Elavl1, which encodes for an RNA-binding protein with roles in transcript stability, was enriched in the premigratory cranial neural crest. Perturbation of Elavl1 resulted in premature neural crest delamination from the neural tube as well as significant reduction in transcripts associated with the neural crest specification GRN, phenotypes that are also observed with downregulation of the canonical Wnt inhibitor Draxin. That Draxin is the primary target for stabilization by Elavl1 during cranial neural crest specification was shown by RNA-sequencing, RNA immunoprecipitation, RNA decay measurement, and proximity ligation assays, further supporting the idea that the downregulation of neural crest specifier expression upon Elavl1 knockdown was largely due to loss of Draxin. Importantly, exogenous Draxin rescued cranial neural crest specification defects observed with Elavl1 knockdown. Thus, Elavl1 plays a critical a role in the maintenance of cranial neural crest specification via Draxin mRNA stabilization. Together, these data highlight an important intersection of post-transcriptional regulation with modulation of the neural crest specification GRN.While neural crest development is known to be transcriptionally controlled via sequential activation of gene regulatory networks (GRNs), recent evidence increasingly implicates a role for post-transcriptional regulation in modulating the output of these regulatory circuits. Using available single-cell RNA-sequencing datasets from avian embryos to identify potential post-transcriptional regulators, we found that  As an embryo develops, different genetic programs become activated to give cell populations a specific biological identity that will shape their fate. For instance, when certain sets of genes get switched on, cells from the outermost layer of the embryo start to migrate to their final destination within the body. There, these ‘neural crest cells’ will contribute to bones and cartilage in the face, pigmented skin spots, and muscles or nerves in the gut.When genes responsible for the neural crest identity are active, their instructions are copied into an ‘RNA molecule’ which will then relay this information to protein-building structures. How well the RNA can pass on the message depends on how long it persists within the cell. Certain RNA-binding proteins can control this process, but it is unclear whether and how this regulation takes place in neural crest cells. In their work, Hutchins et al. therefore focused on identifying RNA-binding proteins involved in neural crest identity.Exploratory searches of genetic data from chick embryos revealed that, even before they started to migrate, neural crest cells which have recently acquired their identity produced large amounts of the RNA-binding protein Elavl1. In addition, these cells did not behave normally when embryos were deprived of the protein: they left the outer layer too soon and then switched off genes important for their identity. Genetic studies of neural crest cells lacking Elavl1 revealed that this effect was due to having lost the RNA molecule produced from the Draxin gene.Introducing an additional source of Draxin into mutant embryos missing Elavl1 was enough to restore normal neural crest behaviour. Further biochemical experiments then showed that the RNA for Draxin decayed quickly in the absence of Elavl1. This suggests that the protein normally allows Draxin’s RNA to persist long enough to pass on its message.These results reveal a new mechanism controlling the identity and behaviour of the neural crest. Since many cancers in adulthood arise from the descendants of neural crest cells, Hutchins et al. hope that this knowledge could lead to improved therapies in the future. Neural crest cells are an essential, multipotent cell population in the vertebrate embryo. During development, these cells undergo coordinated induction, specification, and epithelial–mesenchymal transition (EMT) events to migrate and ultimately form a myriad of tissues, including craniofacial structures, components of the peripheral nervous system, as well as many other derivatives . The traElavl1 as an enriched transcript in newly formed neural crest cells. Elavl1 is a nucleocytoplasmic shuttling protein from the ELAV  family of RNA-binding proteins, which have conserved roles in neural development , we observed significant reduction in the levels of Axud1 (n = 6 embryos), FoxD3 (n = 10 embryos), and Draxin (n = 6 embryos) transcripts compared to contralateral control sides. To parse whether this was a specific effect or a broad defect in neural crest development, we also examined additional neural crest genes and found no significant difference in expression between control or Elavl1 knockdown sides for Pax7 (n = 5 embryos) or Tfap2b (n = 4 embryos), indicating a specific defect in a subset of genes required for cranial neural crest specification.To determine what, if any, role Elavl1 has in cranial neural crest specification, we perturbed Elavl1 function in the early embryo using a translation-blocking antisense morpholino oligo (MO). We electroporated control or Elavl1 MOs bilaterally into gastrula stage chick embryos and analyzed neural crest specification using quantitative fluorescent hybridization chain reaction (HCR) to measure expression of markers of specified neural crest at HH9 . Given Et Draxin . Followin = 4 embryos, 11 sections); however, we found a significant increase in the number of Pax7 + cells that delaminated from the neural tube , and concomitant decrease in the number of Pax7 + cells retained within the dorsal neural tube . Taken together, these data suggest that Elavl1 is required during early cranial neural crest development to regulate specification and prevent premature delamination.We also performed immunostaining for Pax7 in cross-section to assess if Elavl1 knockdown altered neural crest cell number or dorsal neural tube morphology. Interestingly, the total number of Pax7 + cells was unaffected with Elavl1 knockdown , consistent with HCR data  and differential gene expression analysis . Given tand Msx1 . We alsoHCR data . TogetheFoxD3 failed to meet our stringency cutoff during RNA-seq analysis due to low expression at the examined stages, and is likely downregulated with Elavl1 knockdown  or none  followed by quantitative reverse transcription-PCR (qRT-PCR) to pull down endogenous Elavl1 ribonucleoprotein (RNP) complexes. To this end, we incubated lysate generated from wild-type HH9 embryonic heads with magnetic beads coated with either Elavl1 antibody or a rabbit IgG nonspecific control antibody, then eluted bound RNA and performed qRT-PCR, comparing immunoprecipitated RNAs (‘IP’) with RNAs extracted from a fraction of the input lysate (‘Input’). We expected that RNAs bound by Elavl1 would be enriched in the IP compared to the Input, whereas nonspecifically associated RNAs, that is nontargets, would not (Pax7 and FoxD3 mRNAs (nontargets) were neither enriched in the IP, nor significantly different from each other  with Tukey’s post hoc test); however, Draxin mRNA was significantly enriched with Elavl1 IP compared to Pax7 and FoxD3 , wherein in situ fluorescent signal can be detected as puncta only when two proteins are in close proximity (<40 nm), indicating a direct interaction in vivo. Taking advantage of the MS2-MCP reporter system (Draxin 3′-untranslated region (MS2-Draxin 3′-UTR) and performed PLA with antibodies against Elavl1 and GFP , Axud1 , Msx1 , and BMP4  transcripts compared to contralateral control sides , Axud1 (n = 6 embryos), Msx1 (n = 5 embryos), and BMP4 (n = 5 embryos) expression from Elavl1 knockdown  to expression levels not significantly different from control . Taken together, these data indicate that Draxin mRNA is the primary target of Elavl1 in premigratory cranial neural crest and is stabilized via 3′-UTR interaction to maintain neural crest specification.We next asked whether henotype . To thishenotype , and asshenotype  caused bDraxin. We found that loss of Elavl1, and by extension its target, Draxin, interferes with output from multiple nodes of the GRNs required for neural crest specification, including those driven by Wnt and BMP  were purchased locally , and incubated in a humidified 37°C incubator to the specified Hamburger–Hamilton (HH) stage (Fertile chicken eggs (H) stage . Live emH) stage  and stor2/0.5% Triton X-100/0.001% Thimerosal) as described  start codon from nucleotide −20 to +5, and electroporated at [2 mM]. Draxin MO was described previously  was designed to span the eviously , and eleeviously  or pCI-Heviously  to increonstruct . Electroonstruct .For single-cell RNA-seq analysis, raw data from n = 3) before total RNA was extracted using an RNAqueous-Micro kit (Thermo). cDNA libraries  were prepared from 10 ng total RNA per pooled sample using the Takara Bio SMART-Seq v4 Ultra Low Input cDNA kit, according to the manufacturer’s instructions. Single-end RNA-sequencing was performed at the Caltech Millard and Muriel Jacobs Genetics and Genomics Laboratory at a depth of 20 million reads. Reads were processed and differential expression analysis was performed as described , and suspended in explant culture media . Actinomycin D [10 μg/ml] or DMSO (dimethyl sulfoxide) alone (for 0 hr time point) was added to neural fold samples, which were then incubated at 37°C/5% CO2 for 0 hr, 30 min, 2 hr, or 4 hr. Following the specified incubation time, neural fold samples were centrifuged at 300 × g for 3 min to remove media then washed three times with DEPC-treated PBS.Embryos were electroporated bilaterally with either control MO or Elavl1 MO and grown to HH8+/9−. Dorsal neural folds were dissected in DEPC-treated PBS, pooled  to each actinomycin D time point for each target. RNA decay rates were calculated and plotted using one phase decay in Prism9 .Total RNA was then extracted using the RNAqueous-Micro kit (Thermo) and 50 ng of RNA was reverse transcribed using SuperScript III and oligo dT priming. Following reverse transcription, we performed qPCR using FastStart Universal SYBR Green Master (Rox) with cDNA (diluted 1:10) and gene-specific primers (Key Resources Table) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems) in triplicate. Average 2, 0.05% NP-40), blocked in NT-2/5% bovine serum albumin (BSA) for 1 hr at room temperature, then incubated with 5 μg antibody (Elavl1 IgG or Control IgG) in NT-2/5% BSA for 1 hr at room temperature. Following antibody incubation, antibody-coated beads were washed with NT-2 and resuspended in NET-2 , 400 U RNaseOUT, 1× cOmplete, Mini EDTA-free Protease Inhibitor) until addition of cleared lysate.RIP was performed as described , with mig for 4 min at 