Experiments using TUNEL, BrdU, progenitor labeling, and stem cell elimination during regeneration indicate tec-1 limits the survival of newly differentiated neurons. In vertebrates, the Tec kinase family has been studied extensively for roles in immune function, and our results identify a novel role for tec-1 as negative regulator of planarian adult neurogenesis.Negative regulators of adult neurogenesis are of particular interest as targets to enhance neuronal repair, but few have yet been identified. Planarians can regenerate their entire CNS using pluripotent adult stem cells, and this process is robustly regulated to ensure that new neurons are produced in proper abundance. Using a high-throughput pipeline to quantify brain chemosensory neurons, we identify the conserved tyrosine kinase  C. elegans and rare in Drosophila, leaving axon repair as a primary mechanism for healing damage to the CNS and PNS. Mammals undergo adult neurogenesis throughout life, but it is limited to particular brain regions and declines with age . Treatment with a positive control dsRNA targeting ndk, a factor that restricts head regionalization in planarians (cto+ cells in the screen. The screen identified four genes whose inhibition reduced numbers of cto+ cells attained through regeneration (log2FC < −1 and padj <0.05), though several other treatments caused smaller cto+ cell decreases that did not meet the false-discovery corrected statistical cut-off , whose inhibition caused a statistically significant increase in abundance of cto+ cells  can result in production of excess cto+ cells as a consequence of axis transformation  animals, and determined their nearest-neighbor distances and cell body volumes using Fiji/ImageJ  animals, we examined positional control genes whose expression demarcates distinct body territories. ndl-3 and wntP-2 expression was not appreciably altered in tec-1 RNAi treatments that result in excess cto+ cells  is expressed in GABAergic neurons in the ventral-medial and dorsal-lateral CNS  , which have a defined progenitor cell type  animals regenerating a new head sometimes produced disorganized eyes  . Cephalinockdown . ppl-1 ic-1 RNAi , indicatell type , and dd1o+ cells . We obse neurons  and peri neurons  of the ill types . To obtaas study   and control animals simultaneously for cto and either gad or ppl-1, which are expressed in different areas of the brain. Counting two neuron populations at once allowed direct comparisons of the tec-1 RNAi phenotype’s expressivity for each animal. tec-1 inhibition increased numbers of these neuron cell types to the same extent without affecting gad:cto or ppl1:cto cell ratios  animals had features of normal neurons. We noticed that riboprobes detecting cto mRNA primarily stained the cell body, but high magnification imaging revealed some detection of signal in processes extending laterally from these bodies, putatively related to axonal or dendritic regions. A similar proportion of cto+ cells had such processes in control versus tec-1(RNAi) animals (tec-1(RNAi) animals displayed a qualitative increase in size of nerve bundles, particularly evident in the transverse commissural fibers joining the nerve cords along the ventral side of the animal  and calamari  as well as those expressing glucose transporter (glut)  . The funtec-1 exerts its negative regulatory function on neuron abundance. Differentiated cells are produced continually from neoblasts in adult planarians, so that increases to numbers of differentiated cells could either arise from an increase in stem cell-dependent tissue production or through decreases in the death of differentiated cells. Because tec-1 knockdown broadly increased neuronal numbers while decreasing glial numbers, and it is known that neurons and glial cells can arise from common progenitor cells in some contexts in other organisms, we initially hypothesized that tec-1 might control a differentiation switch at some early point in neural specification  animals regenerating a new head, the rate of production of new cto+ cells was not altered at early times (4 days), but abundance of cto+ cells was higher after a week of regeneration (8 days) and reached a maximum point at 16 days. We also confirmed through WISH that tec-1 is expressed broadly in the animal and throughout regeneration from day 2 to day 12 post-amputation without undergoing discernable regeneration-induced expression changes  head fragments undergoing tissue remodeling, the maximal cto+ cell abundance phenotype was observed somewhat earlier, by 4 days, and persisted  animals over a range of timepoints of BrdU pulsing and fixation in regeneration. These data indicate that tec-1 inhibition can increase numbers of neurons without modifying rates of differentiation, pointing instead to a function in controlling neuron survival.To test this possibility, we designed a BrdU labeling strategy to test for possible effects of cendants . In ordecendants . We chosby tec-1 , and thetec-1 controls neuronal survival would predict that tec-1 does not influence the abundance of neural progenitor cells that are the intermediates between pluripotent neoblast stem cells and differentiated neurons  animals over time as regeneration proceeds. To confirm these observations, we examined a subpopulation of piwi-1+ neoblasts expressing the proneural transcription factor pax6  but also within pitx, coe, or pax6A-expressing cells, neoblasts or their differentiating progeny marked with anti-PIWI-1 antibody, and in if-1;cali+ glial cells  phenotype, tec-1 inhibition did not modify numbers of H3P+ mitotically active cells in response to injury  animals did not produce significantly more cto+ cells at day 10 compared to controls but did not undergo the day 12 decrease in cto+ cells. These data confirm that tec-1 does not influence rates of cell production but instead strongly suggest that tec-1 promotes the death of newly born neurons in order to limit the abundance of cells produced through adult neurogenesis  are non-receptor tyrosine kinases similar to Src family kinases. TFKs are perhaps best known for signaling downstream of antigen receptors in hematopoietic differentiation and lymphomas . Howeverpathways . Within pathways . It is ptec-1 for neuron suppression in regeneration and homeostasis in planarians additionally suggests that developmental cell death of neurons might be a common feature of adult neurogenesis across species. Cell death is extensively used throughout the animal kingdom to sculpt the developing nervous system, from nematodes to mammals  incubating in either Multiscreen receiver plates or rectangular 1-well dishes (VWR 73521–420). Stained animals were mounted and imaged at 40x using a fluorescence dissecting microscope to obtain a view of cto expression within the head and also at 25x to obtain a Hoechst-stained view of the entire fragment. A CellProfiler pipeline for automated image analysis was developed to quantify numbers of cto+ cells and also to measure area of the fragment as detected by Hoechst staining. Automated cell counting was optimized by adjusting the fixed detection threshold and object size range. Processed images were manually inspected for proper segmentation and scored manually in the event of visible errors in automated counting. Numbers of cto+ cells scale with animal length, so we normalized cto cell number to the square root of animal area as an approximate of animal length. Log2-fold change of normalized cto cell number was computed for each specimen by comparison to the average of control RNAi treatments, and these values for all fragment types , were binned and plotted in R using ggplot2. T-tests comparing area-normalized cto+ cell numbers between each RNAi condition and RNAi controls were adjusted for false discovery using the Benjamini-Hochberg method as implemented in R.60 putative regulatory molecules were selected by analysis of the planarian transcriptome to identify a set with expression detected in neoblasts after FACS sorting , (into pHomo sapiens (hs), Mus musculus (mm), danio rerio (dr), Drosophila melanogaster (dm), and Caenorhabditis elegans (ce).Protein sequences were aligned using MUSCLE with default settings . MaximumCaenorhabditis elegans unc-22, not present in the planarian genome, served as a negative control. Unless noted otherwise, animals were fed a mixture of liver paste and dsRNA six times in two weeks prior to amputation of heads and tails.RNAi was performed by dsRNA feeding. For RNAi, dsRNA was synthesized from in vitro transcription reactions . dsRNA corresponding to AB_514500), anti-fluorescein-HRP . Hoechst 33342 (Invitrogen) was used at 1:1000 as a counterstain. Colorimetric (NBT/BCIP) assays were performed as described and detected with anti-digoxigenin-AP .Animal fixation, bleaching, and in situ hybridization were performed as previously described . BrieflyFor immunostainings, animals were fixed in 4% formaldehyde. Antibodies against mouse anti-tubulin alpha (1:1000 anti-Tubulin Alpha Neomarkers) or rat anti-BrdU (1:1000 Abcam 6326) and detected with goat anti-mouse HRP  or goat anti-rat HRP  respectively. For histone staining, animals were fixed in Carnoy's solution as described , using ttec-1 primers (5’-GTTTTGATGCTAGAATGTTG-3’ and 5-TTTGACACACATACTCAAAG-3’), with normalization to a ubiquitously expressed gene gapdh detected with gapdh primers (5'-TGGTATTCAATTGACCGATACG-3' and 5'-GATCGATTACACGGCAACTG-3') using the delta-Ct method.For qPCR, total animal RNA was collected using Trizol with a tissue homogenizer, reverse transcribed with oligo-dT primers using Superscript II. qPCR was conducted to detect tec-1 mRNA using For BrdU experiments, the concentration of Montjuic salts were gradually increased to 5x one week before surgery. Animals were treated with 0.0625% N‐acetyl cysteine dissolved in 1x Montjuic salts for one minute, washed with 1xMontjuic salts, and incubated in 25 mg/ml BrdU (Sigma) dissolved in 1x salts containing 3% dimethyl sulfoxide for 4 hr either 1 hr before amputation (day 0 soak), or 2 or 4 days post-amputation (day two soak or day four soak respectively). Animals were then maintained in 5x Montjuic salts until fixation on the day indicated in the experiment . The BrdTerminal uridine nick-end labeling (TUNEL) was performed as described by To measure neuronal persistence after neoblast ablation, animals were fed and cut as described above. Regenerating trunk fragments were irradiated at 6 days post amputation. Irradiations were carried out in a RS2000 Biological Research Irradiator (Rad Source) at 160 kV over 13’30’ for a total, lethal dose of 60 Gy . IrradiaNBT/BCIP-stained animals were imaged with a Leica M210F dissecting microscope and a Leica DFC295, with adjustments to brightness and contrast using Adobe Photoshop. Fluorescent-stained animals were imaged with a Leica TCS SPE confocal microscope or a Leica DM5500B compound microscope.opsin+ photoreceptor neurons were counted using Imaris x64 7.0.0 . Surface module was used to define either opsin+ or tyrosinase+ volume on each eye and then Spots module was used to count nuclei inside these volumes, as previously described  The expression of tec-1RNAi animals were compared in terms of differences