H2A.B genes are testis-expressed short histone H2A variants that arose in placental mammals. Their biological functions remain largely unknown. To investigate their function, we generated a knockout (KO) model that disrupts all 3 H2A.B genes in mice. We show that H2A.B KO males have globally altered chromatin structure in postmeiotic germ cells. Yet, they do not show impaired spermatogenesis or testis function. Instead, we find that H2A.B plays a crucial role postfertilization. Crosses between H2A.B KO males and females yield embryos with lower viability and reduced size. Using a series of genetic crosses that separate parental and zygotic contributions, we show that the H2A.B status of both the father and mother, but not of the zygote, affects embryonic viability and growth during gestation. We conclude that H2A.B is a novel parental-effect gene, establishing a role for short H2A histone variants in mammalian development. We posit that parental antagonism over embryonic growth drove the origin and ongoing diversification of short histone H2A variants in placental mammals.Histone variants expand chromatin functions in eukaryote genomes.  The unusual short histone variant H2A.B is a novel parental-effect gene that plays an important role in early mammalian development. Parental antagonism over embryonic growth resource allocation may have driven the origin and ongoing diversification of short histone H2A variants in placental mammals. Histone proteins package eukaryotic genomes in nucleosomes, which are the basic unit of chromatin. This packaging function is essential for genome regulation and for the transmission of epigenetic information –3. NucleH2A.B, H2A.L, H2A.P, and H2A.Q ) , indicatplicates . ConsistH2A.B-KO animals. We hypothesize that these nucleosomes with smaller footprints correspond to H2A.B nucleosomes in WT animals, which are missing in KO animals  or seminiferous tubule organization between WT and KO males  staining, which intercalates with DNA when sperm nuclei have reduced protamine content. Thus, increased CMA3 staining is an indirect measure of overall genome deprotamination in sperm . We counH2A.B, we tested the effect of H2A.B loss in a fertility assay [n = 4) or KO (n = 6) to age-controlled C57BL/6J females, allowing just 1 mating per male–female pair  in series of crosses, starting with crosses between WT females and KO males. We found that there was a mild but nonsignificant increase in the fraction of resorbed or developmentally delayed conceptuses when compared with WT × WT crosses . This reduction was also observed for placental weights, although with larger standard deviations . Therefore, we infer that the reduced weights of KO × KO embryos might result from growth restriction. Overall, our results imply that H2A.B’s primary biological role appears to be in postfertilization development; embryos have reduced viability and impaired fitness postimplantation in the complete absence of H2A.B.The second striking effect of H2A.B on embryonic fitness became evident upon analyzing surviving embryos. We found that fetal weights of both male and female conceptuses from KO × KO crosses were approximately 30% smaller than those from WT × WT controls or those from KO × WT or WT × KO reciprocal crosses  via crosses between KO females to either WT or KO males . Since the maternal genotypes are identical (KO) in these crosses, we conclude that this difference must solely be due to the different paternal H2A.B genotypes. We thus conclude that H2A.B is a paternal-effect gene [H2A.B is, to our knowledge, the first identified paternal-effect histone variant in mammals.We decided to distinguish between these 2 possibilities by comparing zygotes with identical genotypes, but from genetically distinct parents. We first tested for paternal effects by generating genetically identical KO male embryos  to KO males. To ensure comparisons were influenced by maternal but not zygotic H2A.B genotypes, we focused on identical zygotic genotypes produced from crosses between genetically distinct mothers  and fathers with identical KO genotype. For example, both the HET × KO and KO × KO crosses (dam × sire) produce XKOXKO females and XKOY males. We found no significant difference when comparing fetal or placental weights of these zygotes from the 2 crosses  and female (XXKO versus XKOXKO) embryos produced from genetically identical parents in a HET × KO crosses  analyses to investigate H2A.B expression during oogenesis. We reasoned that H2A.B may follow a similar expression pattern in females as it does in males. If this were the case, expression should be detectable by the leptotene stage of meiosis I, which begins around E14.5 in the female genital ridge [H2A.B, we found a specific induction of H2A.B at the initiation of prophase of meiosis I, until entry into pachytene when it gradually decreased back to 0  and found a significant albeit subtle effect of H2A.B loss on litter sizes and spermatogenesis [H2A.B loss. It is possible that these subtle differences are a result of different genetic backgrounds (FVB/NJArc versus C57BL6/J) or the conditions under which the different mice strains were raised or assayed.In this study, we generated and characterized a triple KO mutant of ogenesis . SurprisH2A.B by investigating the biological consequences of its loss in mice. First, CUT&RUN chromatin profiling with antibodies to specific histone H3 modifications revealed that loss of H2A.B leads to a global increase in the abundance of sub-nucleosomal fragments in haploid spermatids. Closer examination revealed that the sub-nucleosomal fragments do not correspond to specific genomic locations but rather represent a genome-wide pattern of partially unwrapped nucleosomes. This finding might initially appear surprising since H2A.B-containing nucleosomes have been shown to have smaller MNase footprints also suggestive of unwrapping [Instead of an effect on spermatogenesis, we uncovered 3 novel functions of wrapping ,12,17. HH2A.L.2, prevents these changes from occurring in an orchestrated manner, ultimately leading to male sterility [We hypothesize that these alterations in chromatin structure might reveal a role for H2A.B in histone-to-protamine exchange during spermiogenesis –61. Prevterility . Since Hterility –26.H2A.B is expressed during female meiosis [H2A.B is deposited in female germ cells and functions to regulate the chromatin remodeling that occurs following fertilization [Our second novel finding is that  meiosis , and thilization ,66. Evenlization ,42, a prlization . This suH2A.B is both a paternal-effect and a maternal-effect gene. This is the first instance of a histone variant with this dual function. This novel function manifests both at the level of postimplantation embryo development and viability, but not preimplantation development or fertilization success. Although we did not observe differences in birth weights between pups produced from KO × KO and WT × WT crosses under laboratory conditions, we posit that differences in embryonic weight translate to an evolutionarily significant fitness defect in the wild [H2A.B, such that 1 in 10 embryos conceived in KO × KO crosses were inviable. Thus, we can conclude that H2A.B is required for optimal fitness in mice.The third and most significant finding from our study is the discovery that the wild ,34,67,68H2A.B exerts its parental effect? H2A.B seems to be lost after meiosis is completed, in elongating spermatids, and is not found in mature mouse sperm [H2A.B’s effect on splicing regulation or its proposed association with PIWI-related protein might affect the inheritance of specific RNAs required for optimal embryonic development and survival [H2A.B can overcome most but not all of the deleterious effects caused by loss of paternal H2A.B.How does se sperm ,24,42, asurvival ,69. It isurvival . Indeed,survival . HoweverH2A.B during meiosis globally prepares chromatin for proper postfertilization development. Such function has been previously described for chromatin regulators and histones in animals and plants [Instead, our finding of widespread rather than locus-specific alterations of chromatin structure in haploid male germ cells supports an alternative model whereby the deposition of d plants ,57,71–76H2A.B is an important gene required for postimplantation embryonic development in mice, whose function can be redundantly supplied by either parent. However, this model cannot explain either the high rate of turnover of H2A.B including complete loss in different mammalian lineages or the rapid evolution of H2A.B coding sequences. Instead, H2A.B’s unusual evolution, together with its X chromosomal location, is highly suggestive of its involvement in a genetic conflict [H2A.B participation in X-versus-Y meiotic drive, our discovery of a role for H2A.B’s in embryonic development and viability suggests that it might participate in a different form of genetic conflicts, such as parental antagonism [Our findings indicate that conflict ,28,40,77tagonism –40. Paretagonism . In thistagonism ,34.H2A.B meets several but not all tenets of the “classic” parental antagonism model [H2A.B presence ensures maximal fitness for all sired progeny, independent of maternal genotype. Notably, paternal H2A.B increases both fetal and placental weights, consistent with its role as a “demand-enhancer” [H2A.B do not manifest during fertilization, or preimplantation but rather during postimplantation embryonic development, exactly the stage where battles for resource allocation are predicted to occur in utero [H2A.B can eliminate fetal weight differences between progeny sired by WT or KO sires. Thereby, maternal H2A.B acts as capacitor allowing females to exert control over resource allocation to their progeny. Yet, maternal H2A.B does not act as a canonical “demand-inhibitor” as would be expected under the parental antagonism model. Instead, paternal and maternal H2A.B appear to have a synergistic rather than antagonistic effect on promoting embryo fitness; the most severe growth and viability phenotypes in utero are only seen in cases when neither parent encodes H2A.B  allele of H2A.B would be fully acted upon by selection in hemizygous males, even as its female-deleterious effects could be masked in HET females [H2A.B could recurrently arise and rise to high frequency in populations, followed by suppressor mutations that decrease female-detrimental effects to restore female fitness. Unexpectedly, our findings suggest that H2A.B alleles  themselves could evolve such a suppressor role. Thus, X-linkage and parental antagonism explain the rapid evolution and turnover of H2A.B alleles through repeated rounds of selection for optimizing male, followed by female-beneficial function. Moreover, H2A.B might