4°C, washed in RNase-free PBS, and resuspended in polysome lysis buffer -1-piperazineethanesulfonic acid), 0.5% NP-40, 200 U RNaseOUT, 1× cOmplete, Mini EDTA-free Protease Inhibitor). Cells were frozen at −80 °C overnight to complete lysis and reduce adventitious binding. Lysate was then thawed on ice, vortexed, and centrifuged at 14,000 × g for 10 min at 4°C to remove cellular debris, then cleared lysate was added to antibody-coated beads in NET-2. Immediately following addition of cleared lysate, 10% was removed to serve as an Input control. IP reaction was tumbled at room temperature for 1 hr, beads were then washed in NT-2, and RNA was eluted in proteinase K buffer  for 30 min at 55°C and phenol/chloroform extracted.Embryonic heads were dissected in Ringer’s solution, washed in RNase-free PBS, and dissociated in Accumax (Innovative Cell Technologies) for 15 min at room temperature. Following dissociation, cells were pelleted at 2000 × CT (ΔCT = Input CT − IP CT) for Draxin, FoxD3, and Pax7 for Elavl1 and Control IgG RIP samples, then calculated fold enrichment values (=2T− Elavl1 ΔCT(Average Control IgG ΔC)) for each target and replicate.RNA from Input and IP samples was reverse transcribed using SuperScript III and oligo dT priming. Following reverse transcription, we performed qPCR using FastStart Universal SYBR Green Master (Rox) with cDNA (diluted 1:5) and gene-specific primers (Key Resources Table) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems) in triplicate. We determined ΔDraxin 3′-UTR , grown to stage HH9, then fixed at room temperature for 1 hr, washed, embedded, and cryosectioned as described , or MCP-GFP and MS2-escribed . SectionConfocal images were acquired using an upright Zeiss LSM 880 at the Caltech Biological Imaging Facility, and epifluorescence images were acquired using a Zeiss Imager.M2 with an ApoTome.2 module. Images were minimally processed for brightness/contrast and pseudocolored using Fiji  and Adobe Photoshop.Relative fluorescence intensity was determined in Fiji. For each whole mount image, the line tool was used to draw an ROI surrounding the area of neural crest indicated by positive HCR fluorescence for the genes examined. For cross-sections, ROIs were drawn surrounding the neural crest and neural tube based on tissue morphology from nuclear staining. Following background subtraction , integrated density was quantified for the ROIs on the control electroporated (left) and experimental electroporated (right) sides from the same embryo. Relative fluorescence intensity was then calculated by dividing the integrated density measurements for the experimental versus the control side of the same embryo.Pax7 cell counts were performed as described . The limFor PLA analysis, confocal micrographs were processed in Fiji. Following background subtraction , ROIs were drawn surrounding the neural crest and neural tube based on tissue morphology from nuclear staining, then puncta  were counted using ‘analyze particles’ function following global manual thresholding to reduce the appearance of nonspecific puncta outside of electroporated regions.Gallus Axud1 was not available in a public repository, to determine whether the Axud1 3′-UTR contained potential Elavl1-binding sites, we performed 3′-Rapid Amplification of cDNA Ends (3′-RACE) as described  and eight of these clones were sequenced, and the consensus 3′-UTR sequence deposited to GenBank (Accession # ON920861). Primer sequences can be found in the Key Resources Table.Given that the 3′-UTR sequence of escribed . BrieflyDraxin 3′-UTR (GenBank: AB427147.1), Axud1 3′-UTR (GenBank: ON920861), Msx1 3′-UTR (Ensembl: ENSGALT00000024209.4), BMP4 3′-UTR (Ensembl: ENSGALT00000020316.7), Pax7 3′-UTR (Ensembl: ENSGALT00000048594.2), and Tfap2b 3′-UTR (Ensembl: ENSGALT00000026916.6) were predicted using ‘mfold’ web server with default settings . p values are defined in the text, and significance was established with p < 0.05. p values were calculated using Wilcoxon signed-rank, Mann–Whitney, one-sample analyses  confirme In this short report, Hutchins et al. reveal expression of the RNA-binding protein (RBP) HuR in the neural tube and cranial neural crest of chicken embryos. Knock-down of HuR affects expression of Axud1 and FoxD3  and of the Wnt antagonist Draxin previously shown by the authors to regulate neural crest specification and delamination. The authors propose that HuR associates with Draxin mRNA and demonstrate that Draxin overexpression can rescue FoxD3 expression upon HuR knock down. The data is in line with the idea that control of neural crest specification by HuR at least partially involves Draxin mRNA stabilization. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kathryn Cheah as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Lukas Sommer (Reviewer #1).Thank you for submitting your article "RNA-binding protein Elavl1/HuR is required for maintenance of cranial neural crest specification" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this short report, Hutchins et al., reveal expression of the RNA-binding protein (RBP) HuR in the neural tube and cranial neural crest of chicken embryos. Knock-down of HuR affects expression of Axud1 and FoxD3  and of the Wnt antagonist Draxin previously shown by the authors to regulate neural crest specification and delamination. The authors propose that HuR associates with Draxin mRNA and demonstrate that Draxin overexpression can rescue FoxD3 expression upon HuR knock down. The data is in line with the idea that control of neural crest specification by HuR at least partially involves Draxin mRNA stabilization.Essential revisions:As outlined below in the largely unedited Reviewers comments, they suggest a major re-write of the paper.Overall, Reviewer 1 commented that unraveling the gene regulatory network (GRN) of neural crest specification has been the focus of many recent studies. How the GRN is modulated by post-transcriptional control mechanisms, as addressed here, is an important and relevant question. However they feel that the authors missed an opportunity to address this question in a broader manner. Moreover, the evidence that HuR regulates Draxin post-transcriptionally (by mRNA stabilization) has to be strengthened.Overall, Reviewer 2 commented that the main problem about this work is that the central player of the story seems to be a ubiquitous expressed molecule . In the early chicken embryo, the situation does not seem very different, since it is certainly expressed in forming NC/NT, but also in other tissues . Thus reader might conclude that it is not very surprising that when mRNAs are less stable, most tissues, including NC, do not perform as expected. Indeed they have shown that the three genes they have tested  are all down-regulated after Elavl1 knock-down.Major Points:1) According to the Abstract and in the Introduction, the authors' goal of this study was the identification of potential post-transcriptional regulators of neural crest specification. Specifically, they "sought to identify RBPs with early roles in neural crest development". This is an intriguing research question. However, rather than systematically assessing RBPs in their RNA seq datasets, they picked Elav1/HuR for their further experiments. Why? Likely, this was not the only RBP in their RNA seq datasets. Was Elav1 (encoding HuR) the most enriched RBP? These data should be shown and the reason for picking Elav1/HuR should be clarified.2) In Figure 1D', expression of HuR in migratory neural crest is not evident. Transverse section at HH9+ might help revealing HuR expression in migratory neural crest cells.3) The link between HuR and Draxin is rather weak and seems again a bit 'cherry picked'. I recommend a more global assay to identify mRNA targets of HuR (e.g. CLIP seq). As correctly stated in the Discussion, HuR is also known to stabilize Snail1 and other mRNAs with roles in neural crest development, which could all mediate post-transcriptional regulation of neural crest specification and, therefore, mediate the observed phenotypes. Importantly, in Figure 3, enrichment of Draxin mRNA is not very convincing, in particular when compared to its control (non-specific control IP) rather than to Pax7/FoxD3 values (which for reasons unclear to me are highly negative). A positive control should be included (e.g. a previously identified mRNA target of HuR). Can Draxin mRNA binding by HuR be confirmed by other means (such as CLIP seq)? It should be addressed whether Draxin mRNA stability is indeed affected by HuR knock down.4) The Draxin overexpression rescue experiment is nice, but should be extended to other parameters of neural crest specification (the authors only checked FoxD3 expression). What about other markers? What about delamination?5) The authors show that the three genes they have tested  are all down-regulated after Elavl1 knock-down. The experiment shown in Figure 4 somewhat dampens this impression by suggesting that not all those mRNAs bind to Elavl1, but it would give weight to these findings if the authors would i) support this data by checking in silico on the many RNAseq data they have gathered over the years that only a subpopulation of "NC genes" display Elavl1 target sequences in their UTR and ii) verify that some of those genes are unaffected after Elavl1 MO electroporation . This would support the idea that even though Elavl1 may be ubiquitously expressed, its targets are restricted and that we are looking at a regulated biological process and not so much at a house-keeping activity.If these authors can show some specificity in Elavl1 function, this could also be an opportunity to re-write part of the text and convey a more general idea/concept on the importance of RNA stability in biology, something that we use on a daily basis when designing expression vectors, but which has obviously real-life implications.6) It would also help if the authors use Elavl1 gain-of-function approaches to support the data presented here .7) Very little (in fact nothing) is said in the introduction about existing work on full and conditional KO of Elavl1 in mouse , which sends the message that the present work stands on its own. Katsanou's paper is in fact quite extensive and it describes a number of phenotypes that the authors observed in different genetic settings. This has to be included in the present paper (i.e. it should be introduced/discussed upfront), with the aim of convincing the reader that there is still something to be understood about the function of Elavl1 and that the (advantages of the model) chicken embryo can address those questions in a more precise way than the murine model does. Essential Revisions:1) According to the