Motoneurons (MNs) control muscle contractions, and their recruitment by premotor circuits is tuned to produce accurate motor behaviours. To understand how these circuits coordinate movement across and between joints, it is necessary to understand whether spinal neurons pre-synaptic to motor pools have divergent projections to more than one MN population. Here, we used modified rabies virus tracing in mice to investigate premotor interneurons projecting to synergist flexor or extensor MNs, as well as those projecting to antagonist pairs of muscles controlling the ankle joint. We show that similar proportions of premotor neurons diverge to synergist and antagonist motor pools. Divergent premotor neurons were seen throughout the spinal cord, with decreasing numbers but increasing proportion with distance from the hindlimb enlargement. In the cervical cord, divergent long descending propriospinal neurons were found in contralateral lamina VIII, had large somata, were neither glycinergic, nor cholinergic, and projected to both lumbar and cervical MNs. We conclude that distributed spinal premotor neurons coordinate activity across multiple motor pools and that there are spinal neurons mediating co-contraction of antagonist muscles. We are able to walk, run and move our bodies in other ways thanks to circuits of neurons in the spinal cord that control how and when our muscles contract and relax. Neurons known as premotor neurons receive information from other parts of the central nervous system and control the activities of groups (known as pools) of motor neurons that directly activate individual muscles.To bend a joint or move our limbs, the movement of different muscles needs to be coordinated. Previous studies have focused on how premotor neurons activate a pool of motor neurons to contract a single muscle, but it remains unclear if and how some of these premotor neurons can co-activate different pools of motor neurons to control more than one muscle at the same time. Here, Ronzano, Lancelin et al. injected mice with modified rabies viruses labelled with different fluorescent markers to build a map of the premotor neurons that connect to motor neurons controlling the leg muscles.The experiments revealed that many of the individual premotor neurons in the spinal cords of mice connected to different pools of motor neurons. In the upper region of the spinal cord – which is primarily responsible for controlling the front legs – some large premotor neurons activated motor neurons in this region as well as other motor neurons in a lower region of the spinal cord that controls the back legs. This suggests that these large premotor neurons may be important for coordinating muscles contraction within and between limbs.Many neurological diseases are associated with difficulties in contracting or relaxing muscles. For example, individuals with a condition called dystonia experience disorganized and excessive muscle contractions that prevent them from being able to bend and straighten their joints properly. By helping us to understand how the body coordinates the activities of multiple limbs at the same time, the findings of Ronzano, Lancelin et al. may lead to new lines of research that ultimately improve the quality of life of patients with dystonia and other similar neurological diseases. The spinal cord is ultimately responsible for organizing movement by controlling the activation pattern of motoneurons (MNs), which in turn produce appropriate patterns of muscle contractions to produce limb movement. Across any single limb joint, there are fundamentally three types of control – or three ‘syllables of movement’ – possible. The three basic syllables are: (1) changing a joint angle, (2) stiffening a joint, and (3) relaxing a joint. The concatenation of these syllables across joints within and between limbs ultimately produces behaviour .To change a joint angle, MNs innervating synergist muscle fibres are activated whilst those that innervate antagonist muscle fibres are inhibited. This ‘reciprocal inhibition’  is mediaThe other two syllables are less well studied, but it is clear that behavioural joint stiffening requires co-activation of MNs innervating antagonist muscle groups, while joint relaxation would require co-inhibition of these MNs. Co-contraction has largely been thought to result from brain activity , whereasTo identify whether these syllables are produced by spinal circuits, several questions can be asked: Does the spinal cord contain circuits that lead to co-activation or co-inhibition of different pools of MNs – either synergists or antagonists? Does each motor pool have its own dedicated population of premotor INs, and are these INs interconnected in such a way that they can produce contraction of different muscle groups? Or are there populations of INs that project to multiple motor pools in order to effect contraction (or relaxation) of multiple muscles? Indeed, INs that have activity in keeping with innervation of multiple synergists, leading to motor ‘primitives’ or synergies  have beeNormal behaviours in quadrupeds as well as bipeds require coordination of syllables across joints between forelimbs and hindlimbs. This coordination relies on populations of propriospinal neurons projecting in either direction between the lumbar and cervical enlargements . Long deIn the present study, we examine circuits underlying co-activation and co-inhibition in the spinal cord by assessing premotor neurons through the use of RabV tracing techniques . We usedn = 4, two extensor and two flexor pairs). Notably, five MNs were double labelled, most likely due to secondary infection of synaptically connected MNs , one of them being double labelled  or antagonist (n = 3 pairs) pairs of muscles, double-labelled premotor INs were distributed similarly  and extensor (LG) muscles, 260 MNs were labelled  boutons in apposition to L1 , as welln = 4, 2 extensor and two flexor pairs, n = 3 antagonist pairs, In order to maintain posture and stability, trunk muscles are coordinated with hindlimb movements. Neurons in the thoracic cord that are premotor to lumbar MNs have previously been described ; we thusn = 4 synergist and 42/59, n = 3 antagonist pairs, In all animals (7/7), most divergent premotor neurons in the thoracic cord were located in the ipsilateral dorsal quadrant  have been shown to modulate interlimb coordination to provide stability . Given tn = 7, four synergist and three antagonist pairs, n = 4 synergist pairs and 47.9% ± 7.1 % per animal, total of 19/37 neurons, n = 3 antagonist pairs, 2, n = 38 premotor LDPNs) compared to the double-labelled premotor neurons in the thoracic and lumbar cords . On average, the cross-sectional area of divergent cervical LDPNs was comparable to that of cervical MNs  and extensor (LG and MG) MNs were localized throughout the rostro-caudal extent of the ventral cervical cord with an enrichment between C6 and T1 . Of 92 pn = 3 LG injections, n = 3 LG injections, To determine the neurotransmitter phenotype of the premotor LDPNs, we used single ΔG-Rab-mCherry injections in ChAT-Cre;RΦGT mice crossed with mice expressing eGFP under the control of the promoter for the neuronal glycine transporter GlyT2 . GlyT2 iC, cholinergic  and may be detectable at this early postnatal stage  with ΔG-Rab-mCherry, and extensor hindlimb GS with ΔG-Rab-eGFP.2, p = 0.056, n1 = 16 premotor LDPNs infected from both homolateral forelimb and hindlimb injections vs n2 = 38 divergent premotor LDPNs infected from dual hindlimb injections (see above), Mann–Whitney test; Since it has been suggested that LDPNs participate in ipsilateral control of forelimb and hindlimb , we sougGiven the involvement of LPDNs in the diagonal synchronization of forelimb and hindlimb during locomotion , we alsoWhile sharing similar features with the LDPNs infected from dual hindlimb injections, it remains to be determined whether these neurons premotor to hindlimb and forelimb muscles form a homogenous population with the divergent LDPNs.n = 3, Having identified a population of divergent premotor LDPNs with projections from the cervical to the lumbar region, we next investigated whether ascending propriospinal neurons projecting from the lumbar or thoracic segments to cervical MNs could be identified. Following FMs injections, ascending premotor INs were observed throughout the cord . There were very few (<1%) bifurcating (ascending/descending) premotor neurons in the thoracic cord after injections in homolateral GS and FMs . This distribution of lumbar premotor LAPNs is different from that of cervical premotor LDPNs, which were almost exclusively ventral . Of the 117 lumbar premotor LAPNs identified, 10 were also labelled from GS injections, indicating that some neurons projected both to local lumbar MNs as well as to cervical MNs  in the lumbar cord, about half of which were localized in the dorsal ipsilateral quadrant . Of these, however, 10/12 were in the ventral contralateral quadrant , a stiffening of a joint (requiring co-activation of flexors and extensor MNs), and a relaxation of a joint (requiring co-inhibition of flexor and extensor MNs). While neural circuits for reciprocal inhibition have been well studied over many decades , circuitn = 27) were performed according to the Animals (Scientific Procedures) Act UK (1986) and certified by the UCL AWERB committee, under project licence number 70/7621. Homozygous ChAT-IRES-Cre mice . The supernatant was collected and medium was added for another cycle (three cycles maximum), after which the supernatant was filtered (0.45 µm filter) and centrifuged 2 hr at 19,400 rpm (SW28 Beckman rotor). The pellets were re-suspended in phosphate-buffered saline (PBS) and centrifuged together at 21,000 rpm, 4°C, 4 hr in a 20 % sucrose gradient. Pellets of each collection were then re-suspended and stored in 5–10 µl aliquots at –80°C.We used the glycoprotein G-deleted variant of the SAD-B19 vaccine strain rabies virus (a kind gift from Dr M. Tripodi). Modified RabV (ΔG-Rab) with the glycoprotein G sequence replaced by mCherry or eGFP (ΔG-Rab-eGFP/mCherry) was produced at a high concentration with minor modifications to the original protocol . BHK cel5 cells/ml and incubated overnight at 37°C and 10 % CO2 (growth). The virus was prepared for two serial dilutions with two different aliquots and added in the well after an equal volume of medium had been removed  and incubated 48 hr at 35°C and 3 % CO2. The titre was determined from the count of cells in the higher dilution well and was between 109 and 1010 infectious units (IU)/ml.Virus titration was performed on BHK cells plated in 10 % FBS medium at 1.5 × 109 and 1010 IU/ml. The incisions were closed with vicryl suture, and the mice were closely monitored for 24 hr post-surgery. Mice were perfused 9 days after the injections. Due to the proximity of synergist pairs of muscles, prior to spinal tissue processing, we dissected the injected leg and confirmed that there was no contamination of virus across the injected muscles or in adjacent muscles below or above the knee. When injecting FMs, we could not target a single muscle. To visualize which muscles had been infected, we carefully dissected each FM and assess for the presence of fluorescent signal  was given to the neonatal pups (P1–P3) prior to surgery and all procedures were carried out under general isoflurane anaesthesia. After a skin incision to expose the targeted muscle, the virus (1 µl) was injected intramuscularly using a Hamilton injector (model 7652-01) mounted with a bevelled glass pipette (inner diameter 50–70 µm). The mice were injected in TA and PL (ankle flexor pair), LG and MG (ankle extensor pair) for synergist pairs and TA and LG for antagonist