Interestingly, when SU5402 was used to inhibit FGF signaling, neuromast cells expressed much lower levels of the FGF receptor gene, fgfr1, but produced increased levels of Wnt target genes, including ctnnb1, ctnnb2, and tcf7l2, while bFGF treatment produced no alterations in the expression of those genes, suggesting that fgfr1 might restrict Wnt signaling in neuromasts during proliferation. In summary, our analysis demonstrates that both the Wnt and FGF pathways are tightly integrated to modulate the proliferation of progenitor cells during early neuromast development and regenerative cell proliferation after neomycin-induced injury in the zebrafish neuromast.Wnt and FGF are highly conserved signaling pathways found in various organs and have been identified as important regulators of auditory organ development. In this study, we used the zebrafish lateral line system to study the cooperative roles of the Wnt and FGF pathways in regulating progenitor cell proliferation and regenerative cell proliferation. We found that activation of Wnt signaling induced cell proliferation and increased the number of hair cells in both developing and regenerating neuromasts. We further demonstrated that FGF signaling was critically involved in Wnt-regulated proliferation, and inhibition of FGF abolished the Wnt stimulation-mediated effects on cell proliferation, while activating FGF signaling with basic fibroblast growth factor (bFGF) led to a partial rescue of the proliferative failure and hair cell defects in the absence of Wnt activity. Whole-mount in situ hybridization analysis showed that the expression of several FGF pathway genes, including  Studying sensory organs on the skin of zebrafish is revealing details of molecular signaling pathways that may be relevant to our own sensory systems, especially the hair cells of the ear. These cells have fine hair-like structures that move in response to sound waves and help generate electrical signals to the brain that result in perception of sound. Huawei Li and colleagues at Fudan University, Shanghai, China, studied the roles of two well-known cellular signaling pathways in regulating the proliferation of similar sensory hair cells in zebrafish, a commonly used model organism. These pathways involve cell surface proteins that interact with small extracellular molecules to stimulate molecular changes within cells. Learning how the pathways control hair cell generation and multiplication may assist modification of similar systems in humans to study and treat hearing loss. Cell proliferation is a critical process guiding many aspects of auditory organ specification and morphogenesis during embryonic development, and its misregulation is associated with various malformations. However, the precise molecular signaling pathways that control proliferation and their coordination remain unclear.2. Mechanosensory HCs share many structural, molecular, and functional similarities with vertebrate inner ear HCs and thus are an excellent model system for studying HC biology related to hearing and balance and for unraveling their genetic control4. However, unlike mammals, zebrafish retain the ability to quickly regenerate HCs after damage8. Thus, the molecular signaling pathways that guide HC regeneration in the zebrafish lateral line are of great biological and clinical interest.The zebrafish lateral line is a powerful vertebrate model system for the in vivo study of sensory organ developmental biology due to its well-established genetics and ease of in vivo visualization and manipulation. The posterior lateral line (pLL) comprises a series of mechanosensory organs called neuromasts, which lie on the surface of both the head and body. Neuromasts are composed of sensory hair cells (HCs) in the center that are surrounded by nonsensory supporting cells (SCs)11, FGF signaling13, and Wnt/β-catenin signaling11. Most of our current understanding about these pathways is related to early embryonic pLL formation, which entails primordium formation, migration, and deposition stages; however, the precise molecular signaling that initiates and guides cell proliferation and HC regeneration in zebrafish neuromasts remains poorly defined. The Wnt/β-catenin signaling pathway is a well-known signaling cascade that plays central roles in embryonic development and organogenesis by modulating cell migration, proliferation, and specification14. During the early stages of zebrafish pLL development, Wnt/β-catenin signaling is active in the leading part of the primordium and is necessary for cell proliferation and proneuromast formation as the primordium migrates along the body of the organism17. Constitutive activation of canonical Wnt signaling broadly promotes cellular proliferation throughout the primordium but stalls migration during developmental patterning, whereas pharmacological or genetic inhibition of Wnt reduces cellular proliferation, increases cell death, and results in a dramatic truncation of the pLL18. Recently, activation of the Wnt/β-catenin pathway was reported to promote proliferation and increase HC generation in the developing and regenerating zebrafish lateral line19. Conversely, inhibition of canonical Wnt signaling decreases proliferation and arrests neuromast morphogenesis at an early stage of neuromast development. Little is known, however, about the molecular mechanisms underlying this signal transduction during the periods of proliferation and HC regeneration in neuromasts.Previous studies have revealed that the development of the pLL requires complex coordination among diverse molecular signaling pathways, including chemokine signaling20, and FGF is active in the trailing region of the zebrafish pLL primordium, which contains two or three rosette-shaped proneuromasts. FGF acts as a key molecule in promoting the epithelialization and formation of the proneuromasts in the course of the migration of the primordium13, and FGF inhibition leads to a loss of apically constricted rosettes and aberrant migration of the primordium. FGF activation not only induces pro-neuromast formation by initiating the organization of center-oriented epithelial rosettes but also initiates the specification of HC precursors by regulating atoh1a expression13. Although the function of FGF signaling in facilitating pLL morphogenesis has been reasonably well studied, its contribution to proliferation during the establishment of the pLL and to regenerative cell proliferation has not been well addressed.FGF signaling has been shown to regulate cell migration and cell fate changes during embryonic developmentfgf3 and fgf10 genes were Wnt targets during neuromast cell proliferation and acted to modulate FGF activity and that fgfr1, an FGF target gene, acted to repress Wnt activity. Taken together, our findings suggest that the Wnt and FGF signaling pathways are tightly connected to regulate developmental cell proliferation and regenerative cell proliferation in the zebrafish pLL neuromast.In this study, we used pharmacological agonists and antagonists and transgenic zebrafish to regulate Wnt and FGF signaling, and we found significantly more proliferating cells and more HCs and SCs after activation of Wnt or FGF signaling. To further understand whether the Wnt and FGF pathways act synergistically to regulate proliferation, we performed epistasis experiments to verify the mechanisms underlying proliferation in neuromasts. We stimulated Wnt signaling first and then inhibited FGF activity and found that the proliferation of the progenitors induced by Wnt activation disappeared after blocking FGF in Wnt-activated embryos. Conversely, we found that inhibition of Wnt signaling using the DKK1 conditional knockout transgenic line or IWR-1 treatment followed by treatment with basic fibroblast growth factor (bFGF) resulted in significantly more proliferating cells than in the group treated with IWR-1 alone, suggesting that activation of FGF could partly rescue proliferation failure caused by Wnt signaling inhibition. Similar results were observed in HC regeneration experiments. To better understand the interactions of both signaling pathways during cell proliferation in the larval zebrafish neuromast, we performed whole-mount in situ hybridization analysis. We showed that the 21, and embryo ages were marked as hours post fertilization (hpf). The wild-type strain was derived from the AB line, and the Tg(brn3c:mGFP)s356t line was used to visualize HCs. The apcmcr line was a generous gift from Professor Xu Wang. APC genotyping primers were as follows: APCR—CAT GGC TCA CTC TGC ACA; APCWTF—ATA ATG TTG CAG CTG ACC; and APCMTF—ATA ATG TTG CAG CTG ACT. Tg(hsp70l:dkk1b-GFP) offspring were incubated at 42 °C for 5 min at 48 hpf and allowed to recover at 28.5 °C to inhibit Wnt signaling. Approximately 50% of the embryos did not turn green, and these served as controls. To prevent pigment formation, embryos were treated with 0.003% 1-phenyl-2-thiourea  in E3 water from 14 hpf onwards. The larvae were anesthetized in 0.02% MS-222  before fixation. All zebrafish experiments were performed following the institutional guidelines approved by the Institutional Animal Care and Use Committee of Fudan University, Shanghai.Embryos were obtained by natural spawning and developed at 28.5 °C in E3 medium. They were staged according to standard protocolsWe used 1 μM BIO to activate Wnt signaling and 10 μM IWR-1 (Sigma-Aldrich) to inhibit Wnt signaling, and we used 5 μM SU5402  to inhibit FGF signaling and 20 ng/ml bFGF (Invitrogen) to activate FGF signaling. Neomycin sulfate (Sigma-Aldrich) was added to a final concentration of 400 μM, and the 5 dpf larvae were incubated for one hour, followed by three rinses in fresh egg water. The larvae were then allowed to recover at 28.5 °C.For immunofluorescence experiments, 10 mM BrdU (Sigma-Aldrich) was coincubated with the pharmacological treatments described above to label the proliferating cells. Zebrafish larvae were fixed with 4% PFA for 2 h at room temperature and washed three times with PBT-2 (PBS containing 1% Triton X-100). For DNA denaturing, the fixed larvae were treated with 2 N HCl for 30 min at 37 °C followed by three rinses with PBT-2. Before antibody staining, the larvae were incubated in blocking solution (10% donkey serum in PBT-2) for 1 h at 37 °C. The primary antibodies were anti-Sox2 , anti-BrdU , anti-GFP , and anti-Myosin VI . After incubation with primary antibodies overnight at 4 °C, the larvae were washed three times with PBT-2 and then incubated with Alexa Fluor 488-, 594-, and/or 647-conjugated secondary antibodies  for 1 h at 37 °C. After the larvae were washed several times in PBT-2, they were incubated with DAPI  for 20 min to label the nuclei.Fluorescent specimens were examined using a Leica confocal microscope . All images were edited using Adobe Photoshop CS6. The labeled cells in the pLL neuromasts (pLL2–pLL5) were counted in the confocal and fluorescence images using the counting and measuring tools in Adobe Photoshop CS6.22. After the color reaction, the embryos were mounted with 100% glycerol and observed under a bright field microscope (Nikon Instruments). Binding sites were identified as blue-black dots.Digoxigenin-labeled probes were prepared as recommended by the manufacturer . Primers for cloning the examined genes are listed in Table Apoptotic cells in the trunk of the zebrafish were detected by TUNEL assay . After the larvae were rinsed three times with PBT-2, they were incubated with the TUNEL reaction mixture in a humid dark chamber at 37 °C for 1 h and then labeled with DAPI to visualize the nuclei.t-tests