Abstract and in the Introduction, the authors' goal of this study was the identification of potential post-transcriptional regulators of neural crest specification. Specifically, they "sought to identify RBPs with early roles in neural crest development". This is an intriguing research question. However, rather than systematically assessing RBPs in their RNA seq datasets, they picked Elav1/HuR for their further experiments. Why? Likely, this was not the only RBP in their RNA seq datasets. Was Elav1 (encoding HuR) the most enriched RBP? These data should be shown and the reason for picking Elav1/HuR should be clarified.We acknowledge the reviewers’ point, which made us realize that we did not adequately explain why we chose to focus on Elav1/HuR as a post-transcriptional regulator of neural crest development. We now clarify that this is one of the few such genes that we have found enriched in the premigratory neural crest. To better demonstrate this, we have re-analyzed a recently published scRNA-seq dataset . We have reorganized the paper and now include this analysis as a new Figure 1 to further clarify our choice for selecting Elavl1 for follow up.In the revised manuscript, we highlight that several RNA-binding proteins are expressed to varying degrees in cranial neural crest cells. Using gene ontology analysis, we generated a three-way Venn diagram to identify overlap between genes associated with the gene ontology terms “binds RNA,” “regulates translation,” and “stabilizes RNA.” Only three genes, Elavl1, Dazl, and Igf2bp1, were associated with all three, and only Elavl1 is abundant amongst all sorted cranial neural crest cells.We have now included this expanded justification in the text.2) In Figure 1D', expression of HuR in migratory neural crest is not evident. Transverse section at HH9+ might help revealing HuR expression in migratory neural crest cells.We have added a transverse section at HH9+ showing Elavl1 expression in migratory neural crest cells. This is now included in Figure 2F-G.3) The link between HuR and Draxin is rather weak and seems again a bit 'cherry picked'. I recommend a more global assay to identify mRNA targets of HuR (e.g. CLIP seq).We thank the reviewers for raising this good point. To more broadly identify targets ofDraxin, Axud1, Msx1, and BMP4. The downregulation of these transcripts with Elavl1 knockdown was confirmed in vivo using HCR .Elavl1, we performed differential RNA-sequencing analysis on dissected neural folds  for control vs Elavl1 knockdown . We identified 24 differentially expressed genes . Among those genes, only four were known to have roles in neural crest development and were found to be significantly downregulated with Elavl1 knockdown—As correctly stated in the Discussion, HuR is also known to stabilize Snail1 and other mRNAs with roles in neural crest development, which could all mediate post-transcriptional regulation of neural crest specification and, therefore, mediate the observed phenotypes.The reviewers raise an important point. However, Snail1 is not expressed in chick cranial neural crest at these stages :1583-93); rather Snail2 is the predominant Snail family transcription factor at this time and place in chick embryos. Importantly, our newly added RNA-seq data and HCR for Snai2  indicate that, indeed, Snai2 expression is unaffected by Elavl1 knockdown, and is unlikely to be a direct target of Elavl1 at the premigratory stage.Importantly, in Figure 3, enrichment of Draxin mRNA is not very convincing, in particular when compared to its control (non-specific control IP) rather than to Pax7/FoxD3 values (which for reasons unclear to me are highly negative). A positive control should be included (e.g. a previously identified mRNA target of HuR). Can Draxin mRNA binding by HuR be confirmed by other means (such as CLIP seq)?Draxin mRNA over non-targets FoxD3 and Pax7.Thank you for this comment, which made us realize that this experiment needed to be better documented. Accordingly, we have modified the presentation of this RIP data to display Fold Enrichment, in keeping with other studies that have performed RIP experiments for Elavl1 . We hope this better illustrates the enrichment of Draxin mRNA binding by Elavl1 through other means, we have now included a proximity ligation assay experiment  as a secondary means to demonstrate a direct interaction. Proximity ligation is a well-established technique used to demonstrate direct protein-protein interactions. We have now adapted this method to interrogate a protein-RNA interaction. For this experiment, we created an expression construct containing the Luciferase coding sequence followed by MS2 stem loops and the Draxin-3’UTR. When transcribed, and co-expressed with an MCP-GFP construct, the MS2 stem loops in the mRNA transcript are bound by MCP-GFP. Thus, if Elavl1 binds to the Draxin-3’UTR, a proximity ligation assay using antibodies to Elavl1 and GFP will generate fluorescent puncta. Using this approach, we now demonstrate that endogenous Elavl1 physically interacts with the 3’UTR of Draxin .While it would be ideal to use an experiment such as CLIP-seq to confirm direct binding, CLIP-seq (and similar cross-linking experiments) require large amounts of starting material that make this approach unfeasible for embryonic tissue. However, in an effort to provide demonstration of It should be addressed whether Draxin mRNA stability is indeed affected by HuR knock down.Draxin, Axud1, Msx1, and BMP4, since we found using RNA-sequencing that these four genes are all downregulated with Elavl1 knockdown. We also examined RNA decay for Pax7 as a “non-target”. Interestingly, we found that only Draxin mRNA stability was significantly decreased with Elavl1 knockdown .Good point. To quantify changes in mRNA stability with Elavl1 knockdown, we utilized the transcriptional inhibitor actinomycin D followed by qRT-PCR to measure RNA decay for 4) The Draxin overexpression rescue experiment is nice, but should be extended to other parameters of neural crest specification (the authors only checked FoxD3 expression). What about other markers? What about delamination?Axud1, Msx1, and BMP4 (in addition to FoxD3), since we found these transcripts to also be downregulated with Elavl1 knockdown. All of these transcripts were rescued from Elavl1 knockdown with Draxin overexpression . Importantly, Draxin knockdown alone also significantly reduced expression of these same four transcripts . Together these data support the hypothesis that Draxin is the major target of Elavl1 in cranial neural crest specification. These data are included in Figure 5.We extended the rescue experiment to also examine delamination . We found that Draxin overexpression was sufficient to rescue defects in delamination . Further, we also performed HCR for 5) The authors show that the three genes they have tested  are all down-regulated after Elavl1 knock-down. The experiment shown in Figure 4 somewhat dampens this impression by suggesting that not all those mRNAs bind to Elavl1, but it would give weight to these findings if the authors would i) support this data by checking in silico on the many RNAseq data they have gathered over the years that only a subpopulation of "NC genes" display Elavl1 target sequences in their UTR and ii) verify that some of those genes are unaffected after Elavl1 MO electroporation . This would support the idea that even though Elavl1 may be ubiquitously expressed, its targets are restricted and that we are looking at a regulated biological process and not so much at a house-keeping activity.If these authors can show some specificity in Elavl1 function, this could also be an opportunity to re-write part of the text and convey a more general idea/concept on the importance of RNA stability in biology, something that we use on a daily basis when designing expression vectors, but which has obviously real-life implications.Draxin, Axud1, Msx1, BMP4, Pax7, and Tfap2b . Neither Axud1, Pax7 nor Tfap2b contain Elavl1 binding sites, and Msx1 and BMP4 contain only one. Notably, the Draxin-3’UTR contains four high-probability binding sites, all in proximity on the same side of the folded structure.The reviewers raise excellent points. We now include Elavl1 binding analysis for the 3’UTRs of Given that Elavl1 contains three RNA-recognition motifs which are known to cooperatively bind a target RNA , it is possible that, in order for Elavl1 to bind and stabilize an mRNA target, it needs to establish multiple points of contact on the RNA, which may account for the specificity of function we have observed here in neural crest.In addition to the new data figure, we also expand on the above points in the relevant section of the Results.6) It would also help if the authors use Elavl1 gain-of-function approaches to support the data presented here .Draxin; however, we did not observe differences in Draxin levels between the control and Elavl1 overexpressing sides at HH9. We chose not to include these data in the manuscript as we do not think it adds anything to or changes the interpretation of our data or conclusions. This finding is consistent with previous observations in HeLa cells, where reduction but not overexpression of Elavl1 altered type I IFN response :1500-11. doi: 10.1002/eji.201444979).We overexpressed Elavl1 in premigratory cranial neural crest cells (driven by the FoxD3 enhancer NC1) and performed HCR for 7) Very little (in fact nothing) is said in the introduction about existing work on full and conditional KO of Elavl1 in mouse , which sends the message that the present work stands on its own. Katsanou's paper is in fact quite extensive and it describes a number of phenotypes that the authors observed in different genetic settings. This has to be included in the present paper (i.e. it should be introduced/discussed upfront), with the aim of convincing the reader that there is still something to be understood about the function of Elavl1 and that the (advantages of the model) chicken embryo can address those questions in a more precise way than the murine model does.Apologies that we did not sufficiently reference this paper. While our original draft referenced this paper in the Introduction and Results sections with respect to their findings in craniofacial development, which we felt was most pertinent, in the revised manuscript we have now expanded reference to this study in the Introduction. In light of our newly added results, we have also now added reference to this citation in the Discussion, where we describe our findings, how they relate to and build upon the Katsanou paper, and how the chick model enables us to ask these questions in a spatiotemporally controlled manner.