pairs. In hindlimb/forelimb double injections, the LG and MG were both injected with 1 µl of one RabV to increase the number of long projecting cells infected. In addition, 1 µl of the second RabV was injected in FMs see  without gnal see . Three hgnal see .The mice were perfused with PBS (0.1 M) followed by PBS 4 % paraformaldehyde under terminal ketamine/xylazine anaesthesia . The spinal cords were then collected through a ventral laminectomy and post-fixed for 2 hr. The cords were divided into the different parts of the spinal cord , cryoprotected overnight in 30 % sucrose PBS, embedded in optimal cutting temperature compound (Tissue-Tek) and sliced transversally (30 µm thickness) with a cryostat . Sections were incubated with primary antibodies for 36 hr at 4°C and with secondary antibodies overnight at 4°C in PBS double salt, 0.2 % Triton 100-X (Sigma), 7 % donkey normal serum (Sigma). The primary antibodies used were: goat anti-choline acetyltransferase , chicken anti-mCherry , rabbit anti-GFP , guinea pig anti-vGluT2 , and rabbit anti-Lhx1 ; and the secondary antibodies: donkey anti-rabbit Alexa 647 , donkey anti-goat preadsorbed Alexa 405 , donkey anti-rabbit Alexa 488 , and donkey anti-chicken Cy3 . The slides were mounted in Mowiol  and coverslipped  for imaging.x,y) Cartesian system and using the ‘Spots’ function of Imaris. The y-axis was set to the dorso-ventral axis. Positive values were assigned for dorsal neurons in the y-axis and ipsilateral (to the hindlimb injection) neurons in the x-axis. Coordinates were collected on every section and normalized through the cervical, thoracic, lumbar, and sacral parts separately using grey matter borders and fixing the width and the height of the transverse hemisections. To calculate divergence rates, given the high density of premotor INs infected in the lumbar cord all infected premotor INs  were quantified in one of every three sections which further allowed to avoid counting the same cells twice on consecutive sections. In the cervical, thoracic, and sacral regions, all cells were quantified, as their low density allowed for manually excluding premotor neurons found in consecutive sections. Since MNs are big cells localized as a restricted column of the ventral spinal cord, we quantified them on every other sections, to avoid counting the same cell twice on consecutive sections.Images of the entire sections were obtained using a Zeiss LSM800 confocal microscope with a ×20 air objective (0.8 NA) and tile advanced set up function (ZEN Blue 2.3 software). A ×63 oil objective was used for Airy scan imaging of somata and excitatory boutons. Tiles were stitched using Zen Blue and analyses were performed using Zen Blue and Imaris  software packages. Location maps were plotted setting the central canal as  in the  and GraphPad PRISM (version 7.0). To compare cell sectional areas, non-parametric rank tests were used as specified in each related result. The numbers of animals/cells in each experiment and statistical tests used are reported in the figure legends or directly in the text. Results and graphs illustrate the mean ± standard deviation. Statistical significance levels are represented as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns: not significant.All statistical analyses and plots were made using R  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Ronald Calabrese as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Simon Gosgnach (Reviewer #1).Thank you for submitting your article "Spinal neurons innervating multiple local and distant motor pools" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.The three reviewers acknowledge that the paper is of a great interest for researchers working within the field of motor control, and more specifically at the level of the spinal cord. Despite the rather descriptive aspect of the study it might indeed set the foundation for future studies regarding the specific identities and functions of the premotor neurons investigated here. Their detailed comments are described below, but I would like to emphasize a few important points harvested from their reviews:1) You should tone down the part on the V0v origin identification, or better do the appropriate characterization as suggested below;2) You should further discuss the neurotransmitter characterization and eventually address this point experimentally if possible;3) You should further describe the rostro-caudal extension of the labelled pre-motoneurons.4) Also, a functional aspect is clearly missing, the more you can address this in your revised version the more it will strengthen the paper, but we are aware that fully investigating this would require too much time and represents a significant amount of work if the data are not available yet.Reviewer #1 (Recommendations for the authors):1. More detail regarding rostra-caudal location in the spinal cord should be given in all cases. First I think a Figure describing the location of the motor pools for the 4 injected muscles should be included- this could be in the supplementary data or it could go as a panel in Figure 1. This is needed so we have a clear idea of how far the axons of the PMIs project. In my experience a motor pool can span several lumbar segments. More specific information should also be provided regarding the location of the sections which contain the PMIs being described. I realize this is not an exact science after cutting tissue, but given your care in counting every third section, you should have a pretty good idea of which segment houses the neurons. This would be preferred over the current description of "rostral cervical" or equivalent.In the Figures the number of MNs infected with either virus is typically quite low so I assume the sections are not particularly close to the middle of the infected motor pools?2. The amount of raw data shown is light. There is one immuno panel shown per Figure. I realize the take home message comes from the schematics, but it would be nice to see images of spinal sections with more single and double infected cells. Each immuno image contains just one divergent PMI and other than Figure 1 and 6 there are no other cells that seem to be infected with either the red or green expressing virus in the immuno image.3. I get a little uncomfortable with the data interpretation at the back end of the Results- particularly with the section titled Cervical LDPNs are excitatory- this is speculation and should not be in the title. The data simply shows 95% of these cells are not glycinergic.4. Related to point 3 – The next section is titled "cervical premotor LDPNs likely arise from the V0v domain". In addition to V0 and V1 cells, Lhx1 is also expressed postmitotically in the dI2, dI4, dI6, and V2 populations, and even some V3 cells . It is thus extremely speculative to suggest that the V0v cells are the ones we are looking at here. Even more so when we factor in the finding that Lhx1 plays a key role in establishing GABAergic neuronal identity .Incidentally the current thinking on V0v cells is not that they contact MNs directly, but rather disynaptically via an inhibitory IN (see Kiehn 2016 Nat Rev Nsci review). Some V0 and V3 neurons have been shown to project to MNs , but V3 cells also have been shown to project to non MN targets recent (Chopek Cell Rep paper), and V0 cells very possibly have many other downstream targets, this simply has yet to be investigated.I really think this part of the manuscript should not be included due to its preliminary nature. In order to be included there should be some sort of Evx1 staining, or a functional assay in the V0v deficient mice used in the Talpalar 2012 paper in which issues with muscle stiffness are seen in the absence of V0v cells.The reason I have such an issue with this is that the identity of these cells as belonging to the V0v population is so very circumstantial based on the data here. If it were to be published as is, V0v cells would immediately be cited, in the work of others, as key regulators of muscle stiffness. Even in this manuscript – line 398 of the discussion the authors say that a subset of labeled LDPNs "are clearly V0v derived". This is a problem.Reviewer #2 (Recommendations for the authors):Could the author present data or mention that they have tested that pure RΦGT mice injected with virus injected do not labelled any premotor neurons?Reviewer #3 (Recommendations for the authors):– It should be mentioned earlier in the Results that this study was performed in neonates.– Green and blue in the immuno figures  are difficult to differentiate between due to low contrast between the two.– Line 9-11: Divergent is used before it is defined. It works in the initial sentence (line 9) but is a bit deceptive in the next (lines 10-11) as it seems to imply that divergent is referring only to antagonist motor pools.– Lines 37-42: I suggest adjusting the framing of this section. Most of those questions have answers, as mentioned in the last line of the paragraph and in the Discussion. Instead, highlighting that the location and identities of many of these neurons is unknown may better set up the results to follow.– Lines 146-148: It is not clear what the 22% refers to. It may be "where they make up 22% of the neurons labelled"?– The schematic in Figure S4 A implies that the ventral divergent interneuron in cervical cord is a double jump from the forelimb motor pool. It is likely that there is a 'mis-connection' in the schematic – the ventral neuron should synapse with the forelimb motor neurons, rather than the dorsal neuron to the ventral neuron. 1) You should tone down the part on the V0v origin identification, or better do the appropriate characterization as suggested below;We have extensively edited the sections related to the identification of V0v interneurons. We agree with the referees that we do not have sufficient evidence to pinpoint the genetic lineage of the long range descending interneurons and have toned this down considerably. As suggested by Reviewer#1 we performed further experiments using an Evx1 antibody to selectively label V0v interneurons. However, while good labeling was obtained in neonatal (P0) tissue, no labelling was detected in tissue of the age we used in the present study (P9-P10), in line with downregulation of transcription factors during development – a feature that also reduced our sensitivity in the Lhx1 assay, albeit to a lesser extent. To compound our uncertainty, a recent study was published while we were revising our manuscript identifying a class of dI2 derived interneurons with morphological features similar to the LDPNs described in our study. And we note that dI2 INs also express Lhx1. Given this further uncertainty and the reduced level of detection of Lhx1 in more mature tissue, we have toned down any conclusion that may suggest we have identified the cardinal class of divergent LDPNs. But we prefer to keep the data in the manuscript since we give evidence that at least some LDPNs are Lhx1-expressing (and thus likely V0v or dI2 as discussed). Specific changes in the text are detailed in the response to reviewers #1 and #3.2) You should further discuss the neurotransmitter characterization and eventually address this point experimentally if possible;Identifying the transmitter phenotype of all the divergent interneurons would have required a different protocol, e.g. with a genetically (non-cre) labelled mouse. While the excitatory nature of the divergent LDPNs is sufficiently established, divergent premotor INs in the lumbar and thoracic region are certainly both inhibitory and excitatory. We now demonstrate this point further by adding supplementary figures showing double labelled terminals that are positive or