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  1. Author Response

    Reviewer #1 (Public Review):

    The manuscript by Pardo et al. describes the identification and characterization of a novel subpopulation of delta cells that normally resides in the zebrafish pancreatic islet. Using two models of beta cell ablation, the authors demonstrate that this delta cell subpopulation efficiently converts into an insulin/somatostatin co-expressing cell population to restore euglycemia. The study includes robust transcriptome data to determine that this delta cell subpopulation is characterized by the expression of sst1.1 (rather than sst2) and expresses many beta cell genes. Furthermore, the resulting insulin/sst1.1 co-expressing cells represent a long-lived population that are sufficiently functional to restore euglycemia. The study goes on to suggest that inhibition of the p53 pathway compromises formation of the bihormonal population; however this data is not as convincing. Overall, this is a novel study that suggests the existence of heterogeneous delta cell populations in the zebrafish islets and supports previous findings related to adult islet cell plasticity.

    Strengths:

    1. Although several studies have identified heterogeneous populations of islet alpha and beta cells, this is one of the first studies to demonstrate two apparently distinct delta cell populations; the study provides sufficient characterization that it should be easy to test whether a similar population exists in mammals.
    2. Demonstration that the induction of Ins/SST biohormonal cells is triggered in two independent models of beta cell ablation
    3. The use of several different transgenic fish lines to characterize the relative numbers of different islet cell populations in control and ablation conditions.
    4. High quality data, including immunofluorescence images, RNA-seq data and validation studies with appropriate controls.
    5. The extensive use of comparative transcriptome data to validate islet lineage relationships.

    Weaknesses:

    1. Although the data suggests that the newly formed bihormonal cells have sufficient function to rescue the hyperglycemic phenotype, there are no experiments to directly test the functionality of these cells.

    To address the question of the functionality of bihormonal cells, we opted for a complementary approach, ie a glucose tolerance test in adults. We showed that bihormonal cells represent the vast majority (95-99%) of all Insulin-expressing cells throughout the pancreas (see Figure 1 and new Figure 4), thereby minimizing the possibility that a putative population of pure beta cells (SST negative) would significantly contribute to regulate glycemia. The glucose tolerance test reveals that the regulation of blood glucose in regenerated fish after glucose injection is identical to CTL fish (new Figure 4). These observations strongly support that bihormonal cells are the main sources of Insulin in regenerated fish and that they are responsible for blood glucose homeostasis in the near absence of beta cells.

    1. Many of the genes cited as "beta cell specific" are also expressed in delta cells in mouse and human islets - although this could relate to species differences, it causes some confusion and could affect the ultimate interpretation.

    We include now a table Figure3-figure supplement 2.

    1. Although it is clear from the images that are presented in the manuscript that a large number of bihormonal cells arise upon beta cell ablation, the relative numbers of bihormonal cells to monohormonal Sst and insulin cells is not clearly indicated. In some cases, it appears that a large percentage convert, while in others there are only a fraction. One can extrapolate this information from the presented data (ie figure D and E), but it would have been more informative if the direct analysis was provided.

    To be more explicit, we compare now the relative number of bihormonal cells compared to sst1.1 delta-cells in the revised paper (Discussion lines 433-435). Also, for a better representation of the size of each cell population, we present now the absolute number of cells instead of % of islet cells shown in the first version in Figure 1 and new Figure 6. These quantifications reveal that the number of BH cells that are formed after ablation exceeds the number of monohormonal Sst1.1 cells. This indicates a more complex mechanism than simply direct conversion of sst1.1 cells to bihormonal cells, including neogenesis from ducts and proliferation, that we directly address now in the revised manuscript (Figure 4 and Figure 6). See also explanation in our response to Reviewer 3.

    1. The authors only refer to the fact that Pdx1 is known to be expressed in beta and delta cells in a small paragraph in the discussion; it would have been helpful if this information were introduced in the introduction and in the relevant experimental sections.

    We think that presenting Pdx1 in the Introduction section would anticipate too much on the results, so we chose to refer to Pdx1 in the Results and Discussion sections.

    1. The authors make the strong conclusion that sst1 cells directly convert into bihormonal cells based on time lapse imaging. Genetic lineage tracing would be needed to absolutely make this conclusion. The time lapse imaging can only suggest that direct conversion might be occurring.