Multiple recent studies revealed stripes as an architectural feature of three-dimensional chromatin and found stripes connected to epigenetic regulation of transcription. Whereas a couple of tools are available to define stripes in a single sample, there is yet no reported method to quantitatively measure the dynamic change of each stripe between samples. Here, we developed StripeDiff, a bioinformatics tool that delivers a set of statistical methods to detect differential stripes between samples. StripeDiff showed optimal performance in both simulation data analysis and real Hi-C data analysis. Applying StripeDiff to 12 sets of Hi-C data revealed new insights into the connection between change of chromatin stripe and change of chromatin modification, transcriptional regulation, and cell differentiation. StripeDiff will be a robust tool for the community to facilitate understanding of stripes and their function in numerous biological models. A statistical framework accurately detects differential chromatin stripes that regulate gene expression changes between samples. The eukaryotic DNA is folded into hierarchical chromatin domains at multiple genomic scales in the three-dimensional (3D) space. The development of chromosome conformation capture (3C) assay . A key feature of a stripe’s edge is a big difference in the value of ci, j between the left and right sides of the edge, e.g., for the left edge, the values at the right side (located within the stripe) will be larger than the values at the left side (located outside of the stripe). Therefore, we defined a stripe edging signal matrix E = {ei, j}, in which ei, j = ci + 1, j − ci, j  method . We first vertically slid the start point of the window g matrix , step b.) method , step f.) method .mu1 versus mu2 in the two samples at the upward peak and md1 versus md2 at the downward peak, the null hypothesis is that mu1 = mu2 and md1 = md2. The normalized values in a Hi-C contact matrix can be modeled as normally distributed Gaussian random variables  and further adjusted the P values based on the Benjamin-Hochberg method.After the detection of individual stripes in each single Hi-C sample, the third function in StripeDiff defines differential stripes between a pair of samples , step g.ariables , step g.To assess the performance of StripeDiff, we developed SimuStripe, a pipeline to generate simulation data in which stripes were embedded  (fig. S2). Because stripes often co-occur with loops and TADs, SimuStripe further embeds loops and TADs along with stripes. For the simulation data to mimic real Hi-C data, we used real Hi-C data as a template for generation of simulation data so that the distribution of simulated location, size, and average contact frequency in individual TADs, loops, and stripes will be close to those observed in real data. Manual inspection of individual genomic regions in the simulated data confirmed the consistency of stripes, loops, and TADs between the simulated and real data , right. k on the performance of StripeDiff in recapturing the anchor locations of simulated differential stripes. For each tested window size, we repeated the simulation 1000 times, with each time simulated a random genomic region, for which the region size is equal to the window size. Two replicates were simulated for each region in each of two simulated samples. This resulted in four simulated datasets for two samples. We then detected differential stripes between the two samples. We plotted receiver operating characteristic (ROC) curve and calculated the area under the curve (AUC) of ROC curve to evaluate the performance. For comparison, we also customized the edgeR algorithm for differential stripe analysis  and compared it to our StripeDiff algorithm. EdgeR was developed for differential gene expression analysis (P < 1 × 10−16) more distant from the true differential stripes . We manually checked individual differential stripes identified by StripeDiff and observed that many differential stripes were each identified in both replicates . We denoP = 0.0083, Wilcoxon test)  . By manuon test) . This obon test) . In addiIt was reported that the formation of stripe is associated with enhancer activity and RNA polymerase II (Pol II) elongation .By manually inspecting ChIP-seq signals at individual differential stripes, we observed that CTCF, H3K27ac, and Pol II did not always change at the same differential stripe. For instance, a stripe weakened in HUVEC relative to IMR90 could show a reduction of Pol II binding but little change of CTCF binding . We founP value cutoffs to define differential genes (fig. S9B). These results indicated that the stripe formation is positively associated with activation of transcription.As CTCF binding, enhancer activation, and RNA Pol II activity are all associated with transcriptional regulation of gene expression, we further investigated whether the differential stripes defined by StripeDiff are accompanied by changes of gene expression. We identified 7588 differentially expressed genes between HUVEC and IMR90 based on RNA-sequencing (RNA-seq) data from these two cell types. Our analysis indicated that 11.7% of the genes up-regulated in HUVEC relative to IMR90 overlapped with genes in stripes strengthened in HUVEC relative to IMR90, whereas only 1.8% overlap would be expected by chance . SimilarA recent investigation in embryonic stem cells and neural stem cells showed change of stripe during cell lineage specification . We perfTo further understand the dynamic change of stripes during the cell lineage specification, we analyzed Hi-C data generated at six different stages of T cell differentiation from hematopoietic stem and progenitor cell (HSPC) to CD4 and CD8 double-positive (DP) T cell . We applThe chromosomes in the nucleus are hierarchically folded into multiple layers of 3D architectures, such as chromosome territories, compartments, TADs, loops, and stripes , j = 1 : (n − 1)}. For a vertical window k that is l bins long in the column i, where l is a parameter provided by a user, the top and bottom boundaries of the window are j = t and j = w = t + l . If the value of location i is the maximum value when compared to values of the k nearest locations on the left side and k nearest locations on the right side, we will define i as the location of an upward peak. Similarly, we defined a local minimal filter to detect downward peaks. We then further matched an upward peak to a downward peak located within l bins.We denote a matrix of chromatin contact frequency determined by Hi-C as pecified , step a. = t + l , step b.j=twei,j , step c.the plot , step d.s, with the upward and downward peaks of the mean edging signal in a vertical window k being sU and sD, the location of the left and right edges are isU)( and isD), k and mi(sD), k. We define a stripe peak intensity signal for the stripe s in the window k as Ps, k= ∣mi( sU), k − μi( sU)∣ + ∣mi(sD), k − μi(sD)∣, in which i , in which μ and σ are the mean and SD. We note that the parameters μ and σ are assumed to be consistent and independent of i. We assume that the upward and downward peaks of a stripe are both not differential between two samples, i.e., the null hypothesis. Evidence against the null hypothesis is taken as evidence for the difference between the two samples. In a window k that covers the full length of a stripe s in samples 1 and 2, mu1 = mi( sU), k,1 and mu2 = mi( sU), k,2 are height values of the upward peak, and md1 = mi(sD), k,1 and md2 = mi(sD), k,2 are those of the down peak.The normalized values in a Hi-C contact matrix can be modeled as normally distributed Gaussian random variables  − log (mu2), whereas u are the mean and SD of um. Similarly, dm = log (md1) − log (md2), whereas d are the mean and SD of dm. Therefore, the variables that we were testing here are uz and dz. We used uz and dz instead of um and dm, because um and dm could follow different normal distributions. uz and dz represent z scores, which can be both modeled as standard normal distribution .Differential stripe was tested using HWe compared the performance between StripeDiff and edgeR, which were used to calculate the significance of differential chromatin interactions in each stripe between samples. We generated Hi-C simulation data for two conditions, each with two replicates. In edgeR analysis, we calculated the read count in each stripe region, which horizontally started from the left edge and ended at the right edge, and vertically started from the diagonal point and extended to the stipe terminal. Then, we used edgeR to calculate the false discovery rate of difference in read count from each stripe region between samples.We used the overlapping ratio (Jaccard index) cutoff value of 0.5 between the predicted and simulated stripe anchors. The overlapping ratio was calculated as the intersection width divided by the union width between predicted and simulated stripe anchors. The predicted and simulated stripes are considered as overlapped if the overlapping ratio is larger than the cutoff.i, j), we first simulate the contact probability of each TAD as log = Norm(μor), which is a log normal distribution, and μ and σ are the mean and variation of contact probability in each TAD. Next, we select a few TADs to have a loop at the corner of each TAD. We further select TADs to have a stripe at the left or right boundary of each TAD. We also select TADs to each merge with the TAD next to them. These TADs were selected using IMR90 as a template to evaluate similarity to real data or randomly to evaluate performance of StripeDiff. In addition, μ and σ can be calculated from IMR90 data as the template or specified in the following way: μ ata[abs(i − j)] × slope + m, σ = {log [abs(i − j)] × slopeσ + mσ}2. Here, i and j are the smaller and larger genomic coordinates; m, slope, mσ, and slopeσ are the parameters to be specified. It is recommended to set slope as negative and slopeσ as positive values. The resolution is 10 kbp unless a different resolution was explicitly specified. The distribution of simulated stripe length mimics that in the IMR90 Hi-C data unless a length was explicitly specified. We did not simulate interchromosomal reads, so the simulated sequencing depth is the number of intrachromosomal reads.To simulate the count of chromatin contacts (fig. S1), we used the IMR90 Hi-C contact map as a reference. For each pairwise interaction  in the stripe s. We then converted the matrix TM to a 1D vector V = (wv), in which TM to a 2D matrix DM = {wlc}, in which To plot the average read density around a group of stripe anchors, we first categorized the stripes as vertical (left) stripes or horizontal (right) stripes based on the location in the contact map . For eac


Mid-latitude alpine caves preserve a record of past solid precipitation during winter, locally spanning several centuries to millennia. Dating organic macro-remains trapped in ice layers allows the determination of timing and duration of past periods of positive and negative ice mass balance. We present here the largest comparative study of ice cave sites yet published, using Bayesian age-modelling on a database comprising 107 radiocarbon dates, spread over eight caves in the Austrian Alps. We show that periods of positive mass balance coincide with past glacier advances. We find organic and macro-remain rich layers dated to the Medieval Climate Anomaly (between 850 and 1200 CE) marking widespread ice retreat. We demonstrate positive ice mass balance at all studied sites for the Little Ice Age, coinciding with the largest glacier advances in the Holocene between 1400 and 1850 CE. At the sites with records spanning over 2000 years, positive mass balance is also observed during the periods from 300 BCE to 100 CE and 600–800 CE. These subterranean ice deposits show widespread evidence of accelerated negative mass balances in recent years and their record is under imminent threat of disappearing. In the European Alps, modelling studies consistently point towards the near-total loss of glaciers by the end of the 21st Century4. Ice deposited and preserved underground in alpine caves is likewise experiencing a widespread retreat in many parts of the European Alps and mountain regions elsewhere7. Such rock-hosted caves where perennial ice or snow occur are known as ice caves8.Glaciers worldwide have experienced overwhelmingly negative mass balance in the early 21st Century, with thinning rates doubling over the period 2000–20208, no more than a few dozen have been investigated in detail13. Ice deposits in underground caverns are generated by the firnification of snow entering the cave during the cold season or the freezing of water, or both, and preserved thanks to specific ventilation patterns which cool the cave environment15. A growing body of evidence, ranging from monitoring ice-level fluctuations16, radiocarbon-dating of organic matter included in ice17, trace element11 and stable isotope analyses19 demonstrates the clear paleoclimatic potential of