in differentiation 8 days upon amputation. A BrdU pulse was delivered on day 2 to capture to trace the progeny of cells that were in S phase during the pulse (the duration of this pulse is not specified but should be). The result of this experiment is clear – no significant difference in the number of BrdU+ cells in the ppl+ cell population (which was affected in tec-1RNAi much like cto+ cells). While this work supports the conclusion that tec-1RNAi does not affect differentiation of cells in S-phase on day 2, it does not rule out differences in differentiation that may occur prior to or later than this time point. The numbers of progenitors marked by coe, pax6, and pitx were only compared 8 days post amputation, which also does not eliminate the idea that differentiation might be affected by tec-1 at other time points. Is it known that commitment to differentiated fates only begins on day 2 and that differentiation commitment later than day 2 does not contribute to fully differentiated cell types in a significant way? Or that differentiation rates are constant over time as the worms regenerate? Could the original observation that tec-1RNAi animals show larger numbers of cto+ cells later in regeneration be explained by really early (before day 2) differences?2) Control and tec-1 function similarly during regeneration and homeostasis? In Figure 4A, the difference between control and tec-1RNAi increases over time, suggesting more neurons should be removed as regeneration progresses. But the system has to reach a steady state eventually, but there is no data showing this. How and when might the steady state be reached in this case? Is it possible that tec-1RNAi animals just have a delayed peak of neuronal deaths? In this case, the steady states may be similar between control and RNAi. The comparison of neurons between control and RNAi conditions should be performed after the system reaches a steady state.3) Does tec-1RNAi the numbers of many neuronal cell types go up, in particular in the brain region, but the size of the brain doesn't change. It is clearly visible in the staining that the density of the evaluated neuronal cell types is increased. But how does this work? Is the general cell density in the brain region higher? Are the cells smaller? Or are there compensatory losses of other cell types in the region? Could the detected loss in glial cells be sufficient to compensate?4) In the 5) The authors state that there are no changes in polarity or global patterning genes, but Figure 2B shows a clear increase in the number of notum-expressing cells, and Figure 3B clearly shows disorganized photoreceptors. This suggests that there are definitely some – possibly subtle – changes in anterior organization. It would be very interesting to understand whether the change in notum levels could induce this change in responsiveness to apoptotic signals. In addition, there have been some indications that apoptosis is not constant along the A-P axis , and it would be worth taking into consideration whether this might explain some of the observed phenotype.6) Is there an anterior bias in the detected neuronal cell increase? Most of the neuronal cell types are evaluated in the anterior region. Only the bottom panel in Figure 3A shows a change in a more posterior region. Are the effects mostly detected in the anterior, or are neuronal cell increases in the posterior just as strong?tec-1RNAi causes a prolonged differentiation? There is a caveat in terms of Figure 4C showing tec-1's disinvolvement with rate of neuronal differentiation. The regenerating tail fragments were examined at day 8 post-amputation, at which point Figure 4A suggests has only a slight increase in neuronal abundance and thus is statistically unlikely to be noticeable at this stage within a smaller subset of neuronal types. A different time point may address the possibility of delayed differentiation.7) Is it possible that tec-1 in different neuronal populations? Are there any neuronal types within the CNS or PNS that reduce in numbers or are not affected after RNAi? Neuronal types unaffected by tec-1 may bring important insights and hypotheses. Readers would appreciate the authors' insights to these questions. If all neuronal types increase in numbers within a constant brain size, it might suggest that the neurons must reduce their sizes as they experience a cellular overcrowding effect. Is this effect measurable ? Are the changes in number density and cell size consistent? The authors have shown that glial cell number mildly decreases, which is very interesting, but is this change enough to compensate the increase of all neurons? How should we interpret this result – is it explained by the hypothesis of a distinct cell population killing neurons, or might this have any basis in a potential differentiation pathway?8) The phenotype should be characterized and discussed with greater depth. Do the ratios between different neuronal types change? This can be assayed with double FISH. If the ratios have changed, how should we understand the activity of tec-1RNAi animals continue to expand numbers of cto+ cells, whereas control RNAi animals show a decrease in cto+ cells at day 12 relative to day 10. However, Figure 4B showed a statistically significant different in cto+ cells between control and tec-1 RNAi animals on day 10 . If differentiation is not a source of cto+ differences between control and tec-1RNAi animals, one would expect the differences to be the same upon elimination of the contribution of new cells (at least at day 8). What explains the non-significant differences at day 8 and 10 in Figure 5B? Do the results argue that both differentiation and cell survival are affected?9) The choice of timing for the irradiation experiment needs to be explained as well. This experiment shows that in the absence of new cell production, tec-1. The authors' finding that planarian regeneration involves overproduction of neural cells followed by cell death bears overall similarity to the mechanisms of vertebrate development where cell death plays a major role in shaping the nervous system. However, given the lack of demonstrated similarity of function of the ortholog of tec-1 in neural development in vertebrates, the claims of tec-1 being a promising new target for therapeutic interventions are premature.10) The authors report that Tec kinases are known to control inflammation and recovery following traumatic brain injury by regulating macrophages, which does not directly imply that Tec kinase homologs regulate neuronal cell numbers via controlling their survival. Therefore, the next statement (“We suggest that Tec kinases could regulate specific and perhaps conserved functions in adult neurogenesis process in control of neuron abundance”) is not well-supported. There are no data presented that enable the proposal of a putative conserved function for tec-1RNAi is interesting, and the authors should discuss if this could be suggestive of tec-1 being involved in a fate choice – could glial cells and cto+ cells be differentiating from a shared progenitor population?11) Finally, the finding of increased density of glial cells in  Essential revisions:In order to consider this manuscript further, several major issues in terms of the phenotype and interpretation need to be addressed. These are:1) The expression of tec-1 is unclear. While the cell atlas suggests it is broadly expressed and perhaps concentrated in the neuronal progenitors, where is it expressed during regeneration? Does the expression pattern change throughout the regeneration time course? This information is essential, as it should inform the interpretation of phenotypes in terms of whether tec-1 functions autonomously or non-autonomously. Is tec-1 co-expressed with any of the neuronal or progenitor types looked at in the paper? Or is it co-expressed with macrophage/phagocytic markers? If FISH is challenging in whole-mount, it is worth trying in sections and dissociated cells.tec-1 expression in whole animals. Indeed, in confirmation of the scRNAseq data, we find tec-1 FISH signal to be broadly distributed, with co-expression analysis confirming expression in several different neurons, piwi-1+ cells, and glia . We also examine overall tec-1 expression during regeneration  and find it is also broadly expressed during regeneration with no obvious injury-induced expression behavior. The broadness of expression implies that we cannot at present rule out the possibility of tec-1 acting within either neural progenitors, differentiated neurons, or some other cell type to exert its function on neuron numbers, and we clarify this interpretation in the text (Discussion paragraph three).We appreciate this suggestion and now show FISH images of + cells in the ppl+ cell population (which was affected in tec-1 RNAi much like cto+ cells). While this work supports the conclusion that tec-1 RNAi does not affect differentiation of cells in S-phase on day 2, it does not rule out differences in differentiation that may occur prior to or later than this time point. The numbers of progenitors marked by coe, pax6, and pitx were only compared 8 days post amputation, which also does not eliminate the idea that differentiation might be affected by tec-1 at other time points. Is it known that commitment to differentiated fates only begins on day 2 and that differentiation commitment later than day 2 does not contribute to fully differentiated cell types in a significant way? Or that differentiation rates are constant over time as the worms regenerate? Could the original observation that tec-1 RNAi animals show larger numbers of cto+ cells later in regeneration be explained by really early (before day 2) differences?2) Control and tec-1 RNAi animals were compared in terms of differences in differentiation 8 days upon amputation. A BrdU pulse was delivered on day 2 to capture to trace the progeny of cells that were in S phase during the pulse (the duration of this pulse is not specified but should be). The result of this experiment is clear – no significant difference in the number of BrdUppl-1+ cells across all timepoints of regeneration or soaking tested. To support these conclusions, we also conducted an expanded neural progenitor counting experiments across a large range of timepoints in regeneration , and confirm that we could not detect any excess rates of differentiation in tec-1RNAi animals.We have repeated the BrdU experiment by adding multiple time points for BrdU soaking and animal fixation during regeneration in Figure 4B . These experiments did not find any differences between numbers of BrdU+3) Does tec-1 function similarly during regeneration and homeostasis? In Figure 4A, the difference between control and tec-1 RNAi increases over time, suggesting more neurons should be removed as regeneration progresses. But the system has to reach a steady state eventually, but there is no data showing this. How and when might the steady state be reached in this case? Is it possible that tec-1 RNAi animals just have a delayed peak of neuronal deaths? In this case, the steady states may be similar between control and RNAi. The comparison of neurons between control and RNAi conditions