be lost in mammalian species that relieve parental antagonism, for example, by sexual monogamy [H2A.B coincided with complete in utero development that evolved in placental mammals 150 million years ago [Their X-chromosomal linkage could further explain the rapid evolution and gene turnover of  females . As a remonogamy . By reveears ago .H2A.B KOs: gRNA 1: 5′gcctggtggaacagcatctg3′ and gRNA2: 5′cgcaagctccagcaacctgc3′. These sequences have perfect complementarity to all 3 intact H2A.B genes in the mouse genome, with only 1 mismatch in the most 5′ nucleotide of gRNA2 in the H2afb3 paralog (C to G). Using PCR, we synthesized products containing the T7 promoter (5′ upper case in forward primer), the specific gRNA and a common trans-activating crispr RNA (tracrRNA) (upper case in 3′ and reverse primer):Two gRNAs were used for generating gRNA1_Forward: CCTTAATACGACTCACTATAGGgcctggtggaacagcatctgGTTTTAGAGCTAGAAATAGC,gRNA2_Forward: CCTTAATACGACTCACTATAGGcgcaagctccagcaacctgcGTTTTAGAGCTAGAAATAGC, andgRNA_Reverse_primer: AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC.The gRNAs were then in vitro transcribed using the MEGAshortscript T7 kit . gRNAs and Cas9 mRNAs were microinjected into B6;SJLF1/J zygotes at a concentration of 75 ng/uL each gRNA and 100 ng/ul Cas9 mRNA.H2A.B paralog were mated to C57BL/6J strain mice to assess germline transmission. F(1)s were sequenced using primers spanning the whole H2A.B ORF and flanking regions:Founder animals, F(0), with putative mutations in any H2afb3: B1_F:CAAGCCAAACTTTTCTTGAGGATGT, B1_R:TGAGCAGGTCAGCCAAGAAG;H2afb2: B1/2_F:CAGGGAAAACTGTCTTCGAGA, B2_R:GATACTACTAAAGAGAATCAGCTTATAAAGAAGC;H2afb1: B3_F:ACACGGAGAACTGTCTTCAAAG, B3_R:CAGAATCAAATACGGAAACATTTTTCCC.Amplicons were cloned into pCR™4-TOPO TA vector (Thermo-Fisher) and verified by Sanger sequencing .We used PCR genotyping with the same primer pairs to maintain our H2A.B triple KO line. For mutant H2afb3 (B1), we used a forward primer specific to the first deletion . High molecular weight DNA was prepared from tissues collected postmortem on 1 hemizygous male and 1 homozygous sibling female produced from backcross number 7. A total of 5,307 BL6/J specific SNPs were probed by Illumina Infinium BeadChip .Four WT and 6 KO sibling age-matched 8-week-old males (backcross 7) were allowed to mate sequentially with individual C57BL/6J females  for 9 months. Once the female appeared pregnant, she was housed separately. If detected, vaginal plugs were recorded. Live and dead progenies born from pregnant females were recorded along with their sex and genotype. Long-term breeding experiments were performed by mating 1 male and 1 female . Each pair was allowed to produce up to 5 consecutive litters. The litters were genotyped and sexed at weaning.Five pairs of WT and KO sibling males between 2 and 8 months old were used. Testes were dissected, cleaned of fat, and weighed. A portion of 1 testis was dissected, fixed in modified Davidson’s fixative or Bouin’s solution, and stained with H-PAS (Hematoxylin-Periodic Acid Schiff) for histological analysis. For testicular spermatid counts and daily sperm production (DSP), 15 to 40 mg of testis was individually weighed and homogenized in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.1% Triton X-100, using 8 strokes in an all-glass Kontes 15-mL homogenizer. Ten microliters of the homogenate were assayed using a Neubauer phase contrast hemocytometer in duplicate. Total homogenization-resistant spermatids per gram and per organ and DSP were calculated by correcting for the number of squares counted, dilution, volume, weight, and duration of homogenization-resistant spermatids ,82, as pFor motility analyses, spermatozoa were collected from the caudae epididymides from the right side of each animal. We placed the isolated cauda into modified Tyrodes medium with 1% bovine serum albumin , cut the tubule about 6 times with fine microscissors, and allowed sperm to flow out into the medium. The medium was collected, and large pieces were allowed to settle. The suspended spermatozoa were diluted in the same medium as required, and 6 μL was placed into a Leja fixed coverslip slide for observation in a phase contrast microscope at 200× magnification. The percentage of sperm with beating tails was interpreted as % motile.For morphology analyses, caudae epididymidal sperm from 4 pairs of WT and KO males were smeared, and 100 sperm cells were subjected to a multiple anomaly morphology analysis at 1000×. Cells were counted as either “normal,” when no defect was observed or with “abnormal heads,” “bent necks,” or “tails defects” ) at room temperature in the dark for 20 min. Slides were rinsed once in 1× PBS, air-dried for at least 20 min, and mounted in 15 μL of Vectashield with DAPI .Epididymes were collected in Hank’s Buffered Salt Solution from 2 pairs of sibling WT and KO males at 3 months old. Caudal epididymal sperm were allowed to swim out in solution and smeared onto polylysine-coated slides. Slides were then fixed by immersion in 1:1 ethanol:acetone and dried for at least 10 min. CMA3 staining was performed as previously described . BrieflyParaffin-embedded testis cross sections were melted for 45 min at 65°C and deparaffinized in Histoclear twice for 5 min. Slides were then subject to a series of ethanol washes  for 3 min each. Slides were transferred to 1× PBS and subjected to the in situ cell death detection kit (Sigma-Aldrich) according to manufacturer’s instructions. Briefly, slides were proteinase K–treated for 20 min at 37°C in humid chamber, washed, and incubated with TUNEL reagent for 1 h at 37°C in a humid chamber.Germ cell spreads were performed as previously described  except fThe testes and epididymides of 3 pairs of KO and WT males (2 to 3 months old) were dissected, fixed and embedded in paraffin, and sectioned at the Fred Hutchinson Cancer Research Center (FHCRC) Histopathology Core. Tissue composition, structure and count of meiotic and somatic cells of the seminiferous tubule, as well as overall pathology  were assessed using haemotoxylin and eosin (H&E) staining.2, 5% CO2 until 4 days post-insemination and analyzed for progressive development.For in vitro fertilization studies, 26 WT females  were superovulated by Inhibin  followed by hCG , 48 h later. Cumulus–oocyte complexes were collected and in vitro fertilized with sperm from WT or H2A.B KO males  as previously described . The totAll imaging was performed on a Zeiss epifluorescence Axiovert 200M  inverted microscope.6 cells in DMEM 10% FBS. Cells were then washed in PBS and resuspended with Propidium Iodide  for cell sorting on a BD FACSAria II . Haploid cells were collected based on DNA content and red UV shift according to previously published protocols [Live cell sorting was performed as previously described . Brieflyrotocols . Cells wWe used Rabbit anti-H3K4me3 , rabbit anti-H3K27me3 , and rabbit anti-H3K9me3 . Rabbit anti-mouse IgG  was used as a negative control.dx.doi.org/10.17504/protocols.io.ufeetje). Up to 24 barcoded AutoCUT&RUN libraries were pooled per lane at equimolar concentration for paired-end 25 × 25 bp sequencing on a 2-lane flow cell on the Illumina HiSeq 2500  platform at FHCRC Genomics Shared Resource. We aligned paired end reads to the mm10 mouse genome assembly (Genome Reference Consortium) using Bowtie2 version 2.2.5 [For AutoCUT&RUN analyses, 2 WT and KO non-mated sibling pairs (2 months old) were used for replicate experiments. AutoCUT&RUN was performed according to the method previously outlined . Detaileon 2.2.5  with optusegalaxy.org [BAM files were processed using bedTools  to generlaxy.org .Pregnant dams were euthanized 14 days following observation of a reproductive plug. Embryos and placentas were dissected and washed with 1X PBS. For each conceptus, tissues were collected into 1.75 mL microcentrifuge tubes and kept on ice until weighing on a fine scale . Upon dissection, embryos were scored as “severely delayed” if they were properly implanted but displayed no visible vascularization and delayed organ development compared to neighboring E14.5 conceptuses with stereotypical development. Implantation sites with necrotic embryonic or placental structures were scored as resorbed.H2A.B KO animals were performed at the Fred Hutchinson Comparative Medicine animal facility. The facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and complies with all United States Department of Agriculture, Public Health Service, Washington State, and local animal welfare regulations.Animal husbandry and experimentation was reviewed, approved, and monitored under the Institutional Animal Care and Use Committee at Fred Hutchinson Cancer Research Center (Protocol ID: PROTO000050927). Protocols used for the breeding and tissue collection of S1 FigΔH2A.B animals. All H2afb coding loci mapped in mm10 are shown with their coordinates in parentheses. Details on the sequenced deletions for each paralog are shown at the bottom. Scissors indicate the regions of CRISPR/Cas9-induced cuts. Arrows indicates the location of genotyping primers  KO or (larger) WT fragment. H2afb3 primers are specific for the KO allele and do not amplify a band in the WT. HET, heterozygous; KO, knockout; WT, wild-type.(A) Schematic of the CRISPR/Cas9 strategy used to produce (TIFF)Click here for additional data file.S2 FigH2afb paralog ORFs with Sanger traces from the sequencing of H2afb1 (A), H2afb2 (B), and H3afb3 (C) from ΔH2A.B animals. Annotated are ORFs (yellow boxes) gRNAs (purple arrow) and deletions. gRNA, guide RNA; KO, knockout; ORF, open reading frame.Alignment of (TIFF)Click here for additional data file.S3 FigC57BL/6J SNPs in pure WT, H2A.B triple KO males or females are shown . HET, heterozygous; KO, knockout; WT, wild-type.Percentages of HET, homozygous, or non-(TIFF)Click here for additional data file.S4 FigR2 values are shown in red. (E) Percentage of total mapped fragment assigned to chromosome X (blue), Y (orange), or autosomes (blue) (see (A) Schematic of the flow cytometry (scatter plot) and MNase-based profiling (red box) used in this study.  