underground ice archives, as well as their high vulnerability in a changing climate5.Although several thousands of ice caves are known and documented worldwide15. The present study focuses on underground ice deposits in such sag-type caves because they offer the most direct link to atmospheric processes and act as natural snow collectors during the cold season. Leaves, twigs, seeds and cones, or fragments thereof, sourced from the surrounding vegetation are transported into the cave where they may eventually become embedded in the accumulating snow. This process leads to stratified ice bodies typically a few metres to a few tens of metres in thickness, containing inclusions of various origin, organised in layers20. Ice accumulated in this way can be lost through melting or sublimation occurring at the different ice-water, ice-air or ice-rock interfaces. Ice mass balance in sag-type caves is thus a complex function of precipitation type (snow or rain), its temporal distribution and amount, as well as rock and air temperature profiles. Like surface glaciers, this underground ice represents the time integration of a variable annual mass balance21.Caves with a descending morphology, open pits, also known as sag-type, collect winter snow, whose accumulation and subsequent firnification builds an underground ice body22, a task previously achieved using annual layer counting wherever feasible23 or radiogenic 210Pb age determinations for ice deposits no older than about 150 years24. Most studies, however, rely on radiocarbon age determination of wood inclusions for dating underground ice deposits22. The oldest wood macro remains found in ice caves of the European Alps are dated from 3366 to 3030 BCE (95.6%)26, and the oldest currently known sample from an alpine ice cave worldwide, dated from 4256 to 4043 BCE (95.6%), was found in a Pyrenean cave12.Estimates of past mean annual mass balance (MAMB) can be derived from age-depth relationships or chronostratigraphies of underground ice deposits. Placing mass balance changes in a chronological context requires the age of the ice to be determined13 and in other mountain chains of Europe27 and North America28. To date, most published ice cave records focus on single cave ice deposits. For instance, a combination of radiocarbon-dating and dendrochronology was used to reconstruct past periods of positive ice mass balance at St. Livres ice cave9, while the age of the ice in the nearby Monlési Ice Cave was constrained by 210Pb, 14C and anthropogenic inclusions17, both ice caves being located in the Jura Mountains of northern Switzerland. At Hundsalm Ice Cave, western Austria, periods of positive and negative mass balance were bracketed using a 19-sample radiocarbon dataset from wood inclusions13. Similarly, the timing and duration of ice deposition in a sag-type cave of the Julian Alps were constrained with the aid of an 18-sample dataset29. At Dobšínská cave in Slovakia, successive campaigns sampling the remains of bats embedded in the ice body helped constrain the age of the ice deposit to 2600 years31. In the central Pyrenees, the record of 22 radiocarbon samples from A294 cave was shown to span several millennia12, and similarly, the exceptional record of Scărișoara ice cave, Romania, has been the subject of several publications, linking stable isotopes of ice to past winter climate variability during the Holocene32. The ice in Strickler Cavern  was shown to span the past 2000 years based on 26 radiocarbon samples11. There is therefore an increasing need to broaden the database and to compare and integrate records from different sites in order to better understand the response of this part of the cryosphere to climate forcing.Past decades have seen a slowly rising number of studies of chronostratigraphies at cave sites in the Alps26. Many sites also benefit from having been explored and mapped several decades ago, and thus provide a benchmark for more recent ice- and snow-level variations. As such, the Austrian Alps, constituting the eastern part of the European Alps, are ideally suited for such a regional study. Using a total of 107 radiocarbon-dated woody macroremains, this study reports the first detailed chronostratigraphy at eight caves, providing a simultaneous comparison of the ice mass balance dynamics in those caves.Karst regions of the European Alps are particularly conducive to the development of underground ice, in no small part due to the significant amount of snow in winter and the large number of vertical pits developed in carbonate rocks outcropping at elevations over 1500 m a. s. l.26. Within this database, sag-type caves containing a clearly visible and accessible stratigraphy, where the presence of wood debris was attested from previous speleological documentation, made the best targets.Owing to the large limestone plateaus in the Northern Calcareous Alps, where most of the caves are situated at elevations ranging 1500–2100 m a.s.l., more than 1200 ice-bearing caves are recorded in the Austrian cave cadastrePinus mugo), spruce (Picea abies) and larch (Larix decidua). All caves are of the sag-type geometry . At Hundsalm, there is also evidence for positive MAMB13 These four caves were continuously glaciated during the last two millennia. Within the other caves, the radiocarbon data give the earliest evidence of continuous glaciation either in the early LIA The comparison of modern observations with previous exploration reports, topometric surveys and photographs reveals a negative MAMB for the last three decades at least in the majority of the study sites.In total, 107 radiocarbon samples, spread over eight caves and their ice exposures were analysed and placed in a stratigraphic context (Extended Data A). For each cave, an age-depth model was established, thereby providing constraints on the mass balance dynamics at these sites (Extended Data B). At the sites of Guffert, Eisgruben and Bärenloch, the compiled age lists and respective age-depth models reach back before the Common Era and provide strong evidence for positive MAMB prior to the Little Ice Age . In contrast to the section previously sampled near Tiefster Punkt, which lacks a stratigraphic progression13, the eight samples from the northern section are in stratigraphic order . These steeply dipping strata are separated from the subsequent discordant unit by a macro-remain-rich unconformity and the next five samples defining a section of positive MAMB spanning 1300 CE to 1700 CE . In light of this constraint, the uppermost part of the northern section stratigraphy suggests a MAMB of about 4 cm w.e. yr−1. The ice body in this cave is currently experiencing a strongly negative MAMB, as evidenced by the continuous drop in the ice surface of Eisdom in the past decades.We re-sampled the der Fig. . At thatCE Table , ending bottom section (Extended Data B.2.3). Samples BL-C1 to C3 span 200 CE to 100 CE . In the very deepest alcove, 3 m of layered ice are constrained by eight radiocarbon samples . One sample (BL-C24) was rejected from the section (Extended Data B.2.1). The remaining six samples define two main periods of positive mass balance. A large break in the slope of the age model was detected at 1300 CE to 1400 CE.The les Fig.  and one bottom section corresponds to the innermost, small ice alcove at the bottom of the cave, spanning about 140 cm. A total of seven samples were dated from this short section and all were used to build the age model of the bottom section  warranted the construction of a separate model for each of the sections Fig. . The bot up Fig. . A totalbottom section, started at around 400 BCE, spanned about 800 years , due to their relatively young age with respect to the stratigraphy. The Rutschbahn section is comprised of five samples , yielding a much low mean mass balance for that period on the right.The period spanning 600 CE to 700 CE is marked by a seven-fold increase in MAMB recorded on the −1 to about 1.9 cm w.e. yr−1  are evidence of sustained ice retreat bracketing another period of net but clearly discontinuous ice growth spanning the 8th–11th Centuries CE. The deposition of the top half of the stratigraphy, characterised by clear, layered ice with much fewer organic remains started at the turn of the 14th Century CE and continued apparently uninterrupted for at least four centuries Table . The topr−1 Fig. .Figure 435. One sample yielded a modern radiocarbon value (Extended Data B.7) and sample P3 was found to lie outside of stratigraphic order and was thus excluded. The MAMB at Kraterschacht derived from age modelling varies between 1.3 and 1.4 cm w.e. yr−1 . Stratigraphic superposition dictates that this upper part of the ice postdates the Iglu section and was therefore deposited between the late 18th Century CE and present.This 163 m-deep cave hosts a well-dated section lacking unconformities and spanning at least 1390 CE to 1770 CE Fig. . A total17. Cave glaciation likely begun before the oldest age. The starting boundary, however, is a minimum age for the beginning of the current state of cave glaciation.The start and end point of each ice section in the eight studied caves are displayed in Fig. 33 and continuing thereafter.The youngest radiocarbon-dated samples from a section yield an end boundary which cannot be interpreted as marking the cessation of ice deposition, on the contrary. At all sites, the sampling of macro-remains stops when the stratified section transitions upwards into more massive ice and eventually firn and snow. Stratigraphic superposition indicates strongly that ice deposition continued after that last boundary, but with very poor age control and hence, unknown MAMB. The deposition of ice in each cave certainly continued after 1600 CE at Eisgruben, Bärenloch, Guffert and Hochschneid. At Hochschneid, the majority of the ice volume even post-dates the well-dated section found at the bottom. At Bärenloch, the end of the dated, well-layered section is found near the bottom of the cave, again suggesting that more than half of the ice visible in the cave today post-dates this section. At Eisgruben, the undated upper section of the stratigraphy represents nearly 5 m of ice, which post-dates the last sample  saw a drop in MAMB at those two caves, while at Guffert, MAMB was either null or negative. Due to the discontinuous nature of the exposure, we cannot exclude that some of the ice present in Guffert dates from that period. At Eisgruben, this period coincides with the first two of the three large angular unconformities, where a significant amount of ice was lost, altering the geometry of the ice surface. At Bärenloch, there is no evidence for positive MAMB during this period.−1 at Eisgruben, and a positive MAMB is attested at Guffert, with similar values. At both these sites, the character of the ice changes markedly, transitioning upwards into thicker layers containing far fewer woody macro-remains. At Eisgruben, the clean ice section indicates a positive MAMB until the early 1700s CE . The estimated ice loss at Kraterschacht led to a drop in levels of the firn body of almost 20 m in two decades35. Snow-level monitoring of Guffert ice cave also showed a drastic drop of the firn surface of almost 3 m between 2019 and 2021.Only few mass balance records are available for recent decades in these alpine cavesWe therefore report overall negative MAMB during at least the last three decades at most underground ice deposits in this study, especially at low-lying sites.28. Other studies have demonstrated that some alpine caves also host significantly older deposits, reaching back before the start of the Common Era27. Our study provides additional evidence for positive MAMB during the early Common Era, and new evidence for ice growth between 600 to 800 CE at three different field sites. In contrast to previous studies, we have pooled together observations and radiocarbon constraints from eight caves, to draw a more thorough picture of past mass balance dynamics. We are thus able to better detect and discuss the effects of the cave geometry on the volume and stratal organisation of the underground deposits. It is the first time such a comprehensive inter-cave comparison and data integration has been done for ice caves worldwide.Previously published radiocarbon chronologies have provided evidence of widespread positive mass balance in alpine caves of the Northern Hemisphere during the Little Ice Age9, and A29412, which are, like Eisgruben, descending tunnels.Reconstructing a robust chronology in exposed ice cliffs has proven difficult in many cases before, even when abundant radiocarbon dates are available. Firstly, the availability of a continuously exposed section of ice comprising both the oldest ice layers and the youngest seems to be exceptionally rare in sag-type caves. In our study, we identified two main types of geometries: inclined tunnels  and vertical pits . Only at Eisgruben can a regularly layered deposit be accessed from top to bottom without interruption. Hitherto