigate the effects of Rb1 on VC. Our results demonstrated that Rb1 ameliorated calcium deposition and VSMC osteogenic transdifferentiation both in vivo and in vitro. Rb1 treatment inhibited the Wnt/β‐catenin pathway by activating peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), and confocal microscopy was used to show that Rb1 inhibited β‐catenin nuclear translocation in VSMCs. Furthermore, SKL2001, an agonist of the Wnt/β‐catenin pathway, compromised the vascular protective effect of Rb1. GW9662, a PPAR‐γ antagonist, reversed Rb1's inhibitory effect on β‐catenin. These results indicate that Rb1 exerted anticalcific properties through PPAR‐γ/Wnt/β‐catenin axis, which provides new insights into the potential theraputics of VC. PPAR‐γ is expressed in various cell types and regulates certain cellular metabolism and differentiation processes.Ginsenosides are major components of the traditional herbal medicine ginseng, and ginsenosides can be classified into panaxadiols (PPD) and panaxatriols (PPT) by chemical structure. Rb1, the most abundant constituent of panaxadiol, has recently been reported to exert protective properties in vascular diseases and kidney diseases.In view of the demonstration that Rb1 acts as a PPAR‐γ agonist in human umbilical vein endothelial cells (HUVECs) by our previous study,22.1Foetal bovine serum (FBS) and Dulbecco's Modified Eagle's medium (DMEM) were obtained from Gibco‐BRL . β‐Glycerophosphate  and adenine (V900471) were purchased from Sigma‐Aldrich (Merck KGaA). Ginsenoside Rb1 (41753‐43‐9) was obtained from Fleton Natural Products Co., Ltd. The Wnt/β‐catenin agonist SKL2001 (S8320) and PPAR‐γ inhibitor GW9662 (S2915) were from Selleck Chemicals. The Nuclear and Cytoplasmic Protein Extraction Kit (P0027) was from Beyotime Biotechnology.Antibodies against calponin 1 (#17819), runt‐related transcription factor 2 , β‐catenin (#8480), phospho‐β‐catenin (Ser675) (#9567), glycogen synthase kinase‐3β , phospho‐GSK‐3β (Ser9) (#9322) and histone‐H3 (#4499) were from Cell Signaling Technology, Inc. An antibody against α‐smooth muscle actin  was purchased from Novus Biologicals. An antibody against peroxisome proliferator‐activated receptor gamma  was purchased from Santa Cruz Biotechnology. An antibody against glyceraldehyde‐3‐phosphate dehydrogenase  was purchased from ZSGB‐BIO.2.22 sections and placed in a cell culture flask with DMEM containing 4.5 g/L glucose supplemented with 20% FBS, 10 mmol/L sodium pyruvate, 100 U/mL penicillin and 100 μg/mL streptomycin (growing medium). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. Immunocytochemical examination showed positive staining in all cells for α‐smooth muscle actin. Migrated VSMC passages 3‐8 were used for in vitro experiments.Primary rat VSMCs were extracted from 8‐week‐old non‐CKD male Wistar rats. Briefly, the rats were killed by sodium pentobarbital, and the thoracic aorta was dissected out quickly. After removing the adventitia and intima, the artery segment was cut into 1‐2 mmVascular smooth muscle cells were seeded onto a 6‐ or 12‐well dish and incubated with DMEM + 10% FBS , and 10 mmol/L β‐GP (Sigma‐Aldrich) was introduced to induce VSMC calcification for 3‐12 days. The culture medium was replaced every 3 days. For drug‐treated groups, VSMCs were preincubated with different concentrations of Rb1 for 1 hour, followed by β‐GP calcification induction together with Rb1. Furthermore, SKL2001, an agonist of the Wnt/β‐catenin pathway as reported,2.3All animal experimental protocols complied with the Animal Management Rules of the Chinese Ministry of Health  and conformed to the NIH guidelines .Male Wistar rats (160‐180 g) were randomly divided into three groups: control, CKD and CKD with ginsenoside Rb1 (CKD + Rb1), n = 5 per group. For CKD and CKD + Rb1 rats, adenine was administered intragastrically at a dose of 250 mg/kg/d for 2 weeks, followed by 250 mg/kg every other day for 4 weeks to establish CKD models based on the previous report.After 6 weeks, the animals were killed, and rat serum was collected to determine the blood urea nitrogen (BUN), creatinine (Cr), calcium, phosphorus and alkaline phosphatase (ALP) levels by an autoanalyzer . The abdominal arteries were excised for further analysis.2.4Vascular smooth muscle cell calcification was induced using 10 mmol/L β‐GP for 12 days as described above.For artery calcification staining, 3 μm paraffin‐embedded artery sections were stained with alizarin red S solution for 10 minutes after the standard dewaxing procedure. Von Kossa staining of artery sections was also performed to confirm calcification. Dewaxed sections were exposed to a 5% silver nitrate solution and were placed under an ultraviolet light for 2 hours.2.5The Alkaline Phosphatase Assay Kit (A059‐2) and Calcium Assay Kit (C004‐2) were from Nanjing Jiancheng Bioengineering Institute. The ALP activity and calcium content were determined according to the manufacturer's instructions. The results were then normalized by the protein content determined by the BCA Protein Assay Kit (PC0020) from Solarbio.2.63VO4, 0.5 μg/mL leupeptin and 0.1 mmol/L PMSF (R0010) from Solarbio. The supernatant was collected after centrifugation at 12 000 g for 10 minutes at 4°C.Vascular smooth muscle cells or rat arteries were lysed for 30 minutes on ice in lysis buffer containing 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% SDS, 2 mmol/L sodium pyrophosphate, 25 mmol/L β‐glycerophosphate, 1 mmol/L EDTA, 1 mmol/L NaProteins were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes from Millipore. After blocking in 5% nonfat milk for 1 hour at room temperature, the PVDF membranes were probed with primary antibodies against α‐SMA (1:1000 dilution), calponin 1 (1:1000 dilution), RUNX2 (1:500 dilution), β‐catenin (1:500 dilution), phospho‐β‐catenin (Ser675) (1:1000 dilution), GSK‐3β (1:1000 dilution), phospho‐GSK‐3β (Ser9) (1:1000 dilution), PPAR‐γ (1:1000 dilution), histone‐H3 (1:1000 dilution) and GAPDH (1:1000 dilution) overnight. The membranes were then washed with TBS‐T, followed by an incubation with a horseradish peroxidase‐conjugated secondary antibody (1:8000 dilution) (ZSGB‐BIO) for 1.5 hours at room temperature; then, the membranes were developed with chemiluminescence and were stripped and reprobed when necessary.2.7Following the standard procedure, paraffin‐embedded rat artery sections were rehydrated by dimethylbenzene and gradient ethanol. Then, 0.05 mol/L sodium citrate buffer (pH 6.0) was introduced for heat‐mediated antigen retrieval. Slides were submerged in 3% hydrogen peroxide for 10 minutes to remove endogenous peroxidase. After a wash step, the slides were blocked with 10% goat serum  for 30 minutes at 37°C, followed by an overnight incubation with primary antibodies against α‐SMA (1:500 dilution), calponin 1 (1:200 dilution) and RUNX2 (1:100 dilution) at 4°C in a humid box. After 30 minutes of incubation with the appropriate secondary antibody at 37°C, the slides were reacted with DAB solution (ZSGB‐BIO). Haematoxylin was applied to counterstain the nucleus. The tissue sections were visualized under a Nikon Eclipse 80i microscope equipped with a digital camera  and were analysed with Image‐Pro Plus 6.0 software.2.82O2 treatment, the artery sections were permeabilized with 0.3% Triton X‐100  for 15 minutes. After washing with PBS, the slides were then blocked and probed with the appropriate antibodies as described in the IHC procedure. Antibodies against β‐catenin (1:100 dilution) and PPAR‐γ (1:100 dilution) were used in this study. After washing, the slides were incubated with a secondary antibody  for 1 hour at 37°C. The slides were covered by a drop of Fluoroshield Mounting Medium containing 40,6‐diamidino‐2‐phenylindole  before being observed with laser scanning confocal microscopy .After rehydration, heat antigen retrieval, and 3% HFor the VSMC IF procedure, cells were seeded onto coverslips in a 24‐well plate and treated as described above. After fixation with immunostaining fixation solution  for 1 hour at room temperature, VSMCs were blocked, probed with antibodies, stained with DAPI and observed as artery sections.2.9P < .05 was considered significantly different.All experiments were independently repeated at least three times. Data are expressed as the mean ± SEM. GraphPad Prism 6.0 was used to analyse the data and draw figures. Multiple group data were analysed by one‐way ANOVA, followed by Tukey's post hoc test. 33.1P < .01) than the control rats. Moreover, serum calcium levels in CKD rats did not change, whereas the serum phosphorus (P < .01) and ALP (P < .05) levels were increased. Rb1 administration did not ameliorate CKD rats' renal function but lowered the serum phosphorus levels (P < .05).Rat CKD was induced by adenine gavage. As shown in Table P < .05).To assess vascular medial calcification in adenine‐induced CKD rats, alizarin red S staining and von Kossa staining were performed. As shown in Figure P < .01). Notably, Rb1 (40 μmol/L) treatment remarkedly reduced VSMC calcification compared to Rb1 (20 μmol/L) treatment.Primary VSMCs were extracted for in vitro experiments. After positive immunostaining for α‐SMA Figure D, VSMCs Taken together, the results in this section suggest that ginsenoside Rb1 reduces the calcium deposition of CKD‐associated VC both in vivo and in vitro.3.2P < .05). Additionally, the induced expression of RUNX2 in CKD rats was reduced in CKD + Rb1 rats. Western blots further verified these results  or absence (P < .01) of β‐GP , a Wnt/β‐catenin pathway agonist, was observed to promote the nuclear translocation of β‐catenin in VSMCs Figure A. Intere3.6P < .05; Figures The crosstalking between PPAR‐γ and the Wnt signalling pathway has been widely discussed in various diseases.Taken together, Rb1 inhibits the Wnt/β‐catenin pathway by activating the nuclear receptor PPAR‐γ.4In the present study, we demonstrated that ginsenoside Rb1 ameliorated calcium deposition and VSMC phenotype switching in both adenine‐induced CKD rats and in rat VSMCs. Rb1 exerted an anticalcific effect through inhibition of the Wnt/β‐catenin pathway via activating PPAR‐γ, which provides new insights into the potential pharmacological interventions of VC.Vascular calcification poses a great threat to human health due to its high morbidity and mortality.Sufficient evidence has shown that VC involves a pluriform range of pathobiological processes rather than simple calcium deposition, among which, VSMC switching into osteoblast‐like cells plays a critical role. In the present study, we showed that the contractile VSMC markers α‐SMA and calponin were reduced in CKD rat aortas and calcified VSMCs compared to those in the control rats, and we showed that Rb1 increased the expression of α‐SMA and calponin in a dose‐dependent manner. RUNX2, an essential transcription factor in osteoblast differentiation and skeletal morphogenesis,To further explore the underlying mechanism of the anticalcific effect of Rb1, we next focused on β‐catenin/PPAR‐γ crosstalk. Signal transduction via the canonical Wnt pathway includes cytoplasmic stabilization and the nuclear translocation of β‐catenin. Interacting with the T cell factor/lymphoid enhancer factor (TCF/LEF) family, β‐catenin regulates the transcription of downstream genes, thereby governing cellular fate determination processes, such as osteogenic differentiation of MSCs.PPAR‐γ has been reported to regulate glucolipid metabolism and to determine the differentiation of various cell types.et al reported that the functional interaction between β‐catenin and PPAR‐γ involved the TCF/LEF‐binding domain of β‐catenin and a catenin‐binding domain (CBD) within PPAR‐γ.The interplay between PPAR‐γ and the Wnt signalling pathway has been extensively discussed. For example, the PPAR‐γ and Wnt pathways orchestrate the adipogenesis and osteogenesis of MSCs.Nevertheless, insufficiency of this study remains in that, despite being a generally accepted model of CKD, adenine‐induced CKD rats suffered faster weight loss and less extensive VC than clinical CKD patients due to the gavage modelling time being relatively intense and limited, which requires future studies for further exploration.Overall, as illustrated in the schematic diagram Figure C, this sNone.JXP and LHX conceived and designed the experiments. ZP, ZXY, GMQ, WL, ZZH and GR performed the experiments. ZXY, LZW and DM researched the data and contributed to the discussion. ZP and ZXY analysed the data. DHY contributed reagents, materials and analytic tools. ZP and LHX wrote the manuscript. JXP and LHX reviewed and edited the manuscript. Click here for additional data file.


