GLP-1/Notch signaling in Caenorhabditis elegans germline stem cells, we found a single mutation, om40, defining a gene called ego-3. ego-3(om40) causes several defects in the soma and the germline, including paralysis during larval development, sterility, delayed proliferation of germline stem cells, and ectopic germline stem cell proliferation. Whole genome sequencing identified om40 as an allele of hsp-90, previously known as daf-21, which encodes the C. elegans ortholog of the cytosolic form of HSP90. This protein is a molecular chaperone with a central position in the protein homeostasis network, which is responsible for proper folding, structural maintenance, and degradation of proteins. In addition to its essential role in cellular function, HSP90 plays an important role in stem cell maintenance and renewal. Complementation analysis using a deletion allele of hsp-90 confirmed that ego-3 is the same gene. hsp-90(om40) is an I→N conservative missense mutation of a highly conserved residue in the middle domain of HSP-90. RNA interference-mediated knockdown of hsp-90 expression partially phenocopied hsp-90(om40), confirming the loss-of-function nature of hsp-90(om40). Furthermore, reduced HSP-90 activity enhanced the effect of reduced function of both the GLP-1 receptor and the downstream LAG-1 transcription factor. Taken together, our results provide the first experimental evidence of an essential role for HSP90 in Notch signaling in development.In a genetic screen to identify genes that promote  Mutant strains used in this study were: BE63sqt-3(sc63), CB1489him-8(e1489), DG2389glp-1(bn18), DR96unc-76(e911), EL44 unc-32(e189) glp-1(bn18), EL129hsp-90(om40) unc-76(e911)/nT1[unc-?(n754) let-?], EL160 lag-1(om13ts)/nT1[unc-?(n754) let-?]; hsp-90(om40)/nT1[unc-?(n754) let-?], EL301lag-1(om13ts), EL375 sqt-3(sc63) unc-76(e911), EL384 sqt-3(sc63) hsp-90(om118) unc-76(e911)/sdc-3(y52y180) unc-76, EL418 hsp-90(om118) unc-76(e911)/nT1[unc-?(n754) let-?], EL490 unc-39(e257) hsp-90(om40)/unc-39(e257) unc-76(e911), EL660 lag-1(om13ts)/mIs11him-8(e1489), JL48 unc-32(e189) glp-1(bn18); hsp-90(om40)/nT1[qIs51], JL49 hsp-90(om40)/nT1[qIs51], JL54 unc-32(e189) glp-1(bn18); hsp-90(p673)/nT1[qIs51], JT6130hsp-90(p673), TY1470yDf8/nT1[unc-?(n754) let-?], VC914hsp-90(ok1333)/nT1[qIs51], YY216eri-9(gg106). Note: The daf-21 gene name was recently changed to hsp-90. Accordingly, the strains listed here are referred to using hsp-90 rather than daf-21 and ego-3.Worms were maintained on r plates  under str plates . Wild-tyhsp-90 was performed by the feeding method . An E. coli feeding strain expressing GFP dsRNA was used as a negative control.RNAi of om40 non-complementation screens were performed using either UV or EMS mutagenesis in an effort to isolate additional alleles of ego-3. UV mutagenesis was performed as described  unc-76(e911) hermaphrodites were treated with 30 ug/ml trimethylpsoralen (TMP) in M9 buffer for 15 min in the dark and irradiated with a long-wave UV source  at distance of 10 cm for 20 sec. The sqt-3sc63)( mutation confers recessive squat (Sqt) and dominant roller (Rol) phenotypes. Single candidate sqt-3(sc63) ego-3(omx) unc-76(e911)/sqt-3(sc63) ego-3(+) unc-76(e911) F1 hermaphrodites were mated with ego-3(om40) unc-76(e911)/++ males. RolUnc cross-progeny (sqt-3(sc63) ego-3(omx) unc-76(e911)/sqt-3(+) ego-3(om40) unc-76(e911) or sqt-3(sc63) ego-3(+) unc-76(e911)/sqt-3(+) ego-3(om40) unc-76(e911)) were screened for the Ego-3 sterile phenotype, which would presumably be of the genotype sqt-3(sc63) ego-3(omx) unc-76(e911)/+ ego-3(om40) unc-76(e911). The om118 mutation was identified from a total of 30,000 haploid genomes and recovered from fertile sqt-3(sc63) ego-3(om118) unc-76(e911)/+++ Rol non-Unc siblings.ego-3(om40) relative to SNPs as described (unc-39(e257) ego-3(om40) and ego-3(om40) unc-76(e911) chromosomes, generated in the N2 strain background, over chromosomes from the polymorphic wild-type strain, CB4856, and picked unc-39(e257) non-ego-3(om40) and unc-76(e911) non-ego-3(om40) recombinants, respectively. Using data available in WormBase (www.wormbase.org), we identified candidate SNPs in the region and verified their identity by PCR amplification and Sanger sequencing. We isolated homozygous recombinants from each Unc non-Ego recombinant line, PCR amplified the SNP region, and assayed by differential restriction digest or Sanger sequencing, as appropriate. This approach mapped ego-3(om40) to the right of two SNPs in the region covered by cosmid C54G10 and to the left of three SNPs in the region covered by Y50E8A, placing it within a 70 kb interval.We mapped ego-3(om40)/nT1[qIs51], contains ego-3(om40) balanced by a variant of the reciprocal translocation nT1 marked with an integrated myo-2::GFP construct that confers pharyngeal GFP expression. JL49 exhibits pharyngeal GFP and segregates sterile non-GFP ego-3(om40) homozygotes and GFP heterozygotes as the live progeny. The homozygous balancer is recessive lethal, and the strain also segregates a large percentage of nonviable aneuploid progeny.Strain JL49, E. coli strain OP50 or χ1666. Populations were allowed to grow at 20° until dense with growing, well-fed worms of mixed stage, then harvested by M9/Triton X-100 wash and centrifugation. Excess bacterial food was removed by three to five further rounds of washing and pelleting.JL49 was harvested from a starved 60 mm plate using M9 buffer supplemented with 0.01% Triton X-100, pelleted by centrifugation, and replated on 100 mm rich agarose plates (standard NGM recipe with 8X peptone and substituting low-EEO agarose at 1:1 for agar) seeded with lawns of ego-3(om40) homozygotes for whole genome sequencing.The resulting population was flow-sorted using a Copas Biosort 250 with fluorescence detection and Biosorter software version 5.25.2. A sorter screening window was chosen to select worms at the L4 stage or older. This set was further restricted to animals not expressing pharyngeal GFP, using the fluorescence-detection capability of the Copas sorter, resulting in a very highly enriched sample of The sorted homozygotes were replated on standard 60 mm NGM plates, and any remaining GFP animals were removed by hand picking. The contamination rate for GFP animals was very low, approximately 40 GFP animals in 15,000 total sorted worms.ego-3(om40) homozygotes was frozen at -80°. Genomic DNA was prepared using a standard protocol of Proteinase K digestion, treatment with RNAse A and 6 M NaCl, precipitation in isopropanol, washing, and resuspension in distilled water. A sequencing library was generated using the Nextera XT library preparation protocol, and the resulting library was sequenced on an Illumina HiSeq machine in the laboratory of Dr. Corey Nislow . Paired-end reads of 100 bp were generated, resulting in total genome coverage of 57X.The sample of C. elegans reference genome version WS230 (www.wormbase.org) using the short-read aligner Phaster . SNVs were then identified with SAMtools (Single-nucleotide variants (SNVs) were called with the analysis pipeline and filters developed for the Million Mutation Project  unc-76(e911)/nT1[unc-?(n754) let-?] heterozygotes followed by Sanger DNA sequencing. Sequence data suggested the presence of an insertion mutation in hsp-90. To determine unambiguously the location and sequence of the insertion, the region containing the putative insertion was again amplified from ego-3(om118) unc-76(e911)/nT1[unc-?(n754) let-?] heterozygotes, and PCR products were cloned into pUC19 using standard techniques. Plasmid DNA was isolated from 12 independent clones and sequenced. All clones contained either wild-type sequence or the om118 insertion mutation. PCR primer sequences and their locations can be found in Table S3 and Figure S1 in File S1, respectively.The Microscopy was carried out with a Zeiss Axioskop equipped with differential interference contrast (DIC) optics and epifluorescence. For fluorescence visualization of nuclei, intact worms were fixed in -20° methanol, and dissected gonads were fixed with 3% paraformaldehyde. Fixed tissue was incubated for 10-15 min in 0.2 ug/ml DAPI at room temperature and mounted in Vectashield (Vector Laboratories).C. elegans strains and plasmid constructs used in this study are available upon request. The raw sequence data from the WGS have been submitted to the NCBI BioProject (http://www.ncbi.nlm.nih.gov/bioproject) under accession number PRJNA428227 and can be accessed from the Sequence Read Archive  with accession number SRP127800. File S1 contains Figure S1, Locations of primers used to amplify and sequence regions of the hsp-90 gene to identify mutations; Figures S2–S4 in File S1, Sanger DNA sequencing chromatograms of wild-type and mutant sequences; Table S1 in File S1, Gonad transcriptome data for protein-coding genes in the ego-3 region; Table S2 in File S1, Codon and splicing changes detected in ego-3(om40) by whole genome sequence analysis; and Table S3 in File S1, Primers used to amplify and sequence regions of the hsp-90 gene to identify mutations.All glp-1 enhancer screen was to isolate partial loss-of-function alleles of genes that promote GLP-1/Notch signaling ; ego-3(om40) adults do not maintain a population of proliferative germ cells, but instead all germ cells enter meiosis prematurely and undergo gametogenesis (glp-1(+) background, in contrast, ego-3(om40) adults maintain a proliferative germ line. During development, ego-3(om40) alone also causes a number of defects in somatic and germline tissues including delayed development, paralyzed movement (an Unc phenotype), and “early” and “late” germline defects  phenotype appear to be loss-of-function based on analysis of ego-3(om40)/yDf8 individuals (ego-3(om40)/yDf8 animals, suggesting this aspect of the phenotype may not be due to a strict loss-of-function.One goal of the ego-3, we carried out a non-complementation screen using UV/trimethylpsoralen (TMP) mutagenesis. One mutation, ego-3(om118), was identified that fails to complement ego-3(om40) for the germline and somatic developmental defects described above and for glp-1 enhancement. In contrast, ego-3(om118) homozygotes and ego-3(om118)/yDf8 transheterozygotes die as embryos, thus ego-3(om118) appears to be a more severe loss-of-function allele than ego-3(om40). Consistent with this hypothesis, the ego-3(om118/om40)Unc phenotype is more severe than for ego-3(om40) animals, persisting into adulthood rather than being limited to larval stages.To identify additional alleles of ego-3(om40) maps just to the left of unc-61 on LGV  to the right of or very close to the left of daf-21, which was recently renamed hsp-90 (www.wormbase.org). Specifically, 23/23 Unc non-Ego recombinants from an hsp-90(p673)/ego-3(om40) unc-76(e911) strain were hsp-90(p673) unc-76(e911)/ego-3(om40) unc-76(e911). Note that hsp-90(p673) is a recessive gain-of-function mutation causing a dauer-constitutive (Daf-c) phenotype (ego-3(om40) relative to SNPs in the unc-39 to unc-76 interval, further localizing it to an ∼70 kb region containing 14 predicted protein-coding genes