Tgr5‐deficient mouse models. The results clearly show that Tgr5 deficiency can facilitate restoration of the spermatogonia homeostasis and allow faster resurgence of germ cell lineage after exposure to busulfan. TGR5 modulates the expression of key genes of undifferentiated spermatogonia such as Gfra1 and Fgfr2. At the molecular level, the present data highlight molecular mechanisms underlying the interactions among the TGR5, GLIS2, and TP53 pathways in spermatogonia associated with germ cell apoptosis following busulfan exposure. This study makes a significant contribution to the literature because it shows that TGR5 plays key role on undifferentiated germ cell homeostasis and that modulating the TGR5 signaling pathway could be used as a potential therapeutic tool for fertility disorders.Spermatogonial stem cells regenerate and maintain spermatogenesis throughout life, making testis a good model for studying stem cell biology. The effects of chemotherapy on fertility have been well‐documented previously. This study investigates how busulfan, an alkylating agent that is often used for chemotherapeutic purposes, affects male fertility. Specifically, the role of the TGR5 pathway is investigated on spermatogonia homeostasis using in vivo, in vitro, and pharmacological methods. In vivo studies are performed using wild‐type and  The present data show that TGR5 signaling interferes with GLIS2 and TP53 signaling pathways, which control stem cell renewal and survival, explaining its impacts on germ cells following injuries as illustrated using chemodrug exposures. The study makes a significant contribution to the literature because it shows that modulating the TGR5 signaling pathway could be used as a potential therapeutic tool for fertility disorders. However, much remains to be defined regarding the regulation of SSC homeostasis. A property of SSCs is their ability to regenerate following injuries, allowing the recovery of fertility over time. Interestingly, in addition to SSC, the progenitor population of spermatogonia had been observed to participate in recovery following injury. Neurogenin‐3 (Ngn3)‐positive progenitors helped replenish the pool of GFRα1‐positive spermatogonial cells during regeneration.The process of spermatogenesis is responsible for the generation of male gametes and thus, is critical for male fertility. The duration of each stage of spermatogenesis is well defined and in mouse, spermatogonia progress to spermatocytes in approximately 1–2 weeks, which then give rise to spermatids in approximately 2 weeks. Spermatogenesis relies on a balance between the self‐renewal and differentiation of germ cells to enable sperm production.1, 2 Thus, animal models exposed to chemodrugs are important in studying the factors involved in the regenerative capacities of undifferentiated germ cells.Among all the studied models, rodents that have been acutely exposed to busulfan (Bu) is a classic model for studying spermatogenesis, as the germ cell lineage is very sensitive to chemotherapies such as alkylating agents due to their high proliferation rate.7 Thus In mice, a single dose of Bu alters spermatogenesis. The effect of Bu is transient and dose‐dependent; fertility could be restored, but the duration of recovery depends on the dose taken. Therefore, this transient germ cell loss is useful to decipher the mechanisms involved in germ cell homeostasis associated with survival, proliferation, and/or cell differentiation processes. This is important because the deleterious impacts of chemotherapies  on post‐treatment quality of life, especially on fertility, is a major problem for cancer survivors. Thus, understanding the mechanisms involved in germ cell chemosensitivity is key to limit the long‐term deleterious consequences of anticancer treatments.Bu is a chemotherapy molecule commonly used to condition a progenitor cell transplant for the treatment of chronic myeloid leukemia. Treatment could be associated with secondary side effects. Bu treatment results in prolonged azoospermia, which impacts the germ cell lineage.8 In m DNA damages caused by Bu resulted in increased TP53 expression and activation via posttranslational modifications, such as phosphorylation, which are associated with cell apoptosis. This leads to increased permeability of the mitochondrial membrane, leading to the release of cytochrome C and induction of apoptosis. Furthermore, Bu‐treated testes exhibited an increase in lipid peroxidation after 1–2 weeks, suggesting an increase in reactive oxygen species (ROS) production, leading to apoptosis following Bu exposure.Bu acts preferentially by adding an alkyl group between two guanines of the DNA or between a guanine and an adenine. This leads to the formation of intra‐strand DNA bridges, resulting in single strand breaks that block DNA replication and transcription. In the longer time frame, inhibition of cell proliferation and differentiation was observed.9 DNA  Bile acids act through the nuclear receptor FXRα (NR1H4) and the G‐protein‐coupled bile acid receptor . It was initially described that TGR5 binds to the ligand, resulting in the internalization of the receptor into the cytoplasm, leading to an increase in intracellular cyclic AMP (cAMP) concentrations and activation of the protein kinase A signaling pathway. Using TGR5 knockout mice (–/–Tgr5), it was found that several metabolic pathways were under the influence of TGR5 activity, even if the –/–Tgr5 mice did not show major apparent deleterious phenotypes in normal conditions. TGR5 receptor appears to control bile homeostasis, energy expenditure, glucose metabolism, immunity and inflammation.Over the last decades, bile acids have been demonstrated to act as signaling molecules that regulate many physiological functions, including male fertility.16 Bilα. It controls the testicular endocrine function, establishment of spermatogonial stem cells during neonatal life, and adult germ cell survival. Parallelly, signaling pathways of bile acids through TGR5 have been defined in adult mouse testis. The absence of TGR5 protects against the deleterious effects of diet‐supplemented bile acids on the testis and ultimately, on fertility. A diet enriched in bile acids leads to a decrease in fertility associated with testicular defects, without impacting testosterone levels. Initially, bile acids alter the expression of cell‐cell interaction genes such as Connexin‐43 and N‐Cadherin via TGR5. This leads to germ cells detaching from the seminiferous epithelium and the disruption of the blood‐testicular barrier.Within the testis, some roles of bile acids have been associated with the signaling pathways of FXRα.15 It  The offspring of male mice, when exposed to a bile acid‐supplemented diet, exhibited significant perinatal lethality associated with a defect in bile acid homeostasis, reduced postnatal growth, and impaired carbohydrate metabolism in adulthood. These phenotypes are maintained for up to two generations of individuals and appear to be associated with lower levels of DNA methylation in the sperm of individuals that have been exposed to bile acids, compared to control animals. All these alterations are not found in Tgr5 knockout individuals.Bile acids have also been shown to have a deleterious impact on sperm quality.28 The Consistently, analyses of published single‐cell data have shown that TGR5 expression was observed in mouse and human spermatogonia; additionally, pseudotime analyses have determined that TGR5 belongs to a group of genes corresponding to undifferentiated germ cells . TGR5 was also found in a group of early genes along the spermatogonial trajectory, representing a pattern consistent with the SSC specification . These data suggest that within the spermatogonia, TGR5 may be involved in the transition phase from one stage to another, controlling the proliferation and/or differentiation processes. However, the roles of TGR5 in spermatogonia homeostasis have not been studied yet.Together, these findings suggest major roles for TGR5 in testicular physiology and pathophysiology. TGR5 was demonstrated to be expressed in the testicular germ cell lineage in mouse and human.27 ConThis study aimed to decipher the role of TGR5 on the germ cell response to chemotherapy using Bu and subsequent regeneration of the cells. A combination of in vivo, in vitro, and pharmacological approaches was used to define the roles of the TGR5 signaling pathway on spermatogonia homeostasis.22.1Wt) males and Tgr5‐deficient (–/–Tgr5) male mice were administered a single injection of Bu (15 mg/kg). Exposure to Bu had almost no adverse impact on body weight in Wt and –/–Tgr5 male mice   of –/–Tgr5 males produced offspring  of Wt males regained fertility ; whereas almost no impact was observed on –/–Tgr5 males (6.8 pups per litter) compared to their