AutoCUT&RUN normalized fragment counts (blue dots) over all peaks called for the histone modifications H3K9me3 (A), H3K27me3 (B), and H3K4me3 (C). KO counts are shown on the y-axis and WT on the x-axis. Linear regression and lue) see . Y-axis (TIFF)Click here for additional data file.S5 FigH2A.B KO (red), nucleosomal fragments have the size of H2A containing mono-nucleosomes. Note that H2A.B nucleosomes have a smaller MNase footprints than H2A nucleosomes. MNase cleavage is indicated by arrowheads. KO, knockout; MNase, micrococcal nuclease; WT, wild-type.Schematic of fragment size (x-axis) distributions (y-axis) in WT and KO spermatids (replotted from (TIFF)Click here for additional data file.S6 FigR2 values are shown in all 3 cases. KO, knockout; TSS, transcriptional start site; WT, wild-type. Heatmaps of normalized counts  of short fragments (<121 bp) and large fragments (>149 bp) over the top 2,000 low fuzziness nucleosomes in WT or KO samples are shown. Regions are centered on nucleosomes profiled with H3K9me3 (A), H3K27me3 (B), and H3K4me3 (C). (D) Distribution of fragments over the TSS of all + strand RefSeq genes. (E) KO/WT ratios of small/large fragment ratios in peaks defined by H3K27me3, H3K9me3, and H3K4me3 see . Log10 ((TIFF)Click here for additional data file.S7 Fign = 4; KO n = 3). Example images of each morphology are shown on the right. Supporting information can be found as (A) Representative images of testis cross sections of WT and KO males see  (B) Mean(TIFF)Click here for additional data file.S8 Fign is the number of tubules counted. (B) Crossover events measured using staining against the proteins SCP3 and MLH1 (see (A) Fraction of seminiferous tubules with at least 1 TUNEL positive cell in WT (blue) or KO (red) testis cross sections. MLH1 see  in germ (TIFF)Click here for additional data file.S9 FigKO denotes H2A.B KO chrX. (C) Percentage of litters with at least 1 dead pup in long-term breeding  Punnett squares of matings between WT females and WT males (top) or KO males (bottom) in fertility assays (A) or long-term breeding (B). The ratio of female pups (right squares) over male pups (lefts squares) denote the sex ratios. Xding see . Mating (TIFF)Click here for additional data file.S10 Fig(A) Representative images of WT (left panel) and KO (right panel) pachytene cells, from E17.5 germ cell spreads see , stained(TIFF)Click here for additional data file.S11 FigA segment of chr7 is shown, with Refseq genes, and transposon insertions (black bars). Normalized fragment counts across 2 replicates is shown for each AutoCUT&RUN dataset. WT samples (blue tracks) and KO samples (red tracks) are plotted for H3K27me3, H3K4me3, H3K9me3, and IgG. KO, knockout; WT, wild-type.(TIFF)Click here for additional data file.S1 TableTotal number of fragments of sizes <121 bp and >149 bp from equal sampling of total fragments in WT and KO. Chi-squared significance of observed KO counts versus expected counts in WT. KO, knockout; WT, wild-type.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableSEACR, sparse enrichment analysis for CUT&RUN.(XLSX)Click here for additional data file.S4 TableFour WT and 6 KO male siblings were bred sequentially to a total of 28 and 38 females, respectively. Detailed statistics and significance of statistical tests comparing KO to WT are shown. KO, knockout; WT, wild-type.(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 DataRPKM, reads per kb per million.(XLSX)Click here for additional data file.S5 DatachrX, X chromosome; chrY, Y chromosome.(XLSX)Click here for additional data file.S6 Data(XLSX)Click here for additional data file.S7 DataDPS, daily sperm production.(XLSX)Click here for additional data file.S8 DataTUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.(XLSX)Click here for additional data file.S9 Data(XLSX)Click here for additional data file. 17 Sep 2020Attachment106936_1_rebuttal_1863699_qgrtb7.docxSubmitted filename: Click here for additional data file. 28 Sep 2020Dear Harmit, Thank you for submitting your manuscript entitled "Biparental contributions of the H2A.B histone variant control embryonic development in mice" for consideration as a Research Article by PLOS Biology.The revision of your manuscript from Review Commons has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission back to the original reviewers.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. 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We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: -------------------------------------------------------------Reviewers’ commentsRev. 1:In the revised version of their manuscript, the authors have satisfyingly addresses the issues/questions that I had raised in my first review. The study is novel and highly significant; I support its publication without additional changes.NB. I detected two typos: one in Fig S4E title, the other Line 492 .And while re-reading my review I realize I wrote "CUT&TAG" instead of Cut&Run, my apologies for the confusion!Rev. 2:This is a significantly improved version of the manuscript previously submitted to Review Commons. The authors have successfully addressed most of my comments.This is a highly relevant work, giving an important contribution for a better understanding of the developmental roles of short H2A histone variants in placental mammals.Given its relevance to the field, I recommend this manuscript in its current format for publication in PLoS Biology. Yet, the lack of a precise mechanistic explanation for the embryonic roles of H2A.B, should nevertheless be considered before a final editorial decision.Rev. 3:I went through the revised manuscript and feel that the manuscript is now acceptable. 22 Nov 2020AttachmentResponseToReviewers.docxSubmitted filename: Click here for additional data file. 30 Nov 2020Dear Dr Malik,On behalf of my colleagues and the Academic Editor, Laurence D Hurst, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology. 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International Journal of Molecular Sciences. In the corrected version, we revised the title and figure legends in Figures 1, 2 and 5 due to indefinite expressions. As for funding resources, we deleted the NIH grant in light of it having no connection to the research. The following changes are acknowledged, and the changes do not affect the conclusions of the paper.The authors would like to make the following corrections to their paper, published in the 1. We changed a word in the titleChlamydia psittaci PmpD-N modulated Chicken Macrophage Function by Triggering Th2 Polarization and the TLR2/MyD88/NF-κB Signaling Pathway.2. We revised the figure legends of Figures 1, 2 and 5The figure legends should be replaced with the following descriptions:Figure 1. Construction of pEGFP-N1-pmpD-N plasmid and detection of PmpD-N expression. (A) The pmpD-N gene was amplified from genomic DNA of C. psittaci 6BC by PCR. M: Marker (MD103); 1: Negative control; 2: pmpD-N gene (1161 bp). (B) The amplified pmpD-N gene was inserted into the pEGFP-N1 vector to generate pEGFP-N1-pmpD-N. (C) The pmpD-N genes were expressed in HD cells and identified by RT-PCR assay. M: Marker (MD103); 1: DNA product of pEGFP-N1-pmpD-N plasmid transfected into HD11 cells; 2: RNA product of pEGFP-N1 plasmid transfected into HD11 cells. (D) The PmpD-N protein was detected by Western blotting analysis.Figure 2. The effect of PmpD-N on chlamydial load, phagocytic function, and nitric oxide (NO) production in the HD11 cells after treatment with PmpD-N or pEGFP-N1-pmpD-N. (A) The data showed the mean number of chlamydial inclusion forming units (IFU) for each group. (B) Phagocytic activity was calculated as the ratio between the MFI of each group and the net phagocytic value. The data showed the percentage of phagocytic activity (±SD) for cultures for each experiment. (C) The concentration of NO was measured by the Griess method and differences were compared at ** p < 0.01.Figure 5. Detection of NF-κB p65 migration by confocal microscopy. NF-κB p65 nuclear migration in HD11 cells was detected by immunofluorescence microscopy. The p65 unit was stained with Alexa Fluor® 488(green) and nuclei were visualized by DAPI counterstain (blue). Note the diffuse distribution of NF-κB p65 (green) in the nucleus.3. We revised the funding source as follows


Here, we provide evidence for the role of Myo X in Netrin-1-DCC-regulated axon development in developing mouse neocortex. The knockout (KO) or knockdown (KD) of Myo X in cortical neurons of embryonic mouse brain impairs axon initiation and contralateral branching/targeting. Similar axon deficits are detected in Netrin-1-KO or DCC-KD cortical neurons. Further proteomic analysis of Myo X binding proteins identifies KIF13B (a kinesin family motor protein). The Myo X interaction with KIF13B is induced by Netrin-1. Netrin-1 promotes anterograde transportation of Myo X into axons in a KIF13B-dependent manner. KIF13B-KD cortical neurons exhibit similar axon deficits. Together, these results reveal Myo X-KIF13B as a critical pathway for Netrin-1-promoted axon initiation and branching/targeting.Myosin X (Myo X) transports cargos to the tips of filopodia for cell adhesion, migration, and neuronal axon guidance. Deleted in Colorectal Cancer (DCC) is one of the Myo X cargos that is essential for Netrin-1-regulated axon pathfinding. The function of Myo X in axon development SIGNIFICANCE STATEMENT Netrin-1 increases Myosin X (Myo X) interaction with KIF13B, and thus promotes axonal delivery of Myo X and axon initiation and contralateral branching in developing cerebral neurons, revealing unrecognized functions and mechanisms underlying Netrin-1 regulation of axon development. Neurons are highly polarized cells typically with a single axon and multiple dendrites. Axon development is crucial for the establishment of neuronal connections, especially for the connection between different brain regions. Axon development includes the following three main steps: (1) axon specification/initiation during neuronal polarization; (2) axon growth and guidance; and (3) axon branching and presynaptic differentiation. During early development of mammalian cortex, the migrating neurons in the intermediate zone (IZ) transit from multipolar to bipolar, with a leading process toward the pia and a trailing process toward the ventricle. Once localized into the cortical plate (CP), the leading processes will develop into highly branched dendrites and the trailing processes will become long axons projecting to target regions for further bifurcation . EventuaAxon development is regulated by intrinsic factors in neurons as well as by microenvironmental factors or extracellular guidance cues. Netrin-1 belongs to the netrin family of extracellular guidance cues, which is crucial for axon pathfinding . Netrin-in vivo remain to be further elucidated.Myo X, an unconventional actin-based motor protein, is primarily localized at the tips of filopodia or the edges of lamellipodia and membrane ruffles . It undeHere, we present evidence for Myo X interaction with KIF13B to be crucial for Netrin-1-induced axon initiation and branching/targeting in developing mouse cortical brain. Myo X interacts with KIF13B , a kinesin family member that is essential for delivery of phosphatidylinositol -trisphosphate [PIP3] to axons and axon outgrowth . Netrin-f/f mice were generated as previously described (f/f) mice were kindly provided by Yu-Qiang Ding . All the mouse lines indicated above were maintained on a C57BL/6 background for more than six generations. Timed pregnant female mice were obtained by crossing with male mice overnight, and the noon of the following day was designated as embryonic day 0.5 (E0.5) if the vaginal plug was detected.Myo Xescribed , and NetFor immunostaining analysis, the following primary antibodies were used: mouse monoclonal anti-Tau-1 , mouse monoclonal anti-MAP2 , rat monoclonal anti-tubulin  from Millipore; chicken polyclonal anti-GFP  and mouse monoclonal anti-c-Myc  from Abcam. For immunoblotting analysis, the following primary antibodies were used: goat polyclonal anti-DCC  from Santa Cruz Biotechnology; rabbit polyclonal anti-KIF13B  from Thermo Fisher Scientific; mouse monoclonal anti-α-tubulin  and mouse monoclonal anti-GFP  from Sigma-Aldrich; rabbit polyclonal anti-Myc  from Abcam; rabbit polyclonal anti-MyoX was prepared as previously described ; the polyclonal anti-KIF13B antibody was provided by Hiroaki Miki . For immunoprecipitation assay, rabbit polyclonal anti-GFP  was purchased from Thermo Fisher Scientific. Alexa Fluor 488-, 555-, and 647-coupled secondary antibodies against mouse, rat, chicken, or goat and HRP-conjugated secondary antibodies against mouse or rabbit were purchased from Jackson ImmunoResearch. Alexa Fluor 350-phalloidin  and lipofectamine 2000  were obtained from Thermo Fisher Scientific. Wortmannin was from Millipore.Myo X was subcloned into mammalian expression vector (pEGFP-C1) fused with GFP at the N terminus, as described previously  fusion proteins was constructed by ligation into pGEX-6p-1. mCherry-Myo X was kindly provided by Staffan Strömblad .The cDNA of mouse eviously . GFP-MyoThe miRNA expression vectors for Myo X and DCC were generated by the BLOCK-iT Lentiviral miR RNA Expression System (Thermo Fisher Scientific) according to the manufacturer instruction as previously described . The shRd-lysine-coated dishes or glass coverslips at 37°C in a 5% CO2 atmosphere. Four hours later, the medium was changed into Neurobasal medium with 2% B27 supplement and 2 mm Glutamax. For transfection, neurons were electroporated immediately after dissociation using the Mouse Neuron Nucleofector Kit (Amaxa). In brief, 3 × 106 neurons were resuspended in 100 µl of Nucleofectamine solution containing 3 µg of plasmid and electroporated with Program O-003 of Nucleofector.Primary cortical neurons were cultured as described previously . In brieNLT cells and HEK293 cells were grown in DMEM supplemented with 10% FBS and 100 U/ml penicillin-streptomycin. For imaging experiments, 50−70% confluent NLT cells in 12-well plate were transfected with 1.6 μg of indicated plasmids using 3 μl of lipofectamine in DMEM without FBS and antibiotics. For Western blot and coimmunoprecipitation, HEK293 cells were transfected using polyetherimide (PEI). Stable HEK293 cell line expressing human Netrin-1 was used as described previously . The worin utero electroporation (IUE) was conducted as described previously with some modifications  objective at a resolution of 1024 × 1024 pixels. The software ImageJ (FIJI) was used for tracking analysis and kymographic analysis. In brief, the traveling path and velocity of GFP-Myo X puncta were recorded with the “Manual Tracking” plugin by clicking on the GFP-Myo X puncta on the temporal stacks. For kymographic analysis, a segmented line was used to draw a region of interest (ROI) and then the “KymographBuilder” plugin was used to produce kymographs for the selected segments.Transfected neurons were grown on Lab-Tek II Chambered cover glass (Thermo Fisher Scientific) in DMEM supplemented with 10% FBS and antibiotics. For visualizing GFP-Myo X movement, the Lab-Tek II Chambered Coverglass were then fitted into a temperature-controlled chamber on the microscope stage of LSM 710 confocal laser-scanning microscopy (Carl Zeiss) for observation at 37°C in a 5% COThe experiments were performed using the LSM 710 confocal laser-scanning microscopy. Images were acquired with an 63×/1.4 N.A. objective at a resolution of 1024 × 1024 pixels. A region at the proximal axon was bleached with high laser power, and fluorescence recovery was observed for a period of 10 min. For fluorescence recovery after photobleaching (FRAP) analysis, the mean intensity of the bleached area was normalized with the initial fluorescence intensity before bleaching.m Tris, pH 8.0, 10 mm NaCl, 1 mm EDTA, 0.5% NP-40, 1 mm NaF, 1 mm Na3VO4, protease inhibitor cocktail (Roche), 1 mm PMSF]. GFP-hMyoX and its binding partners were immunoprecipitated with anti-IgG (control) or anti-GFP antibodies, respectively. The immunoprecipitates were resolved by 8% SDS-PAGE gel and subjected to silver staining. All unique bands in anti-GFP group at molecular weight from ∼50–250 kDa on the gel were isolated and subjected to liquid chromatography–tandem mass spectrometry (LC-MS/MS) as previously reported (m NH4HCO3. The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a reversed-phase analytical column. The gradient was composed of an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile). The peptides were subjected to nanospray ionization (NSI) source followed by MS/MS in Q Exactive Plus (Thermo Fisher Scientific) coupled online to the ultra-performance LC. The resulting MS/MS data were processed using Proteome Discoverer 1.3. Tandem mass spectra were searched against UniProt database. Trypsin was specified as a cleavage enzyme allowing up to two missing cleavages. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethyl on Cys were specified as fixed modification and oxidation on Met and acetylation modification were specified as variable modifications. Peptide confidence was set at high, and peptide ion score was set >20.Cultured cortical neurons transfected with GFP-hMyoX expression plasmids were lysed 48 h later with lysis buffer [20 mm Tris-HCl, 10 mm NaCl, 1 mm EDTA, 0.5% NP-40). Immune complexes were resolved by SDS-PAGE and subjected to immunoblotting. GST pulldown assay was conducted as described previously . Cell lysates were precleared with GST immobilized on glutathione–Sepharose 4B  and then incubated with the indicated GST fusion proteins (2–5 μg) immobilized on glutathione–Sepharose beads at 4°C overnight with constant rocking. The beads were washed three times with modified RIPA buffer, and bound proteins were resolved by SDS-PAGE and subjected to immunoblotting.Immunoprecipitation was conducted as previously described . Cell lyeviously . Transiet test or one-way ANOVA followed by a protected least significant difference Fisher's post hoc test for multiple comparisons. Statistical evaluations were performed with the software GraphPad Prism version 5.0. The data are presented as the mean ± SEM. p values <0.05 were considered significant.Immunostaining sections and cells were observed under a Zeiss LSM 710 confocal microscope with ZEN 2012 software, and only the brightness, contrast, and color balance were optimized after imaging. The software ImageJ was used to measure fluorescence intensity in all fixed images. The software ImageJ was used to measure fluorescence intensity in all fixed images. In brief, the RGB images were converted into 8-bit grayscale images and inverted to negative images for analysis. After being converted to uncalibrated optical density value, the area of axons, soma, and dendrites was selected with ROI tools and calculated. GFP-Myo X intensity in axons or dendrites was normalized by that in its soma regions. Statistical analyses were performed using either unpaired two-tailed Student's in vivo, we used Cre-LoxP recombination technology in combination with IUE to delete Myo X in portions of projection neurons in developing neocortex. Specifically, we examined the axon development of upper layer (L2–L4) neurons that project within the cortex, mostly via the corpus callosum (CC), and have birthdays at E14.5 to E15.5  embryos at E14.5, and their axon intensity ratio  resulted in a reduction in the axon intensity ratio in the brain, suggesting a role of Myo X in this event. In addition, neuronal migration appeared to be impaired , as reported previously using RNA interference technology  neurons crossed the midline, and their lengths were comparable to those of control axons , suggesting little role, if there is any, for Myo X to play in axon growth or middling crossing. We finally examined axon contralateral branching/targeting in P14 control and Myo X-KO cortical brains. As shown in G and H, axon branching/targeting to the contralateral cortex was detected in control neurons; but, this process was largely impaired in Myo X-KO neurons. These results suggest necessity of Myo X in promoting axon branching/targeting. Together, these results reveal unrecognized roles of Myo X in axon initiation and contralateral branching/targeting in developing cerebral cortex.To investigate the function of Myo X in axon development to E15.5 . IUE wasty ratio  was analty ratio . As showimpaired B,C, as rchnology . We thenol axons E,F, suggf/f) embryos at E14.5. The electroporated brain samples were examined at E18.5 to evaluate the neuronal axon intensity ratio. Indeed, a reduction in the axon intensity ratio of cortical neurons was detected in Netrin-1-KO embryos , revealing a similar role of Netrin-1 as that of Myo X in axon initiation. Neuronal migration was not affected by Netrin-1 KO . We next asked whether Netrin-1-regulated axon initiation depends on Myo X. To this end, plasmids encoding Myc-Netrin-1 and Myo X miRNA were coelectroporated into the E14.5 embryos (D). Netrin-1 ectopic expression restored the axon intensity ratio in Myo X-KD neurons , supporting the view for Netrin-1-Myo X pathway in promoting axon initiation.Given the specific orientation of the trailing processes or nascent axons, we speculate that extracellular signals from ventricular zone (VZ) and subventricular zone (SVZ) modulate intrinsic signaling  for axon initiation and development, and Netrin-1, an upstream Myo X regulator , may be  embryos A,B, reverin-1 KO A,C. We n embryos D. Netrin neurons D,E, suppF,G). Their axons at the age of P14 were examined. Netrin-1 KD in E15.5 cortical neurons had little effect on the axonal contralateral branching/targeting . However, DCC KD impaired axonal contralateral growth and branching . These results, which are in line with our hypothesis, suggest that DCC and Myo X in neurons are necessary to promote axon branching, but Netrin-1, an extracellular cue, regulates axon development in cell-nonautonomous fashion.We then asked whether the Netrin-1–DCC pathway plays a role in axon branching/targeting similar to that of Myo X. Netrin-1 or DCC expression in E15.5 cortical neurons were suppressed by IUE of their