and several non-coding RNA genes (File S1). As none was an obvious candidate for involvement in GLP-1/Notch signaling, we undertook to identify the ego-3(om40) mutation by whole genome sequencing (WGS).We next mapped NA genes . Gonad tego-3(om40) homozygotes identified one sequence variant within the protein-coding region of one gene in the mapped region, hsp-90, which encodes the C. elegans ortholog of the cytoplasmic form of the molecular chaperone HSP90 (Table S2 in File S1) (om40 sequence variant is a T→A transition causing an I→N non-conservative missense mutation (File S1). Subsequently, we amplified and Sanger sequenced all hsp-90 exons from ego-3(om118) to locate the om118 lesion. We discovered a 20 bp non-tandem duplication within the same exon containing the om40 mutation, which is predicted to cause a frameshift and to introduce a premature termination codon (File S1). The inserted sequence occurs only once in the C. elegans wild-type genome and is therefore highly likely to have originated from within the hsp-90 gene. These data are strong evidence that hsp-90 and ego-3 are the same gene.WGS of mutation , which wego-3 and hsp-90 are indeed the same gene, we tested for complementation between ego-3(om40) and hsp-90(ok1333), a deletion and presumed null allele (C. elegans Deletion Mutant Consortium 2012). hsp-90(ok1333) worms have a paralyzed Unc phenotype and arrest development during mid to late larval stages (www.wormbase.org). We crossed ego-3(om40)/nT1[qIs51] males with hsp-90(ok1333)/nT1[qIs51] hermaphrodites at 20°. The nT1[qIs51] balancer chromosome has pharyngeal GFP expression, so non-GFP progeny will be hsp-90(ok1333) homozygous self-progeny and hsp-90(ok1333)/ego-3(om40) cross-progeny. Large numbers of GFP males, presumably ego-3(om40)/nT1[qIs51] cross-progeny, were produced, indicating that the ego-3(om40)/nT1[qIs51] males mated efficiently.To confirm that l allele  (C. eleghsp-90(ok1333) and ego-3(om40) do not complement. A few non-GFP worms survived to adulthood, with most developing into sterile, severe Unc hermaphrodites and severe Unc males. Therefore, ego-3(om40) fails to complement hsp-90(ok1333), and ego-3 is allelic to hsp-90, consistent with our molecular analysis of ego-3 alleles.The vast majority of non-GFP offspring from the cross were paralyzed Uncs and arrested as larvae, as expected if ego-3(om40) larval phenotype resembles the daf-21(ok1333) phenotype in that animals are paralyzed, develop slowly, and some aspects of development arrest during L2-L3 stage  mutants whereas only germline development arrests in ego-3(om40). We interpret the viability and complementation of om40/p673 transheterozygotes  as resulting from the previously reported gain-of-function nature of the p673 allele , which had not been previously reported although the amino acid sequence change for this allele had been (File S1).Additionally, we determined the nucleotide sequence change in om40, om118, and p673 alleles are all predicted to cause amino acid sequence changes affecting the middle domain (MD) of HSP90  of HSP90. The MD is essential for interactions with both client proteins and co-chaperones, and it contains a catalytic loop that contributes to ATP hydrolysis .Based on our molecular and genetic data, we conclude that hsp-90(p673) should not enhance glp-1(bn18), because hsp-90(p673) is a gain-of-function allele (hsp-90(om40) (see above). To test this, we constructed an unc-32(e189) glp-1(bn18); hsp-90(p673)/nT1[qIs51] strain. unc-32(e189) glp-1(bn18); hsp-90(p673) offspring are viable and fertile at 20° and produce a large proportion of dauer offspring, as expected for hsp-90(p673) animals. We also used DIC microscopy to examine the germlines of unc-32(e189) glp-1(bn18); hsp-90(p673) worms raised at 20° to see if hsp-90(p673) enhanced the mild germline proliferation defect in glp-1(bn18). hsp-90(p673) germlines resemble N2 wild-type germlines with extensive germline proliferation (unc-32(e189) glp-1(bn18) worms raised at 20° have substantially smaller germlines because of reduced GLP-1/Notch signaling (unc-32(e189) glp-1(bn18); hsp-90(p673) germlines closely resemble those of unc-32(e189) glp-1(bn18) animals (hsp-90(p673) does not enhance glp-1(bn18). Taken together, our results show that the hsp-90(om40) and hsp-90(p673) alleles both retain substantial HSP-90 activity such that the heteroallelic combination has approximately normal HSP-90 function. We further conclude that only a subset of hsp-90 mutations have the ability to enhance glp-1.We hypothesized that feration . In contignaling   germline proliferation defect is enhanced in hsp-90(om40)/yDf8; glp-1(bn18) animals  and uncoordinated phenotypes were similar in hsp-90(om40)/yDf8; glp-1(+) and hsp-90(om40/om40) mutants, consistent with those defects arising from reduced HSP-90 activity. However, the late germline phenotype, including proximal germline proliferation (Pro phenotype) and delayed gametogenesis, was less severe in hsp-90(om40)/yDf8 than in hsp-90(om40/om40) adults. At the time, we hypothesized this result might indicate that hsp-90(om40) has some gain-of-function character.Our previous study determined that the ego-3 gene has now allowed us to investigate the nature of the om40 allele in more detail. The lethal hsp-90(ok1333) allele appears to be null (as described above) and the yDf8 deletion completely uncovers the hsp-90 gene; therefore, the viability of hsp-90(om40/ok1333) and hsp-90(om40)/yDf8 confirm that hsp-90(om40) is not a null allele. At the same time, similarities among hsp-90(om40), hsp-90(om40/ok1333), and hsp-90(om40)/yDf8 phenotypes suggest that hsp-90(om40) predominantly causes a loss of gene function. To confirm this interpretation and to investigate the nature of the proximal proliferation defect, we knocked down HSP-90 via RNAi and examined the impact on development. Our first strategy was to place L4 wild-type (N2) hermaphrodites onto feeding plates containing bacteria expressing hsp-90 dsRNA at 20° and evaluate their F1 progeny. This strategy partially phenocopied hsp-90(om40), yielding offspring that were sterile, many with very reduced germline proliferation. Knockdown also caused some embryonic lethality in the F1, a phenotype observed in hsp-90(ok1333) and hsp-90(om118), and a protruding vulva phenotype. We observed similar results in unc-32(e189) glp-1(bn18); hsp-90(RNAi) animals with the additional observation of some embryonic lethality. We also performed hsp-90(RNAi) in an enhanced RNAi mutant, eri-9(gg106), and saw the same phenotypes, with the addition that some larvae arrested development. We next placed L1 larvae onto hsp-90 RNAi plates at 20° and examined the adults three days later. In a wild-type background, hsp-90(RNAi) adults were sterile with protruding vulvas and had very reduced germlines; the same results were obtained for unc-32(e189) glp-1(bn18); hsp-90(RNAi) although a smaller proportion of animals had a protruding vulva. eri-9(gg106) hsp-90(RNAi) adults were also sterile, but most animals were Unc and very sick with several having died in early adulthood. In no case did we observe the proximal germline proliferation (Pro) characteristic of hsp-90(om40).Molecular identification of the hsp-90 RNAi at L3 stage, reasoning that allowing some germline development to occur prior to knocking down HSP-90 might increase the chance of observing the Pro phenotype observed in hsp-90(om40) adults (him-8(e1489) strain in order to evaluate both males and hermaphrodites. Untreated him-8(e1489) worms have normal germlines (hsp-90(RNAi) adults produced in this study had germline developmental defects, often resembling either the hsp-90(om40) early or late germline defects (hsp-90(om40) early germline phenotype  phenotype is consistent with previous observations (hsp-90(om40)Pro phenotype (hsp-90(om40)Pro phenotype results from reduced HSP90 activity.As a follow up experiment, we assayed the effect of beginning ) adults . We perfermlines . The maj defects . Approxi n = 52; ; this hsrvations  resemblihenotype . TherefoHSP-90 in promoting Notch signaling, we asked whether hsp-90(om40) enhances the loss of lag-1 function in the germline. LAG-1 is a CSL-type transcription factor active downstream of GLP-1 in the C. elegans germline (lag-1(om13ts), that is fertile at 20° /nT1[unc-?(n754) let-?]; hsp-90(om40)/nT1[unc-?(n754) let-?] strain and evaluated germline proliferation in the hsp-90(om40); lag-1(om13ts) progeny raised at 20°. hsp-90(om40) and lag-1(om13ts) single mutants, each obtained from balanced strains, were assayed in parallel; all animals were stained with DAPI as ∼1 day old adults. As expected based on our previously published data, germline proliferation was retained in 100% of hsp-90(om40) and lag-1(om13ts) single mutants (lag-1(om13ts); hsp-90(om40) adult hermaphrodite germlines contained sperm, but lacked proliferating and meiotic germ cells (lag-1(om13ts); hsp-90(om40) animals apparently divided only a few times, as an average of only 26 sperm were observed per gonad arm , corresponding to an average of 6-7 germ cells. This result is consistent with a severe reduction in GLP-1 signaling in the lag-1(om13); hsp-90(om40) double mutant and further supports our conclusion that HSP-90 activity promotes GLP-1 pathway signaling activity.To further explore the role of germline . Here, w mutants   enhances mutations in both the GLP-1/Notch receptor and the LAG-1 downstream effector demonstrate that HSP-90 activity directly or indirectly promotes GLP-1/Notch signaling and is therefore required for proper germline stem cell function. While HSP90 has long been known to participate in a variety of developmental as well as proliferative/oncogenic signaling pathways in metazoans  and throughout the nematode lifecycle from embryonic through adult stages (In hsp-90(om40) and hsp-90(RNAi) animals are consistent with previously observed strong expression of HSP-90 in the developing and mature C. elegans germline  larval germline phenotype is epistatic to glp-1(oz112gf/q224), a heteroallelic combination that results in germline overproliferation (GLP-1/Notch protein encoded by glp-1(oz112) is constitutively active, yet reduction of HSP-90 activity in glp-1(oz112gf/q224); hsp-90(om40) larvae is sufficient to reduce GLP-1/Notch signaling and produce the hsp-90(om40) phenotype instead. Hence, HSP-90 acts downstream of GLP-1/Notch. Furthermore, the recently reported physical interaction between NICD and HSP90 also supports the germline as a location of HSP-90 action in GLP-1/Notch signaling in C. elegans .The germline phenotypes seen in hsp-90 missense mutations, hsp-90(p673) and hsp-90(om40), which cause non-conservative amino acid substitutions of highly conserved residues in the middle domain HSP-90, do not suggest obvious mechanisms by which they might produce their phenotypic effects. hsp-90(p673) is a recessive gain-of-function mutation producing a dauer-constitutive phenotype (hsp-90(RNAi) experiments, including our own, that fail to show an increase in dauer formation (hsp-90(p673) retains substantial HSP-90 activity and