respective vehicle‐treated controls  was significantly reduced 4 weeks after treatment with Bu in both the e Figure . This pa No difference was observed in the raw number of PLZF+ cells per seminiferous tubule between the Wt and –/‐Tgr5 males in vehicle treated groups , whereas no impact of Bu was observed in –/–Tgr5 mice  marker.25 No u Figure . At 4 weu Figure . These de Figure . This ma–/–Tgr5 males compared to Wt mice.These data support the findings that Bu first affected spermatogonia, resulting in germ cell loss, which was then followed by the regeneration of the germ cell lineage, initiated from the remaining undifferentiated spermatogonia, to give rise to a new wave of spermatogenesis. In addition, these in vivo data suggests that Bu mediated its effects via alterations of the apoptotic and proliferation processes, all of which were minimized in 2.3Tgr5 is expressed in the germ cell lineage; additionally, data from recent single cell analyses showed that TGR5 mRNA expression was observed in mouse and human spermatogonia . This is consistent with the fact that TGR5 must play a direct role within spermatogonia, which explains why –/–Tgr5 mice recovered spermatogenesis faster than Wt males after chemotherapeutic exposure. Next, we wanted to better decipher the roles of TGR5 within the germ cell lineage. However, none of the antibodies in our possession raised against mouse TGR5; therefore, we analyzed the expression of TGR5 by detecting GFP in a mouse TGR5‐T2A‐GFP model expressing the transgene under the control of endogenous mouse Tgr5 promoter. The present results showed that the sequence driving the expression of Tgr5 were connected to the detection of the protein in all germ cell lineage  corresponded to germ cell‐specific knockout males Tgr5 in control DMSO‐treated condition (vehicle: veh.)   of f/–; Cre–/–Tgr5 males did   or an siRNA directed against sly used.27 As No difference was observed in the germ cell apoptotic rate between genotypes in vehicle treated condition . Among the genes identified in the TP53 network, several genes such as Perp, Phlda3, and Mmp24, have been validated using qPCR  of the genes positively regulated by Bu in siCtrl‐transfected cells, with an effect that was counteracted by siTgr5 transfection , using the Crispr/Cas9 technology , in combination with the transfection of a cAMP‐responsive element fused to luciferase , the data supported in vitro findings, showing a lower mRNA accumulation of e Figure 9A. In adWt and –/–Tgr5 mice up to 5 days post‐treatment , the mRNA accumulation of Tgr5 was correlated with the expression of Mmp24 and Perp   Figure . This waGlis2 mRNA accumulation in the testis of –/–Tgr5 males compared to Wt males  and with the Plzf mRNA     and with the expression of Plzf   was correlated with the mRNA level of Stra8  Figure . This hi) Figure  and with) Figure . These dAll these data sustained the idea that TGR5 pathways are associated with regenerative capacity of germ cells in part through the interaction with GLIS2 and TP53 pathways. Our data also support the idea of a putative crosstalk with the Ngn3‐Miwi2 pathway in progenitor cells that could participate to expand the pool of spermatogonial cells with stem cell activity under regenerative conditions.Gfra1; Fgfr2) and progenitors (Ngn3 and Miwi2) in the THY1+ purified spermatogonia.All the present in vivo data supported the in vitro observations. To better decipher the crosstalk between TGR5 and Bu, we performed analyses at 1‐week after Bu exposure, when the number of PLZF+ cells was different between genotypes. To avoid the bias of cellularity induced by Bu between genotypes, we analyzed the expression of markers of SSC  was altered in Wt spermatogonia only at 1 week after treatment and not at 3 days post‐treatment , which acts through TGR5 in GC1spg cells; this was demonstrated using the cAMP‐response element fused to luciferase  could lead to synergistic deleterious effects on germ cell in vivo. Wild‐type males were treated once with Bu at the beginning of the experiment, and received a diet supplemented with 0.5% cholic acid (CA) for 8 weeks. Male mice treated with Bu and sacrificed after 4 weeks showed an almost complete recovery of PLZF+ cell counts Figure . In cont3α, plays a key role in the homeostasis of undifferentiated germ cells as the FXRα knockout mice presented an increased number of undifferentiated germ cells throughout their postnatal life. This was associated with the maintenance of reproductive capacities during aging. Moreover, data obtained from mice have shown that the G‐protein coupled bile acid receptor TGR5 is involved in cholestasis‐induced fertility disorders. Under physiological conditions, no difference was observed between Wt and –/–Tgr5 males. In this study, we used mice treated with Bu as a classical model of transient germ cell depletion to demonstrate that the TGR5 signaling pathway plays critical roles in germ cell homeostasis following injuries.Recently, bile acid homeostasis has been demonstrated to play some roles in controlling male fertility. The nuclear receptor of bile acid, namely FXRtal life.24 Thi Bu led to a decrease in the number of undifferentiated germ cells during the first days following treatment. In turn, this effect on undifferentiated germ cells led to a major impact on spermatocytes, which then affected spermatids and sperm cells, thereby affecting fertility. This confirmed that a Bu‐based study design can help understand the properties of undifferentiated germ cells. It has to be noted that this approach relied on single injection, in contrast to clinical conditions where patients undergo chronic exposure to Bu, in combination with fludarabine or cyclophosphamide. Bu is given at a dose of 3.2 mg/kg, which is repeated over 2 to 4 consecutive days . Thus, the final dose used in this study (15 mg/kg) was consistent with what is used in the clinic, although it is possible that there is a difference between acute and chronic exposure.Regarding the responsiveness to Bu, the present work showed that Bu acted primarily on undifferentiated spermatogonial cells, which was in agreement with the results from other studies.14 Bu Wt and –/–Tgr5 males. Differences are observed between genotypes mainly at the time of germ cell lineage recovery, resulting from the differential ability of –/‐Tgr5 undifferentiated germ cells to proliferate, survive, and/or differentiate. Thus, the present data demonstrate that TGR5 is a key factor for cellular repair and tissue regeneration following injuries.The impact of chemotherapeutic treatments depends on their bioavailability, which depends on their elimination and the various detoxification mechanisms involved. The half‐life of Bu is approximately 3 h, and therefore, it seems unlikely that TGR5 is involved in Bu detoxification as the initial effects of Bu on testicular physiology and on germ cell lineage are almost similar between Gfra1 and Fgfr2 mRNA accumulation, and these effects were lowered in a low TGR5 state. Both FGFR2 and GFRA1 were defined as critical actors of the self‐renewal of spermatogonial stem cells. Moreover, recent data highlighted the role of FGFR2 in inducing germ cell differentiation. All these data support the fact that TGR5 may play a critical role in the maintenance of the SSC pool and in the mechanisms of cell fate and regeneration following injuries, as illustrated here using anti‐cancer therapies. This may explain in part how the germ cells of –/–Tgr5 males were able to rapidly initiate a new wave of spermatogenesis to repopulate the seminiferous tubules and give birth to offspring.Regarding undifferentiated spermatogonia, the present results demonstrated that Bu exposure decreased em cells.44 Mor Moreover, after injury, within the NGN3+ population, MIWI2‐expressing cells exhibited stem cell activity that is essential for the efficient regenerative capacity of the testis. The data from this study identified strong correlations between Glis2 and Tgr5 with Ngn3 and Miwi2, reinforcing the identification of molecular mechanisms resulting in germ cell re‐emergence after chemotherapy‐induced injury. Thus, TGR5 might act on either SCC and/or progenitor spermatogonia, affecting germ cell and fertility recovery after injury.Next to the SCC pool, it has been demonstrated that the NGN3+ spermatogonial progenitor population retain stem cell potential to contribute to regeneration after injury.6 More Therefore, the results of the present study led us to derive a scheme connecting the TGR5 and Bu signaling pathways  production. Indeed, mice treated with melatonin after Bu injection show enhanced spermatogenesis. Melatonin drives the expression of MnSOD (MaNganese SuperOxide Dismutase), which counteracts the apoptosis caused by high levels of Bu‐induced ROS. In parallel, melatonin stimulates