and one-way ANOVA. In all figures, error bars represent the mean ± SEM. p < 0.05 was considered statistically significant, and p < 0.001 was considered highly significant. *Shows the comparison with controls, and # is used when the Wnt and FGF signaling pathways were shown to interact.All statistical analyses were performed with GraphPad Prism 6 Software . Cell counts were analyzed using Student’s ctnnb1, ctnnb2, and tcf7l2. After 24 h of pharmacological treatment, a significant increase in ctnnb1, ctnnb2, and tcf7l2 expression was observed in the 1 μM BIO-treated group, indicating induction of Wnt signaling, whereas 10 μM IWR-1 significantly inhibited the expression of these genes  transgenic line that expresses GFP in the HC membrane23, and the SCs were marked and counted by Sox2 immunostaining. Beginning at 48 hpf, embryos were treated with BIO or IWR-1 in the presence of BrdU for a period of 24 h. Quantification showed significantly more BrdU+ cells, GFP+ HCs, and Sox2+ SCs in the BIO-treated neuromasts than in the DMSO-treated control neuromasts 25, and the second was a heat-shock–inducible dkk1 transgenic line 26. In these experiments, myosin VI was used to label mature HCs. Consistent with the pharmacological results, the numbers of proliferating cells and differentiated cells (HCs and SCs) in neuromasts were significantly higher in Apcmcr larvae relative to those in wild-type (wt Apc) at 72 hpf   Fig. . WISH re28. We, therefore, examined whether FGF signaling plays an important role in neuromast development in the zebrafish pLL. We first conducted a comprehensive expression analysis by means of WISH to examine the expression of known FGF signaling-related genes. In 72 hpf zebrafish larvae, expression of the FGF signaling receptor gene (fgfr1) and target gene (pea3) was clearly detectable within the neuromast  for 24 h, with DMSO treatment as the control. At the highest dose (15 μM), we observed evident larval death (10/12), whereas 5 μM SU5402 did not cause developmental abnormalities or death. We then measured the expression of pea3 and fgfr1, and WISH showed that the expression of both genes was significantly downregulated after 5 μM SU5402 treatment for 24 h compared to expression in DMSO controls  for 24 h, the expression of pea3 and fgfr1 was robustly upregulated compared to the expression in unstimulated neuromasts, and no deformities were detected  and FGF  signaling pathways. As expected, in BIO-treated larvae, the presumed Wnt target genes ctnnb1, ctnnb2, and tcf7l2 were markedly upregulated in the neuromast, which was consistent with sustained activation of Wnt/β-catenin signaling, whereas significant decreases were detected for these genes in the IWR-1 treatment group   larvae with 400 μM neomycin for 1 h, followed by incubation in 6-well plates with or without the presence of different agents for a period of 24 h or 48 h (24 hpa and 48 hpa). We then performed a BrdU (10 mM) incorporation assay and quantified the number of regenerated HCs. The BIO-treated larvae showed significantly more BrdU incorporation in the neuromasts than did DMSO-treated controls at both 24 and 48 hpa . Loss of HCs and SCs following inhibition of Wnt signaling was also partly restored by ectopic FGF activation (Fig. To determine if the increase in proliferation after Wnt activation during regeneration requires FGF signaling, we simultaneously activated Wnt signaling and inhibited FGF signaling using BIO and SU5402, respectively. Indeed, the BIO-induced increase in total proliferation was reduced to below normal levels after the simultaneous addition of SU5402, indicating that the majority of extra BrdUoup Fig. . This cooup Fig. g2, G2, bIn the present study, we used a zebrafish model to gain new insights into the mechanism underlying the control of cell proliferation and fate determination in developing and regenerating neuromasts in the zebrafish pLL, and we provide evidence that activation of the Wnt and FGF pathways is crucial for promoting the proliferation and regeneration of HCs in the pLL. Our current study represents the first analysis indicating that Wnt acts through the FGF signaling pathway to promote progenitor cell proliferation and regenerative cell proliferation in the zebrafish pLL neuromasts. These findings provide strong evidence that manipulation of the Wnt and FGF pathways will be useful for promoting HC regeneration in the mammalian inner ear.34 however, the exact mechanisms through which Wnt signaling stimulates cell proliferation and regeneration under these conditions remain unresolved. Here, we used BIO as a Wnt agonist to activate Wnt signaling in zebrafish neuromasts. Consistent with previous reports, our results indicate that activation of Wnt/β-catenin signaling promotes cell proliferation in developing and regenerating neuromasts. More HCs and SCs were observed in the neuromasts of the BIO-treated group, which suggests that both HCs and SCs were being overproduced and that most of the proliferated SCs differentiated into HCs after BIO treatment. Genetic activation of Wnt/β-catenin signaling phenocopies the pharmacological activation by promoting progenitor cell proliferation and regenerative cell proliferation in neuromasts.Recent studies in zebrafish showed that activating Wnt signaling promotes the proliferation of neuromast progenitors and HC regeneration in both developing and damaged neuromasts;35. Loss of FGF signaling in fgf3 and fgf8 double-deficient zebrafish not only causes failure of placode formation but also blocks HC development36, and Fgf3 and Fgf8 have been shown to act as upstream activators of the atoh1 gene, which is necessary for HC development during distinct inner ear developmental periods, including both the early development of the preotic placode and the later development of the otic vesicle37. Based on these studies, we hypothesized that early neuromast development requires the FGF signaling pathway. To test this hypothesis, we first performed in situ analyses and confirmed the expression of candidate ligands and receptors of the FGF signaling pathway in the neuromast, revealing that this pathway is potentially activated in the developing neuromast. Next, to test whether FGF signaling is involved in the proliferation of neuromast progenitors, we inhibited the FGF signaling pathway using SU5402. The results of our proliferation assay suggested that blockade of FGF signaling inhibited cell proliferation, similar to that observed when the Wnt signaling pathway was inactivated. Given that many similarities between developmental and regenerative processes in the pLL have been identified40, we hypothesized that FGF activation would enhance regenerative proliferation and subsequent new HC generation in the pLL neuromast, similar to the effects of FGF activation during initial development. As hypothesized, neomycin-damaged larval zebrafish exposed to bFGF during recovery periods had significantly more SCs in their neuromasts than neomycin-alone control animals, indicating that increased cell proliferation had occurred, and this was followed by increased HC production. Together, these results suggest that FGF signaling plays a key role in regulating normal neuromast development and HC regeneration by promoting proliferation, similar to that observed for Wnt signaling in HC development and regeneration.A number of studies have indicated the importance of FGF in inner ear development in many speciesTo identify the Wnt-FGF interactions that are required for cell proliferation in larval zebrafish neuromasts, we performed epistasis experiments using a combination of pharmacological and genetic approaches to manipulate Wnt and FGF activity. We compared cell proliferation with activated Wnt signaling alone or with activation of Wnt and inhibition of FGF signaling using the small molecule inhibitor SU5402. While activation of Wnt alone increased cell proliferation and induced greater HC numbers, it was unable to do so in the absence of FGF signaling. To further confirm that FGF functions downstream of Wnt signaling to promote cell proliferation in developing neuromasts, we used IWR-1 to downregulate the Wnt signal and then added bFGF to activate the FGF pathway. Our proliferation assay showed that bFGF treatment encouraged neuromast progenitors to proliferate and partly rescued the cell proliferation phenotype in the absence of Wnt signaling. In neomycin-damaged neuromasts, simultaneous manipulation of Wnt and FGF signaling activities showed that while Wnt signaling is sufficient to drive ectopic cell proliferation and subsequent HC regeneration, this pathway was able to do so only in the presence of functional FGF signaling.fgf3, fgf10, pea3, and fgfr1, whereas their expression in IWR-1-induced Wnt-inhibited neuromasts was significantly reduced. Furthermore, stimulating FGF signaling through the addition of bFGF had no effect on the expression of Wnt components. In addition, no significant changes in the expression of ctnnb1, ctnnb2, or tcf7l2 were observed in the neuromasts of the IWR-1+bFGF group compared to that in the neuromasts of the IWR-1–alone group, indicating that there had been no reactivation of Wnt signaling by bFGF treatment after IWR-1 treatment. Taken together, our results suggest that the pro-proliferative effect induced by Wnt activation was mainly mediated by the FGF pathway, which is consistent with previous reports. For instance, Fgf8 levels are reduced in the absence of Wnt activity in mouse embryos41, while FGF activity is enhanced in β-catenin overexpression embryos43, suggesting that FGF functions downstream of Wnt/β-catenin signaling. Intriguingly, FGF activation did not change the expression of fgf3 or fgf10 in developing neuromasts, and thus, Wnt can be reasonably assumed to activate the FGF signaling pathway through fgf3 and fgf10. We also found that inhibiting FGF signaling with SU5402 resulted in downregulation of fgfr1 and upregulation of fgf10 and fgf3 as well as several Wnt components, suggesting that a feedback mechanism involving Fgfr1 might operate during neuromast proliferation. A number of studies have shown that FGF components, such as fgf3 and fgf10a, have mitogenic activity;46 however, their signaling functions in cell proliferation during neuromast development have not yet been explored. We showed that active Wnt and FGF signaling exerted their function by negatively regulating the cell cycle inhibitor genes p21 and p27 and positively regulating the ccnd1 gene to permit cell proliferation. Sequential induction of Wnt signaling and inhibition of FGFR caused a complete loss of ccnd1 expression and enhanced p21 and p27 expression in the neuromast, similar to SU5402 treatment alone, indicating an essential role of FGF signaling in maintaining the expression of cell cycle genes.In this work, we characterized the expression patterns of the FGF and Wnt pathway components in developing neuromasts in detail. We showed that Wnt activation significantly increased the expression of FGF components such as 49. Therefore, defining which signals act downstream of the Wnt-FGF interactions during proliferation in neuromasts is of great interest.In this study, we showed that the Wnt and FGF pathways cooperate closely to regulate proliferation in the zebrafish neuromast. To fully understand the heterogeneity of the neuromast domain, it is necessary to identify all of the transcriptional targets that are activated or repressed by FGF and Wnt regulation during neuromast proliferation in zebrafish. Recently, many studies have shown that bFGF activates several intracellular pathways, including the RAS/ERK and PI3K/AKT cascadesfgf3 and fgf10 stimulation and that FGF signaling inhibits the Wnt pathway through the Wnt inhibitor fgfr1. Our findings contribute to studies on HC biology and provide a new avenue for understanding the signaling mechanisms that regulate cell proliferation during mammalian inner ear development.In summary, we have investigated the cross talk between the Wnt and FGF signaling pathways during cell proliferation and regeneration in the zebrafish neuromast and have shown that FGF activation enhances proliferation and promotes subsequent cell differentiation in the developing and regenerating neuromasts of the zebrafish pLL. Furthermore, we provide evidence that Wnt signaling activates FGF activity via Supplementary Figure LegendSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Table S1