with the biochemical and biophysical analysis of bacterially-expressed wild-type and E292K (corresponding to hsp-90(p673)) C. elegansHSP-90 showing that this mutation only slightly reduces function of the protein (hsp-90(p673). Specifically, HSP-90(E292K) mutant protein is slightly reduced at 25°, but not at lower temperature. Moreover, the E292K substitution does not seem to alter the structural stability of the protein nor its binding of two HSP-90 co-chaperones, p23 (encoded by ZC395.10) and AHA-1, although binding to the STI-1 co-chaperone was slightly reduced (The molecular identities of the two known hsp-90(om40), hsp-90(om40), leading the authors to conclude that hsp-90(om40) might have both loss- and gain-of-function features. For example, the Pro phenotype is less severe in hsp-90(om40/null) adults, a result usually interpreted to mean that a phenotype is a consequence of a gain-of-function. However, nearly all hsp-90(om40) phenotypes , including Pro, are recapitulated by creating hsp-90(om40) transheterozygotes with two severe loss-of-function alleles, hsp-90(om118) and hsp-90(ok1333) and by hsp-90(RNAi) knockdown (hsp-90(om40), which causes an I461N substitution, is a loss-of-function mutation and that the I461N substitution reduces HSP-90 activity.With respect to et al. 2013; hsp-90 alleles and with hsp-90(RNAi) are not surprising and are likely in part a consequence of disrupting both HSP-90’s interaction with any of a number of co-chaperones and the folding of at least several of the hundreds of client proteins with which HSP90 assists (https://www.picard.ch/downloads/Hsp90interactors.pdf). Furthermore, co-chaperones display a range of tissue expression patterns, including in C. elegans, suggesting that even wild-type HSP-90 activities will vary in different tissues (hsp-90(om40). For example, a curious aspect of the hsp-90(om40) phenotype is a reversal of larval germline mitotic arrest and severe Unc defects when mutant animals reach late larval stages and adulthood (HSP-90(I461N) has impaired interactions with co-chaperones and/or client proteins expressed in larval germline and muscle, but has increased activity in older animals because of differences in co-chaperones and client proteins produced during late larval and adult stages.HSP90 is assisted in its protein folding function of a plethora of clients by more than 20 different co-chaperones  and hsp-90(RNAi) worms is proliferation in the proximal region of the germline (Pro phenotype) leading to formation of a proximal germline tumor. How does reduction of HSP-90 function, which appears to reduce GLP-1/Notch signaling and therefore reduce distal germline mitotic proliferation, paradoxically promote proximal germline mitotic proliferation? Whatever the mechanism, it seems to involve GLP-1/Notch signaling because proximal tumor formation is suppressed by the loss of glp-1 function (hsp-90(om40) and hsp-90(RNAi) hermaphrodites, mitotic arrest during larval germline development delays the entry of germ cells into meiosis and exposes them to a proposed latent sheath cell niche, thereby stimulating their proliferation via GLP-1/Notch signaling, as described for mutations in several other genes (C. elegans are possible candidates for activating proximal proliferation in males as well as in hermaphrodites (HSP-90 chaperone system, including the roles and expression patterns of specific co-chaperones, should help to illuminate the complex functions of HSP-90 reported here.In wild-type er genes . It is nhrodites . Furtherwww.g3journal.org/lookup/suppl/doi:10.1534/g3.118.300551/-/DC1.Supplemental Material is available online at Click here for additional data file.


Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt) and extends lifespan. Here we report that transaldolase  deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.Mitochondrial dysfunction can increase oxidative stress and extend lifespan in  C. elegans. The reasons for these pro-longevity effects have been elusive, but one current model is that adaptive responses to mitochondrial inhibition promote organismal health and stress resistance. Here, we report an intriguing example of mitochondrial stress induced by inhibition of a cytosolic metabolic pathway that extends lifespan in worms. We find that inhibition of the pentose phosphate pathway, which is essential for cytosolic redox homeostasis, affects multiple parameters of mitochondrial function and activates a starvation-like response that promotes longevity through recycling of damaged cellular components and induction of the enzyme flavin-containing monooxygenase 2. These results establish novel links between the pentose phosphate pathway, mitochondrial function, redox homeostasis, and organismal aging.There are a growing number of studies linking mitochondrial dysfunction to enhanced longevity, especially in the nematode  C. elegans, where inhibition of mitochondrial respiration by mutation or knockdown of numerous electron transport chain (ETC) components usually increases lifespan ), SJ4143 (zcIs17[ges-1p::gfpmt]), BX113 (waEx15 [fat-7p::gfp + lin-15(+)]), MAH235 (sqIs19 [hlh-30p::hlh-30::gfp + rol-6(su1006)]), KAE9 (eft-3p::fmo-2 + h2b::gfp + Cbr-unc-119(+)), and VS20 (hjIs67 [atgl-1p::atgl-1::gfp + mec-7::rfp]) were obtained from the Caenorhabditis Genetics Center . The atfs-1(tm4525) and hlh-30(tm1978) strains were obtained from the National BioResource Project .RB967  protocol , 488ex500-550em [1.5–5.5 ns], and 405ex450-470em [7–11.5 ns] ("autofluorescence" channel). The fluorescence lifetime of HyPer is extremely short (<4ns), so by using an emission wavelength far from that of HyPer and a much longer lifetime gating [7–11.5ns], the autofluorescence channel contains no signal from HyPer. This simplifies the unmixing problem allowing us to determine R is ratio of the autofluorescence as measured in each separate HyPer channel to the autofluorescence as measured in the autofluorescence channel. This ratio is determined using age matched wild-type animals fed EV(RNAi) and imaging in all three channels. The ratio is then determined by fitting a linear model to values for each pixel in all channels for multiple animals. To ensure that the any variation in imaging parameters or laser function is corrected for, this process is repeated for each of the three experimental groups. The HyPer fluorescence was then determined by removing the autofluorescence contribution to each HyPer channel by the following: FHyPer = FHyPer Raw − (R * FAF in AF). This allowed the autofluorescence to be removed pixel-by-pixel for each animal. The 488ex500-550em channel displayed negligible autofluoresence after fluorescence lifetime gating, so this was only done for the 405ex500-550em channel. The final HyPer redox values were determined as For imaging the HyPer reporter, we removed the animal autofluorescence by performing linear unmixing similar to previous work . Rather escribed .p-values were calculated using the Wilcoxon rank-sum test.Synchronized eggs or L1 larvae were grown on NGM plates containing 4 mM IPTG, 25 μg/ml carbenicillin and seeded with RNAi bacteria. At the L4/young adult stage, worms were transferred to plates with 50 μM FUDR to prevent hatching of progeny. When necessary, worms were transferred to new plates with fresh bacteria. Lifespans were performed at 25°C for the majority of experiments, unless otherwise noted in the text and figures. Cohorts were examined every 1–3 days using tactile stimulation to verify viability of animals. Animals that displayed vulval rupture were included in analysis, since it is an age-related phenotype , 91. Aniin vivo oxygen consumption in C. elegans, we utilized the Seahorse X24 Bioanalyzer (Seahorse Biosciences) as previously described . Proteins of interest were detected by immunoblot using anti-GFP , anti-p-JNK , anti-p-p38 , anti-p-KGB-1 (a gift from Drs. Naoki Hisamoto and Kunihiro Matsumoto), and anti-alpha-tubulin  antibodies at a 1:1000 dilution in 5% BSA TBS-T.S1 Fig(A) Mean relative fluorescence of hsp-6p::gfp animals grown on PPP RNAi. Fluorescence is calculated relative to EV(RNAi) controls . (B) N2 fed EV(RNAi) , N2 fed tald-1(RNAi) , atfs-1(gk3094) fed EV(RNAi) , atfs-1(gk3094) fed tald-1(RNAi) . Lifespans were performed at 20°C, with pooled data from two independent experiments shown. (C) N2 fed EV(RNAi) , N2 fed cco-1(RNAi) , atfs-1(gk3094) fed EV(RNAi) , atfs-1(gk3094) fed cco-1(RNAi) . Lifespans were performed at 20°C, with pooled data from five independent experiments shown.(TIF)Click here for additional data file.S2 Fig(A) N2 fed EV(RNAi) , N2 fed tald-1(RNAi) , daf-16(mu86) fed EV(RNAi) , daf-16(mu86) fed tald-1(RNAi) . Lifespans were performed at 25°C, with pooled data from three independent experiments shown. (B) N2 fed EV(RNAi) , N2 fed tald-1(RNAi) , aak-2(ok524) fed EV(RNAi) , aak-2(ok524) fed tald-1(RNAi) . Lifespans were performed at 25°C, with pooled data from three independent experiments shown. (C) N2 fed EV(RNAi) , N2 fed tald-1(RNAi) , glp-1(e2141) fed EV(RNAi) , glp-1(e2141) fed tald-1(RNAi) . Lifespans were performed at 25°C, with pooled data from three independent experiments shown. (D) N2 fed EV(RNAi) , N2 fed tald-1(RNAi) , hif-1(ia4) fed EV(RNAi) , hif-1(ia4) fed tald-1(RNAi) . Lifespans were performed at 25°C, with pooled data from three independent experiments shown. (E) N2 fed EV(RNAi) , N2 fed cco-1(RNAi) , hif-1(ia4) fed EV(RNAi) , hif-1(ia4) fed cco-1(RNAi) . Lifespans were performed at 25°C, with pooled data from three independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in (TIF)Click here for additional data file.S3 Fig(A) mtDNA content (nd-1/act-3 DNA) in L4 tald-1(RNAi) or cco-1(RNAi) animals does not change . (B) mtDNA content (nd-1/act-3 DNA) in adult day 1 tald-1(RNAi) or cco-1(RNAi) animals does not change .(TIF)Click here for additional data file.S4 Fig(A) RNAi knockdown of mitochondrial fusion and fission factors alters intestinal mitochondrial morphology. ges-1p::gfpmt reporter animals were imaged and max intensity projections of five z-slices are presented. RNAi knockdown of tald-1 alters mitochondrial morphology independent of (B)pdr-1, (C)fzo-1, and pink-1. For (B), pdr-1(gk448) mutants were used. ges-1p::gfpmt reporter animals were imaged and max intensity projections of five z-slices are presented. Scale bar, 10 μm.(TIF)Click here for additional data file.S5 Figcco-1, but not tald-1 reduces the (A) extinction coefficient and (B) time of flight of C. elegans. N2 animals were grown on RNAi bacteria for 3 days from hatching, washed off plates, and analyzed using the COPAS BIOSORT. In this figure, statistics are displayed as: * p<0.05, ** p<0.01, *** p<0.001.RNAi knockdown of (TIF)Click here for additional data file.S6 Fig(A) Representative INR images of HyPer animals grown on tald-1(RNAi) or cco-1(RNAi). Two images for each condition are shown to emphasize variability in HyPer oxidation across individual worms and consistent effects of tald-1(RNAi) on oxidation of the reporter. (B) Confocal image quantification of HyPer reporter . In this figure, statistics are displayed as: * p<0.05.