Vagal sensory neurons (VSNs) form an important body-to-brain connection, navigating visceral organs along the rostral–caudal axis of the body and crossing the surface–lumen axis of organs into appropriate tissue layers6. The brain can discriminate numerous body signals through VSNs, but the underlying coding strategy remains poorly understood. Here we show that VSNs code visceral organ, tissue layer and stimulus modality—three key features of an interoceptive signal—in different dimensions. Large-scale single-cell profiling of VSNs from seven major organs in mice using multiplexed projection barcodes reveals a ‘visceral organ’ dimension composed of differentially expressed gene modules that code organs along the body’s rostral–caudal axis. We discover another ‘tissue layer’ dimension with gene modules that code the locations of VSN endings along the surface–lumen axis of organs. Using calcium-imaging-guided spatial transcriptomics, we show that VSNs are organized into functional units to sense similar stimuli across organs and tissue layers; this constitutes a third ‘stimulus modality’ dimension. The three independent feature-coding dimensions together specify many parallel VSN pathways in a combinatorial manner and facilitate the complex projection of VSNs in the brainstem. Our study highlights a multidimensional coding architecture of the mammalian vagal interoceptive system for effective signal communication.Interoception, the ability to timely and precisely sense changes inside the body, is critical for survival Single-cell profiling of vagal sensory neurons from seven organs in mice and calcium-imaging-guided spatial transcriptomics reveal that interoceptive signals are coded through three distinct dimensions, allowing efficient processing of multiple signals in parallel using a combinatorial strategy. As a key body–brain axis in interoception, VSNs in the nodose and jugular ganglia transmit numerous signals from visceral organs in the respiratory, cardiovascular, gastrointestinal, endocrine and immune systems into the brainstem6. Signals communicated through VSNs are precisely discriminated in the brain for highly specific responses5, yet how this is achieved remains unclear. Extensive data show that VSNs are highly heterogeneous in multiple characteristics, including developmental origins, electrical properties, response patterns, molecular identities, terminal morphologies, sensory mechanisms, anatomical connections and physiological roles19. However, despite the accumulation of a large amount of data over the past seven decades describing the complexity of VSN characteristics, little is known about how such heterogeneity and diversity facilitates interoceptive coding at a systems level.Sensing the body’s internal state is a critical life-ensuring process that maintains physiological homeostasis, provides motivational drivers and shapes our thoughts and emotions20. Similarly, the same bioactive signal (such as serotonin) released from different tissue layers within the intestine communicates different organ information to the brain21. We reasoned that a faithful coding of these features in VSNs is necessary for accurate signal discrimination in the brain. Here we developed several techniques to determine whether and how these essential features of interoceptive signals are coded in VSNs.The physiological role of an interoceptive signal can be specified by three important features: visceral organ; tissue layer; and stimulus modality. Stimuli that differ in these features represent different body changes. For example, stretching the arterial wall implicates an increase in blood pressure, whereas stretching the stomach wall signals food ingestion23. Whether a viscerotopic map exists in the nodose ganglion, where most visceral-organ-innervating VSNs are located6, is a subject of debate27. Using retrograde adeno-associated virus (AAVrg), we show that VSNs labelled from various visceral organs are largely non-overlapping  along the spinal cordWe therefore hypothesize that visceral organs are coded by specific genes in VSNs. We developed a single-cell sequencing approach named ‘Projection-seq’ for unbiased, high-throughput genetic and anatomical dissection of complex neural circuits Fig. . EngineePhox2b+ placode-derived nodose VSNs18  were grouped into 12 subpopulations (A–L) and 52 clusters (A1–L2) on the basis of differentially expressed genes (DEGs) using Seurat and visualized using uniform manifold approximation and projection (UMAP)  Fig. . VSNs laAP) Fig. . The disAP) Fig. . VSN cluAP) Fig. , showingAP) Fig. . VSNs thAP) Fig. . Togethe32. Of note, it seems that VSNs are primed for the functions of their target organs. For example, the stomach contains several anatomically and functionally distinct subregions: the proximal region similar to the oesophagus and the distal region similar to the intestine33. Accordingly, UPB-stomach+/UPB-oesophagus+ VSNs are genetically similar to oesophagus VSNs, whereas UPB-stomach+/UPB-duodenum+ VSNs are closer to duodenum VSNs , cardiovascular (heart), gastrointestinal  and exocrine–endocrine (pancreas) systems Fig. . Gut VSN6. Our discovery of a genetic ‘visceral organ’ trajectory is in accordance with this long-standing observation, suggesting that VSNs might follow morphogen gradients to their target spots. Indeed, signalling gradients formed by secreted morphogens help to establish an anterior–posterior patterning34 and specify peripheral targets for vagal motor neurons35. Together, our data show that instead of a topographic organization, VSNs use a genetic trajectory to code visceral organs along the body’s rostral–caudal axis.Notably, we further identified a genetic trajectory that represents visceral organs along the body’s rostral–caudal axis Fig. . The relGpr65, Sst, Trpv1, Drd2 and Agtr1a  and examined the innervation of tdTomato+ sensory fibres in whole-mount cleared organs. Notably, VSNs along this second trajectory project to different tissue layers   Fig.  and calced organ . The tis17, it is unclear how they contribute to interoceptive coding. Systematic analyses of VSN terminals within the lung, heart, oesophagus, stomach, duodenum and colon in Vglut2tdT mice revealed that there is a marked similarity between many stereotypical VSN ending types in the same tissue layer across visceral organs ; and (6) bud endings wrapped around specialized sensory epithelial cell clusters. Our data thus suggest that VSN ending structures are predominantly organ-independent but tightly associated with tissue layers.We then asked whether VSNs form organ-specific ending structures in various tissue layers. Although VSN endings have been extensively characterized in individual organsans Fig. , includi+ VSNs by nodose infection of AAV-FLEX-tdTomato in corresponding Cre mice; and (3) determined the sensory structures formed by DEG+ VSNs in the target organ or region using whole-organ clearing and volumetric imaging , we (1) identified enriched VSN clusters and their DEGs on the basis of Projection-seq data; (2) labelled DEGing Fig. . With thing Fig. , showing38. Together, our results reveal a two-dimensional genetic representation of the body’s internal space in VSNs that codes the precise anatomical location of interoceptive signals  biological processes for neuron development, cell–cell signalling and synaptic signalling or ion transportation  mice. VSN identity was then determined post hoc in cryo-sectioned nodose ganglia using RNAscope against 21 marker genes that together faithfully represent VSN subpopulations  responsive VSNs were unambiguously registered from six mice  Fig. . First, ice Fig. . VSNs reice Fig. . The vCaPiezo2+ VSNs (55.1%) were activated significantly faster than Piezo2− K/L-VSNs . Uts2b+Vip+Glp1r+Cckar+ G-VSNs represented a large fraction (39.2%) of intestinal stretch responders   Fig. , suggestTo better quantify the relationship between VSN characteristics, we systematically mapped all characterized connections among the five VSN characteristics Fig. . In this47. Consistently, VSN central projections labelled using AAVrg-tdTomato from respiratory, cardiovascular and digestive systems were largely segregated, whereas vagal afferents from functionally related organs terminated in more overlapping areas  guidelines. All mice were age- and gender-matched adults (older than 8 weeks) and no differences between sexes were observed.Glp1r-ires-Cre (029283), Gpr65-ires-Cre (029282), Npy2r-ires-Cre (029285), P2ry1-ires-Cre (029284), Agtr1a-Cre (030553), Calb2-ires-Cre (010774), Nts-Cre (017525), Vglut2-ires-Cre (016963), Sst-ires-Cre (013044), Twist2-Cre (008712), Vip-ires-Cre (010908), Piezo2-GFP-ires-Cre (027719), Trpv1-Cre (017769), Pvalb-Cre (017320), Vglut1-ires2-Cre (023527), Chat-ires-Cre (031661), lox-ChR2 (024109), lox-tdTomato (007914), and Snap25-2A-GCaMP6s-D (025111) were from the Jackson Laboratory. Drd2-Cre (032108-UCD) mice were from the Mutant Mouse Resource and Research Center (MMRRC). lox-L10-GFP mice were described before12.Wild-type C57BL/6J (000664), 12–1013 viral genomes per ml) were generated at the UNC Vector Core. Plasmids have been deposited to Addgene.UPB sequences were cloned from coding sequences of hChR2 , hM3Dq  and hM4Di  and inserted right before the SV40 poly (A) of AAVrg-CAG-tdTomato-WPRE-SV40  using the In-Fusion HD Cloning Kit . Projection-seq AAVs (at titre > 10UPB sequences (5′–3′):UPB-oesophagus :ACAGCACCATCCTCAACTCCACCAAGTTACCCTCATCGGACAACCTGCAGGTGCCTGAGGAGGAGCTGGGGATGGTGGACTTGGAGAGGAAAGCCGACAAGCTGCAGGCCCAGAAGAGCGTGGACGATGGAGGCAGTTTTCCAAAAAGCTTCTCCAAGCTTCCCATCCAGCTAGAGTCAGCCGTGGACACAGCTAAGACTTCTGACGTCAACTCCTCAGTGGGTAAGAGCACGGCCACTCTACCTCTGTCCTTCAAGGAAGCCACTCTGGCCAAGAGGTTTGCTCTGAAGACCAGAAGTCAGATCACTAAGCGGA.UPB-stomach :ATGGACTATGGCGGCGCTTTGTCTGCCGTCGGACGCGAACTTTTGTTCGTTACTAATCCTGTGGTGGTGAACGGGTCCGTCCTGGTCCCTGAGGATCAATGTTACTGTGCCGGATGGATTGAATCTCGCGGCACGAACGGCGCTCAGACCGCGTCAAATGTCCTGCAGTGGCTTGCAGCAGGATTCAGCATTTTGCTGCTGATGTTCTATGCCTACCAAACCTGGAAATCTACATGCGGCTGGGAGGAGATCTATGTGTGCGCCATTGAAATGGTTAAGGTGATTCTCGAGTTCTTTTTTGAGTTTAAGAATCCCTCTATGCTCTACCTT.UPB-duodenum :AATGGCAGCTCGGGCAATCAGTCCGTGCGCCTGGTCACGTCATCATCCCACAATCGCTATGAGACGGTGGAAATGGTCTTCATTGCCACAGTGACAGGCTCCCTGAGCCTGGTGACTGTCGTGGGCAACATCCTGGTGATGCTGTCCATCAAGGTCAACAGGCAGCTGCAGACAGTCAACAACTACTTCCTCTTCAGCCTGGCGTGTGCTGATCTCATCATAGGCGCCTTCTCCATGAACCTCTACACCGTGTACATCATCAAGGGCTACTGGCCCCTGGGCGCCGTGGTCTGCGACCTGTGGCTGGCCCTGGACTGCGTGGTGAGCAACGCCTCCGTCATGAACCTTCTCATCATCAGCTTTGACCGCTACTTCTGCGTCA.UPB-colon :ATCGATGGGCCTTAGGGAACTTGGCCTGTGACCTCTGGCTTGCCATTGACTGCGTAGCCAGCAATGCCTCTGTTATGAATCTTCTGGTCATCAGCTTTGACAGATACTTTTCCATCACGAGGCCGCTCACGTACCGAGCCAAACGAACAACAAAGAGAGCCGGTGTGATGATCGGTCTGGCTTGGGTCATCTCCTTTGTCCTTTGGGCTCCTGCCATCTTGTTCTGGCAATACTTTGTTGGAAAGAGAACTGTGCCTCCGGGAGAGTGCTTCATTCAGTTCCTCAGTGAGCCCACCATTACTTTTGGCACAGCCATCGCTGGTT.UPB-pancreas :AAGATGGCAGGCCTCATGATTGCTGCTGCCTGGGTACTGTCCTTCGTGCTCTGGGCGCCTGCCATCTTGTTCTGGCAGTTTGTGGTGGGTAAGCGGACGGTGCCCGACAACCAGTGCTTCATCCAGTTCCTGTCCAACCCAGCAGTGACCTTTGGCACAGCCATTGCTGGCTTCTACCTGCCTGTGGTCATCATGACGGTGCTGTACATCCACATCTCCCTGGCCAGTCGCAGCCGAGTCCACAAGCACCGGCCCGAGGGCCCGAAGGAGAAGAAAGCCAAGACGCTGGCCTTCCTCAAGAGCCCACTAATGAAGCAGA.UPB-lung :ACAGGACACCGGGTGCAGTGGCTGCGCTATGCAGAGTGGCTGCTCACTTGTCCTGTCATCCTTATCCGCCTGAGCAACCTCACCGGCCTGAGCAACGACTACAGCAGGAGAACCATGGGACTCCTTGTCTCAGACATCGGGACTATCGTGTGGGGGGCTACCAGCGCCATGGCAACCGGCTATGTTAAAGTCATCTTCTTTTGTCTTGGATTGTGCTATGGCGCGAACACATTTTTTCACGCCGCCAAAGCATATATCGAGGGTTATCATACTGTGCCA.UPB-heart :AGGACACTTCCAATGAGTCCAGCTCAGGCAGTGCCACCCAGAACACCAAGGAACGCCCAGCCACAGAGCTGTCCACCACAGAGGCCACCACGCCCGCCATGCCCGCCCCTCCCCTGCAGCCGCGGGCCCTCAACCCAGCCTCCAGATGGTCCAAGATCCAGATTGTGACGAAGCAGACAGGCAATGAGTGTGTGACAGCCATTGAGATTGTGCCTGCCACGCCGGCTGGCATGCGCCCTGCGGCCAACGTGGCCCGCAAGTTCGCCAGCATCGCTCGCAACCAGGTGCGCAAGAAGCGGCAGATGGCGGCCCGGGAGCGCAAAGTGACACGAACGATCTTTGCCATTCTGCTGGCCTTCATCCT.51 represents a powerful genetic tool in VSN studies54. AAVrg-tdTomato and AAVrg-GFP were purchased from the UNC vector core. AAVrg-FLEX-tdTomato (28306-AAVrg), AAVrg-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (84445-AAVrg) and pAAVrg-hSyn-Con/Fon hChR2(H134R)-EYFP (55645-AAVrg) were purchased from Addgene. All viruses contained 1012–1013 viral genomes per ml, and 0.05% Fast Green FCF  was occasionally used to facilitate visualization.Retrograde adeno-associated virus (AAVrg)−1) and buprenorphine (1.5 mg kg−1). Lung: virus  was injected through a tracheal catheter into the lung using a Hamilton syringe. Heart: the mouse was ventilated  using a mouse ventilator  via an intubated angiocatheter. The heart was gently exposed via thoracotomy. Virus (5 μl) was injected intramurally (20 nl per second) using a Nanoject III injector at multiple sites covering most of the area of the heart. Stomach, duodenum, colon and pancreas: the target organ was gently exposed via an abdominal incision. Virus was injected intramurally at multiple sites covering most of the area . Oesophagus: the cervical oesophagus underneath the trachea was surgically exposed via a neck incision. The abdominal oesophagus and the oesophageal sphincter were gently exposed via an abdominal incision. Virus was injected into oesophageal muscularis and serosa layers .In all surgeries, mice were anaesthetized with 1–2% isoflurane on a heating pad, followed by subcutaneous injections of meloxicam  were mixed and co-injected into the stomach in wild-type mice  and reverse-transcribed into cDNA using the SuperScript IV First-Strand Synthesis System . Primer sets used : CATCGATACCGTCGACACAGGACACCGGGTGCAGTG (forward); CTGCTCGAAGCGGCCGCTGGCACAGTATGATAACCCTCG (reverse). UPB4 (5′–3′): CATCGATACCGTCGACATCGATGGGCCTTAGGGAAC (forward); TGCTCGAAGCGGCCGCAACCAGCGATGGCTGTGCC (reverse).17. Approximately 5,000–10,000 VSNs were loaded in each channel of the 10X microfluidic device to target 3,000–6,000 cells as an output of one sample. Single-cell cDNA libraries were prepared at the Yale Center for Genomic Analysis (YCGA) and sequenced using an Illumina NovaSeq S4 sequencer at 150–300 million reads to achieve a fine sequencing depth of 30,000–50,000 reads per cell. For Projection-seq, 30 age- and gender-matched wild-type mice (divided into four samples) were first injected with different Projection-seq AAVs into thoracic and abdominal organs . Vagal ganglia were collected seven days later. Of the examined VSNs, 29.5% (102/346) were tdTomato+. VSNs were sequenced as mentioned above.For control scRNA-seq, vagal ganglia (left and right) were collected from 40 age- and gender-matched C57BL/6J wild-type mice (10 mice per sample) and VSNs were acutely isolated and enriched using previously described methods55, and 42 cell clusters identified using the top 30 principal components (PCs) were visualized using UMAP56  were re-clustered into 67 populations with the top 100 PCs  were re-clustered into 52 clusters with the top 100 PCs and visualized with UMAP separately , and consistently more Sprr1a+ VSNs were observed after AAVrg injection  or a custom mouse genome reference with additional sequence information of UPBs (Projection-seq) using the Cell Ranger software v.3.0.2 (10X Genomics). The following quality control metrics were applied to filter low-quality cells in scRNA-seq: number of genes per cell > 500; number of genes per cell < 8,000; percentage of mitochondria genes < 10%. A total of 56,575 cells were sequenced . Control scRNA-seq and Projection-seq data were then integrated and processed using the R package Seurat v.3tely Fig.. DEGs fotely Fig.. VSN cluPrdm12+ (2.5%) neural-crest-derived jugular VSNs and 4,791 out of 14,590 (32.8%) Phox2b+ placode-derived nodose VSNs59 were recognized as UPB-positive (expression level > 0.8), suggesting that VSNs retrogradely labelled from the seven major visceral organs in our study mainly originate from the nodose but not the jugular ganglia. We therefore focused on nodose VSNs. After removing E-VSNs, 3,539 out of 4,609 UPB-marked nodose VSNs (76.8%) expressed a single barcode , and 740 out of 4,609 (16.1%) VSNs expressed were marked by two UPBs. A correlation matrix . DEGs for organ-specific VSNs, or between thoracic  and abdominal  VSNs were identified using the Wilcoxon rank-sum test . For example, Pou4f1, which is essential for DRG neuron specification32, is preferentially expressed in lung VSNs  using the Upstream Analysis (Upstream Regulators) module. For cell–cell interaction analysis between organ-innervating VSNs and various organ cell types , lung63 , colon64 , duodenum65  and pancreas60 . Cell–cell interactions between organ-innervating UPB+ VSNs and indicated organ cell types  were then analysed using the CellPhoneDB66  Python package. GO pathway analyses of DEGs in heart, lung, gut and pancreas VSNs, and along the tissue trajectory .Further analysis was performed for the Projection-seq dataset. A total of 42 out of 1,701 organ . The organ position score for VSN clusters  as the weight value for each organ, expressed as Σ(Porgan-cluster × Positionorgan)/ΣPorgan-cluster. The organ trajectory score of an organ-specific VSN , and the tissue layer index for DEG+ VSNs  × Indextissue)  Fig. .12. In brief, mice were anaesthetized with 1–2% isoflurane and maintained on a heating pad. Both left and right vagal ganglia were surgically exposed. A virus mix containing a 1:1 dilution of AAV9-FLEX-tdTomato and AAV5-CAG-GFP with 0.05% (w/v) Fast Green FCF was injected  using a Nanoject III injector (Drummond). Mice were euthanized four weeks after surgery for tissue collection (see ‘Histology and immunochemistry’).Vagal ganglia injection was performed as previously describedRNAscope HiPlex assays were performed following the manufacturer’s protocol (Advanced Cell Diagnostics). Vagal ganglia were acutely dissected and freshly frozen in cryo-embedding medium (OCT). Cryosections  were cut using a cryostat (Thermo Fisher Scientific), mounted onto Superfrost Plus slides (Thermo Fisher Scientific), and stored at −80 °C until use. Slides were immediately immersed into fresh 4% paraformaldehyde (PFA) in RNase-free PBS for 60 min at room temperature, followed by dehydration with 50%, 70% and 100% ethanol. Samples were then digested with protease IV for 30 min at room temperature. After hybridization with designed probes , followed by 25 ml cold 4% PFA. Fast Green FCF injected organs  and transcardially perfused with 15 ml cold PBS (pH 7.4) containing 10 U mlFor brain samples, 40-μm cryosections were mounted onto Superfrost Plus slides. Cryosections were washed (3× PBS), permeabilized , blocked ) and incubated with primary antibodies  diluted in blocking buffer for 2 h at room temperature. Then, the slides were washed (3× PBST), and incubated with fluorophore-conjugated secondary antibodies , 1:1,000, Jackson ImmunoResearch) diluted in blocking buffer for 2 h at room temperature. After incubation, the samples were washed (3× PBST), and mounted with Fluoromount-G with DAPI before imaging with the Leica SP8 confocal microscope.72 and stained with the following protocol unless specifically mentioned. In brief, the dissected organ was immersed into 1/2-water-diluted reagent-1  with shaking at 37 °C for 3–6 h, followed by reagent-1 (R1) with shaking at 37 °C for 7 days. R1 was replaced fresh every two days. Next, the tissue was washed (3× PBS/0.01% NaN3), blocked  and incubated with primary antibodies  in blocking buffer with shaking for 7 days at room temperature. Samples were then washed  and incubated with fluorophore-conjugated secondary antibodies diluted in blocking buffer with shaking for five days at room temperature. After antibody incubation, the samples were washed and immersed in 1/2-PBS-diluted reagent-2  overnight at room temperature, and then reagent-2 (R2) at 37 °C for 2 days. The samples were finally immersed in oil for at least 1 h and flattened to approximately 500 μm in a custom-built imaging chamber and imaged using the Leica SP8 confocal microscope as described above, with a 10× objective  or a 40× objective . Some heart samples were similarly processed but not flattened and imaged with a LaVision Vltramicroscope II light-sheet microscope at the CNNR Imaging Core at Yale University , 1:1,000) overnight, respectively, and treated with R2 for one day. Cleared ganglia were imaged with the Leica SP8 confocal microscope.tdT-GCaMP6s mice were continuously anaesthetized (1–2% isoflurane/oxygen) during the experiment. A tracheal tube was inserted for air injection. The upper oesophagus and pyloric sphincter were cannulated and flushed with saline multiple times to remove residual food particles. The duodenum (around 0.5 cm below the pyloric sphincter) was cannulated with a bundle of six PE-10 tubing for separate delivery of saline, water and glucose (1 M), Ensure , 10× PBS and 150 mM HCl (pH = 0.84). Left vagal ganglia were exposed and immobilized on a stable platform12. During calcium imaging, a series of stimuli were delivered to the same mice in the following sequence: (1) lung inflation for 20 s with 600 ml min−1 flow , twice with a 2-min interval; (2) small intestine stretch via fast injection of 600 μl saline through the duodenal cannula; stomach stretch with 100 μl, 300 μl and 600 μl saline through the oesophageal cannula for 30 s (duration precisely controlled by closing or opening of the pyloric cannula); (4) small intestine infusion with 100 μl saline, water, 1 M glucose, Ensure, 10× PBS and 150 mM HCl in sequence with a 3-min interval between infusions. GCamp6s fluorescence was measured from two focal planes 15 μm apart using a two-photon microscope . The imaging frequency for each plane was 1.72 s per frame. At the end, electrical stimulation was applied to the vagus nerve and a z-stack of the ganglia was taken for both GCaMP and tdTomato  signals for cell registration.Gpr65Trpa1 (R1T1), Runx3 (R1T2), Uts2b (R1T3), Gabra1 (R1T4), Slit2 (R1T5), Kcng1 (R1T6), Piezo2 (R1T7), Ddc (R1T8), Vip (R1T9), Trpv1 (R1T10), tdTomato (R1T11), Gpr65 (R1T12), Chodl (R2T1), Glp1r (R2T2), Grm5 (R2T3), Slc17a7 (R2T4), P2ry1 (R2T5), Tmc3 (R2T6), Car8 (R2T7), Nts (R2T8), Cckar (R2T9) and