Wnt signalling mediates complex cell-cellinteractions during development and proliferation. Annexin A8 (AnxA8), a calcium-dependent phospholipid-binding protein, and canonical Wnt signalling mechanisms have both been implicated in retinal pigment epithelial (RPE) cell differentiation. The aim here was to examine the possibility of cross-talk between AnxA8 and Wnt signalling, as both are down-regulated upon fenretinide (FR)-mediated RPE transdifferentiation. AnxA8 suppression in RPE cells via siRNA or administration of FR induced neuronal-like cell transdifferentiation and reduced expression of Wnt-related genes, as measured by real-time PCR and western blotting. AnxA8 gene expression, on the other hand, remained unaltered upon manipulating Wnt signalling, suggesting Wnt-related genes to be downstream effectors of AnxA8. Co-immunoprecipitation revealed an interaction between AnxA8 and β-catenin, which was reduced in the presence of activated TGF-β1. TGF-β1 signalling also reversed the AnxA8 loss-induced cell morphology changes, and induced β-catenin translocation and GSK-3β phosphorylation in the absence of AnxA8. Ectopic over-expression of AnxA8 led to an increase in active β-catenin and GSK-3β phosphorylation. These data demonstrate an important role for AnxA8 as a regulator of Wnt signalling and a determinant of RPE phenotype, with implications for regenerative medicine approaches that utilise stem cell-derived RPE cells to treat conditions such as age-related macular degeneration. In vivo, retinal pigment epithelial (RPE) cell phenotype is sustained by the retinal microenvironment. However, once removed from the retina and placed in culture, RPE cells dedifferentiate within a few rounds of division, losing signature characteristics such as pigment granules and expression of genes such as MerTk and RPE65. The widely used human RPE cell line, ARPE19, is typical in this respect, though several studies have shown that under appropriate culture conditions ARPE19 cells will re-adopt a more mature phenotype that includes restoration of pigment granules and expression of key RPE-associated genes3. Interest in RPE de-differentiation has also been driven by the need to understand the process in proliferative vitreoretinopathy (PVR) where epithelial mesenchymal transition (EMT) plays a key role in the pathogenesis of this condition. More recently, interest in RPE cell differentiation and maturation has intensified with advances in regenerative medicine that utilize RPE cells derived from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells6. RPE cells derived from ES or iPS cells exhibit many characteristics of mature fully differentiated RPE cells, and first-in-man transplantation studies in dry and wet age-related macular degeneration (AMD) have yielded encouraging results10. Key to these clinical advances is a better understanding of the signaling pathways that regulate and maintain RPE cell phenotype.12. In our investigations into the mechanisms underlying the RPE response to FR, we found that FR-mediated RPE cell transdifferentiation is dependent on, and mediated by, AnxA8 downregulation13, demonstrating a key role for this phospholipid- and calcium-binding protein in maintaining the plasticity of the RPE cell phenotype.The plasticity of RPE cells in culture is evident from studies showing that not only can they dedifferentiate, but they can also transdifferentiate. Thus, low doses of the retinoic acid (RA) derivative fenretinide (FR) inhibit RPE cell proliferation and induce a neuronal-like phenotype13, raising the question of whether cross-talk occurs between AnxA8 and Wnt signaling. Canonical Wnt signaling maintains cell fate specification and proliferation in diverse mammalian cell types15 and it occurs upon binding of secreted Wnt proteins to Frizzled receptors and their co-receptors, lipoprotein receptor-related proteins (LRP)-5 and 6. This inactivates glycogen synthase kinase (GSK)-3β, leading to accumulation of non-phosphorylated β-catenin in the cytosol16. β-Catenin is then translocated to the nucleus to promote ECF/LEF-1 mediated expression of Wnt target genes. In the absence of Wnt, β-catenin is degraded by a complex consisting of GSK-3β, axin, protein phosphatase 2a, adenomatosis polyposis coli and casein kinase 1α.A microarray analysis performed on FR-transdifferentiated RPE cells revealed down-regulation of AnxA8 and suppression of several genes involved in Wnt signalingHere, we report that RPE phenotype is critically dependent on canonical Wnt signaling, and that this in turn is regulated by AnxA8. We thus identify a novel signaling nexus that has implications for strategies aimed at preventing dedifferentiation and at yielding mature RPE cells from ES or iPS cells.17. We recently found that AnxA8 was down-regulated in ARPE-19 cells induced towards a neuronal lineage following treatment with FR, and showed that AnxA8 down-regulation is both necessary and sufficient for RPE transdifferentiation13. FR-induced AnxA8 loss also correlated with decreased expression of the Wnt-related genes Frizzled-1, Frizzled-4 and Wnt2b  cells, and in cells treated with Wnt signaling activators or inhibitor Figs.  and S3A.To investigate possible physical association between AnxA8 and components of the Wnt signalosome, we performed co-immunoprecipitation experiments and observed interactions between AnxA8 and both β-catenin and active β-catenin in ARPE19 cells Fig. . The intWe next investigated how interactions between AnxA8, β-catenin and GSK-3β influence Wnt signaling. We therefore examined cytosolic and nuclear pools of β-catenin and GSK-3β in the presence and absence of siRNA for AnxA8, and with or without cell stimulation with Wnt3a and TGF-β1 in human ARPE19 Fig.  and in pWe then over-expressed AnxA8 in RPE cells using an AnxA8-GFP construct to examine Wnt signaling in the presence of exogenous AnxA8. Transient transfection with AnxA8-GFP led to increased levels of active β-catenin protein. However, total β-catenin levels were unchanged and there was no change in the GSK-3β phosphorylation status. TGF-β1 signaling induced an increase in active β-catenin only Fig. . As expe13, consistent with roles for AnxA8 in the phenotypic regulation of other cell types19. Here we found that exposure of RPE cells to FR also led to down-regulation of several key genes involved in canonical Wnt signaling, including β-catenin, Wnt2b, Wnt3a, Frizzled-1 and Frizzled-4, whereas in contrast GSK-3β expression was raised. RA derivatives are well known to suppress canonical Wnt signaling in different cancer cell lines by destabilization of β-catenin21. There is also a positive correlation between cell differentiation and increased GSK-3β-mediated degradation of phosphorylated β-catenin in the presence of all-trans RA23. Interestingly, we observed an almost identical shift in Wnt-related genes in AnxA8-depleted RPE cells, suggesting that AnxA8 is required for the maintenance of Wnt signaling in RPE cells.In this study and previous work, we have shown that in RPE cells, FR-induced neuronal transdifferentiation is mediated at least in part by suppression of AnxA824, but is elevated during the latter stages. Demonstrating that AnxA8 is required to maintain Wnt signaling prompted us to ask whether there is a reciprocal interdependency in which β-catenin or GSK-3β may similarly be required for AnxA8 expression. However, whilst depletion of AnxA8 led to down-regulation of Wnt signaling, we found that neither stimulation nor inhibition of Wnt signaling had any impact on RPE morphology or AnxA8 expression in ARPE19 and primary porcine RPE cells, placing AnxA8 activity upstream of Wnt signaling.Here we observed a decrease in Wnt gene expression in cultured RPE cells within two days of applying AnxA8 siRNA (not shown), and after four days cells adopted neuronal-like cell morphology. Canonical Wnt signaling appears to have phased effects on neuronal differentiation. Thus, Wnt signaling is suppressed during the initial phase of neuronal differentiation26. TGF-β1 has been shown to stimulate nuclear translocation of β-catenin27, and to upregulate several Wnts and thereby stabilise β-catenin28. TGF-β1 further seems to be capable of controlling osteoblast differentiation in mouse C2C12 cells29.To understand how AnxA8 exerts its influence over Wnt signaling, we asked whether it interacts with components of the Wnt signaling machinery, and observed that AnxA8 physically associates with β-catenin. Several studies have shown that cross-talk between TGF-β signaling and Wnt signaling pathways plays an important role in the regulation of certain developmental events, and here we observed that the AnxA8/β-catenin association was reduced when RPE cells were exposed to TGF-β1. The cooperative regulation by Wnt signaling and TGF-β1 is mediated by an association between Smads and TCF/LEF in the nucleus, and results in the synergistic activation of specific target genes30. The cytoplasmic accumulation of β-catenin and its nuclear translocation have consequences for gene expression, which in some cases have been implicated in pathological conditions such as AMD31. In AnxA8-depleted RPE cells, we observed a decrease in several β-catenin target genes, including Otx2 and Mitf, two major transcription factors with roles in RPE development. In the context of PVR, Han and colleagues found that laser photocoagulation activates Wnt signaling, leading to increased RPE proliferation and epithelial to mesenchymal transition through elevated Otx2 and Mitf32.Here we have shown that AnxA8 functions as a regulator of canonical Wnt signaling in RPE cells. Suppression of AnxA8 led to a significant decrease in the key Wnt signaling protein β-catenin and its active form in both cytosol and nucleus. Concomitantly, increasing phosphorylation of GSK-3β at serine-9 leads to a reduction in GSK-3β-mediated phosphorylation of β-catenin, and therefore to an activation of Wnt signaling33, but here we observed only marginal increases in β-catenin upon exposure of RPE cells to TGF-β1. However, cells depleted of AnxA8 showed a significant increase in β-catenin and active β-catenin when stimulated with TGF-β1. We also observed an increase in the phosphorylation of GSK-3β in TGF-β1-treated AnxA8-suppressed RPE cells. This is consistent with reports that TGF-β1 increases the expression of the ser9-phosphorylated inactive form of GSK-3β in human lung fibroblasts27. We also found that TGF-β1 blocked AnxA8 siRNA-induced neuronal transdifferentiation, which is in line with data describing that inhibition of GSK-3β suppresses RA-mediated differentiation of ES cells34. TGF-β1 was reported to stimulate a physical association between GSK-3β and Smad3, thereby facilitating β-catenin translocation36. However, it is well known that TGF-β1 signaling is highly context-dependent. It can induce Wnt signaling in pulmonary fibroblasts37 and inhibit photoreceptor differentiation through down-regulation of the canonical Wnt pathway in human Müller glia38.TGF-β1 signaling has been shown to increase β-catenin expression and nuclear translocation in various cell types39. Wnt signaling also participates in ES cell differentiation towards RPE cells40 where it seems to control self-renewal and spontaneous differentiation of naïve human ES cells. Decreased Wnt signaling activity was found to inhibit differentiation of ES cells42, suggesting that AnxA8-mediated changes in Wnt signaling might potentially have an impact on stem cell fates. A better understanding of the interplay between AnxA8 and Wnt signaling could be important in the development of stem cell-based regenerative therapies for RPE replacement in diseases such as AMD.In summary, we have identified a novel role for AnxA8 in the regulation of RPE phenotype that is mediated through canonical Wnt signaling. This is consistent with the observation in the regenerating chick retina that β-catenin prevents RPE cells from entering the cell cycle, whereas loss of β-catenin leads to dedifferentiation and proliferation43 as well as primary pRPE were cultured in Dulbecco’s modified Eagle’s medium . Media was substituted with 100 U/ml penicillin/streptomycin , 2 mM L-glutamine (Gibco) and 10% foetal bovine serum . Incubators were kept at 37 °C and 5% CO2.The human ARPE-19 cell line (ATCC number CRL-2302)2 and treated with 1 µM FR  or vehicle  in DMEM and 1% FBS for 7 days each day. Phase images were taken (Leica DC200) and cells were processed for gene and protein analysis.RPE cells were plated at 2,200 cells/cm44. β-catenin  and GSK-3β siRNA  were used at 10 nM. Allstars negative control siRNA (Qiagen) was used at respective concentrations accompanying each experiment. RPE cells were transfected in P/S-free media for 48 h, before the transfection mix was replaced with full media for another 48 h. Phase images were taken, and cells were processed for real-time PCR  transfection reagent (Polyplus transfection). 