negative for VGlut2 and are localized throughout the thoracic and lumbar cord . Further work will be required to understand whether divergent excitatory and inhibitory premotor interneurons are segregated and whether they belong to homogeneous cardinal classes or not.3) You should further describe the rostro-caudal extension of the labelled pre-motoneurons.We have added new figures where the distribution of premotor interneurons is shown along the rostrocaudal axis for lumbar, thoracic and sacral cord .4) Also, a functional aspect is clearly missing, the more you can address this in your revised version the more it will strengthen the paper, but we are aware that fully investigating this would require too much time and represents a significant amount of work if the data are not available yet.The question on the function of these LDPNs is clearly one of our top priorities. Our manuscript is focused on the discovery of these peculiar cells, but determining their function is a necessary next step. The finding of a precise anatomical location of LDPNs and the availability of tracing methods will now allow us to access and manipulate these cells, but this is clearly a separate project and in our opinion could not be reduced to an appendage to an already dense manuscript.Reviewer #1 (Recommendations for the authors):1. More detail regarding rostra-caudal location in the spinal cord should be given in all cases. First I think a Figure describing the location of the motor pools for the 4 injected muscles should be included- this could be in the supplementary data or it could go as a panel in Figure 1. This is needed so we have a clear idea of how far the axons of the PMIs project. In my experience a motor pool can span several lumbar segments. More specific information should also be provided regarding the location of the sections which contain the PMIs being described. I realize this is not an exact science after cutting tissue, but given your care in counting every third section, you should have a pretty good idea of which segment houses the neurons. This would be preferred over the current description of "rostral cervical" or equivalent.We have now added as supplementary figures the rostro-caudal distribution of divergent premotor INs of lumbar segments, and the rostral-caudal distribution of all premotor INs of the thoracic and cervical regions . We also provided the number of motoneurons infected from each pair of muscles injected, a number that we note is underestimated due to the toxicity of the virus for starter cells (supplementary file 1).In the Figures the number of MNs infected with either virus is typically quite low so I assume the sections are not particularly close to the middle of the infected motor pools?In Figure 1, the section presented is close to the middle of the infected motor pools. Indeed, 2 MNs infected on one single section, as in the example of Figure 1, represents the typical count of MNs within a single section within the labelled motor column. This is due partly to the sparseness or rabies infection, but, more importantly, to the fact that each section is only 30 μm thick. We have also added supplementary figures  showing more examples of lumbar sections following injections in the different motor pools. Amongst the examples shown, there are up to 8 MNs infected on a single section, but this is a rare occurrence .2. The amount of raw data shown is light. There is one immuno panel shown per Figure. I realize the take home message comes from the schematics, but it would be nice to see images of spinal sections with more single and double infected cells. Each immuno image contains just one divergent PMI and other than Figure 1 and 6 there are no other cells that seem to be infected with either the red or green expressing virus in the immuno image.On Figure 1, we highlighted the 3 divergent interneurons on the image shown. Due to the relative low density of divergent premotor neurons and the thinness of the sections, it is impossible to have more divergent neurons in a single section, particularly in the thoracic and cervical cord. To provide a broader perspective of divergent premotor neurons, we added more examples of stained sections from lumbar, thoracic, and cervical spinal cord showing divergent neurons. These are presented in figure supplements 1,2,3 linked to figure 1, 2, and 3.3. I get a little uncomfortable with the data interpretation at the back end of the Results- particularly with the section titled Cervical LDPNs are excitatory- this is speculation and should not be in the title. The data simply shows 95% of these cells are not glycinergic.We have modified the title of this section: “Cervical LDPNs are neither inhibitory nor cholinergic” (line 201) since only 1/21 cervical LDPNs was eGFP positive and none of them were ChAT positive.4. Related to point 3 – The next section is titled "cervical premotor LDPNs likely arise from the V0v domain". In addition to V0 and V1 cells, Lhx1 is also expressed postmitotically in the dI2, dI4, dI6, and V2 populations, and even some V3 cells . It is thus extremely speculative to suggest that the V0v cells are the ones we are looking at here. Even more so when we factor in the finding that Lhx1 plays a key role in establishing GABAergic neuronal identity .Incidentally the current thinking on V0v cells is not that they contact MNs directly, but rather disynaptically via an inhibitory IN (see Kiehn 2016 Nat Rev Nsci review). Some V0 and V3 neurons have been shown to project to MNs , but V3 cells also have been shown to project to non MN targets recent (Chopek Cell Rep paper), and V0 cells very possibly have many other downstream targets, this simply has yet to be investigated.I really think this part of the manuscript should not be included due to its preliminary nature. In order to be included there should be some sort of Evx1 staining, or a functional assay in the V0v deficient mice used in the Talpalar 2012 paper in which issues with muscle stiffness are seen in the absence of V0v cells.The reason I have such an issue with this is that the identity of these cells as belonging to the V0v population is so very circumstantial based on the data here. If it were to be published as is, V0v cells would immediately be cited, in the work of others, as key regulators of muscle stiffness. Even in this manuscript – line 398 of the discussion the authors say that a subset of labeled LDPNs "are clearly V0v derived". This is a problem.We thank the reviewer and agree that our evidence for the identification of V0 interneurons is not definitive. While preparing the revision, we attempted to identify Evx1 derived interneurons using a specific antibody (a gift from Columbia University). However, while we obtained good staining in young (P0) tissue, no staining was observed in P9 tissue, the age at which the tissue was analyzed in this paper. Having tried many different concentrations and incubation times, with similar results, we conclude that Evx1 expression is downregulated, even more than Lhx1  and therefore, a posteriori identification of the long descending projection neurons is not possible. Only genetic labelling could resolve the issue, but this would not be possible since we do not have the relevant mouse line. To compound our uncertainty, a recent study was published while we were revising our manuscript identifying a class of dI2 derived interneurons with morphological features similar to the LDPNs described in our study. And we note that dI2 INs also express Lhx1. Given this further uncertainty and the reduced level of detection of Lhx1 in more mature tissue, we have toned down any conclusion that may suggest we have identified the cardinal class of divergent LDPNs and re-written the related section, (now starting at line 219). But we prefer to keep the data in the manuscript, since we give evidence that at least some LDPNs are Lhx1-expressing (and thus likely V0v or dI2 as discussed). See also answer to reviewer #2 related to the same point.Reviewer #2 (Recommendations for the authors):4) Could the author present data or mention that they have tested that pure RΦGT mice injected with virus injected do not labelled any premotor neurons?We tested for leaks of the rabies glycoprotein by performing injection of an EnvA pseudotyped rabies virus in heterozygous RΦGT mice. We chose to use a pseudotyped rabies in order to test for ectopic expression of both the rabies glycoprotein and the TVA receptor . The example data are shown in our preprint in which we describe a similar approach . In summary, following the injections we observed scant motoneuron labelling , but no interneurons. Since we used pseudotyped rabies, the infection of some motoneurons shows that there is some ectopic expression of TVA in motoneurons that gave rise to motoneuron infection due to the extremely high affinity of EnvA for the TVA receptor. However, no trans-synaptic labelling was observed, indicating that any leak of the G protein, was not sufficient to sustain transsynaptic jumps. We now state this at the end of Methods section on intramuscular injections (lines 504-508).Reviewer #3 (Recommendations for the authors):– It should be mentioned earlier in the Results that this study was performed in neonates.This has been clarified and is now mentioned line 95 of the results.– Green and blue in the immuno figures  are difficult to differentiate between due to low contrast between the two.Colors are unfortunately difficult to pick when taking into account the many ways people can actually see them. We spend far too much time in lab meetings discussing colours. We had to pick two colors that everyone could easily differentiate for eGFP (green) and mCherry (pink), while keeping colors close to their fluorescent emission spectrum to avoid misunderstanding. Also, since we are focusing on double labelled neurons, the colocalisation of the two colors needed to give a completely different tint (white in our case). Blue remained the only option as the fourth color so that it could readily be differentiated from the three others. It is true that blue is more difficult to visualize, however, since blue is used for motoneuron staining, which is not the focus of the study, we thought that the combination used was the best option.– Line 9-11: Divergent is used before it is defined. It works in the initial sentence (line 9) but is a bit deceptive in the next (lines 10-11) as it seems to imply that divergent is referring only to antagonist motor pools.Divergent is now defined clearly in the abstract before its use.– Lines 37-42: I suggest adjusting the framing of this section. Most of those questions have answers, as mentioned in the last line of the paragraph and in the Discussion. Instead, highlighting that the location and identities of many of these neurons is unknown may better set up the results to follow.We have included the referee’s suggestion and, while keeping the list of questions, we have stressed that what is not known are the location and identities of the interneurons mediating synergies (lines 46-48).– Lines 146-148: It is not clear what the 22% refers to. It may be "where they make up 22% of the neurons labelled"?This has been clarified (line 164).– The schematic in Figure S4 A implies that the ventral divergent interneuron in cervical cord is a double jump from the forelimb motor pool. It is likely that there is a 'mis-connection' in the schematic – the ventral neuron should synapse with the forelimb motor neurons, rather than the dorsal neuron to the ventral neuron.We thank the reviewer for this comment and apologize. Indeed, we had made a mistake in the schematic of what is now Figure 6—figure supplement 1A. This has now been corrected.