(TIF)Click here for additional data file.S7 Fig(A) JNK-1 is not required for daf-2(RNAi) lifespan extension. N2 fed EV(RNAi) , N2 fed daf-2 (RNAi) , jnk-1(gk7) fed EV(RNAi) , jnk-1(gk7) fed daf-2(RNAi) . Lifespans were performed at 25°C, with pooled data from three independent experiments shown. (B) KGB-1 is not required for daf-2(RNAi) lifespan extension. N2 fed EV(RNAi) , N2 fed daf-2 (RNAi) , kgb-1(um3) fed EV(RNAi) , kgb-1(um3) fed daf-2(RNAi) . Lifespans were performed at 25°C, with pooled data from four independent experiments shown. (C)hsp-6p::gfp reporter induction in tald-1(RNAi) or cco-1(RNAi) animals is not prevented from jnk-1(gk7) and kgb-1(um3) mutations. Scale bar, 200 μm. (D) Mean relative fluorescence of hsp-6p::gfp reporter animals. Fluorescence is calculated relative to N2 EV(RNAi) controls . (E) Oxygen consumption rate decreases independent of JNK-1 and KGB-1 from tald-1(RNAi) or cco-1(RNAi). OCR was measured using the Seahorse XF Analyzer and normalized to animal number  and kgb-1(um3) animals, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in p<0.05, ** p<0.01, *** p<0.001.(TIF)Click here for additional data file.S8 Fig(A)hsp-6p::gfp reporter induction in tald-1(RNAi) or cco-1(RNAi) animals is not prevented by the pmk-1(km25) mutation. (B) Mean relative fluorescence of hsp-6p::gfp reporter animals in the context of the pmk-1(km25) mutation. Fluorescence is calculated relative to N2 EV(RNAi) controls . (C) PMK-1 is partially required for daf-2(RNAi) lifespan extension. N2 fed EV(RNAi) , N2 fed daf-2 (RNAi) , pmk-1(km25) fed EV(RNAi) , pmk-1(km25) fed daf-2(RNAi) . Lifespans were performed at 25°C, with pooled data from three independent experiments shown. (D) NSY-1 is partially required for daf-2(RNAi) lifespan extension. N2 fed EV(RNAi) , N2 fed daf-2 (RNAi) , nsy-1(ag3) fed EV(RNAi) , nsy-1(ag3) fed daf-2(RNAi) . Lifespans were performed at 25°C, with pooled data from four independent experiments shown. (E) MAPKs in C. elegans are activated by H2O2 treatment. Western blot analysis was performed on protein lysates isolated from animals exposed to 10 mM H2O2 for either 0, 5, 15, 30, or 60 minutes in M9 media. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in p<0.05, ** p<0.01, *** p<0.001.(TIF)Click here for additional data file.S9 Fig(A) Pumping rate per minute was measured for individual animals, with each dot representing an individual . In this figure, statistics are displayed as: * p<0.05, ** p<0.01, *** p<0.001.(TIF)Click here for additional data file.S10 Fig(A) RNAi knockdown of cco-1 increases fmo-2p::mCherry reporter induction in cells proximal to the anterior bulb. fmo-2p::mCherry reporter animals were grown on RNAi bacteria from hatching and imaged 4 days later using fluorescent microscopy. (B) Mean relative fluorescence of fmo-2p::mCherry reporter animals in the context of jnk-1(gk7) and kgb-1(um3) mutations. Fluorescence is calculated relative to N2 EV(RNAi) controls . (C) Mean relative fluorescence of hsp-6p::gfp reporter animals in the context of the fmo-2(ok2147) mutation. Fluorescence is calculated relative to N2 EV(RNAi) controls . In this figure, statistics are displayed as: * p<0.05, ** p<0.01, *** p<0.001.(TIF)Click here for additional data file.S1 Tablep-values are shown for condition comparison* or genotype to N2 comparison** and calculated using Wilcoxon Rank-Sum test.(XLSX)Click here for additional data file.S2 TableForward and reverse primer sequences are listed for each gene set tested.(XLSX)Click here for additional data file.


Arabidopsis thaliana. We identify factors that enable SE development in PRC2-depleted shoot and root tissue and demonstrate that the establishment of embryogenic potential is marked by ectopic co-activation of crucial developmental regulators that specify shoot, root and embryo identity. Using inducible activation of PRC2 in PRC2-depleted cells, we demonstrate that transient reduction of PRC2 activity is sufficient for SE formation. We suggest that modulation of PRC2 activity in plant vegetative tissue combined with targeted activation of developmental pathways will open possibilities for novel approaches to cell reprogramming.Many plant cells can be reprogrammed into a pluripotent state that allows ectopic organ development. Inducing totipotent states to stimulate somatic embryo (SE) development is, however, challenging due to insufficient understanding of molecular barriers that prevent somatic cell dedifferentiation. Here we show that Polycomb repressive complex 2 (PRC2)-activity imposes a barrier to hormone-mediated transcriptional reprogramming towards somatic embryogenesis in vegetative tissue of  Somatic embryogenesis provides the strongest support for plant cell totipotency but reprogramming of non-reproductive tissue is problematic or even impossible in many plant species. Here we show that the activity of Polycomb Repressive Complex 2 (PRC2) constitutes a major barrier to hormone-mediated establishment of embryogenic competence in plant vegetative tissue. We identify a conservative set of transcription factors whose expression coincides with the establishment of embryogenic competence in vegetative tissue, among which are key developmental regulators of root, shoot and embryo development. We show that lowering the PRC2-imposed barrier combined with activating hormone treatments establishes embryogenic competence in different tissue types, which opens possibilities for novel strategies to plant cell identity reprogramming. Plant cells have long been recognized for their capacity to become pluripotent and to enter various differentiation pathways in response to chemical or mechanical stimuli. This is exemplified by ectopic organ formation and entire plant regeneration from vegetative tissue in response to plant hormone and/or stress signaling, which is widely used for clonal propagation of horticultural species and as a developmental model system , 2.Arabidopsis thaliana (Arabidopsis), SEs can be efficiently produced by applying the synthetic auxin 2,4-dichlorophenoxyacetic acid  to immature zygotic embryos [WUSCHEL (WUS))[PLETHORA 4/BABY BOOM (PLT4/BBM))[PLT5/EMBRYO MAKER (PLT5/EMK))[AGAMOUS-LIKE 15 (AGL15))[LEAFY COTYLEDON genes LEC1[LEC2[SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1))[The most extensive reprogramming establishes high level of pluripotency or even totipotency that allows somatic embryogenesis—the development of ectopic embryos from somatic cells in a process that is independent of gamete formation, fertilization or seed development . Somatic embryos . In addiEL (WUS)), the AP2LT4/BBM)) or PLT5/LT5/EMK)), the MAD AGL15)), 12 or tenes LEC1 or LEC2[LEC1[LEC2, or by t), 12 or  (SERK1))–19. This (SERK1)), 17. Molpickle [Polycomb repressive complexes (PRCs) are well-studied epigenetic executors of developmental phase transitions and cell fate specification in animals and plants –22. By epickle , 31, sugHere we show that absence of PRC2 by itself is not sufficient to achieve full cell dedifferentiation required for somatic embryogenesis but that PRC2-depleted somatic cells respond to external hormone and stress treatments by becoming competent for somatic embryogenesis. We define a set of morphological, anatomical and molecular features of SEs that distinguish them from neutral lipid-containing structures that spontaneously develop in PRC2 mutant plants. By combining stable and transient PRC2-depletion with hormone treatments, we demonstrate that cell fate can be reset and somatic embryogenesis induced in PRC2-depleted shoot and root tissue. We propose that lowering the PRC2-imposed epigenetic barrier combined with hormonal stimuli allows ectopic co-activation of key developmental regulators and establishment of embryogenic potential in plant vegetative tissue.In Arabidopsis, external application of the synthetic auxin 2,4-D to immature ZEs induces reprogramming and development of SEs , 16. SomCLF and SWN (clf swn) spontaneously develop poorly differentiated shoot organs and ectopic finger-like protrusions that ectopically accumulate neutral lipids [clf swn [CLF CLF and SF or SWN , its losretarded .clf-29 swn-3 (further clf swn) plants do not produce seeds and mutant phenotypes become visible only after germination [clf swn seedlings were selected from the progeny of clf/+ swn/- plants 7 days after induction of germination. We exposed separated shoot and root explants of 7, 14 or 21 day-old wild-type, clf, swn and clf swn plants to 5 μM 2,4-D for different periods of time followed by 7-day culture on hormone-free medium  and relatively short induction time (50–70 hours) offered the opportunity to establish an experimental model system to address processes associated with somatic embryogenesis.clf swn-derived SEs from SE-like structures  and shoot apical meristem (SAM) identity  in wild-type seedlings, ZEs and SEs to clf swn seedlings, SEs and SE-like structures clf swn-dPLT4/BBM   targets –40, tranCLV3pro::GUS and WOX5pro::NLS-GUS [clf swn. CLV3pro::GUS was expressed in a single focus in the apical part of wild-type ZEs as expected  that alt plants . We obsen plants . PartialGR lines . Consequreatment , similarreatment . In the reatment , S4F Figreatment , demonstreatment . Togetheclf swn-derived SEs in the presence or absence of dex with plants regenerated from wild-type ZE-derived SEs , LEC1, LEC2, FUSCA 3 (FUS3) or ABSCISIC ACID INSENSITIVE  were among the upregulated genes. Among the genes down-regulated in treated clf swn compared to wild type (clf swn (clf swn resulted in the down-regulation of 240 genes related to metabolic processes and oligonucleotide transport (MONOPTEROS (MP), TARGET OF MONOPTEROS 6 (TMO6) [PLT2, WOX5, or root-specific LATERAL ORGAN BOUNDARY DOMAIN genes  [clf swn samples. The GO category response to oxidative stress was enriched among the up-regulated genes (DORNRöSCHEN / ENHANCER OF SHOOT REGENERATION 1 (DRN/ESR1) and DRN-LIKE (DRNL/ESR2) [We searched for gene expression signatures associated with the embryogenic competence. 