150 pM of the following AnxA8 siRNA sequences were used: humanAnxA8 siRNA-1, 5′-CAG CCU UUC GGU CUU CUA UTT-3′; humanAnxA8 siRNA-2, GCG UGA UGG GAC CCU GAU ATT-3′; humanAnxA8 siRNA-3, 5′-GCC CUU AUG UAC CCG CCA UTT-3′CHX (30 µg/ml) was added to ARPE-19 cells treated with AnxA8 siRNA and FR at 6, 4, 2 and 0 hours before end of treatment. Cells were processed for western blotting as described in the Supplementary Information.To analyse whether Wnt signalling has an effect on AnxA8 expression, Wnt activators or inhibitor, respectively, were added to ARPE19 and to pRPE cells. 10 nM Wnt3a (Abcam) or 2.5 µM SB216763 (Tocris Bioscience) were added every other day for 7 days to activate Wnt signalling, while 50 ng/ml DKK-1 (Life Technologies) was added 1 hour prior to FR treatment to inhibit Wnt signalling. Phase images were taken, and cells were processed for real-time PCR and Western blotting.Subconfluent ARPE19 cells were harvested in 1 ml of ice-cold lysis buffer  on ice for 30 min. The lysate was spun down at 10,000 g at 4 °C for 15 min, and the supernatant was incubated with 50 µl Dynabeads Protein A (Life Technologies) for 30 minutes at 4 °C. Beads were pelleted at 2500 g for 3 minutes at 4 °C and an aliquot of the pre-cleared lysate was stored at −20 °C. 5 µg of rabbit anti-AnxA8 (Avira) or equivalent amounts of rabbit IgG (Sigma) was added to the pre-cleared lysate and incubated for 1 hour at 4 °C. Beads were added and incubated for an additional hour. Beads were centrifuged at 2500 g for 1 min and washed with lysis buffer 5 times. Beads were diluted in 50 µl of sample buffer containing 2% sodium dodecyl sulfate (SDS), 25% glycerol, 12.5% 0.5 M Tris, 0.02% dithiothreitol and 0.2% bromophenol blue, and analysed for β-catenin using Western blotting analysis.et al. (35). Briefly, cells were lysed on ice with a cytoplasmic lysis buffer . After 10 min on ice, cells were spun down for 15 sec. The supernatant was diluted in sample buffer, whereas the pellet was washed twice in cytoplasmic lysis buffer. The pellet was then lysed in nuclear lysis buffer  for 30 min on ice. After centrifuging the nuclear proteins for 20 min at 14,000 rpm at 4 °C, the supernatant was diluted in sample buffer. Samples were boiled for 5 min and subjected to SDS-PAGE.Nuclear and cytoplasmic protein levels were analysed according to Jian Subconfluent ARPE19 cells were transfected using Lipofectamine LTX Plus transfection reagent (Life Technologies). According to the manufacturer’s instructions, 2.5 µg AnxA8-GFP or GFP plasmid DNA was used to transfect a dish of 8.55 cm² for 5 h at 37 °C. Cells were washed and cultured in 10% FBS-containing DMEM for another 48 h. TGF was added in serum-free DMEM for 24 h, and cells were harvested for Western blot anaylsis.Data were presented as mean and standard deviations. Student T-test was performed for normally distributed data to analyse differences between two groups (Excel). One-way ANOVA and Tukey posthoc were applied for normally distributed data to evaluate differences between more than two groups. Statistically significant differences were marked with an asterisk (*).Supplementary information.


Microneedles (MNs) have been extensively developed over the last two decades, and highly efficient drug delivery was demonstrated with their minimal invasiveness via a transdermal route. Recently, MNs have not only been applied to the skin but also to other tissues such as blood vessels, scleral tissue, and corneal tissue. In addition, the objective of the MN application has been diversified, ranging from drug delivery to wound closure and biosensing. However, since most MN fabrication methods are expensive and time-consuming, they are inappropriate to prototype MNs for various tissues that have different and complex anatomies. Although several drawing-based techniques have been introduced for rapid MN production, they fabricated MNs with limited shapes, such as thin MNs with wide bases. In this study, we propose a three-step thermal drawing for rapid, prototyping MNs that can have a variety of shapes and can be fabricated on curved surfaces. Based on the temperature control of polymer bridge formation during thermal drawing, the body profile and aspect ratios of MNs were conveniently controlled, and the effect of temperature control on the body profile of MNs was explained. Thermally drawn MNs with different shapes were fabricated both on flat and curved surfaces, and they were characterized in terms of their mechanical properties and insertion into vascular tissue to find an optimal shape for vascular tissue insertion. The first microneedle (MN) arrays were developed by microfabrication using dry reactive ion etching . This siRecently, MNs have been applied not only to the skin but also to other organs, such as blood vessels ,14,15, eIn addition, the objective of the MN application has been diversified, ranging from drug delivery to wound closure  and bios6/O2 chemicals and the BOSCH process were often combined [Since the first MN was developed out of silicon, most MN fabrication methods have been based on conventional microfabrication techniques which can be mainly divided into dry ,28,29,30combined ,28 for acombined . A spaticombined . Severalcombined , electrocombined , magnetocombined , and cencombined  lithograThese drawing-based approaches can rapidly produce a high aspect ratio (A/R) in MN structures. However, at the same time, this limits the shape of MNs fabricated by the drawing approaches to be only very thin and tall. This often leads to premature bending or collapse of polymeric MNs during their insertion into most tissue barriers. For successful MN insertion into tissues, it is critical to fabricate optimized, custom-shaped MNs for each type of tissue. Most MNs have been fabricated on a flat, two-dimensional (2D) substrate to build MN patches for their applications to skin. Drawing approaches were also limited to the fabrication of MNs on a flat substrate made of the same polymer as the drawn MNs. Thus, more versatile drawing techniques that can fabricate various MN shapes on flat or non-flat surfaces, are necessary to make the approach clinically meaningful in diverse medical applications.In this study, we propose a three-step thermal drawing technique to rapidly fabricate polymeric MNs that have a wide range of shapes on various surfaces. In particular, it is discussed how temperature differences between a MN substrate and drawing metal pillar can influence the body shapes of thermally drawn MNs. By controlling the thermal drawing parameters, MNs with different A/Rs were fabricated to investigate how the shapes of MNs influenced their insertion into vascular tissue. The structural integrity of thermally drawn MNs with different shapes was compared before and after their insertion into vascular tissue. The shape of the tissue wound was also analyzed using optical coherence tomography (OCT) imaging.w = 268,000 Da, Samyang Biopharmaceuticals, Pangyo, Korea), was used to fabricate an MN array by a three-step thermal drawing. Rhodamine B  was purchased from Samchun Chemical Inc. . Stainless steel, micropillar array structures were fabricated by electrical discharge machining (EDM). Each micropillar had a height of 300 µm and a diameter of 260 µm. The pillars were formed in a 3 × 3 array with 700 µm interval. A medical balloon from Fogarty® Occlusion Catheters  was used to fabricate MNs on its surface. In order to perform ex vivo insertion tests for MN penetration of vascular tissue with respect to the different A/R of MNs, an abdominal aorta from New Zealand white rabbits (3.0~4.0 kg) was harvested in the operating room.The biodegradable polymer, poly(lactic-co-glycolic)acid 90:10  made by hot-pressing was used to fabricate an MN array by a three-step thermal drawing system. A custom-built system based on a probe station  was comprised of a hot and cold chuck on a three-axis stage, a precisely machined stainless steel micropillar, and a stereomicroscope A. The teIn this study, we focused on the real-time transitional phenomenon of a polymer bridge when the PLGA50/50 film was drawn by a micropillar B. Our thTlow) is below the glass transient temperature (Tg) of the polymer, the viscosity of the polymer at the contact surface is relatively high, and the polymer bridge forms a convex shape (Tmed = Tg), the polymer starts to wet the micropillar surface to a higher degree. This results in the concave profile of the drawn polymer bridge (Tg (Thigh), the drawn polymer has a low viscosity similar to that of a liquid, and the wetting of the polymer at the surface of the micropillar becomes more significant. This leads to the formation of an inverted concave curvature to the polymer bridge . Then, while in contact with the micropillar, the heated polymer substrate is drawn by about 1/10 of the target height  are equally applied to have uniform viscosity in the drawn polymer bridge. This polymer bridge then has a linear profile while being stretched to the target length. The tip of a MN is sharpened by the final step of ‘Tip Drawing’  is typically longer than usual transfer drawing . Depending on the duration of the contact dwelling , the morphology of the polymer bridge between the substrate and the micropillar was controlled like the previous contact drawing of the 3-step thermal drawing. If the Δt is short, the flowability of the polymeric bridge is reduced; then, MNs are formed with a convex curvature, which eventually leads to the formation of bullet-shaped MNs . For the ex vivo insertion tests, a rabbit abdominal aorta was harvested and cut into samples with a width of 20 mm and a length of 5 mm. The prepared tissue samples were then fixed to a customized jig at both ends only, without sagging. Each MN was directed downwards and attached to an auto z-axis. The MN was aligned at the center of the tissue sample and gradually lowered until the MN tip penetrated to a depth of 300 µm from the surface of the vascular tissue at a rate of 10 µm/s. After removal of the MN from the tissue, the vascular tissue samples were fixed for 4 