    See our response to point 1 of Essential revisions and explanation and experimental exploration of alternative mechanisms.

    1. The inhibition of p53 appears to only cause a relatively small decrease in the number of bihormonal cells (from ~20 to ~15), somewhat undermining the conclusion that p53 promotes the formation of this cell population.

    To augment the data on p53, we present now validations of the activation in the islet of the p53 pathway by in situ hybridization with ccng1 and mdm2 (shown now in Figure 6G), two established p53 target genes that were identified in our transcriptomes. We also explore the cell cycle signatures.

    We decided to remove the experiment with pifithrin alpha. Indeed, using different timely treatments with the p53 inhibitor pifithrin alpha, we obtained two opposite responses: one that confirms the results shown in the first version of the paper (a decrease of bihormonal cells that is moreover paralleled by an increase of sst1.1:GFP cells), the other showing an increase. We think that p53 acts at different levels, possibly in monohormonal sst1.1 delta cells and in bihormonal cells and the understanding of these observations would be the focus of another project.

    Reviewer #2 (Public Review):

    This is an interesting and potentially exciting manuscript that reports, based on a series of zebrafish reporter lines, that there exists a subset of delta cells that can rapidly assume partial beta cell-like identity following beta cell ablation. This conversion correlates with the restoration of (near) normal glucose levels within 3 weeks. The major strengths are a series of technically well executed experiments that report an interesting observation of two discernable populations of delta cells. These populations are supported by transcriptome data, which validate the differences between these populations established using FISH or immunofluorescence. Major weaknesses are the lack of lineage tracing of delta cells and questions on the mechanisms underlying the origins of the bihormonal cells reported in this paper. The observation of the rapid appearance of bihormonal cells is potentially exciting and important. However, directionality of the conversion is insufficiently established. The conversation of delta to beta cells needs to be supported by direct lineage tracing. The alternative explanation that these cells are surviving beta cells that turn on somatostatin expression cannot be ruled out on the basis of the current experiments. The authors tend to extrapolate too much from their transcriptome data and subsequent pathway analyses to make claims that would be better supported by additional experiments, or toned down. The authors are right to point out the major differences in zebrafish beta cell regenerative potential and plasticity compared to mammalian models, but this diminishes the credibility of the claims of translational potential. There is value in conducting careful experiments into islet cell plasticity in a zebrafish without having to make a promise of direct translational relevance.

    All these points have been addressed.

    This paper suggests the presence of two sets of delta cells, marked by Sst1.1 and Sst1.2. The Sst1.1 cells are marked by GFP in a Sst1.1:GFP transgenic reporter. This reporter clearly is not selective for Sst1.1 cells only, as a majority of delta cells expresses GFP at dimmer levels and is Sst2 positive. This is in good agreement with the lower - but not absent - Sst1.2 and Sst2 mRNA profiles in Figure 4, but complicates the claim that it is specifically Sst1.1 delta cells that convert into bihormonal cells. An overlay between Sst1:1 and Sst1:2 or Sst2 mRNA to demonstrate that it is specifically the Sst1:1 expressing delta cells that become INS positive (Figure 1B) would help. Formal lineage tracing of the Sst1:1 delta cells is the accepted way to solidify support for this claim, but such data are absent from this paper.

    Unfortunately, we did not succeed in performing genetic lineage tracing of the sst1.1 delta-cells.

    However, we now explored alternative cellular origins of bihormonal cells such as the ducts and proliferation (new Figure 4 and Figure 6). We toned down our previous conclusion that ruled out beta cells as an origin of bihormonal cells (Figure 2).

    To follow the suggestion of Reviewer 3, we provide now the comparative expression by double fluorescent ISH of sst1.1 and sst2 mRNA with the insulin mRNA (performed in larvae, see new Figure 2C). The overlays show that insulin is coexpressed with sst1.1 specifically, but not with sst2. This demonstrates that bihormonal cells express selectively the sst1.1 somatostatin gene and provides support, though still does not demonstrate, to the hypothesis that it is specifically the Sst1:1 expressing delta cells that become INS positive.