Since its introduction, the zebrafish has provided an important reference system to model and study cardiovascular development as well as lymphangiogenesis in vertebrates. A scientific workshop, held at the 2018 European Zebrafish Principal Investigators Meeting in Trento  and chaired by Massimo Santoro, focused on the most recent methods and studies on cardiac, vascular and lymphatic development. Daniela Panáková and Natascia Tiso described new molecular mechanisms and signaling pathways involved in cardiac differentiation and disease. Arndt Siekmann and Wiebke Herzog discussed novel roles for Wnt and VEGF signaling in brain angiogenesis. In addition, Brant Weinstein’s lab presented data concerning the discovery of endothelium-derived macrophage-like perivascular cells in the zebrafish brain, while Monica Beltrame’s studies refined the role of Sox transcription factors in vascular and lymphatic development. In this article, we will summarize the details of these recent discoveries in support of the overall value of the zebrafish model system not only to study normal development, but also associated disease states. Zebrafish has been utilized over the past years to successfully study cellular and genetic processes regulating the development of cardiac, vascular, and lymphatic development . Among tFurthermore, zebrafish are amenable to both forward genetic approaches , and reverse genetic studies (such as TALEN and CRISPR/Cas9) . Cre/loxin vivo techniques to study action potential propagation or changes in intracellular calcium concentrations during each heartbeat. High-speed optical mapping techniques are currently limited to an ex vivo analysis /PKA signalosome. They further showed that the AKAP/PKA signalosome is an essential component of Wnt11 signaling regulating channel function during the establishment of intercellular electrical coupling gradients in the developing zebrafish heart.Pertaining to the functional patterning of the developing myocardium, Dr. D. Panáková expanded on already reported data showing the Wnt11 non-canonical pathway regulating the intercellular electrical coupling in the developing zebrafish heart through attenuation of the L-type calcium channel (LTCC) conductance . The LTCdspa and dspb) Tiso’s team could specifically phenocopy the ultrastructural features and cardiac frequency defects of AC8. Moreover, the availability of zebrafish lines, which are able to report in vivo the activation of a series of signaling pathways , out of nine considered, that are specifically altered in AC8-like conditions . Interesin vivo  is one of the main signaling molecules important for blood vessel growth and homeostasis y251 transgenic zebrafish line for visualizing and investigating lymphatic vascular development y251 transgenic line, the Weinstein lab researchers discovered that in addition to lymphatics, this transgenic line also displays strong EGFP expression in a novel cell population on the surface of the zebrafish brain  formation was unaffected and they questioned the role of Sox18 in the regulation of lymphatic development in zebrafish. Dr. Monica Beltrame presented at the meeting data gathered in her lab on an independent sox18-null mutant, generated through the Zebrafish Mutation Project at Sanger . A slight perturbation of VegfC signaling exacerbated TD defects in a genotype-dependent manner, unraveling differences in TD formation even in sox18 heterozygotes versus wild types. Remarkably, an upregulated expression of sox7 in the posterior cardinal vein was observed in sox18 mutants, thus possibly explaining the milder lymphatic phenotypes with respect to the knockdown of sox18, which was not affecting sox7 expression  occurring in the cardiovascular compartment. To date, several zebrafish lines have been generated to identify and perturb in vivo different sub-cardiac compartments, such as the endocardium, myocardium, epicardium, valves, or conduction system as well as specific vascular components and cell types including arteries, veins, lymphatic vessels, mural, and endothelial cells , elegantly demonstrating that both factors act through the same receptor (Kdrl) to induce sprouting, vessel formation, and vascular-specific gene expression. Finally, using the transgenic line Tg(mrc1a:eGFP), Dr. Weinstein’s group could efficiently analyze a new population of perivascular cells in vivo (In this perspective, during the EZPM2018 in Trento, a spectacular series of new interesting systems have been presented, all worth to be reported and added to the available toolkit. In the context of new mutant and transgenic line production, the research group of Dr. Beltrame presented a new allele for Sox18, which allowed to further elucidate the role of this key factor in the development of lymphatic vessels in vertebrates. Dr. Siekmann’s research group has produced new mutant lines for the two  in vivo .In the field of cardiovascular signaling, Dr. Tiso’s group presented the systematic application of pathway-specific transgenic lines for the identification of drugs with therapeutic effects in cardiac pathology. Specifically, a form of arrhythmogenic cardiomyopathy was modeled in a set of different zebrafish lines reporting signaling pathways activated in the cardiac compartment. This signaling-based screen allowed identifying the involved cascade, rapidly passing to the test of pathway-specific compounds, and the identification of a Wnt agonist as a potential therapeutic drug . The rolin vivo, will soon ensue. The dysregulation of ion channels, such as LTCC, can result in arrhythmias as well as cardiac hypertrophy, both of which are underlying pathologies leading to sudden cardiac death. Future studies, in which modulation of functional inputs plays a crucial role, will therefore also accelerate novel avenues for future therapies regarding common cardiac diseases, including cardiac hypertrophy, heart failure, and arrhythmias.Zebrafish is a long-standing model to study heart morphogenesis. With the growing number of genetic tools and technological advances, probing the functional inputs and their related physical stimuli, such as mechanical or calcium cues in greater detail Many questions still remain open in the field of vascular and lymphatic studies in zebrafish. Besides VEGF and Wnt, other signaling pathways are increasingly studied in vascular development, such as the Hippo pathway . Similardesideratum for researchers working in the field to also include perivascular cells, such as mural cells.We envision a new interactive atlas of zebrafish vascular and lymphatic anatomy using modern technologies, such as light sheet microscopy and fluorescent markers of different colors. It would be a It can be expected that in the coming years the growing application of single-cell sequencing technologies and the use of reporter pathway lines, with conditional modulation, combined with mutant lines for new regulators of cardiac as well as vascular components, will allow a highly specific genetic dissection of each process, with thus far unprecedented precision in the cardiovascular research field. We expect that cutting-edge high throughput approaches, such as single-cell RNA sequencing and metabolomics will be feasible in the near future . We enviThe datasets generated for this study are available on request to the corresponding author.All authors contributed to the writing of the manuscript. MMS coordinated the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors NT.


Emotions have traditionally been considered crucial in the development of functional neurological disorder, but the evidence underpinning this association is not clear. We aimed to summarize evidence for association between functional neurological disorder and emotions as formulated by Breuer and Freud in their conception of hysterical conversion. Based on a systematic literature search, we identified 34 controlled studies and categorized them into four groups: (i) autonomic arousal, (ii) emotion-motion interactions, (iii) social modulation of symptoms, and (iv) bodily awareness in FND. We found evidence for autonomic dysregulation in FND; convergent neuroimaging findings implicate abnormal limbic-motor interactions in response to emotional stimuli in FND. Our results do not provide enough empirical evidence for social modulation of the symptoms, but there is a clinical support for the role of suggestion and placebo in FND. Our results provide evidence for abnormal bodily awareness in FND. Based on these findings, we propose that functional neurological symptoms are forms of emotional reactions shaped into symptoms by previous experience with illness and possibly reinforced by actual social contexts. Additional research should investigate the effect of social context on the intensity of functional neurological symptoms and associated brain regions. Functional neurological disorders (FND) refer to patients with neurological symptoms in the absence of neurological disease. These symptoms have also been labeled as “hysterical,” “psychogenic,” “non-organic,” or “medically unexplained.” FND are common in neurology wards with the levels of disability similar to epilepsy or multiple sclerosis . DespiteSeveral etiological models of the disorder have been proposed throughout the history with varying degree of suggested psychological function of the symptoms . WhereasSome authors question the importance of emotions in the etiology of the disorder, because psychological stressors and emotional dysregulation are not always apparent in FND . The chaStudies on Hysteria. In their seminal work, Breuer and Freud  Is there evidence for higher sensitivity to emotional distress in FND? (2) Is there a relationship between processing of emotion and motor behavior in FND? (3) Are functional neurological symptoms modulated by social context in a similar way as emotional expressions? (4) Is bodily awareness reduced in FND?In the review, we focus on the two most prevalent subtypes of FND: psychogenic non-epileptic seizures (PNES) and functional movement disorders (FMD). Even though there is a debate on whether these two classes of symptoms constitute separate disorders (they differ in a few respects such as age at symptom onset and comorbidities) the functional neurological symptoms commonly co-occur and unifying pathophysiology is therefore likely .We aim to bring together biomedical and psychosocial perspectives on FND. While the biomedical perspective highlights abnormalities in a person's psychological and brain functioning, the psychosocial perspective stresses that the illness occurs in individuals with a personal history within an interactional matrix including their family, health care system, and cultural context such that the symptoms may have meaning other than as signs of