Oral cancer is one of the most common human malignancies, and its incidence is increasing worldwide. In particular, oral squamous cell carcinoma (OSCC) is characterized by high rates of proliferation, invasiveness, and metastasis. Currently, standard treatment for OSCC includes surgical removal, chemotherapy, and radiotherapy; however, the survival rate of patients with OSCC remains low, thus new therapies are needed. It has been proven that excessive NLRP3 inflammasome activation and apoptosis alteration may contribute to oral cancer progression. This study aimed to investigate the effect of BAY-117082, an NLRP3 inflammasome inhibitor, in an in vitro and in vivo xenograft model of oral cancer. In vitro results revealed that BAY-117082 at concentrations of 5, 10, and 30 µM was able to reduce OSCC cell viability. BAY-117082 at higher concentrations significantly reduced NLRP3, ASC, caspase-1, IL-1β, and IL-18 expression. Moreover, Bax, Bad, and p53 expression were increased, whereas Bcl-2 expression was reduced. Furthermore, the in vivo study demonstrated that BAY-117082 at doses of 2.5 and 5 mg/kg significantly decreased subcutaneous tumor mass, and also reduced NLRP3 inflammasome pathway activation. Therefore, based on these results, the use of BAY-117082 could be considered a promising strategy to counteract oral cancer progression, thanks its ability to modulate the NLRP3 inflammasome and apoptosis pathways. Oral cancer is one of the most common malignancies in the world . Oral caRecent studies demonstrated that the NLRP3 inflammasome pathway is upregulated in OSCC animal models and OSCC patients , suggestMTT assay was used to assess CAL27, HSC-2, and SCC-4 cell viability following 24 h of treatment with BAY-117082 at different concentrations . Our results show that BAY-117082 treatment only at concentrations of 5, 10, and 30 µM significantly reduced CAL27, HSC-2, and SCC-4 cell viability in the same way, as shown in Based on the MTT results, we decided to investigate in another analysis only BAY-117082 at concentrations of 5, 10, and 30 µM, because these represented the most cytotoxic concentrations. Furthermore, since BAY-117082 showed similar effects on cell viability in all three cell lines, we decided to continue to investigate its effect on only the CAL27 cell line, because it represented a frequently used cell line in the field of OSCC ,19.Inflammasome is a cytoplasmic multi-protein complex that regulates innate immunity response through ASC and caspase-1 activation . NLRP3 iOnce activated, NLRP3 inflammasome induces the release of pro-inflammatory cytokines IL-1β and IL-18, which can contribute to cancer progression ,22. ThusApoptosis is a physiological process of programmed cell death that is essential for normal tissue development and cell hemostasis . DysreguRecent studies highlighted the role of pro-apoptotic p53 protein in modulating various cellular processes such as metabolism, metastasis, and communication within the tumor microenvironment . p53 defOur results demonstrate that the control group was characterized by low p53 expression, while treatment with BAY-117082 at concentrations of 10 and 30 µM significantly increased pro-apoptotic p53 expression E. To evaluate the effect of BAY-117082 on the growth of OSCC cells in vivo, the CAL27 xenograft model was established in nude mice. In this context, our results show that treatment with BAY-117082 at doses of 2.5 and 5 mg/kg significantly reduced subcutaneous tumor mass and neutrophilic infiltration in a dose-dependent manner A–C. MoreTo confirm the effect of BAY-117082 on the NLRP3 inflammasome pathway, we decided to also investigate this pathway in the xenograft model by Western blot analysis. The results show that the control group was characterized by high expression of NLRP3, ASC, and caspase-1; however, treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to significantly reduce their expression . Moreover, we decided to investigate, by immunohistochemistry staining, the expression of IL-1β, a pro-inflammatory cytokine related to cancer progression. Our results show that the control group was characterized by marked expression of IL-1β, while treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to reduce its expression in a dose-dependent manner , confirmAccumulating evidence has suggested that the nuclear factor-κB (NF-κB) signaling pathway plays a critical role in oral carcinogenesis ,25. It hCancer development and its response to therapy are strongly influenced by innate and adaptive immunity, which can promote or attenuate tumorigenesis with opposite effects on the therapeutic outcome . It has Apoptosis plays a key role in cancer progression, therefore targeting apoptosis could be a relevant therapeutic approach in anti-cancer drug development . Thus, wKi-67 is a DNA-binding nuclear protein involved in cell proliferation , and is Oral squamous cell carcinoma (OSCC), the most common oral cancer, arises from the mucosal lining of the oral cavity, with an incidence of 450,000 new cases per year . Major rAlthough the pathophysiology of oral cancer remains unclear, in vivo and in vitro studies have demonstrated that aberrant and excessive NLRP3 inflammasome activation significantly contributes to the initiation and progression of oral cancer ,20. MoreFirst of all, we evaluated the cytotoxic effect of BAY-117082 at different concentrations in an in vitro model of OSCC using CAL27, HSC-2, and SCC-4 cell cultures. Clearly, our results demonstrate that BAY-117082 treatment only at higher concentrations significantly reduced CAL27, HSC-2, and SCC-4 cell viability in the same way. Many papers have proven that NLRP3 inflammasome plays a key role in oral carcinogenesis ,21. NLRPHowever, recent studies have demonstrated that excessive and aberrant NLRP3 inflammasome activation may contribute to oral cancer progression ,20. ThusOnce activated in response to intracellular stimuli, NLRP3 inflammasome promotes the proteolytic processing of pro-IL-1β and pro-IL-18 into their bioactive forms IL-1β and IL-18, respectively . OverexpAmong the mechanisms involved in cancer pathogenesis, apoptosis also has a fundamental role ,35. ProgDespite the promising results obtained with an in vitro model on CAL27 cell culture, we decided to construct an in vivo xenograft model of oral cancer to confirm the beneficial effect of BAY-117082. Our data demonstrate that BAY-117082 treatment significantly reduced subcutaneous tumor mass and tumor necrosis compared to the control group in a dose-dependent manner. Additionally, treatment with BAY-117082 significantly decreased tumor burden and tumor weight compared to the control group in a dose-dependent manner, without changing the animals’ weight. Moreover, considering the key role of NLRP3 inflammasome in oral carcinogenesis , we alsoIt has been demonstrated that the NF-κB signaling pathway has a fundamental role in oral cancer pathogenesis . NF-κB iIn addition, NF-κB plays a critical role in regulating the activation and differentiation of innate immune cells and inflammatory T cells . In a maFurthermore, to confirm the data obtained in the in vitro model with regard to the apoptosis pathway, we decided to also investigate the effect of BAY-117082 on programmed cell death in the xenograft model, demonstrating that it was able to significantly increase pro-apoptotic Bax protein expression, while anti-apoptotic Bcl2 and Bcl-xL were significantly reduced following BAY-117082 treatment in a dose-dependent manner, in contrast to cell proliferation. Another important hallmark of cancer is cell-cycle dysregulation . During Therefore, in this study we decided to investigate Ki-67 expression, showing that the control group was characterized by elevated Ki-67 expression, whereas treatment with BAY-117082 was able to significantly reduce its appearance. Thus, based on the results obtained, using BAY-117082 could be considered as a valid therapeutic strategy to reduce or counteract OSCC progression thanks its ability to modulate the NLRP3 inflammasome and apoptosis pathways. However, further investigations are needed to better understand the involvement of these pathways in oral carcinogenesis. 2.Human OSCC cell lines CAL27, HSC-2, and SCC-4 were obtained from ATCC . Cells were grown in Dulbecco’s Modified Eagle’s Medium  for CAL27 and Minimum Essential Eagle’s Medium  for HSC-2 and SCC-4 cells, supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 100 U/mL penicillin and 100 μg/mL streptomycin  at 37 °C with 5% CO4 cells/well to a final volume of 150 μL. After 24 h, cells were treated with BAY-117082 (Sigma-Aldrich®) for 24 h at increasing concentrations 0.1, 0.5, 1, 3, 5, 10, and 30 μM dissolved in PBS. Then, cells were incubated at 37 °C with MTT (0.2 mg/mL) for 1 h. The medium was removed and cells were lysed with dimethyl sulfoxide (DMSO) (100 µL). The extent of reduction in MTT to formazan was quantified by measuring optical density at 550 nm with a microplate reader.A 3--2,5-diphenyltetrazolium bromide (MTT) assay was used to assess cell viability, as previously described . CAL27, Control group (Ctr): Human OSCC cell lines CAL27, HSC-2, and SCC-4BAY-117082 0.1 μM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 0.1 μM for 24 hBAY-117082 0.5 μM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 0.5 μM for 24 hBAY-117082 1 μM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 1 μM for 24 hBAY-117082 3 μM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 3 μM for 24 hBAY-117082 5 μM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 5 μM for 24 hBAY-117082 10 μM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 10 μM for 24 hBAY-117082 30 μM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 30 μM for 24 h6 CAL27 cells were plated in 6-well plates  and incubated with BAY-117082 for 24 h [g for 15 min at 4 °C. Protein concentration was estimated by the Bio-Rad protein assay using bovine serum albumin as standard. Samples were heated at 95 °C for 5 min, and the same amounts of protein were separated on 12% SDS-PAGE gel and blotted to PVDF membrane (Immobilon-P). Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-NLRP3 , anti-ASC , anti-caspase-1 , anti-IL-1β , anti-IL-18 , anti-Bax , anti-Bcl2 , and anti-Bad . Then, membranes were incubated with peroxidase-conjugated bovine anti-mouse or goat anti-rabbit or anti-mouse IgG  for 1 h at room temperature. To ascertain that the blots were loaded with equal amounts of proteins, they were also incubated in the presence of the antibody against β-actin protein for cytosolic fraction  and Lamin A/C for nuclear fraction . The signals were detected with a chemiluminescence detection system reagent according to the manufacturer’s instructions . The relative expression of protein bands was quantified by densitometry with Bio-Rad ChemiDoc  using ImageLab software.For the Western blot, 1 × 10for 24 h . Then, c2HPO4, pH 7.4), permeabilized in 0.2% Triton X-100/PBS, and blocked with 10% bovine albumin serum. Cells were incubated overnight (O/N) at 4 °C with primary antibodies: anti-p53  and anti-caspase-1 . After being washed in PBS, cells were incubated with secondary antibody Alexa Fluor 488 goat anti-mouse  for 1 h at 37 °C. Sections were washed in PBS and 2 μg/mL 4′,6′-diamidino-2-phenylindole  in PBS was added for nuclear staining. Sections were observed and photographed at 40× magnification using a Leica DM2000 microscope . Immunofluorescence assay was performed as previously described by Donaldson . CAL27 cBALB/c nude male mice were obtained from Jackson Laboratory  and housed in microisolator cages under pathogen-free conditions with 12 h light/12 h dark. Animals were fed with a standard diet and water ad libitum. This study was approved by the University of Messina Review Board, under project identification code 137/2017-PR, released on 9 February 2017. Animal care was in compliance with Italian regulations on the protection of animals used for experimental and other scientific purposes (DM 116192) as well as EU regulations (OJ of EC L 358/1 18 December 1986).6 CAL27 cells per tumor in 0.2 mL of PBS and 0.1 mL Matrigel  as previously described [The xenograft tumor model was established by subcutaneously inoculating 3 × 10escribed . After t3, mice were treated with BAY-117082 at doses of 2.5 and 5 mg/kg every 3 days according to [2 × L/2, where W and L represent minor and major length. After 30 days, mice were sacrificed and tumors were excised and processed for analysis. After 1 week of tumor induction, mice were divided randomly into 3 groups. When tumor size reached about 200–300 mmrding to . BAY-117Control group (vehicle): weekly intravenous (IV) administration of salineControl group + BAY-117082 2.5 mg/kg: intraperitoneal administration of BAY-117082 2.5 mg/kg dissolved in PBSControl group + BAY-117082 5 mg/kg: intraperitoneal administration of BAY-117082 5 mg/kg dissolved in PBSMice were randomly divided into 3 groups:n = 14 mice for each technique. The minimum number of mice for each technique was estimated with one-way fixed effects ANOVA with G-power software. This statistical test generated a sample size equal to Histological evaluation was performed as previously described by Paterniti et al. . Tumor sImmunohistochemical localization was carried out as previously described by Scuderi et al. . Tumor sw/v) dried nonfat milk in buffered saline (PM) for 45 min at room temperature and subsequently probed with specific antibodies: anti-NLRP3 , anti-ASC , anti-caspase-1 , anti-Bax , anti-Bcl2 , anti-Bcl-xL , anti-NF-κB , and anti-IκBα  in 1× PBS, 5% w/v dried nonfat milk and 0.1% Tween-20 (PMT) at 4 °C overnight. Membranes were incubated with peroxidase-conjugated goat anti-mouse IgG secondary antibody  or peroxidase-conjugated goat anti-rabbit IgG secondary antibody  for 1 h at room temperature. To establish that blots were loaded with equal amounts of proteins, they were also incubated in the presence of the antibody against β-actin protein . Signals were revealed with an enhanced chemiluminescence (ECL) detection system reagent according to the manufacturer’s instructions . The relative expression of protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS+ software and standardized to β-actin levels as an internal control.Protein levels in tumor samples were quantified as previously described . CytosolAll compounds and other chemicals were obtained from Sigma-Aldrich . All stock solutions were prepared in non-pyrogenic saline .t-test analysis. All values are indicated as mean ± standard error of the mean (SEM) of N observations. Data were analyzed with GraphPad Prism 7.04 software using In conclusion, the results obtained offer new insights into the role of the NLRP3 inflammasome and apoptosis signaling pathways in oral carcinogenesis, showing that the use of BAY-117082, a strong NLRP3 inflammasome inhibitor, could represent a potential therapeutic treatment to counteract or reduce the progression of OSCC, which has been showing an increasing mortality rate.