Calca (R2T11). The following criteria were used to categorize VSN subpopulations  to generate successive in vivo imaging planes that resemble RNAscope sections. tdTomato− neurons were then registered on the basis of their relative distance and depth to their neighbouring tdTomato+ cells  was defined as the average GCaMP6s fluorescence over a 10-frame period  before stimulus induction and neuronal activity was calculated as ΔF/F. Cells were coded as responsive to a given stimulus if the maximum GCaMP6s fluorescence was more than 100% above baseline during stimulus period . Peak response was identified as the maximum ΔF/F within the stimulus period. To compare adaptation rates in lung stretch-sensitive VSNs, GCaMP6s traces were aligned at activation frame  reached 10% of the peak response  reached 10% of peak response.Regions of interest were manually extracted from GCaMP images. The stimulus induction frame was set as 0 unless specifically mentioned. Baseline signal . For quantitative analyses of MEs and IMAs in the indicated gastrointestinal regions; alveoli, longitudinal and patch-terminals in the lung; and varicose endings and IMAs in the heart .Four types of gut VSN ending types, three types of heart VSN ending types and five types of lung VSN ending types were classified on the basis of their morphologies and locations using whole-mount preparations in Vglut2ice Fig. ; n = 4–7Piezo2), H4 (Drd2) and I3/I4 (Agtr1a). In Piezo2tdT mice, most cardiac afferents were varicose surface endings. In Drd2tdT mice, cardiac afferents densely innervated myocardium with both branched and parallel IMAs. Agtr1a+ VSNs predominantly formed flower-spray endings in the heart and the aortic arch. Our results thus reveal the identity of various VSN cardiac ending types  bud ending wrapping around NEBs  near the bronchial bifurcation with branch endings  and examined IMAs around pyloric sphincters in P2ry1tdT and Calb2tdT mice. Most (75.0%) of the P2ry1+ IMAs around the oesophageal sphincter were pIMAs and all Calb2+ IMAs were cIMAs , H2 (Vip) and C4 , whereas all afferent types in Vglut2tdT mice were enriched in region 8 around the pyloric sphincter. Both Sst+ and Gpr65+ VSNs formed MEs on the stomach, as reported16, with Sst+ endings mainly in the antrum and Gpr65+ endings more evenly distributed across the stomach, indicating that F1-VSNs form MEs. Both Glp1r+ and Piezo2+ VSNs predominantly formed IGLEs, suggesting that C4-VSNs form stomach IGLEs. Vip+ VSNs exhibited pIMA morphology in region 8, suggesting that H2-VSNs also form pIMAs  for each ending type  in each stomach region , calculated as the innervation intensity of corresponding ending types in various stomach regions normalized by the innervation intensity of the same ending type in the entire stomach in Vglut2tdT mice  of each ending type  in each stomach region  was calculated as the percentage of corresponding dual-labelled VSNs forming this ending type normalized to the percentage of stomach-UPB single-labelled VSNs forming this ending type, expressed as Σ/Σ. We then calculated the total variance between anatomically and Projection-seq-derived innervation intensities in all stomach regions across all ending types, expressed as Σ (SFnME- − AFnME-)2 + Σ (SFnpIMA- − AFnpIMA-)2 + Σ (SFncIMA- − AFncIMA-)2 + Σ (SFnIGLE- − AFnIGLE-)2. The trial with the lowest variance was defined as the best fit. AFafferent type-stomach region and SFafferent type-stomach region for this condition were plotted together , Vip (H3/G2/G5), Glp1r (G2/G5), and Agtr1a (I5) , suggesting that both F- and G-VSN clusters form MEs. By contrast, Agtr1a+ neurons representing I5-VSNs primarily formed IGLEs in the duodenum , C5–C8 VSNs also form oesophageal IMAs. Probably owing to low infection efficiency, no apparent oesophageal ME or IGLE clusters were revealed. Notably, among all Cre lines examined, afferents in NTStdT mice preferentially formed oesophageal IGLEs , expressed as (number of connections − minimum number of possible connections)/(maximum number of possible connections − minimum number of possible connections), for each pair of characteristics. We next calculated the statistical variance of connections per variable for both characteristics (v1 and v2). As both variances contribute to the correlation equally, the correlation index was finally calculated as (1 − C)/((v1 + 1) × (v2 + 1)).Correlations among various VSN characteristics, including 7 visceral organs , 4 tissue layer types , 6 VSN ending types , 11 VSN subpopulations and 4 response patterns , were plotted in Fig. Mouse line and organ combinations were selected on the basis of Projection-seq anterograde tracing results: lung alveoli ending , lung NEB ending , oesophageal IGLE , oesophageal IMA , heart IMA , stomach IGLE , stomach mucosal ending , stomach IMA , duodenal IGLE , duodenal mucosal ending  and colon IGLE . Organ injection of AAVrgs is described in ‘AAV infection of visceral organs’. Brain processing and imaging is described in ‘Histology and immunochemistry’.Bregma-subnucleus) was calculated as average fluorescence in the sub-nucleus minus background fluorescence measured in a region in the sub-nucleus with no fluorescence-labelled vagal fibres; (2) the area of each sub-nucleus (ABregma-subnucleus) was measured; (3) the total fluorescence (TFBregma) was calculated as Σ (FIBregma-subnucleus × ABregma-subnucleus); and (4) the percentage innervation (PI) of a sub-nucleus at a certain Bregma was calculated as FIBregma-subnucleus × ABregma-subnucleus/TF × 100. The correlation variance between VSNs labelled from various visceral organs 2 and a phylogenetic tree was generated based on the correlation variance matrix using the seqlinkage function in MATLAB.Area innervated by vagal afferents retrogradely labelled from various visceral organs at different Bregma levels  were measured using Fiji (ImageJ) , heart (n = 3); varicose: heart (n = 5), aorta (n = 6); pIMA: colon (n = 3), heart (n = 6); cIMA: stomach (n = 4), oesophagus (n = 3); epithelial: stomach (n = 4), lung (n = 5); bud: lung (n = 5), oesophagus (n = 3); Extended Data Fig. n = 3; Extended Data Fig. n = 2; Extended Data Fig. n = 3; Extended Data Fig. n = 3; Extended Data Fig. n = 2; Extended Data Fig. n = 3; Extended Data Fig. n = 2; Extended Data Fig. n = 4; Extended Data Fig. n = 5; Extended Data Fig. n = 2; Extended Data Fig. n = 4; Extended Data Fig. n = 4; Extended Data Fig. n = 4; Extended Data Fig. n = 3), stomach (n = 4), duodenum (n = 3), colon (n = 3), aorta (n = 6); Extended Data Fig. n = 3; Extended Data Fig. n = 4; Extended Data Fig. Piezo2 (n = 9), Drd2 (n = 6), Agtr1a (n = 3), Npr2r (n = 2); Extended Data Fig. n = 4; Extended Data Fig. Npy2r (n = 3), Trpv1 (n = 5), P2ry1 (n = 6), Agtr1a (n = 4), Piezo2 (n = 9), Vglut1 (n = 4), Pvalb (n = 3), Twist2 (n = 3); Extended Data Fig. P2ry1 (n = 4), Calb2 (n = 3), Glp1r (n = 5), Vip (n = 5), Gpr65 (n = 7), Sst (n = 5), Agtr1a (n = 12); Extended Data Fig. Glp1r (n = 3), Agtr1a (n = 4); Extended Data Fig. Trpv1 (n = 4), Agtr1a (n = 3); Extended Data Fig. Nts (n = 5), Trpv1 (n = 3), Agtr1a (n = 4), Gpr65 (n = 3); Extended Data Fig. n = 5; Extended Data Fig. n = 5; Extended Data Fig. n = 3), heart (n = 3), oesophagus (n = 2), stomach (n = 3), duodenum (n = 3), colon (n = 3), pancreas (n = 3); Extended Data Fig. n = 3 for all groups.Representative images and experiments were repeated independently in multiple mice with similar results: Fig. Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-022-04515-5.Supplementary Figure 1Original source image for electrophoresis, related to Extended Data Fig. 2b. Red box indicates how the gel was cropped for the final figure.Reporting SummarySupplementary Table 1Detailed information for RNAscope probes.


However, the mechanisms underlying the regulation of FTO and its subsequent impact on the regulation of the epitranscriptome remain to be further elucidated. Here, we demonstrate that FTO expression is downregulated and inversely correlated with poor survival of lung adenocarcinoma patients. Mechanistically, Wnt signaling induces the binding of EZH2 to β-catenin. This protein complex binds to the LEF/TCF-binding elements at the promoter region of FTO, where EZH2 enhances H3K27me3 and inhibits FTO expression. Downregulated FTO expression substantially enhances the m6A levels in the mRNAs of a large number of genes in critical pathways, particularly metabolic pathway genes, such as MYC. Enhanced m6A levels on MYC mRNA recruit YTHDF1 binding, which promotes MYC mRNA translation and a subsequent increase in glycolysis and proliferation of tumor cells and tumorigenesis. Our findings uncovered a critical mechanism of epitranscriptome regulation by Wnt/β-catenin-mediated FTO downregulation and underscored the role of m6A modifications of MYC mRNA in regulating tumor cell glycolysis and growth.FTO removes the N6-methyladenosine (m The abundance and effects of m6A on RNA depend on the dynamic and integrated regulation by “writers” and “erasers”, which add and remove the methylation, respectively, and “readers”, which recognize the modification8. The identified writers include methyltransferase-like (METTL) 3/14, Wilms tumor 1-associated protein (WTAP), RNA binding motif protein 15/15B (RBM15/15B), and KIAA1429, whereas erasers include fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5). YT521-B homology (YTH) domain-containing proteins , heterogeneous nuclear ribonucleoprotein (HNRNP) protein families, and IGF2 mRNA binding proteins (IGF2BP) families are regarded as readers9. FTO, as the first m6A demethylase identified11, regulates the m6A modification of critical genes in different types of cancer, such as glioblastoma12, acute myeloid leukemia (AML)14, cervical squamous cell carcinoma (CSCC)15, and breast cancer16. However, the mechanisms underlying the regulation of FTO and its subsequent impact on the regulation of the epitranscriptome remain to be further elucidated.N6-Methyladenosine : normal rabbit IgG (# 2729) (for immunoprecipitation and ChIP), β-catenin (#8480) (for immunoblotting and immunoprecipitation), FTO (#31687) (for immunoblotting and RIP), EZH2 (#5246) (for immunoprecipitation and ChIP), TCF4 (#2569) (for ChIP), H3K27me3 (#9733) (for ChIP), H3K9me2 (#4658) (for ChIP), HK2 (#2867) (for immunoblotting and IHC), and Ki-67 (#9449) (for IHC). A mouse monoclonal antibody against tubulin (T9026) (for immunoblotting) was purchased from Sigma-Aldrich . Rabbit antibodies recognizing c-Myc (ab32072) (for immunoblotting and IHC), Wnt-3a (ab219412) (for IHC) and FTO (ab124892) (for IHC) were purchased from Abcam . Rabbit antibody recognizing YTHDF1 (17479-1-AP) (for RIP and immunoblotting) was purchased from Proteintech . RIPA lysis and extraction buffer (89901) and Pierce IP lysis buffer (87787) were purchased from Thermo Fisher Scientific . Protein A/G plus-agarose (sc-2003) was purchased from Santa Cruz Biotechnology . Wnt-3a (5036-WN) was obtained from R&D Systems . Cycloheximide (CHX) (HY-12320) and nitro