underlying psychological or brain pathology . These tWe use recently developed Bayesian predictive coding framework applied to emotion and interoception –15 to inThe predictive coding model of brain function is a powerful neurobiological framework postulating that the brain uses its generative model of the world to make predictions about causes of sensory data . The preThe predictive coding framework applied to interoception puts the homeostatic regulation and sensory consequences of the regulation, i.e. interoception, at the core of the mind and brain architecture . In thisIn humans, social interaction serves in the regulation of homeostasis . Because* OR affect*). Reference lists of relevant articles were also searched. The results were assessed for inclusion using the publication title and abstract. Studies were included if they conformed to the following inclusion and exclusion criteria. Studies were included if (i) they reported on patients with functional neurological disorder described as functional, non-organic, psychogenic, hysterical, or conversion disorder; (ii) they reported data comparing cases with at least one control group; (iii) we included studies in adult as well as pediatric populations; (iv) we focused our review on experimental studies so we excluded studies that used only self-reports with exception of studies on life events where self-reporting cannot be avoided. The exclusion of self-reports is motivated by the assumption that self-reports targeting emotional processing demand certain capacity for introspection which may be diminished in the studied population of FND patients; (v) neuroimaging studies were only included if they also yielded behavioral or physiological data.We searched the databases PubMed and Web of Knowledge from 2007 to June 2018. Search terms were  AND  study, Fiess et al.  found thEven during action generated without emotional stimulus, Voon et al.  reportedThe reviewed articles revealed task-based co-activations between limbic structures and motor areas, especially the SMA, in FND. The SMA is activated by a range of tasks that require motor planning ; it has Beyond the known subcortical-motor pathways that mediate automatic and stereotypical motor behaviors in animals and humans in reaction to threat , 82, theAlthough the motor system has been thus far studied mostly apart from the limbic system, and there is a lack of evidence for a specific meaning of limbic-SMA interactions, a few reviewed studies suggested a possible role of the SMA in transforming emotional experience into motor actions. In their FND research, Voon et al.  proposedTwo inherent features of emotional expressions are that they influence the behavior of others and are also influenced by social context . For exaSerious “escape events” preceding FND onset  and incrWe draw another support for the social modulation of functional neurological symptoms from placebo effect and hypnotic suggestibility research in FND. Recent evolutionary accounts of animal signaling propose that symptoms of illness may have a signaling function with the goal of shaping the behaviors of conspecifics . In thisataques de nervios is a phenomenon similar to PNES that is common in Latin American societies; it includes fainting, trembling, or convulsions that people use to communicate distress as a way to elicit social support  or a set of behaviors provoking symptoms in the case of nocebo . These mHigher hypnotic suggestibility reported in FND and strikingly similar neural correlates in experiments matching functional symptoms with clinical analogs created by suggestion  are in tSeveral reviewed studies provide evidence for abnormal interoceptive awareness in FND. Ricciardi et al.  reportedThe present review sought to summarize support for the hypothesis that functional neurological symptoms are emotional expressions of distress. We derived our hypothesis from the conception of conversion hysteria postulated by Freud and Breuer  and seleThe reviewed experimental research studies provide evidence for higher levels of affective excitation in FND postulated by Breuer and Freud . Such exFrom the developmental point of view, emotional abuse and neglect, evidenced in FND by several studies, is potentially harmful also for the proper development of interoceptive brain networks. Lack of attunement between child and dismissive caregiver may cause models of internal bodily processes to be inefficient in predicting sensory inputs. Interoceptive signals then become imprecise and orient child more to external aspect of the body in the state of higher arousal. Later on, when primed by experience with illness or injury, the higher precision of exteroceptive inputs relative to interoceptive inputs may lead to symptom onset as falsely inferred cause of emotional distress.The finding of abnormal limbic-motor interactions in a reaction to emotional stimuli seems to be consistent in the reviewed literature. However, there is a debate about the meaning of the limbic-motor interactions among researchers. Two main hypotheses for the finding have been suggested in the reviewed literature: (i) defense mechanism akin to freezing behavior in animals , 31, andBlakemore et al.  interpreVoon et al.  propose We can only suggest that the proposed explanatory models for the FND are not mutually exclusive and further speculate that the symptoms serve as protective mechanisms patients use to cope with arousing situations  and these mechanisms are shaped by previous experience with illness or injury. We also propose that the onset of functional neurological symptoms may be motivated by perceived or expected social reactions .In our review, we identified only two studies that indirectly examined social modulation of functional neurological symptoms. However, there is  evidence for the influence of suggestion and placebo on the intensity of functional neurological symptoms. Functional neurological symptoms seem to be sensitive to motivational factors that a patient receive from his immediate environment. As illness or injury is embedded in social system , illness behavior may be shaped by behavior of others. In childhood, abnormal behaviors similar to the symptoms of a disease may be one of the limited ways how to elicit nurturance in a psychologically absent caregiver. Bowlby  suggesteSeveral reviewed studies showed evidence for impaired interoceptive awareness in FND. Edwards et al.  has alreIn the review, we showed that emotional processing is an important factor in the etiology of FND. Taken together, we conclude that limbic-motor interactions evidenced in FND may reflect learned emotional behaviors of an individual with low interoceptive  awareness and we interpret functional neurological symptoms as forms of complex affective reactions to stress similar to emotional expressions. Future studies should focused on examining brain activations in FND patients in response to stimuli relevant for the disorder, such as attachment narratives or autobiographical information. Moreover, exploring an effect of social context on the intensity of functional neurological symptoms could provide new information about the function of the symptoms.The literature search for the study was managed by PS. The first draft of the review was written by PS and MB. TK and MS critically revised and commented on the manuscript and figures. All the authors substantially contributed to and have approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thus, upon integration, HTLV-1 randomly inserts a vCTCF-BS into the host genome. vCTCF-BS studies to date have focused primarily on HTLV-1 chronically infected or tumor-derived cell lines. In these studies, HTLV-1 was shown to alter the structure and transcription of the surrounding host chromatin through the newly inserted vCTCF-BS. However, the effects of CTCF binding in the early stages of HTLV-1 infection remains unexplored. This study examines the effects of the vCTCF-BS on HTLV-1-induced in vitro immortalization and in vivo viral persistence in infected rabbits.Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATL) and the neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The exact mechanism(s) through which latency and disease progression are regulated are not fully understood. CCCTC-binding factor (CTCF) is an 11-zinc finger, sequence-specific, DNA-binding protein with thousands of binding sites throughout mammalian genomes. CTCF has been shown to play a role in organization of higher-order chromatin structure, gene expression, genomic imprinting, and serve as a barrier to epigenetic modification. A viral CTCF-binding site (vCTCF-BS) was previously identified within the overlapping Hbz gene expression were not significantly different between HTLV-1 and HTLV-1∆CTCF-infected rabbits throughout a 12 week study. However, HTLV-1∆CTCF-infected rabbits displayed a significantly decreased HTLV-1-specific antibody response compared to HTLV-1-infected rabbits.HTLV-1 and HTLV-1∆CTCF LTR-transactivation, viral particle production, and immortalization capacity were comparable in vitro. The total lymphocyte count, proviral load, and Mutation of the HTLV-1 vCTCF-BS does not significantly alter T-lymphocyte transformation capacity or early in vivo virus persistence, but results in a decreased HTLV-1-specific antibody response during early infection in rabbits. Ultimately, understanding epigenetic regulation of HTLV-1 gene expression and pathogenesis could provide meaningful insights into mechanisms of immune evasion and novel therapeutic targets. Human T-cell leukemia virus type 1 (HTLV-1) is the first discovered human retrovirus with an estimated 5–10 million individuals infected worldwide . HTLV-1 Recently, a CCCTC-binding factor (CTCF) binding site was identified within the HTLV-1 provirus . CTCF isUnlike HIV-1, HTLV-1 integration appears to have no strong integration site predilection , 14. ThuTax) and HTLV-1 basic leucine zipper factor (Hbz). Tax is considered the primary oncogene of HTLV-1. Tax drives proviral transcription via transactivation of the 5′ HTLV-1 long terminal repeat (LTR) and has been shown to promote cellular proliferation via dysregulation of multiple pathways including activation of NF-κB and cyclin dependent kinases 2/4 [High HTLV-1 proviral load in asymptomatic carriers is recognized as a risk factor for the development of both ATL and HAM/TSP , 17. Thuases 2/4 . Hbz hasases 2/4 . It has ases 2/4 , 20. ChaThe aims of this study are to determine the effects of vCTCF-BS ablation on in vitro immortalization capacity via a co-cultivation assay and in vivo persistence, utilizing the NZW rabbit as a model for early infection. Our results indicate that abrogation of CTCF binding to the vCTCF-BS does not alter in vitro immortalization capacity or in vivo persistence, but does significantly decrease the in vivo HTLV-1-specific antibody response when compared to appropriate HTLV-1 controls.Hbz gene. However, the vCTCF-BS mutations do result in changes to p12, a sense transcribed HTLV-1 accessory gene. Previous reports have shown that mutant ablation of p12 (ATG changed to GTG) had no measurable effect on HTLV-1 transforming capacity of primary human T-cells in cell culture nor infectivity and persistence in inoculated rabbits [p12 gene product with multiple substitutions and potentially confounding results, an additional mutation was introduced in p12, immediately upstream of the vCTCF-BS mutations. This mutation results in deletion of the carboxy terminal 23 amino acids of p12 . HTLV-1∆CTCF contains several point mutations within the consensus vCTCF-BS while