    The model is presented as a 'beta cell ablation' model, but there are some concerns with the flow of islet cells between islet cell populations immediately following ablation and during recovery that require clarification. The beta cell population size measures between 25-35% of islet cells (Figure 1D/Figure 1Suppl2). If these cells are all ablated acutely, this should immediately lead to significant increase in the remaining non-beta cell populations, including Sst1:1 delta cells. However, this is not observed as Sst1:1 GFP+ cells are steady as a fraction of total islet cell number (Figure 1F). Instead, the population that is increased at 3 days following ablation is the mCherry-GFP double positive cell population, which accounts for approximately half of the loss of beta cells. The scenario that a portion of beta cells is not actually ablated but is instead converted into a bi-hormonal state is insufficiently explored as detailed below. If the rapid appearance of these cells were indeed attributable to the conversion of GFP cells into co-positive cells, this should have been reflected in the data of Figure 1F. However, the GFP population appears to be neither increasing to reflect the loss of beta cells, or decreasing in response to the co-expression of mCherry. In Figure 5, a drop in GFPhigh cells specifically is shown, but this reflects only a potential 5% shift of islet cell numbers from GFPhigh to potentially bihormonal cells. The live imaging data in Figure 5B are not helping as there is simply not enough spatial and axial resolution to place the mCherry signal in GFP+ cells. If both processes are balancing each other out to maintain steady numbers of GFP+ delta cells, this implies rapid proliferation of GFP positive delta cells to replenish the delta cells that become bihormonal, or the rapid proliferation of bihormonal cells shortly after they arise. Either of these scenarios should be readily demonstratable.

    This ablation model has been shown to lead to a massive destruction of beta cells through apoptosis (Curado et al, 2007) (Bergemann et al, 2018). In line with the loss of beta cells, the total number of cells (new Figure 1G), shows a downward shift after ablation. We also quantified islet cells in situ on paraffin section in Figure 6-figure supplement 1. Due to the difficulty to detect INS+ or mCherry after ablation (very low expression in bihormonal cells), we used Pdx1 as a proxy for beta and sst1.1 delta and bihormonal cells. The decrease of Pdx1+ nuclei we observed is consistent with the extent of the loss of β-cells.

    Together with the fact that we do not detect a lot of spared beta cells after ablation by lineage tracing, all these observations support that we have an efficient model of ablation. Despite this efficient ablation, we nevertheless observed some bihormonal cells derived from pre-existing beta cells (Figure 2G and close-up in E’) and now openly discuss this possible cellular source.

    We realized that our initial representation in terms of percentages “% of cells / islet” was misleading. For a more accurate representation of population size, we now present in this revised manuscript the absolute number of cells (instead of %) detected for each population (per fish), as this reflects the real size of the populations present in the dissected tissue, which contain all cells of the main islet, and make easier the comparisons between conditions and cell types.

    As pointed out by the Reviewer, the respective size of GFP monohormonal, bihormonal and beta cell populations indicate that the flow between islet cells (and potentially with non-islet cell types) is too complex to infer directionality of conversion. While ~3300 beta cells are lost and ~1500 bihormonal cells are gained, there are only ~900 monohormonal sst1.1 delta cells before ablation (GFPhigh), which is inferior to the number of BH cells formed after ablation. This suggest multiple origins of bihormonal cells, and/or proliferation. In the revised manuscript, we consider the following scenarios: i) the contribution of non-ablated beta cells to bihormonal cells (Figure 2), ii) neogenesis from ducts (Figure 4) and iii) expansion of GFP and/or bihormonal cells by proliferation (Figure 6). We discuss these results lines 433-447). These mechanisms are not mutually exclusive and are compatible with a “direct” conversion sst1.1 delta cells.

    The presumption is that new beta cells are formed, and this is based in part on lineage tracing data using the zsYellow label in conjunction with an inducible beta cell specific Cre driver strain. It is not clear why this experiment was done in developing embryos instead of during the adult stage where the original observation of the appearance of bihormonal cells that is associated with normalization of glucose levels was made. It appears that in that crucial lineage tracing experiments, the authors are ambiguous about the use of mCherry to detect beta cells after ablation. They describe beta cells as mCherry+ beta cells in the text, while they indicate in the legend and figure labels to have used INS antibody staining to detect these cells. The punctate staining that is different from the mCherry staining elsewhere in the manuscript certainly is compatible with the use of an INS antibody, but raises the question why mCherry was not used to detect beta cells which is what was used throughout the rest of the paper. This is relevant as the lack of zsYellow positivity is interpreted as a sign of beta cell neogenesis. However, these cells might have lost zsYellow precisely because they were killed and have lost their fluorescence lineage markers, including mCherry, but are still detectable by INS immunofluorescence as they have not been cleared from the islet tissue.