hours in a 10 % formaldehyde solution and dehydrated. The vessel samples were imaged using high-resolution OCT to visualize the MN marks within vascular tissue. The MNs used for the insertion tests were carefully examined via optical microscopy.Optical coherence tomography (OCT) imaging was performed at an acquisition speed of 20 fps. After scanning the region of MN insertion, the images were stacked up for three-dimensional (3D) OCT images. Using the 3D OCT images, XZ and YZ cross-sectional images of MN-inserted vascular tissues were obtained from the surface to a depth of 250 μm. Thereafter, the crack sizes formed by MN insertion were estimated by the ImageJ program (NIH). Mechanical deformation of MN against vascular tissue insertion was examined with an optical microscope.t-test (2-tailed). p < 0.05 was considered to be significant. One-way ANOVA and post hoc Bonferroni tests were used to compare incision depth, incision width, and full width at half depth of a rabbit abdominal aorta with each A/R of the MNs.Results were presented as means ± standard error and evaluated by the student Three-step thermal drawing for rapid prototyping MNs with various shapes was investigated with the representative three types of MNs as described in w/w) rhodamine B for visualization was heated up to 200 °C. A micropillar heated at 200 °C was placed in contact with the bulk PLGA and then drawn up to form a droplet of the polymer melt at the end of the micropillar. This PLGA, droplet-attached micropillar was aligned and placed in contact with the medical balloon surface to make a polymeric bridge between the surface of the balloon and micropillar. The dwell time (Δt) was adjusted to control the curvature of the polymer melt between the micropillar and the balloon surface similarly to the contact drawing step of the three-step thermal drawing. As shown in t was short (ten s), MNs were formed as a bullet shape (t becomes longer (20 s)  B. Since r (20 s) C. MNs onAlthough MNs with high A/Rs are expected to have easier tissue insertion than MNs with low A/Rs, they may be vulnerable to bending or buckling. Therefore, we performed ex vivo tests A with MNMNs with A/R from 1.5 to 5.0 were applied to a rabbit abdominal aorta similarly to the previous ex vivo insertion study. OCT was employed to monitor the shape of the tissue wound caused by MN application. As shown in To quantify the geometry of insertion wounds for each MN A/R, the insertion depth, width, and full width at half depth of ex vivo inserted tissues were measured from OCT images B. MNs wiIn this study, we demonstrated rapid prototyping of MNs for vascular drug delivery application. MNs fabricated by a three-step thermal drawing were applied for perivascular drug delivery in previous works ,14,15. S6 and O2 gases used to form the plasma. The wet etching process can also fabricate both cavities  and the original  shape of an MN. Based on a common process to make trenches in a semiconductor process, a (100) wafer with square mask patterns can be etched with a KOH solution to fabricate pyramidal MN cavities tapered at 54.72º [In order to apply MNs to various tissues other than the skin, preliminary experiments using various shapes of MNs are required for the effective insertion into each organ or tissue. Research using dry etching for MN fabrication reported 150 µm tall MNs with 45~50 µm diameter , 250~300t 54.72º . Howevert 54.72º ,33,34. TDrawing lithography can also be a solution for rapid prototyping of MNs since it takes less than a few minutes to fabricate the MNs. However, MN shapes that can be fabricated by the method are still limited. Ultra-high A/R MNs were produced to have a length of up to 2 mm and over 100 A/R . DropletTo control the MN shape during three-step drawing, we utilized temperature-dependent polymeric behavior. As shown in Although there is a range of MN shapes that other techniques can produce, the MN shapes and the range of A/R were generally limited. In addition, there have been few guidelines about how to control MN shapes in their fabrication process. In light of this, three-step thermal drawing is simpler to set up than other fabrication methods described above, and MNs can be rapidly prototyped with various shapes and A/Rs ranging from 1.5 to 7.0. Furthermore, transfer three-step thermal drawing still controls the shape of MNs and allows simultaneously polymeric MNs to be fabricated on curved and flexible surfaces like medical balloons, which would be able to enhance the applicability of MN for biomedical usage in the future.In this study, we introduced three-step thermal drawing for rapid prototyping MNs for vascular tissue insertion. The principles of the three-step thermal drawing were described. We demonstrated fabrication of MNs with an A/R ranging from 1.5 to 7.0 within a few minutes. In addition, MNs were also fabricated onto a curved and flexible balloon surface for vascular drug delivery. MNs fabricated by the three-step thermal drawing were applied to vascular tissue for perivascular drug delivery. The shape of biodegradable MNs was optimized to insert into a rabbit aorta by mechanical insertion tests and optical coherence tomography analysis.


Dlk1-Dio3 locus produces multiple long non-coding RNAs (lncRNAs) from the maternally inherited allele, including Meg3  in the mammalian genome. Although this locus has well-characterized functions in stem cell and tumor contexts, its role during neural development is unknown. By profiling cell types at each stage of embryonic stem cell-derived motor neurons (ESC~MNs) that recapitulate spinal cord development, we uncovered that lncRNAs expressed from the Dlk1-Dio3 locus are predominantly and gradually enriched in rostral motor neurons (MNs). Mechanistically, Meg3 and other Dlk1-Dio3 locus-derived lncRNAs facilitate Ezh2/Jarid2 interactions. Loss of these lncRNAs compromises the H3K27me3 landscape, leading to aberrant expression of progenitor and caudal Hox genes in postmitotic MNs. Our data thus illustrate that these lncRNAs in the Dlk1-Dio3 locus, particularly Meg3, play a critical role in maintaining postmitotic MN cell fate by repressing progenitor genes and they shape MN subtype identity by regulating Hox genes.The mammalian imprinted  When a gene is active, its DNA sequence is ‘transcribed’ to form a molecule of RNA. Many of these RNAs act as templates for making proteins. But for some genes, the protein molecules are not their final destinations. Their RNA molecules instead help to control gene activity, which can alter the behaviour or the identity of a cell. For example, experiments performed in individual cells suggest that so-called long non-coding RNAs (or lncRNAs for short) guide how stem cells develop into different types of mature cells. However, it is not clear whether lncRNAs play the same critical role in embryos.Meg3. Further experiments showed that motor neurons in mouse embryos that lack Meg3 do not correctly silence a set of genes called the Hox genes, which are crucial for laying out the body plans of many different animal embryos. These neurons also incorrectly continue to express genes that are normally active in an early phase of the stem-like cells that make motor neurons.Yen et al. used embryonic stem cells to model how motor neurons develop in the spinal cord of mouse embryos. This revealed that motor neurons produce large amounts of a specific group of lncRNAs, particularly one called Meg3 regulates the activity of Hox genes in motor neurons, research could now be directed toward investigating whether lncRNAs help other tissues to develop in a similar way.There is wide interest in how lncRNAs help to regulate embryonic development. With this new knowledge of how  Investigations of the gene regulatory networks involved in cell-type specification during embryonic development have been protein-centric for decades. However, given the prevalence of high-throughput sequencing analyses of mammalian genomes, it is now appreciated that non-coding RNAs (ncRNAs) account for at least 50~80% of transcriptomes . Regulatcis- and trans-acting gene silencing. For example, the lncRNA Airn directly represses the paternally-expressed Igf2r gene in cis for the maintenance of ESC differentiation  and forelimb-innervating MNs . Finally, another set of mutually exclusive Hox proteins, such as Hox5 and Hox8 expression in the Foxp1on LMC, further controls the rostral and caudal motor pool identity, which directs motor pools to either innervate proximal or distal muscles in the forelimb  differentiation as a paradigm to assess these interactions. Although spinal cord development is one of the best characterized processes in the central nervous system (CNS) , how lncforelimb .Hox chromatin is demarcated into discrete domains controlled by opposing RC patterning signals , Wnt, and fibroblast growth factors (FGFs)) that trigger rapid and domain-wide clearance of H3K27me3 modifications deposited by PRC2  component Bmi1 at brachial level causes ectopic expression of Hoxc9 and subjects LMC neurons to a thoracic preganglionic column (PGC) fate. Conversely, elevation of Bmi1 represses Hoxc9 at thoracic level and subjects PGC neurons to an LMC fate . These o by PRC2 . More spidentity . TogetheDlk1-Dio3 locus are highly enriched in postmitotic MNs. The Dlk1-Dio3 locus contains three protein-coding genes  from the paternally inherited allele, and multiple lncRNAs and small ncRNAs are derived from the maternally inherited allele, including Meg3, Rian (containing 22 box C/D snoRNAs), as well as the largest miRNA mega-cluster in mammals . Interestingly, all of the ncRNAs are regulated by a common cis-element and epigenetic control, resulting in a presumable large polycistronic transcription unit , MN progenitors , and postmitotic MNs  by fluorescence-activated cell sorting (FACS). Simultaneously, spinal interneurons (INs) derived from low conditions were collected and Hb9::GFPoff cells were sorted at day 7 as controls , implying that lncRNAs might play specific roles in each cell type during ESC~MN differentiation.As epigenetic landscape remodelling and the cell fate transition during MN differentiation are well characterized , we tookinto MNs , and we controls . Next, wcontrols , and carcontrols  to discocontrols . Severalcontrols . Our appproaches  , histone modifications (H3K4me3 and H3K27ac), and chromatin accessibility (ATAC-seq) across the Meg3-Rian-Mirg locus from previously published studies (matΔIG-DMR) ESC line, which leads to simultaneous silencing of all maternally-expressed lncRNAs in the Meg3-Rian-Mirg locus but leaves the MN-TF binding site intact  is enriched in the mantle zone of the developing spinal cord during development and is gradually enriched in postmitotic MNs (Isl1/2on cells) after E12.5; (2) has no preference for columnar MN subtypes, as revealed by Foxp1 (LMC-MNs) and Lhx3 (MMC-MNs) immunostaining; and (3) exhibits rostral high  and caudal low (lumbar) asymmetry after E12.5  as nuclear marker, and Kcnq1ot1 as a chromatin-associated RNA control). Together, these findings suggest that lncRNAs in the Dlk1-Dio3 locus are postmitotic MN-enriched, and that they are directly activated by MN-TFs and RA/RAR. At a cellular level, Meg3 is highly enriched in MN nuclei and is chromatin-associated, indicating a potential function in chromatin regulation.Finally, to characterize the abundance and subcellular localization patterns of gulation . FurtherDlk1-Dio3 locus of ESCs  of endogenous PRC2 complex components , as well as the PRC2 cofactor Jarid2, from ESC~MNs specifically retrieves Meg3, Rian and Mirg RNA, whereas the nuclear ncRNA Rnu1 and the lncRNA Malat1 were not captured by Ezh2, Jarid2, or Suz12  and found that two isoforms, v1Meg3 and v5Meg3 (Ensemble mm10 database), are predominantly expressed in Hb9::GFPon MNs . These results suggest that these Meg3 isoforms directly interact with PRC2/Jarid2 complexes and might facilitate association of the PRC2 complex with Jarid2.While several previous reports have revealed that interactions between Jarid2 and ncRNAs regulate PRC2 recruitment to chromatin, including lncRNAs in the  of ESCs , the rol of ESCs . Surprisell type . Moreoveor Suz12 . Given td miRNAs  and thatipotency , imprintipotency , and PRCipotency , we focuFPon MNs . Moreovetiation.   