avoiding introduction of mutations to the opposite-strand coding sequence of the  rabbits . HoweverTo determine the capacity of HTLV-1∆CTCF or HTLV-1p12Stop mutant proviral clones to synthesize viral proteins, direct viral replication, and induce cellular immortalization, stable 729 cell transfectants expressing the proviral clones were isolated and characterized. Each stable transfectant contained complete copies of the provirus with the expected mutations (data not shown). To monitor the virion production in these mutant stable transfectants, the concentration of p19 Gag in the culture supernatant of isolated cell clones was quantified by ELISA revealing similar levels of virion production irrespective of wild-type or mutant virus produced Fig. a.Fig. 3H4) were cocultured with tenfold dilutions of virus-producing cells in a 96-well plate assay. Since this assay is very stringent, slowly growing or non-dividing cells are eliminated very quickly and the percentage of surviving wells is an accurate measure of the immortalization efficiency of viruses; PBMCs cocultured with parental 729 uninfected cells as a negative control results in no growth. Data presented in Table We next assessed the capacity of the HTLV-1 mutant viruses to immortalize human PBMCs in coculture assays. Freshly isolated human PBMCs cocultured with lethally irradiated 729.HTLV-1, 729.HTLV-1∆CTCF, or 729.HTLV-1p12Stop in the presence of 10 U/ml of human IL-2 showed very similar progressive growth patterns consistent with the HTLV-1 immortalization process Fig. b. PBMCs To evaluate the role of CTCF in vivo, we compared the abilities of 729, 729.HTLV-1, 729.HTLV-1∆CTCF, or 729.HTLV-1p12Stop cell lines to transmit virus to rabbits, which is an established model of infection and persistence , 23. RabAn important parameter to HTLV-1 infection in vivo is the immune response, particularly specific antibody response to the virus. Plasma was isolated from whole blood samples throughout the study and subjected to a qualitative immunoblot assay and/or quantitative HTLV-specific ELISA. Qualitatively, there appeared to be no significant difference in HTLV-1-specific antibody response throughout the course of the study . However, in an attempt to recapitulate the findings of Satou et al., HTLV-1, HTLV-1p12Stop, and HTLV-1∆CTCF immortalized human peripheral blood leukocytes . While subjectively decreased, the difference in p30 expression between PBL.HTLV-1∆CTCF and PBL.HTLV-1 was not significant (p 0.175). The statistically significant and subjective changes in p30 expression are supportive of the previous report by Satou et al. Moreover, Gag/Pol expression was utilized as another measure of sense transcription. Gag/Pol expression was detectable at week 2 and maintained a steady state throughout the study .The HTLV-1 vCTCF-BS was only recently identified, and its role in HTLV-1 replication and/or pathogenesis remains to be defined . ResearcOur in vitro LTR-transactivation data and short-term proliferation and immortalization assays indicated that loss of CTCF binding site was not sufficient to disrupt the capacity of the virus to infect, transcribe, induce proliferation, and/or immortalize primary T-lymphocytes. Therefore, similar to the phenotypes of HTLV-1 open reading frame (ORF) I, II and Hbz protein deletions , 26, 27,Hbz and Gag/Pol) and proviral loads were variable in individual rabbits with no significant difference between HTLV-1, HTLV-1p12Stop or HTLV-1∆CTCF persistently infected rabbits. Despite the level of variability, gene expression data displayed trends concurrent with a previous work that examined HTLV-1 gene expression kinetics during early infection of rabbits; Hbz expression increased over time with proviral load [Hbz and Gag/Pol transcript levels in vivo. It is important to note that at several time points during the in vivo study, proviral load and gene expression values in HTLV-1p12Stop infected rabbits were subjectively lower than those in rabbits infected with HTLV-1. A previous study utilized both the NZW rabbit and macaque models of HTLV-1 persistence to investigate the effects of p12 on infectivity [p12 knockout proviral clone  showed no decrease in HTLV-1 viral persistence. In the current study, p12 was truncated by 23 amino acids, as opposed to the previously described complete knockout. While unlikely, these differences in p12 manipulation could have contributed to in vivo alterations in proviral load and gene expression.HTLV-1∆CTCF and HTLV-1p12Stop inoculated rabbits became efficiently infected with HTLV-1. Viral gene expression  293T cells and 729. B cells were cultured in Dulbecco’s modified Eagle medium (DMEM) and Ivscoe’s DMEM, respectively. The culture media was supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 μg/mL), and 2 mM glutamine. hPBMCs and rPBMCs were isolated from freshly-collected whole blood using the Ficoll-Paque™ PLUS or Ficoll-Paque™ PREMIUM  density gradient medium, respectively. Isolated hPBMCs were cultured in RPMI 1640 medium and supplemented with 20% FBS, 10 U/mL of recombinant human interleukin-2 , glutamine, and antimicrobials as described above. All cells were cultured at 37 °C in a humidified atmosphere of 5% COR gene and has been previously described [Hbz gene. However, the vCTCF-BS mutations do result in changes to p12, a sense transcribed HTLV-1 accessory gene. In lieu of producing a p12 gene product with multiple substitutions and potentially confounding results, an additional mutation was introduced in p12, immediately upstream of the vCTCF-BS mutations that results in deletion of the carboxy terminal 23 amino acids of p12  and proteinase inhibitor cocktail in the presence and absence of the CTCF antibody for 30 min at room temperature. Protein bound DNA was separated from unbound DNA in a 6% polyacrylamide gel in TBE buffer and transferred to a nylon membrane. DNA was then cross-linked to the membrane with the UV Stratalinker 1800 (Stratagene) for 1 min. After 15 min blocking, the membrane was incubated with streptavidin–horseradish peroxidase conjugate in blocking buffer for 15 min and then exposed to the substrate solution. Biotin labeled DNA was detected by using Chemidoc XRS + molecular imager (Bio-Rad). The sequences of target DNA oligos: (1) DNA with wild type CTCF binding site: ATGCGTTTCCCCGCGAGGTGGCGCTTTCTCCCC. (2) DNA with mutated CTCF binding site: ATGCGTTTCCCCTCTCGGTGGAGCTTTCTCCCC. The LTR-1-Luc and thymidine kinase (TK)-Renilla reporter plasmids were described previously [EMSA was performed by using Light Chemiluminescent EMSA kit (Thermo Scientific) and following the manufacturer’s protocol with some modification. Briefly nuclear extract of 293T cells transfected with the plasmid overexpressing human CTCF protein was incubated with biotin labeled target DNA in 1 × binding buffer containing 5 mM MgCLeviously .®-2020 transfection reagent  according to manufacturer’s instructions. HEK293T cells were co-transfected with 1 μg of HTLV-1, HTLV-1∆CTCF, HTLV-1 p12Stop, or empty (pcDNA3) plasmids along with 100 ng of LTR-1-Luc and 20 ng of TK-Renilla reporter plasmids. An HTLV-1 p19 Gag enzyme-linked immunosorbent assay  was performed with supernatant collected 48 h post-transfection. Transfected cells were also harvested at the time of supernatant collection. Cell pellets were lysed and HTLV-1 LTR-transactivation was measured via luciferase assay according to the manufacturer’s protocol  [Renilla luciferase.HEK293T cells were transfected using Mirus TransITose, CA) . Assays Stable 729 HTLV-1 producer cell clones were generated by nucleofection of 729.B cells with 2 µg of HTLV-1, HTLV-1∆CTCF, or HTLV-1p12Stop plasmid using an Amaxa Cell Line Nucleofector™ Kit V in accordance with the manufacturer’s suggested protocols . Nucleofected cells were then subjected to G418 selection . An HTLV-1 ELISA was used to confirm p19 Gag production in G418 selected cell lines. Cell lines with p19 Gag production were then single cell selected via limiting dilution. HTLV-1∆CTCF and HTLV-1 p12Stop mutations were confirmed via Sanger sequencing (see Methods: PCR and quantitative PCR). p19 Gag ELISAs were performed on single cell clones and those with comparable p19 Gag production were selected for coculture immortalization assays.6 729. B cells and 729 HTLV-1 producer cell clones  were lethally irradiated (100 Gy) and cocultured with freshly-isolated hPBMCs (2 × 106) in the presence of IL-2 . Three randomly selected wells from each coculture were counted weekly via trypan blue exclusion and p19 Gag concentration was measured by ELISA at weeks 3, 6, 10, and 14. Wells with continuous hPBMC expansion in conjunction with p19 Gag expression in the presence of IL-2 were scored as immortalized. HTLV-1∆CTCF and HTLV-1p12Stop immortalized hPBMCs were checked for reversions via Sanger sequencing (see “4 cells/well) were co cultured with tenfold dilutions  of lethally irradiated 729.HTLV-1, 729.HTLV-1∆CTCF, or 729.HTLV-1p12Stop in the presence of 10 U/ml of human IL-2 in a 96 well plate (48 replicates each). The number of wells which became immortalized were determined by microscopy.1 × 107 lethally irradiated (100 Gy) 729 producer cell clones or 729.B control cells. A portion of lethally irradiated cells (1 × 106) were maintained in cell culture to assess p19 Gag production 24 h post-irradiation and ensure cell death. Blood was drawn via the central auricular artery pre-infection (week 0) and at weeks 2, 4, 8, and 12 post-infection. rPBMCs and plasma were isolated from freshly-collected blood using Ficoll-Paque™ PREMIUM  density gradient medium. Whole blood samples were analyzed for CBC at each time point . rPBMCs or plasma were assessed for proviral load, HTLV-1 gene expression, and HTLV-1-specific antibody response, as described below. Sanger sequencing of the vCTCF-BS was performed at week 12 to monitor for viral reversions. All animal procedures were performed in accordance with a protocol approved by University Laboratory Animal Resources (ULAR) of The Ohio State University.After a 2 week acclimatization period, fourteen-week-old, intact, male, specific pathogen-free New Zealand White rabbits (Crl:KBL (NZW); Charles River Laboratories, Wilmington, MA) were inoculated via the lateral ear vein with 1 × 10DNA isolation from 729 HTLV-1 producer cell clones and coculture-immortalized hPBMCs was performed using the Qiagen DNeasy Kit . Standard PCR followed by Sanger sequencing for vCTCF-BS mutation verification was performed for each newly-generated producer cell clone and coculture-immortalized hPBMCs (hPBMCs collected at week 16 of coculture assay). vCTCF-BS primer sets . Proviral load was quantitated with probe-based qPCR using 250 ng of rPBMC gDNA, Bio-Rad iQ™ Supermix, and the escribed . DNA isoHbz and Gag/Pol gene expression was quantified via cDNA synthesis followed by a pre-amplification and qPCR. cDNA synthesis was performed using the SuperScript™ IV First-Strand Synthesis System with 250 ng rPBMC RNA and appropriate-RT controls . Pre-amplification was performed using the SsoAdvanced™ PreAmp Supermix with 2 μL of rPBMC cDNA. The pre-amplification pool included primers for Hbz, Gag/Pol, and rGAPDH in accordance with the manufacturer’s protocol (primers listed in Table Hbz), pre-amplified products destined for rGAPDH qPCR were diluted 1:50 in order to obtain acceptable Ct values. A 45-cycle qPCR was performed in duplicate with appropriate minus-RT controls using Bio-Rad iQ™ Supermix with 2 μL of pre-amplified cDNA and Hbz or rGPADH primer/probe sets described in Table 10 dilutions of the Hbz standard listed in Table 6 rGAPDH. A mixed model analysis