blue tetrazolium chloride (HY-15925) were purchased from MedChem Express . Agar (1182GR500) was purchased from BIO FROXX .http://xena.ucsc.edu/welcome-to-ucsc-xena/). Gene transcription estimates for each gene were analyzed with RNA-Seq using Expectation Maximization (RSEM) software.The clinical records and RNAseqV2 level 3 gene-level lung adenocarcinoma data were downloaded from TCGA (Forty pairs of frozen tissues for RNA extraction and 83 pairs of frozen tissues for immunohistochemistry (IHC) were obtained from patients with lung adenocarcinoma who underwent radical resections in the Department of Thoracic Surgery of the Cancer Hospital, Chinese Academy of Medical Sciences. The clinical features of the patients are summarized in Table S17. Section of lung adenocarcinoma TMA was stained with an antibody against FTO. The tissue sections were quantitatively scored according to the percentage of positive cells and staining intensity as described previously18. The following proportion scores were assigned to the sections: 1, 0–1%; 2, 2–10%; 3, 11–30%; 4, 31–70%; and 5, 71–100%. The staining intensity was rated on a scale of 0–3: 0, negative; 1, weak; 2, moderate, and 3, strong. Then the proportion and intensity scores were combined to obtain a total score  as described previously18.Eighty-three pairs of frozen tissues from lung adenocarcinoma patients were formalin-fixed and paraffin-embedded. The tissue microarray (TMA) was constructed as previously described2 at 37 °C in a humidified incubator. And all these cells were routinely tested for mycoplasma. When cells were 50% confluent, the medium was replaced with a fresh medium containing 0.5% serum for 1 day, and then Wnt-3a was added at a final concentration of 60 ng/ml for cell stimulation.H322 and HEK 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin. H358 cells were grown in RPMI 1640 supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin. Cells were cultured in 5% CO19. The relative mRNA expression levels were calculated by the 2−ΔΔCt method with normalization to ACTB or GAPDH; the PCR primers are listed in Table STotal RNA was isolated with TRIzol reagent  according to the manufacturer’s instructions. RNA (1000 ng) was reverse-transcribed into cDNA with a RevertAid First Strand cDNA Synthesis kit (Thermo); SYBR Green-based qRT-PCR was performed using a 7900HT fast real-time PCR system , as described previously20. The shRNA sequences are listed in Table SPlasmids containing transgenes and packaging plasmids were cotransfected into HEK 293T cells using Lipofectamine 3000 . Viruses were collected and concentrated after 48 h. When tumor cells reached 50%-60% confluence, we infected the cells with concentrated virus and then selected them by antibiotic treatment21.Extraction of proteins with a modified buffer from cultured cells was followed by immunoprecipitation and immunoblotting with antibodies, as described previously22. The primers are listed as follows:ChIP assays were performed using the SimpleChIP® Enzymatic Chromatin IP kit  according to the manufacturer’s instructions, as described previouslyFTO promoter Forward: GTTATCCTTCTTTGCTCACTATGC;binding site within the FTO promoter Reverse: CTGAGGAAGTGAACTGAGCTC.binding site within the 6A reporter assays, the DNA fragments of MYC-CDS containing the wild-type m6A motifs, as well as the mutated motifs (m6A was replaced by T) were inserted into the Xhol site of the pMIR-REPORT luciferase reporter vector. Dual-luciferase reporter assays were performed, as described previously in HEK 293T cells23.For the promoter-reporter assay, the wild-type DNA oligos of the FTO promoter and the mutated oligos with three LEF/TCF-binding element (TBE) deletions were inserted into the upstream region of the firefly luciferase of the PGL4.1 vector. For mSequences of wild type FTO promoter:CTTTGCTCACTATGCTTCACTTA CATTATTCTTTACTTTCCTCGAACCCCCCATACCCTTGTCTTGCTCAAGG CCTTTGTATTAGCTGGTTCCTTAATCTTTGGAGCTCAGTTCACTTCCTCA GACAGGTTTTCCCTGACCATCCTATGTTAGAGTAGTCTTCCTTACATTTC TTCACTGTTTATTTCTTTTCTTTTCTTTTTTTTTTTTTTGAGACAGGGTC TTGCTATGTTGCCCGGGCTGGCCTTGAATTCATGGGTTCAAGTGATCCTC CCACCTCAGCCTCCCGAGTAGCTGGAACTACATGTGCGTGCCACCAAGCA TGGCTTGTATCTCTTATAGCAACTGCCTCTATCTGAAGTTATCAGATAAA ATTATTGTTTGTCTCCACTAAAAAAGGATAAACATCTTGAGACGGGTATT AGTCTTGTTCACAACTGTTCAGGAACAGTGCCTGGTACAGGGTGGGAACC AACATTAATATTTATTGAATGATTGGCTGTGCGTGGTGGCTCACACCTGT AATCCCAGCACTTTGCGAGGCCGAGACGGGCGGCTGACTTCAGGCCAGGA GTTCGAGACCAGCCTGGCCAACATGATGAAACCCTGTCTCTACTAAAAAT ACAAAAATTAGCTGGGTGTGGTGGCACACGCCTGTAATCCCAGCTACTCG GGAGGCTGAGGCAGAAGAATCGCTTGAACCTGGGAGGAGGAGTTTGCAGT AAGCTGAGGTCTTACCACTGCACTCCAGCCTGGGCAACGGAGCAAGAACC TGTCACACACACAAAAAAAAGAATAAAGAAAAAATATTTATTGAATGAAT AAATGAATATCAGGTACTGAGATTAAAATGGCAAGCAAAACCCCCGCCTT TATGAAGCTAGCAAGTTATGGAGGTAATCACATGATAAACAAATAATATA TAATTAAGCAAACAATAGACCACTCAGGAGGTTTAGGGTCTACCAACCAA CTCCTAATCCAGGGCAAATGAGCAAACTGTGTTAGGGACCTACAACTTGC AGGATCTGGATAGAGATGGCAATTAGCAGCATCAACTCTCACCTTCATGG CTGGGATATAACATTTCAAATTGGTCCTGGACGTGGGGATAAAGGGCGGC CTGTGATTCAGGCCTGAGGATGTGGAGGTGTCTTGGGCTGGGCTGCTTTC ACGCCAGCAGAACTCCAGGGCCAACTCCAGGGCCTTCTCCAGGCGGCAGA GCGGACCCTAGGACCCCGGCCCGCGCTGCAGTGGGGAGGGTCAGCAACCT CCACCCACCCTCATCCTCCCCCATCCTCCCGGGTACTCACCGTGCCACTG GCCCTGCAGCTAGCTACCGTTGCTATAGCGCCGACAGCGTGGCGGGCGGC TGGCCGAGAGGAGCACGGGAGAAACATGGCAGGCTCCCGTAGCCTCCTGG GAAATGTAGTTCTCCTTGGACTCTAGCCTGTTTGCTCGCGGGGTAGCGGA CTCATTTATGCTTGGTGTTATGATTGTAACTAAGAATCCTGGAGTGAGCT GGTTACAAAGTGAGCCCGACTTTCCATGGATGCACCATCCTAGAGTGCACGGAAGTACTCCTATAGAAAAGGTCAATTTTTAGGATCCTGTTGACACATA GGCCCGTGTATGAAAATGATTAGTTTTCCATGACAGAGTTAAGGTCACTT TAAAAATAATAATGATGATGATGATGATGATGGTGTTAACATTCATTGAA CGCTTACTATGTGCCAGGTACTGTTCTAAGTGTTCTGTTATAGGAATGAA GTGTCTCACCATATCCTTGTGAGGCTGTTACTCAAATGATTCCTGCTTTA CAAATGAGGAAGCTGAGACACAGATTAGTTAACTCACTTAAGGTGGTAGT TGAAAGTATTAATAGTTGTGTCTGGTGATATTTTTGGTTGTCACAACAAG GAAGCGGGATGCTACTGGCATCTAGTGAGTAGAGGCCATGGATGATGCTA AATATCTTACAGTGCTTAGGAGACATAATAATGAATTATCCAGCCCAAAA TGTTAATAATAGTGTAGAAGCTGAAAAACCCTGCACAATGCTGCAATGCC TCTCCAACACCATCTTATGTTATCCTTSequences of truncated FTO promoter:GGAAGTACTCCTATAGAAAAGGTCAATTTTTAGGATCCTGTTGACACATA GGCCCGTGTATGAAAATGATTAGTTTTCCATGACAGAGTTAAGGTCACTT TAAAAATAATAATGATGATGATGATGATGATGGTGTTAACATTCATTGAA CGCTTACTATGTGCCAGGTACTGTTCTAAGTGTTCTGTTATAGGAATGAA GTGTCTCACCATATCCTTGTGAGGCTGTTACTCAAATGATTCCTGCTTTA CAAATGAGGAAGCTGAGACACAGATTAGTTAACTCACTTAAGGTGGTAGT TGAAAGTATTAATAGTTGTGTCTGGTGATATTTTTGGTTGTCACAACAAG GAAGCGGGATGCTACTGGCATCTAGTGAGTAGAGGCCATGGATGATGCTA AATATCTTACAGTGCTTAGGAGACATAATAATGAATTATCCAGCCCAAAA TGTTAATAATAGTGTAGAAGCTGAAAAACCCTGCACAATGCTGCAATGCC TCTCCAACACCATCTTATGTTATCCTTACTATGCTTCACTTA CATTATTCTTTACTTTCCTCGAACCCCCCATACCCTTGTCTTGCTCAAGG CTAGCTGGTTCCTTAATCTCAGTTCACTTCCTCA GACAGGTTTTCCCTGACCATCCTATGTTAGAGTAGTCTTCCTTACATTTC TTCACTGTTTATTTCTTTTCTTTTCTTTTTTTTTTTTTTGAGACAGGGTC TTGCTATGTTGCCCGGGCTGGCCTTGAATTCATGGGTTCAAGTGATCCTC CCACCTCAGCCTCCCGAGTAGCTGGAACTACATGTGCGTGCCACCAAGCA TGGCTTGTATCTCTTATAGCAACTGCCTCTATCTGAAGTTATCAGATAAA ATTATTGTTTGTCTCCACTAAAAAAGGATAAACATCTTGAGACGGGTATT AGTCTTGTTCACAACTGTTCAGGAACAGTGCCTGGTACAGGGTGGGAACC AACATTAATATTTATTGAATGATTGGCTGTGCGTGGTGGCTCACACCTGT AATCCCAGCACTTTGCGAGGCCGAGACGGGCGGCTGACTTCAGGCCAGGA GTTCGAGACCAGCCTGGCCAACATGATGAAACCCTGTCTCTACTAAAAAT ACAAAAATTAGCTGGGTGTGGTGGCACACGCCTGTAATCCCAGCTACTCG GGAGGCTGAGGCAGAAGAATCGCTTGAACCTGGGAGGAGGAGTTTGCAGT AAGCTGAGGTCTTACCACTGCACTCCAGCCTGGGCAACGGAGCAAGAACC TGTCACACACACAAAAAAAAGAATAAAGAAAAAATATTTATTGAATGAAT AAATGAATATCAGGTACTGAGATTAAAATGGCAAGCAAAACCCCCGCCTT TATGAAGCTAGCAAGTTATGGAGGTAATCACATGATAAACAAATAATATA TAATTAAGCAAACAATAGACCACTCAGGAGGTTTAGGGTCTACCAACCAA CTCCTAATCCAGGGCAAATGAGCAAACTGTGTTAGGGACCTACAACTTGC AGGATCTGGATAGAGATGGCAATTAGCAGCATCAACTCTCACCTTCATGG CTGGGATATAACATTTCAAATTGGTCCTGGACGTGGGGATAAAGGGCGGC CTGTGATTCAGGCCTGAGGATGTGGAGGTGTCTTGGGCTGGGCTGCTTTC ACGCCAGCAGAACTCCAGGGCCAACTCCAGGGCCTTCTCCAGGCGGCAGA GCGGACCCTAGGACCCCGGCCCGCGCTGCAGTGGGGAGGGTCAGCAACCT CCACCCACCCTCATCCTCCCCCATCCTCCCGGGTACTCACCGTGCCACTG GCCCTGCAGCTAGCTACCGTTGCTATAGCGCCGACAGCGTGGCGGGCGGC TGGCCGAGAGGAGCACGGGAGAAACATGGCAGGCTCCCGTAGCCTCCTGG GAAATGTAGTTCTCCTTGGACTCTAGCCTGTTTGCTCGCGGGGTAGCGGA CTCATTTATGCTTGGTGTTATGATTGTAACTAAGAATCCTGGAGTGAGCT GGTTACAAAGTGAGCCCGACTTTCCATGGATGCACCATCCTAGAGTGCAC6A sites:MYC-CDS with wild-type mGGACTTGTTGCGGAAACGACGAGAACAGTTGAAACACAAACTTGAACAGCTACGGAACTCTTGTGCGGTAGTTATCCTTAAAAAAGCCACAGCATACATCCTGTCCGTCCAAGCAGAGGAGCAAAAGCTCATTTCTGAAGA6A sites:MYC-CDS with the mutated mGGTCTTGTTGCGGAAACGACGAGATCAGTTGAATCACAATCTTGATCAGCTACGGATCTCTTGTGCG.GTAGTTATCCTTAAAAAAGCCACAGCATACATCCTGTCCGTCCAAGCAGAGGAGCAAAAGCTCATTTCTGAAGA5 cells were seeded in a 6-well plate and maintained in a medium with 10% FBS for different periods of time. The cells were harvested and counted24.2 × 1025. Briefly, for the bottom layer of agar, we deposited the mix of 1% agar and 2× medium into each well of a six-well plate. For the upper layer of agar, we deposited the mix of 0.6% agar and a suspension of cells in each well (10000 cells/well). The cells were cultured for 21 days in a 37 °C humidified cell culture incubator and then were stained with nitroblue tetrazolium chloride solution (200 μl/ well) and incubated overnight at 37 °C.The assay was performed as described previously26.Migration and invasion assays were performed in the chamber coated with or without Matrigel matrix  according to the manufacturer’s instructions as previously described6 Cells were seeded in 6 cm plates. After incubation for 24 h, the culture medium was replaced and the cells were arrested at the S phase by adding thymidine (2 mM) for 24 h. The thymidine was then removed, and the cells were washed by PBS and cultured in a fresh culture medium for 3 h to release cells before nocodazole treatment (100 ng/ml) for 12 h to arrest cells in the G2/M phase. Then, the cells were washed with PBS and cultured in a fresh medium to release cells27.2 × 10g for 5 min, followed by washing with PBS. The cells were then fixed in 1 ml ice-cold 70% ethanol at 4 °C for 24 h and centrifuged at 500 × g for 5 min. After being washed with PBS, the fixed cells were fully resuspended in 1 ml of 2 M HCl/0.5% Triton X-100 solution and incubated at room temperature for 30 min. The cells were spun down and neutralized in 1 ml of 0.1 M Na2B4O7 solution . Then, the cells were spun down and washed with cold PBS containing 1% BSA and incubated with mouse anti-BrdU  at 4 °C for 12 h. Afterward, centrifuged cells were washed with cold PBS containing 1% BSA and incubated with Alexa Fluor 647-conjugated goat anti-mouse IgG  in room temperature for 1 h. Finally, the cells were spun down and washed with cold PBS containing 1% BSA, followed by resuspended in 300 μl of PBS containing 5 μg/ml DAPI for 15 min at room temperature, and then cell cycle progression was analyzed by flow cytometry.One hour before harvesting cells at each time point, BrdU  (20 μM) was used to label the cells. The cells were collected by centrifugation at 500 × 18.Cells were seeded in culture dishes, and the medium was changed when cells reached 50% confluence. After incubation for 12–24 h, the culture medium was collected. The glucose levels were detected by a glucose colorimetric assay kit , and the lactate levels were detected by a lactate colorimetric assay kit  according to the manufacturer’s instructions, and values were calculated as previously described6 cells were injected subcutaneously into the flank regions of female BALB/c nude mice (4–5 weeks). The width (W) and length (L) of the tumors were measured every three days, and the volume (V) of each tumor was calculated using the formula V = (W2 × L/2). For lung colonization assays, 1 × 106 cells were injected into the tail vein of female NOD/SCID mice (6–7 weeks), and 6 weeks later the lung was removed and fixed with 10% formalin. Fixed lung tissues were embedded in paraffin and cut into consecutive sections. These sections were stained by hematoxylin and eosin (H&E)28. All animal experiments were approved by the Animal Care and Use Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences.Mice were randomized into several groups. For the subcutaneous implantation model, 1 × 10A Magna RIP kit  was used to perform the RIP assays. A sufficient number of H322 cells were lysed by RIP lysis buffer, and the supernatant of the RIP lysate was incubated with specific antibodies on beads overnight at 4 °C. After washing, RNA was extracted and analyzed by qRT-PCR.6A-seq was performed by Cloudseq Biotech Inc.  according to the published procedure with slight modifications1. Briefly, fragmented mRNA was incubated with an anti-m6A polyclonal antibody  in IPP buffer for 2 h at 4 °C. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher) at 4 °C for an additional 2 h. The bound RNA was eluted from the beads with N6-methyladenosine  in IPP buffer, and then the RNA was extracted with TRIzol reagent (Thermo Fisher) following the manufacturer’s instructions. Purified RNA was used for RNA-seq library generation with the NEBNext® Ultra™ RNA Library Prep kit (NEB). Both the input sample without immunoprecipitation and the m6A IP samples were subjected to 150 bp paired-end sequencing on an Illumina HiSeq 4000 sequencer.m29. After that, clean reads of all libraries were aligned to the reference genome by Hisat2 software (v2.0.4)30. Methylated sites on RNAs (peaks) were identified by MACS software31. Differentially methylated sites were identified by diffReps32. These peaks identified by both software and overlapped with exons of mRNAs were selected. Raw counts of mRNA sequencing were got by HTSeq software (v0.9.1) and normalized by edgeR software.Paired-end reads were harvested from Illumina HiSeq 4000 sequencer and were quality-controlled by Q30. After 3′ adapter-trimming and low-quality reads removal by cutadapt software (v1.9.3), clean reads of input libraries were aligned to reference genome (UCSC HG19) by STAR software6A kit  was used according to the manufacturer’s instructions. m6A enrichment was analyzed by qPCR with specific primers, and data were normalized to input. Primer sequences were as follows:Total RNA was isolated with a miRNeasy kit  with DNase I digestion. mRNA was extracted from total RNA using a GenElute mRNA Miniprep kit . Then, a Magna MeRIP mMYC Forward: TTGCGGAAACGACGAGAACA;MYC Reverse: TCATAGGTGATTGCTCAGGACA.33.Cycloheximide (CHX) was added to the cell culture at a final concentration of 100 μg/ml for 10 min to stop translation. Cells were washed with cold PBS and lysed with lysis buffer. The cell lysate was loaded onto the top of a 10–50% sucrose gradient tube immediately, and the tube was centrifuged at 36,000 rpm for 3.5 h at 4 °C. The sample was separated into 12 fractions by a fraction collector and measured at 254 nm. RNA was extracted by TRIzol and subjected to qPCR analysis. The relative expression of MYC in each fraction was normalized to GAPDH, as well as to the inputt-tests to compare means between two groups; p < 0.05 was considered significant.All data are expressed as the mean ± SD. We used two-tailed Student’s 35. As a major type of non-small-cell lung carcinoma, lung adenocarcinoma (LUAD) remains one of the most aggressive and fatal tumor types36. To determine the expression of FTO in lung adenocarcinoma, we analyzed TCGA data and revealed that FTO expression levels were much lower in lung adenocarcinoma tissues than in their adjacent normal tissues  accounts for 85% of cases, is the most commonly occurring cancer and the leading cause of cancer deathues Fig. . To valiues Fig. . Consistues Fig. C, D. Kapues Fig. . These rTo determine the role of FTO in lung adenocarcinoma progression, we depleted FTO expression in H322 Fig.  and H358We next subcutaneously injected H358 cells with or without FTO depletion into athymic nude mice. As shown in Fig. FTO promoter sequence with PROMO software and identified three potential LEF/TCF-binding elements (TBE)  assays demonstrated that Wnt stimulation increased the binding of TCF4 to the promoter region of FTO  Fig. S that areFTO Fig. . LuciferRNA Fig.  and protRNA Fig.  levels oion Fig. . These r42. ChIP analyses with anti-H3K27me3 and anti-H3K9me2 antibodies showed that Wnt stimulation significantly enriched H3K27me3  27 (H3K27me3) and dimethylation of histone H3 K9 (H3K9me2) are marks of transcription repressionme3 Fig.  but not me3 Fig.  in the Tnin Fig.  and the ion Fig. , as deteion Fig. . These rWe next examined the clinical relevance of Wnt signaling component expression and FTO expression by TCGA analysis. We found that expression of a positive regulator of Wnt signaling Frizzled (FZD)9 and DVL1 was negatively correlated with FTO expression whereas expression of negative regulators of Wnt signaling APC, GSK3β, and AXIN2 was positively correlated with FTO expression in lung adenocarcinoma Fig. . These r6A RNA sequencing (m6A-seq) on H322 cells with or without FTO depletion. We showed that m6A peaks primarily occurred in a sequence context as GGAC (P = 3.5e10–41)  Fig. S and were1) Fig. S, which aays Fig. , which iion Fig. . MethylaRNA Fig. , FTO ovels Fig. S. Consistlls Fig. . These r6A regulation on c-Myc expression, we constructed a luciferase reporter gene with an integrated CDS sequence containing the WT or mutated m6A sites from the 3′ end of MYC mRNA , but not in subunits smaller than 40S, 40S, and 60S   Table S. As expe) Table SA, B. Not) Table SC, D. Cor) Table S, S5E, la) Table S, S5F, ce) Table S, migrati) Table S, S5J of 48. Of note, the FTO depletion-enhanced these protein expressions were largely reduced by c-Myc depletion (Fig. We next subcutaneously injected H358 cells with or without FTO depletion or combined FTO and c-Myc depletion into athymic nude mice. We showed that FTO depletion-promoted tumor growth was blunted by c-Myc depletion Fig. . Immunohion Fig. . In addiion Fig. , furtherion Fig. . These r6A modification from mRNAs, plays instrumental roles in the regulation of many cellular functions7. However, how FTO is transcriptionally regulated, especially by oncogenic signaling, remains to be explored. We demonstrated here that FTO expression was downregulated in lung adenocarcinoma and positively correlated with overall survival. Depletion of FTO enhanced tumor cell proliferation, anchorage-independent growth, migration, invasion, and tumor formation in mice. Of special interest, we found that Wnt signaling, which is vital in lung adenocarcinoma tumorigenesis51, induced the binding of β-catenin/TCF/LEF to the TBEs of the FTO promoter region and suppressed FTO expression. Mechanistically, we showed that WNT signaling induced the binding of EZH2 to β-catenin, leading to an EZH2-dependent H3K27me3 increase at the FTO promoter region for inhibition of FTO expression. Downregulation of FTO expression enhanced m6A levels on mRNAs in a number of signaling pathways, including metabolic pathways, in which MYC is a master regulator of gene expression for glycolysis45. Enhanced m6A levels on MYC mRNA recruited the binding of YTHDF1 and enhanced MYC mRNA translation. FTO depletion and YTHDF1-dependent upregulation of c-Myc promoted tumor cell glycolysis, growth, migration, and invasion and accelerated tumor growth in mice and tumor metastasis to the lung (Fig. In addition to epigenetic regulation of gene expression, regulation of the epitranscriptome exerts another critical layer to regulate protein expression. FTO, a key demethylase that removes the mMYC gene transcription can be upregulated directly by β-catenin/TCF/LEF45. FTO downregulation can upregulate Wnt signaling by increasing m6A modification in FZD10 mRNA52. In addition, m6A-dependent and YTHDF2-dependent decreases in MYC transcript stability in leukemia cells were reported14. We showed that Wnt stimulation suppressed FTO expression, therefore inducing m6A-dependent and YTHDF1-dependent increases in MYC mRNA translation. Given that FTO globally regulates the expression of many genes, which can be signaling context-dependent and cancer type-dependent16, the differential and distinct regulation of m6A in mRNAs downstream of the FTO exhibit wide variation and may elicit distinct cellular activities. Our finding that FTO downregulation induced by Wnt/β-catenin signaling enhanced c-Myc expression through upregulation of m6A of MYC mRNA and subsequent YTHDF1 binding revealed a novel mechanism by which lung adenocarcinoma promotes glycolysis and growth. The discovery of the novel regulation of c-Myc expression may lead to an alternative approach for the therapeutic treatment of lung adenocarcinoma.Supplementary figure legendsSupplementary table 1Supplementary table 2Supplementary table 3Supplementary table 4Supplementary table 5Supplementary figure 1Supplementary figure 2Supplementary figure 3Supplementary figure 4Supplementary figure 5