to retrieve Jarid2 from ESC~MNs (Meg3 knockdown (KD) did not affect the protein abundance of Ezh2/Jarid2, but we did observe that it undermined the interaction between Ezh2 and Jarid2, suggesting that Meg3 facilitates this interaction (Meg3 isoforms have differing abilities to facilitate Ezh2/Jarid2 binding by generating two locus-defined Tet-ON-inducible Meg3 ESCs (v1iMeg3 and v5iMeg3). Upon doxycycline induction, both Meg3 isoforms were induced ~20–50 fold, yet the abundance of Ezh2/Jarid2 remained unaffected  (matΔIG-DMR ESCs (Meg3 and used a knockdown approach to prevent impairment of DMR sites. Both shRNAs reduced the expression of Meg3 by an average of ~90% compared to endogenous levels in ESC~MNs (Meg3 KD ESCs (referred to as H6 and K4 hereafter) that had the best ESC morphology for further experiments. Verification by qPCR indicated that the expressions of two other lncRNAs, Rian and Mirg, from the maternal allele of the Dlk1-Dio3 imprinted locus were all significantly down-regulated in Meg3 KD MNs, whereas paternal genes were unaffected  . To overatΔ ESCs , we furt ESC~MNs . As negaaffected . This final unit .Meg3 KD MNs. We observed a trend of global down-regulation of H3K27me3 in both independent experiments of Meg3 KD MNs, most likely a reflection of compromised Ezh2/Jarid2 interaction  and majority of caudal Hox genes (Hox8~13) were up-regulated with a concomitant down-regulation of the H3K27me3 epigenetic landscape (Meg3 KD ESC line (I6) and confirming that all Meg3 KD ESC~MNs exhibited imbalanced expression of 3' and 5' Hox genes across entire Hox clusters  the binding patterns of Ezh2/H3K27me3 would display concordant tendency in MNs; and (2) the bindings of Ezh2/Jarid2 to the gene loci of progenitor and caudal Hox genes in MNs would be compromised upon the loss of Meg3. Consistent with our prediction, we verified that (1) the epigenetic landscapes of H3K27me3 in the progenitor and caudal Hox genes uncovered here are concordant with Ezh2 enrichment revealed by a previous study that used the same ESC~MN differentiation approach to generate cervical Hoxa5on MNs  and caudal Hox  genes were concomitantly reduced  Upo reduced . Taken amatΔIG-DMR ESC~MNs, we further scrutinized the MN phenotype in matΔIG-DMR embryos. Consistent with previous studies, matΔIG-DMR embryos died soon after E16 , and Irx3on cells  for postmitotic MNs (Is11/2on) along the entire RC axis of the ventral spinal cord in the matΔIG-DMR embryos (on and Isl1(2)on MNs were comparable between the control and matΔIG-DMR embryos at E10.5 , thoracic (Hoxc9), and lumbar (Hoxd10) Hox levels. We observed comparable numbers of cells expressing respective Hox proteins at each segmental level between control and mutant embryos (on) and limb-innervating MNs (Foxp1on) were also largely unaffected in the matΔIG-DMR embryos . Taken together, the switching of Hoxa5on to Hoxc8on in MNs of matΔIG-DMR embryos is not transient, which leads to a concomitant change in motor pool fate  is not affected in either Δ/ΔRian or Δ/ΔMirg ESCs. In the Δ/ΔRian ESC, expressions of Meg3 and Mirg were relatively unaffected, whereas that of Rian was compromised. Conversely, only expression of Mirg was impaired significantly in the Δ/ΔMirg ESCs, but expressions of Meg3 and Rian manifested minimal changes in that cell line  this locus is conserved between human and mouse , which makes it one of the best systems for uncovering the physiological functions of lncRNAs . In thisnd mouse ; (2) sevppressor ; and (3)fication , which pDlk1-Dio3 locus are highly expressed in the CNS, their functions during neural development are largely unknown (Meg3 (a Dlk1-Dio3 locus-derived lncRNA), we uncovered that: 1) many adjacent progenitor genes were significantly up-regulated; and 2) the rostral Hox genes were significantly down-regulated, with a concomitant increased expression of caudal Hox genes in ESC~MNs. This phenotype was recapitulated in matΔIG-DMR embryos, in which Hoxc8 expression is expanded in otherwise Hoxa5on MNs. Several reports have identified that certain lncRNAs can shape the Hox epigenetic landscape by cis and trans modulation  and Kdm6b (Jmjd3), are reactivated in postmitotic MNs (data not shown). Although the function of H3K27me3 demethylase reactivation in postmitotic MNs remains unknown, these findings raise the possibility that the epigenetic memory of the H3K27me3 landscape in postmitotic MNs might still need to be ‘actively’ maintained to counterbalance H3K27me3 demethylase activity. Enrichment of postmitotic MN lncRNAs might therefore bridge and scaffold the PRC2/Jarid2 interaction and activity to maintain MN epigenetic memory by repressing progenitor genes and also carve MN subtype identity by repressing caudal Hox genes   . This rental MNs . Our resntal MNs , or morental MNs . Moreovental MNs , they ma-/-Suz12 ESCs maintained with a low amount of H3K27me3 can differentiate into MNs, albeit with a significant increase in the expression of caudal Hoxc6 and Hoxa7 compared to wild-type cells (Meg3-Rian-Mirg exhibited 50% concordance (3953/7474) with the up-regulated genes in -/-Suz12 spinal MNs (data not shown). Together, these results strongly endorse the critical function of PRC2/lncRNA in perpetuating the postmitotic cell fate of cervical Hoxa5on MNs.A previous study reported that hypomorphic pe cells . Interespe cells . This oupe cells . In thisHotair are proposed to scaffold the PRC2 complex and guide it to specific genome loci  Meg3 might function in cis to regulate its imprinting status (Meg3 knockout mouse lines have previously been generated either by deletion of the first five exons plus approximately 300 bp of the adjacent upstream promoter region of Meg3 (~5.9 kb)   or by de of Meg3 . Both of KO line  exhibite KO line  presenteMeg3 acts as a scaffold for the PRC2/Jarid2 complex, it is still possible that Rian and Mirg could independently and/or synergistically function with Meg3 to contribute to the Hox-mediated MN subtype switching we observed in the MatΔIG-DMR mutants. To investigate this possibility, we instead generated two independent targeted deletion ESC lines of Meg3 downstream lncRNAs, Δ/ΔRian and Δ/ΔMirg. Consistent with previous results  and microdeletions affecting IG-DMR and/or MEG3-DMR of maternal origin result in a unique human phenotype manifested as a small bell-shaped thorax, coat-hanger-like appearance of the ribs, abdominal wall defects, placentomegaly and polyhydramnios. One hallmark of patients with this disease, termed ‘Kagami-Ogata syndrome’ (KOS) (IG-DMR give rise to this phenotype.Consistent with the imprinting status of the e’ (KOS) , is thatmatΔIG-DMR embryos, we previously observed extra ossification at the sites where the 6th to 8th ribs attach to the sternum, similar to the malformed thorax in KOS patients  lines. This scenario might lead to failure of these iPSCs to form viable mice  and non-effective scrambled sequence (TR20003) were used to create stable knockdown lines of Meg3 within the Hb9::GFP ESCs. We used two different shRNA sequences to knockdown Meg3. Additionally, stable infected ESCs were selected by puromycin. Single ESC clones with good morphology and only presenting knockdown efficiencies >90% were picked for further expansion and characterization.The Gapdh was used as a control for normalization. For GeneChip expression analysis, RNA was purified and amplified using the Qiagen RNAeasy kit and a one-color Low Input Quick Amp Labeling Kit (Agilent Genomics) and hybridized to a SurePrint G3 Mouse GE 8×60K Microarray. Differentially-expressed genes were defined by ranking all probes according to Moderated t-test and a fold-change threshold ≥2 (p<0.001).ESCs or embryoid bodies were harvested for total RNA isolation by Trizol (Thermo Scientific). For qPCR analysis, total RNA from each sample was reverse transcribed with Superscript III (Thermo Scientific). One-tenth of the reverse transcription reaction was used for subsequent qPCR reactions, which were performed in triplicate with three independent experimental samples on a LightCycler480 Real Time PCR instrument (Roche) using SYBR Green PCR mix (Roche) for each gene of interest. We followed a previously published protocol to perform ChIP-seq for ESC~MNs . Four miImmunohistochemistry was performed on 15 μm cryostat sections as described . PrimaryGapdh (mRNA in cytoplasm), Rnu1 (snRNA in nucleus), and Kcnq1ot1 (a known chromatin-associated lncRNA) were used as quality controls to verify fractionation.We followed a previously published protocol to extract subcellular fractions of RNA . We used2) was added and the mix was shifted to room temperature for 20 min to allow proper secondary structure formation. Folded RNA was then mixed with 1 mg of ESC protein nuclear extract in RIP buffer  and incubated at 4°C for one hour. Twenty µL Dynabeads M-280 Streptavidin (Invitrogen) were added to each binding reaction and further incubated at room temperature for one hour. Beads were washed briefly five times and boiled in SDS buffer, and the retrieved protein was detected by standard Western blot analysis.In vitro-transcribed biotin-labelled RNAs were generated by the Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Promega). Templates were treated with RNase-free DNase I (Promega) and the reaction mix was purified with Oligo Clean and Concentrator . Biotinylated RNA (3 μg) was heated to 65°C for 10 min and then slowly cooled down to 4°C. After that, RNA structure buffer . Samples were incubated on ice for 10 min, followed by centrifugation at 2,000 rpm for 10 min. The cloudy supernatant cytoplasmic fraction was removed. After washing twice , the cell pellets were resuspended in the same volume of high salt buffer , and rotated for 30~60 min at 4°C. Then cell pellets were centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was incubated overnight at 4°C with antibody and pre-cleared Protein-G beads (depending upon the antibody) to immunoprecipitate endogenous protein against the specific antibody used. We collected 10% of cleared supernatant as input. Subsequently, IP-protein beads were washed three times with PBS and 0.01% Tween-20, each for 5 min at 4°C. IP-proteins and their interacting partners were eluted from beads in 6X reducing loading buffer at 70°C for 15 min. Finally, samples were cooled down to room temperature and spun briefly to collect condensation. Standard Western blot procedures were applied using anti-Jarid2  or anti-Ezh2  antibodies. Blots were developed using HRP-conjugated anti-rabbit or -mouse antibodies, depending on the species of the primary antibody. Signals were developed and filmed by enhanced SuperSignal West Femto Maximum Sensitivity Substrate . All exposures were done using hyper film.Meg3 smFISH probes were purchased from Stellaris. Images were captured with a Delta Vision microscopy system.ESC~MNs were cultured and harvested on slides. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature, permeabilized for 5 min on ice in PBS with 0.5% Triton X-100, and then rinsed in 70% EtOH for subsequent RNA FISH. Slides and coverslips were kept in 70% EtOH at 4°C until staining. Slides were then washed in wash buffer (10% deionized formamide in 2X SSC) for 5 min and incubated in a dark room at 37°C for at least 4 hr with 1 μL of probe stock solution and 100 μL of hybridization buffer . RIP was performed with the RNA-Binding Protein Immunoprecipitation Kit  according to the manufacturer’s protocol with some modifications. ESC~MNs were dissociated at a concentration of 2 million cells/mL and treated with 0.3% formaldehyde in ice-cold PBS for 10 min at 37°C. Glycine/PBS was added to a final concentration of 0.125 M and each sample was incubated for 5 min at room temperature. After crosslinking, ten million cells were washed twice with cold PBS and then suspended in 100 μL RIP lysis buffer (with the addition of protease inhibitor and RNase inhibitor). The lysate was incubated on ice for 5 min and centrifuged at 14,000 rpm for 10 min at 4°C. Ezh2, Jarid2, and Suz12 antibodies were added for respective IP reactions and then incubated in RIP buffer (0.5 M EDTA/RNase inhibitor) for 3 hr to overnight at 4°C. Samples were washed at least five times with RIP washing buffer. RIP beads were resuspended in RIPA buffer (RIP washing buffer +10% SDS +protease K) to reverse crosslinking at 56°C for 30 min. RNA samples were extracted and qPCR was performed as described above. Isolated proteins before proteinase K treatment were collected from the beads and verified by Western blot analysis. Data on retrieved RNAs was calculated from the RT/input ratio for each experiment.t-tests were used for comparisons between experimental samples and controls. Statistical significance was defined as * p<0.05 and ** p<0.01 by Student’s t-test.All statistical analyses were generated with GraphPad Prism 6 (GraphPad Software). The values are shown as mean ± SD, as indicated. Student’s Adapter contamination in the paired-end reads was removed using PEAT , and thegenomecov functionality supported in the BEDTools suite (−5) in each ChIP-seq sample with the corresponding input library and then overlapping peaks were merged using MAnorm  for quantification of axon arborization. Regions of interest were segmented for detection of individual neurons. Motor nerve terminals were semi-automatically traced using the filament tracer wizard from a defined starting point. The AutoPath (no loops) algorithm was selected. Seed points detected from background signals were manually removed. Disconnected segments were removed by indicating the maximum gap length, and background subtraction was applied for noise removal. The ‘Filament No. Dendrite Terminal Points’ tool automatically calculated the number of motor nerve terminals. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Dlk1-Dio3 Locus-Derived LncRNAs Perpetuate Postmitotic Motor Neuron Cell Fate and Subtype Identity" for peer review at eLife. Your article is being evaluated by two peer reviewers, and the evaluation is being overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor.Thank you for sending your article entitled "Summary:Dlk1-Dio3 locus, which produces three protein-coding genes  from the paternally inherited allele and multiple lncRNAs  and small ncRNAs from the maternally inherited allele. Although this locus has been studied for years, the authors have discovered a new role for the Dlk1-Dio3 locus-derived lncRNAs in post-mitotic motor neurons (MNs). The authors find that Meg3 is induced in MN differentiation in response to retinoic acid and that Meg3 affects PRC2-Jarid2 complex assembly. Meg3 knockdown cause H3K27me3 depletion and gene induction at several 5' Hox genes and the authors nicely show that this is associated with altered motor neuron identity and nerve targeting in vivo. Through a combination of elegant biochemical approaches in mESC-derived MNs, the authors find that these lncRNAs function through PRC2 (via gene repression) and have a dual role: (a) repression of progenitor/non-neural fate, and (b) establishment of MN subtype identity via repression of caudal Hox genes. This work represents a significant contribution as it aims to uncover the role of the lncRNAs of the mammalian imprinted Dlk1-Dio3 locus in neuronal development. In addition, the authors devote significant effort into translating the in vitro mESC-derived MN findings to the mouse spinal cord. That being said, the authors should address the following concerns.The function of most lncRNAs in nervous system development remains largely unknown. This study by Yen et al. aims to address this knowledge gap by focusing on the imprinted Essential revisions:Dlk1-Dio3 locus-derived lncRNAs (Meg3) repress progenitor genes in MNs. However, it has not been demonstrated that Dbx1 de-repression occurs in post-mitotic MNs of IG DMR mice. This point could be further consolidated by discovering additional progenitor genes that are de-repressed in vivo in IG DMR mice. The list generated via RNA-seq on mESC-derived MNs contains additional candidates .1) A major conclusion of this paper is that Meg3 knock-down in mESC-derived MNs, the authors show effects on progenitor genes and Hox genes. However, it remains to be shown in vivo whether loss or knock-down of Meg3 results in similar effects. They indirectly conclude that this is the case by using the IG DMR mice , but in these mice concomitant up-regulation of paternal coding genes occurs , including Dlk1 (known to affect MN development). In addition, the IG DMR mESC-MNs show massive differences in gene expression that are not found in Meg3 KD ESC-MNs . These differences could contribute to the phenotypes observed in IG DMR spinal cords. This concern could be addressed by a more targeted approach to knock-out or knock-down (shRNA) Meg3 in the mouse spinal cord. Along the same lines, I do not understand the argument made in Discussion  about not using the available mouse KO lines for Meg3. The Meg3 KO mice represent a more elegant/targeted approach to perturb Meg3 compared to IG DMR mice.2) By performing Meg3 interacts with PRC2 components and Jarid2. If this model is correct, we can make 2 predictions: (a) ChIP for PRC2  or Jarid2 and subsequent qPCR will reveal binding to progenitor genes and Hox genes. (b) And if Meg3 is required for Ezh2/Jarid2 interaction, then PRC2/Jarid2 binding to DNA should be compromised in Meg3 KD ESC-derived MNs. The authors have the necessary expertise for this experiment.3) Through elegant biochemistry, the authors show that Meg3 interaction with Ezh2 and Jarid2: The authors find that genetic manipulation of Meg3(V5) isoform affects the co-IP of Ezh2 and Jarid2, and conclude that Meg3 facilitates Ezh2-Jarid2 complex assembly directly. This is a major claim of the paper that is not fully substantiated. What is the copy number of Ezh2, Jarid2, and Meg3? Is the copy number of Meg3 sufficient to mediate this effect? Are there independent binding sites on Meg3 for different subunits of the PRC2-Jarid complex, and how can the authors rule out indirect effects of Meg3 on cell state that affects PRC2 complex assembly?4) Likewise, Meg3 RNA to retrieve Ezh2 from MN cell extract, and then conclude this demonstrates "direct interaction" between Meg3 and Ezh2 . This is incorrect as the extract contains a multitude of proteins. UV crosslinking in cells or in vitro reconstitution with purified components is needed to make such a conclusion.5) The authors use biotinylated Dlk1-Dio3 locus somewhat complicates the interpretation of these results. The use of Meg3(v5) transgene to rescue is informative, but this was only done for a subset of experiments. The authors should take this into account more in the Discussion.6) The presence of multiple long and small regulatory RNAs from the Major concerns that do not require experiments:Meg3 facilitates interaction of the PRC2 complex with Jarid2 in MNs”, the authors conclude that Meg3 facilitates binding of PRC2 and Jarid2 in post-mitotic MNs. Also, this argument could be strengthened by providing evidence that Jarid2 is expressed in vivo in spinal MNs. Similarly, it appears that Meg3 knock-down was performed at the ESC stage, not at the progenitor MN or post-mitotic MN stage? The reason for this should be clearly stated in Results because this point is raised in Discussion  but with little justification.1) Why were the experiments shown in Figure 3C-G done on ESC and not ESC-derived MNs? At minimal, an explanation must be provided. This is important because at the end of the subsection “Meg3, which is highly expressed in rostral spinal cord, (ii) it will also help the reader in Discussion  when the authors talk about cervical MNs.2) The authors compare data from other studies performed in ESC-derived MNs. However, it is not clearly stated whether all these studies generate the same "type" of MNs. Do all these previous studies generate mESC-derived MNs of "rostral" identity? If yes, this should be added in the text because: (i) it makes sense then that by profiling these MNs the authors found HOTAIR  could be discussed .3) In Discussion, similarities and differences between the mechanism described by the authors and previous studies on the lncRNA Meg3 is enriched in rostral MNs. However, in Figure 4—figure supplement 1E, Meg-3 knock-down results in loss of rostral Hox gene expression and gain of caudal Hox gene expression. The authors should at least discuss how Polycom-mediated gene repression results in these 2 different effects on Hox gene expression, especially in light of the fact that these results are not phenocopied in vivo with the IG-DMR mice.4) The authors find that Reviewer #1:Meg3 imprinted lncRNA in motor neuron differentiation. The authors find that Meg3 is induced in MN differentiation in response to retinoic acid, and that Meg3 affects PRC2-Jarid2 complex assembly. Meg3 knock down cause H3K27me3 depletion and gene induction at several 5' Hox genes, and the authors nicely show that this is associated with altered motor neuron identity and nerve targeting in vivo. The strength of this work is the use of rigorous genetic models to validate the model that lncRNA regulates Hox loci in trans and thereby alter neuronal cell positional identity. The mechanistic details are not yet fully clear, which the authors should be more cautious in their interpretations.Yen et al. present an integrated analysis of the Meg3 interaction with Ezh2 and Jarid2: The authors find that genetic manipulation of Meg3(V5) isoform affects the co-IP of Ezh2 and Jarid2, and conclude that Meg3 facilitates Ezh2-Jarid2 complex assembly directly. This is a major claim of the paper that is not fully substantiated. What is the copy number of Ezh2, Jarid2, and Meg3? Is the copy number of Meg3 sufficient to mediate this effect? Are there independent binding sites on Meg3 for different subunits of the PRC2-Jarid complex, and how can the authors rule out indirect effects of Meg3 on cell state that affects PRC2 complex assembly?1) Meg3 RNA to retrieve Ezh2 from MN cell extract, and then conclude this demonstrates "direct interaction" between Meg3 and Ezh2 . This is incorrect as the extract contains a multitude of proteins. UV crosslinking in cells or in vitro reconstitution