should be performed after the system reaches a steady state.tec-1 has a common role in negatively regulating neurogenesis, with each particular assay influencing the kinetics of response.We now include a longer time course showing that the steady state is reached by day 14 and the phenotype persisted through at least 28 days of regeneration . Based on observing similar overall phenotypes in regeneration or homeostasis assays, we suggest 4) In the tec-1 RNAi the numbers of many neuronal cell types go up, in particular in the brain region, but the size of the brain doesn't change. It is clearly visible in the staining that the density of the evaluated neuronal cell types is increased. But how does this work? Is the general cell density in the brain region higher? Are the cells smaller? Or are there compensatory losses of other cell types in the region? Could the detected loss in glial cells be sufficient to compensate?cintillo cells from confocal stacks and made measurements of cell volume and the nearest-neighbor distances for each cell. This approach found that in tec-1 RNAi, cintillo cells were more numerous and slightly smaller, and also that they are more densely packed with respect to each other . We suggest that increased density along with reduced size accounts for how more neurons physically occupy apparently similar brain volumes in tec-1RNAi.To consider this, we performed 3D segmentation of 5) The authors state that there are no changes in polarity or global patterning genes, but Figure 2B shows a clear increase in the number of notum-expressing cells, and Figure 3B clearly shows disorganized photoreceptors. This suggests that there are definitely some – possibly subtle – changes in anterior organization. It would be very interesting to understand whether the change in notum levels could induce this change in responsiveness to apoptotic signals. In addition, there have been some indications that apoptosis is not constant along the A-P axis , and it would be worth taking into consideration whether this might explain some of the observed phenotype.notum+ cells. These excess cells were found to be within a previously described set of notum+chat+ cells of the brain , which we interpret to be consistent with the supported role of tec-1 to limit numbers of multiple types of neurons. We suggest based on our analysis that tec-1 likely acts in an independent process from other known head patterning genes , because tec-1 inhibition primarily influences neuron cell density versus head regionalization.To address this, we used FISH to more carefully detect and pinpoint the excess tec-1 could be part of the previously characterized A-P axis apoptotic pathway. To test this, we searched the recently published scRNAseq planarian cell atlas to find a neuron subtype that was broadly distributed but disperse enough that total animal cell quantifications could be carried out confidently. Of the tested neuron subtypes, we found one marker  that was strongly sensitive to tec-1 inhibition. In tec-1RNAi animals, we found an equivalent increase in dd2223+ neuron cells in anterior versus posterior body regions . Therefore, our findings suggest tec-1 can exert its influence independent of A-P axis position, so we suggest it is likely part of a distinct pathway from factors specifically influencing anterior versus posterior cell survival.We appreciate the suggestion to consider whether 6) Is there an anterior bias in the detected neuronal cell increase? Most of the neuronal cell types are evaluated in the anterior region. Only the bottom panel in Figure 3A shows a change in a more posterior region. Are the effects mostly detected in the anterior, or are neuronal cell increases in the posterior just as strong?dd2223, labeling nonciliated neuron cluster#32 identified by Fincher et al., 2018) that was strongly sensitive to tec-1 inhibition. In tec-1RNAi animals, we found an equivalent increase in dd2223+ neuron cells in anterior versus posterior body regions . Therefore, tec-1 can exert its influence independent of AP axis position.Similar to above, to test this, we searched the recently published scRNAseq planarian cell atlas to find a neuron subtype that was broadly distributed but disperse enough that total animal cell quantifications could be carried out confidently. Of the tested neuron subtypes, we found one marker (7) Is it possible that tec-1 RNAi causes a prolonged differentiation? There is a caveat in terms of Figure 4C showing tec-1's disinvolvement with rate of neuronal differentiation. The regenerating tail fragments were examined at day 8 post-amputation, at which point Figure 4A suggests has only a slight increase in neuronal abundance and thus is statistically unlikely to be noticeable at this stage within a smaller subset of neuronal types. A different time point may address the possibility of delayed differentiation.coe, pax6A, pitx), we found that both control and tec-1RNAi animals undergo a similar reduction of progenitor numbers during regeneration. There was no significant difference in progenitor numbers between control and tec-1RNAi animals at all timepoints throughout regeneration (day 2-day 12) . Therefore, we do not find evidence for tec-1 inhibition prolongs a period of higher differentiation in regeneration.We tested the possibility of delayed differentiation through an extended set of measurements over time  and for 3 different neural progenitors during regeneration. In all three cases (8) The phenotype should be characterized and discussed with greater depth. Do the ratios between different neuronal types change? This can be assayed with double FISH. If the ratios have changed, how should we understand the activity of tec-1 in different neuronal populations? Are there any neuronal types within the CNS or PNS that reduce in numbers or are not affected after RNAi? Neuronal types unaffected by tec-1 may bring important insights and hypotheses. Readers would appreciate the authors' insights to these questions. If all neuronal types increase in numbers within a constant brain size, it might suggest that the neurons must reduce their sizes as they experience a cellular overcrowding effect. Is this effect measurable ? Are the changes in number density and cell size consistent? The authors have shown that glial cell number mildly decreases, which is very interesting, but is this change enough to compensate the increase of all neurons? How should we interpret this result – is it explained by the hypothesis of a distinct cell population killing neurons, or might this have any basis in a potential differentiation pathway?cto+ neurons to other neuron types in Figure 3—figure supplement 3. These revealed no alteration of cto:gad or cto:ppl1 cell ratios after tec-1RNAi, suggesting tec-1concordantly affects the abundance of multiple neuron types.Using double-FISH, we now include experiments to examine the ratio of tec-1RNAi phenotype led us to focus on cell types for which total body enumeration could be performed at a high degree of precision. Of the 5 genes we found to meet this criteria, 4 underwent tec-1 dependent increases : dd2223, dd3733, spp-4, dd17258) and the other did not change in abundance after tec-1RNAi (dd2723). Altogether, our analysis now finds 10 of 13 neuron types increased after tec-1RNAi, and 3 of 13 did not change (note we found none that decreased in number). Those unaffected by tec-1 inhibition did not have any known discernable commonality in terms of location or distribution . These findings, in combination with the broad expression of tec-1, prevented us from identifying a best hypothesis at this time for why some neurons are affected by tec-1 and some are not.To expand the analysis of the phenotype we examined five additional neuron subtypes identified in the recent scRNAseq cell atlas for planarians. Though planarians may have ~50 different types of neurons, the effect size of the tec-1(RNAi) animals physically pack in space, given for example that overall brain size is not affected. To address this, we performed 3D segmentation on cintillo-stained animals and computed volume and nearest neighbor distances between the cells . This approach found that tec-1 inhibition indeed results in overcrowding of cto+ neurons, leading to higher neuron densities and smaller volumes of cto+ cells. Perhaps as extra neurons grow, some process constrains overall tissue volume, leading to these cells attaining reduced sizes. Based on the separation of regions occupied by glia and the affected neuron cell types, we believe the crowding effect to be a separate phenomenon than loss of glial abundance or size. In combination with additional experiments showing lack of support for increased differentiation of neurons , our analysis suggests tec-1 is unlikely to function in a switch to control glial-vs-neuron differentiation.We appreciate the suggestion to determine how extra neurons in 9) The choice of timing for the irradiation experiment needs to be explained as well. This experiment shows that in the absence of new cell production, tec-1 RNAi animals continue to expand numbers of cto+ cells, whereas control RNAi animals show a decrease in cto+ cells at day 12 relative to day 10. However, Figure 4B showed a statistically significant different in cto+ cells between control and tec-1 RNAi animals on day 10 . If differentiation is not a source of cto+ differences between control and tec-1 RNAi animals, one would expect the differences to be the same upon elimination of the contribution of new cells (at least at day 8). What explains the non-significant differences at day 8 and 10 in Figure 5B? Do the results argue that both differentiation and cell survival are affected?tec-1 excess cto+ cell phenotype was somewhat variable across experiments, dependent on feeding schedule and fragment type . For the irradiation experiment, we chose trunk fragments because they can survive for longer as compared to irradiated tail fragments. In those fragments, we never observed the excess cto+ phenotype at d8 but only observed it emerge at later times – by day 12 in head regeneration, the phenotype was always observed. We repeated the experiment in Figure 5B three times and observed in each case the tec-1RNAi phenotype in irradiated animals by day 12. Our interpretation of the BrdU labeling and progenitor counting experiments is that we were unable to observe any effect of tec-1 inhibition on differentiation.We chose this timing in order to attempt to generate a pulse of new neurons whose behavior could be assessed over time. In contexts of head regeneration, the exact timing of the onset of the 10) The authors report that Tec kinases are known to control inflammation and recovery following traumatic brain injury by regulating macrophages, which does not directly imply that Tec kinase homologs regulate neuronal cell numbers via controlling their survival. Therefore, the next statement (“We suggest that Tec kinases could regulate specific and perhaps conserved functions in adult neurogenesis process in control of neuron abundance”) is not well-supported. There are no data presented that enable the proposal of a putative conserved function for tec-1. The authors' finding that