Accumulating evidences supported that knock-down of DHCR24 is linked to the pathological risk factors of AD, suggesting a potential role of DHCR24 in AD pathogenesis. However, the molecular mechanism link between DHCR24 and tauopathy remains unknown. Here, in order to elucidate the relationship between DHCR24 and tauopathy, we will focus on the effect of DHCR24 on the tau hyperphosphorylation at some toxic sites. In present study, we found that DHCR24 knock-down significantly lead to the hyperphosphorylation of tau sites at Thr181, Ser199, Thr231, Ser262, Ser396. Moreover, DHCR24 knock-down also increase the accumulation of p62 protein, simultaneously decreased the ratio of LC3-II/LC3-I and the number of autophagosome compared to the control groups, suggesting the inhibition of autophagy activity. In contrast, DHCR24 knock-in obviously abolished the effect of DHCR24 knock-down on tau hyperphosphrylation and autophagy. In addition, to elucidate the association between DHCR24 and tauopathy, we further showed that the level of plasma membrane cholesterol, lipid raft-anchored protein caveolin-1, and concomitantly total I class PI3-K (p110α), phospho-Akt (Thr308 and Ser473) were significantly decreased, resulting in the disruption of lipid raft/caveola and inhibition of PI3-K/Akt signaling in silencing DHCR24 SH-SY5Y cells compared to control groups. At the same time, DHCR24 knock-down simultaneously decreased the level of phosphorylated GSK3β at Ser9 (inactive form) and increased the level of phosphorylated mTOR at Ser2448 (active form), leading to overactivation of GSK3β and mTOR signaling. On the contrary, DHCR24 knock-in largely increased the level of membrane cholesterol and caveolin-1, suggesting the enhancement of lipid raft/caveola. And synchronously DHCR24 knock-in also abolished the effect of DHCR24 knock-down on the inhibition of PI3-K/Akt signaling as well as the overactivation of GSK3β and mTOR signaling. Collectively, our data strongly supported DHCR24 knock-down lead to tau hyperphosphorylation and the inhibition of autophagy by a lipid raft-dependent PI3-K/Akt-mediated GSK3β and mTOR signaling. Taking together, our results firstly demonstrated that the decrease of plasma membrane cholesterol mediated by DHCR24 deficiency might contribute to the tauopathy in AD and other tauopathies. Previous studies found that there is a significant reduction in the expression of new gene in specific vulnerable brain regions in Alzheimer's disease (AD) patients, which was named selective AD indicator 1 (Seladin-1), or 3β-hydroxysterol-Δ24 reductase (DHCR24)  and mammalian target of rapamycin (mTOR) signaling, which are involved in the abnormal hyperphosphorylation of some tau sites in tauopathy. In our experiment, we will focus on the effect of DHCR24 on the tau hyperphosphorylation at some sites such as Thr181, Ser199, Thr231, Ser262, Ser256, Ser396, and autophagy, in order to elucidate the relationship between DHCR24 downregulation and tauopathy.DHCR24 is known as the key synthetase in cholesterol synthesis, which catalyzes the final step of cholesterol synthesis via the Bloch pathway, or converts lanosterol to cholesterol at the first step in the Kandutsch-Russell pathway , AKt (#4691), Phospho-Akt (T308) (#13038), Phospho-Akt (Ser473) (#4060), PI3 Kinase p110α (#4249), GSK-3β (#12456), Phospho-GSK-3β(Ser9) (#5558), mTOR (#2983), Phospho-mTOR (#2971), Tau (#46687), Phospho-Tau (Thr181) (#12885), Phospho-Tau (Ser199) (#29957), Phospho-Tau (Ser396) (#9632), LC3B (#3868), P62/SQSTM1 (#23214), GAPDH (#2118), secondary antibodies anti-rabbit IgG, HRP-linked antibody (#7074), Anti-rabbit IgG (H+L) (#5366), Anti-mouse IgG, HRP-linked antibody (#7076) were purchased from Cell Signaling Tech (USA), Primary antibodies against Caveolin-1 (ab2910), Phospho-Tau (Ser262) (ab131354) were purchased from British abcam Company, Primary antibodies against Phospho-Tau (Thr231) (4137) was purchased from China ABclonal Company. Primers were purchased from Sangon Biotech . Propidium Iodide (abs9105), Methyl-β-cyclodextrin (abs42021762), Filipin III (abs42018484) were purchased from China Absin Company.SH-SY5Y cells were obtained from Chinese Academy of Sciences  and kept in DMEM F12  with 10% fetal bovine serum . Following ATCC protocols, all cells were cultured in a 5% CO2 humidified incubator at 37°C.With regard to lentivirus transfection, lentivirus at multiplicity of infection (MIO) about five mediated DHCR24 shRNA, DHCR24 cDNA, and NC infected into the SH-SY5Y cells. At 48 h after infection, the medium was replaced with fresh complete growth medium containing the appropriate concentration of puromycin (6 μg/mL). At 3 to 4 days after infection, the selected cells were used for experiments. Note: ShRNA target sequence was as follow: (5'-GCATCATCCCTGCCAAGAAGT-3'); The empty vector sequence was as follow: (5'-TTCTCCGAACGTGTCACGT-3'), cDNA sequence was as follow: (5' -ATGGAGCCCGCCGTGTCGCTGGCCGTGTGCGCGCTGCTCTTCCTGCTGTGGGTGCGCCTGAAGGGGCTGGAGTTCGTGCTCATCCACCAGCGCTGGGTGTTCGTGTGCCTCTTCCTCCTGCCGCTCTCGCTTATCTTCGATATCTACTACTACGTGCGCGCCTGGGTGGTGTTCAAGCTCAGCAGCGCTCCGCGCCTGCACGAGCAGCGCGTGCGGGACATCCAGAAGCAGGTGCGGGAATGGAAGGAGCAGGGTAGCAAGACCTTCATGTGCACGGGGCGCCCTGGCTGGCTCACTGTCTCACTACGTGTCGGGAAGTACAAGAAGACACACAAAAACATCATGATCAACCTGATGGACATTCTGGAAGTGGACACCAAGAAACAGATTGTCCGTGTGGAGCCCTTGGTGACCATGGGCCAGGTGACTGCCCTGCTGACCTCCATTGGCTGGACTCTCCCCGTGTTGCCTGAGCTTGATGACCTCACAGTGGGGGGCTTGATCATGGGCACAGGCATCGAGTCATCATCCCACAAGTACGGCCTGTTCCAACACATCTGCACTGCTTACGAGCTGGTCCTGGCTGATGGCAGCTTTGTGCGATGCACTCCGTCCGAAAACTCAGACCTGTTCTATGCCGTACCCTGGTCCTGTGGGACGCTGGGTTTCCTGGTGGCCGCTGAGATCCGCATCATCCCTGCCAAGAAGTACGTCAAGCTGCGTTTCGAGCCAGTGCGGGGC CTGGAGGCTATCTGTGCCAAGTTCACCCACGAGTCCCAGCGGCAGGAGAACCACTTCGTGGAAGGGCTGCTCTACTCCCTGGATGAGGCTGTCATTATGACAGGGGTCATGACAGATGAGGCAGAGCCCAGCAAGCTGAATAGCATTGGCAATTACTACAAGCCGTGGTTCTTTAAGCATGTGGAGAACTATCTGAAGACAAACCGAGAGGGCCTGGAGTACATTCCCTTGAGACACTACTACCACCGCCACACGCGCAGCATCTTCTGGGAGCTCCAGGACATTATCCCCTTTGGCAACAACCCCATCTTCCGCTACCTCTTTGGCTGGATGGTGCCTCCCAAGATCTCCCTCCTGAAGCTGACCCAGGGTGAGACCCTGCGCAAGCTGTACGAGCAGCACCACGTGGTGCAGGACATGCTGGTGCCCATGAAGTGCCTGCAGCAGGCCCTGCACACCTTCCAAAACGACATCCACGTCTACCCCATCTGGCTGTGTCCGTTCATCCTGCCCAGCCAGCCAGGCCTAGTGCACCCCAAAGGAAATGAGGCAGAGCTCTACATCGACATTGGAGCATATGGGGAGCCGCGTGTGAAACACTTTGAAGCCAGGTCCTGCATGAGGCAGCTGGAGAAGTTTGTCCGCAGCGTGCATGGCTTCCAGATGCTGTATGCCGACTGCTACATGAACCGGGAGGAGTTCTGGGAGATGTTTGATGGCTCCTTGTACCACAAGCTGCGAGAGAAGCTGGGTTGCCAGGACGCCTTCCCCGAGGTGTACGACAAGATCTGCAAGGCCGCCAGGCACTGA-3').Total RNA was extracted using RNAiso Plus  according to the manufacturer's instructions. Reverse transcription was performed using a PrimeScript™ RT Master Mix  with specific primers see . ReactioSH-SY5Y cells seeded in twelve-well-plate were incubated for 12-20 h, followed by washing three times with PBS, then fixed with 4% paraformaldehyde at room temperature for 30 min, followed by washing three times with PBS.0.5% Triton X-100 was adopted to permeate cells for 20 min. Cells were blocked with 5% bovine serum Albumin (BSA) in PBS at room temperature for 1 h, then incubated with the primary antibody at 4°C overnight followed by three times washing with PBS. Samples were incubated in secondary antibody in the dark for 1 h followed by stained with DAPI (0.5 μg/mL) for 5 min at room temperature. Finally, cells were observed and the fluorescent images were captured by a confocal microscope .After designated treatment, lysates were generated by placing the cells in SDS lysis buffer containing a cocktail of protease and phosphatase inhibitors. BCA was performed to determine total protein concentrations, which were normalized to 1 mg/mL for all samples. Samples were then prepared in sample buffer and heated to 100°C for 8 min. Equal amounts of lysates were fractionated using 8–12% sodium dodecyl sulfate polyacrylamide gels and were then electrotransferred onto nitrocellulose membranes. Gels were run at a constant voltage (80V) for 2–3 h for maximum separation. Wet transfer was performed for 120 min at constant current (250 mA) using polyvinylidenedifluoride membrane presoaked in methanol. The membrane was blocked in 5% milk in 0.2% TBST. The membrane was then washed three times in TBST for 10 min each. After overnight incubation at 4°C with the primary antibodies  the blots were washed and exposed for 1 h at room temperature to corresponding HRP-conjugated secondary antibodies. Chemiluminescent  detection was then used to detect the expression of each protein; GAPDH level served as internal loading controls.Cells were grown on coverglass bottom dishes. In order to allow a better visualization of intracellular cholesterol pools, cells were treated with 10 mM methyl-β-cyclodextrin  for 30 min at 37°C to remove cholesterol from the plasma membrane. Cells were then fixed for 10 min at RT with 4% paraformaldehyde in phosphate-buffered saline (PBS). Cells were incubated with a solution of filipin  for 30 min. Finally, after two washes, cells were counterstained with propidium iodide  for 5 min. Acquiring images with a confocal laser scanning microscope equipped with a 380 nm optically pumped semiconductor laser . Whole-cell cholesterol staining with filipin did not require MβCD treatment.SH-SY5Y cells were treated by stationary liquid which contained 3% glutaraldehyde and 0.22 mmol/L sucrose phosphate buffer for 4 h, then rinsed three times with 0.1 M phosphoric acid rinsing solution for 10–15 min, and fixed with 1% citric acid for 2 h. Next, stepwise ethanol dehydration, followed by epoxy resin embedding, overnight in a 30°C oven, then in a 60°C oven for 12 h. Using an ultra-thin slicer, 50-to 100-nm-thick slices were cut. Using uranium acetate and lead nitrate double staining, a transmission electron microscopy  was used to examine autophagosomes in SH-SY5Y cells.P-values were determined using one-way ANOVA. Significance was defined as P < 0.05.Statistical analysis was performed using SPSS statistical software. All data were expressed as mean ± SD from at least three independent experiments. To explore the influence of DHCR24 on tauopathy in Alzheimer's disease, a transient knock-down of DHCR24 by lentivirus-mediated shRNA (knock-down group) and knock-in of DHCR24 by lentivirus-mediated cDNA (knock-in group) in the SH-SY5Y cell line was performed. DHCR24 shRNA, DHCR24 cDNA were well-expressed in SH-SY5Y cells, as indicated by GFP (green) under fluorescence microscopy . TransduSo far, it is still not to elucidate the potential relationship between DHCR24 and tau hyperphosphorylation. Increasing data supported that the hyperphosphorylation of tau at several sites, such as Thr181, Ser199, Thr231, Ser262, Ser356, and Ser396, which are tightly associated with tauopathy in AD and other tauopathies  and western blot analysis. Autophagy is an intracellular degradation pathway essential for cellular and energy homoeostasis, involving in the clearance of misfolded proteins and damaged organelles   and Phospho-Akt (Thr308) were also markedly lowered by DHCR24 knock-down in SH-SY5Y cells. In contrast, the DHCR24 knock-in obviously increased the expression level of total class I PI3-K and the Phospho-Akt (Ser473 and 308), leading to the enhancement of PI3-K/Akt signaling. Thus, we considered that the decrease of PI3-K expression induced by DHCR24 knock-down could directly inhibited the phosphorylation activation of Akt, leading to the inhibition of PI3-K/Akt signaling. In addition, previous study suggested that brain cholesterol deficiency induced by silencing DHCR24 was associated with reduce of membrane cholesterol and altered membrane cholesterol-rich lipid-raft composition including the dysfunction of caveolae in cholesterol-rich lipid-raft microdomains, resulting in the markedly Akt inactivation, which is partly consistent with our outcomes (Lu et al., /caveolae may lead to the alteration of raft-dependent functional protein kinase, such as PI3-K kinase (Noticeably, in our study, K kinase . TogetheIn present paper, we also further showed that the DHCR24 knock-down could induce overactivation of GSK3β and mTOR kinase activity by the inhibition of PI3-K/Akt. Conversely, the DHCR24 knock-in could promote the activation of PI3-K/Akt signaling, and simultaneously inhibition of GSK3β and mTOR signaling. Furthermore, GSK3β was one of the first identified substrates of the heavily studied oncogenic kinase Akt, phosphorylation by which inhibits GSK3β activity. Akt activation