2664 genes were down-regulated and 2890 genes up-regulated in wounding- and 2,4-D-treated oped SEs . The uproped SEs  and incloped SEs . To idenarehouse . This esild type , overrepild type , suggest(clf swn . The treransport  and the ransport . The uprransport . Among t6 (TMO6) , PLT2, W and 29) . Finallyed genes . The upred genes , for exaNL/ESR2) , 52.clf swn shoot apex sample upon induction of SEs (clf swn in response to the inductive treatment are significantly enriched for PRC2-target (H3K27me3-marked) genes compared to the genome-wide average [clf swn background are PRC2 targets. This finding is consistent with the notion that loss of PRC2 by itself does not cause activation of these target genes unless additional signals such as hormones are present.Together, we identified a non-redundant set of 485 TF genes whose expression marks the n of SEs . These Tn of SEs . The mai average   root clf swn sot stele . Within e tissue . Next, wwn shoot  is condiPLT4/BBM  in both ype root . These rype root .clf swn root explants  by the general absence of vascular connection to the parental explant, and (ii) by the development of correctly localized and functional root and shoot apical meristems . Here, ot stele . Second,explants . It is preatment , the numLEC1, LEC2, PLT4/BBM, AGL15 or WUS, which induce the vegetative-to-embryonic transition or retention of embryogenic potential [DRN/ESR1, DRNL/ESR2 or WUS, which induce ectopic shoot development [PLT2, which induces ectopic root development [LBD16, LBD18 and LBD29, which induce callus formation [clf swn samples [swn-3 (SALK_050195)[WOX5pro::NLS-GUS (42) and CLV3pro::GUS (NASC N9610)[clf-29 swn-3 by crossing and F3 seedlings were used. The ABI3pro::GUS and DR5::GUS marker lines were generated by crossing ABI3pro::GUS [DR5::GUS [clf-29/+ swn3/- and F2 seedlings were used. clf-29/+ swn3/- CLF-GR plants were created by transformation of a pCLF::CLF-GR construct [clf-29/+ swn/- plants by floral dip. 18 independent transgenic lines were obtained, from which two lines with least CLF activity and most pronounced clf swn-like phenotypes in the absence of dex were selected for further experiments [The strong PRC2 mutants alleles K_021003) and swn-SC N9610) reporterpro::GUS  and DR5:DR5::GUS  with clfonstruct  into clferiments , 73.-2 s-1, 22°C and 8 hrs dark, 20°C). The following plant growth regulators were used at concentrations specified in each experiment: 2,4-Dichlorophenoxyacetic acid , abscisic acid , 1-naphthylacetic acid , indole-3-butyric acid , dexamethasone (Sigma-Aldrich #D1756) was used at 10 μM. All chemicals were dissolved in DMSO which was used in mock treatments.As a standard, seeds were surface sterilized using 70% and 90% ethanol, placed on ½ strength MS medium  containing 1% (w/v) sucrose and 0.8% (w/v) agar (standard MS) and were stratified for 48 h. Plants were grown under long-day growth conditions -treated controls were included in all experiments. After the specified induction times, the explants were kept on hormone-free plates for another 7 days after which percentage of SE-forming explants was calculated. Each experiment was repeated 2 to 4 times using at least 25–30 explants as indicated in the figure legends.For SE induction from seedlings, seeds were surface sterilized using ethanol, placed on standard MS medium, stratified for 48 h and grown under long-day conditions as specified above. SE induction was carried out under the same conditions as described for ZEs using 14 DAG Embryonic lipids were visualized by 20-minute staining with Sudan Red 7B (Sigma-Aldrich #46290) as described . GUS staPP2A (AT1G13320) was used as reference gene. All experiments were performed at least twice using biological replicates and technical triplicates.RNA from seeds was extracted using the hot borate buffer method modified after  for det. RNA froclf swn shoot apex explants were exposed for 55 hours to different combinations of treatments as shown in www.r-project.org). For additional details on sequencing and data analysis, see Wild-type and Unless stated otherwise, all experimental data was analyzed using unpaired two-tailed Student’s t-test. Letters above bars in bar charts represent statistical significance level where at least three biological replicates were analyzed (same letter–same significance level).S1 Text(DOCX)Click here for additional data file.S1 Fig(A) Examples of the clf swn phenotype after 3 weeks of growth under standard conditions. Embryonic lipids (red) are visualized by staining with Sudan Red 7B. Scale bar = 2 mm. (B) Expression of genes encoding the subunits of the EMF and VRN PRC2 complexes in early bent cotyledon-stage zygotic embryos (ZE) and in germinating seeds. Graphs show means ±SEM, N = 2 biological replicates. (C) Efficiency of somatic embryogenesis in germinating Arabidopsis wild-type (WT), clf and swn embryos. Graphs show means ±SEM, N = 4 biological replicates, 90–100 seeds/ZE per experiment. Identical letters above columns indicate lack of a statistical significant difference (p>0.05).(TIF)Click here for additional data file.S2 Fig(A) 2,4-D treatment induces somatic embryo (SE) formation in the shoot of clf swn plants independent of the plant age. Quantification of efficiency of SE formation in shoot explants of 7, 14 and 21-DAG wild-type (WT), clf, swn and clf swn plants. To control for more efficient 2,4-D effects in clf swn, WT, clf and swn explants were treated for 7 days and clf swn explants for 60 hours with 5 μM 2,4-D. Graphs show means ±SEM, N = 3 biological replicates with 30–50 explants/experiment. Red numbers above bars indicate the percentage of mock (DMSO)-treated explants forming SEs. (B) Frequency of vascular attachment to parental explant distinguishes somatic embryos (SEs) from somatic embryo-like structures (SE-like). Percentage of SE and SE-like structures connected by vascular tissue with the parental explant was determined in wild type and clf swn. Individual structures were defined as those that emerged from the parental explant following the treatment and could be clearly separated from the parental explant. Graphs show means ±SEM, N = 3 biological replicates, 25–35 explants per experiment. Example dark-field and DIC microscopy images of a shoot-like SE-like structure and an explant with SEs are shown. Scale bar = 100 μm. White arrowheads point to the vascular tissue. (C) The effect of different auxins on the efficiency of SE formation in clf swn. 2,4-D, 2,4-dichlorophenoxyacetic acid; IBA, indole-3-butyric acid; NAA, 1-naphthaleneacetic acid. Graphs show means ±SEM, N = 3 biological replicates with 25 explants/experiment. Identical letters above columns indicate lack of a statistical significant difference (p>0.05). Red number above bars in (A) and (B) indicate the percentage of SE formation ±SEM in mock (DMSO)-treated explants. H-F—hormone-free medium, h—hour, d—day.(TIF)Click here for additional data file.S3 FigWUS and STM, root markers WOX5 and SCR, and PLT3 and PLT4 were tested. Graphs show means ±SEM, N = 2 biological replicates.Shoot markers (TIF)Click here for additional data file.S4 Fig(A) Phenotype of plants segregated from CLF/- swn/- CLF-GR parental plants at 7 and 21 days after germination (DAG) grown in the absence of dexamethasone (dex). While CLF swn CLF-GR display the expected wild-type (WT)-phenotype similar to swn single mutants, clf swn CLF-GR plants display a severe clf-like phenotype, which was, however, milder than the phenotype of clf swn indicating that CLF was partly active even in the absence of dex.(B) Phenotype of 14-dag segregated plants in the absence and presence of 10 μM dex. Left: image of segregating T2 plants grown in the absence of dex. Asterisks mark clf swn CLF-GR plants. Right: quantification of frequency of clf-like phenotypes in a population of 14-DAG plants segregated from a CLF/- swn/- CLF-GR parent in the presence or absence of dex. In the absence of dex, the observed frequency of the severe clf-like phenotype does not significantly differ from the expected 25% , indicating that the clf swn CLF-GR genotype was correctly distinguished. In the presence of 10 μM dex, the severe clf-like phenotype is restored to wild type, indicating that the transgene is functional.(C) Example of phenotypes of 6-week-old T2 plants segregated from CLF/- swn/- CLF-GR parental plants. Similar to observations on seedlings (A), the mild phenotype of adult clf swn CLF-GR plants indicates partial activity of CLF in the transgenic plants even in the absence of dex.(D) Chromatin immunoprecipitation (ChIP)-based quantification of the relative H3K27me3 enrichment at the PRC2 target genes LEC1 and ABI3 in the absence or presence of 10 μM dex in clf swn CLF-GR plants. The higher abundance of H3K27me3 in clf swn CLF-GR compared to clf swn indicates partial PRC2 activity in clf swn CLF-GR even in the absence of dex and the increase of the enrichment in the presence of 10 μM dex demonstrates the functionality of the transgene. The H3K27me3 enrichment pattern corresponds with the relative gene expression level in (E) and with the phenotype difference between clf swn CLF-GR plants grown in the absence or presence of 10 μM dex. Graphs show means ±SEM of three technical triplicates. The ChIP experiment was repeated 2 times with similar results.(E) Expression of the PRC2 target genes LEC1 and ABI3 that are commonly up-regulated in clf swn in the clf swn CLF-GR transgenic plants. In the absence of dex, LEC1 and ABI3 are less up-regulated in clf swn CLF-GR than in clf swn consistent with partial activity of CLF in the transgenic plants even in the absence of dex. The lower expression in dex-treated plants indicates functionality of the transgene. A PP2A gene (AT1G13320) served as reference. Graphs show means ±SEM of technical triplicates.(F) Somatic embryos develop from the leaf axils of clf swn CLF-GR plants after 5 μM 2,4-D treatment in the absence but not in the presence of 10 μM dex. Examples of phenotypes quantified in (TIF)Click here for additional data file.S5 Fig(A) Longer but not short exposure to 2,4-D can partially substitute for wounding. Comparison of SE efficiency after 60-hour or 7-day of the 5 μM 2,4-D-treatment. Bars represent means ±SEM, N = 4, 25–30 explants each. (B) The effect of methyl jasmonate (MeJA) on clf swn SE induction. Bars represent means ±SEM, N = 3, 30 explants each. Identical letters above columns in  indicate lack of a statistical significant difference (p>0.05). (C-F) RNA-sequencing of samples with different embryogenesis potential. (C) Schematics of RNA-sequencing experiment setup. Black arrowheads indicate the 2 sampling time points. Images demonstrate the dissected shoot apexes sampled. Scale bar = 1 mm. (D) Gene ontology (GO) categories enriched more than 2-fold among the 2890 genes up-regulated and more than 3-fold among the 2664 genes down-regulated in 2,4-D- and wounding-treated clf swn compared to wild type ((E) Gene ontology (GO) categories enriched more than 2-fold among the 1451 genes up-regulated and 240 genes down-regulated by the 2,4-D and wounding treatment in clf swn ((F) Gene ontology (GO) category enriched more than 2-fold among the 139 genes up-regulated and 35 genes down-regulated specifically in the wounding- and 2,4-D-treated clf swn samples (ild type . (E) Gen clf swn . (F) Gen samples . WT–wild(TIF)Click here for additional data file.S6 Fig(A) Most prominent biclusters using the set of TF genes up-regulated in 2,4-D and wounding-treated clf swn compared to treated wild type explants (clf swn-WA/ WT-WA) ((B) Most prominent using the set of TF genes up-regulated in response to the combined 2,4-D and wounding treatment in clf swn  (/ WT-WA) . Expressf swn-D) . Express(TIF)Click here for additional data file.S7 Figclf swn shoot explants compared to untreated wild-type control   control  for whic(TIF)Click here for additional data file.S8 FigABI3pro::GUS transgene  is shown.2-hour GUS-staining in seedlings that carried the (TIF)Click here for additional data file.S9 FigDR5::GUS transgene  is shown.2-hour GUS-staining in seedlings that carried the (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.


