Urban agriculture has become a common form of urban land use in European cities linked to multiple environmental, social and economic benefits, as well as to diversified forms . Social acceptance will determine the development of urban agriculture and specific knowledge on citizens’ perception is required in order to set the basis for policy-making and planning. The ecosystem services provided by urban agriculture can be determinant in this process. The goal of this paper is to evaluate the social acceptance and the perceived ecosystem services of urban agriculture in the city of Bologna , as an example of a Southern European city. In particular, we evaluated the preferences for urban land uses, for different typologies of urban agriculture and for the resulting products, the perceived provision of ecosystem services and the willingness to engage in new initiatives. A survey that investigated these topics  was performed on the citizens of Bologna (n = 380) between October and November 2016. Results showed that urban agriculture is widely accepted by the inhabitants of Bologna, particularly regarding vegetable production. Although intensive farming systems were the least preferred forms to be implemented in Bologna, citizens highly accepted a large variety of urban agriculture goods, with preference for those obtained from plants as compared to animal products. The willingness-to-pay for urban food products was mostly the same as for conventional ones, although the participants recognised the social values, proximity and quality of the former. Socio-cultural ecosystem services were perceived as more valuable than environmental ones. Policy-making recommendations can be extracted from the results to facilitate the development of urban agriculture plans and policies. The significance of Urban Agriculture (UA) is grounded on its potential to address two of the major global challenges, those of responding to increasing urbanization and ensuring food security . This ismigrant gardens  emerged as a way to counterbalance the negative side-effects of industrialization and urbanization. Allotment gardens sprouted in lands belonging to the public administration, factories or religious communities. The availability of urban grown food from these gardens was even more important in the first half of the 20th century, especially during the world wars, when towns were isolated from the countryside and food scarcity occurred : Besides exploring the general preferences for urban land use, we asked participants about their acceptance and approval of different UA types. The types in question ranged from low-tech to high-tech solutions of UA, embracing different levels of market orientation, production intensity and location. The results revealed that the participants highly appreciated UA on vacant land, as well as gardens for social purposes and activities taking place in the peri-urban areas. Aquaponics and vertical farms had a higher level of acceptance than growing activities on urban rooftops (such as rooftop gardens or rooftop greenhouses). Nevertheless, all the proposed UA types were broadly accepted and over 50% of the participants highly appreciated them, with intensive agriculture reaching the lowest level of acceptance .To explore participants’ views on different production orientations, we asked about the level of acceptance of various potential UA production orientations ) [Q5]. The results demonstrated that environmentally friendly production orientations, such as resource efficient or organic production were well supported, whereas intensive crop production, the use of GMOs and the intensive production of animals were strongly rejected .rs = -.242, p = .003) and a lower preference for extensive production . Regarding age, older participants preferred intercultural gardens  and gardens on vacant land  more than younger participants. However, a closer look at the effect sizes (rs) indicated that there are only small effects of the sociodemographic attributes on the preferences.The statistical analysis of the sociodemographic attributes of the participants showed that the preference for peri-urban parks and pick-your-own gardens as potential UA typologies was significantly higher in the female group. However, the effect size (r) was very weak for both results according to the Mann-Whitney-U-Test . For the variables income and age, Spearman correlation test showed that the preference for public parks and extensive production was significantly related to income. People in higher income groups had a higher preference for public parks  or even less (1%)– 78% price, on average–. The motrs = -.076, p = .423) and income  on the willingness to pay. Concerning the risks associated with UA products, female participants were less concerned about air contamination  and soil contamination . The higher income groups significantly perceived less risk due to air contamination  than the lower income groups.The data showed no significant