Mangifera casturi Kosterm., a mango plant from Kalimantan Selatan, Indonesia, has limited genetic information, severely limiting the research on its genetic variation and phylogeny. We collected M. casturi’s genomic information using next-generation sequencing, developed microsatellite markers and performed Sanger sequencing for DNA barcoding analysis. These markers were used to confirm parental origin and genetic diversity of M. casturi hybrids. The clean reads of the Kasturi accession were assembled de novo, producing 259 872 scaffolds (N50 = 1 445 bp). Fourteen polymorphic microsatellite markers were developed from 11 040 microsatellite motif-containing sequences. In total, 58 alleles were produced with a mean of 4.14 alleles per locus. Microsatellite marker analysis revealed broad genetic variation in M. casturi. Phylogenetic analysis was performed using internal transcribed spacers (ITS), matK, rbcL, and trnH-psbA. The phylogenetic tree of chloroplast markers placed Kasturi, Cuban, Pelipisan, Pinari, and Hambawang in one group, with M. indica as the female ancestor. Meanwhile, the phylogenetic tree of ITS markers indicated several Mangifera species as ancestors of M. casturi. Thus, M. casturi very likely originated from the cross-hybridization of multiple ancestors. Furthermore, crossing the F1 hybrids of M. indica and M. quadrifida with other Mangifera spp. may have generated much genetic variation. The genetic information for M. casturi will be a resource for breeding improvement, and conservation studies. Mangifera casturi Kosterm., or Kalimantan mango, is an endogenous fruit plant in Kalimantan Selatan, Indonesia; it is classified as extinct in the wild according to the IUCN Red List1. M. casturi belongs to the Mangifera genus within the Anacardiaceae family2 and is classified as a common ancestor of the Mangifera species in Indonesia3. M. casturi is proposed to be the natural hybrid of M. indica and M. quadrifida according to single nucleotide polymorphisms (SNPs) analysis4. In Kalimantan Selatan, M. casturi are known by various local names, such as Kasturi, Cuban, Pelipisan, Pinari, and Rawa-rawa  in nucleotide repositories such as the NCBI, and one SRA study (SRP183190) reported.7. Also, from NGS data, it is easier to obtain genetic information such as microsatellite markers, which are superior to other markers like RAPD and AFLP and are already used in other Mangifera species9. Microsatellite markers can determine distinct variations at the level of species as they are codominant; as a result, they are widely used in population and genetic studies10. Microsatellite markers have also been used to determine the genetic variation in M. indica11. Although microsatellites are important for taxonomy and the study of genetic conservation, no M. casturi-specific microsatellite markers have been reported.Recently, sequencing has advanced significantly from Sanger sequencing to next-generation sequencing (NGS). For example, whole-genome sequencing can produce comprehensive genomic information on a speciesrbcL, matK12, and trnH-psbA13, internal transcribed spacers (ITS), and second internal transcribed spacers (ITS2) from nuclear ribosomal DNA14, have been widely used for phylogenetic analysis at various taxonomic levels. These DNA barcoding markers from chloroplast regions can also be determined at the genus or family level because of their inheritance from a maternal ancestor. On the other hand, the ITS region can determine the barcoding of the paternal and maternal ancestor. However, DNA barcoding sequences for M. casturi have not been recorded in any public database. As a result, there have been no phylogenetic studies of M. casturi using DNA barcoding to achieve accurate identification at the taxonomy level.In recent years, Sanger sequencing approaches have been utilized for DNA barcoding. DNA barcoding methods based on chloroplast regions, such as M. casturi using NGS and Sanger and to analyze and determine the genetic variation among the M. casturi hybrids. Microsatellite markers were used to assess genetic variation among M. casturi hybrids, Kasturi, Cuban, Pelipisan, and Pinari. Furthermore, at a higher taxonomy level, phylogenetic analysis of M. casturi hybrids and Mangifera species was performed using DNA barcoding. In addition, there is no clear information and proof about the genetic variation and relationship among M. casturi hybrids. In this study, we propose a candidate ancestor from a natural hybridization of M. indica and M. quadrifida.This study aimed to collect genomic information from M. casturi DNA was obtained with high-throughput sequencing using an Illumina HiSeq 4000 system with paired end 150 bp reads. The raw data were registered in the DDBJ with accession number DRA011022. Clean reads were obtained via filtering, and 10.95 Gbp of de novo genome assembly was performed using a Ray Assembler. We obtained 259 872 scaffolds with an N50 value of 1 445 bp and a maximum scaffold length of 144 601 bp  ranged from 0 for 4 markers  to 0.62 for the mc8693 locus with a mean Ho of 0.26. The expected heterozygosity (He) ranged from 0.40 for the mc167596 locus to 0.80 for the mc230178 locus, with a mean of 0.65. The fixation index (FST), reflecting the degree of genetic differentiation, ranged from 0.08 to 1.00 with an average of 0.60 per locus. Lastly, Shannon’s information index ranged from 0.69 and 1.75 with a mean of 1.21.Eight samples, namely Pelipisan, Cuban, Pinari, Kasturi, Rawa-rawa, M. casturi accessions in a natural population, as it is dissimilar to other Mangifera species.The mc230178 and mc58089 loci produced seven alleles from eight samples, while the mc122955 and mc88387 loci produced two alleles. Some loci, namely mc176197, mc21672, and mc88075, displayed the same alleles between Kasturi and Cuban. In the mc88387 locus, only the Kasturi sample was not amplified; thus, this locus might have a null allele in Kasturi. Therefore, mc88387 locus can be used to identify M. foetida), Pelipisan, Pinari, M. indica in one cluster, while Kasturi and Cuban in the second cluster, and Rawa-rawa and M. quadrifida in the third cluster.Furthermore, principal coordinate analysis was performed using the GENALEX 6.501, indicating that 33.85% of the variance within the microsatellite data was graphed by the first axis, and 19.43% by the second axis Fig. . AdditioM. quadrifida and Rawa-rawa were placed in the same clade. All the M. casturi accessions were in the same clade as M. indica and Hambawang (M. foetida). Cuban were most closely related to M. indica. However, Kasturi and Pelipisan were in the same clade where some loci showed similar alleles. Thus, these accessions had a closer genetic relationship to each other than to Cuban. However, Pinari also exhibited distinct genetic differences from the other M. casturi accessions, even though Cuban was quite distant from other M. casturi accessions.The result was also presented in a UPGMA dendrogram Fig. . M. quadmatK, rbcL, and trnH-psbA  and were deposited in the DDBJ Nucleotide Sequence Submission System under the accession number LC602976- LC602993. However, the matK, rbcL, and trnH-psbA sequences from other Mangifera species were downloaded from the public nucleotide database of NCBI , M. griffthii, M. quadrifida, M. kemanga, M. torquenda, and M. sumatrana.On the other hand, the ITS phylogenetic tree produced three large groups: Indica 1, indica 2, and a group containing Kasturi, Cuban, Pelipisan, and Pinari. Hambawang was included in the indica 2 group. Meanwhile, Pinari was placed in a sub-group with Mangifera genus originates from southeast Asia and has polyembryonic seeds, derived from gametes or nucellar cell components2. Most Mangifera flowers are either hermaphrodites or males32; thus, self-crossing can occur in various species. However, self-incompatibility in the Mangifera genus has been reported in several types of mangos33, suggesting that various Mangifera species can cross-hybridize34. As a result, cross-hybridization in the natural populations has produced many interspecies, including M. odorata (Kuini), a natural hybrid between M. indica and M. foetida9.The 36. Microsatellites in Mangifera species that were previously identified in M. indica have been useful in the genetic analysis of genus Mangifera and its related genera38. In This study, genetic analysis has revealed markers in 14 microsatellite loci and that different allele sizes have arisen from four accessions of M. casturi namely Kasturi, Cuban, Pelipisan, and Pinari. The expected heterozygosity (He) value of the microsatellite markers used in the M. casturi analysis ranged between 0.40 and 0.80, with an average of 0.65, which indicated that the highly informative microsatellite markers could be employed in genetic diversity studies of M. casturi. In this study, a high level of genetic variation was discovered in M. casturi accessions, likely arising