the expression of SIRT1, which participates in TP53 deacetylation, leading to TP53 degradation and resulting in cellular resistance to apoptosis. Although we did not detect lipid peroxidation or ROS‐related gene clusters in the RNA‐seq approach, the involvement of such a mechanism cannot be completely excluded because busulfan was found to modulate the level of acetylated‐TP53 (ac‐TP53) and this impact was lowered in siTGR5‐transfected cells  were sterile. When spermatogenesis was restored, Bu‐treated Tgr5–/– males, which were then bred with an untreated female after 20 weeks, gave birth to a normal number of offspring per litter without major impact on the number of fetuses that died in utero. However, it is conceivable that Bu may have an impact on sperm quality, which could translate into an impact on the offspring. The fact that TGR5‐mediated apoptosis leads in part to delayed healing does not necessarily exclude it from participating in a protective mechanism to avoid DNA damage in germ cells and thus safeguard gamete quality. Thus, although it is beyond the scope of the present study, many experiments remain to be performed to get a better idea of the beneficial impact of positive or negative modulation of TGR5 signaling on sperm quantity and quality. We believe that this issue will need to be further evaluated using relevant models in future experiments.The results of this study demonstrate that Tgr5 invalidation counteracts testicular defects and degradation of sperm quality, which is induced by a cholic acid‐supplemented diet with a major metabolic impact on multiple generations of individuals. This suggests that Tgr5 plays an important role in sperm quality, and this effect appears to be dependent on the de novo DNA methyltransferase enzyme DNMT3B. A diet supplemented with cholic acid leads to a decrease in Dnmt3b expression through TGR5 in germ cells, resulting in a decrease in DNA methylation. Thus, before proposing the modulation of TGR5 in the clinic to safeguard fertility after chemotherapy, it is essential to realize its impact on sperm quality and offspring over several generations in experimental models. This will allow us to avoid defects in sperm quality that could lead to the transgenerational transmission of abnormalities.The qualitative impact on spermatozoa is relevant to the continuation of this study, as it is known that ividuals.28 ThiTgr5 in mice minimized the effects of Bu on spermatogenesis, which raised the question of the impact of TGR5 in response to other chemotherapies. Interestingly, the data showed that the impact of the lack of Tgr5 was also observed using other chemodrugs such as treosulfan, hepsulfam, and cyclophosphamide. In contrast, Tgr5 invalidation did not alter the response of GC1spg cells to cisplatin. It is interesting to note that cisplatin gives rise to double DNA strand breaks, whereas Bu, cyclophosphamide, and treosulfan generate single strand DNA breaks. These data must help to further define the underlying molecular mechanisms associating the lack of Tgr5 to protection against chemodrugs.The role of TGR5 in response to Bu was evaluated because it is a commonly used molecule in the study of spermatogenesis. The results showed that the invalidation of d breaks,49 wheNext to the protective effect of the lack of TGR5 to minimize the impacts of chemodrugs, the present data show that co‐exposure to Bu and bile acids (endogenous ligands of TGR5) lead to synergic deleterious effects on testicular physiology in vivo. These results were obtained using a cholic acid supplemented diet concomitant with Bu treatment, and they showed that the presence of high levels of bile acids could increase the effectiveness of the treatment by leading to greater and prolonged cell death. This additive effect between Bu and TGR5 activation was confirmed on GC1spg cells. These data support the idea that in the pathophysiological conditions of cancer‐treatment using chemotherapy, it is critical to consider pre‐existent hepatotoxicity. It could lead to a more permanent effect of the chemotherapy on germ cell capacity to recover. As highlighted here, the activation of TGR5 by its agonists led to repression of GLIS2 expression and activation of TP53. This molecular mechanism must explain the crosstalk between TGR5‐GLIS2 and TP53 Figure . These d Microbiota is involved in the synthesis of secondary BA, which are endogenous ligands of TGR5. The potential association between TGR5 and BA produced by IM will have to be studied to better understand how microbiota might impact male fertility following treatments with anticancer drugs.All the data highlighted the major role of bile acid signaling pathway through TGR5 in germ cell lineage. Interestingly, recent publications demonstrated that the reduction of the richness and/or diversity of intestinal microbiota (IM) in the context of exposure to anticancer drugs (such as Bu), negatively impacted testicular physiology, leading to reduced sperm production.52 Mic–/– males after Bu exposure and the prolonged effect of Bu when combined with a TGR5 agonist demonstrate that this pathway is involved in controlling germ cell fate. Specifically, these results identify the key involvement of TGR5‐mediated modulation of GLIS2‐ TP53 pathways in the regulation of stem cell renewal and survival gene expression.In conclusion, the present work defines the hitherto unidentified roles of the TGR5 on the physiology of spermatogonial stem cells and progenitor cells. The early germ cell resurgence in Tgr5 The present work work helps to further understand fertility disorders in the context of cancer treatments and provides new perspectives for developing therapeutic solutions targeting TGR5 for fertility disorders to promote germ cell lineage regeneration. Among chemotherapies, alkylating agents induce long‐term testicular dysfunction and impair fertility. However, apart from the cryopreservation of sperm, which is not always possible, little progress has been made to preserve male fertility following cancer treatments, particularly chemotherapy. Therefore, the side effects of cancer treatments on the quality of life, including the maintenance of fertility, is a key issue. It is therefore important to pursue research programs to decipher the underlying molecular mechanisms to improve cancer treatments while minimizing their long‐term side effects, particularly on fertility. This is a public health issue, because with advances in the effectiveness of cancer treatments, patient survival rates have increased, and it is estimated that 1 in 530 young adults aged 20 to 40 years is a cancer survivor; this relative number is expected to increase in the coming decades.These data are relevant because it has been shown that 69% of patients who underwent chemotherapy  before hematopoietic stem cell transplantation had an increased risk of persistent azoospermia.54 The4−/−Tgr5 mice used have been previously described. The mice used in this study were maintained in a C57BL/6J background.C57Bl/6J mice were purchased from Charles River Laboratories . Mice were acclimated at least 2 weeks before experiments. The Tgr5 floxed mice and the model driving the expression of the recombinase‐CRE under the control of the Vasa‐promoter. The hTGR5‐T2A‐GFP mouse line was previously described.To better decipher the role of TGR5 within the germ cell lineage, specific germ cell knock‐out mice were generated using the oxed mice58 andMice were housed in temperature‐controlled rooms with 12 h light/dark cycles. Mice had ad libitum access to food and water. The refinement is based on the housing and monitoring of the animals as well as the development of protocols that consider the animal welfare. This has been achieved by enriching the cages (cardboard tunnel and mouse houses). The mice were housed in social groups with cage sizes that complied with the legislation according to the number of mice.12‐week‐old mice were exposed to busulfan, hepsulfam or cyclophosphamide  and organs were harvested at several time points after from 1 day up to 20 weeks after exposure. The number of animals per group was defined on independent experiments to validate the reproducibility of results.To minimize the confounders, several independent experiments were done, and groups of animals were not sacrifice in the same order from one experiment to another. These protocols have already been performed in adult mice where no suffering was reported in the time frame of our experiments. However, we have performed daily monitoring of the animals in the first few days following the injection. Wehave monitored food consumption, the possibility of diarrhea. We have checked for signs of suffering activity disorders or physiological changes. No signs of suffering were reported.This study was conducted in accordance