influence of gender , age , “Contact with nature and spiritual experience”  and “Urban aesthetics and cultural/art inspiration” . For all three of these the effect size (rs) was small. While age had no significant influence on the evaluation of ES, income influenced the evaluation of “Education and training”  and “Contribution to political realization” , which was lower among the higher income groups.Gender differences in the valuation of ES were found for three ecosystem services. The female level of evaluation was significantly higher than that of the male participants for “Improvement of local climate”  . In partrs = -.195, p = .017), animal management  and noise from vegetable production  than the lower income groups.Concerning the risks that are associated with UA, statistical tests showed that the female participants perceived less risks of noise from vegetable production  and noise from animal production . All calculations indicated a low effect size. While age had no effect on the risk perception, the higher income groups saw significantly less risk from competition with other roof uses (The citizens of Bologna showed a high engagement in UA initiatives and 98% of the respondents were willing to participate . Using t2 (1) = 9.758, p = .002. Based on the odds ratio, the odds that women would participate in education and training on UA was 1.92 times higher than the odds that men would participate. Similar results were found for funding UA projects (x2 (1) = 8.050, p = .005), where the odds that women would fund projects were 2.69 times higher than for the male participants of the survey. However, the effect size was low for both calculations . While income had no effect on the results, age was significant. The younger participants had a significantly lower willingness to participate in UA by producing food than the older age groups . The results indicated a small effect size.There was a significant association between gender and the respondents’ willingness to participate in education and training xDespite the growing importance of UA worldwide in recent years , more thConcerning the participation in UA, respondents were highly willing to commit themselves 98%, , althougThe acceptance of certain urban animal products was also high  and highest in honey and aquaponic products. Urban honey production is currently facing an exponential growth, which is mainly associated with the growing public debates about bees and beekeeping, the diversification of beekeeping opportunities, and new actors and motivations for beekeeping . In a reThe sustainability of food production was also highly appreciated by the survey participants in Bologna, who mainly referred to it with the concepts of resource efficiency and organic production  65,66],,66,65,66http://www.thf-berlin.de/).In this section, we compare the results obtained in the city of Bologna with the previous study of Specht et al.  in BerliPublic parks were the preferred urban green infrastructures in both cities. On the other hand, the inhabitants of Bologna rejected animal-related landscapes and meadows, whereas Berliners refused the high-tech and intensive UA options  the most. Although the implementation of rooftop gardens was the preferred option in Berlin, they showed a lower level of acceptance in the city of Bologna, where forms of intensive UA were the least valued options. On the contrary, the citizens from both cities agreed in the preference for resource-oriented and organic farming and in the rejection of GMOs and intensive livestock production, highlighting the prevalent environmentally-friendly discourse of UA in Europe.Concerning the market options for UA products, Berliners showed a lower willingness-to-buy (22–50%) than the Bolognese (69–92%). Vegetables were the preferred product in Berlin, while seedlings and specialties were prioritized in Bologna. Nevertheless, some animal products  were the least preferred options in both cities, underlining the low acceptance of animal production in UA. While the most important requirement for UA products in Berlin were the production protocol  and the high quality, Bologna’s inhabitants appreciated more the social benefits and the local nature of UA. In both cities, the food safety risk due to urban pollution was considered significant.As for the contributions of UA to the city and its citizens, the improvement of the urban image was highlighted in both studies. While Berliners most valued the potential environmental improvements resulting from UA, Bologna citizens placed in the foreground the social benefits associated with the new uses of urban spaces . These results suggest that while the environmentally-friendly discourse dominates in Berlin, the socially-oriented UA is also in the spotlight in Bologna.The evaluation of the ES of UA by the citizens of Bologna showed some similarities and some divergences when compared to a recent study of urban gardens in Barcelona (Spain) . First, However, several