    The genetic tracing of beta cells was performed in larvae. The experimental details are now shown in Figure 2D. CRE recombination by 4-OHT was induced at 6 dpf before ablation at 7 dpf and the larvae were analysed at 14 dpf. We opted for larval stages since bihormonal cells appear at any stage and young small animals are more amenable to fast and efficient inducible CRE recombination (Hans et al, Plos One, 2009; Mosimann et al, Development, 2011).

    We thank the reviewer for highlighting the discrepancy about the INS/mCherry antibodies. It is indeed an anti-Insulin detection with typical punctate staining that is shown Figure 2E and quantified in Figure 2F-H, and not anti-Cherry, because of species incompatibility between antibodies in the immunodetection assay (both Cherry and zsYellow antibodies are from rabbit while INS is made in guinea pig). We have rectified in the Figure and in the corresponding text and legend.

    We think that, were the INS protein to persist in the ablated islet, its presence specifically in sst1:GFP+ cells is consistent with our transcriptomic data and with true expression of the insulin gene in bihormonal cells rather than with persistence of killed beta cells.

    However, we agree that the absence of zsYellow lineage marker as a sign of neogenesis was overinterpreted. Indeed, we clearly detect some (5.8 cells, 12% of all INS+) INS+ zsYellow+ cells (Figure 2E and E’) confirming the persistence of some traced beta cells. In fact, 4 of the 5.8 cells are sst1.1GFP+, indicating that preexisting beta cells become bihormonal. For this reason, we do not rule out anymore the beta cell origin of bihormonal cells.

    Although it is possible that the number of spared beta cells (and beta-derived bihormonal cells) is underestimated as some beta cells could have escaped excision of the Lox cassette before ablation (therefore, surviving beta cells would be zsYellow negative), we would like to stress that the ablation efficiency is very good and does not favour (but yet does not exclude) a huge contribution of beta cells to bihormonal cells.

    In the revised paper, we tone down our conclusions and consider alternative origins and mechanisms of bihormonal cells.

    The enrichment of Sst1.1 mRNA in biohormonal cells is an important piece of data that should be included instead of 'not shown'. The same is true for the statements that ROS, lack of insulin signaling and hyperglycemia all do not drive INS expression in Sst1.1 cells, which amplifies concerns that the appearance of bihormonal cells is contingent on the administration of beta cell toxins.

    We include now our “data not shown”. See new Figure 1-figure supplement 1 and Figure 6-figure supplement 2.

    To relate the interesting observation on biohormonal beta cells in zebrafish to human pancreas biology, the authors point at single cell sequencing data and then claim that 'the occurrence of SST+ and INS+ beta cells in mammals remains largely undocumented'. It strikes me that there must be dozens of papers that show high quality insulin and somatostatin co-labeling in human, primate and rodent pancreas with no evidence of clear colocalization (unless following severe beta cell ablation, see Chera et al., 2014). That actually is clear documentation of their absence.

    We realize that this point was not clear. By referring to scRNAseq data, our goal was to suggest that some Ins+ Sst+ cells could be detected at the mRNA level while we admit that there is poor, if any, evidence of naturally occurring bihormonal cells at the protein level in mammals. This part was too speculative and we removed it.