with current regulations and standards approved by Institut National de la Santé et de la Recherche Médicale Animal Care Committee and by the animal care committee .Each male was mated at night with a female C57Bl6J (Charles River) for 10 days. Mating was monitored daily for vaginal plugging to determine if mating had occurred. After 17 days of gestation, the mating efficiency was inspected and the number of pups per litter was counted.The testes were collected, fixed in 4% PFA and embedded in paraffin, and 5 µm‐thick sections were prepared and stained with H&E. on 5 µm sections of testis fixed in paraformaldehyde 4%. Raw numbers of TUNEL positive cells are given for each vehicle groups in dedicated graphs and then the impact of pharmacological molecules is compared to vehicle groups. For that purpose, vehicle groups of each genotype have been arbitrarily set at 1 and the results are expressed as the relative number of TUNEL positive cells.TUNEL experiments were performed, as previously described27 on 5 µm sections were mounted on positively charged glass slides (Superfrost plus), deparaffinized, rehydrated, treated for 20 min at 93–98°C in 0.01 M citric buffer‐tween 0.1% (pH 6), rinsed in osmosed water (2 × 5 min), and washed (2 × 5 min) in Phosphate‐buffered saline (PBS). Immunohistochemical studies were conducted according to the manufacturer's recommendations. Slides were then counterstained with Hoechst medium (1 mg/mL) and then mounted on PBS/glycerol (50/50). The antibodies used are reported in Table For some experiments, raw numbers of positive cells are given for vehicle‐treated groups  in dedicated graphs and then the impact of molecules is analyzed regarding respective vehicle group of each genotype. For that purpose, the vehicle groups of each genotypes have been arbitrarily set at 1. GC1spg Tgr5 knockout cells (GC1spgKO‐Tgr5) were generated as following:GC1spg cells  were used as previously described.27 GC1http://crispor.tefor.net/. The sequences used are for guide‐1: GGCTGCGCAAGTGGCGGTCC and for guide‐2: GCCGGAACCATCAGGGCTAC. Then, the guides were introduced on PX458: pSpCas9(BB)‐2A‐GFP (PX458) (Addgene Plasmid #48138)  and PX459: pSpCas9(BB)‐2A‐Puro (PX459) V2.0 (Addgene Plasmid #62988). pSpCas9(BB)‐2A‐Puro (PX459) was a gift from Feng Zhang . Guides were cloned on vectors at the BbsI restriction site.For the generation of the GC1spg Tgr5 knockout cells, using Crispr/CAS9 approach .59 GuiGC1spg cells were plated on 6‐well plates and transfected, using Jet PEI (Ozyme) with 1 µg of guide 1 in PX458 (expressing pfg) and 2 µg of guide 2 in PX459. 24 h after transfection, using GFP expression, GC1spg cells were sorted by FACS (BD FACSMelody™ Cell Sorter from BD Biosciences) and plated as individual cell in a 96‐well plate in GC1spg conditioned medium (DMEM and 1% SVF). The clones were then validated by sequencing and genotyping. Primer sequences for PCR are giver in Table KoTgr5 cells, cells were plated in 6 well plates. 24 h (24 h) after plating, cells were treated with vehicle , INT777  or busulfan 200 × 10−6 m, cyclophosphamide (75 × 10−6m), hepsulfam (3 × 10−6m), treosulfan (10 × 10−6m). For cisplatin (5 × 10−6m), vehicle used was NaCl 0.09% (1/1000). Then, cells were harvested at different time points, and messenger RNA (mRNA) or protein extractions were performed.For non‐transfected GC1spg or GC1spgTgr5 and the control siRNA were previously used (see). The siRNA directed against Glis2 was purchased to DarhmaconTM (SMART pool siRNA L‐042208‐01‐0005). The siRNA directed against Tgr5 or Glis2, as well as control siRNA, siCtrl (si Gfp), were transfected at 5 ng per well.GC1spg cells were transfected with small interfering RNA (siRNA) using interferin  in six‐well plates (50 000 cells per well). The siRNA directed against used (see27). TGC1spg cells were transfected with Jet‐PEI  in six‐well plates . The plasmid vector of GLIS2  or empty vector were transfected at 200 ng per well.−6 m), cyclophosphamide (75 × 10−6m), hepsulfam (3 × 10−6m), treosulfan (10 × 10−6m), NaCl 0.9% (1/1000) or cisplatin (5 × 10−6m).24 h after the transfection cells were starved for 12 h and treated for 6–24 h later with either DMSO (1/1000) or busulfan  or OA , and 24 h later with either DMSO (1/1000) or busulfan (200 × 10−6 m).Cells were pretreated with vehicle , INT‐777  in 24‐well plates. cAMP‐RE‐luc construct (360ng) was transfected together with expression plasmid encoding for mouse TGR5 (40ng). The quantity of DNA was maintained constant by addition of empty pCMX vector to a total amount of 1000ng of DNA per well. Twenty‐four hours after transfection, cells were treated with vehicle, INT‐777 (12.5 or 25 µM), or OA (25 µM). Cells were harvested 24 h later and assayed for luciferase. Luciferase values were normalized to protein quantity.cAMP‐response element‐luciferase (cAMP‐RE‐luc) reporter plasmid has been previously described.60 GC1−6 m) or vehicle (1/1000) for 24 h and then cells were exposed to vehicle (DMSO 1/1000) or Bu (200 µM) for 24 h. Cells were then incubated with BrdU (10 × 10−6 m) for the one last hour. Then cells were washed with PBS1X and fixed with methanol. The detection of BrdU was performed using primary antibody anti‐Bromodeoxyuridine  revealed with specific Alexa488‐coupled secondary antibody.To define proliferation rate, cells were pretreated with INT‐777 . The real‐time PCR measurement of individual cDNA was performed using SYBR green dye  to measure duplex DNA formation with the Eppendorf‐Realplex system. For each experiment, standard curves were generated with pools of cDNA from cells with different genotypes and/or treatments. The results were analyzed using the ΔΔct method. Primers sequences are reported in Table Proteins were extracted from tissues using RIPA lysis buffer completed with protease inhibitors . Antibodies were suspended in Tris‐buffered saline, 0.1% Tween, and 5% BSA. The list of antibodies used is given in the Supporting Information (Table GC1spg cells transfected with siRNA‐Ctrl or siRNA‐Tgr5 and treated with vehicle (1/1000) or busulfan (200 × 10−6 m). Starting from RNA, all preparations were made using the IGBMC platform (Illkirch). The mRNA‐seq libraries were sequenced (1 × 50 b).The experiment was performed on  and the BoWtie2 v2.1.0 aligner. Only uniquely aligned reads were retained for further analysis.Reads were mapped onto the mm10 assembly of the mouse genome using Hisat2 v2.1.061 and3 using gene annotations from Ensembl release 77.Quantification of gene expression was performed using HTSeq v0.5.4p363 usi Comparison between groups was performed using the method proposed previously define implemented in the DESeq2 Bioconductor library (DESeq2 v1.0.19). Resulting p values were adjusted for multiple testing using the method of Benjamini and Hochberg.Read counts were normalized across libraries with the method proposed by Anders and Huber.64 Comp < 0.01) that were differentially expressed after Bu exposure in cells transfected with siCtrl or with siTgr5.We generated lists of genes  .Testis cell suspensions from adult Wt or Tgr5l sorting67 usiFor some in vivo experiment raw number of positive cells is given for each vehicle groups in dedicated graphs and then to analyze the impact of pharmacological molecules compared to vehicle groups; vehicle groups of each genotype have been arbitrarily set at 1.All numerical data are represented as mean ± SEM. Significant difference was set at P < 0.05.n) for each statistical analysis, Number and type of replicates  are reported in the figure legends.Sample size (Differences between groups were determined by t‐test or two‐way ANOVA. Multiple comparisons were made with Holm‐Sidak's test. Spearman correlation test was used for some analysis.Statistical analyses were done using Sigmastat3 software.The authors declare no conflict of interest.All authors agree with this publication. T.L. and V.D.H. performed and designed the experiments. T.L., H.H., M.M., G.M., d.H.A., D.‐S.C., R.Y., S.J.‐P., P.A., and S.K. performed the experiments. T.L., H.H., R.Y., B.C., and V.D.H. participated to write the paper.