is highly dependent on the class I PI3-K, and in turn promte the GSK3β at Ser9 phosphorylation and inhibition of GSK3β activity (Buller et al., In addition, the alteration of GSK3β and mTOR signaling plays a crucial role in tauopathy, which directly or indirectly participated in regulation of tau phosphorylation (Balaraman et al., Dysfunction of autophagy mechanism has been proposed to play a critical role in AD and other neurodegenerative disorders (Zare-Shahabadi et al., de novo synthetic ability and mainly rely on lipoprotein-conjugated cholesterol produced by Astrocytes (Dietschy and Turley, Noteworthily, Ledesma et al. revealed that the neuronal membrane cholesterol have a significant reduction, the loss of neuronal membrane cholesterol and anomalous raft microdomains, which contribute to excessive amyloidogenesis in AD patients (Abad-Rodriguez et al., In conclusion, in present study, our results further showed that DHCR24 could control the rate of cholesterol synthesis and cholesterol homeostasis by the Bloch pathway and/or K-R pathway. As a synthetase heavily involved in cholesterol synthesis, DHCR24 knock-down significantly lead to a reduction of cholesterol in plasma membrane and intracellular compartments, resulting in the cholesterol loss in SH-SY5Y cells. Furthermore, the decrease of plasma membrane cholesterol mediated by DHCR24 deficiency might contribute to the tauopathy in AD and other tauopathies, at least partly by membrane lipid raft-mediated signaling mechanism . Taken tThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.HZ designed the study and wrote the manuscript. XB contributed to the experimental work about tau phosphorylation and autophagy. JW and MZ contributed to the transfection work of SH-SY5Y cells. YX, LD, and KY provided their experimental assistance and TEM work. JZ and JB contributed to analysis of data. YZ and GX provided their technical assistance. All authors have read the manuscript and provided the input.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The importance of histone variant H2A.Z in transcription regulation has been well established, yet its mechanism-of-action remains enigmatic. Conflicting evidence exists in support of both an activating and a repressive role of H2A.Z in transcription. Here we report cryo-electron microscopy (cryo-EM) structures of nucleosomes and chromatin fibers containing H2A.Z and those containing canonical H2A. The structures show that H2A.Z incorporation results in substantial structural changes in both nucleosome and chromatin fiber. While H2A.Z increases the mobility of DNA terminus in nucleosomes, it simultaneously enables nucleosome arrays to form a more regular and condensed chromatin fiber. We also demonstrated that H2A.Z’s ability to enhance nucleosomal DNA mobility is largely attributed to its characteristic shorter C-terminus. Our study provides the structural basis for H2A.Z-mediated chromatin regulation, showing that the increase flexibility of the DNA termini in H2A.Z nucleosomes is central to its dual-functions in chromatin regulation and in transcription. Chromatin is a dynamic structure that has an important regulatory role in controlling the genomic DNA accessibility and various nuclear processes. One mechanism used by the cell to influence chromatin structure and function is through histone variant exchange, which confers the nucleosome with new chemical and physical properties. With the lack of details and insights into how histone variants influence chromatin structure and function, the molecular mechanism of variant-mediated gene expression is subject to much speculation.Drosophila, Tetrahymena and mouse   dimer and the (H3–H4) tetramer and in the nucleosome surface around the acidic patch   while twle (NCP) ,15. An eic patch . The latThe DNA template containing twelve tandem repeats of 167-bp 601 Widom sequence was a kind gifts from Dr Craig Peterson. The DNA template containing twelve tandem repeats of 208-bp 601 Widom sequence was a kind gift from Dr Ed Luk. Large-scale plasmids were purified as previously described . To geneThe sequences for the 167-bp and 208-bp Widom sequence are listed as following with the 601 sequence underlined:CTGGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCTGTGCATGACTAGAT167-bp DNA repeat: ATCCCGCCCTGGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATACATCCTGTGCATGTATTGAACAGCGACCTTGCCGGAGT.208-bp DNA repeat: ACTTATGTGATGGACCCTATACGCGGCCGCCCTGGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCAGATATATGCATCCTGTGCATGTATTGAACAGCGACCTTGCCGGAGT.Modified 208-bp DNA repeat with NsiI site introduced: ACTTATGTGATGGACCCTATACGCGGCCGCCXenopus laevis histones H2A, H2B, H3 and H4 were expressed in BL21 (DE3) pLysS E. coli cells and purified as previously described . The H2A.Z.1 gene was sub-cloned into pET LIC expression vector. The H2A.Z mutant in the acidic patch includes residue substitution D97N, S98K, G106Q and Glycine insertion after I100. The H2A.Z L1 loop mutant was constructed with residues in H2A.Z replaced by those from the H2A L1 loop . These mutations were introduced using Q5 site-directed mutagenesis kit . The H2A.Z M6–M7 cassette mutant was constructed by swapping residues G93–G120 with the corresponding H2A sequence (residue N90-P118). The H2A.Z C-terminus mutant was constructed by replacing H2A.Z amino acid G123-V128 with the corresponding H2A sequence (amino acid T121-K130). The H2A.Z C-terminus-extended mutant was constructed by replacing H2A.Z sequence from amino acid G93-V128 with the corresponding sequence in H2A from amino acid N90-K130. These mutants were obtained by gene synthesis (Synbio Technologies). Both wild-type and mutant H2A.Z were expressed in BL21 (DE3) E. coli cells and purified using the same procedure as the canonical histones.escribed . Mouse Hin vitro using salt dialysis as previously described  of the chromatin fiber was screened using negative-stained EM.A 1:1 optimal ratio of octamer:DNA for nucleosome reconstitution and for array reconstitution was determined empirically through the electrophoretic mobility shift assay (EMSA). Induction of chromatin fiber formation and purification were done through the addition of Mgd method . BrieflyP ≤ 0.05. Two-way Anova test and graphical representation was done using Prims 5 software.250 nM of 208 bp nucleosome was mixed with 45 U of restriction enzyme (HinfI or NsiI) in Cutsmart buffer (NEB)  in total volume of 45ul and incubated at 37°C. Samples were collected every 15 min (5 ul), quenched with 8ul of stop buffer  and incubated at 50°C for 20 min for deproteination. Samples were examined on an 8% Native-PAGE gel and stained with SYBR GOLD. Digestion results were analyzed using ImageJ software version 1.53e. Two-way Anova test was used to determine the level of significant difference at For the reactions mentioned in Figure P value.Fitting was done using the Solver add-in in Excel over 5 iterative cycles. The rate constants  under 8°C and 100% humidity. Aliquots of 3.5 μl of the nucleosome were applied to glow-discharged QUANTIFOIL grids (R1.2/1.3 – 400 mesh), blotted for 4–5 s and plunged into liquid ethane cooled by liquid nitrogen. Grids were stored in liquid nitrogen until they were imaged.2 per micrograph  in counting mode using EPU software, giving a pixel size of 1.12 Å at the specimen level. Defocus values range from 0.9 to 3 μm. Each movie was dose-fractionated to 78 frames with a dose rate of ∼0.69 e/pixel/s. Total exposure time was 60 seconds, corresponding to a total dose of 33 e/ Åcrograph .2. For the H2A fiber, part of the dataset was collected with the same Titan Krios microscope using the condition mentioned above, and the rest of the dataset was collected using the Talos Arctica microscope operating at 200 keV with a Falcon 3EC direct electron detector at counting mode with a nominal magnification of 73 000×, giving a physical pixel size of 1.4 Å/pixel. Each movie in the Arctica dataset was dose-fractionated to 78 frames with a dose rate of ∼0.75 e/pixel/s. Total exposure time was 60 s, corresponding to a total dose of 30 e/ Å2 per micrograph. The micrographs from the Krios dataset were scaled to the same pixel size as the Arctica dataset, before the two datasets were merged . The histone core from mouse H2A.Z nucleosome structure (PDB: 1F66) and the 601 DNA from the NCP structure (PDB: 6FQ5) were combined to generate the initial template used for refinement. This template was manually fitted into the density map in UCSF Chimera . followeThe nominal resolution of the overall reconstructions of chromatin fibers is limited, due to the motions of individual nucleosome relative each other within the fiber, and thus it is intrinsically difficult to improve. However, the positions of nucleosome N3–N10 in the multi-body-refined maps is sufficiently clear to fit the nucleosome crystal structures. The fiber models were built by rigid-body docking eight crystal structures of H2A.Z nucleosome (PDB ID: 1F66) or canonical nucleosome (PDB ID: 3LZ0) into the cryo-EM density map of the corresponding fibers using UCSF Chimera . The linIn Figure Xenopus histone H2A or mouse histone variant H2A.Z.1 were reconstituted using recombinant proteins , even though it appears faster than the H2A nucleosome control (P = 0.015)  Figure . Concurr) Figure  is simil) Figure . In the ) Figure . For the) Figure . These ret al. showed that variant histone H2A.Z facilitates the intramolecular folding of nucleosome arrays into 50S chromatin fibers in vitro, while simultaneously inhibiting the formation of a highly condensed structure  and γ (27.5 º) are larger than those of the H2A fiber (β = 12º and γ = 9.7º). The average shift d between units is shorter for H2A.Z fiber (59.5 Å) than for the H2A fiber (61 Å). Using these parameters, we built two 30-nm fiber models, each containing 24 tandem repeats of 167-bp 601 Windom sequence    . Overall2+) , with a ) Figure , while t) Figure . Bendingin-vitro DNA accessibility assay. Several recent cryo-EM studies of histone variants such as CENP-A . Though the molecular details of these interactions and how their interplays contribute to chromatin higher-order folding are less clear, our model is generally consistent with an ic patch . The morLinker histones H1 are known as an important factor for stabilizing the higher-order chromatin structure  and for in vitro. Based on these results, we propose a model for H2A.Z-mediated transcription regulation where the function of H2A.Z is context-dependent  and the Protein Data Bank (https://www.rcsb.org/). EM maps are deposited at the Electron Microscopy Database for the canonical nucleosome (EMD-23632), H2A.Z nucleosome (EMD-23626), H2A fiber (EMD-23631), and H2A.Z fiber (EMD-23630) respectively. The protein coordinate of H2A.Z nucleosome is deposited at the Protein Data Bank (PDB ID: 7M1X).The data that support the findings of this study are openly available in the Electron Microscopy Database (gkab907_Supplemental_FileClick here for additional data file.