planarian regeneration involves overproduction of neural cells followed by cell death bears overall similarity to the mechanisms of vertebrate development where cell death plays a major role in shaping the nervous system. However, given the lack of demonstrated similarity of function of the ortholog of tec-1 in neural development in vertebrates, the claims of tec-1 being a promising new target for therapeutic interventions are premature.We appreciate this suggestion to the presentation. We clarified our reference to the study noted above, that it found an increase in brain cell survival  in an ischemic brain injury model after pharmacological inhibition of the Tec-family kinase BTK in mammals (Discussion paragraph five).tec-1 to suppress neurogenesis. Given that some components of neurogenesis are broadly conserved, we think it is worthwhile to raise the idea that future studies could address this possibility. In addition, we now tone down statements of the concept for a potential use for tec-1 inhibition to enhance neurogenesis . We hope our revised text makes clear that we do not present evidence for a conserved function in this study:We also modified the text suggesting that our study necessarily implies the existence of a conserved activity of tec-1 as negative regulator of planarian adult neurogenesis.”Abstract: “In vertebrates, the Tec kinase family has been studied extensively for roles in immune function, and our results identify a novel role for Discussion paragraph five: “If the ability of Tec Kinases to limit neuron production ultimately proves to be conserved, they would be potential targets for enhancing neural repair in other species.”Discussion paragraph four: “It is possible that Tec kinases could regulate conserved functions in adult neurogenesis processes in control of neuron abundance.”11) Finally, the finding of increased density of glial cells in tec-1 RNAi is interesting, and the authors should discuss if this could be suggestive of tec-1 being involved in a fate choice – could glial cells and cto+ cells be differentiating from a shared progenitor population?tec-1RNAi. We have added text discussing the hypothesis of a tec-1 functioning in a neuron-glial differentiation switch, but also how our experiments would suggest against this model because of finding no evidence for tec-1 inhibition to increase rates of neuron differentiation .We appreciate this suggestion, as it was indeed our initial hypothesis leading us to examine the glia in


Ionic liquids are novel solvents of interest as greener alternatives to conventional organic solvents aimed at facilitating sustainable chemistry. As a consequence of their unusual physical properties, reusability, and eco-friendly nature, ionic liquids have attracted the attention of organic chemists. Numerous reports have revealed that many catalysts and reagents were supported in the ionic liquid phase, resulting in enhanced reactivity and selectivity in various important reaction transformations. However, synthetic chemists cannot ignore the stability data and intermolecular interactions, or even reactions that are directly applicable to organic reactions in ionic liquids. It is becoming evident from the increasing number of reports on use of ionic liquids as solvents, catalysts, and reagents in organic synthesis that they are not totally inert under many reaction conditions. While in some cases, their unexpected reactivity has proven fortuitous and in others, it is imperative that when selecting an ionic liquid for a particular synthetic application, attention must be paid to its compatibility with the reaction conditions. Even though, more than 200 room temperature ionic liquids are known, only a few reports have commented their effects on reaction mechanisms or rate/stability. Therefore, rather than attempting to give a comprehensive overview of ionic liquid chemistry, this review focuses on the non-innocent nature of ionic liquids, with a decided emphasis to clearly illuminate the ability of ionic liquids to affect the mechanistic aspects of some organic reactions thereby affecting and promoting the yield and selectivity. Ionic liquids (ILs) are low-melting molten salts composed entirely of ions, and many of them are liquids at room temperature ,3,4,5,6.et al., switching from an organic solvent to an ionic liquid often results in a significant improvement in catalytic performance e.g. rate acceleration, (enantio)selectivity improvement and an increase in catalyst stability [NO3] (m.p. 12 °C) was discovered in 1914 [OH] on Michael addition reactions  cation is not completely inert . They fo2O [NTf2] and [bdmim][PF6] produced less than 10% deuterium exchange at C2 methyl group at room temperature for one week time and only [bmim][PF6] was found to be deuterium exchanged rapidly (50% in 1 h) and our [b-3C-im][NTf2] gave no detectable exchanges even after one week at ambient temperature [NTf2] ionic liquid is chemically stable to base (t1/2 ~ 6 days). Among all tested ionic liquids, [b-3C-im][NTf2] ionic liquid was most stable to solvent deuterium isotope exchange while the previously reported [bmim][NTf2] and [bdmim][NTf2] was deuterium exchangeable instantaneously and in 30 min, respectively, at ambient temperature.Our [b-3C-im] is far more chemically stable than the previously reported [bmim][PF6], [bdmim][NTf2], and [bdmim][PF6] .From the results of solvent deuterium exchange experiments, we therefore concluded that the new ionic liquid [b-3C-im].,76.75,76et al. anticipated that imidazolium-based ionic liquids would be a good choice of solvent for the Baylis–Hillman reaction [6] than in acetonitrile. The reactions were, however, low yielding. Soon after Afonso’s paper was published, Aggarwal et al. quickly pointed out that the ionic liquids used were all [bmim]-based and, in the presence of bases such as DABCO or 3-hydroxyquinuclidine, the imidazolium moiety in ionic liquids could be deprotonated at its C2 position and the resulting nucleophile directly reacted with and thereby consumed the aldehyde    90]..via form2 was heated in [bmim][Br] in the absence of the alkene and aryl halide, under reaction conditions. They observed rapid color changes from dark brown to red and then to yellow and the products formed were analyzed by NMR. The characteristic chemical shifts of the olefin protons (δ 7 in the 1H-NMR spectrum) and the carbene carbons (δ 160–170 in the 13C- NMR spectrum) suggested the presence of a mixture of palladium carbene complexes. Supporting this formation of Pd carbene complexes, other species such as Ir carbene complexes (as well as hydrogenated species) are also obtained in reactions with imidazolium salts [BF4] . Wong et -based RTILs in organozinc catalysed Negishi couplings met with failure; on the other hand, when a new ionic phosphine ligand 5 was incorporated for the reaction performed in a biphasic solvent system of toluene and [bdmim][BF4], this palladium catalyzed cross-coupling reaction proceeded smoothly giving high isolated yields  (or PdCl2) nanoparticles proceeds under ultrasound irradiation in the ionic liquid [bbim][BF4]  bromochlorides or dibromides are obtained from trans-olefins while cis-olefins give the corresponding threo-  adducts  are less expensive to manufacture on an industrial scale, and are also an efficient and recyclable media for Pd-mediated cross-coupling reactions ,108. PILet al.  found thet al. ,64 are pet al.  such as t al. -, . Some of these salts are potential candidates for use as electrolytes in high-energy storage devices , [PF6], [BF4], [CF3SO3] or [(CF3SO2)2N], which in some regard limits their ‘greenness’. The presence of halogen atoms may cause serious concerns if the hydrolytic stability of the anion is poor  or if a thermal treatment of spent ionic liquids is required. In both cases, additional effort is needed to avoid the liberation of toxic and corrosive HF or HCl into the environment.The stability of the anions involved in ionic liquids plays a critical role in certain applications or their toxicity. Typical ionic liquids consist of halogen containing anions such as  ionic liquids that are, in fact, catalyzed by adventitious HF. Thus, a significant impact of IL anions is the release of HF containing compounds for which considerable care should be taken while using PF6 containing ionic liquids. Thus, ILs should be treated with caution due to their unexplored toxicity and/or stability [et al. identified 1-butyl-3-methylimidazolium fluoride hydrate crystallographically as a decomposition product that was obtained from hydrolytic degradation of [bmim][PF6] during purification process [Cl], [bmim][BF4], [bmim][PF6], urea ammonium nitrate (UAN), and decylmethylimidazolium tetraphenyl-borate under ultrasound conditions and reported that, upon sonication, all of the imidazolium ionic liquids darkened from colorless to amber while UAN did not undergo a color change [1H-NMR analysis of [bmim][Cl] and [bmim][BF4] also indicated the appearance of new peaks in the imidazole region. Imidazolium ionic liquids produced gases/vapors that contained trace amounts of light hydrocarbons and nitriles consistent with the decomposition of imidazoles. Thus, the ionic liquids do decompose under ultrasonic conditions.Suslick r change . 1H-NMR dT of quaternary ammonium ILs give a wide range for a particular pair of cation and anion  and the corresponding alkyl compound. In a prelude to the study of the effects of R4N+X- on ammonium perchlorate  decomposition, they interpreted that the amine (R3N) formed by decomposition of IL can interact with the HCIO4 produced during the thermal decomposition of AP, thus forming an amine perchlorate [Previously, Sheikh studied the thermal decomposition of Rchloride . Prasad ecreases . It is wchlorate ,156.et al. with KMnO4 [3 [2 and H2O, while the anions were not oxidized. They observed that ILs undergo decomposition under the effect of ozone in aqueous solution more efficiently. Dynamically growing application of ILs induces misgivings as to their utilization when the real effects of cations and anions are unrevealed.As organic salts, ionic liquids are prone to oxidative degradation, particularly with vigorous oxidizing agents has been demonstrated by Pernak th KMnO4  and O3 [KMnO4 [3 . They obet al. reported that the radiation stability from pulse radiolysis studies indicate that in ILs composed of aromatic cations (such as imidazolium and pyridinium), the electrons rapidly attach to the ring to form the corresponding neutral radicals [Shkrob radicals . In anotradicals .Our review emphasizes the urgent need for understanding the fundamental chemistry of ionic liquids as their incompatibility is now being revealed gradually. Most repeated justifications that ILs are inert and do not interact with the reagents is only an assumption until its properties are clearly studied. Unique and hidden chemistry of ILs limits their application in certan synthetic reactions. ILs, like all other solvents are chemicals which have intrinsic reactivity and thus the acidic nature, instability of anions, thermal stability, and degradation with oxidising agents has always to be kept in mind while choosing ionic liquids as reaction media.