from repetitive interspecific hybridization. In Petunia, microsatellite markers have determined genetic differentiation and hybrid identification39. The accessions of Kasturi, Cuban, and Pelipisan were more closely related than Pinari. Kasturi and Cuban are very similar in fruit size. However, morphologically, the fruit shape of Kasturi is more oval than Cuban. In contrast, a Pelipisan fruit is more oval and slightly larger than Kasturi and Cuban. Pinari has the largest fruit size among the M. casturi accessions. Lastly, Pinari is classified into the M. casturi group by the locals, based on its purplish skin similar to that of other M. casturi accessions 4.Microsatellite markers have been used successfully to determine genetic variation among many plantsJuglans regia and J. cathayensis indicated a rare phenomenon and backcrosses between hybrids and either of the parental species40. In addition, Kuini (M. odorata), a natural hybrid between M. indica and M. foetida was revealed by AFLP analysis to represent a simultaneous backcross between the F1 hybrids of Kuini and M. foetida9. On the other hand, SNP analysis using double-digest restriction-site-associated DNA (ddRAD)4, revealed M. casturi to be a natural hybrid between M. indica and M. quadrifida, whereas their F1 hybrid was a backcross with M. indica. Morphologically, M. casturi is very close to M. quadrifida, with the same purplish skin and small fruit size4. Therefore, M. casturi has several types known as Kasturi, Cuban, Pelipisan, and Pinari. These hybrids are believed to be hybrids between M. indica and M. quadrifida and backcrosses between hybrids and either of the ancestors.Intraspecies genetic variation can occur because of multiple cross-hybridizations among several species. Using microsatellite markers, hybridization between Garcinia mangostana), microsatellite markers indicate cross-hybridization with multiple ancestors, including G. malaccensis, G. celebica, and G. porrecta22. In this study, microsatellite analysis results showed that four accessions of M. casturi, namely Kasturi, Cuban, Pelipisan, and Pinari had allelic differences in all the microsatellite loci. However, allele sharing between the four accessions was detected in the mc8693 locus with an allele size of 160/182, indicating that these accessions were derived from the same ancestor, M. indica. In contrast, the allele differences in the microsatellite loci suggested that the four M. casturi accessions underwent cross-hybridization with multiple ancestors. In Oaks (Quercus spp.), microsatellites indicate sharing most alleles in their hybrids than recurrent gene flow41.In an allopolyploid plant such as mangosteen  region has also been indicated as a barcoding regionM. casturi hybrids. Based on genomic data, we have identified that M. casturi, which is endemic to Kalimantan Selatan, consists of 4 types, namely Kasturi, Cuban, Pelipisan, and Pinari. In Addition, based on the combination of microsatellite data, and DNA barcoding, M. casturi hybrids are natural hybrids between M. indica and M. quadrifida. Moreover, the genomic data represent an important genetic resource for breeding and improving the characteristics of this local mango in the future. The habitat of M. casturi is severely threatened; as a result, it is classified as extinct in the wild. Thus, more intensive conservation efforts are necessary. Moreover, since M. casturi varieties have never been confirmed or registered by authorities, the results of this study can help breeders and the local government to officially document this local mango and one of their elite germplasm.In this study, the genomic data revealed the genetic variation and ancestral origin of M. casturi accessions were collected from the Banjar district, Kalimantan Selatan, in the southern region of Kalimantan, three Mangifera species were collected from Banua Botanical Garden  and Dramaga, Bogor, West Java (M. indica) . 15, and clean reads were filtered using the Fastp version 0.20.1 (https://github.com/OpenGene/fastp) with default parameters16. Clean reads were assembled using a Ray version 2.1.0 (https://github.com/sebhtml/ray) with default parameters (-k 63 -minimum-contig-length 200)17 under the Maser Platform (https://cell-innovation.nig.ac.jp/). 18. The assessment of genome assembly was performed to check the assembled contig quality using BUSCO version 3.0.2 (https://busco.ezlab.org). 19 on the Maser Platform with default parameters.ca) Fig. . To analhttps://webblast.ipk-gatersleben.de/misa/). 20 from M. casturi scaffolds, the parameters being set to the following minimum repeat levels: six for two bases, and five for three, four, five, and six bases. The difference between microsatellite motifs was 100 bases. The microsatellite motif-containing sequences were selected based on parameters; (1) the flanking region are at least 150 bp long in both directions (2) microsatellite repeats have the longest repeat motifs. The primer was designed using the web version of Primer 3 with default parameters21.Microsatellite markers were extracted using the MISA version 2.1 . A Type-it microsatellite PCR kit (Qiagen) was used to analyze the microsatellite markers. PCR master mix was prepared with a mixture of 3.2 μL RNase-free water, 5 μL 2 × Type-it Multiplex PCR Master Mix, 0.4 Q solution, 0.2 μL of 10 μM forward primer, and 0.2 μL of 10 μM reverse primer. PCR was performed using a SimpliAMP Thermo Cycler (Applied Biosystems). The PCR conditions were as follows: initial conditions of PCR pre-denaturation at 95 °C for 5 min, followed by 32 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 1 min 30 s, extension at 70 °C for 30 s, and final extension at 60 °C for 30 min. The amplicons were checked using 1% electrophoresis gel in TAE buffer for 20 min at 100 v. Before loading the sample into QIAxcel capillary electrophoresis (Qiagen), the sample was diluted twice and then run using a QIAxcel DNA High Resolution Kit (Qiagen). Allele size data were confirmed and processed manually using QIAxcel ScreenGel version 1.4.0 .Genomic DNA was isolated using the modified CTAB method, with a slight modificationhttps://biology-assets.anu.edu.au/GenAlEx/) for each microsatellite marker, including the number of alleles (Na) per locus, and both observed (Ho), and expected (He) heterozygosity, fixation index (FST), and Shannon's information index (I). The principal coordinate analysis (PCoA) via Covariance matrix with data standardization was also performed using GENALEX 6.501. The microsatellite data were processed using the Phylip version 3.695 (https://evolution.genetics.washington.edu/phylip.html) with the unweighted pair group method and arithmetic mean (UPGMA) method. The resulting dendrogram was edited using the program MEGA-X Software (https://www.megasoftware.net)23.Descriptive statistics were calculated using GENALEX version 6.501 27. PCR barcoding was performed using KOD Plus (Toyobo) according to the manufacturer's protocol. The PCR products were cleaned using ExoSAP-IT PCR Product Cleanup Reagent (Applied Biosystems). Then, PCR sequencing was carried out with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), followed by purification using a BigDye XTerminator Purification Kit (Applied Biosystems) according to the manufacturer protocol. The sequencing products were performed using a 3500 Genetic Analyzer (Applied Biosystems). Sequence data were analyzed using Sequencing Analysis Software version 6.0 , and the data were processed with ATGC-MAC version 7 (https://www.genetyx.co.jp) and MEGA-X software (https://www.megasoftware.net). 23.For the DNA barcoding analysis, we used three chloroplast genes: 28. The Mangifera sequences of matK, rbcL, and trnH-psbA, and ITS complete sequences were downloaded from NCBI  from Plant Quarantine Division of National Agency for Agricultural Quarantine in Banjarbaru, South Kalimantan following permit approvals from South Kalimantan Natural Resources Conservation Agency/BKSDA of the Ministry of Environment and Forestry of the Republic of Indonesia (KLHK) as agency in charge of managing conservation areas including protected plant in the territory, particularly the nature reserve forests  and national park. The M. casturi samples from Banjar, South Kalimantan as herbaria voucher (received by Agung Sriyono) were duplicated and stored in Banua Botanical Garden, Province of South Kalimantan, Banjarbaru, Indonesia.All experiments were performed in accordance with relevant guidelines and regulations. The experimental research has complied with Bogor Agricultural University research regulation (No: 11/SA-IPB/P/2016 on research and publication ethics), and the field study was in accordance with the national legislations of Indonesian Law Number 5/1990 on biological diversity conservation and Indonesia Law Number 11/2013 on the ratification of the Nagoya Protocol. Supplementary Information 1.Supplementary Information 2.