with purified components is needed to make such a conclusion.2) The authors use biotinylated Dlk1-Dio3 locus somewhat complicates the interpretation of these results. The use of Meg3(v5) transgene to rescue is informative, but this was only done for a subset of experiments. The authors should take this into account more in the Discussion.3) The presence of multiple long and small regulatory RNAs from the Reviewer #2:Dlk1-Dio3 locus, which produces three protein coding genes  from the paternally inherited allele and multiple lncRNAs  and small ncRNAs from the maternally inherited allele. Although this locus has been studied for years, the authors have discovered a new role for the Dlk1-Dio3 locus-derived lncRNAs in post-mitotic motor neurons (MNs). By using a well-established in vitro differentiation protocol to generate MNs from stem cells, the authors first provide a mini-resource  of lncRNAs that show cell-type specificity during different stages of MN development . This resource revealed that the Dlk1-Dio3 locus-derived lncRNAs  are enriched in post-mitotic MNs in vitro, and further demonstrated that Meg3 is expressed in developing mouse spinal MNs in vivo . Through a combination of elegant biochemical approaches in mESC-derived MNs, the authors find that these lncRNAs function through PRC2 (via gene repression) and have a dual role: (a) repression of progenitor/non-neural fate, and (b) establishment of MN subtype identity via repression of caudal Hox genes.The function of most lncRNAs in nervous system development remains largely unknown. This study by Yen et al. aims to address this knowledge gap by focusing on the imprinted Dlk1-Dio3 locus in neuronal development. In addition, the authors devote significant effort into translating their in vitro mESC-derived MN findings to the mouse spinal cord. However, I do have several major concerns that moderated enthusiasm.This work represents a significant contribution as it aims to uncover the role of the lncRNAs of the mammalian imprinted Major concerns that require experiments:Dlk1-Dio3 locus-derived lncRNAs (Meg3) repress progenitor genes in MNs. However, it has not been demonstrated that Dbx1 de-repression occurs in post-mitotic MNs of IG DMR mice. This point could be further consolidated by discovering additional progenitor genes that are de-repressed in vivo in IG DMR mice. The list generated via RNA-seq on mESC-derived MNs contains additional candidates .1) A major conclusion of this paper is that Meg3 knock-down in mESC-derived MNs, the authors show effects on progenitor genes and Hox genes. However, it remains to be shown in vivo whether loss or knock-down of Meg3 results in similar effects. They indirectly conclude that this is the case by using the IG DMR mice , but in these mice concomitant up-regulation of paternal coding genes occurs , including Dlk1 (known to affect MN development). In addition, the IG DMR mESC-MNs show massive differences in gene expression that are not found in Meg3 KD ESC-MNs . These differences could contribute to the phenotypes observed in IG DMR spinal cords. This concern could be addressed by a more targeted approach to knock-out or knock-down (shRNA) Meg3 in the mouse spinal cord. Along the same lines, I do not understand the argument made in Discussion  about not using the available mouse KO lines for Meg3. The Meg3 KO mice represent a more elegant/targeted approach to perturb Meg3 compared to IG DMR mice.2) By performing Meg3 interacts with PRC2 components and Jarid2. If this model is correct, we can make 2 predictions: (a) ChIP for PRC2  or Jarid2 and subsequent qPCR will reveal binding to progenitor genes and Hox genes. (b) And if Meg3 is required for Ezh2/Jarid2 interaction, then PRC2/Jarid2 binding to DNA should be compromised in Meg3 KD ESC-derived MNs. The authors have the necessary expertise for this experiment.3) Through elegant biochemistry, the authors show that Major concerns that do not require experiments:Meg3 facilitates interaction of the PRC2 complex with Jarid2 in MNs”, the authors conclude that Meg3 facilitates binding of PRC2 and Jarid2 in post-mitotic MNs. Also, this argument could be strengthened by providing evidence that Jarid2 is expressed in vivo in spinal MNs. Similarly, it appears that Meg3 knock-down was performed at the ESC stage, not at the progenitor MN or post-mitotic MN stage? The reason for this should be clearly stated in Results because this point is raised in Discussion  but with little justification.1). Why the experiments shown in Figure 3C-G were done on ESC and not ESC-derived MNs? At minimal, an explanation must be provided. This is important because at the end of the subsection “Meg3, which is highly expressed in rostral spinal cord, (ii) it will also help the reader in Discussion  when the authors talk about cervical MNs.2) The authors compare data from other studies performed in ESC-derived MNs. However, it is not clearly stated whether all these studies generate the same "type" of MNs. Do all these previous studies generate mESC-derived MNs of "rostral" identity? If yes, this should be added in the text because: (i) it makes sense then that by profiling these MNs the authors found HOTAIR  could be discussed .3) In Discussion, similarities and differences between the mechanism described by the authors and previous studies on the lncRNA Meg3 is enriched in rostral MNs. However, in Figure 4—figure supplement 1E, Meg-3 knock-down results in loss of rostral Hox gene expression and gain of caudal Hox gene expression. The authors should at least discuss how Polycom-mediated gene repression results in these 2 different effects on Hox gene expression, especially in light of the fact that these results are not phenocopied in vivo with the IG-DMR mice.4) The authors find that  Essential revisions:1) A major conclusion of this paper is that Dlk1-Dio3 locus-derived lncRNAs (Meg3) repress progenitor genes in MNs. However, it has not been demonstrated that Dbx1 de-repression occurs in post-mitotic MNs of IG DMR mice. This point could be further consolidated by discovering additional progenitor genes that are de-repressed in vivo in IG DMR mice. The list generated via RNA-seq on mESC-derived MNs contains additional candidates .matΔIG-DMR embryos. In revised new Figure 5—figure supplement 1A, we first verified that the expression of Meg3 is still lacking in the developing spinal cord of E14.5 matΔIGDMR embryos. We then checked the progenitor proteins Pax6, Irx3and Dbx1 (data not shown for Dbx1 as Dbx1 antibody did not work well for immunostaining). Compared to the control littermates, we observed a significant increase in the percentage of Pax6on , and Irx3on cells  for postmitotic MNs (Isl1/2on) along the entire rostrocaudal axis of the dorsal spinal cord in the matΔIG-DMR embryos . However, Hb9on and Isl1(2)on MNs were comparable between the control and matΔIG-DMR embryos at E10.5 . Although dorsal progenitor genes were aberrantly up-regulated in the postmitotic MNs, production of MNs remained relatively unaffected, suggesting that the generation of MNs is still intact with the co-expressed progenitor/postmitotic genes in the matΔIG-DMR embryos . This outcome is consistent with the postmitotic expression of Meg3 and its function to maintain the silenced epigenetic state of progenitor genes.We thank the reviewer for this suggestion to strengthen our conclusion that progenitor genes are derepressed in the onHb9on cells in the matΔIG-DMR embryos , ectopic Pax6 was manifested at partial penetrance  and several caudal Hox protein (including Hox9 and 10) were not shown to display significant changein vivo , consistent with the finding that removal of Ezh2 from MN progenitors has no detectable impact on segmental Hox expression in the spinal cord (Hoxc6 and Hoxc9) . This result also suggests that a compensatory PRC1-mediated function in vivo might make up for the loss of Meg3-mediated epigenetic maintenance in segmental MNs . Our results are not entirely unexpected as many potent miRNA/lncRNA KO mice reflect either only partial phenotype penetrance , or more severe phenotypes upon genetic or environmental stresses . Moreover, many miRNAs/TFs function as repressors to silence progenitor/neighboring interneuron genes , they may also constitute a coherent loop with lncRNAs to safeguard the terminal postmitotic cell fate with a fail-safe control. This part is now added in the Discussion subsection “A fail-safe mechanism to guard MN epigenetic landscape”, last paragraph.Although we observed a complete 100% penetrance of Hoxa5-c8 cell fate switch in the rostrobrachial segments and an increase co-expression of Irx32) By performing Meg3 knock-down in mESC-derived MNs, the authors show effects on progenitor genes and Hox genes. However, it remains to be shown in vivo whether loss or knock-down of Meg3 results in similar effects. They indirectly conclude that this is the case by using the IG DMR mice , but in these mice concomitant up-regulation of paternal coding genes occurs , including Dlk1 (known to affect MN development). In addition, the IG DMR mESC-MNs show massive differences in gene expression that are not found in Meg3 KD ESC-MNs . These differences could contribute to the phenotypes observed in IG DMR spinal cords. This concern could be addressed by a more targeted approach to knock-out or knock-down (shRNA) Meg3 in the mouse spinal cord. Along the same lines, I do not understand the argument made in Discussion  about not using the available mouse KO lines for Meg3. The Meg3 KO mice represent a more elegant/targeted approach to perturb Meg3 compared to IG DMR mice.Meg3 KO as both Meg3 KO mice lines manifested loss of maternal Rian and Mirg lncRNA expression.In particular, the Japanese Meg3 KO mice in which ~10 kb is deleted, including the Meg3-DMR region and the first five exons of Meg3 , also causes aberrant expression of paternal genes. This outcome is consistent with our results that most, if not all, lncRNAs in the Meg3-Rian-Mirg locus are reduced upon Meg3 KD and in IG-MatΔDMR. Therefore, it remains technically challenging to obtain a specific Meg3 KO without abrogating the expression levels of downstream lncRNAs. We hope the reviewer can appreciate that it is a longstanding challenge in the field to dissect the role of maternal lncRNAs in the regulation of Dlk1Dio3 locus imprinting control , and that several labs have attempted to tackle the topic but failed to generate a clear answer. We now explain our rationale more clearly in the revised Discussion .We appreciate the reviewer’s comment and apologize for not more clearly explaining our rationale. We did not use Meg3-Rian-Mirg locus are reduced upon Meg3 KD and in MatΔIG-DMR , it remains puzzling if these ncRNAs work independently or synergistically in this locus to regulate MNs. To further parse this question, we generated two single lncRNA Δ/ΔRianand Δ/ΔMirg ESCs respectively by using CRISPR-Cas9 mediated approaches . The design of the targeted deletion regions of Rian and Mirg followed two previous studies , which led to a 23 kb deletion in the Δ/ΔRian ESC and a 20 kb deletion in the Δ/ΔMirg ESC . We first verified that the paternal gene  is not affected in either Δ/ΔRianor Δ/ΔMirg ESCs. In the Δ/ΔRianESCs, expressions of Meg3 and Mirg were relatively unaffected, whereas that of Rian was compromised. Conversely, only expression of Mirg was impaired significantly in the Δ/ΔMirg ESCs, but expressions of Meg3 and Rian manifested minimal changes in that cell line . Upon