Lgals8/Lac-Z knock-in mice lacking Gal-8 expression have higher polarization toward Th17 cells accompanied with decreased CCR6+ and higher CXCR3+ regulatory T cells (Tregs) frequency. These conditions result in exacerbated MOG35-55 peptide-induced EAE. Gal-8 eliminates activated Th17 but not Th1 cells by apoptosis and ameliorates EAE in C57BL/6 wild-type mice. β-gal histochemistry reflecting the activity of the Gal-8 promoter revealed Gal-8 expression in a wide range of CNS regions, including high expression in the choroid-plexus. Accordingly, we detected Gal-8 in human cerebrospinal fluid, suggesting a role in the CNS immune-surveillance circuit. In addition, we show that MS patients generate function-blocking anti-Gal-8 antibodies with pathogenic potential. Such antibodies block cell adhesion and Gal-8-induced Th17 apoptosis. Furthermore, circulating anti-Gal-8 antibodies associate with relapsing-remitting MS (RRMS), and not with progressive MS phenotypes, predicting clinical disability at diagnosis within the first year of follow-up. Our results reveal that Gal-8 has an immunosuppressive protective role against autoimmune CNS inflammation, modulating the balance of Th17 and Th1 polarization and their respective Tregs. Such a role can be counteracted during RRMS by anti-Gal-8 antibodies, worsening disease prognosis. Even though anti-Gal-8 antibodies are not specific for MS, our results suggest that they could be a potential early severity biomarker in RRMS.Galectin-8  is a member of a glycan-binding protein family that regulates the immune system, among other functions, and is a target of antibodies in autoimmune disorders. However, its role in multiple sclerosis (MS), an autoimmune inflammatory disease of the central nervous system (CNS), remains unknown. We study the consequences of Gal-8 silencing on lymphocyte subpopulations and the development of experimental autoimmune encephalitis (EAE), to then assess the presence and clinical meaning of anti-Gal-8 antibodies in MS patients.  Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS) that damages myelin and axons in the brain and spinal cord , 2. MostGalectins are a family of glycan-binding proteins that has emerged as strong modulators of adaptive and innate immunity . These lGal-8 is a tandem-repeat galectin that, unlike other galectins, possesses unique high affinity for α-2,3-sialylated glycans in its N-terminal carbohydrate recognition domain (CRD), which is linked by a short peptide to a C-terminal CRD bearing another glycan specificity , 28. Gal+ and CCR6+ Treg frequencies. Strikingly, MS patients generate neutralizing anti-Gal-8 antibodies that can block the Gal-8 immunosuppressive function and impact upon RRMS prognostic. Patients recently diagnosed with RRMS and harboring anti-Gal-8 antibodies in serum, before establishing the treatment, develop a more aggressive disease within just one year of evolution, as shown by monitoring the Expanded Disability Status Scale (EDSS). Overall, the evidence suggests that Gal-8 plays an immunosuppressive function that can be counteracted by anti-Gal-8 antibodies in RRMS leading to worse evolution. Although Gal-8 function-blocking antibodies are not specific to MS, as they can be found in other autoimmune diseases [Here we study: 1) How the absence of Gal-8 function in a Gal-8 KO mice impinges upon lymphocyte subpopulations and EAE severity; 2) The effect of Gal-8 treatment on EAE of wild type mice; 3) The presence and clinical meaning of autoantibodies against Gal-8 in patients with MS. Our results indicate a protective immunosuppressive role of Gal-8 against CNS autoimmunity involving apoptotic elimination of activated Th17 cells and opposite dysbalances of CXCR3diseases , their pThe Scientific Ethics Committee CEC-MED-UC and the Science Ethics Committee for Animal and Environmental Care of the Pontificia Universidad Católica de Chile (PUC) approved the informed consent voluntarily given by MS patients and all animal procedures, respectively.MS patients were monitored by trained neurologists in the Center of Multiple Sclerosis PUC, between 2011 and 2013, during standard clinical visits, by collecting demographic data, relevant clinical history, neurologic assessments, including the Expanded Disability Status Scale (EDSS), relapse symptoms and Magnetic Resonance Imaging (MRI) using 1.5 Tesla MRI with 5 mm thick slices and 2.5 mm separation between each slice. Clinically relevant EDSS worsening implied changes from 0 to 1.5 or increments of 1 point within the range of 1 to 5.5 scores. Blood samples were obtained from RRMS patients before starting treatment with any disease-modifying drugs (DMD). Patients with progressive forms were not receiving any DMD or immunosuppressant drugs at least 6 months before the blood samples. No steroid treatment was used in RR, SP or PP patients for at least 4 weeks prior to the blood sample. Patients were followed up for an average of one year, controlled every 3 to 6 months, or more frequently (e.g. during relapses), as needed. All patients accepted and signed the informed consent approved by the Scientific Ethics Committee CEC-MED-UC.TM (Bv421)-conjugated anti-CXCR3 (Clone CXCR3-173), PE-conjugated anti-CD44 (clone IM7), APC-conjugated anti-CD25 (clone PC61) and APC/Cyanine dye 7 (APC/Cy7)-conjugated anti-CD62L (clone MEL14) were purchased from Biolegend . PerCP-conjugated anti-CD4 (clone RM4-5), PECy7-conjugated IFN-γ (clone XMG1.2) were purchased from eBioscience. Alexa 647-conjugated IL-17A (clone TC11-18H10.1), PE-conjugated anti-IFN-γ and Alexa 488–conjugated anti-IL-17 were purchased from BioLegend. Alexa 488-conjugated anti-CD8a (clone 53–6.7), alexa 647-conjugated anti-CD19 (clone 1D3), PE-conjugated anti-CD11c (clone HL3), PE-conjugated anti-IL4 (11B11), Alexa 647-conjugated anti-IL17A (clone TC11-18H10), PE/Cy7-conjugated anti-IFN-γ (cloneXMG1.2) were purchased from BD Biosciences-Pharmingen. For T cell activation, anti-mouse CD28 (clone 37.51) and anti-mouse CD3 (clone 145-2C11) were purchased from BD Bioscience.To determine expression levels of key surface and intracellular molecules associated with different T-cell phenotypes, splenocytes were immunostained with different combinations of the following fluorochrome-conjugated monoclonal antibodies (mAbs) at room temperature for 30 min: allophycocyanin (Apc)-conjugated anti-FoxP3 (clone FJK-16S) and R-Phycoerythrin (PE)-conjugated anti-RORγt (clone BSD), both from eBioscience . Otherwise, PerCP/Cyanine dye 5.5 (PerCP/Cy5.5)-conjugated anti-CCR6 (clone 29-2L17), PE/Cyanine dye 7 (PE/Cy7)-conjugated anti-CD4 (clone GK1.5), Brilliant-Violet 421Ionomicina, brefeldin A and PMA (Phorbol Myristate Acetate) were purchased from Sigma-Aldrich. GST-Gal-8 expressed in bacteria was isolated by affinity chromatography and Gal-8 was released by thrombin treatment as described .Lgals8/Lac-Z knock-in  mice were generated from C57BL/6NTac mice engineered in Regeneron Pharmaceuticals Inc., New York, using Velocigene technology for replacing the entire coding region of the mouse Lgals8 gene  with LacZ lox-Ub1-EM7-Neo-lox Cassette containing the LacZ gene that encodes β-galactosidase [Lgals8 KO mice and PCR genotyping assay, including the predicted PCR products and the primers, are available at the Velocigene website (www.velocigene.com/komp/detail/14305). IL-17A-GFP reporter mice in the C57BL/6J background, which express EGFP under the control of the IL-17A promoter, were purchased from Jackson Laboratories .Mice were mantained under conditions of strict confinement, which included automatic control of temperature (21°C) and photoperiod (12 h light / 12 h dark). tosidase . DetailsLgals8-/- and Lgals8+/+ mice by immunization of an emulsion containing 150 μg of MOG35-55 peptide (MOGp)  and 500 μg of Mycobacterium tuberculosis extract (Difco Lab.) in incomplete Freund’s adjuvant oil. In addition, mice received 200 ng of pertussis toxin  on day 0 and 2 post-immunization (p.i.). Clinical progression of EAE symptoms were monitored daily using the following scale: 0, no clinical signs; 1, loss of tail tone; 2, flaccid tail; 3, incomplete paralysis of one or two hind legs; 4, complete hind limb paralysis; 5, moribund ; 6, death. In some experiments, EAE was induced in wild-type C57BL/6J mice and simultaneously treated (i.p.) with either 100 μg recombinant Gal-8 or PBS (control group) during 20 consecutive days. Topical diclofenac (gel 1%) was used to alleviate animal suffering. The use of local analgesia rather than systemic analgesia was favored to avoid unwanted influences on immunological and inflammatory parameters under study. The animals were euthanized by cervical dislocation after CO2 sedation.EAE was induced in 8-12-week-old Brains were fixed by perfusion in 4% paraformaldehyde (PFA) in PB buffer (0.1 M phosphate buffer pH 7.4) and processed for β-gal histochemistry as described , 42. Cro+ T cell subpopulations were analyzed in stimulated cells by incubating with 50 ng/ml PMA, 500 ng/ml ionomycin, and 10 μg/ml brefeldin A for the last 4 h of culture. Cells were stained with Zombie Aqua Fixable Viability kit (Biolegend) in PBS followed by staining for cell-surface