published ice cave chronostratigraphies where the complete exposure is accessible include the sites of St. Livres13 initially ran into similar difficulties but nevertheless identified a continuous exposure with dated macro-remains in normal stratigraphic order. Making stratigraphic sense of separate outcrops was equally a challenge at the ice deposit of Strickler Cavern (northern US)11. At all these sites, the ice outcrops are discontinuous and the retreat of a crevasse between ice and bedrock allows seasonal snow to fall in, further obscuring the stratigraphy. The ice bodies of vertical pits have a complex geometry dictated by the shape of the host cave. Liquid precipitation, dripwater and rain falling directly onto the ice or firn deposit form holes several metres deep below the firn surface, melting previously deposited snow. Crevasses forming on the side of the ice body during the melting season are also the first to be refilled following new snowfalls. In descending tunnels where most of the ice body is sheltered from the open sky, deposits are less prone to disturbance. Such a geometry thus provides the best chance for preserving a minimally disturbed ice record.Predominantly vertical geometries are seldom associated with single continuous ice outcrops. At Bärenloch, we were forced to divide the analysis of the record in three different (but overlapping) ice exposures. At Hochschneid, the top-most ice stratigraphy is obscured by seasonal ice formation. In Kraterschacht and Großer Naturschacht, the outcrops are also located near the base of the caves, and the ice sections grade into massive snow with faint to non-existent layering, and little woody material. At Guffert, the two separate sections bracket two periods of positive MAMB with no temporal overlap. The initial study of HundsalmOwing to the lag between the time of wood growth, its transport into the cave, and finally, its inclusion into the ice stratigraphy, the radiocarbon date provides a maximal age for the ice layer. This lag may reach several decades for wood from old trees, but is likely shorter for small twigs or other similarly fragile organic macro-remains, as most samples in this study. Finally, the transport and storage of these organic inclusions within the cave before their embedding within the ice body depends on cave geometry. In the studied sag-type caves, this time is likely short as ice /snow occurs near vertically underneath the respective entrances. Nonetheless, too-old wood detected with the outlier analysis of the age-model is thus interpreted to have been stored in the cave before its incorporation within the ice deposit. Howevern material falling in the caves at times of positive mass balance is likely embedded quickly after being introduced into the cave, eventually appearing as sporadic macro-remains found in clean ice.deep and the middle section, as the age offset never exceeds 250 years  radiation in the cave environment, or the relatively small influence of summer air temperaturesEiszungendom section provides evidence for cave glaciation as early as ≈ 3700–3400 BCE. Owing to the lack of further age constraints, we conclude that the period spanning 3500–0 BCE did not lead to the complete deglaciation of Eisgruben. Notably, this period of very slightly positive MAMB coincides with a nearly continuous two-millennium-long period of glacier recession in the Swiss Alps41. The onset of cave glaciation was coeval with the beginning of a 5000 year-long trend of decreasing summer air temperatures in the Alps42.The oldest dated ice section from sag-type caves is located at Eisgruben ice cave, where the thinly layered 45. In the eastern Alps, the Pasterze and Gepatsch glaciers also exceeded their 1940’s extent between 300 and 400 CE 4)46. The sections in Bärenloch, Guffert and Eisgruben document an overall positive MAMB during the period from 300 to 400 CE , a period of improved climate in Europe with reduced glacier extents in the Alps in particular43. It also coincides with a gap in the mass balance records of Hundsalm and Guffert  were used to assess decadal ice variations at long-known sites.The caves were surveyed using a calibrated laser disto X2 with an accuracy better than 0.05 m for lengths less than 30 m, and less than 0.5° for azimuth and inclination. Wherever recent disto-based surveys (2016–2018) were already available, only a partial remapping was undertaken, to account for both 1) new discoveries and 2) snow/ice level fluctuations opening new areas of cave hitherto inaccessible. Otherwise, new maps were produced by the authors. Original exploration reports and photographs hosted on the Spelix database  at the CHRONO Centre, Queens University Belfast, following acid–alkali pre-treatment and graphitisation procedures56. Ages were calculated according to Stuiver and Polach57 and calibrated using IntCal2039, further age modelling was carried out using OxCal 4.458. Conventional radiocarbon ages are reported with a 1σ error  was computed from sample to sample with a tape measure. Bringing the 2D distributed samples to a 1D height model was achieved by projecting the distance between samples onto a line to the ice perpendicular to the layers. To account for the possibility of material being remobilised and thus out-of-sequence relative to the stratigraphy, outlier treatment is incorporated in the model, using the OxCal Outlier_Model, a Student-t test with 5 degrees of freedom, with prior outlier probability set at 0.05. A first section was thus modelled, and according to the posterior probability of each date being outlier, a new section, omitting detected outliers (whether too-old or too-young samples) was then modelled. The relevant age model definitions are given in the Extended Data B.Dated samples for each continuous section were modelled as OxCal P_Sequenceh) during periods spanning several decades to centuries. This value is quoted in other studies as the ice accumulation rate20. The mean annual mass balance over a unit area (MAMB), expressed in kg m−2, is closely related to the change in height h, expressed in cm yr−1. To convert to water-equivalent units59, we multiply h[L T−1] by the dimensionless ratio k = ρfirn/ρwater , with a ρfirn = 0.87 ± 0.18 g cm36.Age-depth models based on radiocarbon dating usually exhibit monotonic relationships. In the absence of layer counting, changes of annual mass balance cannot be resolved at any higher resolution than the multi-decadal scale. We used the first derivative of such an age-model function to estimate of the mean annual change in height of the ice body (Supplementary Information 1.Supplementary Information 2.


Like many social behaviors, aggression can be rewarding, leading to behavioral plasticity. One outcome of reward-induced aggression is the long-term increase in the speed in which future aggression-based encounters is initiated. This form of aggression impacts dendritic structure and excitatory synaptic neurotransmission in the nucleus accumbens, a brain region well known to regulate motivated behaviors. Yet, little is known about the intracellular signaling mechanisms that drive these structural/functional changes and long-term changes in aggressive behavior. This study set out to further elucidate the intracellular signaling mechanisms regulating the plasticity in neurophysiology and behavior that underlie the rewarding consequences of aggressive interactions. Female Syrian hamsters experienced zero, two or five aggressive interactions and the phosphorylation of proteins in reward-associated regions was analyzed. We report that aggressive interactions result in a transient increase in the phosphorylation of extracellular-signal related kinase 1/2 (ERK1/2) in the nucleus accumbens. We also report that aggressive interactions result in a transient decrease in the phosphorylation of mammalian target of rapamycin (mTOR) in the medial prefrontal cortex, a major input structure to the nucleus accumbens. Thus, this study identifies ERK1/2 and mTOR as potential signaling pathways for regulating the long-term rewarding consequences of aggressive interactions. Furthermore, the recruitment profile of the ERK1/2 and the mTOR pathways are distinct in different brain regions. Society has historically viewed aggressive behavior as maladaptive. However, aggression is a natural behavior commonly displayed throughout the animal kingdom and is essential for the allocation of resources, fitness and mental health ,2,3,4. FAs in other species and across both sexes, aggressive interactions are rewarding in female Syrian hamsters based on observations that females will display an increase in the amount of time spent in environments associated with aggressive experiences and will seek out social stimuli to engage in aggressive interactions ,8. TheseAggressive interactions in female hamsters result in neuroplastic changes within the nucleus accumbens (NAc). These include long-term increases in mushroom-like dendritic spines on medium spiny neurons (MSNs) and an increase in postsynaptic density protein 95 (PSD-95), metabotropic glutamate receptor 5 (mGluR5), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) protein . The actThese dramatic changes in synaptic structure and excitatory neurotransmission are most likely due to short-term changes in cell transcription and translation ,13,14. IF = 5.876, p = 0.020, df = 1, 40) . See For female Syrian hamsters that had 2 consecutive days of aggressive behavior testing, there was no change in the latency to initiate an attack in the first compared to the second aggressive behavior test session B. Howeve= 1, 40) C. SpecifTo determine if aggressive experience results in changes in the phosphorylation and expression of regulators of FMRP in the nucleus accumbens, we examined the phosphorylation of S6K (70 + 85 kDa), which in turn phosphorylates FMRP, and the expression of PP2Ac, which dephosphorylates FMRP. Ten minutes following either the second or the fifth consecutive day of aggressive experience there was no change in the phosphorylation of S6K in the nucleus accumbens compared to control females that had no social interaction experience A. There H = 8.607, p = 0.014, df = 2) , and females that had 5 consecutive days of aggressive experience . However, there was no change in the overall protein expression of ERK1/2 in the nucleus accumbens 10 min following either 2 or 5 consecutive days of aggressive experience compared to controls  (42 + 44 kDa) and mammalian target of rapamycin (mTOR). Ten minutes following 2 consecutive days of aggressive experience there is an increase in the phosphorylation of ERK1/2 in the nucleus accumbens ( df = 2) B comparecontrols C. Ten micontrols .H = 6.377, p = 0.041, df = 2) compared to control females   D.d = 1.178, p = 0.026, df = 17)  A. There df = 17) B. Finalldf = 17) . Western blot analysis revealed that 10 min following either 2 or 5 consecutive days of aggressive experience there was no change in the phosphorylation of FMRP in the nucleus accumbens or prefrontal cortex compared to control females that had no social interaction experience . For effThe goal of this study was to advance our understanding of the intracellular signaling mechanisms that underlie the neurological and behavioral adaptations resulting from the rewarding effects of aggressive interactions. There was a progressive decrease in the latency to display aggression in female Syrian hamsters that experienced 5 aggressive interactions. There is an increase in the phosphorylation of ERK1/2 in the nucleus accumbens 10 min following the second aggressive interaction. There is also a decrease in the phosphorylation of mTOR in the medial prefrontal cortex 10 min following the second aggressive interaction.Along a parallel pathway, the closure of this translational event is driven by a delayed and strong increase in the phosphorylation of the kinase S6K, which drives the re-phosphorylation  of FMRP . FurtherWe present evidence that there is an increase in the phosphorylation of ERK1/2 in the nucleus accumbens 10 min following aggressive interactions. Interestingly, this effect was observed following the second aggressive interaction, but returned to baseline for hamsters that experienced five aggressive interactions. There was no change in the phosphorylation of mTOR, S6K, FMRP, or the enzymatic activity of PP2Ac. Although it did not reach significance, a trend for a decrease in the phosphorylation of S6K in the nucleus accumbens was noted. There was a significant increase in the ratio of the phosphorylation of ERK1/2 to the phosphorylation of S6K for hamsters that experienced two and five aggressive interactions compared to no aggressive interaction controls. ERK1/2 is a promising candidate for neural plasticity underlying aggression escalation due to its very well studied role in numerous fundamental cell functions ,29,30. FWe also observed a decrease in the phosphorylation of mTOR in the medial prefrontal cortex 10 min following the second aggressive