differentiation, Meg3 KD MNs showed ectopic up-regulated expressions of progenitor and caudal Hox genes . In contrast, expression of Hoxa5 remained unchanged in the Δ/ΔRianand Δ/ΔMirg MNs, while Pax6 and Hoxc8 expressions were restored to similar levels to control ESC~MNs . These results indicate that Meg3 might be the major regulatory lncRNA responsible for the observed MN phenotype displayed by the matΔIG-DMR embryos .As our current and previous results indicated that the expressions of most lncRNAs in the Meg3 might be the major regulatory lncRNA and largely accounts for the observed MN phenotype observed in the matΔIG-DMR embryos, we cannot fully exclude the possibility of synergistic effects of the Meg3-Rian-Mirg locus. We discuss this possibility with new paragraphs in our Discussion section as follows:Although our new results indicate that Meg3-Rian-Mirg lncRNA cluster“Combinatorial and individual roles of the Meg3-Rian-Mirg locus over the past decade , but definitive results remain elusive. […] We anticipate that a detailed map of individual and synergetic lncRNA/miRNA functions during neural development attributable to this imprinted locus will be uncovered in the near future.”There have been multiple efforts to dissect the functions of the maternally-expressed lncRNAs in the 3) Through elegant biochemistry, the authors show that Meg3 interacts with PRC2 components and Jarid2. If this model is correct, we can make 2 predictions: (a) ChIP for PRC2  or Jarid2 and subsequent qPCR will reveal binding to progenitor genes and Hox genes. (b) And if Meg3 is required for Ezh2/Jarid2 interaction, then PRC2/Jarid2 binding to DNA should be compromised in Meg3 KD ESC-derived MNs. The authors have the necessary expertise for this experiment.Pax6, Irx3, Dbx1, and Neurog2) and caudal Hox genes. This information has been added into new Figure 4—figure supplement 2A, B.As previous studies have elegantly performed Ezh2/Suz12 ChIP-seq in ESC~MNs (using the same approach applied in this study), we have checked these available ChIP-seq data  and revealed that Ezh2 enrichment peaks are concordant with our H3K27me3 binding sites in the progenitor  and caudal Hox  genes is concomitantly reduced .We also verified that upon 4) Likewise, Meg3 interaction with Ezh2 and Jarid2: The authors find that genetic manipulation of Meg3(V5) isoform affects the co-IP of Ezh2 and Jarid2, and conclude that Meg3 facilitates Ezh2-Jarid2 complex assembly directly. This is a major claim of the paper that is not fully substantiated. What is the copy number of Ezh2, Jarid2, and Meg3? Is the copy number of Meg3 sufficient to mediate this effect? Are there independent binding sites on Meg3 for different subunits of the PRC2-Jarid complex, and how can the authors rule out indirect effects of Meg3 on cell state that affects PRC2 complex assembly?5) The authors use biotinylated Meg3 RNA to retrieve Ezh2 from MN cell extract, and then conclude this demonstrates "direct interaction" between Meg3 and Ezh2 . This is incorrect as the extract contains a multitude of proteins. UV crosslinking in cells or in vitro reconstitution with purified components is needed to make such a conclusion.Points #4 and 5 raise three related issues:Meg3 and Ezh2/Jarid2 direct?a) Is the interaction between Meg3 bind to other proteins in the PRC2 complex via indirect interactions?b) Does Meg3 copy number sufficient to mediate the pervasive PRC2/Jarid2 complex?c) Is To address these issues, we reshaped our conclusions and performed additional experiments as follows:MEG3 via PAR-CLIP, a strong indication of direct interaction . We discuss this study more clearly in our revised manuscript (subsection “Regulatory mode of Meg3 for the PRC2/Jarid2 complex”).1) Dr. Danny Reinberg’s laboratory has shown that JARID2/EZH2 retrieved Meg3 and revealed its abundance in postmitotic MNs . The PRC2 complex methylates histones for epigenetic silencing and associates with thousands of protein-coding and noncoding RNAs. The impact of RNA binding on PRC2 recruitment and activity were elegantly shown by previous studies . Based on our analysis of the RNA-seq data generated from our ESC~MN approach, we have added an analysis to our revised manuscript to show that the copy number of Meg3/Rian is much more abundant than that for many other cis- and trans-acting lncRNAs known to bind the PRC2 complex, including Xist, Kcnqtlot1, and Hotair .2) Previously, we performed both qPCR and single molecule FISH on Meg3 binds to other PRC2 proteins indirectly to contribute to PRC2/Jarid2 recruitment, activity control, and targeting mode. This topic represents a big challenge in the lncRNA/epigenetics field. Our aim in this study is to illustrate the importance and unexpected functions of lncRNAs in the well-known imprinted Dlk1-Dio3 locus during neural development. We now highlight in our revised manuscript that future experiments, including Meg3 ChIRP-seq/MS and Ezh2/Jarid2 CLIP-seq, will help reveal the comprehensive interaction and targeting mode of the Meg3/PRC2/Jarid2 interactome. Our revised discussion of this topic is as follows:3) We also understand that even with the above-described results, it is not possible to completely rule out that Meg3 for the PRC2/Jarid2 complex“Regulatory mode of Meg3 ChIRP-seq analyses, together with Ezh2/Jarid2 CLIP-seq, at all stages of ESC~MN differentiation might aid in identifying the context-dependent/independent lncRNA-mediated PRC2 targeting strategy.”The role of lncRNAs in modulating PRC2 function is well documented, but very dynamic; from recruitment, complex loading and activity control to gene targeting . […] Future systematic 6) The presence of multiple long and small regulatory RNAs from the Dlk1-Dio3 locus somewhat complicates the interpretation of these results. The use of Meg3(v5) transgene to rescue is informative, but this was only done for a subset of experiments. The authors should take this into account more in the Discussion.Meg3 in the region of Meg3-Rian-Mirg is a longstanding question. Given that Meg3 is more than 15 kb and has more than 10 variants, it is very challenging to address this issue. Although it is not a perfect approach, we instead generated two single lncRNA Δ/ΔRianand Δ/ΔMirgknockout ESC lines using CRISPR-Cas9-mediated approaches to address this issue . Details are specified in point #2 above.In fact, unraveling the essential role of Major concerns that do not require experiments:1) Why were the experiments shown in Figure 3C-G done on ESC and not ESC-derived MNs? At minimal, an explanation must be provided. This is important because at the end of the subsection “Meg3 facilitates interaction of the PRC2 complex with Jarid2 in MNs”, the authors conclude that Meg3 facilitates binding of PRC2 and Jarid2 in post-mitotic MNs. Also, this argument could be strengthened by providing evidence that Jarid2 is expressed in vivo in spinal MNs. Similarly, it appears that Meg3 knock-down was performed at the ESC stage, not at the progenitor MN or post-mitotic MN stage? The reason for this should be clearly stated in Results because this point is raised in Discussion  but with little justification.We apologize for the confusion and typo. All of the experiments in Figure 3 were performed on ESC~MNs, rather than ESCs. We have corrected this error in the revised manuscript.Meg3 in postmitoic MNs simply due to the technical difficulty of performing lentivirus-mediated knockdown in embryoid bodies (EBs), which are notoriously challenging to infect given their 3D spherical shape. We have therefore added the following sentence to our revised Discussion:We did not knockdown Meg3 is also highly expressed in ESCs , we plan to generate a targeted Meg3 floxed allele mouse line in the future that will allow us to specifically knockout Meg3 in MNs and recover the potential function of Meg3 in cell-type specific contexts.”“Given that 2) The authors compare data from other studies performed in ESC-derived MNs. However, it is not clearly stated whether all these studies generate the same "type" of MNs. Do all these previous studies generate mESC-derived MNs of "rostral" identity? If yes, this should be added in the text because: (i) it makes sense then that by profiling these MNs the authors found Meg3, which is highly expressed in rostral spinal cord, (ii) it will also help the reader in Discussion  when the authors talk about cervical MNs.on in the revised manuscript as follows:We appreciate the reviewer’s comments. We now specifically define our ESC~MNs as being cervical Hoxa5Hox genes uncovered here are concordant with Ezh2 enrichment revealed by a previous study that used the same ESC~MN differentiation approach to generate cervical Hoxa5on MNs  .”“Consistent with our prediction, we verified that 1) the epigenetic landscapes of H3K27me3 in the progenitor and caudal on MNs.”“Together, these results strongly endorse the critical function of PRC2/lncRNA in perpetuating the postmitotic cell fate of cervical Hoxa53) In Discussion, similarities and differences between the mechanism described by the authors and previous studies on the lncRNA HOTAIR  could be discussed .We thank the reviewers for reminding us of these two landmark studies. Although these two papers were discussed and cited in our original submission, we now re-emphasize them in our revised manuscript as they inspired our study.New revised Discussion:Hotair are proposed to scaffold the PRC2 complex and guide it to specific genome loci , it is tantalizing to hypothesize that Meg3 might manifest the dual functions of scaffolding the PRC2/Jarid2 complex and guiding it to specific loci in different cell contexts. […] Generating compound lncRNA mutants that can scaffold the PRC2/Jarid2 complex will shed light on this topic.”“Given that lncRNAs such as 4) The authors find that Meg3 is enriched in rostral MNs. However, in Figure 4—figure supplement 1E, Meg-3 knock-down results in loss of rostral Hox gene expression and gain of caudal Hox gene expression. The authors should at least discuss how Polycom-mediated gene repression results in these 2 different effects on Hox gene expression, especially in light of the fact that these results are not phenocopied in vivo with the IG-DMR mice.Hox cluster (Hox1~5), while Hox6~13 remain repressed. The transcriptional partitioning of the Hox cluster is mirrored at the level of chromatin. As previously described , and also shown in this study, H3K27me3 decorates the entire Hox cluster in ESCs. Upon differentiation into MNs, H3K27me3 becomes restricted to the caudal segment . It is still not clear how the “repressive epigenetic state of caudal Hox” is maintained in the rostral  MNs, as the PRC2 complex does not manifest a differential expression pattern along the RC axis of the spinal cord. In this study, we found that Meg3 is enriched in the rostral  MNs, and is able to scaffold the PRC2/Jarid2 complex for the robust catalytic activity of depositing H3K27me3. We speculate that Meg3 might be able to further help PRC2/Jarid2 to target to the caudal Hox loci to maintain its repressive H3K27me3 state. Upon Meg3 KD in the cervical MNs, the silent H3K27me3 in caudal Hox loci is compromised. Therefore, the caudal Hox genes  become ectopically up-regulated. The rostral genes are likely repressed by the well-known “posterior prevalence” effect, while the ectopically up-regulated caudal Hox proteins silence the rostral Hox genes directly.It is an interesting point. ESC~MNs exposed to RA activate the rostral portion of the