with a Bonferroni correction was performed in weeks 8 and 12 to determine statistical significance. A p < 0.0083 was considered a statistically significant change.p30 gene expression was quantified via cDNA synthesis followed by qPCR. cDNA synthesis was performed using the SuperScript™ IV First-Strand Synthesis System with 1 µg HTLV-1, HTLV-1∆CTCF, or HTLV-1p12Stop immortalized PBL RNA and appropriate-RT controls . A 45-cycle qPCR was performed in duplicate with appropriate minus-RT controls using Bio-Rad iQ™ Supermix with 2 μL of cDNA and p30 or human GAPDH (hGPADH) primer/probe sets . p30 primer/probe set were previously described by Li et al. [hGAPDH quantification utilized Integrated DNA Technologies (IDT) PrimeTime® Predesigned qPCR Assay Hs.PT.39a.22214836 in conjunction with a custom IDT gBlock gene fragment; GCGCCGCTGCGGGCCGAGCCACATCGCTCAGACACCATGGGGAAGGTGAAGGTCGGAGTCAACGGATTTGGTCGTATTGGGCGCCTGGTCACCAGGGCTGCTTTTAACTCTGGTAAAGTGGATATTGTTGCCATCAATGACCCCTTCATTGACCTCAACTACATGGTTTACATGTTCCAATAT . The qPCR conditions were as follows: 95 °C for 3 min followed by 45 cycles of 95 °C for 15 s, 60 °C for 30 s. Total copy number was determined using a standard curve generated by duplicate samples of log10 dilutions of the p30 standard previously described by Li et al. [6hGAPDH. One way ANOVA with multiple comparisons was used for statistical analysis with significance denoted by p < 0.05.In vitro i et al. . hGAPDH i et al. . Copy nuGag/Pol gene expression for HTLV-1, HTLV-1∆CTCF, and HTLV-1p12Stop at weeks 4, 8, and 12 post-infection. A statistically significant correlation is denoted by p < 0.05.A Pearson Correlation was performed between the HTLV-1-specific antibody response and The HTLV antibody response was assessed qualitatively in a representative rabbit from each condition via a modified MP Diagnostics HTLV Blot 2.4 Western Blot Assay protocol . The supplied alkaline phosphatase conjugated goat anti-human immunoglobulin gamma (IgG) was substituted for an alkaline phosphatase conjugated goat anti-rabbit IgG . Plasma from each condition was diluted 1:10.After qualitative assessment of representative rabbits, HTLV-1-specific antibody response was quantified for all rabbits using a modified Avioq HTLV-1/2 Microelisa System protocol . The supplied horseradish peroxidase (HRP) conjugated goat anti-human IgG was substituted for an HRP conjugated goat anti-rabbit IgG . Rabbit plasma was diluted 1:500 to obtain absorbance values within the linear range of the assay.6. Mixed model analyses with a Bonferroni correction were performed in weeks 8 and 12 (HTLV-1-specific) or 2 and 12  to determine statistical significance. A p < 0.0083 was considered a statistically significant change.Total rabbit IgG was quantified using the Abcam Rabbit IgG ELISA Kit in accordance with the provided protocol . Plasma samples were diluted 1:1 × 10Additional file 1: Fig. S1. Ablation of HTLV-1 CTCF-binding site does not qualitatively decrease HTLV-1-specific antibody response. The HTLV antibody response was assessed qualitatively at 0, 4, and 12 weeks post-inoculation in a representative rabbit from each condition via a modified MP Diagnostics HTLV Blot 2.4 Western Blot Assay protocol . The supplied alkaline phosphatase conjugated goat anti-human immunoglobulin gamma (IgG) was substituted for an alkaline phosphatase conjugated goat anti-rabbit IgG . Plasma from each condition was diluted 1:10. Reactive HTLV-1 proteins are labeled on the left. rgp46-1 (HTLV-1-specific recombinant envelope surface protein); p53 (Gag precursor); p24 (capsid protein); p19 (matrix protein); GD21 (recombinant transmembrane envelope protein). “Serum control” denotes serum immunoglobulin levels among.Additional file 2: Fig. S2. Ablation of the HTLV-1 CTCF-binding site significantly decreases HTLV-1 p30 expression in vitro in HTLV-1∆CTCF immortalized PBLs when compared to HTLV-1p12Stop, but not when compared to HTLV-1. p30 gene expression was assessed via qPCR. RNA was isolated from HTLV-1-immortalized (PBL.HTLV-1), HTLV-1∆CTCF-immortalized  PBLs. cDNA was synthesized from 1 µg of RNA, then a 45-cycle qPCR was performed in duplicate using 2 µL of cDNA per reaction and a p30-specific primer/probe set. Total copy number was determined using a standard curve generated by duplicate samples of log10 dilutions of p30 standard . Copy numbers were normalized to 106human GAPDH (hGAPDH). Bars represent mean log10p30 copy normalized to hGAPDH. Error bars indicated standard deviation. PBL.HTLV-1∆CTCF showed a significant decrease in p30 gene expression when compared to PBL.HTLV-1p12Stop (p 0.025). While subjectively decreased, the difference in p30 expression between PBL.HTLV-1∆CTCF and PBL.HTLV-1 was not significant (p 0.175). One way ANOVA with multiple comparisons was used for statistical analysis with significance denoted by values with p < 0.05.Additional file 3: Fig. S3. There is a positive correlation between HTLV-1-specific antibody response and Gag/Pol gene expression in HTLV-1∆CTCF-infected rabbits. A Pearson Correlation was performed between the HTLV-1-specific antibody response and Gag/Pol gene expression for HTLV-1 (A), HTLV-1p12Stop (B), and HTLV-1∆CTCF (c) at weeks 4, 8, and 12 post-infection. A statistically significant correlation (p < 0.05) was not found at any time point, but HTLV-1∆CTCF showed a strong positive correlation between HTLV-1-specific antibody response and Gag/Pol gene expression at weeks 8 and 12. Comparatively, HTLV-1 and HTLV-1p12Stop had weakly positive to negative correlations at weeks 8 and 12. While not statistically significant, this finding may suggest that the decrease in HTLV-1-specific antibody response for HTLV-1∆CTCF at week 12 may be the result of decreased Gag/Pol gene expression.


We reported that human T cell leukemia virus 1 (HTLV-1) infection is positively associated with atherosclerosis. Recent evidence has revealed a close association of periodontitis with atherosclerosis, endothelial dysfunction, and disruption of the microcirculation. However, the association between HTLV-1 and advanced periodontitis has not been investigated to date. Since hematopoietic activity is closely linked to endothelial maintenance activity and is known to decline with age, we hypothesized that the state of hematopoietic activity influenced the association between HTLV-1 and advanced periodontitis in elderly participants.A cross-sectional study was performed including 822 elderly participants aged 60–99 years who participated in a dental health check-up. Advanced periodontitis was defined as a periodontal pocket ≥ 6.0 mm. Participants were classified as having low or high hematopoietic activity according to the median values of reticulocytes.HTLV-1 infection was positively related to advanced periodontitis among participants with lower hematopoietic activity (lower reticulocyte count), but not among participants with higher hematopoietic activity (higher reticulocyte count). The adjusted odds ratio  considering potential confounding factors was 1.92 (1.05–3.49) for participants with a lower reticulocyte count and 0.69 (0.35–1.36) for participants with a higher reticulocyte count.Among elderly participants, the association between HTLV-1 infection and advanced periodontitis is influenced by hematopoietic activity. Since hematopoietic activity is associated with endothelial maintenance, these findings provide an efficient tool for clarifying the underlying mechanism of the progression of periodontitis among elderly participants. Human T cell leukemia virus 1 (HTLV-1) is an oncogenic retrovirus affecting individuals worldwide, although the southern part of Japan is known as a highly endemic region for HTLV-1 infection . The majIn addition, recent studies have revealed that periodontitis is closely associated with atherosclerosis , endotheIn addition, bone marrow activity (hematopoietic activity) has been recently identified to be associated with endothelial maintenance –17; retiTo evaluate this hypothesis, we conducted a cross-sectional study to examine the influence of hematopoietic activity (assessed by reticulocyte counts) on the association between HTLV-1 and advanced periodontitis among 822 elderly participants that received an annual dental health check-up from 2016 to 2018.n = 4) or laboratory data (n = 3) available, or if they had less than 10 remaining teeth (n = 1078). Since anemia  has been reported to be associated with periodontitis −1.094 × (age)−0.287 × (0.739 for women).Blood samples were collected into EDTA-2K tubes and siliconized tubes, and all measurements were conducted according to standard automated laboratory procedures at SRL, Inc. . The parameters tested included red blood cell (RBC) count, reticulocyte count, Hb level, and serum creatinine level. Reticulocyte count was calculated with the following formula: reticulocytes . Mean values for the left and right CIMT were calculated with automated digital edge-detection software  according to a previously described protocol . The valA chemiluminescent enzyme immunoassay (CLEIA) kit was used to detect the presence of HTLV-1  according to the manufacturer’s instructions.Trained dentists performed a periodontal examination according to a modified method of the Third National Health and Nutrition Examination Survey . Probing4 cells/μL for men and 4.926 × 104 cells/μL for women) was set as the cutoff point for classifying the participants into those with low and high hematopoietic activity. The differences in mean values or proportions of reticulocyte levels were analyzed in relation to HTLV-1 status. Significant differences were evaluated using analysis of variance (ANOVA) for continuous variables and chi-squared test for proportion data.The median value for the reticulocyte count  and 95% confidence intervals (CIs) to determine the association between HTLV-1 infection and advanced periodontitis. Since aging influences the hematopoietic activity, which in turn influences erythropoietin production, the participants were stratified according to their hematopoietic activity (maintained or reduced hematopoiesis) based on the level of reticulocytes (high or low).Periodontitis risk factors with a direct influence on the intra-oral environment, such as smoking  and numbIn addition, hemoglobin level is known to be associated with renal function  as well p < 0.05 were regarded as statistically significant.All statistical analyses were performed with the SAS system for Windows . Values of Among the total 822 participants with a mean ± standard deviation (SD) age of 71.2 ± 6.7 years, 140 (17.0%) showed an HTLV-1-positive status and 181 (22.0%) were diagnosed as having advanced periodontitis; 411 participants had low hematopoietic activity .Table As shown in Table Sensitivity analysis according to the quartile of reticulocyte levels demonstrated associations between HTLV-1 infection and advanced periodontitis, similar to the main results.The major finding of the present study is that HTLV-1 infection is positively associated with advanced periodontitis only among elderly participants with lower hematopoietic activity (a lower reticulocyte count).A previous case-controlled cross-sectional study including 42 periodontally healthy individuals, 64 HTLV-1-seronegative individuals presenting with chronic periodontitis, and 36 individuals with chronic periodontitis with HTLV-1-seropositive HTLV-1-associated myelopathy (HAM)/symptomatic tropical spastic paraparesis (TSP) revealed that HTLV-1 may play a critical role in the pathogenesis of periodontal disease through deregulation of the local cytokine network [In the present study, we