interaction. Similar to the increase in the phosphorylation of ERK1/2 in the nucleus accumbens, this effect was not observed for hamsters that experienced 5 aggressive interactions. There was no change in the phosphorylation of ERK1/2, S6K, FMRP, or the enzymatic activity of PP2Ac in the medial prefrontal cortex. Although it did not reach significance, a trend for an increase in the phosphorylation of ERK1/2 in the medial prefrontal cortex was noted. The phosphorylation of mTOR positively regulates the phosphorylation of the FMRP, the FMRP kinase S6K, and negatively regulates the activity of the FMRP phosphatase PP2Ac ,34,35. TFMR1), there is elevated phosphorylation of ERK1/2, but not change in the phosphorylation of mTOR is observed in the neocortex. While elevated phosphorylation of mTOR, but no change in the phosphorylation of ERK1/2 is observed in the hippocampus in FMR1 KO mice [FMR1 KO mice there are increases in protein synthesis in the hippocampus, and this increased protein synthesis is blocked with mGluR5 or ERK1/2 inhibitors. An mTOR inhibitor had no effect on protein synthesis. ERK1/2 inhibition also blocked the increase in the seizure susceptibility associated with FMR1 KOs [FMR1 KO is associated with an increase in protein synthesis and the phosphorylation of ERK1/2 [Previous studies have also reported divergent effects of the ERK1/2 and mTOR pathways on protein synthesis. These effects are also brain region specific. In mice with the genetic knockout of the gene that codes for FMRP  and intracellular signaling events  were purchased from Charles River Laboratories  at approximately 60 days of age (120–130 g). Subject females were housed individually while intruder males were pair-housed in polycarbonate cages . These semi-natural housing arrangements maximize aggression in female hamsters, while decreasing aggression in male intruders [Adult female (n = 30) and male Syrian hamsters (n = 16)  or 5-day (n = 11) period. Hamsters were weighed before behavior testing and the average body weight was balanced between treatment conditions (mean ~145 g per hamster per group). A two-step process was used to verify scoring (live scoring followed by video scoring). Each session was scored for two main criteria: (1) the latency to the subject’s first attack and (2) the total number of attacks. Attacks were operationally defined as the subject biting, or clearly attempting to bite, the intruder male. Intruder males were used at most every other day and for each test individual females were paired with a different male to minimize the likelihood that submission by the male would influence the behavior of the females [Ten minutes following the second or fifth aggression test, females were rapidly decapitated without anesthetic. Control hamsters that did not experience social interaction also had tissue collected at this time. Coronal sections (approximately 1–2 mm) containing the nucleus accumbens (NAc), caudate putamen (CPu) and medial prefrontal cortex (mPFC) were taken with the aid of a brain matrix, and bilateral tissue punches (1 mm diameter) were immediately collected from each area , referenoC for 20 min . Finally, the supernatant was aspirated (~80–100 μL) and placed in a fresh tube. Approximately 6 μL per sample was used for protein analysis and the rest was stored in a −80 °C freezer until used for Western blots. Protein was quantified using Bio-Rad protein DC assay . Samples were run in triplicate at 20, 10 and 6.7 dilution factors (1.0–3.0 μL of sample and 19.0–17.0 μL of 1% SDS). Standards were run in duplicate in eight serial dilutions of 1.5–0.05 mg/mL (Bio-Rad Laboratories). To prepare tissue for Western blot analysis, flash frozen tissue was first transferred into 1.5 mL protein lysate tubes with metallic beads. 100 μL of ice-cold RIPA buffer (250 mM NaCl and 50 mM Tris pH 8) and 1 μL of HALT phosphatase and 1 μL of HALT protease inhibitors  were added to each lysate tube. Tissue samples were then homogenized in a bullet blender  for 6 min. Twenty-one μL of detergent master mix  and 5 mL of 10% sodium deoxycholate) was then added to tubes with homogenized tissue. Samples were then rotated for 45 min with a sample mixer at 4 °C. Samples were centrifuged at 4For each subject 30 μg of total protein were added to a micro-centrifuge tube with 7.5 μL of 4× Laemmeli buffer  and RIPA buffer for a total volume of 30 μL and heated for 10 min on a 90 °C hot plate. Samples were then loaded into a 12–15% polyacrylamide gradient gel in 1% SDS running buffer . Proteins from the gel were then transferred to a nitrocellulose membrane . After transfer, membranes were then blocked in a 50:50 dilution of Immobilon Block—PO Phosphoprotein Blocker  and TBST for 1 h. Membranes were then incubated overnight in primary antibodies diluted in TBST as indicated in Different lanes were treated with either 30 or 15 μg of total protein to confirm that the antibodies were within the dynamic range of intensity expression . To confData were analyzed using GraphPad Prism 8.3.0 for Windows. Statistical outliers were removed using the ROUT method . All data were examined to determine if the assumptions of parametric statistical tests were met: normality (Bartlett’s test) and equal variance (Brown-Forsythe test). If assumptions were not met, a Kruskal–Wallis (non-parametric) test was performed . All data are presented as mean ± standard deviation (SD).t-tests or repeated-measures (RM) ANOVAs for linear trend were used to detect significant differences in aggressive behaviors following repeat aggressive experience across testing days (days 1 through 2 or 1 through 5 for the two corresponding groups), p < 0.05 [Behavioral analyses: Paired p < 0.05 . p < 0.05.Western blot analyses: For protein analyses, significant differences between groups were detected using one-way ANOVAs with LSD post hoc tests, We present data demonstrating that 10 min following two consecutive days of aggressive experience there is an increase in the phosphorylation of ERK1/2 in the nucleus accumbens, and a decrease in the phosphorylation of mTOR in the medial prefrontal cortex in female Syrian hamsters. Interestingly, these phosphorylation events are not observed in hamsters following five consecutive days of aggressive experience. A decrease in the latency to attack is observed after five days of aggressive experience, but not in the hamsters that received only two days of aggressive experience. We hypothesize that activation of mGluR5 by glutamate binding results in the rapid phosphorylation of ERK1/2, which increases the enzymatic activity of PP2Ac and inhibits the phosphorylation of S6K . Thus, iThus, the transient increase in the phosphorylation of ERK1/2 in the nucleus accumbens and the decrease in the phosphorylation of mTOR in the medial prefrontal cortex may underlie the translational changes in these reward brain regions that drive the long-term morphological (mushroom-like spines) and thus electrical  adaptations underlying aggressive experience ,11. Fina


Recently, the strategy of multivalency has been widely employed to design glycosidase inhibitors, as glycomimetic clusters often induce marked enzyme inhibition relative to monovalent analogs. Polyhydroxylated pyrrolidines, one of the most studied classes of iminosugars, are an attractive moiety due to their potent and specific inhibition of glycosidases and glycosyltransferases, which are associated with many crucial biological processes. The development of multivalent pyrrolidine derivatives as glycosidase inhibitors has resulted in several promising compounds that stand out. Herein, we comprehensively summarized the different synthetic approaches to the preparation of multivalent pyrrolidine clusters, from total synthesis of divalent iminosugars to complex architectures bearing twelve pyrrolidine motifs. Enzyme inhibitory properties and multivalent effects of these synthesized iminosugars were further discussed, especially for some less studied therapeutically relevant enzymes. We envision that this comprehensive review will help extend the applications of multivalent pyrrolidine iminosugars in future studies. Iminosugars, containing an endocycling nitrogen atom that effectively mimics carbohydrates by facilitating the reversible and competitive inhibition of their processing enzymes, have generated much attention in recent years as targets in the treatment of a wide range of illnesses  ,3,4,5,6.N-alkylated DNJ derivatives are approved drugs, N-hydroxyethyl-1-deoxynojirimycin , which is involved in a rare metabolic disorder amino-methane 97 and tetravalent scaffold 95 (1.0 equiv.) in the presence of CuSO4 (0.3 equiv.) and sodium ascorbate (0.6 equiv.) in THF/H2O a, followed by purification through flash column chromatography and size-exclusion chromatography Sephadex LH-20 (H2O) to separate the excess azide 87, gave the pyrrolidine iminosugar clusters 98 (48%) and 96 (76%) in good yields. In addition, inhibitory performance against a panel of glycosidases was evaluated among the nona-, tetra-, and trivalent iminosugars 83, 96, and 98, as well as monovalent references DAB and its derivative 90.Similar to the method for synthesizing compound 6 and 98 . Microwa99 and 101) and two different tri- or hexavalent alkynyl spacers (97 and 102) was exploited to give the target clusters (103–106). It was found that spacers containing aromatic moieties in multivalent inhibitors showed excellent inhibition against octameric BglA (µM range) compared to the similar monomeric BglB. Moreover, a modest multivalent effect was detected for the hexavalent inhibitor 106.Two years later, Moreno Vargas and co-workers prepared four multivalent pyrrolidine iminosugars for GH1 β-glucosidases A and B (BglA and BglB) to continue their study on binding modes and key determinants responsible for the inhibitory effect displayed by pyrrolidine-based clusters . Therefo107 reported by the same group, the protected azide 99 was obtained in good yield by reacting with the aromatic alkyne 108 . Re. Re, and 5 and 6 , the effy Robina , Behr [1y Robina , and Wony Robina . To furtN-glycans in cells. Such processes are, for instance, involved in the treatment of cancers and lysosomal diseases [98, 96, and 83, 83  analysis, nuclear magnetic resonance (NMR), and molecular dynamic studies were carried out to elucidate the binding mode of the multivalent iminosugars and α-mannosidases. NMR studies showed the existence of specific interactions of the multivalent ligands with JBMan, which presumably take place within the enzyme active site. TEM studies indicated that the binding mode would probably be intermolecular cross-linking, due to the formation of ligand–JBMan aggregates. It is worth noting that a remarkable selectivity of iminosugars  for Golgi α-mannosidase IIb (GMIIb) over lysosomal α-mannosidase II (LManII), two biologically relevant enzymes , was observed. The interesting selectivity appeared particularly relevant for selective application of multivalent compounds in anticancer therapy without the undesirable side effect of mannosidosis syndrome. Subsequently, scaffold resorcinarene was employed to explore the role of both the conformability and the valency of multivalent iminosugars to therapeutically relevant target GMIIb [147, 148, 149, 150, 149 had stronger inhibition, for example, towards GMIIb  compared to its monovalent reference 90 . Computational studies suggest that the binding mode should be the rebinding process, since the resorcinarene ligands bind the dimer of the JBMan by coordination of one Zn ion at a time. From these results, we can know that the multivalent effect of pyrrolidine iminosugars on α-mannosidases has a great relationship with valency. Generally, higher valency iminosugars usually exhibit better inhibitory activities. The multivalent effect is also affected by the type of linker and the conformation of the scaffold. In addition, the proposed binding modes—cross-linking and aggregation, and bind and recapture  and iduronate-2-sulfatase (IDS) could cause diseases of mucopolysaccharidoses: Morquio A syndrome and Hunter disease, respectively [83 , confirming that multimerization of DAB epitopes generates potent GALNS inhibitors. Comparing the inhibitory activities of 79 and 82 with 83 and 84, we can learn that the ligand topology strongly affected the affinity of the DAB-based multivalent iminosugars for GALNS. Divalent iminosugar 64D (64D exhibited remarkable inhibition of human β-N-acetylglucosaminidase (hOGA) in the nanomolar range -nonavalent iminosugar 133  for the first time . A decreectively ,131,132.19.7, 79  and 82  would improve glycosidase inhibition efficiency and selectivity, two major problems currently existing. Despite the many challenges, we hope that, with the information presented in this review, researchers in this field will continue to explore multivalent effects based on pyrrolidine for developing new glycosidase inhibitors, as well as for candidates for advanced clinical trials or markets.