Morphological analysis also reveals that neurogenic niches in the adult mouse brain contain cells with nuclear shapes highly similar to those observed during in vitro neurogenesis from hPDLSCs. Our results provide additional evidence that it is possible to differentiate hPDLSCs to neuron-like cells and suggest the possibility that the sequence of events from stem cell to neuron does not necessarily requires cell division from stem cell.Understanding the sequence of events from undifferentiated stem cells to neuron is not only important for the basic knowledge of stem cell biology, but also for therapeutic applications. In this study we examined the sequence of biological events during neural differentiation of human periodontal ligament stem cells (hPDLSCs). Here, we show that hPDLSCs-derived neural-like cells display a sequence of morphologic development highly similar to those reported before in primary neuronal cultures derived from rodent brains. We observed that cell proliferation is not present through neurogenesis from hPDLSCs. Futhermore, we may have discovered micronuclei movement and transient cell nuclei lobulation coincident to  Understanding the mechanisms that underlie the temporal and spatial control of neuronal polarization is not only important for neurobiology3, but also for nerve regeneration in injured nervous systems4.Neurons are highly polarized cells exhibiting functionally and structurally distinct processes called axons and dendrites2. The establishment of polarity in cultured rodent neurons are divided into different stages6.The process of neuronal polarization has been studied for decades using dissociated rodent postnatal cerebellar granule neurons and embryonic hippocampal pyramidal neurons in culturein vitro start as rounded spheres that spread lamellipodia (stage 1). These spheres appear symmetrical, extending and retracting several immature neurites of a similar length (stage 2). Elongation of a single process, that which presumably becomes the axon, breaks this symmetry (stage 3). The next step involves the remaining short neurites morphologically developing into dendrites (stage 4). The last step (stage 5) in neuronal polarization from dissociated pyramidal neurons in culture is the functional polarization of axon and dendrites, including dendritic spine formation and axon branch formation5. Dissociated granule neurons also present a lamellipodia after attaching to the substratum (stage 1). These spheres extend a unipolar process at a single site on the plasma membrane (stage 2) followed by extension of a second process from the opposite side of the cell body, resulting in a bipolar morphology (stage 3). One of the two axon elongates futher and start branching (stage 4), and shorter dendritic processes develop around the cell body (stage 5)6.Upon isolation, dissociated pyramidal neurons retract their processes, so their neuronal development 6, less is known about the process of neuronal polarization in human cells8.Although much progress has been made in the knowledge of how rodent neurons establish their polarity11.The major barrier to studying human neurons is the inaccessibility of living tissue, therefore an enormous effort has been made in this study to derive neurons from human stem cells13. NCSCs can be isolated not only from embryonic neural crest, but also from fetal and adult neural crest-derived tissues14.Neural crest stem cells (NCSCs) are a migratory cell population that generate numerous cell lineages during development, including neurons and glia15. Isolation and characterization of multipotent stem cells from the human PDL have been previously described17.The periodontal ligament (PDL) is a connective tissue surrounding the tooth root that contains a source of human NCSCs which can be accessed with minimal technical requirements and little inconvenience to the donor18, we showed that human adult periodontal ligament (hPDL) tissue and hPDL-derived cells express marker genes of stem cells and neural crest cells. In vitro, hPDL-derived cells differentiate into neural-like cells based on cellular morphology and neural marker expression. In vivo, hPDL-derived cells survive, migrate and expressed neural markers after being grafted to the adult mouse brain. Futhermore, some hPDL-derived cells graft into neural stem cell (NSC) niches such as the ventricular-subventricular zone (V-SVZ) of the anterolateral ventricle wall and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. It is important to mention that the hPDLSCs located in the NSC niches show neural stem morphology.In previous publication19 and displayed inward currents conducted through voltage-gated sodium (Na+) channels and spontaneous electrical activities after neurogenic differentiation21. Therefore, the neural crest origin15 and neural differentiation potential both in vitro and in vivo24, make human periodontal ligament stem cells (hPDLSCs) interesting for investigating stem cell to neuron differentiation mechanisms.Moreover, hPDLSCs expressed ion channel receptorsHere, we show that hPDLSCs-derived neural-like cells display a sequence of morphologic development highly similar to those reported before in primary neuronal cultures derived from rodent brains during neurogenesis, providing additional evidence that it is possible to differentiate hPDLSCs to neuron-like cells.in vitro neurogenesis, without being related to cell proliferation. We observed that small DNA containing structures may move within the cell to specific directions and temporarily form lobed nuclei.We observed that cell proliferation is not present through neurogenesis from hPDLSCs. In fact, the cell shape of hPDLSCs is reset and start their neuronal development as round spheres. Futhermore we may have discovered a transient cell nuclei lobulation coincident to in vitro neurogenesis from hPDLSCs. We suggest the possibility that neuronal differentiation from NSCs may also occur during in vivo adult mammalian neurogenesis without being related to cell proliferation.Morphological analysis also reveals that the V-SVZ of the anterolateral ventricle wall and the SGZ of the hippocampal dentate gyrus in the adult mouse brain contains cells with nuclear shapes highly similar to those observed during 25. Dental and oral-derived stem cells displayed spontaneous expression of neural marker β-III tubulin, even without having been subjected to neural induction26. Western blot analysis verified the expression of β-III tubulin in hPDLSCs  is considered the gold standard to confirm apoptosisove Fig. . Therefo3  have been described in two neurogenic niches, the V-SVZ of the anterolateral ventricle wall and the subgranular zone SGZ of the hippocampal dentate gyrus49.In the V-SVZ, putative aNSCs (type B cells) divide to give rise to intermediate progenitor cells (type C cells), which divide a few times before becoming neuroblasts (type A cells). The neuroblast then migrate into the olfactory bulb and differentiate into distinct types of neurons53. Neuroblast, as polarized cells, then migrate, guided by the leading process, along SGZ and differentiate into dentate granule neurons55.In the SGZ, putative aNSCs divide to give rise to intermediate progenitor cells which exhibit limited rounds of proliferation before generating polarized neuroblast58. Type-B cells have irregular nuclei that frecuently contain invaginations and irregular contours of the plasma membrane. Type-C cells nuclei contained deep invaginations and these cells are more spherical. Type-A cells have elongated cell body with one or two processes and the nuclei are occasionally invaginated43. Importantly, some studies have shown that murine and human V-SVZ have segmented nuclei connected by an internuclear bridge45. Although it has been suggested that lobed nuclei connected by an internuclear bridge are associated with quiescence in aNSCs45, we observed that this kind of nuclei may be associated to nuclear movement within the cell during initial phases of neurogenesis, without being related to cell proliferation.Previous ultrastructure and immunocytochemistry studies show that the V-SVZ stem cell niche contains cells with different morphologies and irregular nuclei66. Adult SGZ NSCs (type 1 cells) have irregular contours of the plasma membrane, and differences in heterochromatin aggregation has been also observed53. Futhermore, adult SGZ NSCs (type 2 cells) had an irregularly shaped nucleus67. Importantly, one study also found that many cultured hippocampal neurons have irregular nuclei or even consisted of two or more lobes connected by an internuclear bridge68.In addition, previous reports also shown irregular shaped nuclei in the adult SGZ55. Despite the advantages for the detection of adult neurogenesis using exogenous nucleotide analog administration or endogenous cell cycle markers, in addition to retroviral transduction, cell stage and lineage commitment markers, recent findings indicate that some observations interpreted as cell division could be false-positive signals72. The main method used to labed new neurons has been the incorporation of the thymidine analogs into the genome of dividing cells during S-phase of the cell cycle, but nevertheless thymidine analogs such as tritiated thymidine, BrdU, CldU and IdU may also be incorporated during DNA turnover or DNA repair72. Infection with retrovirus is another method used to label new neurons however, retroviral vectors not specifically infect dividing cells71 and also, it has even been observed that postmitotic pyramidal neurons can also be labeled by fused infected microglia73. Although the expression of endogenous cell cycle proteins is also used to label new neurons, recent findings indicate that cell cycle proteins expression is not necessarily related to cell division72. Proliferating cell nuclear antigen is also invoved in DNA repair74. Positivity of the proliferation marker KI-67 in noncycling cells has also been observed75.It has commonly been assumed that adult neurogenesis occurs progressively through sequential phases of proliferation53. Importantly, the existence of non-proliferative neuronal precursors in several brain areas has also been observed77. Moreover, the self-renewal and multipotent properties demonstrated by NSC in vitro78 have not been clearly demonstrated in vivo79.These findings indicate that there is a lack of a reliable definitive method to label new neurons. In addition, it is important to note that almost none of these labeled new born neurons show mitotic chromosomes or mitotic spindle to really confirm that adult neurogenesis occurs progressively through sequential phases of proliferationCollectively, these results suggest the possibility that the sequence of events from aNSCs to neuron may also occur without being related to cell proliferation. It would therefore be interesting to examine whether SVZ and SGZ intermediate progenitor cells represent different stages of neurogenesis without being related to cell proliferation.in vitro and in vivo82. Future analysis will be necessary to study if hPDLSCs convert directly to neural-like cells or if they go through neural progenitor stage first. Our results suggest the possibility that hPDLSCs could be use to advance knowledge of the cellular plasticity.It is known for many decades that adult cells can change their indentity throught spontaneous dedifferentiation, differentiation and transdiferentiation 86. We observed that the nuclei of hPDLSCs during initial phases of neurogenesis are highly similar to those reported in immune cells. Thus, we suggest the possibility that multilobed nuclear structures may be associated to nuclear movement within the cell. Therefore, it would also be interesting to examine whether these putative madure cells also represent different stages of haematopoietic stem cell differentiation without being related to cell proliferation.Beyond the central nervous system, the presence of lobed nuclei has been reported in most blood and immune cells, but the functional significance of multilobed nuclear structures is not yet known88. Fibrillarin and lamin A/C proteins were detected in these small DNA containing structures.One of the most important discoveries in this work is the observation that small DNA containing structures may move within the cell to specific directions and temporarily form lobed nuclei. These small DNA containing structures displayed a spherical or ovoid shape, and it seems that some of them are connected to the main body of the nucleus by thin strands of nuclear material. It is important to note that some DNA containing structures are highly similar to nuclear envelope-limited chromatin sheets (ELCS)89. Although these nuclear anomalies have been associated with chromosomal instability events during mitosis92, recent reports showed generation of micronuclei during interphase95. These findings call into question that micronuclei, nucleoplasmic bridge and nuclear bud does necessarily generated during mitosis96. Moreover, a high frequency of human mesenquimal stem cells with nuclear bud, micronuclei and nucleoplasmic bridge was detected under normal in vitro culture97. Therefore, the mechanisms that lead to extra-nuclear bodies formation and their biological relevance are still far from been understood99.It is known for many decades that chromatin particles can appear in the cellular