differences were found in the evaluation of environmental ES. While the provisioning services  were the least valued environmental ES in Barcelona, they were the highest ranked by the citizens of Bologna. However, while food provision was more valued than medicinal and aromatic plants provision by the practitioners of Barcelona gardens, the opposite was observed in the Bologna study. In a similar vein, prevention of soil erosion and maintenance of soil fertility was the most valued environmental ES in the Barcelona study, while placed in the worse ranking positions by Bologna citizens. Apart from these divergences, pollination was one of the most valued environmental ES in both systems. Overall, socio-cultural ES were the most valued among UA initiatives although the ranking of ES, particularly for environmental ES, could be influenced by the type of users  and the local context.Comparing the results presented herein with the evaluation of the ES provided by home gardens in rural areas , it was The statistical assessment unveiled some socio-demographic factors that can be key for future policy-making. Social acceptance differences regarding gender, age and income level of the participant were significant for some of the aspects evaluated in this survey. In particular, gender, age and income can determine the preference for certain typologies of UA. Furthermore, gender and income level were also relevant in the evaluation of ES. These results suggested that UA planning shall include a participatory process where different citizens can provide inputs in the design and implementation of UA initiatives in Bologna, with the aim of encompassing these diverse preferences and perceived impacts of UA. On the other hand, the perception of risks and the type of activities that the participants would be willing to join showed also correlation with the socio-demographic characteristics. Thus, communication and awareness campaigns from local authorities aiming at affecting the social acceptance of UA may use these results to target certain population groups and topics.This study was based on a rigorous sampling approach. Nevertheless, by focusing on Bologna as a case study, the results were not representative of the entire population of Italy or even Southern Europe. It would be opportune to repeat the survey using a larger sample from all over Italy or even beyond, to include different local realities and socio-demographic differences. The repetition of the survey questionnaire, which was previously used in Berlin (Germany), allowed to draw some conclusions on common observations in both case studies. However, while the same questionnaire was applied in both cities, the sampling method in Berlin was based on a less structured, non-probability sampling approach and was characterized by a disproportionate number of urban, younger (20 to 40 years old) participants. This created a weakness with regards the comparability of the results. Nonetheless, both existing studies showed some common results and future studies on consumers’ preferences and acceptance can build on these results to generate hypotheses. As pointed out by Specht et al. , social This paper contributes to an increased knowledge of the perception and acceptance of UA in Europe. We surveyed the inhabitants of Bologna, a landmark city in Southern Europe regarding the evolution and diversity of UA projects in the last decades. In particular, we evaluated the social acceptance in a comprehensive way, including the potential implementation of UA forms and the potential acceptance of the resulting products by identifying the preferences for urban land uses, UA typologies as well as UA products, investigating the perceived provision of ES by the citizens of Bologna and evaluating their willingness to engage in new UA initiatives. The outcomes resulted in the evaluation of new aspects of UA and with new data, which was compared and discussed with previous results in a broader European context.The inhabitants of Bologna showed a wide acceptance of UA uses and products in their city, indicating that new initiatives might be positively received. While UA was conceptualized as multifunctional and diverse, animal production–including meadows -, rooftop agriculture and intensive farming were the least preferred. This result was in line with the low acceptance of intensive techniques and GMOs. As potential consumers, UA products were largely accepted although only a third of the population demonstrated a willingness-to-pay higher prices for UA products over conventional ones. Social ecosystem services were the most valued, as in other similar studies for urban and home gardens. The provision of food and medicinal plants were highlighted over the other environmental ES tied to UA.Comparing Berlin and Bologna, specific trends regarding the acceptance of UA in Europe were observed. A diverse and multifunctional UA is appreciated in Europe, demonstrating the importance of