shRNAs, respectively. The knock-down efficiency of Netrin-1 or DCC shRNA was examined by Western blot F,G. Theiargeting H,I. Howeranching H,J. ThesA). Upon Netrin-1 stimulation, an obvious increase of GFP-Myo X in Tau-1-positive axons with a slight decrease of GFP-Myo X in MAP2-positive dendritic neurites were detected (A–D), suggesting a role of Netrin-1 in regulating GFP-Myo X distribution in axons and dendrites.To understand how Netrin-1 regulates the function of Myo X in axon development, we examined the effect of Netrin-1 on exogenous Myo X (GFP-Myo X) distribution in cultured neurons. To our surprise, GFP-Myo X was largely distributed in the soma and the tips of dendrite-like filopodia, but was low in Tau-1-positive axonal compartments in the absence of Netrin-1 A. Upon Ndetected A–D, suggE). At 3 d in vitro (DIV3), neurons exhibited a type of polarized morphology with a very long axon and multiple short dendrites. The longest neurite, likely to be the axon (E–G), supporting the view for Netrin-1 to enhance GFP-Myo X movement. In contrast to axonal GFP-Myo X, GFP-Myo X in dendrite-like filopodia exhibited a puncta pattern (H). Time-lapse imaging and analyzing the motility of GFP-Myo X in these filopodia showed both extension and retraction movements of GFP-Myo X puncta in control neurons . Upon Netrin-1 stimulation, the traveling path and the average velocity of GFP-Myo X puncta were all reduced , with an increase in the percentage of stationary puncta . Since Myo X motility is coupled with the filopodia movement and MyoX undergoes forward and reverse movement along filopodia . In the absence of Netrin-1, Myo X mutants containing the motor domain showed a distribution pattern similar to that of full-length Myo X, with predominant localizations in the soma and dendritic filopodia . However, in the presence of Netrin-1, Myo X deletions in the second PH domain (Myo XΔPH2) or in the FERM domain (Myo XΔFERM) abolished Netrin-1-induced axonal distribution . These results suggest a requirement of both PH and FERM domains in Myo X for Netrin-1-induced axonal distribution of Myo X.To further understand how Netrin-1 regulates the axonal distribution of Myo X, we first mapped domains in GFP-Myo X that are necessary for this event. Myo X is a multidomain containing unconventional myosin, containing 3 PH domains, an MyTh4 domain, and a band in the FERM domain in the C terminus, in addition to the motor domain in the N terminus , and neurons suppressing DCC expression by its shRNA failed to response to Netrin-1 in targeting Myo X to axons .As the FERM domain in Myo X binds to DCC , we thusFig. 5m), an inhibitor of PI3K that blocks the production of PIP3 and PIP2. Such an inhibition abolished Netrin-1-induced axonal distribution of Myo X (F–H), supporting a role for PI3K activation and its products, PIP3/PIP2, in this event.PH domains of Myo X are known to bind to multiphosphoinositols, including PIP2, PIP2, and PIP3 . The secof Myo X F–H, suppA). Interestingly, KIF13B  was identified (A). Given that anterograde transport is powered by kinesin family motor (B). As shown in B and C, terminal regions of KIF13B , but not the N terminus (KIF13B1-557), were detected in Myo X immunoprecipitates. Further analysis of their interaction identified that the C-terminal domain KIF13B1532-1826 is involved in the interaction with Myo X.To further investigate the underlying mechanisms of Netrin-1-stimulated Myo X anterograde transport, we attempted to identify additional Myo X binding proteins by use of the LC-MS/MS to sequence the proteins associated with the Myo X immunoprecipitates. Specifically, GFP-hMyoX (GFP-tagged headless MyoX) was transfected into cultured cortical neurons. The cell lysates were immunoprecipitated with anti-IgG (as a control) or anti-GFP antibody. The immune complexes were resolved by SDS-PAGE and subjected to silver staining. All specific bands were subjected to LC-MS/MS. Five hundred eighty-six proteins were detected in the immunoprecipitates with anti-GFP antibody, including previously reported binding partners, such as actin, α-tubulin, Arp2/3 complex, and E-cadherin A. Intereentified A. Given ly motor , we thusly motor . We thenly motor B. As sho1532-1826 fusion protein was produced (C), which was used to pull down lysates expressing various GFP-Myo X mutants . hMyoX is an abbreviation of headless MyoX, which contains amino acids from 860 to 2062. hMyoXKK1225/6AA means that the 1225/1226 lysine was further mutated to alanine. These two lysines are located in the second PH domain and are required for Myo X binding with PIP3. Note that only hMyo X and hMyo XKK1225/6AA were pulled down by GST-KIF13B1532-1826, suggesting the requirement of the second and third PH domains of Myo X for its binding to the C-terminal domain in KIF13B (C). By this assay, the effective Myo X binding region in KIF13B was further mapped to the last 74 aa in its C terminus (D). It is noteworthy that while the site KK1225/6 in Myo X is critical for binding to PIP3 P3 , it was E, GFP-Myo X was colocalized with Myc-KIF13B in filopodia tips in NLT cells and in axon and dendrite-like filaments in cultured cortical neurons. In addition, their interaction was reconfirmed by coimmunoprecipitation analysis of exogenously expressed GFP-Myo X and Myc-KIF13B (F), as well as endogenous KIF13B with Myo X in primary neuronal lysates (G). Interestingly, Netrin-1 stimulation increased Myo X–KIF13B interaction in neurons (G). Together, these results suggest that Myo X interacts with KIF13B, implicating KIF13B in Netrin-1-induced Myo X distribution in axons.Finally, we examined Myo X–KIF13B interaction by coimmunostaining analysis. As shown in Figure 6c-KIF13B F, as wel lysates G. Intere neurons G. TogethA–C). In line with this view was the observation by the FRAP assay that the recovery of GFP-Myo X after photobleaching in axons was sped up by KIF13B expression (D–F). Furthermore, the effect of KIF13B on GFP-Myo X distribution was examined by time-lapse imaging analysis. As shown in Figure 7G, GFP-Myo X puncta exhibited high motility in dendrite-like neurites. Such actin-based motility of GFP-Myo X was decreased in neurons coexpressing KIF13B (G–I).To investigate the function of KIF13B in Netrin-1-induced Myo X anterograde transportation, we first examined whether the distribution of GFP-Myo X in Tau-1-positive axons was affected by KIF13B expression. Indeed, the expression of KIF13B was sufficient to increase the localization of GFP-Myo X in axons and decrease the localization of GFP-Myo X in dendrites in the absence of Netrin-1A–C. In lpression D–F. Furtg KIF13B G–I.A,B). GFP-Myo X with KIF13B shRNA or control shRNA were cotransfected into neurons treated with or without Netrin-1. In control neurons (GFP-Myo X with control shRNA), the distribution of GFP-Myo X in axons was increased and in dendrites was decreased by Netrin-1 stimulation (C–E). However, such an increase of the axonal distribution of Myo X was abolished in neurons cotransfected with KIF13B shRNA . Moreover, KIF13B KD in embryonic mouse cortical neurons significantly suppressed GFP-Myo X distribution in axon-like neurites . Together, these results suggest that KIF13B is not only sufficient, but also necessary for Netrin-1-induced Myo X distribution in axons. In line with this view, the distribution of KIF13B in axons was increased by Netrin-1 (H–J).Second, we determined whether KIF13B was necessary for Netrin-1-induced Myo X axonal distribution. A plasmid encoding KIF13B shRNA was generated, which selectively suppressed KIF13B expression A,B. GFP-mulation C–E. Howe3B shRNA C,D. Moreneurites F,G. TogeNetrin-1 H–J.in vivo. To test this speculation, KIF13B shRNA and Myo X miRNA were transported by IUE into the progenitor cells of cortical neurons in E14.5 mouse embryos, and their brain sections at E18.5 were examined (A). As shown in A and B, KIF13B KD resulted in a decrease in axon intensity ratio in the cortical brains, an impairment in axon initiation similar to that of Myo X-KD neurons. In addition, the percentage of Myo X- or KIF13B-deficient neurons into CP was decreased compared with that of control neurons . Furthermore, we analyzed the polarization of neurons in IZ and defined the longest neurites as axons by the following two criteria: the length of the longest neurite is >50 μm and is two times more than that of the second longest one. Based on these criteria, the percentages of polarized neurons in both Myo X-KD and KIF13B-KD groups were decreased  and their axons were also shorter . These results suggest that KIF13B plays a role similar to that of Myo X in axon initiation.The important role of KIF13B in Netrin-1-induced Myo X axonal distribution led us to speculate that that KIF13B plays a role similar to that of Myo X in Netrin-1-induced axonal initiation and targeting examined A. As sho neurons A,C. Furtecreased D,E and t shorter D,F. ThesG,H). However, at P14, the axonal contralateral branches were severely diminished in KIF13B-KD neurons compared with those of controls . Together, these results suggest that KIF13B is necessary to promote axon initiation and branching/targeting in developing cortical neurons, providing additional support for KIF13B as an important mediator for Netrin-1-induced and Myo X-regulated axon initiation and targeting.We next determined whether KIF13B regulates axon projection and branching as Myo X does. To this end, KIF13B was suppressed in E15.5 embryos by IUE of its shRNA (GFP). At neonatal age , the neurons electroporated mostly migrated into cortical L2/3 pyramidal neurons whose axons project to the contralateral side via CC . Just asFig. 