cytoplasm and they are referred to as micronuclei, nucleoplasmic bridge and nuclear budIn this study, we show that there can be a relationship in the formation of the nuclear bud, micronuclei, nucleoplasmic bridge and nuclear envelope-limited chromatin sheets. Collectively, these results suggest the possibility that the interphase cell nucleus may can reversibly disassembled into functional subunits that may moved independently within the cell, if necessary.101. In addition, alterations in nuclear morphologies are closely associated with a wide range of human diseases, including cancer102. hPDLSCs could facilitate an understanding of the mechanisms regulating nuclear morphology in response to cell shape changes and their functional relevance104.Multinuclear cells are commonly found in various human organs including heart, liver, salivary glands, muscle and endometrium, but their functional advantage remains unknownMethods were carried out in accordance with the relevant guidelines and regulations. The experimental protocols were approved by the Institutional Review Board of the Miguel Hernández University of Elche (No. UMH.IN.SM.03.16) and the signed informed consent was obtained from all patients before the study. The authors declare that all experiments on human subjects were conducted in accordance with the Declaration of Helsinki. All protocols and care of the mice were carried out according to the guidelines of the European Communities Council Directive of 24 November 1986 (86/609/EEC). The authors further attest that all efforts were made to minimize the number of animals used and their suffering.18. Human premolars were extracted and collected from three different healthy adult donors undergoing orthodontic therapy in Murcia dental hospital (Spain). hPDL was scraped from the middle third region of the root surface. After washing the extracted PDL with Ca and Mg-free Hank’s balance salt solution , hPDL was digested with 3 mg/ml type I collagenase  and 4 mg/ml dispase II (Gibco) in alpha modification minimum essential medium eagle (α-MEM)  for 1 h at 37 °C. The reaction was stopped by the addition of α-MEM. PDL derived from different subjects were pooled together to obtain single cell suspensions by passing the cells through a 70 μm strainer . Cells were centrifuged, and the pellet was resuspended in in serum-containing media , composed of α-MEM supplemented with 15% calf serum (Sigma), 100 units/ml penicillin-streptomycin (Sigma) and 2 mM l-glutamine (Sigma). The cell suspension was plated into six-well multiwell plates  and incubated at 37 °C in 5% CO2. The cells at passage 3–4 were then used for the following experiments. We used µ-Dish 35 mm, high Grid-500 (Ibidi) for live cell imaging. Numeric marks on the bottom of each dish allow users to identify the location of cells.Dissociated cell cultures of hPDL tissue were prepared as previously describedTo induce neural differentiation, cells were cultured in serum-free media , consisting in Dulbecco’s modified Eagle’s medium/F12  supplemented with bFGF , EGF , glucose , N2-supplement (Gibco), 2 mM l-glutamine (Sigma), and 100 units/ml penicillin-streptomycin (Sigma). Cells at passage 3–4 were allowed to adhere to the plates overnight. Media was removed the following day and neural induction media was added directly to the cells. Neural induction media were changed every 3 days until the end of the experiment (2 weeks).18, we showed that several stem cell and neural crest cell markers are expressed in human adult periodontal ligament (hPDL) tissue and hPDL-derived cells.In previous publication18. Cells were plated onto collagen IV (Sigma) coated plastic or glass coverslips, and maintained in basal media or neural induction media. Cells were rinsed with PBS and fixed in freshly prepared 4% paraformaldehyde . Fixed cells were blocked for 1 h in PBS containing 10% normal horse serum (Gibco) and 0.25% Triton X-100 (Sigma) and incubated overnight at 4 °C with antibodies against: β-III-tubulin , Tau , MAP2 , Connexin-43 , Synaptophysin , Synapsin1 , Fibrillarin  and Lamin A/C  in PBS containing 1% normal horse serum and 0.25% Triton X-100. On the next day, cells were rinsed and incubated with the corresponding secondary antibodies: Alexa Fluor® 488 , Alexa Fluor® 594 , biotinylated anti-rabbit , biotinylated anti-chicken . Cell nuclei were counterstained with DAPI . Alexa Fluor 488® phalloidin was used to selectively stains F-actin (Molecular Probes). Data are representative of ten independent experiments per condition.A standard immunocytochemical protocol was used as previously described18. hPDL-derived cells  were harvested using trypsin/EDTA (Gibco), washed twice with PBS, resuspended in RIPA lysis buffer (Millipore) in the presence of protease inhibitors  and PMSF 1 M (Abcam) for 30 min at 4 °C. Protein concentration was determined using the bradford protein assay (Sigma-Aldrich). Proteins were separated in 8% SDS-polyacryamide gel (PAGE-SDS) and transferred to a nitrocellulose membrane (Whatman). PageRuler™ Prestained Protein Ladder (Thermo Scientific) has been used as size standards in protein electrophoresis (SDS-PAGE) and Western-Blotting. After transfer, nitrocellulose membranes were stained with Ponceau S solution (Sigma-Aldrich) to visualize protein bands. Blots were then incubated over-night at 4 °C with rabbit antibody against β-III-tubulin . Secondary antibody was used at 1:7000 for peroxidase anti-mouse Ab . Immunoreactivity was detected using the enhanced chemiluminescence (ECL) Western blot detection system (Amersham Biosciences Europe) and LuminataTM Forte (Millipore corporation) using ImageQuant LAS 500 Gel Documentation System . The molecular weight of β-III-tubulin is approximately 55 kDa.A standard Western Blott protocol was used as previously described18. Experiments were carried out according to the guidelines of the European Community (Directive 86/609/ECC) and in accordance with the Society for Neuroscience recommendations. Animals used in this study were 12-week-old immune-suppressed mouse , housed in a temperature and humidity controlled room, under a 12 h light/dark cycles, with ad libitum access to food and water. The animals were anesthetized and intracardially perfused with freshly prepared, buffered 4% PFA . Brains were removed, post-fixed for 12 hr in the same fixative at 4 °C and dehydrated in 30% sucrose solution at 4 °C until sunk. 30 µm thick coronal sections were collected using a freezing microtome. Serial sections were used for DAPI staining. Free-floating sections were incubated and mounted onto Superfrost Plus glass slides (Thermo Scientific). The slides were dried O/N and coverslipped with mowiol-NPG .A standard immunohistochemistry protocol was used as previously describedAnalyses and photography of visible and fluorescent stained samples were carried out in an inverted Leica DM IRB microscope equipped with a digital camera Leica DFC350FX (Nussloch) or in confocal laser scanning microscope Leica TCS-SP8. Digitized images were analyzed using LASX Leica confocal software. Z-stacks of confocal fluorescent images were also analyzed to calculate the nuclear volume by using ImageJ software.We used Photoshop software to improve the visibility of fluorescence images without altering the underlying data. The fluorescent photographs presented in this study are original fluorescence images and inverted images (negatives) that are produced from the original fluorescence images.Cells were plated onto collagen IV (Sigma) coated glass coverslips and maintained in basal media or neural induction media. Cells were treated with fixative for 20 minutes. Coverslips were postfixed in 1% osmium tetroxide for 1 hour and dehydrated in graded ethanol washes. The coverslips were allowed to dry at a conventional critical point and were then coated with gold-palladium sputter coated. Coverslips were view on a Jeol 6100 scanning electron microscope.hPDL-derived cells were harvested using trypsin/EDTA (Gibco) and were treated with fixative for 60 minutes. Cells were postfixed in osmium tetroxide solution, dehydrated embedded in resin. Ultrathin sections (70–90 nm) were cut, stained with lead citrate, and examined under Jeol 1011 and Philips Tecnai 12 transmission electron microscopes.Supplementary information


DNA replication is an essential process occurring prior to cell division. Cell division coupled to proliferation ensures the growth and renewal of a large variety of specialized cell types generated during embryonic development. Changes in the DNA replication program occur during development. Embryonic undifferentiated cells show a high replication rate and fast proliferation, whereas more differentiated cells are characterized by reduced DNA synthesis and a low proliferation rate. Hence, the DNA replication program must adapt to the specific features of cells committed to different fates. Recent findings on DNA synthesis regulation in different cell types open new perspectives for developing efficient and more adapted therapies to treat various diseases such as genetic diseases and cancer. This review will put the emphasis on recent progress made in this field. Replication origin activation in space and time (or replication program) is set by a sequence of events, starting already at the end of mitosis, lasting through G1 phase, and ending in S phase when DNA replication is activated. These coordinated events ensure that the full genome will be replicated before mitosis since a faithful DNA synthesis is a prerequisite for genome integrity maintenance2. DNA replication initiates from thousands of replication origins scattered along the chromosomes. Origins acquire the competence to replicate in a step called “licensing” that involves formation of a pre-replication complex (pre-RC), including loading of the replicative helicase MCM2-7 , Cdc6, and Cdt1 proteins onto chromatin18. Two hexamers of the MCM2-7 helicase complex then are loaded in an inactive state prior to S phase 29. Notably, this population of shared origins tends to initiate early during S phase, whereas cell type–specific origins initiate during late S-phase and are linked with compacted chromatin marks28. These observations underline the relationship between chromatin modifications with the cellular context in origin selection.It is now clear that most replication origins being constitutively activated across multiple metazoan cell types (hereafter named “shared origins”) are associated with unmethylated CpG islands, G-rich elements, transcriptional start sites, and histone modifications related to open chromatin marks  cells, depletion of the histone H1 perturbs the landscape of replication origin activation37. Because chromatin environment changes during cell fate commitment and in different cell types, it is generally assumed that the DNA replication program is coordinated with transcription to avoid transcription–replication conflicts and thus preserve genomic integrity. This seems particularly crucial during the onset of developmental programs and cell lineage specification. InCaenorhabditis elegans, origins from rapidly replicating pluripotent embryos coincide with open chromatin regions of highly transcribed genes, whereas establishment of the new transcriptional program, occurring at later embryonic stages when cell differentiation begins, correlates with the reorganization of replication initiation sites38.Two independent studies describing reconstitution of DNA replication initiation events1 compared with their differentiated counterparts, which impacts on the licensing step. ES cells recruit much more MCM2-7 to the chromatin than tissue-specific stem cells or progenitor cells39. This excess of origins that remain dormant during S phase appears to be important to maintain pluripotency41 and could also protect the genome against DNA replication stress occurring in ES cells42.Permissive chromatin features for rapid activation of replication origins could influence the cell cycle length. ES cells have a shorter G1 length, suggesting that pre-RC binding is developmentally regulated. Indeed, quantitative single-cell analysis performed in human cells by Matson and colleagues demonstrated that origins are licensed faster in pluripotent cells compared with their isogenic differentiated counterparts and that loading of the MCM2-7 helicase slows down as G1 duration is extended43. The authors revealed that high expression of the licensing factor Cdt1 was important for fast MCM2-7 loading rates, thus enabling ES cells to rapidly license origins prior to the G1-to-S phase transition. Similar to the need for additional dormant origins, fast licensing kinetics seems essential to ensure pluripotency in ES cells and induced pluripotent stem cells. This observation was corroborated by Carroll and colleagues in intestinal stem cells from adult tissues44. The authors demonstrated that licensing is interconnected with the proliferative commitment of stem cells. They found that Lgr5+ intestinal stem cells, which contribute primarily to the renewal of the intestinal epithelium, reside mostly in a G1, unlicensed state, although they express MCM2 and other proliferative markers to a level equivalent to the licensed population. Interestingly, this unlicensed state correlates with an elongated cell cycle that could be considered as a backup mechanism to sustain