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Slipped capital femoral epiphysis (SCFE) is a common problem treated by pediatric orthopedic surgeons. A 13-year-old male presented with right-sided hip pain. Both sides, symptomatic and asymptomatic, were treated with a single 7.3-mm screw. The patient returned with symptoms to the bilateral hips 16 months after the procedure. He was treated with removal of hardware and revision fixation with a good outcome. We report a rare case of fixation failure in bilateral SCFE with an excellent outcome. We highlight the importance of quick recognition of failure before displacement and a strategy for hardware removal. Slipped capital femoral epiphysis (SCFE) affects on average nearly 11 out of 100,000 children with a male predominance . BilaterA 13-year-old male presented to our outpatient pediatric orthopedic clinic with a one to two-week history of right-sided progressive hip pain with ambulation. According to reports, the patient probably had a fall a few weeks prior but the pain was possibly noted for some time before. On physical examination, the patient was noted to have external rotation to 60 degrees on a bilateral hip examination. He had -5 degrees of internal rotation bilaterally. He had pain in his groin on the right side greater than the left. He was able to ambulate but had a mildly antalgic gait on the right side. He had referred knee pain on the right side as well. Anteroposterior (AP) pelvis and frog-leg bilateral hips were performed. The frog leg is shown in Figure Radiographs were notable for right-sided physeal widening most notable on the frog lateral view, and slight inferior displacement of the epiphysis relative to the metaphysis on the AP. The left hip was otherwise unremarkable. Labs were drawn at this appointment that showed a low vitamin D level, but a normal thyroid-stimulating hormone. The patient had a bone age hand assessment of 13 years of age. He was provided crutches and a wheelchair to reduce weight-bearing. He was scheduled for surgery for in situ screw fixation of the right proximal femur and prophylactic fixation of the left proximal femur. Radiographs were taken in the hospital which showed no changes from prior.He was taken to the operating room where two 7.3 mm × 85 mm fully threaded cannulated screws were placed transphyseal into the central portion of bilateral femoral heads with no complications. No formal capsular decompression was performed. He was discharged with no complications on postoperative day one with notable improvement in his hip pain on range of motion assessment. The patient was seen as an outpatient twice once at two weeks and then at nine months with no complaints of pain and radiographs that appeared unchanged, as shown in Figure 2 during this time frame. It was described as being milder compared to the initial presentation but present. This was not precipitated by any traumatic event. His physical examination was mostly benign with a normal range of motion and minimal to no pain on walking. Radiographs taken at this time revealed bilateral screw failure with the progression of displacement  was used to remove the retained distal portion of the screws bilaterally Figure .Three Synthes cannulated screws were placed bilaterally on the right, all three were 6.5-mm fully threaded screws in new screws tracts, and on the left, there were two 6.5-mm fully threaded and one 7.3 mm screw. The 7.3-mm screw was placed in the same start point as the prior screw due to the lack of available space for a new screw trajectory in that hip.Postoperatively, he was seen by the pediatric endocrinology team which ordered a more extensive endocrine workup which showed a continued vitamin D deficiency despite supplementation. They also recommended an outpatient workup with a dual-energy X-ray absorptiometry scan and the possibility of a pediatric genetic consult. He was cleared for discharge by physical therapy, orthopedics, and endocrinology on postoperative day two with no complications.The patient missed his initial postoperative appointment but presented at the three-month follow-up. According to the family report, the patient was compliant with non-weight-bearing for the first six weeks but progressively began to weight bear thereafter. His pain had completely improved with full painless flexion of bilateral hips, internal rotation of 5 degrees, and a painless gait. His radiographs showed intact hardware with no signs of failure.At the 12-month follow-up, the patient was shown to be well-healed proximal femoral physis with a remodeled femoral neck on the left walking without pain Figure .Percutaneous placement of cannulated screws continues to be a mainstay for the treatment of SCFEs. However, the construct and placement of screws may need some rethinking. There have been risk factors regarding the progression of a slip without hardware failure , as wellThere are theoretical risks with multiple screw placements such as chondrolysis due to inadvertent joint penetration and prominence. However, multiple studies have also shown a biomechanical advantage to multiple screws . It has If screw failure is noted early intervention is recommended. Even if screws initially only appear to be bent, the screws fracture on retrieval attempt, as occurred in our patient’s case. Prior case reports have suggested leaving the failed screw and supplementing with additional screws around the prior screw. We believe that extraction of the failed screw is best to allow easier placement of multiple new screws for greater stability. For cannulated screws, the conical extracting device is easy to place in the prior screw tract with fluoroscopic guidance. The reverse threads allow engagement of the distal screw segment and removal. When planning screw removal, our preferred technique is to provisionally pin the SCFE with an additional guidewire while removing the screw to prevent the possibility of further displacement due to the torque levels seen while using the conical extractor. If possible, using a wire that is compatible with the cannulated system of choice would allow for quick placement of a new screw after extraction of the failed screw. We opted for multiple screws at the second surgery for greater stability and to increase the chances of epiphysiodesis to occur at the proximal femoral physis. If revising a failed screw, the biomechanical advantage of multiple screws is preferable to just upsize a prior screw.Though rare, biomechanical failure of centrally placed screws in a mild stable SCFE is possible. Host factors and patient compliance may have an impact on failure, but there is a need for further investigation regarding the biomechanics of cannulated screw failure. If a failure occurs, it is our recommendation to extract the broken or bent screw and place multiple screws for stability and increased likelihood to lead to completion epiphysiodesis of the proximal femoral physis.


Taken together, these site-specific PTM maps and the dynamic changes of site-specific collagen PTMs in ECM during heart regeneration will open up new avenues to decode ECM remodeling and may lay the foundation to tinker the cardiac regeneration process with new approaches.Cardiac fibrosis-mediated heart failure (HF) is one of the major forms of end-stage cardiovascular diseases (CVDs). Cardiac fibrosis is an adaptive response of the myocardium upon any insult/injury. Excessive deposition of collagen molecules in the extracellular matrix (ECM) is the hallmark of fibrosis. This fibrotic response initially protects the myocardium from ventricular rupture. Although in mammals this fibrotic response progresses towards scar-tissue formation leading to HF, some fishes and urodeles have mastered the art of cardiac regeneration following injury-mediated fibrotic response. Zebrafish have a unique capability to regenerate the myocardium after post-amputation injury. Following post-amputation, the ECM of the zebrafish heart undergoes extensive remodeling and deposition of collagen. Being the most abundant protein of ECM, collagen plays important role in the assembly and cell-matrix interactions. However, the mechanism of ECM remodeling is not well understood. Collagen molecules undergo heavy post-translational modifications (PTMs) mainly hydroxylation of proline, lysine, and glycosylation of lysine during biosynthesis. The critical roles of these PTMs are emerging in several diseases, embryonic development, cell behavior regulation, and cell-matrix interactions. The site-specific identification of these collagen PTMs in zebrafish heart ECM is not known. As these highly modified peptides are not amenable to mass spectrometry (MS), the site-specific identification of these collagen PTMs is challenging. Here, we have implemented our in-house proteomics analytical pipeline to analyze two ECM proteomics datasets  of the zebrafish heart during regeneration (post-amputation). We report the first comprehensive site-specific collagen PTM map of zebrafish heart ECM. We have identified a total of 36 collagen chains (19 are reported for the first time here) harboring a total of 95 prolyl-3-hydroxylation, 108 hydroxylysine, 29 galactosyl-hydroxylysine, and 128 glucosylgalactosyl-hydroxylysine sites. Furthermore, we comprehensively map the three chains  of collagen I, the most abundant protein in zebrafish heart ECM. We achieved more than 95% sequence coverage for all the three chains of collagen I. Our analysis also revealed the dynamics of prolyl-3-hydroxylation occupancy oscillations during