Individuals with greater genetic risk for Schizophrenia or Bipolar Disorder are more vulnerable to the adverse effects of birth at early gestational age on brain development, as assessed by cognition at age four. Better understanding of gene-environment interactions will inform more effective risk-reducing interventions for this vulnerable population.Preterm birth is an extreme environmental stress associated with an increased risk of later cognitive dysfunction and mental health problems. However, the extent to which preterm birth is modulated by genetic variation remains largely unclear. Here, we test for an interaction effect between psychiatric polygenic risk and gestational age at birth on cognition at age four. Our sample comprises 4934 unrelated individuals . Genome-wide polygenic scores (GPS’s) were calculated for each individual for five different psychiatric pathologies: Schizophrenia, Bipolar Disorder, Major Depressive Disorder, Attention Deficit Hyperactivity Disorder and Autism Spectrum Disorder. Linear regression modelling was used to estimate the interaction effect between psychiatric GPS and gestational age at birth (GA) on cognitive outcome for the five psychiatric disorders. We found a significant interaction effect between Schizophrenia GPS and GA ( Preterm infants have higher rates of cognitive impairment, cerebral palsy, autism spectrum disorders and psychiatric disease5, not all of which can be explained by the degree of prematurity and severity of clinical problems around the time of birth.Preterm birth is an extreme perinatal stress and is associated with a significantly increased risk of later cognitive dysfunction and psychiatric disease. Preterm birth is common, accounting for around 11% of all births and is a leading cause of infant mortality and morbidity worldwideMagnetic resonance imaging studies indicate that preterm brain injury is a multifactorial disorder which encompasses alterations to cortical and subcortical gray matter and white matter. These studies have contributed to our understanding of the pathophysiology underlying neurodevelopmental disability and cognitive impairments in the preterm population, however, a great deal remains unknown. Specifically, the extent to which genetic factors play a role in the variable cognitive outcomes of preterm infants and the possible interaction between genetic risk and the environmental exposure to preterm birth has not been examined in detail. Here, we explore whether common genetic variation is important in determining an individual’s susceptibility to the environmental stress of prematurity and thus plays a role in the diverse neurodevelopmental and cognitive outcomes we observe in preterm infants.6. We therefore anticipate that gene-environment interactions involving these complex pathologies will be most effectively studied by looking at multiple genetic variants rather than focussing on a single genetic locus. As samples sizes have grown, genome-wide association studies (GWAS) have become increasingly informative, allowing detection of small effects of single nucleotide polymorphisms (SNPs). Using the summary statistics from these studies it is possible to generate individual-specific genotypic scores to predict phenotypic variance, genome-wide polygenic scores (GPS). A genome-wide polygenic score, which is an aggregate of trait-related effect sizes of SNPs across the genome in independent samples can be used to test the predictive power of multiple genetic variants simultaneously. Genome-wide polygenic scores provide a useful approach to exploring gene-environment interactions in complex traits.Research into the genetics of psychiatric disease has shown it to be polygenic; psychiatric disorders are influenced by many genetic variants, each of small effect7. Under this notion, individuals with a greater polygenic risk for psychiatric disease, when born preterm, would be more likely to suffer adverse developmental consequences. We showed that in a cohort of preterm infants, psychiatric GPS was negatively associated with lentiform volume; genetic predictors of neuropsychiatric disease increase vulnerability to abnormal lentiform development after perinatal stress.In previous work, we hypothesised that genes associated with psychiatric disease might increase vulnerability to abnormal brain development in infants subjected to the environmental stress of prematurityWe now extend this work by looking at the possible influence of genome-wide polygenic risk for psychiatric disease in preterm individuals, as measured by their cognitive abilities in early childhood. In a large cohort of unrelated twins, we test for a possible gene-environment interaction between the GPS’s for five different psychiatric disorders (Autism Spectrum Disorder (ASD), Attention Deficit Hyperactivity Disorder (ADHD), Bipolar Disorder, Major Depressive Disorder and Schizophrenia) and gestational age at birth on cognition at age four.Linear regression modelling was used to test for a possible gene-environment interaction between the GPS’s for five different psychiatric disorders  and gestational age at birth on cognition at age four. Genome-wide polygenic scores were adjusted for the first five ancestry principle components, genotype chip and plate and each regression model included the covariates sex and socio-economic status (SES) (Eq. ).Genotype, phenotype and covariate information was available for 4934 individuals . Of these 2066 were born before 37 weeks completed gestation and 918 were born at or below 34 weeks completed gestation. A table showing the number of individuals at different gestational age cut-offs is included in the Supplementary Information  and an interaction effect for gestational age at birth and Bipolar genetic risk . There was no evidence for an interaction effect between gestational age at birth and genetic risk for Autism Spectrum Disorder, ADHD or Major Depressive Disorder. For both Schizophrenia and Bipolar Disorder, the effect of gestational age at birth on cognitive outcome was greater for those individuals with a larger psychiatric genetic risk burden. Results for the full regression model for all five psychiatric pathologies are given in Supplementary Table Results indicated a significant interaction between psychiatric genetic risk and gestational age at birth for two of the five pathologies investigated Table  after Boβ = 0.038, se = 0.013, p = 7.07 × 10–3) and gestational age at birth and Bipolar genetic risk  on cognitive outcome at four. Further, there was no evidence for an association of cognition with twin birth order and the addition of this as a covariate to our model did not significantly alter the results. There remained a significant interaction effect of gestational age at birth and Schizophrenia genetic risk  and gestational age at birth and Bipolar genetic risk  on cognitive outcome at four.Removing the covariate socio-economic status from the model did not markedly alter the results. There remained a significant interaction effect of gestational age at birth and Schizophrenia genetic risk .–3). The adverse effect of birth at earlier gestational age (< = 34 weeks) on cognition is greater for individuals with a higher genetic risk burden for Schizophrenia than it is for those with the lowest risk  including sex and socio-economic status as covariates. None of the psychiatric genetic risk scores showed a significant association with cognition at age four .The similar results obtained for both Bipolar Disorder and Schizophrenia are consistent with evidence for shared genetic aetiology for these disorders. In our cohort there is a significant correlation between genetic risk for Bipolar Disorder and Schizophrenia  and stressful life events, provides an early example of such a study. The authors showed that the level of depressive symptoms experienced by individuals in response to stressful life events was dependant on the allele they carried at this polymorphism.Gene–environment interactions are the result of individuals responding differently to environmental stimuli, depending on their genotype. Many studies have explored gene-environment interactions for psychiatric pathologiesWhilst early studies largely focussed on single polymorphisms, evidence suggests that a polygenic approach is likely to be more fruitful in understanding how genetic risk for psychiatric pathologies may increase susceptibility to environmental risk factors. More recent studies frequently investigate gene-environment interactions on a genome-wide scale, leveraging results from genome-wide association studies to construct polygenic scores. It is hoped that this approach could reveal interactions that may have gone undetected using candidate SNP analysis.15 and early life adversity17 with a few studies exploring perinatal factors. Studies with a perinatal focus include work indicating a significant interaction between maternal CMV infection and a polymorphism in the CTNNA3 gene on schizophrenia risk18 and work indicating that low birth weight predicts poorer academic and physical performance in individuals at high risk of Schizophrenia but not for those at low risk19.Gene-environment interaction studies exploring Schizophrenia have largely focussed on stressful life events13. The majority of work on Bipolar Disorder has been using candidate genes and to the best of our knowledge there have been no previous studies using summary statistics for Bipolar Disorder from the Psychiatric Genomics Consortium.Studies exploring Bipolar Disorder have followed similar themes to those for Schizophrenia focussing on environmental exposure to stressful life events20.Preterm birth itself may be a genetically inherited trait. However, it is difficult to know the extent to which this could contribute to confounding in this work. Despite evidence for a modest contribution of genetics to the risk of preterm birth, to-date, there exist few robust genetic associations identified through genome-wide association studies for prematurity. A large recent study identified two intergenic loci associated with preterm birth but failed to replicate these resultsPreterm birth is complex with heterogenous aetiology. Data that would allow us to distinguish between different aetiologies such as information on maternal infection or maternal pathology were not available in this study. It is possible that genetic risk alleles that confer an increased risk of Schizophrenia or Bipolar Disorder might also confer an increased risk of pathologies associated with preterm birth . Whilst we believe that such shared genetic aetiology, if present, is unlikely to fully explain our results, we cannot exclude the possibility that it could introduce confounding.21. That said, we cannot exclude the possibility that we are underpowered to detect such an association.We have explored the possibility that genetic risk for psychiatric disease is associated with gestational age at birth within our cohort. For the five psychiatric pathologies examined in this study we found no significant association between genetic risk for psychiatric disease and gestational age at birth . As children grow older, smaller differences in ability might be easier to measure but they will similarly be subjected to a larger diversity of additional environmental exposures.Finally, this study focused on cognitive outcome at age four. We hypothesised that the interaction between genetic risk for psychiatric disease and gestational age at birth would explain a significant fraction of the variability in cognitive outcome at age four. Work looking at preterm outcomes has shown stability of impairment in early childhood and confirmed that the most commonly observed impairment is in developmental or cognitive functionIn summary, individuals with greater genetic risk for either Schizophrenia or Bipolar Disorder were shown to be more vulnerable to the negative effects of birth at earlier gestational age on cognitive outcome. The pathophysiology underlying preterm neurodevelopmental impairment may therefore be vulnerable to genetic variability associated with adult psychiatric disease.23. Written informed consent was obtained from parents prior to data collection. Project approval was granted by King’s College London’s Research Ethics Committee for the Institute of Psychiatry, Psychology and Neuroscience (PNM/09/10-104) and the research was performed in compliance with the Declaration of Helsinki.Participants were drawn from the Twins Early Development Study (TEDS). Between 1994 and 1996 TEDS recruited over 15,000 twin pairs born in England and Wales, who have been assessed in multiple waves across their development up until the present date. Roughly 10,000 twin pairs still actively contribute to TEDS, providing genetic, cognitive, psychological and behavioural data. TEDS participants and their families are representative of families in the UKThis study included 4934 unrelated individuals of European ancestry from the TEDS cohort. The dataset was subject to standard exclusions. This included exclusion of individuals with serious medical conditions which would likely affect their ability to take part in the TEDS assessments or conditions known to be associated with significant cognitive impairment. It also included exclusion of individuals in whom extreme circumstances were identified in the perinatal period such as prolonged hospital admission following birth. An initial filtering was then undertaken to select a cohort of unrelated individuals; this involved selection of all the unpaired twins plus one twin from each genotyped pair. Individuals were retained that had genotyping for the GPS analysis, data for cognitive outcome at age four, recorded gestational age at birth and recorded parental socio-economic status.The final sample comprised 4934 individuals. Of these, 1858 were from monozygotic (MZ) twin pairs and 3076 were from dizygotic (DZ) twin pairs (1596 same-sex DZ pairs and 1480 opposite-sex DZ pairs). 