Tricholusia ni insect pupae as natural bioreactors, which are programmed by recombinant baculovirus vectors. Co-infecting the insect pupae with two baculovirus vectors expressing the RHDV GI.1- and RHDV GI.2-derived VP60 proteins, we obtained chimeric VLPs incorporating both proteins as determined by using serotype-specific monoclonal antibodies. The resulting VLPs showed the typical size and shape of this calicivirus as determined by electron microscopy. Rabbits immunised with the chimeric VLPs were fully protected against a lethal challenge infection with the two RHDV serotypes. This study demonstrates that it is possible to generate a dual cost-effective vaccine against this virus using a single production and purification process, greatly simplifying vaccine manufacturing.The VP60 capsid protein from rabbit haemorrhagic disease virus (RHDV), the causative agent of one of the most economically important disease in rabbits worldwide, forms virus-like particles (VLPs) when expressed using heterologous protein expression systems such as recombinant baculovirus, yeasts, plants or mammalian cell cultures. To prevent RHDV dissemination, it would be beneficial to develop a bivalent vaccine including both RHDV GI.1- and RHDV GI.2-derived VLPs to achieve robust immunisation against both serotypes. In the present work, we developed a strategy of production of a dual-serving RHDV vaccine co-expressing the VP60 proteins from the two RHDV predominant serotypes using CrisBio technology, which uses  Lagovirus genus within the Caliciviridae [Rabbit haemorrhagic disease virus (RHDV) is the causative agent of a highly infectious and fatal disease affecting domestic and wild rabbits ,2,3. It iviridae . The posiviridae ,7. Additiviridae ,9. The viviridae .Commercially available vaccines are still traditional inactivated forms of the original pathogens , which aIn the course of its evolution, RHDV has thus far diverged to form six recognised genotypes , all higEscherichia coli [Saccharomyces cerevisiae [Trichoplusia ni pupae as natural bioreactors [The VP60 capsid protein expressed in heterologous recombinant systems forms characteristic VLPs, which are protective in immunised rabbits. VP60-derived VLPs have been obtained in hia coli , Saccharrevisiae , plants revisiae ,27, and revisiae ,29,30 anrevisiae ,32,33,34reactors  to increSpodoptera frugiperda Sf21 cell line was cultured at 28 °C in TNM-FH culture media  supplemented with 10% heat-inactivated foetal bovine serum , gentamicin at 50 µg/mL ), and Antibiotic-Antimicotic 1× ). Recombinant baculoviruses (rBacs) were generated using the TopBac expression cassette [® II Reagent (Thermo Fisher Scientific) and following the manufacturer’s instructions.The cassette ,37 as doBaculovirus stocks were titered on Sf21 cells using the 6-well plate format. Subconfluent Sf21 cells were infected with 10-fold serial dilutions of recBac stocks for 1 h at 28 °C. Subsequently, inocula were removed and cells were overlayed with an insect cell media-agarose mix at 1% agarose final concentration  and further incubated for 3–5 days. Monolayers were stained with neutral red to facilitate plaque counting.The improved baculovirus vector system TopBac was used for the generation of recombinant baculoviruses. The construction of recombinant baculoviruses was carried out essentially as described in , with thRHDV GI.1 Isolate AST89 Genbank accession number Z29471MEGKARTAPQGEAAGTATTASVPGTTTDGMDPGVVATTSVVTAENSSASIATAGIGGPPQQVDQQETWRTNFYYNDVFTWSVADAPGSILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTGDPGLVPTLVLSVYNNLINPFGGSTSAIQVTVETRPSEDFEFVMIRAPSSKTVDSISPAGLLTTPVLTGVGNDNRWNGQIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFADIDHRRGSASYPGSNATNVLQFWYANAGSAIDNPISQVAPDGFPDMSFVPFNGPGIPAAGWVGFGAIWNSNSGAPNVTTVQAYELGFATGAPGNLQPTTNTSGSQTVAKSIYAVVTGTAQNPAGLFVMASGVISTPSANAITYTPQPDRIVTTPGTPAAAPVGKNTPIMFASVVRRTGDVNATAGSANGTQYGTGSQPLPVTIGLSLNNYSSALMPGQFFVWQLTFASGFMEIGLSVDGYFYAGTGASTTLIDLTELIDVRPVGPRPSKSTLVFNLGGTANGFSYV*RHDV GI.2—previously RHDVb/2 isolate Nav10/11 accession number KM878681MEGKARTASQGETAGTATTASVPGTTTDGMDPGVVATTSVVTTENASTSIATAGIGGPPQQVDQQETWRTNFYYNDVFTWSVADAPGNILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTGNPGLVPTLVLSVYNNLINPFGGSTSAIQVTVETRPSEDFEFVMIRAPSSKTVDSISPADLLTTPVLTGVGTDNRWNGEIVGLQPVPGGFSTCNRHWNLNGSTFGWSSPRFAAIDHDRGNASYPGSSSSNVLELWYASAGSAADNPISQIAPDGFPDMSFVPFSGTTVPTAGWVGFGGIWNSSNGAPFVTTVQAYELGFATGAPSNPQPTTTTSGAQIVAKSIYGVATGINQATAGLFVMASGVISTPNSSAITYTPQPNRIVNAPGTPAAAPIGKNTPIMFASVVRRTGDINAEAGSTNGTQYGAGSQPLPVTVGLSLNNYSSALMPGQFFVWQLNFASGFMELGLSVDGYFYAGTGASATLIDLSELVDIRPVGPRPSTSTLVYNLGGTTNGFSYV*8 pfu (plaque-forming units)/mL were reached. Baculovirus stocks were stored at −80 °C containing 5% glycerol. The correct expression of VP60 proteins from recombinant baculoviruses was confirmed by detection of proteins using specific sera by Western blot analysis.The recombinant baculoviruses produced were subjected to two rounds of clonal selection by plaque formation, and after virus propagation, infective titres of approximately 10Trichoplusia ni  insects were reared following a previously described methodology [hodology . BrieflyThe crude protein extracts were obtained by homogenising the frozen biomass in the adequate protein extraction buffer. The resulting suspension was subjected to the downstream processing  to obtaiw/v) aqueous uranyl acetate. Micrographs were recorded with an EM 2000 Ex microscope . For the quantitation analysis of VLP stability, samples obtained from GI.2 (N11-) and GI.1 (AST) infections or from co-infections with the two viruses under different conditions were analysed at day 0 and 60 of storage at 4 °C. Samples were diluted to a protein concentration of 25 mg/mL to obtain an even distribution of the capsids on the electron microscopy grid and an easily countable number of capsids per image at the suitable magnification. Three grids were prepared per sample and images were taken in four different areas of each grid. We quantified the capsids with ImageJ tools in 15 pictures.Electron microscopy analyses were performed by conventional means. Briefly, purified VLPs (approximately 5 µL) were applied to glow-discharged carbon-coated grids for 2 min. Samples were negatively stained with 2%  was added. All washing steps were carried out in triplicate using PBS-Tween (0.05% PBS-Tw). For peroxidase-labelled antibodies, the Pierce™ ECL Western Blotting Substrate system was used for detection (Thermo Fisher Scientific).2SO4. Absorbances were read at 450 nm using Varioskan Flash microplate reader and Skanit software.ELISA were carried out in flat bottomed 96-well plates . Plates were coated with 100, 50, or 10 ng of Mab (varying concentrations are indicated in the results section) in each well in PBS (1 mM a pH 7.3 with sodium azide 0.05% PBS-AS). Coating plates were incubated overnight at 4 °C. The next day the supernatant was removed, and plates were blocked with 1% BSA in PBS-AS for 1 h at 37 °C. Plates were washed 3 times with 200 µL PBS-Tw per well. Antibodies were added in volumes of 100 µL per well and incubated for 1 h at 37 °C followed by 3 washing steps as previously described. Secondary antibodies at the indicated dilutions were incubated for 1 h at 37 °C. Prior to revelation, plates were washed 3 times in PBS-Tw. For reaction development TMB  (Sigma) (100 µL per well) was added and incubated at room temperature in the dark until the reaction was stopped using 100 µL 2M HNew Zealand White rabbits were supplied by San Bernardo Farm . Prior to vaccination, sera were collected from the marginal ear vein of all animals to confirm the absence of RHDV antibodies.Two protection experiments were carried out. In general terms, rabbits were vaccinated with chimeric RHDV VLPs and in experiment 1; 7 days post vaccination they were challenged with RHDV GI.2 and in experiment 2; 7 days post vaccination they were challenged with classic RHDV GI.1 isolate RHDV AST89.Group A: Non-vaccinated controls.Group B: 20 µg chimeric VLPs.Group C: 40 µg chimeric VLPs.Group D: 5 µg RHDV G1 VLPs + 5 µg RHDVb VLPs.Experimental challenge 1: 30-day-old rabbits were divided into 4 groups (Groups A–D), each of 5 animals. Each group was vaccinated with the following VLPs administered in a total volume of 0.5 mL:Group E: Non-vaccinated controls.Group F: 40 µg chimeric VLPs.Experimental challenge 2: 2-month-old rabbits were divided into 2 groups (Groups E and F), each of 5 animals. Each group was vaccinated with the following VLPs administered in a total volume of 0.5 mL:Rabbits were kept under observation throughout the experiment and housed in pairs in a biosecurity level 2 laboratory. L. europaeus GI.2 virus, common name RHDV-Gal08/13, with 10 LD50. Groups E and F were challenged 7 days post vaccination with RHDV isolate L. europaeus RHDV GI.1 RHDV-Ast89 (Genbank accession number Z49271). Rabbits were infected on day 7 post vaccination with 150 hemagglutination units/0.5 mL (as determined using O- and B-type human erythrocytes). The virus was administered subcutaneously in the cervical area as a dilution of clarified infected liver homogenates. Euthanasia of surviving rabbits was carried out by intravenous injection with Dolethal following the manufacturer’s instructions .Two virus isolates were used in this study. Groups A–D were challenged 7 days post vaccination with isolate Symptoms of RHDV infection, such as nervous, respiratory, and digestive signs, were monitored and recorded daily. Blood samples were collected on days 0 and 7 post vaccination, and from all surviving rabbits 7 days post challenge. Serum was separated after clotting by centrifugation and stored at −20 °C until analysis. After euthanasia, rabbits were necropsied, and gross lesions were recorded. Samples of liver were directly frozen at −80 °C for total RNA extraction using the GenElute Mammalian Total RNA Miniprep Kit , and RHDV GI.2 VP60 detection and quantification were obtained by specific RT-qPCR .n = 5) from each group.Rabbit serum RHDV antibody levels were tested by enzyme-linked immunosorbent assay (ELISA) using the commercially available INgezim RHDV kit  following the manufacturer’s instructions. The absorbance was measured at 450 nm in a microplate reader  and data were analysed using the Skanit software Version 2.4.5 (Thermo Fisher Scientific). Negative and positive control sera were included in all plates. Results are expressed as mean optical densities ± SD . To determine if VLPs obtained after co-infecting with the two baculoviruses were formed by a single VP60 protein or were chimeras of the two VP60 proteins, we designed ELISA and dot blot-based sandwich analyses using monoclonal antibodies capable of recognising one or the two VP60 proteins. To determine the consequences of the co-infection conditions on the chimeric VLP stability, we compared the VLP stability after 2 months of storage at 4 °C. For this analysis, VLPs were produced using GI.1/GI.2-derived particles at baculovirus infection ratios of 1:1, 1:3, and 3:1. In parallel, we performed the same analysis using GI.2 (N11-)- and GI.1 (AST)-derived VLPs produced independently. After this storage period, we observed a dramatic degradation (>70%) of GI.2 (N11)-derived VLPs, indicative of their low stability . This ra5 viral RNA copies per microliter—one rabbit in group B), indicating a solid protection conferred by the chimeric VLPs. The negative control groups showed 80% mortality after infection with RHDV GI.1 or RHDV GI.2 viruses and all animals were positive for virus genome detection. Viral RNA detection ranged between 6.03 × 105 and 5.81 × 106 copies per microliter for animals in group A.To analyse the protection conferred by the chimeric VLPs against the two predominant RHDV serotypes, we used a mixture of GI.2 (N11-) and GI.1 (AST89)-derived VLPs at a single previously determined protective dose and two doses of chimeric VLPs to immunise rabbits. As expected, the mixture of 5 µg of both VLPs induced protection against a lethal dose of RHDV GI.2 virus, showing no interference of the GI.1 (AST89)-derived VLP in this protection. Two doses of chimeric VLPs of 20 and 40 µg were also used to determine the protection ratios against RHDV GI.2, and only 40 µg was used to protect against an RHDV GI.1 virus. A high protection rate of 80% against RHDV GI.2 was achieved with the 20 µg dose of chimeric VLP, and a rate of 100% was achieved with a dose of 40 µg against both lethal viruses , demonstAntibodies generated against RHDV were analysed using a commercial ELISA from sera extracted from all rabbits on day 0 (pre-vaccination) and from surviving animals on day 14 post vaccination . As expected, all sera were negative for anti-RHDV antibodies on day 0. Seven days post challenge, the majority of surviving animals elicited an immune response as indicated by rising Ab levels . OverallDevelopment of efficacious recombinant subunit vaccines is essential, especially for causal agents for which a cell culture capable of supporting its propagation has not been identified. This is the case for RHDV, which is the causative agent of one of the most economically important diseases in rabbits worldwide. Commercially available vaccines are still traditional inactivated viruses collected from organs of artificially infected rabbits, which is unacceptable from both the safety and ethical points of view. The production of a new-generation vaccine against RHDV needs to meet two main requirements: first, cost-efficiency in production to make it commercially viable, and second, the vaccine has to induce a broad protection against RHDV GI.1 and RHDV GI.2 viruses as cross-protection is only partial at best ,40.Caliciviridae. The productivity of this protein is generally good in most studied systems, but the development of a subunit vaccine based on the VLP that this protein forms is commercially difficult for the production and regulatory-associated costs, considering the limited number of vaccine doses applied in the domestic rabbit population worldwide. In addition, any vaccine against this virus has to be dual because of the potential of two circulating virus serotypes today, RHDV GI.1 and RHDV GI.2. In the present work, we attempted to produce RHDV VLPs in one of the most cost-efficient methods developed to date based on the use of Trichoplusia ni insect chrysalises (CrisBio technology), and also to develop a single production process (upstream and downstream) of a chimeric VLP-based vaccine containing the critical antigens from both predominant RHDVs circulating today.Virus-like particles have received considerable attention due to their potential application in human and veterinary vaccines against infectious diseases. VLPs are highly immunogenic and are extremely safe. In the case of RHDV, the capsid protein forms characteristic VLPs when expressed in different systems as for the other members of the RHDV has been previously described as a versatile platform for foreign B-cell epitope display, inducing protective humoral immune responses . It has In the absence of a widely available tissue culture system that can sustain replication of human noroviruses, VLPs have been used as a surrogate to study the capsid’s structural features and as immunogens to elicit protective humoral responses . The mosTrichoplusia ni larvae [Trichoplusia ni Lepidoptera is highly susceptible to AcMNPV-derived vectors, and therefore, any common baculovirus can be used for industrial or experimental production. This technology is also linearly scalable, facilitating their implementation for large quantities of vaccine doses in a record time.RHDV GI.1 VLPs have been previously produced in i larvae  and pupai larvae . Recentli larvae . The proIn the present work, we also used the TopBac expression cassette to generate the baculovirus vectors ,37. The In the present work, we demonstrated the feasibility of producing a vaccine against RHDV that incorporates two different capsid proteins derived from two virus subtypes and confers broad protection in immunised rabbits. The chimeric RHDV VLPs were produced in a single upstream and downstream production process, significatively reducing the complexity of a binary vaccine. In addition, the combination of TopBac baculovirus vector and CrisBio technology allows for a cost-efficient production required for a vaccine directed to domestic rabbits to make it commercially viable.


The authors wish to introduce the following corrections to their article .OLLAS::RPA-1 is incorrect. The correct sequence is:1) The DNA sequence for the TTCCCCAATTTTTATGTATCTGTTTCAGATAGTGAAAGATGTCCGGATTCGCCAACGAGCTCGGACCACGTCTCATGGGAAAGGCGGCAATTCACATCAATCACGATGTCTTCAATAArpa-1 was indicated to be placed on chromosome I, when its correct position is on chromosome II.2) In some places in the list of Strains The published article has been updated. These changes do not affect the results, discussion and conclusions presented in the article.