heart regeneration at these sites. Moreover, quantitative site-specific analysis of lysine-O-glycosylation microheterogeneity during heart regeneration revealed a significant ( Cardiac fibrosis leading to heart failure has remained one of the major causes of death worldwide . Extraceet al. have predicted 58 collagen genes contributing towards the zebrafish matrisome (1164)  .Apart from proline hydroxylation, lysine hydroxylation, and glycosylation are also critically important for the proper functioning of collagens. In collagen chains, lysine residues occurring within the Gly-Xaa-Lys motif are likely to be hydroxylated by different family members of the lysyl hydrolase; lysyl hydroxylase 1 (LH1), LH2, and LH3. Once hydroxylated, these hydroxylysines (HyK) can further be O-linked glycosylated by the addition of monosaccharides and/or disaccharides by specific glycosyltransferases . Lysine In the context of zebrafish cardiac tissue regeneration, the dynamics of the site-specific collagen PTM network are still unexplored. Therefore, to comprehensively map the site-specific collagen PTMs constituting the myocardium ECM and to understand the dynamics of collagen PTMs during the regeneration process, we found an appropriate proteomics study performed by et al. established the zebrafish myocardium ECM enrichment protocol by decellularizing zebrafish ventricular tissue samples    was used for liquid chromatography separation. Nanoflex (Thermo) was used as an ESI source and LC-MS/MS was done on Q-exactive HF mass spectrometer (Thermo) with HCD fragmentation and the data-dependent acquisition opted for dataset “PXD011627”. For dataset “PXD010092” a nano HPLC system (Proxeon) coupled with Maxis Impact (Bruker) Q-TOF mass spectrometer was used and each sample of regenerating and sham model heart ventricle was analyzed using LC-MS in duplicates. Furthermore, healthy human heart sample (ECM) mass-spectrometry data from Barallobre-Barreiro D028908) , and micD002488)  was reanet al (PX028908) and 6 (.raw) files from Padmanabhan et al. (PXD002488).The publicly available datasets “PXD011627” and “PXD010092” were downloaded from ProteomeXchange. The complete *.d folders for “PXD010092” were shared by Prof. Angel Raya’s research group. Dataset “PXD011627” contains 9 (.raw) files and Dataset “PXD010092” contains 8 (.d) files, corresponding to two files  each for sham, 7-, 14- and 30-days post-amputation samples. Dataset “PXD010092” was not compatible with search engines because (.d) files are not readily accepted by many search engines. To overcome this, (.d) files were converted into. mgf and. mzML format using MSConvert with turbocharger filter. These converted files were further used as inputs by search engines MyriMatch  and MSFrDanio rerio uniprot database having 62,593 entries, downloaded on 20 October 2020. For the PXD028908 dataset, a general database search was performed on raw MS data (*.raw files) with a complete Homo sapiens uniprot database having 20,396 entries, downloaded on 16 February 2021. For the PXD002488 dataset, a general database search was performed on raw MS data (*.raw files) with a complete Mus musculus uniprot database having 17,090 entries, downloaded on 04 December 2021. Precursor ion tolerance was set at 10 ppm and fragment ion m/z tolerance was allowed up to 20 ppm for zebrafish and human data. However, for the mice dataset, a fragment ion tolerance of 50 ppm was used. Carbamidomethylation (+57.0236) on cysteine was used as static modification and methionine oxidation (+15.9949) and hydroxyproline (+15.9949) were used as dynamic modifications with up to 4 maximum dynamic modifications per peptide. A maximum of 2 missed cleavages were allowed for fully tryptic digestion. MyriMatch searches were performed to generate *.pepXML files for respective datasets. These *.pepXML files were further grouped for PSM matches, peptide, and protein group identification using IDPicker with <1% FDR. After a general database search, the identified list of ECM proteins from zebrafish and humans was exported from IDPicker as a *.FASTA database. This *.FASTA database  was used as a subset database to perform an in-depth collagen PTM search. In the subset database search, up to 4 missed cleavages for fully tryptic digestion were allowed with a similar precursor (10 ppm) and fragment ion tolerance (20 ppm) tolerance. For mice MS data, a fragment ion tolerance of 50 ppm was used. As mentioned previously,  facility at the Indian Institute of Technology (IIT)- Mandi. First, a general database search was performed on raw MS data (*.raw files) of dataset “PXD011627” with a complete viously, , a “Gly-Danio rerio, 62,593 entries, downloaded on 20 October 2020) as in the case of the MyriMatch general search. Precursor tolerance and fragment ion tolerance were kept at 50 and 25 ppm, respectively. PSM, peptide, and protein level FDR were kept at < 1%, with a maximum of three missed cleavages of fully tryptic peptides. Carbamidomethylation (+57.0236) on cysteine was used as static modification and methionine oxidation (+15.9949), hydroxyproline (+15.9949), N-terminal acetylation (+42.010565), hydroxylysine , galactosyl-hydroxylysine (+178.047738) and glucosylgalactosyl-hydroxylysine  were used as dynamic modifications with up to 5 maximum dynamic modification per peptide. IDPicker was used for grouping the *.pepXML output file generated from MSFragger searches by controlling FDR at <1% level for PSMs, peptides, and proteins. Similar to MyriMatch-based workflow, pLabel was used for manual inspection and validation for assigning a specific PSM to identify site-specific collagen PTM containing peptides.mzML files were used for MSFragger mediated database search. In MSFragger based pipeline, the decoy database was generated by a philosopher from the same uniprot database  from the results of the MyriMatch database search were exported from IDPicker. These spectral counts were normalized with the total spectral counts of each raw MS file ID. The normalized spectral counts corresponding to unique protein groups were further used to quantitate the relative abundance of collagens in wild-type and regenerating zebrafish hearts. Additionally, the same normalized spectral counts data for collagens from MyriMatch database search results were used to generate a heatmap of relative collagen expression during regeneration. Collagen chains identified with ≥3 normalized spectral counts were only considered for the heatmap representation.1 based targeted extraction pipeline as described previously (Database search results (*.pepXML files) generated by either MyriMatch and/or MSFragger were parsed through PeptideProphet (TPP pipeline module) for importing the probability scores (0-1) . After peviously .p value < 0.05 was considered statistically significant.GraphPad prism was used for statistical analysis for calculating the dynamics of different collagen site-specific PTM level quantitation. ANOVA was performed and a Myocardium extracellular matrix (ECM) consisting of collagens as a major component, is sought to be one of the key molecules involved in the remodeling process during zebrafish heart regeneration . CollageTo identify and quantitate the abundance of collagen chains present in wild-type zebrafish heart ECM, a dual database search engine-based (MyriMatch and MSFragger) comprehensive strategy was implemented. MyriMatch  and MSFrThe cardiac regeneration process involves ECM remodeling. Being the most abundant component of ECM , levels inhouse collagen PTM identification pipeline  and nine sites  of 3-HyP in COL5A1 and COL5A2a, respectively. COL5A2a was then further mapped with 11 sites  of glucosyl-galactosyl hydroxylysines. Only one galactosyl-hydroxylysine site (K1344) was identified in COL5A1. As previously shown in the zebrafish genome (from fin tissue) analysis (470 in COL6A2)  of glucosyl-galactosyl-hydroxylation were identified. Surprisingly, 24 glucosyl-galactosyl-hydroxylation sites were identified in COL4A2. A total of 4  and 3  prolyl-3-hydroxylation sites were identified in COL4A1 and COL4A2, respectively. It appeared that COL4A2 is way more glycosylated than COL4A1 in the zebrafish cardiac ECM compared to mice (225). Additionally, many other sites of prolyl-3-hydroxylations, lysyl-hydroxylation and O-glycosylation sites of lysine have been identified in various other collagen types  and hydroxylation (on lysine and proline residues) have been shown to be crucial for embryonic development, assembly of ECM fibrils, and cell-matrix interactions . Collagepipeline  with a dpipeline . We detepipeline  present pipeline . This hipipeline . Howeveranalysis , no type COL6A2) . Althoug to mice . InteresCollagen I is the highest abundant ECM protein present in the zebrafish hearts . The com176, P551, P755, P800, P869, P881, P911, P1103, P1106, and