Although the global environmental impact of Laurentide Ice-Sheet destabilizations on glacial climate during Heinrich Events is well-documented, the mechanism driving these ice-sheet instabilities remains elusive. Here we report foraminifera-based subsurface (~150 m water depth) ocean temperature and salinity reconstructions from a sediment core collected in the western subpolar North Atlantic, showing a consistent pattern of rapid subsurface ocean warming preceding the transition into each Heinrich Event identified in the same core of the last 27,000 years. These results provide the first solid evidence for the massive accumulation of ocean heat near the critical depth to trigger melting of marine-terminating portions of the Laurentide Ice Sheet around Labrador Sea followed by Heinrich Events. The repeated build-up of a subsurface heat reservoir in the subpolar Atlantic closely corresponds to times of weakened Atlantic Meridional Overturning Circulation, indicating a precursor role of ocean circulation changes for initiating abrupt ice-sheet instabilities during Heinrich Events. We infer that a weaker ocean circulation in future may result in accelerated interior-ocean warming of the subpolar Atlantic, which could be critical for the stability of modern, marine-terminating Arctic glaciers and the freshwater budget of the North Atlantic. The mechanism driving past Laurentide Ice-Sheet instabilities remains elusive Here, the authors present a sediment record from the subpolar western North Atlantic and show that massive warming of the upper interior ocean was the likely trigger for repeated collapses of the Laurentide Ice-Sheet and iceberg discharge into the North Atlantic, known as Heinrich Events. Although the impact of Heinrich Events on global climate has been studied thoroughly during the last decades6, the driving mechanism behind the episodic iceberg-discharge during Heinrich Events is a matter of ongoing debate10. A commonly discussed hypothesis is that Heinrich Events were initiated by the periodic unstable flow of the LIS, controlled by internal ice-sheet oscillations under otherwise stable environmental conditions, known as “binge-purge” hypothesis7. It is assumed that the massive freshwater release during Heinrich Events initiated strong disruptions of the Atlantic Meridional Overturning Circulation (AMOC)3 and surface-ocean cooling of the North Atlantic4. However, surface ocean temperature and ice-rafted debris (IRD) proxy data from the high latitudes of the North Atlantic indicate that surface-ocean cooling occurred hundreds to thousands of years earlier than ice-rafting events in the North Atlantic10. Moreover, proxy data of AMOC strength show that deep ocean-circulation weakened prior to Heinrich Events in the North Atlantic6. Other studies propose a strong impact of weaker overturning circulation on LIS dynamics and the onset of Heinrich Events13. Based on numerical modelling simulations, a reduced AMOC would lead to strong subsurface warming and rapid retreat of ice-shelves around the Labrador Sea that is followed by the destabilization of the LIS during Heinrich Events13. Proxy data of bottom-water temperatures show that the mid-depth North Atlantic indeed warmed prior to Heinrich Events and thus seem to support this mechanism14. Nevertheless, proxy records reflecting subsurface ocean temperature variability near the Labrador Sea, close to the grounding line of marine-based portions of the LIS, are not available yet, hampering the evaluation of subsurface ocean warming as the trigger of Heinrich Events.The deposition of ice-rafted debris layers in the glacial North Atlantic (known as Heinrich Events) provides evidence for a substantial freshwater release via melting icebergs in response to past instabilities of the Laurentide Ice Sheet (LIS)Neogloboquadrina pachyderma sinistral (N. pachyderma sin.) as a proxy for subsurface temperatures (subSSTMg/Ca at ~150 m water depth) from site GeoB18530-1  of N. pachyderma sin. allow calculating the regional ice-volume-corrected oxygen isotopic composition of seawater (δ18Oivc-sw) as a proxy for salinity (see “Methods”). The chronostratigraphy of sediment core GeoB18530-1 is based on 20 accelerator mass spectrometer 14C ages spanning the last 35,000 years  its location close to the exit of the Labrador Sea within the IRD-belt16, the North Atlantic gateway of the main iceberg trajectory associated with Heinrich Events  thnts Fig. ; and . The IRD layers are characterized by elevated Ca/Sr ratios as expected from the high content of detrital carbonate are originating from Paleozoic limestone and dolostone from Hudson Bay and Hudson Strait2 . The opposing temperature trends are particularly evident prior to Heinrich Events as well as the Younger Dryas and we conclude that subsurface water masses must be very well isolated from the surface ocean of the subpolar Atlantic during these intervals  temperature variability and AMOC strength yields three key observations Fig. . First, ars Fig. . Moreoveals Fig. .Fig. 4Co24  at GEOMAR, Helmholtz-Centre for Ocean Research in Kiel. The levels of detection typically vary for each cation, ranging from 0.001 to 0.1 µg/ml. Mg/Ca was normalized to the ECRM 752–1 standard  and drift-corrected. Regular analyses of the ECRM 752–1 standard yield an analytical error of ±0.01 mmol/mol for Mg/Ca.For Mg/Ca analyses, ~100 individuals of the foraminiferal species 38. Six out of 110 samples showed elevated Al/Ca values (>0.2 mmol/mol), however, are inconspicuous in the Mg/Ca value compared to neighbouring Mg/Ca values with no sign of potential contamination  of N. pachyderma sin. were measured with Thermo Scientific MAT 253 mass spectrometers equipped with an automated Kiel IV Carbonate Preparation Device at MARUM, University of Bremen and at GEOMAR, Helmholtz-Centre for Ocean Research in Kiel. The δ18O isotope values were calibrated versus the NBS19  carbonate standard and an in-house standard (“Standard Bremen”). The long-term analytical precision is 0.06‰ for δ18O. Results were calibrated to the VPDB scale, given in per mille (‰) relative to the VPDB.Stable isotopes , 0.18 °C after calibration to Eq. 42) and 4.32 °C after calibration to Eq. 43). Consequently, we considered the calibration after ref. 42 as appropriate to convert the Mg/Ca ratio to subsurface temperatures.Comparison of converted core-top Mg/Ca temperature to instrumental temperaturesN. pachyderma sin.42. Consequently, we checked whether changes in the assumed habitat depth may afflict the robustness of our results. To do so, we calculated the temperature gradient between 50 and 200 m water depth (∆T200-50) using modern instrumental data20 from a water column profile near-site GeoB18530-1  and full Heinrich conditions in the western subpolar North Atlantic44. However, we note that reconstructed subsurface ocean warmings preceding Heinrich Events in our time series and thus, do not reflect full Heinrich conditions. Nevertheless, we investigated the effect of a possible change in seasonality assuming a maximum shift in seasonality from April to September, considering modern hydrographic data20. The difference in subSST150m between April (7.2 °C) and September (7.45 °C) is 0.25 °C at our core site  considering changes in global δ18Osw due to continental ice-volume variability using the relative sea-level curve of ref. 45. To remove the temperature effect from the δ18Oivc-sw record we applied the temperature versus δ18Ocalcite equation of ref. 46:We calculated the regional ice-volume-corrected δ18Oivc-sw were converted from V-PDB into Vienna Standard Mean Ocean Water (V-SMOW) scale according to equation of ref. 47:The calculated δ18Oivc-sw pointing to fresh  ocean conditions.Low (high) values of δ18Oivc-sw calculations considering the uncertainty in performed Mg/Ca and δ18O measurements, the uncertainty of applied Mg/Ca calibration as well as the error involved in the relationship between salinity and δ18Osw. We obtained an error of ~0.38 for reconstructions of δ18Oivc-sw. A previous study reported similar error propagations for δ18Osw of 0.36 (+/−0.02%) derived from δ18OCalcite and Mg/Ca-temperature reconstructions of N. pachyderma sin. in the subpolar Pacific48.We performed an error propagation analysis to assess the error of the δ2 area from the split core surface of the archive halves. The split-core surface was scanned three times with different settings for light , medium  and heavy  elements. To avoid contamination of the XRF measurement unit the core surface was covered with a 4 µm thin SPEXCerti Prep Ultralene foil. The core scanner unit includes a Canberra X-PIPS Silicon Drift Detector  with 150 eV X-ray resolution, a Canberra Digital Spectrum Analyser DAS 1000 and an Oxford Instruments 100 W Neptune X-ray tube with rhodium (Rh) target material. Raw data spectra were processed by Analysis of X-ray spectra by the Iterative Least square software (WIN AXIL) package from Canberra Eurisys.X-ray fluorescence core-scanning measurements of GeoB18530-1 were performed with an Avaatech XRF Core Scanner at the MARUM, University of Bremen. Elemental intensities were obtained at 1 cm resolution over a 1.2 cmN. pachyderma sin. (19 samples) and one mixed sample (Globigerina bulloides and N. pachyderma sin.) from core GeoB18530-1 (Supplementary Table 49. Radiocarbon ages were calibrated using the IntCal20 calibration curve50 and modelled local reservoir ages51 provided through the toolbox PaleoDataView52. To assess the uncertainty of Mg/Ca temperatures, we combined 2000 noisy proxy realisations with the 2000 BACON age models, interpolated the resulting 2000 time series to the median ages of all sampling depths and calculated the 95% confidence envelope for the Mg/Ca SSTs. We further re-calibrated the reported radiocarbon ages from site MD01-246124 following the same procedure as described for core GeoB18530-1.The age model for gravity core GeoB18530-1 is based on 20 accelerator mass spectrometry radiocarbon dates from samples of Supplementary InformationPeer Review File