specific UA policies for its development. The discourse of environmentally-friendly and resource efficiency was prevalent in both cities, although the citizens of Bologna also stressed the social nature and values of UA. Therefore, two main narratives were observed in European cities which could be addressed by policy-makers. In both cases, participants showed a broad acceptance of UA typologies and products, demonstrating the potential market success and diversification options of UA. Further research on other geographical areas might provide a more global vision of the acceptance of UA.The results highlighted some policy-making recommendations for local authorities, which could be extended to similar European cities. First, more communication and dissemination activities regarding UA are needed, as the population of Bologna showed a lower awareness on the topic. Second, the citizens of Bologna showed a high willingness to engage in new UA projects, as both consumers and users. Thus, local authorities could promote not only the development of community-oriented UA activities but also the design of specific policies and norms for the development of UA businesses which will support entrepreneurs in overcoming current barriers and protect against perceived risks. Finally, the statistical assessment highlighted some differences between target groups which can set the basis for more precise policy-making regarding UA.S1 File(DOC)Click here for additional data file.S2 File(DOC)Click here for additional data file.S3 File(XLSX)Click here for additional data file.


HTLV-1 infects over 20 million people worldwide and causes a progressive neuroinflammatory disorder in a subset of infected individuals called HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The detection of HTLV-1 specific T cells in the cerebrospinal fluid (CSF) suggests this disease is immunopathologically mediated and that it may be driven by viral antigens. Exosomes are microvesicles originating from the endosomal compartment that are shed into the extracellular space by various cell types. It is now understood that several viruses take advantage of this mode of intercellular communication for packaging of viral components as well. We sought to understand if this is the case in HTLV-1 infection, and specifically if HTLV-1 proteins can be found in the CSF of HAM/TSP patients where we know free virus is absent, and furthermore, if exosomes containing HTLV-1 Tax have functional consequences.p = 0.0001). We were able to observe HAM/TSP patient CSF (12/20) containing Tax+ exosomes but not in HTLV-1 seronegative MS donors (0/5), despite the absence of viral detection in the CSF supernatant (Fisher exact test p = 0.0391). Furthermore, exosomes cultivated from HAM/TSP PBMCs were capable of sensitizing target cells for HTLV-1 specific CTL lysis.Exosomes that were positive for HTLV-1 Tax by Western blot were isolated from HAM/TSP patient PBMCs (25/36) in ex vivo cultures by trapping exosomes from culture supernatants. HTLV-1 seronegative PBMCs did not have exosomes with Tax (0/12),  contains supplementary material, which is available to authorized users. Infecti T cells . HAM/TSP T cells , 6. Whilgression , 8, HTLVgression , 10.Similar to other viruses that establish persistent infections, HTLV-1 is adept at replicating while evading the immune system; it tightly controls the expression of viral genes in cells to escape potential immune recognition , 12. How+CD25+ T cells, including Tregs, also are the primary reservoir of HTLV-1 in infected individuals [Exosomes are unilamellar extracellular vesicles that originate from the endosomal compartment in all cells . Exosomeividuals , 36. As ividuals , 38, ass+CD25+ T cells, HTLV-1 is capable of infecting several other cell types of the immune system, including dendritic cells, CD8+ T cells, and monocytes [In addition to CD4onocytes –41. As eonocytes , 42, an onocytes –46. HTLVonocytes , howeveronocytes , 49. In onocytes .® particle-isolated exosomes from HAM/TSP patient PBMCs contained HTLV-1 Tax protein. Importantly, HTLV-1 Tax protein was found in exosomes directly isolated from HAM/TSP patient cerebrospinal fluid (CSF) supernatant previously found to be negative for free HTLV-1 virus, indicating the presence of HTLV-1 viral antigen in the non-cellular fraction. Furthermore, HAM/TSP exosomes could sensitize uninfected target cells for lysis by HTLV-1 specific cytotoxic T lymphocytes (CTLs). Collectively, the incorporation of HTLV-1 products into exosomes may represent a mechanism by which viral antigens could be transported to the CNS and be targeted by virus-specific immune responses associated with the immunopathogenesis of HAM/TSP. In addition, the detection of exosomes containing viral antigen may be a biomarker of viral exposure particularly in sites  that are absent of cell-free virus.Here we demonstrated that Nanotrap® (NT) particles (NT80) has been shown in capturing viral protein-containing exosomes