P1148) are evolutionarily conserved in mice  3-HyP sites in the COL1A1a from zebrafish heart ECM. In zebrafish heart ECM, COL1A1a harbors numerous unique 3-HyP sites close to the N-terminal propeptide cleavage sites , spanning across the triple-helical region as well. Two clusters of “Gly-Pro-Pro” motif present close to C-terminal propeptide were found to be 3-hydroxylated . The presence of many new 3-HyP sites compared to human and mouse hearts is itself a unique finding in the zebrafish COL1A1a  and C terminal propeptide cleavage sites  were determined by sequence alignment, corroborating the previous report . We deteCOL1A2 is the third contributing collagen chain in the collagen 1 triple helix of zebrafish heart ECM. COL1A2 is a little smaller than the other 2 chains of collagen 1 triple helix. It consists of 1,352 amino acids. We performed a similar propeptide cleavage site sequence alignment match of human COL1A2 with zebrafish COL1A2 . We dete1164) of COL1A1 is associated with osteogenesis imperfecta. Mutation in the prolyl-hydroxylases can cause the formation of dysfunctional collagen (without a 3-HyP site) as found in osteogenesis imperfecta disease conditions  involved in osteogenesis imperfecta had been previously shown to be fully 3-hydroxylated in mouse skin fibroblast (1148) control cardiac ECM is only about half (48%) 3-hydroxylated  of COL1A1a decreased at 30 DPA compared to control and 7 DPA occupancy (707 occupancy level was increased (7.5–20%) at 30 DPA regenerated zebrafish heart compared to sham. This increased occupancy may favor the assembly of ECM by enhancing collagen fibril formation by forming a water bridge with the available free carbonyl group of the subsequent collagen chain. However, a decrease in occupancy of other 3-HyP sites may favor the reduced interaction of deposited collagen molecules in ECM thereby favoring the transition to regeneration from the fibrotic milieu. Although, the exact functional role of these site-specific 3-HyPs needs to be experimentally assessed. A total of five 3-hydroxylation sites  occupancy levels were quantitated in COL1A1b. Out of these, 4 sites  showed the trend of increased occupancy level at 30 DPA regenerated zebrafish heart. We quantitated the occupancy level of 4 sites in COL1A2. Most of these sites were having <10% prolyl-3-hydroxylation. Interestingly, we identified a cluster of 3-HyP sites  in COL5A2a to be highly 3-hydroxylated (24%) after 7 DPA samples compared to only 1.7% occupancy in control. The occupancy of this 3-HyP cluster showed dynamic changes at 14 DPA (55%) and further reverting to 30% at 30 DPA, in the regenerated zebrafish heart. The site-specific variation in the occupancy level of 3-hydroxyproline could be due to varied gene expression of specific enzymes expressed in the zebrafish heart during post-amputation regeneration. A recent report from Sanchez-Iranzo et al. showed the expression of prolyl-3-hydroxylase-3 (P3H3) and prolyl-3-hydroxylase-4 (P3H4) decreased at 7 days post cryoinjury , in the zebrafish heart. However, in the same RNA seq analysis, the expression level of prolyl-3-hydroxylase-1 (P3H1) and prolyl-3-hydroxylase-2 (P3H2) were found to be increased  in the zebrafish heart at 7 days post-cryoinjury model. Following the regeneration in the same model till 60 days post-cryoinjury, the expression level of P3H2 and P3H4 increased  and the expression of P3H1 and P3H3 decreased  respectively highlighting the dynamic changes in the expression of these isoforms of prolyl-3-hydroxylases. However, the enzyme-substrate specificity for these different isoforms of prolyl-3-hydroxylases is not known yet. Thus, ascertaining a direct correlation between the expression level of one enzyme isoform with the substrate (occupancy level of site-specific modifications) specificity in this dataset is difficult. But, the varied expression of these enzymes during the post-cryoinjury regeneration model in zebrafish heart further supports the specific regulation of site-specific dynamic prolyl-3-hydroxylation changes in collagens deposited in the zebrafish heart ECM during regeneration. The dynamics of prolyl-3-hydroxylation occupancy in collagen molecules deposited in ECM during zebrafish heart regeneration are reported here for the first time.Identification of prolyl-3-hydroxylation sites in collagen chains of heart ECM provided new insights into the tissue-specific variation of collagen molecules providing structural support in ECM. Latest studies have revealed that the absence of prolyl-3-hydroxylation in one site , and 4 GG-HyK  sites were identified. Three of these sites showed microheterogeneity in glycosylation patterns. Similarly, a total of 21 HyK, 6 G-HyK , and 9 GG-HyK  sites were identified in COL1A2 chain for the first time from zebrafish heart ECM. Four of these sites showed microheterogeneity in different glycosylation moieties . Overall, our analysis mapped the different lysine O-glycosylation sites in three different chains of collagen 1 chains. Collagen 1A1a was found to be less deposited during the regeneration of the zebrafish heart by our as well as Garcia-Puig et al. analysis  sites. Hydroxylation of K1017 resulted in an early elution of this peptide at ∼42 min in the C18 column-based separation. Further, the glucosyl-galactosyl-hydroxylysine form of K1017 was further eluted earlier (at 41.2 min) than the hydroxylation form. We determined the microheterogeneity of this site (K1017) across control, 7-day post-amputation (DPA), 14 DPA, and 30 DPA samples across zebrafish heart regeneration. Interestingly, the glucosyl-galactosyl-hydroxylation of K1017 increased significantly (p < 0.01) to 15.4% in the 30 DPA regenerated heart as compared to control (2.74%), (3.35%) 7 DPA. and 14 DPA (10.5%) samples (1017 site was evident by a decrease in the HyK level during regeneration (846 (COL1A1a) to be specifically present in a full glycosylated form (either G-HyK or GG-HyK). The GG-HyK846 was decreased at 30 DPA regenerated zebrafish heart. We also quantitated the microheterogeneity of three lysine sites  in COL1A1b and 2 sites in COL1A2   of COL1A1a is found to be significantly increased during the regeneration process. Taken together, our analysis will widen new avenues to dig deeper in understanding the role of collagen PTMs during zebrafish heart regeneration.In conclusion, our analysis presents the first comprehensive mapping and dynamics of site-specific collagen PTMs from the crude extracellular matrix proteins in wild-type and regenerating zebrafish hearts. We optimized an in-house dual database search engine-based mass-spectrometry data analysis strategy to efficiently identify long post-translationally modified collagen peptides from crude ECM analysis. We identified the highest number of collagen chains (36) present in the zebrafish heart ECM. Our analysis revealed that the expression of collagens gets altered during regeneration. Global characterization of zebrafish heart ECM revealed the identification of site-specific PTMs  in 23 different collagen chains. These are the first comprehensive site-specific collagen PTM maps of zebrafish cardiac ECM highlighting some novel sites. Further, we present the first comprehensive PTM maps of three chains  of collagen 1. Our analysis also identified the conserved 3-hydroxyproline sites occurring in the COL1A1 chain in humans, mice, and zebrafish heart ECM. This finding highlights the importance of these conserved 3-HyP sites involved in the basic function of collagens in humans, mice, and zebrafish myocardium. Moreover, we established that site-specific PTM (Prolyl-3-hydroxylations and lysyl O-glycosylation) changes in collagen chains are correlated with the regeneration process. Glycosylation on one site (GG-HyK