found further evidence that HTLV-1 infection is positively associated with advanced periodontitis among participants with low reticulocyte levels, even though most of the participants were asymptomatic.Periodontitis has been shown to be closely associated with atherosclerosis , endotheAging is known to be associated with a decline of bone marrow activity (reduction in hematopoietic activity) –21 and aIn addition, HTLV-1 infection is reported to be positively associated with atherosclerosis . TherefoFurthermore, a significant interaction was observed between HTLV-1 status and hematopoietic activity level (low vs. high) with respect to advanced periodontitis. These results also support the abovementioned mechanism by indicating that hematopoietic activity levels could act as a determinant of the association between HTLV-1 and advanced periodontitis.Some potential limitations of this study warrant consideration. Although the age-related endothelial dysfunction should have an influence on the association between HTLV-1 infection and advanced periodontitis, no data on endothelial function were available to directly assess this relationship. Thus, further analyses that include endothelial function-related data such as flow-mediated dilation  will be Owing to the lack of knowledge regarding the determination of the exact cutoff point, the median values of reticulocytes were used for the present analysis. Further investigation is necessary to clarify whether the balance between endothelial damage and endothelial repair that contributes to endothelial maintenance can help to determine the exact cutoff point for the present associations. In addition, because this was a cross-sectional study, causal relationships could not be established. Nevertheless, the association identified provides new insights into the potential mechanisms mediating the relationship between HTLV-1 and periodontitis, highlighting the importance of maintaining hematopoietic activity for the overall health of elderly participants.HTLV-1 infection is positively associated with advanced periodontitis among elderly participants with lower hematopoietic activity (a lower reticulocyte count) but not among elderly participants with higher hematopoietic activity (a higher reticulocyte count). Since elderly participants with higher hematopoietic activity should have higher tissue-repairing activity  with a p


TSLP, interleukin 7 receptor (IL7R), and TSLPR. Three common variants in the TSLP/TSLPR axis were significantly associated with CAD in a Chinese Han population . Reporter gene analysis demonstrated that rs3806933 and rs6897932 could influence TSLP and IL7R expression, respectively. Furthermore, the “T” allele of rs3806933 might increase plasma TSLP levels . In a stepwise procedure, the risk for CAD increased by nearly fivefold compared with the maximum effect of any single variant . In addition, the epistatic interaction between TSLP and IL33 produced a nearly threefold increase in the risk of CAD in the combined model of rs3806933TT-rs7025417TT . Our study illustrates that the TSLP/TSLPR axis might be involved in the pathogenesis of CAD through upregulation of mRNA or protein expression of the referenced genes and might have additive effects on the CAD risk when combined with IL-33 signaling.The thymic stromal lymphopoietin (TSLP)/TSLP receptor (TSLPR) axis is involved in multiple inflammatory immune diseases, including coronary artery disease (CAD). To explore the causal relationship between this axis and CAD, we performed a three-stage case-control association analysis with 3,628 CAD cases and 3,776 controls using common variants in the genes  Genetic factors have been demonstrated to be equally important as environmental factors in the pathogenesis of coronary artery disease (CAD). Based on family and twin studies, researchers have estimated that the heritability of CAD is between 40 and 60% . Much pr+ and CD8+ T cells /TSLP receptor (TSLPR) axis plays an important role in the regulation of a broad spectrum of inflammatory immune response-related diseases, including asthma and CAD. The key proteins of this axis are as follows: TSLP, a four-helix bundle cytokine targeting a variety of inflammatory immune cells, such as dendritic cells (DCs), B cells, mast cells, regulatory T cells, and CD4 T cells ; and the T cells –15. Unde T cells . TSLPR m T cells . Most re of TSLP . These rntiation , 19. Howntiation . In in vgression . Howevergression .Therefore, we performed the following studies to uncover the potential causal genes for the influence of the TSLP/TSLPR axis in the pathogenesis of CAD: (1) selecting all the tag SNPs covering the key genes of the TSLP/TSLPR axis; (2) performing a three-stage case-control genetic association study for CAD based on Chinese Han population with a large sample size; the selected tag SNPs were tested in stage 1 discovery study and then the positive associations from stage 1 discovery study were further tested in stage 2 validation study and stage 3 replication study; (3) testing correlations between the significant variants for CAD and the expression levels of their genes, by a reporter gene analysis or a circulation level study; and (4) constructing the interaction model and detecting the effect size of the causal variants in the contribution to the development of CAD.IL1RL1 gene, which encodes the receptor of IL-33, might influence the circulation level of soluble ST2 (sST2). The exact mechanism might be that the missense variants in IL1RL1 regulate the expression of IL33 and the interaction between IL-33 and its receptor ST2L, and then significantly increase the circulating level of sST2  between common variants in the genes regulated by each other might have quite different functions in populations with different genetic backgrounds, thereby leading to a quite different effect size or direction on the risk of the diseases. In 2013, we reported that variants in the IL-33 signaling pathway might influence the development of CAD by regulating the expression levels of key genes of the pathway . The Fra of sST2 . Other s of sST2 –27.IL33 and TSLP in the pathogenesis of CAD.These results indicated that variations in genes in the IL-33 and TSLP signaling pathways might contribute to epistatic interactions and then affect the develop of conditions such as CAD. In the present study, we investigated the potential epistatic interaction between The total sample size was 7,404, with 3,628 CAD cases and 3,776 controls (GeneID database) , 28–30. This study followed the guidelines set forth by the Declaration of Helsinki and passed the review of the Ethics Committee of Tongji Medical College, and Huazhong University of Science and Technology. All study participants have signed a written informed consent form.r2 or D′ was more than 0.8 between the SNPs ; (2) the threshold of the minor allele frequency was greater than 0.05. Tag SNPs, such as rs3806933 and rs2289276 in TSLP and rs1494558, rs1494555, rs7737000, and rs6897932 in IL7R were selected , 4 variants  in TSLPR were selected for the association analysis . A High-Resolution Melt system with Rotor-gene 6000 software (Corbett Life Science) was used for genotyping, which was validated by direct DNA sequencing analysis , 28–30. TSLP, two plasmids of TSLP-C and TSLP-T were generated with the pGL3-basic vector  in the human HELA cell line, and three independent experiments were performed.We performed the reporter gene assay to investigate whether the variants could regulate their gene expression. For the promoter variant rs3806933 in c vector . In addiA total of 336 CAD patients were selected from the replication cohort for analysis of the association between rs3806933 genotypes and the plasma level of TSLP. The plasma samples were frozen at –80°C and tested in less than 2 months for TSLP concentration with an enzyme-linked immunosorbent assay kit (eBioscience Inc.).TSLP; GRS2 comprised SNP rs7025417 of IL33; and GRS3 comprised the both SNPs rs3806933 of TSLP and rs7025417 of IL33.Genetic risk scores were analyzed using a Cox regression model , adjusting for the conventional risk factors of CAD to obtain survival forecasts and calculate the C indices. GRS1 comprised SNP rs3806933 of P < 0.01 in the text, figures, and figure legends indicates statistical significance.All the control cohorts were examined using the Hardy–Weinberg equilibrium (HWE) test (PLINK version 1.06). The allelic and genotypic association analyses were performed using chi-square tests with Pearson’s 2 × 2 and 2 × 3 contingency tables (SPSS version 17.0). Under the genotypic model, the interaction analysis was carried out by a logistic association analysis with the conventional risk factors for CAD as covariates . StatistThe CAD patients were more likely to have hypertension and diabetes mellitus than the control subjects. Moreover, in all studied populations, the age, body mass index (BMI), total cholesterol, and low-density lipoprotein cholesterol levels were much higher in CAD patients than in controls, and the high-density lipoprotein cholesterol level was significantly lower in CAD patients than in controls Table .T in TSLP and rs6897932T in IL7R, rs3806933T in TSLP and rs7025417T in IL33 under the genotypic model) . The statistical power was also more than 70% for the two interaction analyses (rs3806933c model) .P > 0.001). In stage 1, the allelic frequencies of rs3806933T in TSLP, rs6897932T in IL7R, and g.19646A>GA in TSLPR were significantly different between cases and controls  after adjustment for the conventional risk factors  in the validation cohort, which passed Bonferroni correction. In stage 3, the associations between the three variants and CAD were confirmed in the replication cohorts  after adjustment for covariates of CAD, which passed Bonferroni correction  were also identified for CAD in the combined cohort under an additive model after applying Bonferroni correction .Combining the three cohorts together, a meta-analysis with 3,628 CAD cases and 3,776 controls was performed. In the combined cohorts: 3,339 CAD cases and 3,569 controls were genotyped successfully for rs3806933; 3,330 CAD cases and 3,761 controls were genotyped successfully for rs6897932; 3,628 CAD cases and 3,776 controls were genotyped successfully for g.19646A>G. The above three variants all were significantly associated with CAD in the combined cohort . The genotypic association results also were not significant with adjusted P values of more than 0.05 under any one of the three models  for these variants . Therefore, we discarded these variants in the next two stages.The allelic association results between rs2289276 in TSLP which might regulate the expression of TSLP. Using luciferase assay, we found that compared to the empty pGL3-basic vector group, the luciferase activity increased in both the construct TSLP-T and construct TSLP-C groups, and the luciferase activity increased more for the construct TSLP-T than for TSLP-C  showed considerably lower luciferase activity than the plasmid IL7R-C . It was concluded that the allele changes of rs3806933 in TSLP and rs6897932 in IL7R could both regulate the expression of their genes, whereas g.19646A>G in TSLPR might not.Rs3806933 was located in the promoter region of n = 237, R2 = 0.175, P < 0.01; Figure P < 0.01; Figure TSLP and as the number of risk allele “T” of rs3806933 increased, the circulating level of TSLP increased.Among the 336 CAD patients, the detection rate of plasma TSLP concentrations was 70.5% . All the detectable samples were included in this study. Linear regression analysis revealed a significant