9In this study, we present evidence that Netrin-1 increases axonal targeting of Myo X in neurons. This event is essential for axon initiation and contralateral branching, but not midline crossing. Further mechanistic studies suggest that Netrin-1 increases Myo X interaction with KIF13B, thus promoting axonal transport of Myo X, axonal initiation, and branching/targeting. These results reveal a new mechanism underlying Netrin-1-regulated axon pathfinding.Xenopus cranial neural crest cells  or KO (by Cre-Loxp combination) decreased the axon intensity ratio, suggesting a deficit in axon initiation . Morphology analysis of individual neurons suggested an impairment of axon genesis (D–F). In addition to axon initiation, Myo X KD or KO also impaired axon terminal branching/targeting . This function of Myo X is in line with the observations that Myo X plays an important role in regulating axon filaments and actin filaments in the leading margin of growth cones, which are responsible for perceiving extrinsic guidance factors or adhesive signals and producing traction for axon terminal elongation toward its target . These results together suggest that DCC and Myo X in neurons play a cell-autonomous role in promoting axon branching, while Netrin-1, as an extracellular cue, regulates axon development in a nonautonomous way. Although Netrin-1-DCC-Myo X was involved in axon branching, it is not the only pathway underlying axon branching. It is of interest to note that Netrin-1 promotes exocytosis and plasma membrane expansion for axon branching via TRIM9 release of SNAP25 and SNARE-mediated vesicle fusion . These results suggest a neuronal DCC-Myo X-independent mechanism for axon midline crossing.As an unconventional Myosin family protein, Myo X is widely expressed and implicated in multiple cellular functions in different cell types, including Netrin-1-induced neurite outgrowth and growth cone guidance; Zhu et al., 2007), BMP6-dependent filopodial migration and activation of BMP receptors , and migst cells . While ist cells , the in itiation E, 9A. Mo genesis D–F. In aargeting C,I. Thiss target . Interess target F,G. These fusion . It wille fusion F,G. ThesAxons contain abundant microtubules, although their growth cones have enriched actin filaments . How is in situ and single-cell RNA-seq databases on the websites. These results implicate that Netrin-1/DCC signaling in local microenvironment surrounding newborn neurons is important for axon development. In line with this view, we found that the axon initiation was slowed down by Netrin-1 KO in the local region , and Netrin-1 overexpression diminished Myo X deficiency-induced axon initiation deficit. Netrin-1 was knocked out in neural stem cells, as well as their progeny, including neurons and glial cells, which likely reduce Netrin-1 in the local environment. Therefore, the defect in Netrin-1-KO neurons could be because of loss of the autocrine and/or local paracrine effect of Netrin-1. How does Netrin-1 rescue the axon initiation deficit in Myo X-deficient neurons? It is noteworthy that neurons with Myo X KD by its miRNA, unlike Myo X KO, still contained low levels of Myo X. Upon Netrin-1 overexpression, it may promote the axonal transportation of the remaining Myo X and thus diminish the axon initiation defect. Alternatively, Netrin-1 may increase axonal initiation in a Myo X-independent manner, as multiple downstream signals can be activated by Netrin-1.Netrin-1/DCC signaling is involved in many aspects of axon development, including axon outgrowth and guidance, growth cone steering, and axon branching. The canonical model for the function of Netrin-1 in axon guidance is that Netrin-1 acts as a long-range diffusible guidance cue, centered in the midline , attracting or repulsing axons for their midline crossing. Recent studies have shown that Netrin-1 is produced not only in the midline, but also in neural progenitor cells in the VZ, and is deposited on the pial surface as a haptotactic adhesive substrate, where it guides DCC-positive axon growth . In devel region A,B, and In light of our results, we speculate the existence of the DCC–Myo X–KIF13B complex. Netrin-1 may increase the complex formation by generating more PIP3, which binds to Myo X, changes Myo X conformation for DCC and KIF13B binding, and then undergoes anterograde transport along microtubules . The mot


However, their role in neuronal subtype specification remains largely unknown. Using three different co-culture setups, we show that microglia-secreted factors enhance dopaminergic differentiation of somatic and induced pluripotent stem cell-derived human neural stem cells (NSCs). The effect was consistent across different NSC and microglial cell lines and was independent of prior microglial activation, although restricted to microglia of embryonic origin. We provide evidence that the effect is mediated through reduced cell proliferation and decreased apoptosis and necrosis orchestrated in a sequential manner during the differentiation process. tumor necrosis factor alpha, interleukin-1β, and insulinlike growth factor 1 are identified as key mediators of the effect and shown to directly increase dopaminergic differentiation of human NSCs. These findings demonstrate a positive effect of microglia on dopaminergic neurogenesis and may provide new insights into inductive and protective factors that can stimulate   •Differentiating NSCs in co-culture with microglia enhance dopaminergic differentiation•The effect is restricted to microglia of embryonic origin•Microglial TNFα, IL-1β, and IGF1 are key mediators of the effect In this article, Schmidt and colleagues show that differentiating human NSCs in co-culture with microglia enhance dopaminergic differentiation. The effect is consistent across different NSC and microglial cell lines but restricted to microglia of embryonic origin. TNFα, IL-1β, and IGF1 are identified as key mediators of the effect, providing new insights into factors stimulating dopaminergic differentiation. Neurogenesis is a complex process comprising several steps that require regulation by the microenvironment. Neural stem cell (NSC) proliferation and differentiation, migration of neuroblasts to their appropriate location, survival of immature and mature neurons, and construction of synaptic connectivity thus all rely on extrinsic cues .Microglia are immune cells in the central nervous system (CNS) that originate from hematopoietic progenitors of the yolk sac and start to colonize the developing brain as early as the fourth gestational week . As micrStudies suggest that the neurogenic effect of microglia is dependent on their activation state and is mediated by their cytokine release . FollowiMicroglia are widely, but not uniformly, distributed throughout the CNS. In the rodent brain, they are present in higher density in many brain regions containing dopaminergic neurons or dopaminergic projections, such as the substantia nigra, striatum, hippocampus, and the olfactory system, compared with surrounding structures . This suin vitro. We found that, independent of the microglial cell type and NSC lines, co-culturing NSCs with microglia during differentiation increased the content of dopaminergic neurons in the cultures. This effect was limited to microglia of embryonic origin and was not influenced by prior microglial activation. Microglial-secreted TNFα, IL-1β, and IGF1 were identified as key mediators, and recombinant TNFα, IL-1β, and IGF1 were shown to directly increase dopaminergic differentiation.To address the role of microglia and their activation state in dopaminergic neurogenesis, we investigated whether microglia-secreted factors enhanced dopaminergic differentiation of somatic and induced pluripotent stem cell (iPSC)-derived human NSCs L NSCs  were either grown in BV2 microglia-conditioned medium or directly  or indirectly (separated by semi-porous membrane inserts) co-cultured with BV2 microglia (+) neurons was significantly increased in all groups compared with control , only the direct and indirect co-culture setups revealed a significant increase in the yield of TH+ neurons  and a more differentiated morphology for neurons in the indirect co-culture group compared with control  tension (3%–5%) rather than atmospheric O2 tension (20%), which is the standard for most cell culture incubators, enhances the dopaminergic differentiation outcome , the vesicular monoamine transporter 2 (VMAT2), and the midbrain-specific transcription factors pituitary homeobox 3 (PITX3) and homeobox protein engrailed-1 (EN1) genes. The expression of the aromatic amino acid decarboxylase (AADC) and the midbrain dopaminergic LIM homeobox transcription factor 1-alpha (LMX1A) was not significantly changed. Midbrain characteristics of the TH+ neurons were also confirmed by FOXA2 immunostaining  compared with control; however, it was only significant for the CHME group -culture A and 5B.henotype C and 5D.henotype E and 5F.L cells  generate TH+ neurons of the midbrain dopaminergic phenotype . This difference might be explained by overexpression of the anti-apoptotic protein Bcl-XL in the hVM1-Bcl-XL line as the effect of the microglia was partly apoptosis mediated.Although the positive effect on dopaminergic differentiation was consistent across the investigated microglia and NSC lines, the effect on the general neurogenesis varied. For the hVM1-Bcl-Xin vitro culturing, as such procedures have been shown to induce transient activation of microglia  and hNS1 (forebrain NSC line) are described elsewhere  C57BL/6j mice . Cell isNSCs were exposed to microglia-secreted factors during differentiation by three different co-culture setups: exposure to BV2 microglia-conditioned media, direct co-culture, or co-culture using semi-porous membrane inserts A.L cells were seeded in this medium at day 0 of differentiation. One-hundred percent media change was performed every third day using newly prepared conditioned medium mixed in the same way. The control group was differentiated in unconditioned HNSC.100 medium.Microglia-conditioned medium was generated by culturing BV2 microglia in HNSC.100 medium for 3 days and collecting the medium through a 0.22-μm filter (Sarstedt) before mixing 1:1 with new unconditioned medium. hVM1-Bcl-XL cells were seeded together with BV2 microglia cells in a 1:50 ratio, and differentiation was performed as described in the For direct co-culture, hVM1-Bcl-XL and hNS1 differentiations and at day 5 for iPSC-NSC differentiation. The control groups received empty inserts.For co-culture using semi-porous membrane inserts (Merck), NSCs were seeded and allowed to attach for half an hour before inserts were placed and microglia seeded on the inserts in a 1:5 ratio relative to the NSCs. Differentiation was performed as described in the Immunocytochemistry, image analysis, and western blotting were performed as described in Escherichia coli O111:B4 (Sigma) or 20 ng/mL IL-4 (Peprotech) for 24 h in serum-free RPMI-1640 medium prior to co-culture setup (BV2 microglia were activated with either 100 ng/mL LPS from re setup . ActivatL cells and iPSC-NSCs were differentiated with addition of human recombinant TNFα (Sigma), IL-1β (R&D), or IGF-1 (R&D) during the first part of differentiation. Details are provided in hVM1-Bcl-XAnalysis was performed in GraphPad Prism version 6.0  using two-tailed unpaired Student's t test, multiple t test, one-way ANOVA, or two-way ANOVA as appropriate. Data are presented as mean ± SEM, and p values < 0.05 were considered statistically significant.Conceptualization, S.I.S., H.B., and M.M.; Methodology and Investigation, S.I.S., H.B., L.R., J.B.A., D.H., A.A., A.W., S.G.L., M.J., and J.O.; Resources, A.M.S., M.D.S., B.W.K., T.O., and M.M.; Data Curation, S.I.S; Writing – Original Draft, S.I.S., H.B., K.F., and M.M.; Writing – Review & Editing, all authors; Funding Acquisition, T.O., A.M.S. and M.M.