PTEN tumor suppressor. In the present study, we investigated the causative gene of CS in a family of PTEN (phosphatase and tensin homolog deleted on chromosome 10) -negative CS patients. Whole exome sequencing analysis revealed AMBRA1 (Autophagy and Beclin 1 Regulator 1) as a novel candidate gene harboring two germline variants: p.Gln30Arg (Q30R) and p.Arg1195Ser (R1195S). AMBRA1 is a key regulator of the autophagy signaling network and a tumor suppressor. To functionally validate the role of AMBRA1 in the clinical manifestations of CS, we generated AMBRA1 depletion and Q30R mutation in hTERT-RPE1  using the CRISPR-Cas9 gene editing system. We observed that both AMBRA1-depleted and mutant cells showed accumulation in the S phase, leading to hyperproliferation, which is a characteristic of hamartomatous lesions. Specifically, the AMBRA1 Q30R mutation disturbed the G1/S transition of cells, leading to continuous mitotic entry of mutant cells, irrespective of the extracellular condition. From our analysis of primary ciliogenesis in these cells, we speculated that the mitotic entry of AMBRA1 Q30R mutants could be due to non-functional primary cilia that lead to impaired processing of extracellular sensory signals. Additionally, we observed a situs inversus phenotype in ambra1-depleted zebrafish, a developmental abnormality resulting from dysregulated primary ciliogenesis. Taken together, we established that the AMBRA1 Q30R mutation that we observed in CS patients might play an important role in inducing the hyperproliferative potential of cells through regulating primary ciliogenesis.Cowden syndrome (CS) is a rare autosomal dominant disorder associated with multiple hamartomatous and neoplastic lesions in various organs. Most CS patients have been found to have germline mutations in the  Cowden syndrome (CS), also known as multiple hamartoma syndrome, is a rare genetic autosomal dominant disorder affecting 1 in 200,000 people ,4. The mWe investigated the causative gene of CS in a family: the father and two daughters were diagnosed with CS, while the mother and one daughter were not affected. We performed whole exome sequencing (WES) analysis on all members of the family and identified AMBRA1 as the top candidate. AMBRA1 could be a good candidate from the genetic results, but its functional significance needs to be examined.AMBRA1, a key adaptor protein of the autophagy signaling network, is a tumor suppressor and mutated in a wide range of tumors ,7. In adIn our study, AMBRA1 Q30R mutant hTERT-RPE1 cells showed hyperproliferation, disturbed G1/S transition, and abnormal primary ciliogenesis. Primary cilia are immotile extracellular sensory organelles that are dynamically regulated during cell cycle progression. Primary cilia assemble in non-dividing cells of the G0/G1 phase and disassemble upon cell cycle entry . Therefohttps://j-rdmm.org/; accessed on 29 November 2018). As part of these projects, we aimed to identify the candidate gene for one CS family. The affected individuals of this study were the father and two daughters of the family (The IRUD (Initiative on Rare and Undiagnosed Diseases) and J-RDMM (Japan Rare Disease Models and Mechanisms Network) are ongoing multi-center research projects supported by the Japan Agency for Medical Research and Development  and R1195S (c.G3585C). Q30R, an AMBRA1 variant, exhibited the most deleterious effect according to the in silico pathogenicity prediction C, locate−/−)  entering the S and G2/M phases was significantly higher than that of the wildtype  . When wewildtype A,B, indipe cells C.AMBRA1 Q30R mutation affects cell proliferation similar to AMBRA1 depletion, we introduced the Q30R mutation in exon 2 of AMBRA1 to hTERT-RPE1 cells using the CRISPR-Cas9 gene editing system  which entered the G0 phase  which entered the cell division phases including G1 and S+G2/M   (To confirm the association of ambra1a depletion with the  0.0001) F. On thesitus inversus phenotype in ambra1a morphants but not in ambra1b morphants suggests that these two genes play different roles in zebrafish embryogenesis, with ambra1a being involved in the orientation of visceral organs, which could plausibly be through regulating primary cilia. This confirms that AMBRA1 function is tightly correlated with ciliogenesis. However, we could not observe any lesion similar to a hamartoma in the ambra1a knockdown zebrafishes.Our observation of the AMBRA1 Q30R as a novel candidate germline mutation of Cowden syndrome. The Q30R germline mutation, located in the highly conserved N-terminal region of AMBRA1, has not been documented previously. The N-terminal region of AMBRA1 is characterized by three WD40 domains which act as protein interaction scaffolds mediating the interaction of AMBRA1 with a wide range of proteins [In the present study, the overview of which is presented in proteins . Deletioproteins . This si+/−, −/−, Q30R A/G, Q30R G/G) confirmed the hyperproliferative potential of the AMBRA1 Q30 mutation, which could possibly be responsible for the benign self-limited growth of hamartomatous lesions in CS patients [+/− cells apparently showed a higher proliferation rate than the wildtype cells, which is consistent with previous reports of increased tumorigenesis in monoallelic AMBRA1 mouse embryonic fibroblast cells [AMBRA1 as a haploinsufficient tumor suppressor, sensitive to the gene dosage.Our observation of increased proliferation in AMBRA1-perturbed cells  and G1/S transition. We suggest that AMBRA1−/− cells could have lost G0/G1 transition control, while AMBRA1 Q30R mutant cells lost G1/S phase transition control. The mutants, AMBRAQ30R A/G and Q30R G/G, showing uncontrolled proliferation, albeit the unfavorable extracellular condition, signifies the pathogenicity and irreversible hyperproliferative ability of Q30R mutation and its possible influence on benign hamartoma and malignant transformation in CS patients.However, in nutrient-deprived conditions, wildtype and AMBRA1AMBRA1 depletion and AMBRA1 Q30R mutation could induce hyperproliferation through regulating cyclinD1, we analyzed cyclinD1 expression in AMBRA1+/−, AMBRA1−/−, AMBRAQ30R A/G and Q30R G/G cells  and the ciliogenesis phenotype. The heterozygous AMBRA1Q30R A/G cells showed ciliary assembly in nutrient-adequate conditions, and an irregular structure and multiciliation upon nutrient deprivation, while on the contrary, the homozygous AMBRA1Q30R G/G cells showed no ciliation. It is not uncommon for a single mutation in highly pleiotropic genes that are involved in more than one function and have multiple protein interactions to result in very different phenotypes depending upon the alleles involved [AMBRA1 Q30R and R1195S using AlphaFold2 demonstrated that both the mutations did not induce a significant structural change in the AMBRA1 tertiary structure . After participants gave informed consent, we performed whole exome sequencing using genomic DNA of both the affected and unaffected members of the CS family. The detailed procedure of WES is presented in the Supporting Information. We prioritized novel germline variants (i) based on their potential to alter protein structure or function  and (ii) those that were not reported in dbSNP and the 1000 Genomes Project, (iii) excluding common variants found in both the affected and unaffected family members and including germline mutations observed in all three probands, (iv) based on PhyloP, Polymorphism Phenotyping v2 (Polyphen-2) pathogenicity scores and GERP conservation scores. We predicted the structural changes induced by the missense variants (p.Q30R and p.R1195S) of our candidate gene AMBRA1 using AlphaFold2. Detailed protocol of the 3D-structural analysis is presented in the AMBRA1 Q30R editing (c.A89G), we designed a guide RNA -low glucose supplemented with 10% heat-inactivated fetal bovine serum and 1% Penicillin–Streptomycin. We created AMBRA1-depleted hTERT-RPE1 cells using the CRISPR-Cas9 system by deleting approximately 1093 bp between intron 6 and exon 7 . For AMBuide RNA  targetin10% PAGE . The AMB10% PAGE . To confquencing . Details+/− and AMBRA1−/− knockouts and AMBRA1Q30R A/G and AMBRA1Q30R G/G mutants in the cell cycle phases through flow cytometry using a BD Accuri flow cytometer  with Ki67 staining using an anti-Ki67 antibody  and DNA staining using PI. The fractions of cells in different cell cycle phases were calculated using FCS express software . We analyzed the distribution of AMBRA1+/− and AMBRA1 −/− knockout and AMBRA1Q30R A/G and AMBRA1Q30R G/G mutant cells in both nutrient-sufficient and starved conditions through immunocytochemistry using rabbit anti-arl13B primary antibody , a primary cilia marker. The immunostained cells were imaged under a Deltavision Personal DV fluorescent microscope . Quantification analysis of the nucleus and primary cilia was performed using Volocity 6.5 software . We observed the frequency of primary cilia in AMBRA1Detailed protocols of the flow cytometry and immunocytochemistry are presented in the ambra1 codes for two paralog nonredundant genes, ambra1a and ambra1b [ambra1 CRISPANTS and morphants were analyzed using a Leica M165 FC microscope . Details of the CRISPR/Cas9 and morpholino experiments on zebrafish are provided in the All zebrafish experiments were carried out with permission from the Committee for Animal Care and Use of the National Institute of Genetics, Japan . Adult zebrafish were maintained on a 13 h light/11 h dark cycle. Embryos were kept at 28° until processing for phenotypic analysis. In zebrafish,  ambra1b . We mani+/−, AMBRA1−/−, AMBRA1Q30R A/G, and AMBRA1Q30R G/G with the AMBRA1 wildtype cells and Fisher’s exact test to compare the situs inversus phenotype of ambra1a morphants with the wildtype zebrafish were performed using GraphPad prism V.9. .Statistical analyses such as ANOVA to compare AMBRA1