association between the rs3806933 genotypes and the plasma TSLP levels in the subjects with a detectable TSLP level  in association with CAD than the single variants did  indicated significant associations with a P value of 3.67 × 10−4 for the type of rs3806933_TT/rs7025417_TT, which provided a nearly threefold increase in the risk of CAD  or only an SNP in the single gene  by a Cox regression model. Our results showed that, after adjusting for the conventional risk factors of CAD, GRS3 increased the C index by 3.1% as compared to GRS1 and by 0.3% as compared to GRS2  for CAD. Furthermore, reporter gene analysis showed that rs3806933 in TSLP and rs6897932 in IL7R could regulate the mRNA expression of their respective genes. In addition, the circulating level study also confirmed that the variant rs3806933 in TSLP could influence the circulating level of TSLP protein. These results indicate that variants in the TSLP/TSLPR axis might affect the risk of CAD through upregulating mRNA or protein expression, and the variants or key genes are likely to be causal risk factors for CAD.In our study, we discovered three genetic risk variants  might interact with each other in a biologically relevant way and the crosstalk between TSLP and IL-7Rα might greatly enhance the risk of CAD.In 2000, Pandey et al. and Park et al. demonstrated that IL-7Rα, in combination with TSLPR, can increase the binding affinity of TSLP for its receptors, which might promote the downstream signaling of TSLP, thus improving its biological efficacy , 15. In  protein , 33. In  of TSLP . In the TSLP is moderately associated with CAD and rs6897932 in IL7R is not associated with CAD in the European ancestry (TSLP and rs6897932 in IL7R might be the specific cis-expression quantitative trait loci (eQTLs) for CAD in our Chinese Han population. This inconsistency between the results may be explained by the heterogeneity of the eQTL in different diseases or populations with different ancestries  and then influence the risk of asthma (−/− mice and LDLR−/− mice developed less severe atherosclerotic, which implied a proatherogenic function of Th2 inflammation (TSLP and IL33 was much higher than those of the single genes. Considering our findings together, we hypothesized that variants in the TSLP/TSLPR axis might regulate the expression of the key genes in the pathways as well as the cytokines and their receptors involved in the development of CAD by a positive feedback effect, which could also be called an epistatic effect. This positive feedback loop among IL-33, TSLP, and their receptors might increase CAD risk through facilitating the Th2-skewed inflammation. Further functional studies are needed to confirm our hypothesis.In 2013, we reported that variants in the IL-33 signaling pathway might influence the development of CAD by regulating the expression of key genes of the pathway and then influenced the risk of CAD . IL-33 wf asthma . In the f asthma . The posf asthma . Howeverf asthma ; when taammation , 50. In In conclusion, this study indicated that the TSLP/TSLPR axis might affect the CAD risk through upregulating the reference genes’ mRNA expression or protein secretion, and the TSLP and IL-33 signaling pathways might have an epistatic effect on the risk of CAD. These results might provide a novel point of view for the prevention and treatment of CAD based on targeting the mechanisms of the epistatic effect between the related cytokines.We declare that all the data supporting the findings of this study are available within the article and the Supplementary Information files and can be obtained from the corresponding authors upon reasonable request.This study followed the guidelines set forth by the Declaration of Helsinki and passed the review of the Ethics Committee of Tongji Medical College, and Huazhong University of Science and Technology. All study participants signed a written informed consent form.Conceived and designed the experiments: XT and XC. Performed the experiments and analyzed the data: S-FN, L-FZ, and QF. Contributed reagents/materials/analysis tools: S-FN, L-FZ, QF, Y-HL, H-SZ, Q-WC, FW, T-TT, NX, C-QX, J-YZ, Y-ZL, W-JZ, Z-PZ, JJ, Y-YL, TX, DW, C-CW, J-JF, H-BX, JY, QY, P-YW, S-HT, Q-LL, Q-XL, JQ, BL, GW, Y-XW, YY, X-PY, YH, QW, XC, and XT. Wrote the paper: S-FN. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fibroblast Growth Factor 10 (FGF10) is a multifunctional mesenchymal-epithelial signaling growth factor, which is essential for multi-organ development and tissue homeostasis in adults. Furthermore, FGF10 deregulation has been associated with human genetic disorders and certain forms of cancer. Upon binding to FGF receptors with heparan sulfate as co-factor, FGF10 activates several intracellular signaling cascades, resulting in cell proliferation, differentiation, and invasion. FGF10 activity is modulated not only by heparan sulfate proteoglycans in the extracellular matrix, but also by hormones and other soluble factors. Despite more than 20 years of research on FGF10 functions, context-dependent regulation of FGF10 signaling specificity remains poorly understood. Emerging modes of FGF10 signaling regulation will be described, focusing on the role of FGF10 trafficking and sub-cellular localization, heparan sulfate proteoglycans, and miRNAs. Systems biology approaches based on quantitative proteomics will be considered for globally investigating FGF10 signaling specificity. Finally, current gaps in our understanding of FGF10 functions, such as the relative contribution of receptor isoforms to signaling activation, will be discussed in the context of genetic disorders and tumorigenesis. Fgf10) gene has been identified in all examined vertebrates (The Fibroblast Growth Factor 10 (tebrates . It belotebrates  which watebrates .Fgf10 knockout (KO) mice die at birth with defects in multiple organ development, including the limb, lung, kidney, salivary gland and adipose tissue    and lacrs (ALSG) ; chronics (ALSG)  and certs (ALSG) , pancreas (ALSG) , and gass (ALSG)  cancers s (ALSG) .Fgf10 expression and activity are modulated in different cellular contexts. Mechanisms underlying FGF10-dependent control of signaling specificity will be discussed and novel technologies to study the multiple roles of FGF10 will be introduced.Fibroblast Growth Factor 10 activates key intracellular signaling pathways in several cell types leading to the modulation of organ branching and cell proliferation, differentiation, and migration during development; wound healing and tissue repair; maintenance of stem cells compartment; and cancer cell invasion and proliferation . Here, wFgfr). Fgfr1–3 are alternatively spliced into “b” and “c” isoforms, which differ in their extracellular domain and ligand binding specificity and which are expressed by epithelial or mesenchymal cells, respectively  residues are trans-autophosphorylated. The sequential order of tyrosine residue phosphorylation has been reported for FGFR1 . The catFibroblast Growth Factor receptors engage multiple signaling pathways via adaptor proteins Figure . FGF-regThese results suggest context-dependent and fine-tuned modulation of downstream signaling pathways upon FGF10 binding to its receptors. FGF10 acts as a canonical FGFR ligand in recruiting the adaptor proteins FRS2, GRB2, or PLCγ to the receptor, but it can also induce the formation of cell type-specific signaling complexes . Either structural rearrangements of the receptor or the presence of cell type specific co-activators may explain these two different modes of FGF10 signaling initiation.Fibroblast Growth Factor receptors signal through ERK1/2 during development . For insThe role of FGF10 in signaling regulation through PI3K and downstream kinases like protein kinase B (AKT) is less clear Figure . The PI3Even though the role of other kinases in FGF10 signaling specification remains to be determined, FGF10 is a versatile growth factor that enables epithelial cell growth and migration via ERK1/2 and controls cell survival via PI3K/AKT signaling during development and in several pathological conditions.Fibroblast Growth Factor 10 is known to control cellular outputs through several transcription factors Figure . FGF10 pAs FGF10 activates both ERK1/2 and PI3K all the known transcription factors that depend on these kinases   should pAmong several other important players, we will focus on four families of proteins with a context-specific role in FGF10 signaling Figure .Hes1 in the adult small intestine (Neurogenic locus notch homolog protein 1 (NOTCH1) is a receptor controlling cell signaling via several ligands and mechanisms . FGF10 antestine .The functions of WNT ligands, which bind to the frizzled family of seven transmembrane receptors , are modSonic hedgehog protein (SHH), which plays crucial roles during vertebrate development  , Fgf10 es Figure .in vivo.Heparan sulfate proteoglycans are a family of glycoproteins composed of a variety of heparan sulfate moieties (HS) attached to a core protein which play critical roles during organ branching and morphogenesis . CleavagFgf10, whereas prolactin significantly induced Fgf10 gene expression during pregnancy , which are crucial regulators of gene expression . The mirormation . More reormation  and thatormation .Fgf10 expression and activity regulation has clearly emerged, a conundrum in FGF10 signaling still remains: how is FGF10 signaling specificity controlled in different cellular contexts? The importance of subcellular localization in modulating specific aspects of FGF10 responses will be discussed in the next section.Although a comprehensive picture of The FGF10 receptor FGFR2b is internalized via clathrin-coated pits into intracellular vesicles (early endosomes) Figure , and theFgf10 gene  . In a tyWe suggest a few proteomic approaches to study FGF10 signaling specificity in an unbiased manner:(1) Given the lack of available isoform-specific antibodies, FGF10 signaling can be compared in cells or organs expressing exclusively FGFR1b or 2b, using CRISPR-Cas9-based techniques or transgenic mice, by quantitative interactomics combined with phosphoproteomics. This will (a) increase our understanding of the relative contribution of FGFR1b and 2b isoforms to FGF10 response, as the phenotype of the FGFR2b (but not FGFR1b) KO mice resembles that of FGF10 KO mice ; (b) dis(2) System-level analysis of cellular signaling in response to different ligands for FGFR1-2b based on quantitative phosphoproteomics would reveal whether or not cellular responses are encoded by the identity of the ligand Figure . Two of (3) MS-based organelles proteomics has been recently used to map protein subcellular localization . FGF10 sFibroblast Growth Factor 10-dependent responses range from cell proliferation, migration, and invasion, to multi-organ development, cancer or genetic disease progression. Despite the enormous increase in our understanding of FGF10 signaling and regulation, gaps in our knowledge of FGF10 specificity depending on cellular and extracellular environment still exist. Systems biology approaches, including MS-based quantitative proteomics or high-content microscopy (not discussed here due to space limitations) will contribute to a full understanding of FGF10 signaling. Moving toward personalized treatments for human diseases, this knowledge will be fundamental to develop novel therapies. For instance, recombinant FGF10 or antibodies against FGF10 may be developed to modulate FGF10 signaling depending on cellular context.All authors listed have contributed to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.