markers using flurophore-conjugated monoclonal antibodies against mouse CD4, CD19, CD8, CD11c, CD25, CD44, CD62L, CXCR3 and CCR6. For intracellular staining, cells were washed, fixed, and permeabilized with Cytofix/Cytoperm kit (BD Biosciences-Pharmingen) and incubated simultaneously with fluorophore-conjugated antibodies against IFN-γ, IL-4, IL-17, Foxp3 and RORγt. Samples were acquired with BD FACSVerse or FACSCantoTM II (BD) flow cytometers and data was analyzed with FlowJo V10 software or CellQuest Pro software (BD Biosciences).Splenocytes were isolated as described , 44 fromin vitro, naive (CD62L+ CD44-) CD4+ T-cells from IL-17A-GFP reporter or from wild-type mice were purified by cell sorting, resuspended at 0.5x106 cells/ml in IMDM medium supplemented with antibiotics/antimycotics (Gibco) and Gentamycin (Gibco), and incubated (200 μl/well) in 96-well plates preactivated with anti-CD3 (50 μl/well at 2 μg/ml) at 37°C, 90% humidity and 5% CO2. Cells were incubated for 3 days with mouse IL-6 , mIL-1ß (20 ng/ml), human TGF-β1 , anti-CD28 (2 μg/ml) and blocking antibodies anti-IFNγ, anti-IL-4 and anti-IL-2 (5 μg/ml each). Afterwards, cells were washed, resuspended in fresh medium and incubated for additional 4 days in 96-well plates with mIL-6 (25 ng/ml) and hTGF-β1 (5 ng/ml). All cytokines (Biolegend) used were carrier free. Differentiated Th17 cells were purified by cell sorting based on IL-17A expression (GFP+), resuspended in supplemented IMDM and incubated (1x105 cells/well) in 96-well plates pre-activated with anti-CD3 and anti-CD28 (50 μl/well at 2 μg/ml each) in the presence or absence of Gal-8 (20 μg/ml) for 72 h. The extent of apoptosis/necrosis was determined with a commercial kit  analyzing death cells by FACS, in the CD4+ GFP+ gated population corresponding to ≥70% of total cells, as observed in FSC versus SSC analysis. Gal-8-induced cell death of Th17 lymphocytes was analyzed with a similar protocol, except that naive CD4+ T-cells were isolated from WT C57BL/6 mice, differentiated Th17 cells were purified by using a commercial kit (from Biolegend) based on the surface expression of IL-17. Cell death (Annexin V versus 7-AAD) was analyzed in the CD4+ gated population, which corresponded to ≥90% of total cells, accordingly to FSC versus SSC analysis.To analyze apoptosis of Th17 cells differentiated Western blot analysis was performed using recombinant Gal-8 to screen sera of patients with MS for anti-Gal-8 antibodies, as previously described in patients with SLE or AR . The samCSF collected for diagnostic purposes from individuals studied for diplopia, vertiginous syndrome (C7), cephalea, febrile syndrome and meningitis was analyzed for the presence of Gal-8 by immunoblot, using a rabbit anti-Gal-8 polyclonal antibody produced in our laboratory . CSF  and centrifuged at 400 x g for 30 min at room temperature. Cells in the opaque interface were collected, diluted twice with 50 ml of PBS, centrifuged at 250 x g for 10 min and resuspended in RPMI 1640 medium containing 10% FBS.Cover slips were coated overnight at 4°C with 10 μg/ml of Gal-8 in the absence or presence of anti-Gal-8 autoantibodies, as described , 38, 46.t-test as indicated in figure legends.The presence of anti-Gal-8 autoantibodies in RRMS, PPMS and SPMS patients was analyzed respect to various clinical, radiological and CSF variables using Mann-Whitney U test (with Tukey multiple comparisons) and SPSS Statistics 19.0 software. Yates chi-square corrected test and Odds ratio were calculated with VassarStats. EAE disease scores were analyzed with Mann–Whitney U test or one-way ANOVA Kruskal Wallis with Dunn post-test using Prism 5 software (GraphPad Software). Other analyses were made with two tailed non-paired Student’s Lgals8 disruption affects EAE severity. We evaluated the development, progression and CNS inflammation in Lgals8-/- and Lgals8+/+ littermates immunized with MOG35-55 peptide (MOGp). The two mouse groups had similar EAE incidence . However, Lgals8-/- mice displayed a faster disease onset and developed a more severe disease during the chronic phase  lymphocytes, dendritic cells, and CD4+ T-cells subsets, including naive (CD4+CD25- or CD4+ CD62L+ CD44-), effector (CD4+ CD62L- CD44+) and memory (CD4+ CD62L+ CD44+) T-cells  after anti-CD3/anti-CD28 activation  [- CCR6+) Tregs [+) in Lgals8-/- mice splenocytes compared to control mice  , 10 or T+) Tregs  lymphocyrol mice . We also+/+ mice . Therefoin vitro and activated them with anti-CD3/anti-CD28. Annexin V/7-AAD staining showed that Gal-8 induced apoptosis of these Th17 activated cells  cassette reporter gene replaces the entire Gal-8 gene with LacZ, thus offering the possibility to assess the activity of the corresponding promoter by β-gal histochemistry [Although peripheral events tailoring the immune system contribute to MS and EAE pathology, most of the autoimmune pathogenic condition unfolds inside the CNS . In our hemistry –42. Thishemistry , displayThe results showing an immunosuppressive and protective role of Gal-8 against EAE prompted us to assess whether patients with MS generate blocking-function anti-Gal-8 antibodies, as previously reported in LES and AR patients , 46. UsiTo evaluate whether MS patients generate function-blocking anti-Gal-8 autoantibodies we performed two assays. The established assay of cell adhesion to Gal-8-coated coverslips, which assesses glycan-mediated interaction of Gal-8 with integrins , 38, shoAs the presence of anti-Gal-8 neutralizing antibodies might mimic the condition of Gal-8 silencing that exacerbates EAE, we next explored the impact of these antibodies on the clinical course of MS. We studied 58 patients, 36 with recent diagnosis of RRMS and 22 with a progressive disease (8 with SPMS and 14 with PPMS). The results show that 33% (19/58) of these patients have anti-Gal-8 antibodies. Interestingly, 90% of the patients bearing Gal-8(+) sera corresponded to the RRMS phenotype. Within the group of RRMS patients, 17 out of 36 had Gal-8 autoantibodies (47%). In contrast, only 9% of patients with progressive forms (2/22) had anti-Gal-8 autoantibodies  comparing RRMS versus progressive MS) . We prevWe next analyzed whether anti-Gal-8 antibodies associate with worse prognosis in RRMS patients. Before starting the DMD treatment, RRMS patients with or without Gal-8 autoantibodies showed no differences in age, gender, age at onset, disease duration, EDSS, and presence of baseline gadolinium-enhanced T1 lesions . HoweverConsidering our previous results in mice, the association of anti-Gal-8 antibodies with severity in RRMS patients might be due to an interference with the immunosuppressive role of Gal-8. Within the RRMS group of patients these antibodies constitute a potential early prognostic marker.+ and CCR6+ Tregs. We then show that patients with MS generate function-neutralizing Gal-8 antibodies that can counteract the immunosuppressive Gal-8 function. Our clinical analysis disclosed an association of circulating anti-Gal-8 antibodies with worse evolution in newly diagnosed and still untreated RRMS patients, showing EDSS worsening within the first year of follow up. Thus, anti-Gal-8 antibodies emerge as a potential early prognostic biomarker.Our results demonstrate a crucial role of Gal-8 and its function-blocking antibodies in the pathogenesis of MS. We first show that Gal-8 exerts an immunosuppressive protective influence against EAE, as revealed by both the enhanced disease developed in Gal-8 KO mice and the ameliorating effect of Gal-8 treatment. The mechanism likely involves a modulating action on the balance of Th1 and Th17 cell polarization and differentiation of their respective CXCR3+ Tregs and decreased CCR6+ Tregs frequencies, developing exacerbated EAE accompanied by an increased polarization toward Th17 cells after MOG immunization. In vitro, Gal-8 reduces the Th17 cell response to re-stimulation with MOG only in splenocytes from MOG-immunized Gal-8 KO but not from wild-type mice. Th1 cells are not affected. This suggests that Gal-8 eliminates activated Th17 but not Th1 cells during in vivo immunization. In congruency, we found that Gal-8 induces apoptosis of anti-CD3/anti-CD28 activated Th17 cells in vitro. The relative contribution of Th1 versus Th17 cells in MS pathogenesis remains contentious [+ and CCR6+ Tregs, configuring an immunologic context prone to develop an exacerbated EAE, support the notion that both Th17 and Th1 functions are compromised in the pathogenesis of this disease.We show that Gal-8 KO mice at steady state display increased CXCR3tentious . Dysregutentious –51, whiltentious –54. Althtentious , 50, 55,tentious , 56. Ourin vitro differentiated Th17 cells, as we corroborated here in a different setting, and to promote the differentiation of Tregs with ameliorating effects on EAU [+ T cell subpopulations remained unchanged. These Gal-8-polarized Tregs expressed CTL-4 and IL-10 markers of immune suppression activity and most of them lacked neurophilin expression, suggesting a peripheral rather than thymic origin [in vitro, modulating the known roles of IL-2 and TGF-β receptors in this process [An immunosuppressive role for Gal-8 