2066 of the 4934 individuals were born at less than 37 weeks completed gestation and 918 were born at or below 34 weeks gestation.https://www.wtccc.org.uk/ccc2/). The remaining 3190 individuals were genotyped on HumanOmniExpressExome-8v1.2 arrays at the Molecular Genetics Laboratories of the Medical Research Council Social, Genetic Developmental Psychiatry Centre, using DNA that was extracted from saliva samples.Genotyping was undertaken at two separate timepoints with two different genotyping platforms. 1744 of the 4934 individuals were genotyped on the Affymetrix GeneChip 6.0 SNP array at Affymetrix, Santa Clara  using buccal cell DNA samples. Genotypes were generated at the Wellcome Trust Sanger Institute  as part of the Wellcome Trust Case Control Consortium 2  through the Sanger Imputation Service before merging genotype data from both platforms .After quality control, 635,269 SNPs remained for Affymetrix GeneChip 6.0 genotypes, and 559,772 SNPs for HumanOmniExpressExome genotypes. Genotypes from the two platforms were separately phased using EAGLE22 > 0.1) and excluding high linkage disequilibrium genomic regions.The final data contained 7,363,646 genotyped or well imputed SNPs. Principal component analysis was performed on a subset of 39,353 common (MAF > 5%), perfectly-imputed (info = 1) autosomal SNPs, after stringent pruning to remove markers in linkage disequilibrium (r26. The cognitive ability measure was computed as the mean of the four standardised elements.The standardised cognitive ability measure at four was a composite of ‘Parent Report of Children’s Abilities’ (PARCA) including both a parent-reported and parent-administered element and an assessment of verbal and non-verbal ability. The parent administered assessment included an odd-one-out task, design drawing task and puzzle task and the parent-reported assessment examined conceptual knowledge. Non-verbal ability was assessed with a 48-item vocabulary test and verbal ability with a grammar assessment which looked at length of sentences and correct use of both ‘-est’ words and ‘but’. The parent administered assessments have been validated in a subsampleSocio-economic status is a composite of maternal age at birth of eldest child, the mean score of maternal and paternal highest education level, as well as the respondent’s (mother or father) occupation, administered by the Standard Occupational Classification 2000  when the child was two, which was the first age of contact.We calculated genome-wide polygenic scores, which are the SNP effect-size weighted sums of the number of trait-associated alleles. The SNP weights were derived from summary statistics for the largest GWA studies available for five psychiatric disorders at time of computation .The genome-wide polygenic scores were calculated using the software LDpredpsych × GA) on cognitive outcome at age four, for five different psychiatric pathologies  separately (Eq. (28. Interaction terms for psychiatric genome-wide polygenic score (GPSpsych) with sex and socio-economic status as well as interaction terms for gestational age at birth (GA) with sex and socio-economic status were included to properly control for possible confounding effects29.Linear regression modelling was used to estimate the effect of the gene-environment interaction (GPSely (Eq. ). Prior psych × GA on cognitive outcome. We therefore explored whether the estimate of the coefficient β3 of the interaction term deviated significantly from zero. P-values lower than the significance level α = 0.05/5 = 0.01 were considered significant to account for the family-wise error rate using the Bonferroni method, correcting for the five psychiatric pathologies explored. Calculations were implemented with “lm” function in R (https://www.r-project.org/). Standard errors for the beta regression coefficients were estimated using the bootstrap technique with 10,000 bootstrap samples with random sampling of individuals with replacement.The aim of this analysis was to investigate the effect of the interaction term GPSAs a further exploration of our data, we looked at the extremes of the data undertaking a two-way analysis of variance (ANOVA) comparing mean cognition between the extreme quintiles of polygenic risk for individuals born at or below 34 weeks gestation and term-born (> = 37 weeks) individuals for the psychiatric pathologies that yielded significant results in our initial analysis. Similar analysis for the non-significant pathologies is presented in the Supplementary Information .2\documFor completeness we also explored a possible association between cognition at four and genetic risk for each of the five psychiatric pathologies examined, details can be found in the Supplementary Analysis . The research was performed in compliance with the Declaration of Helsinki.Supplementary Information.


We conducted formative research using in-depth interviews to identify preferences for and anticipated responses to receiving thank you notes and lay summaries of aggregate results among caregivers and adolescent participants of pragmatic pediatric studies conducted by the National Institute of Health-sponsored Pediatric Trials Network. We analyzed the data using qualitative thematic analysis. Nearly all participants said receiving a thank you note would make them feel valued, appreciated, and proud because they contributed to science. Similarly, nearly all participants said that receiving a lay summary of research results would make them aware of their role in improving the lives of children, feel like they are an active partner in research, and believe that researchers want to keep them informed. Participants also said that receiving a thank you note or lay summary may motivate them to participate in future research. Providing thank you notes as part of study participation should become a standard clinical trial practice, similar to the practice of providing lay summaries. •Participants want to be thanked for their contributions to clinical research.•Providing thank you notes and lay summaries demonstrate to caregivers and adolescent participants that researchers value and appreciate their contributions.•Providing thank you notes and lay summaries may also increase caregivers’ and adolescent participants’ interest in participating in future research studies. Sharing lay summaries  of aggreWe engaged two stakeholder groups of PTN studies—caregivers/parents and adolescent participants—in formative research on their preferences for and anticipated reactions to receiving thank you notes and aggregated lay summaries of pediatric clinical research results of pragmatic studies. Findings from this study allow stakeholder data to guide PTN decisions about how, when, where, and what to provide in thank you notes and lay summaries.Here, we describe a subset of the study findings and focus on the perceived value of thank you notes and lay summaries as they are likely applicable to studies outside of PTN. Findings on the preferred content of thank you notes and lay summaries are presented elsewhere.2We conducted a qualitative descriptive study  with carDuring the one-on-one interviews, we asked participants to view several mock versions of thank you notes and lay summaries of PTN study results and describe their expectations of and anticipated reactions if they had received these materials as part of their PTN study participation. We also asked about a pre-identified list of potential reactions if not initially mentioned by the participant. We conducted the interviews by telephone, and they were audio-recorded with participant permission, transcribed, and analyzed using applied thematic analysis .Using NVivo 12 , two anaThe Duke University Health System Institutional Review Board (IRB) reviewed and approved the study, waiving informed consent for the caregiver interviews. For adolescent interviews, adolescents provided their oral informed assent and their caregivers provided parental permission.33.1We interviewed 27 participants (24 caregivers and 3 adolescents) of diverse race, ethnicity, education level, employment, geographic location, and disease state .3.2[Receiving a thank you note] would make me feel appreciated … like I made a difference in something. —Adolescent, age 14[Receiving a thank you note] would give me a sense of purpose. You're not just a piece of data. You've got something else going on. You're involved. —Caregiver, age 47Most participants said caregivers and adolescent participants should receive thank you notes because the notes demonstrate gratitude for their research contributions. Nearly all said receiving a thank you note would make them feel valued, appreciated, and proud because they contributed to science. Participants said:I think that [caregivers] should be thanked. It's hard on us being caregivers. We don't get to clock out. It's a daily struggle. It just feels good to know that there are people out there that support you. They feel for you when it comes to each individual situation.[Caregivers] can be thanked, and maybe know that your child's research is very significant. It's just imperative that [caregivers] feel like they have contributed to something. We are truly grateful that we could help someone else's child. To know that the study helped prevent another child from going through the different adverse effects, that really means a lot to us.Some caregivers expressed that their efforts caring for children with severe illnesses are often unrecognized, and a thank you note would be a source of encouragement. A 26-year-old caregiver said:As a parent of a child [with many health issues] … I think [receiving a thank you note] would encourage me to participate in more studies, to know that my kid helped someone find out information.Some also felt that providing a thank you note may promote altruism and interest in participating in future studies, including among adolescents. A 39-year-old caregiver explained:Participant preferences varied on the timing of providing thank you notes, and some suggested the length of the trial should be considered. For example, one thank you note can be provided at the end of shorter trials and two notes for longer trials—one after enrollment and another at the end. Some participants cautioned on losing sentiment with providing too many thank you notes.3.3[Receiving a lay summary] would make me feel good. Because a lot of times, when you [participate in] these studies in the past, I don't know what came of that. I think [getting a lay summary] gives you more closure than anything. If you actually have it printed out in front of you, and [even though] you're not named individually, you personally know how you answered questions … this is what I contributed to. —Caregiver, age 45[I would] definitely [take part in other research] because they are involving you in the study. They are keeping you in the loop basically. And next time, you're like, “Okay, I'll enroll my child in other studies because they always keep me informed.” —Caregiver, age 31Most participants said caregivers should receive a lay summary of the research results, and about one-third said adolescents should. Participants stressed the importance of informing caregivers of the results, and some explained that caregivers can decide if and when to inform their child. Nearly all said receiving a lay summary would make them aware of their role in improving the lives of children, feel like they are an active partner in research, believe that researchers want to keep them informed, and motivated to participate in future research. Participants said:I'd be more likely [to take part in future research] if I knew the results of one study and was thanked. Knowing that the research team actually cared that you participated, and cared that you found out the results of the study, no matter how long it takes, [makes] you feel like your participation mattered.Most also expressed value in providing a lay summary years after their individual participation is over, acknowledging the long clinical trial timeline, and noted their expectation to receive individual-level results. A 37-year-old caregiver said:4Our findings provide new evidence suggesting that participants want to be thanked for their participation in research and value the acknowledgement. Our findings also contribute to existing evidence on the importance of lay research summaries by demonstrating the value and appreciation that participants place on receiving such information for pragmatic pediatric clinical research. Because of the anticipated participant response and ease of provision, we recommend that participant thank you notes become an expected study procedure in all clinical research similar to the provision of lay summaries.Participants’ narratives also suggest that providing thank you notes and lay summaries may motivate future research participation. Research has shown that receiving a lay summary of research findings increases interest in participation in future research ; such daDr Corneli conceptualized and designed the study, designed the data collection instruments, drafted the initial manuscript, and reviewed and revised the manuscript.Dr. Zimmerman conceptualized and designed the study, and critically reviewed the manuscript for important intellectual content.Mr. Perry designed the study, coordinated and supervised data collection, collected data, carried out the analyses, and critically reviewed the manuscript for important intellectual content.Dr. Benjamin conceptualized the study, obtained funding, and critically reviewed the manuscript for important intellectual content.All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.The authors have no conflicts to disclose.10.13039/100000071National Institute of Child Health and Human Development (NICHD) contract (HHSN275201000003I) for the Pediatric Trials Network (PI Danny Benjamin). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This work was funded under the