proliferative fate decisions and tissue maintenance. Nonetheless, the link between chromatin structures and adaptation to G1 length in such a context remains to be determined.Besides changes in the kinetics by which MCM2-7 is loaded onto chromatin, licensing control adapts to G1 length with licensing kinetics has been observed during oncogenic transformation. Overexpression of oncogenes, such as cyclin E and c-MYC, shortens G1 and forces cells to engage S phase earlier with incomplete licensing. Consequently, the replication program is perturbed, leading to accumulation of replication stress46. Origin usage following oncogene activation was recently investigated genome-wide. The authors found that in these conditions new initiation zones, which were normally suppressed by transcription in G1, appear in intragenic regions47. These results also confirm an observation indrosophila showing that active transcription modulates MCM2-7 distribution48. Nonetheless, how RNA polymerases inactivate and redistribute MCM2-7 remains to be determined. It has been reported that DNA translocases and RNA polymerases in yeast are able to push MCM2-7 along DNA, but similar processes have not yet been described in metazoans49. An emerging picture from these studies is that cell cycle length, local chromatin structure, and active transcription can modulate the extent of origin licensing.Uncoupling of G50. Chromatin environment contributes to licensing as described previously through MCM2-7 loading but also to origin selection through MCM2-7 activation52. The dynamics of origin activation during S phase follow a spatio-temporal order known as replication timing  are prone to form G-quadruplexes (G4s). Their presence in the genome is associated with origin activity54. Nucleosome organization at the proximity of origins was reported to modulate both origin licensing (as described above) and MCM2-7 helicase-dependent activation steps of initiation55 in yeast and more recently in mammals37. Different histone modifications and certain histone modifiers have been assigned in origin selection and the dynamics of their activation across S phase60.During S phase, origins are activated by the combined activities of cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) kinases  targetin timing . Replica61. More recently, a great deal of investigation has focused on the contribution of chromatin regulators in controlling the timing of activation through chromatin organization. The telomere-associated protein Rif1 interferes with DDK-dependent phosphorylation of MCM2-7 and modulates nuclear architecture by anchoring heterochromatin63. Rif1 might organize replication-timing domains through its association with G4s to repress pre-RC activation until late S-phase65. The ORC-associated protein ORCA/LRWD1 establishes a repressive chromatin environment at a subset of origins, thus priming them for late replication66. The replication-initiation determinant protein RepID initiates replication in a sequence-specific manner by interacting with a subgroup of origins67.Several histone-modifying enzymes such as the methyltransferase PR-Set7 and the acetyltransferase HBO1 in complex with BRPF3 have been reported to have a role in activation of particular replication origins71. These constitute structurally distinct chromatin domains covering several megabases where DNA interactions are favored. TADs show a strong correlation with replication timing domains in which several replication units are concomitantly activated. Nuclear reorganization that accompanies differentiation and the establishment of developmental programs result in changes in replication timing of the human genome73; notably, alterations in replication timing have been detected in several diseases75.Modulation of origin activation in a cell type–specific manner depends upon global changes in chromosomal architecture and mainly occurs within large chromosome domains known as Topologically Associating Domains (TADs)77. Origins that are activated in a cell type–dependent manner appear to replicate late in S phase28. Histones and chromatin modifications described above impose a crosstalk between replication and transcription and are interconnected during differentiation and development38 although this relationship remains enigmatic.Therefore, flexibility in origin activation seems critical to accommodate the dynamic changes in the transcriptional program and genome repositioning . Indeed,78. Moreover, perturbations in DNA replication during development can cause epigenetic changes  potentially inherited by the next generations79.Origin activation dynamics that determine replication timing therefore might play a role in establishing local and global chromatin structure to facilitate the cellular response to the differentiation process. Remarkably, establishment of TADs during early development in mammals requires DNA replication but not transcription80.Chromatin organization immediately following fertilization in vertebrates is particularly critical for DNA replication initiation and this occurs in a transcription-independent manner following fertilization85. The Sld3 vertebrate homolog Treslin/TICRR, a CDK target that acts as a binding site for TopBP1 and Mdm2 binding protein MTBP (both proteins being required for GINS-CDC45 recruitment), was proposed to link chromatin acetylation to DNA replication initiation efficiency and timing in different cancer cell lines86. This observation shows that Rif1 is not the only known site-specific regulator of DNA replication initiation and timing. Likewise, a function in origin efficiency has been assigned to the Treslin partner MTBP, probably through its ability to localize Treslin near G4 structures87. Finally, Rif1 accessibility to chromatin was recently implicated in determining the onset of late replication during embryonic development88.The capacity to selectively modulate origin usage in a cell type–specific manner suggests that the proteins involved in origin activation might play specific functions. Indeed, replication initiation proteins, and their availability during S phase, can be involved in dictating specificity in origin activationin vitro reconstitution assays using human proteins will undoubtedly bring to light new exciting concepts in this field.Overall, these studies suggest a functional interaction between components of the replication machinery with chromatin modifiers leading to reorganization of the genome architecture during development. In turn, reorganization of the chromatin architecture defines cell type–specific transcriptional programs that feedback on the availability of replication proteins but equally on the replication timing by shaping chromatin architecture in specific domains such as TADs. Hence, genetic and epigenetic features set during developmental transitions may function as selective ways to repress or activate licensing or firing (or both) of a subset of origins, potentially within replication timing domains. Precise origin mapping at the single-molecule level in a single cell  completed by90. Lengthening of the total cell cycle time is achieved mostly by extension of the G1 phase and moderately the S phase91.A large variety of cell types critical for tissue function are formed during early embryonic development, when cell commitment first takes place. The proliferative state decision is variable for each cell type and may influence cell cycle duration. Consequently, steps required for proliferation, such as genome duplication, must be tightly regulated with cell cycle progression to maintain homeostasis. For instance, embryonic cell division in metazoans exhibits dramatic lengthening of the cell cycle at the onset of gastrulation, a stage required for cell type specialization and embryo patterning93. Analysis of the replication machinery at the forks by isolation of proteins on nascent DNA (iPOND) allows comparison of protein abundance at replication forks in different contexts. It was shown that replisomes of pluripotent stem cells contain a particular protein network that accommodates a high proliferative capacity with short cell cycle phases and reduced endogenous DNA replication stress94. Factors involved in DNA repair, such as mismatch repair, but equally pluripotency and epigenetic inheritance factors, such as the NuRD-HDAC complex, were found to be enriched at replication forks. Interestingly, independent studies confirmed the requirement of a NuRD complex for DNA replication during early embryonic development95. ES cells seem to require additional factors at the replication forks to cope with DNA replication perturbations. Thus, Filia-Floped was identified by iPOND as a new protein complex involved in resolution of stalled forks during a normal S-phase in mouse ES cells compared with their differentiated counterparts96. It is likely that strategies set in stem cells to ensure fast DNA replication may also be exploited in other biological contexts requiring fast proliferation, including very early embryogenesis and tumorigenesis. For example, the Rad18 E3 ubiquitin ligase, a master regulator of translesion DNA synthesis, is an abundant component of the DNA replication machinery duringXenopus early development and confers to the replisome the ability to hijack DNA lesions and suppress the DNA damage response, ensuring fast cell cycle progression. Remarkably, Rad18 was found to be highly expressed in glioblastoma cancer stem cells97.A variation in replisome composition following DNA replication perturbations or between different cell types was unraveled thanks to recent advances in quantitative proteomics at the replication forksa la carte” regarding the biological features set by specific cell types. Consistent with this idea, replication of specific genome locations like telomeres, common fragile sites, or centromeres has been shown to involve particular DNA replication partners at the vicinity of the DNA replication fork to ensure stability of those genomic regions100.Overall, these recent observations converge toward the idea that replisome composition can be selected “101. Metazoan origins lack consensus sequences and are associated with different structural features that confer cell type–specific replication. Additional mechanisms operate to fine-tune pre-RC assembly, origin activity, and replisome composition. This allows cells to coordinate a fast replication program with their fate, contributing to genome integrity maintenance despite DNA replication perturbations often observed during tumorigenesis and development. Identification of various factors involved in origin selection and their activity in a cell type–specific manner is a great source of interest. Such factors may often be deregulated during cancer development; thus, their targeting might constitute effective anti-cancer therapies.A defective DNA replication program is the source of several pathologies during development (Meier-Gorlin syndrome) and during the adult life (carcinogenesis)in vitro reconstitution assays using vertebrate proteins, and genomic approaches on single cells should largely contribute to breakthrough findings in the field.Nevertheless, these discoveries are only the tip of the iceberg as limited information is currently available regarding their regulation. Biochemistry, electron cryomicroscopy analysis,CDK, cyclin-dependent kinase; DDK, Dbf4-dependent kinase; ES, embryonic stem; G4, G-quadruplex; iPOND, isolation of proteins on nascent DNA; ORC, origin recognition complex; pre-RC, pre-replication complex; TAD, topological associated domain


We recently reported dynamic epigenetic markers of fibrosis detectable in patients’ plasma that may have utility in non-invasive diagnosis and staging of fibrosis in patients with chronic liver disease.The original study posed several important outstanding questions: (1) Can ccfDNA methylation be used as a biomarker of fibrosis in liver diseases of other aetiologies? (2) Does the presence of hepatocellular carcinoma (HCC) alter the biomarker in plasma? (3) Does presence of fibrosis in other organs generate similar biomarker profiles?In the present letter, we answer these questions and demonstrate the broader utility of DNA methylation at three CpG dinucleotides within PPARγ promoter in several new patient cohorts . Employi10.1136/gutjnl-2017-315668.supp1Supplementary file 1We next determined if hypermethylation is specific to fibrosis of liver origin. To this end, we quantified ccfDNA methylation in a cohort of patients with limited and diffuse systemic sclerosis (SSc) who have various combinations of skin, lung and kidney fibrosis, but no hepatic fibrosis.This important validation study supports our original hypothesis that hypermethylation at the PPARγ gene promoter is a marker for fibrotic progression of chronic liver disease and holds true for viral, alcoholic and metabolic disease aetiologies. As fibrosis in other organs does not generate a similar epigenetic signature, it is likely that the PPARγ hypermethylation specifically reflects a liver pathology. The ability to detect and quantify hypermethylation at the promoter of the PPARγ in ccfDNA as a new liquid biomarker that specifically reports the fibrotic progression of liver diseases of multiple aetiologies offers the potential for a cost-effective blood-based liquid biomarker of liver fibrosis.