has been originally suggested by its apoptotic effect on Jurkat T cells and CD3/CD28-activated human peripheral T cells . Such ans on EAU . In a mos on EAU  showed tc origin . Gal-8 a process . These a process , as well+Tregs, which should compromise Th1-mediated functions [+ T cell population suggest a Treg polarization distinct from the immunosuppressive-competent Tregs differentiated under a Gal-8 environment [+ Tregs found enriched in RRMS patients [+ Tregs play protective roles under transient inflammatory stimuli [+ Tregs to that of healthy controls and increases the frequency of Th17 suppressive Tregs [However, our results reveal a more complex role of Gal-8 on Treg homeostasis than previously appreciated from studies showing Gal-8-promoted Treg differentiation , 31. At unctions . Indeed,unctions , 58, migironment , 31. In patients . Interes stimuli , 10, 60, stimuli –54. RRMS stimuli . Furtherve Tregs . TherefoThe high expression levels of Gal-8 found in the choroid plexus of mouse brain and the presence of Gal-8 in human CSF suggest a direct role of Gal-8 in the CNS immune-surveillance circuit. The immune-surveillance circuit includes subarachnoid regions bathed in CSF and the deep cervical lymph nodes where dura lymphatic vessels drain CSF from the brain , 61, 62.In addition, our results suggest that an impaired Gal-8 immunosuppressive role in MS, mimicking the autoimmune CNS enhanced condition of Gal-8 KO mice, might occur within an autoimmune context that generates anti-Gal-8 neutralizing antibodies similar to those described in SLE , 37–39. A pathogenic role of antibodies in MS pathogenesis has long been debated . AgainstSeveral endogenous galectins probably contribute to the attenuation of autoimmune-inflammation in the CNS through both redundant and complementary pathways. Gal-1- and Gal-9-deficient mice develop more severe EAE due to selective expansion of antigen-specific Th1 and Th17 cells and to increased immunogenicity of dendritic cells , 83. In Galectins also offer new therapeutic opportunities. Gal-1 and Gal-9 enhance the frequency and immunosuppressive capacity of Treg cells , 85 and In conclusion, our results point to Gal-8 as an endogenous immunosuppressor that limits the autoimmune attack on the CNS, which can be blocked by antibodies leading to worse prognosis. Our clinical results together with a plausible pathogenic mechanism suggest that circulating anti-Gal-8 antibodies might be an early prognostic maker in RRMS and prompt to consider a therapeutic use of this lectin in patients bearing anti-Gal-8 antibodies.S1 TableLgals8+/- mice with positive reaction for β-gal histochemistry are listed as regions considered to express Gal-8.Brain sections of the heterocygous knock-in (PDF)Click here for additional data file.S2 TableRRMS patients before starting the DMD treatment, with or without Gal-8 autoantibodies in sera, share similar characteristics of age, gender, age at onset, disease duration, EDSS, and presence of baseline gadolinium-enhanced T1 lesions.(PDF)Click here for additional data file.S1 File+/+ versus Lgals8-/- mice, induced by immunization of an emulsion containing 150 μg of MOG35-55 peptide (MOGp) in 8-12-week-old Lgals8-/- and Lgals8+/+ mice. Daily monitored clinical scale of EAE symptoms: 0, no clinical signs; 1, loss of tail tone; 2, flaccid tail; 3, incomplete paralysis of one or two hind legs; 4, complete hind limb paralysis; 5, moribund 6, death. Data was used to establish daily progression of EAE Click here for additional data file.S2 File+/+ (WT) and Lgals8-/- (KO) mice. +), B cells (CD19+), CD8+ T cells and dendritic cells (CD11c+). + T cell subpopulations Th1, Th2 and Th17. For polyclonal T cell activation, splenocytes were grown in the presence of 1 μg/ml of αCD3 /μCD28 antibodies for 72 h. For the last 4 h of culture cell were stimulated incubating with 50 ng/ml PMA, 500 ng/ml ionomycin, and 10 μg/ml brefeldin A. Tables show frequencies for Th1, Th2 and Th17 in Lgals8+/+ (WT) and Lgals8-/- (KO) mice in untreated condition (UN) or under polyclonal activation (aCD3/28). Lgals8+/+ (WT) and Lgals8-/- (KO) mice analyzed by FACS: (A) Dendritic cells (CD11c+), B cells (CD19+), CD8+ T cells and different CD4+ T cells subsets, naïve (CD44-CD62L+), effector (CD44+CD62L+), memory (CD44+CD62L-) and total cells analyzed in the subset of viable CD4+ CD25- T cells. Results from 4–8 independent experiments show that Gal-8 deficit favors selective Th17 cell differentiation upon polyclonal activation.(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 FileLgals8+/+ (WT) and Lgals8-/- (KO) mice analyzed at steady state for total Tregs (Foxp3+), CXCR3+ and CCR6+ frequency in the Treg (Foxp3+ CD4+) population. Upper Table: Total Tregs obtained from five WT and five Gal8-KO mice (upper table). Middle Table: CXCR3+CCR6-. Bottom Table: CXCR3-CCR6+. Analysis from three WT and three Gal8-KO mice. Data show that galectin-8 deficit increases the frequency of total Tregs and CXCR3+ Tregs.Splenocytes isolated from (PDF)Click here for additional data file.S5 FileEAE was induced in wild-type C57BL/6J mice and simultaneously treated (i.p.) with either 100 μg recombinant Gal-8 or PBS (control group) during 20 consecutive days. EAE symptoms monitored daily using the following scale: 0, no clinical signs; 1, loss of tail tone; 2, flaccid tail; 3, incomplete paralysis of one or two hind legs; 4, complete hind limb paralysis; 5, moribund 6, death.(PDF)Click here for additional data file.S6 FileNumber of adherent cells counted in six randomly selected fields after incubation with anti-Gal-8 autoantibodies from three positive  and three negative patients . Anti-Gal-8 autoantibodies inhibit cell adhesion.(PDF)Click here for additional data file.S7 FileIn vitro differentiated Th17 cells from IL-17A-GFP reporter mice were purified based on IL-17A expression (GFP+) and incubated with Gal-8 (20 μg/ml) in the presence of lactose, sucrose or anti-Gal-8 antibodies affinity purified from pooled serum of MS patients. Numbers are the frequency of Annexin V+ 7AAD+ cells of the treated sample relative to the frequency of Annexin V+ 7AAD+ cells of untreated control, representing apoptosis, from three independent experiments. Anti-Gal-8 autoantibodies inhibit Gal-8-induced apoptosis of Th17 cells.(PDF)Click here for additional data file.S8 File+ ; Spinal MRI Gd+ ; DMD treatment ; Final EDSS (EDSS at follow-up); ARR ; Follow-up .Clinical characteristic of 17 RRMS patients anti-Gal-8(+) and 19 RRMS patients anti-Gal-8(-): N (number of patient); Gender (1 is man and 0 is woman); Age ; Age at onset MS (age at first symptoms in years); Diagnostic delay (time between age of onset and age of MS diagnosis); Disease duration (years between onset of MS and age at sample assay); Basal EDSS ; Brain MRI Gd(PDF)Click here for additional data file.


Sequence analyses revealed the presence of an IncI1/ST103-ISEcp1-blaCTX-M-166-orf477 plasmid region and of diverse antibiotic resistance and virulence-acquired genes.We report here the draft genome sequence of the CTX-M-166-harboring O6:H16 sequence type 48 (ST48)- Escherichia coli strain recovered in May 2014 from a 6-week-old Gallus gallus broiler flock from an industrial poultry unit in the central region of Portugal, carrying a new amino acid substitution, compared with CTX-M-1.Animals are considered potential reservoirs of antimicrobial-resistant bacteria . In thisE. coli LV13072 was extracted using DNeasy blood and tissue kit (Qiagen) and quantified using Qubit 1.0 fluorometer (Invitrogen). The Nextera XT DNA sample preparation kit (Illumina) was used to prepare sequencing libraries from 1 ng of genomic DNA, according to the manufacturer’s instructions. WGS was performed using 250-bp paired-end reads on a MiSeq (Illumina). Sequence reads were trimmed and filtered according to quality criteria and de novo assembled into contigs by means of CLC Genomics Workbench 8.5.1 (Qiagen), as previously described  detected 4,982 coding sequences (CDSs), 13 noncoding RNAs (ncRNAs), four complete rRNAs, and 68 tRNAs.The In silico antimicrobial resistance analyses using ResFinder version 2.1 (blaCTX-M-166 [contig 249] and blaTEM-1 [contig 63]), aminoglycosides (strA-strB [contig 289]), tetracycline [tet(A)-type (contig 20)], sulfonamides (sul2 [contig 289]), and trimethoprim (dfrA14-type [contig 257]). Seven virulence factors were also detected using VirulenceFinder version 1.5 (iss (contig 232), gad (contigs 29 and 125), astA (contig 123), iroN (contig 232), iha (contig 102), mchF-type (contig 57), celb-type (contig 46), and cma-type (contig 318) genes.sion 2.1 , with a  contig 6), aminogPlasmidFinder version 1.3 and pMLST version 1.4 tools , a CTX-M-1 IncI1 plasmid found on a UK pig farm (The blaCTX-M-166 and functional characterization of CTX-M-166 β-lactamase aiming to assess the potential impact of this new variant in veterinary settings, particularly under pressure caused by antibiotic exposure.The information presented herein will enable further studies about the genetic background of MCBU00000000. The version described in this paper is version MCBU01000000.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no.