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  2. Evaluation Summary:

    This manuscript describes the presence of two discernable populations of pancreatic delta cells in a zebrafish model. One of these subsets of delta cells is suggested to facilitate the regeneration of functional beta cell mass following beta cell ablation. This observation is of interest to investigators studying islet regeneration.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  3. Reviewer #1 (Public Review):

    The manuscript by Pardo et al. describes the identification and characterization of a novel subpopulation of delta cells that normally resides in the zebrafish pancreatic islet. Using two models of beta cell ablation, the authors demonstrate that this delta cell subpopulation efficiently converts into an insulin/somatostatin co-expressing cell population to restore euglycemia. The study includes robust transcriptome data to determine that this delta cell subpopulation is characterized by the expression of sst1.1 (rather than sst2) and expresses many beta cell genes. Furthermore, the resulting insulin/sst1.1 co-expressing cells represent a long-lived population that are sufficiently functional to restore euglycemia. The study goes on to suggest that inhibition of the p53 pathway compromises formation of the bihormonal population; however this data is not as convincing. Overall, this is a novel study that suggests the existence of heterogeneous delta cell populations in the zebrafish islets and supports previous findings related to adult islet cell plasticity.

    Strengths:
    1. Although several studies have identified heterogeneous populations of islet alpha and beta cells, this is one of the first studies to demonstrate two apparently distinct delta cell populations; the study provides sufficient characterization that it should be easy to test whether a similar population exists in mammals.
    2. Demonstration that the induction of Ins/SST biohormonal cells is triggered in two independent models of beta cell ablation
    3. The use of several different transgenic fish lines to characterize the relative numbers of different islet cell populations in control and ablation conditions.
    4. High quality data, including immunofluorescence images, RNA-seq data and validation studies with appropriate controls.
    5. The extensive use of comparative transcriptome data to validate islet lineage relationships.

    Weaknesses:
    1. Although the data suggests that the newly formed bihormonal cells have sufficient function to rescue the hyperglycemic phenotype, there are no experiments to directly test the functionality of these cells.
    2. Many of the genes cited as "beta cell specific" are also expressed in delta cells in mouse and human islets - although this could relate to species differences, it causes some confusion and could affect the ultimate interpretation.
    3. Although it is clear from the images that are presented in the manuscript that a large number of bihormonal cells arise upon beta cell ablation, the relative numbers of bihormonal cells to monohormonal Sst and insulin cells is not clearly indicated. In some cases, it appears that a large percentage convert, while in others there are only a fraction. One can extrapolate this information from the presented data (ie figure D and E), but it would have been more informative if the direct analysis was provided.
    4. The authors only refer to the fact that Pdx1 is known to be expressed in beta and delta cells in a small paragraph in the discussion; it would have been helpful if this information were introduced in the introduction and in the relevant experimental sections.
    5. The authors make the strong conclusion that sst1 cells directly convert into bihormonal cells based on time lapse imaging. Genetic lineage tracing would be needed to absolutely make this conclusion. The time lapse imaging can only suggest that direct conversion might be occurring.
    6. The inhibition of p53 appears to only cause a relatively small decrease in the number of bihormonal cells (from ~20 to ~15), somewhat undermining the conclusion that p53 promotes the formation of this cell population.

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  4. Reviewer #2 (Public Review):

    This is an interesting and potentially exciting manuscript that reports, based on a series of zebrafish reporter lines, that there exists a subset of delta cells that can rapidly assume partial beta cell-like identity following beta cell ablation. This conversion correlates with the restoration of (near) normal glucose levels within 3 weeks. The major strengths are a series of technically well executed experiments that report an interesting observation of two discernable populations of delta cells. These populations are supported by transcriptome data, which validate the differences between these populations established using FISH or immunofluorescence. Major weaknesses are the lack of lineage tracing of delta cells and questions on the mechanisms underlying the origins of the bihormonal cells reported in this paper. The observation of the rapid appearance of bihormonal cells is potentially exciting and important. However, directionality of the conversion is insufficiently established. The conversation of delta to beta cells needs to be supported by direct lineage tracing. The alternative explanation that these cells are surviving beta cells that turn on somatostatin expression cannot be ruled out on the basis of the current experiments. The authors tend to extrapolate too much from their transcriptome data and subsequent pathway analyses to make claims that would be better supported by additional experiments, or toned down. The authors are right to point out the major differences in zebrafish beta cell regenerative potential and plasticity compared to mammalian models, but this diminishes the credibility of the claims of translational potential. There is value in conducting careful experiments into islet cell plasticity in a zebrafish without having to make a promise of direct translational relevance.