Most type 2 diabetes-associated genetic variants identified via genome-wide association studies (GWASs) appear to act via the pancreatic islet. Observed defects in insulin secretion could result from an impact of these variants on islet development and/or the function of mature islets. Most functional studies have focused on the latter, given limitations regarding access to human fetal islet tissue. Capitalising upon advances in in vitro differentiation, we characterised the transcriptomes of human induced pluripotent stem cell (iPSC) lines differentiated along the pancreatic endocrine lineage, and explored the contribution of altered islet development to the pathogenesis of type 2 diabetes.q < 0.01, log2 fold change [FC] > 1) were assigned to the stages at which they were most markedly upregulated. We used these data to characterise upstream transcription factors directing different stages of development, and to explore the relationship between RNA expression profiles and genes mapping to type 2 diabetes GWAS signals.We performed whole-transcriptome RNA sequencing of human iPSC lines from three independent donors, at baseline and at seven subsequent stages during in vitro islet differentiation. Differentially expressed genes  were notably enriched in genes differentially expressed at the posterior foregut stage (q = 0.002), as calculated by gene set enrichment analyses. In a complementary analysis of enrichment, genes differentially expressed in the final, beta-like cell stage of in vitro differentiation were significantly enriched  for genes within the credible intervals of type 2 diabetes GWAS loci.We identified 9409 differentially expressed genes across all stages, including many known markers of islet development. Integration of differential expression data with information on transcription factor motifs highlighted the potential contribution of The present study characterises RNA expression profiles during human islet differentiation, identifies potential transcriptional regulators of the differentiation process, and suggests that the inherited predisposition to type 2 diabetes is partly mediated through modulation of islet development.Sequence data for this study has been deposited at the European Genome-phenome Archive (EGA), under accession number EGAS00001002721.The online version of this article (10.1007/s00125-018-4612-4) contains peer-reviewed but unedited supplementary material, which is available to authorised users. Our understanding of the genetic contribution to pathogenesis of type 2 diabetes has been greatly facilitated by genome-wide association studies (GWASs). These have identified over 100 genomic regions showing a robust association to disease risk .Most functional follow-up of GWAS signals has involved studies in adult islets and/or a variety of beta cell lines, but there is mounting evidence that some of the implicated genetic variants influence islet development . For exaUntil recently, restricted access to human fetal material constrained the study of islet development to murine models. However, key differences between human and murine islet development , togetheIslet differentiation protocols are rapidly improving , 10 and www.stembancc.org) (see ESM Human induced pluripotent stem cell (iPSC) lines from three independent individuals without diabetes were obtained from the StemBANCC consortium  was received from the National Research Ethics Service South Central Hampshire A research ethics committee (REC 13/SC/0179).2, and passaged as single cells every 3–4 days or when confluent. In vitro differentiation involved the timely addition of recombinant growth factors and small molecules to sequentially generate cells representing key developmental stages of the endocrine pancreas: definitive endoderm, primitive gut tube, posterior foregut, pancreatic endoderm, endocrine progenitors, endocrine-like cells and beta-like cells. The differentiation protocol was carried out as described by Rezania and colleagues  and pancreas/duodenum homeobox protein 1 [PDX1]); and endocrine-like cells   as the transcriptome reference, using STAR v.2.5 . Mo. Mo20]. Enrichment analysis was implemented in two ways: as a hypergeometric test in R  or using the gene-scoring function in MAGENTA  followedFor the hypergeometric test, we analysed the differentially expressed genes from each differentiation stage for enrichment in genes mapping to type 2 diabetes or fasting glucose GWAS signals, which were defined as protein-coding and lincRNA genes located within specified distance bins  surrounding the credible intervals for trait-associated loci. Credible intervals were defined by the boundaries of the 99% credible sets of variants  from DIAp values for each gene was tested in GSEA  , and theGSEA ESM .To determine whether the differentiated cells followed normal islet development, we profiled gene expression patterns across iPSC and seven subsequent developmental stages in lines from three independent donors  differentiated in parallel. Each iPSC line successfully generated cells recapitulating key developmental stages of the endocrine pancreas as confirmed by the expression of known marker genes from developing and adult beta cells  and ‘hormone transport’ (q = 2.0 × 10−5).Principal component analysis of the transcriptome showed that the beta-like cells generated in the current study clustered more closely with in vivo-matured islet-like cells  than celMAFA, which was completely absent in our previous differentiation protocol, and INS, whose high expression indicates the correct differentiation towards the last stage of beta cell development.Overall, cells generated in this study, compared with those previously reported , are morq < 0.01, absolute log2FC > 1) across all stages, ranging in number from 623 in the primitive gut tube stage to 2773 in iPSCs  and ‘regulation of insulin secretion’ in beta-like cells (q = 2.0 × 10−4).To characterise the transcriptomic landscape of each developmental stage in the in vitro-differentiated cells produced in this study, we assigned significantly differentially expressed genes to the stage at which they were most upregulated: if expression peaked in iPSCs, the gene was assigned to that stage see . We deteGATA6, for example, was differentially expressed at the definitive endoderm stage , whereas GATA4 was differentially expressed in posterior foregut cells ; the later expression of GATA4 could contribute to the less severe phenotype of individuals carrying GATA4 vs GATA6 mutations [The expression patterns of monogenic diabetes genes can point towards stages at which disruption of islet development has long-term consequences for glucose homeostasis. Of 28 genes implicated in monogenic or syndromic diabetes , 24 wereutations , 30.LMNA, for example, encodes a nuclear membrane protein involved in chromatin structure and nuclear stability; it has been implicated in the function and development of many tissues [LMNA mutation is mostly driven by altered adipose tissue deposition and insulin resistance [LMNA expression during in vitro islet differentiation  may indicate an additional impact on islet development [The differentiation model used in this study also sheds light on the developmental role of monogenic diabetes genes with lesser described roles.  tissues . The diasistance . Howeverelopment .HNF1B, which showed iRegulon enrichment of its targets at the primitive gut tube stage (NES 3.0–5.7 [see SMAD7 [ID2 [GGCX) [The developmental competence of differentiating cells is in part driven by a subset of transcription factors that initiate and regulate changes in response to external stimuli, as highlighted by the many monogenic diabetes genes that are also transcription factors. To identify potential upstream transcriptional regulators active at each stage of islet development, we performed a WGCNA and determined the enrichment of transcription factor binding motifs and ChIP sequencing signals near differentially expressed genes using iRegulon see . This an–5.7 see ). Some oee SMAD7 , ID2 [352 [GGCX) .REST peaks in the intermediate steps of in vitro differentiation and declines at the endocrine-like cell and beta-like cell stages, with reciprocal expression patterns seen among its predicted targets. These targets include genes encoding neurexins  and subunits of the glutamate receptor channels  implicated in insulin exocytosis [REST to the same cluster as TCF7L2 and other genes from the Wnt signalling pathway, such as TCF7, TCF3 and TCF12 [Analysis of the sets of stage-specific differentially expressed genes also highlighted the targets of transcription factors with less-well studied roles in human islet development. For example, expression of the transcriptional repressor ocytosis , 38. Cornd TCF12 . This pand TCF12 . These dnd TCF12  stages ond TCF12 , 42.TCF7L2 maps to the type 2 diabetes-associated locus with the largest common effect on disease risk [TCF7L2 expression . The expression of several other Wnt family members also peaks at the posterior foregut stage; these include the coactivator CREBBP, the binding sites of which are significantly enriched in type 2 diabetes-associated loci [HHEX, which maps to a prominent type 2 diabetes-risk locus and is implicated in foregut development [TCF7L2 and HHEX loci may influence early expression of these genes, which could affect development in multiple metabolic tissues. This view is supported by cellular and murine studies indicating that TCF7L2 regulates beta cell development and function [Hhex is essential for the differentiation of the posterior foregut into the liver in mice [ase risk . Analysited loci , and HHEelopment . In the elopment . Allelesfunction , includifunction , as wellfunction . Similar in mice , yet is  in mice .Thus, several key functional candidates mapping within type 2 diabetes GWAS signals, in addition to those which overlap known monogenic diabetes genes, appear to be active during this early critical window of pancreatic development. Studying these and other diabetes-relevant genes in stem cell-derived models can help to decipher the role of multiorgan developmental effects on pathogenesis of diabetes. By integrating the differential expression data with genomic annotations on transcription factor binding and clustering of longitudinal expression, we identified novel potential regulators orchestrating gene expression patterns within the different developmental stages. Such transcriptomic analysis can also illuminate the mechanisms of action for monogenic diabetes genes and inform the search for novel MODY genes that influence the same pathways.Most of the more than 100 type 2 diabetes susceptibility loci identified to date [q = 0.002, Fig. q = 0.001) if the GWAS genes also implicated in monogenic diabetes  per stage, we compared the most differentially expressed genes at each stage for enrichment among type 2 diabetes GWAS loci; this analysis highlighted the beta-like cell stage  after monogenic diabetes genes had been excluded.We first concentrated on genes whose coding sequence was at least partly contained within 99% credible intervals from type 2 diabetes GWAS fine-mapping efforts on the basis that these represented a set of genes likely to be substantially enriched for type 2 diabetes effector transcripts see . Of the p value =0.049; Fig. p value =0.302). We repeated the enrichment test using a subset of 15 type 2 diabetes GWAS loci for which the evidence from physiological studies points most emphatically to risk of type 2 diabetes mediated via reduced insulin secretion  after excluding the monogenic diabetes genes and those within the same credible interval. Using the same approach of sampling from the hypergeometric distribution, we also detected enrichment for genes mapping to credible intervals for 16 loci significantly associated with fasting glucose  based approaches that allow their experimental manipulation in in vitro systems . Stem ceESM(PDF 1364 kb)ESM Table 4(XLSX 2730 kb)ESM Table 5(XLSX 627 kb)ESM Table 6(XLSX 4224 kb)ESM Table 7(XLSX 295 kb)ESM Table 8(XLSX 45 kb)ESM Table 9(XLSX 206 kb)ESM Table 10(XLSX 16 kb)