The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3′ to 5′ direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3′-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging.Sulfolobus solfataricus. A relatively high proportion (17–19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5′ parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3′-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5′-ends of RNAs was detected.To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3′-5′ direction.In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5′-ends of genes suggests simultaneous binding of both RNA ends by the The online version contains supplementary material available at 10.1186/s12864-020-07200-x. Sulfolobus solfataricus is a crenarchaeon with a growth optimum at 80 °C to 85 °C and pH 2 to 4 -ATP were performed. Complexes were resolved on a denaturing neutral-pH SDS-polyacrylamide gel electrophoresis , and transferred to a nitrocellulose membrane. Protein-RNA-complexes were visualized by autoradiography on an X-ray film at − 80 °C. Complexes of adequate size were excised from the membrane and RNA was isolated by proteinase K treatment. iCLIP library preparation was performed as described in [All following steps were performed as described in . In brie3′-RNA linker (L31): P-UGAGAUCGGAAGAGCGGUUCAG-Puromycin.Reverse transcription primers :R1clip P-NNAACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC.R6clip P-NNCCGGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC.R9clip P-NNGCCANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC.R10clip P-NNGACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC.R13clip P-NNTCCGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC.R14clip P-NNTGCCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC.Following oligonucleotides were used:Rrp4 Replicate 1: primer R1 - BC1: NNNGGTTNN.Rrp4 Replicate 2: primer R9 - BC4: NNNTGGCNN.Rrp41 Replicate 1: primer R10 - BC2: NNNGGTCNN.Rrp41 Replicate 2: primer R13 - BC5: NNNCGGANN.Trx Replicate 1: primer R14 - BC3: NNNGGCANN.Trx Replicate 2: primer R6 - BC6: NNNCCGGNN.These reverse transcription primers add the following barcodes to the 5′-end of the CLIP-tag sequences :The control Trx2, which was directly adjacent to the exosome samples on the membrane Fig. d, had soTwo independent bioreactor cultures were used for the iCLIP experiment. Samples from these cultures and from a third bioreactor culture were used for RNA-Seq. No rRNA depletion was performed for any of the samples. Total RNA was sequenced by Vertis Biotechnologie AG, Freising, Germany.http://hannonlab.cshl.edu/fastx_toolkit/index.html) were used for read processing. Due to bad sequencing quality of the first nucleotide, it was removed using fastx_trimmer (−f 2). Reads were subjected to a two-step quality filtering using fastq_quality_filter (−q 5 -p95 and -q 20 -p 90). The random barcode was utilized to remove PCR duplicates via fastx_collapser. 3′ adapter sequences were removed by fastx_clipper (−a TGAGATCGGA) and manually trimming of remaining nucleotides using awk (4.1.3). Fastx_trimmer (−f 3) was used to remove the first random barcode (2 nt). Separation of reads according to the experimental barcode was done with fastx_barcode_splitter.pl (−-bol --exact). Remaining barcodes (6 nt) were subsequently remove using fastx_trimmer (−f 7). Resulting sequences were mapped to the S. solfataricus P2 genome  via bowtie2 (2.3.4) (−-sensitive) [Reads resulting from Illumina sequencing (75 nt) carried a nine nucleotide barcode at the 5′ end. Barcodes were composed of a three-nucleotide random barcode, a four-nucleotide experimental barcode and a final two-nucleotide random barcode. Several tools from the FASTX Toolkit  , 51, sepnsitive)  and bedtnsitive) .S. solfataricus. Therefore the occurrence of circRNAs was investigated. For the detection of circRNAs interacting with the archaeal exosome, we applied a strategy similar to the one of Danan et al. [Sulfolobus, a prokaryotic organism lacking introns in protein-coding genes. The central idea of circRNA detection is the identification of a circularization junction. Sequencing of such a junction will result in a chimeric read, mapping to the target genome in a non-linear pattern. Briefly, detection of circRNAs harbouring a ligation site can therefore be performed by identification of two adjacent blast results in a single read aligning in a permuted way to the reference genome.A major part of the non-aligning reads contained significant amounts of sequence fragments from n et al.  that wasS. solfataricus P2 sequence acting as a reference, whereby a first blast result with a minimum length of 20 nt was obtained. For further analysis blast results were split according to strandedness. Furthermore, only those hits were kept that fulfilled the requirement of a non-aligned sequence within the read 5′ or 3′ to the blast result. Those reads were subsequently subjected to a second blastn search . The blast results obtained had to fulfil two requirements: They were not allowed to be more than 4000 nt apart from the position of the first blast hit and only single positional results were permitted. This allowed for the combination of the second fragments with their firstly identified counterparts.For circRNA detection only unaligned reads were used, as circularization junctions can only be contained in the non-aligning reads. The reads were subject to a blastn (2.2.31+)  search  . CIRI2 rS. solfataricus sequence were identified. Potential circRNAs were excluded.As with the detection of circRNAs, reads harbouring a 3′ attachment synthesized by the exosome can only be found within the set of non-aligning reads. In theory reads carrying a 3′ attachment must consist of a 5′ section, which maps perfectly to the organism’s genome while there 3′ attachment does not, preventing alignment of the entire read. Therefore detection of 3′ attachments shared considerable similarities with circRNA detection. Reads with a 5′ end alignment to the The nucleotide composition of the 3′ attachments of those reads was statistically assessed for all bases as well as for each position within the attachments. Data were summarized by custom Python 3 scripts and plotted using R (3.4.2).S. solfataricus was obtained from the DOOR database  [To evaluate the binding preference of the protein complex, iCLIP crosslink sites had to be determined. To consider only those reads with a high mapping quality and unique mapping position, reads with a MAPQ-score below 20 were removed (samtools view -q 20) . Replica0.07.31) . All genThe coverage ratio between iCLIP and RNA-Seq mappings for all coding sequences (CDS) and intergenic regions (IGRs) was assessed as follows: firstly a bed file containing all IGRs was creating using bedtools complement (v2.27.1)  and the Motif discovery for a predominant crosslink peak was performed using meme (−dna -nmotifs 1 -minw 4 -maxw 10) (4.12.0) . Ten basSulfolobus solfataricus P2 (NCBI Reference Sequence: NC_002754.1) [A-content, AG-content and the length of the longest poly(A) stretch were determined for each coding sequence (CDS) based on the RefSeq genome annotation of 02754.1)  using cuAdditional file 1: Figure S1. Uncropped images. Figure S2. CircRNAs identified in the exosome iCLIP analysis of S. solfataricus.Figure S3. Distribution of bases in RNA-tails detected in the aRrp41-iCLIP of the archaeal exosome. Figure S4. Poly(A) stretches in RNA are bound by the exosome. Figure S5. Global analysis of RNAs enriched by coimmunoprecipitation with the archaeal exosome by iCLIP. Figure S6. Antisense RNAs as preferred substrates of the archaeal exosome. Figure S7. Binding of the archaeal exosome to 5’and 3’parts of selected genes. Figure S8. Low affinity of the archaeal exosome to the abundant mRNA tmoA (SSO_RS06040 gene encoding toluene-4-monooxygenase system protein). Figure S9. Distribution of read counts in the genome of S. solfataricus was analyzed with respect to biotype RNA. Figure S10. Clustering of read counts at the 5′-and 3′-end of protein coding genes. Figure S11. Clustering of crosslink sites at the 5′ and 3′ end of predicted operons. Figure S12. Distribution of crosslink sites around the ends of annotated genesAdditional file 2:. iCLIP and RNA-Seq statistics.Additional file 3:. Mapped crosslink sites.Additional file 4: CircRNAs identified in the iCLIP analysis of the S. solfataricus exosome.Additional file 5: RNA-tails with mapped 5′-sequence, detected by iCLIP of the archaeal exosome.Additional file 6: Nucleotide content of the linear non-mapped cDNA reads in the iCLIP analysis of S. solfataricus.Additional file 7: List of gene products, A-content, AG-content and the length of the longest poly(A) stretch for each coding sequence.Additional file 8: Poly(A) stretches in CDS and in intergenic regions (IGRs), and binding of the archaeal exosome detected by iCLIP.Additional file 9: iCLIP and RNA-Seq counts of annotated genes included in Fig.