    This paper suggests the presence of two sets of delta cells, marked by Sst1.1 and Sst1.2. The Sst1.1 cells are marked by GFP in a Sst1.1:GFP transgenic reporter. This reporter clearly is not selective for Sst1.1 cells only, as a majority of delta cells expresses GFP at dimmer levels and is Sst2 positive. This is in good agreement with the lower - but not absent - Sst1.2 and Sst2 mRNA profiles in Figure 4, but complicates the claim that it is specifically Sst1.1 delta cells that convert into bihormonal cells. An overlay between Sst1:1 and Sst1:2 or Sst2 mRNA to demonstrate that it is specifically the Sst1:1 expressing delta cells that become INS positive (Figure 1B) would help. Formal lineage tracing of the Sst1:1 delta cells is the accepted way to solidify support for this claim, but such data are absent from this paper.

    The model is presented as a 'beta cell ablation' model, but there are some concerns with the flow of islet cells between islet cell populations immediately following ablation and during recovery that require clarification. The beta cell population size measures between 25-35% of islet cells (Figure 1D/Figure 1Suppl2). If these cells are all ablated acutely, this should immediately lead to significant increase in the remaining non-beta cell populations, including Sst1:1 delta cells. However, this is not observed as Sst1:1 GFP+ cells are steady as a fraction of total islet cell number (Figure 1F). Instead, the population that is increased at 3 days following ablation is the mCherry-GFP double positive cell population, which accounts for approximately half of the loss of beta cells. The scenario that a portion of beta cells is not actually ablated but is instead converted into a bi-hormonal state is insufficiently explored as detailed below. If the rapid appearance of these cells were indeed attributable to the conversion of GFP cells into co-positive cells, this should have been reflected in the data of Figure 1F. However, the GFP population appears to be neither increasing to reflect the loss of beta cells, or decreasing in response to the co-expression of mCherry. In Figure 5, a drop in GFPhigh cells specifically is shown, but this reflects only a potential 5% shift of islet cell numbers from GFPhigh to potentially bihormonal cells. The live imaging data in Figure 5B are not helping as there is simply not enough spatial and axial resolution to place the mCherry signal in GFP+ cells. If both processes are balancing each other out to maintain steady numbers of GFP+ delta cells, this implies rapid proliferation of GFP positive delta cells to replenish the delta cells that become bihormonal, or the rapid proliferation of bihormonal cells shortly after they arise. Either of these scenarios should be readily demonstrable.

    The presumption is that new beta cells are formed, and this is based in part on lineage tracing data using the zsYellow label in conjunction with an inducible beta cell specific Cre driver strain. It is not clear why this experiment was done in developing embryos instead of during the adult stage where the original observation of the appearance of bihormonal cells that is associated with normalization of glucose levels was made. It appears that in that crucial lineage tracing experiments, the authors are ambiguous about the use of mCherry to detect beta cells after ablation. They describe beta cells as mCherry+ beta cells in the text, while they indicate in the legend and figure labels to have used INS antibody staining to detect these cells. The punctate staining that is different from the mCherry staining elsewhere in the manuscript certainly is compatible with the use of an INS antibody, but raises the question why mCherry was not used to detect beta cells which is what was used throughout the rest of the paper. This is relevant as the lack of zsYellow positivity is interpreted as a sign of beta cell neogenesis. However, these cells might have lost zsYellow precisely because they were killed and have lost their fluorescence lineage markers, including mCherry, but are still detectable by INS immunofluorescence as they have not been cleared from the islet tissue.

    The enrichment of Sst1.1 mRNA in biohormonal cells is an important piece of data that should be included instead of 'not shown'. The same is true for the statements that ROS, lack of insulin signaling and hyperglycemia all do not drive INS expression in Sst1.1 cells, which amplifies concerns that the appearance of bihormonal cells is contingent on the administration of beta cell toxins.

    To relate the interesting observation on biohormonal beta cells in zebrafish to human pancreas biology, the authors point at single cell sequencing data and then claim that 'the occurrence of SST+ and INS+ beta cells in mammals remains largely undocumented'. It strikes me that there must be dozens of papers that show high quality insulin and somatostatin co-labeling in human, primate and rodent pancreas with no evidence of clear colocalization (unless following severe beta cell ablation, see Chera et al., 2014). That actually is clear documentation of their absence.

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