Siraitia grosvenorii (Swingle) was used in Traditional Chinese Medicine to reduce inflammation and facilitate bowel movement. The bioactive components of this plant show hypolipedimic, antidiabetic, antifibrotic activity and have been used against pancreatic cancer. Here, we examined whether mogroside IIE, a major bioactive component of unripe S. grosvenorii fruit, can protect against AP. We found that mogroside IIE decreased the activity of trypsin and cathepsin B induced by cerulein plus lipopolysaccharide (LPS) in the pancreatic acinar cell line AR42J and primary acinar cells in a dose- and time-dependent manner. Mogroside IIE treatment decreased the levels of serum lipase and serum amylase in mice injected with cerulein plus LPS without influencing inflammation significantly. A multi-cytokine array revealed that mogroside IIE decreased the level of interleukin 9 (IL-9) in AP mice. Exogenous IL-9 eliminated the mogroside IIE induced reduction of trypsin and cathepsin B activity and reversed the inhibition of cytosolic calcium and modulation of autophagy mediated by mogroside IIE. An IL-9 receptor antibody neutralized the effect of IL-9, restoring mogroside IIE activity. The mogroside IIE targeted IL-9 may partially arise from Th9 cells. Taken together, we provide experimental evidence that mogroside IIE ameliorates AP in cell models and mice through downregulation of the IL-9/IL-9 receptor pathway.The incidence of pancreatitis (AP) is increasing and there is no specific treatment available. Intracellular digestive enzyme activation is a key event in the pathogenesis of AP downstream of cytosolic calcium overload and impaired autophagy.  Siraitia grosvenorii (Swingle) is used in TCM as a general anti-inflammatory agent as well as bowel movement facilitator and a laxative (S. grosvenorii (Swingle) have also been regarded as hypolipedimic, anti-fibrotic and anti-diabetic (S. grosvenorii (Swingle) bioactive components showed benefits in pancreatitic cancer models  fruit (Acute pancreatitis (AP) is a common inflammatory disease without specific treatment . In Tradlaxative . The actdiabetic , which mr models . Thus, de) fruit  can protIntrapancreatic trypsinogen activation is central in the pathogenesis of AP. It is believed to be initiated by calcium overload or impaired autophagy . IndepenIL-9 is mainly secreted from T cells, and it is involved in type 2 immunity and regulates allergic inflammation . IL-9 isE on molecular markers of AP, focusing on pancreatic enzyme activity, cell calcium concentration, and systemic cytokine release. Out of 23 cytokines tested, we found a very significant reduction in IL-9 in the presence of mogroside IIE. Hence, a large part of our work was dedicated to elucidate the effect of mogroside IIE on IL-9 pathway and its role in AP.In our study we examined the effect of mogroside IIE (HPLC ≥98%) was purchased from Chengdu Man Si Te company  (http://www.cdmust.com/). Cerulein and 3-MA were purchased from MCE. LPS was purchased from Sigma. IL-9 was purchased from Origene. The IL-9 receptor antibody was purchased from SAB. β-actin and p62 antibodies were purchased from PTG. The LC3 antibody was purchased from Sigma. Fura-2 AM was purchased from Sigma. Fluro-4 AM was purchased from Solarbio.Magoside II2. Pancreas tissue was separated from C57BL/6 mice. Collagenase was used to digest pancreatic acinar cells. After filtration, primary pancreatic acinar cells were cultured in F12K (Gibco) supplemented with 10% FBS and used for subsequent experiments. The Ethical Committee on Animal Experiments at Guilin Medical University approved all animal care and experimental procedures, which were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals .AR42J cells  were cultured in Dulbecco's modified Eagle's medium  containing 10% fetal bovine serum  and 1% penicillin–streptomycin (Solarbio). All cultures were maintained in a 37°C incubator with 5% COE (M) (20 μM) was added for the indicated durations, or different concentrations of M were added for 6 h with C + L. IL-9 (10 ng/ml) was added to cells in the presence of C + L + M (20 μM) (C + L + M + IL9) for 6 h. The autophagy inhibitor 3-MA (2 mM) was added to cells with C + L or C + L + IL9 for 6 h. The IL-9 receptor antibody (2.5 μg/ml) was added to cells with C + L + M + IL9 for 6 h.AR42J cells or primary acinar cells were seeded in 12-well plates. Cells were treated with solvent (Control) or 200 nM cerulein plus 10 ng/ml LPS (C + L) for 6 h. Mogroside IIE (10 mg/kg) was injected once after the first injection of cerulein. C + L + Mshort group: similar to the C + L + Mlong group but mogroside IIE was given after the final cerulein injection. C + L + Mlong + IL9 group: similar to the C + L + Mlong group and IL-9 (10 mg/kg) was intraperitoneally injected after the first injection of cerulein. Mice were sacrificed 6 h after the final injection of saline or cerulein. Sera were collected to measure lipase and amylase. Pancreas tissues were lysed to measure the activity of trypsin and cathepsin B.Female C57BL/6 mice, 6–8 weeks old, 20 g, were purchased from SLAC . Mice were housed in clean environment and were deprived from food before any treatments (n = 6 in each group). Control group: mice were intraperitoneally injected with saline seven times at one-hour intervals, and solvent was given after the first saline injection. C + L group: cerulein (50 μg/kg) were intraperitoneally injected seven times, and LPS (10 mg/kg) was injected once after the final cerulein injection. C + L + Mlong group: cerulein and LPS treatments were the same as the C + L group, mogroside IIE and IL-9 treatment , mice were sacrificed. Spleens were ground. The homogenate was filtered using a 200-mesh cell sieve and centrifuged. Cell pellets were washed with PBS. After centrifugation, the cell pellet was incubated with red blood cell lysis buffer . Each sample was adjusted to a concentration of 1 × 106 cells/well. These cells were incubated with specific antibodies, including APC-CD4  and PECy7-CD8  and fixed and stained with a PE-IL9 antibody . Th9 cells are CD4+/CD8−/IL9+, and flow cytometry was used for analysis.After mogroside IIPancreatic acinar cells were lysed and incubated with butoxycarbonyl-Gln-Ala- Arg-7-amido-4-methylcou-marin hydrochloride (Sigma) in trypsin reaction buffer  at 37°C for 30 min. The fluorescence intensity of the reaction solution, which reflects trypsin activity, was measured  in a fluorescence microplate reader. Purified trypsin protein was added to the reaction mixture separately as the standard. In some experiments, trypsinogen activation was detected using the trypsin fluorescent substrate Rhodamine 110, bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine amide) (Sigma). Briefly, 50 μM of this molecule was added to cells for 20 min, and cells were imaged with microscopy. The fluorescence intensity reflecting trypsin activity was qualified using ImageJ software.Cells or tissue were lysed. Cathepsin B activity was measured according to the Enzymatic Assay of Cathepsin B protocol provided by Sigma, Inc. Briefly, cell lysates were incubated with cathepsin B substrate Nα-CBZ-Arg-Arg-7-amido-4-methylcoumarin at 37°C, and the fluorescence intensity was determined at 440 nm in a fluorescence microplate reader under excitation at 348 nm. Purified cathepsin B protein was added to the reaction mixture separately as the standard.Fura-2 AM was added to acinar cells according to the protocol provided by the manufacturer. Once indicated compounds were added, fluorescence was detected with spectrophotometer (Molecular Devices) with excitation wavelengths of 340 and 380 nm and emission at 505 nm. Dynamic changes in cytosolic calcium were monitored by using the Fura-2 340/380 nm fluorescence ratio. Endpoint calcium was also detected with Fluo-4 AM using a microscope (Themo EVOS M5000). After the indicated treatments, acinar cells were stained with Fluo-4 AM, imaged and quantified.Serum lipase was measured using an automatic-chemistry analyser .Formalin-fixed pancreas samples were processed, and 4-µm thick paraffin sections were stained with hematoxylin and eosin (H&E). In this study, histopathological changes, including edema, necrosis and infiltration of inflammation cells were qualified according to the criteria described in Luminex Mouse Cytokine/Chemokine Magnetic Bead Panel (MCYTOMAG-70K-23) was used, and cytokines were detected according to the manufacturer's instructions.SPSS 19.0  was used for statistical analyses. One-way analysis of variance or Student's t test was used to evaluate the statistical significance.E on trypsin activity was investigated first. AR42J cells and primary pancreatic acinar cells were cultured, and mogroside IIE was added for 1, 3, or 6 h. The results showed that the compound inhibited trypsin in a time-dependent manner  treatment. Mogroside IIE reduced the activity of trypsin and cathepsin B (E at low concentrations (5 or 10 μM) significantly decreased cerulein plus LPS-induced trypsin activation without showing obvious inhibition on the background level of trypsin activity , eotaxin, IL-1β, IL-6, IL-9, keratinocyte cytokine (KC), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), and macrophage inflammatory protein 1β (MIP-1β), RANTES and TNFα, are increased significantly in AP mice . Previous report showed that cytokine could activate trypsinogen or induce digestive enzyme  deactivation was related to IL-9. We performed a recovery experiment to investigate this hypothesis. IL-9 protein was injected into mogroside IIE-treated AP mice, and the activities of related digestive enzymes were detected.Mogroside IIe enzyme . TherefoE, but this phenomenon was reversed by IL-9 injection , and it is the predominant saponin component  and have similar molecular structures to mogroside IIE. These two compounds protect mice from LPS-induced acute lung injury , the recruitment program for the Affiliated Hospital of Guilin Medical University, and the Science and Technology Planned Project in Guilin . The present study was also supported by the Lijiang Scholar Award in Guilin (2017-004), the High Level of Innovation Team and Outstanding Scholars Program in Colleges and Universities in Guangxi (2017-38-08), and the Guangxi Distinguished Experts Special Fund (2019-13-12).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.