from HTLV-1 infected cell line supernatants, while other particles were shown to be more specific for viral capture (NT86) [® technology. NT80 + 82 particles pulled down exosomes from both cell lines, regardless of HTLV-1 infection status, as demonstrated by the presence of Alix, CD63, and Actin  , 51. Howe (NT86) . Therefo® particles can be used to concentrate exosomes to equal or greater levels than those isolated from 10 times the same starting material by conventional ultracentrifugation.Next, exosomes nanotrapped from several T cell lines were assessed for CD81, another exosomal marker. As shown in Fig. ® particles in exosomal capture, samples were assessed for acetylcholinesterase (AchE) activity before and after NT80 + 82 concentration [To further characterize the efficiency of Nanotrapntration , 55. Pri9 109 nm vesicles along with 5 × 108 311, 477 and 583 nm vesicles (left panel). NTs alone showed a population of particles sized 215–600 nm in size, as expected (middle panel). However, the resultant nanotrapped exosomes and NTs after pulldown were denoted by peaks from 215 to 600 nm (right panel). Additionally, a population of ~ 3.5 × 109 145 nm vesicles had been pulled from the initial tissue culture supernatant. Analysis of the vesicles from pre- and post-NT supernatants showed that small (< 150 nm) vesicles were reduced by nanotrapping, while medium (200–300 nm) and large (> 300 nm) vesicles increased after nanotrapped vesicles were removed from the media. This may indicate the selective nanotrapping of small vesicles (exosomes). Collectively, these results support nanotrapping with NT80 + 82 particles as an efficient method for exosome isolation.Samples were additionally analyzed by nanoFACS, a technique which quantifies vesicles by forward and side scatter  . As seenAs shown in Fig. 3H-thymidine uptake assay. Maximum proliferation was seen after 5 days in culture, as demonstrated by peak 3H-thymidine uptake by representative HAM/TSP patients compared to an ND  as a potential source of exosomes in HAM/TSP, since HTLV-1 primarily infects immune cells . Exosome+CD25+) are both prolific producers of exosomes and a primary target for HTLV-1 infection [+CD25+ and CD8+CD25+ T cell subsets, implicating Treg cells as a predominant producer of exosomes in activated PBMC T cells.HAM/TSP patients are associated with an activated immune response, and here we showed that PBMCs from HAM/TSP patients produce exosomes during active proliferation Fig. e, f. Givnfection , 34, 36,+ T cells [− activated ND PBMCs, endogenously stimulated HAM/TSP CD4+CD25+ produce more exosomes than CD4+CD25− cells  are likely one of the main culprits in the generation of exosomes during both T cell activation and HTLV-1 infection in HAM/TSP patients.As HTLV-1 in HAM/TSP patients is known to predominantly infect and activate CD4 T cells , we nextlls Fig. h. Howeve+) exosomes were produced from cultured PBMC of HTLV-1 seronegative controls (0/12) .Previously, it has been shown that HTLV-1 infected cell lines produce exosomes that contain Tax protein . Likewis+ as detectable by Western blot . Interestingly, the detection of exosomes containing Tax from HAM/TSP CSF did not correlate with the total numbers of exosomes isolated (data not shown). As it is well established that HTLV-1 is a cell-associated virus and restricted to cells in the CSF [HAM/TSP is a neuropathologically mediated disorder and therefore the detection of exosomes in the CSF is of significant interest. Cell-free CSF was obtained from 4 HAM/TSP patients and 5 HTLV-1 seronegative multiple sclerosis (MS) control patients and directly trapped by NT80 + 82 to be analyzed by Western blot for the presence of HTLV-1 Tax. Measurable, but variable exosome levels were detected from both HAM/TSP and MS CSF samples as quantified by CD81 ELISA reactivity  with Tax+ exosomes in the CSF were also positive for Tax in exosomes from cultured ex vivo PBMCs (data not shown). For CSF samples from HAM/TSP patients that were negative for Tax in the CSF, 3/8 were also negative in the exosomes isolated from ex vivo PBMCs (data not shown). These results suggest that Tax+ exosomes in the CSF could reflect levels of systemic infection.Since subsets of HAM/TSP patient PBMCs Fig. b and CSF+ exosomes were immunologically functional. Therefore, we investigated the potential of exosomes produced by HAM/TSP PBMCs to sensitize targets for HTLV-1 Tax-specific CTL lysis. HTLV-1 Tax is highly immunogenic and HTLV-1 Tax-specific CTLs have been detected in brain and spinal cord from HAM/TSP biopsy and autopsy cases [− exosomes from representative NDs (n = 4) did not sensitize targets for CTL-mediated lysis  of tissue culture supernatant yielded ~ 18% specific lysis, while nanotrapping from 500 μL (green closed square) yielded 15% specific lysis of targets and exosomes isolated from 250 μL (green closed triangle) of starting tissue culture supernatant yielded 10% specific lysis of targets at an E:T of 20:1. Each sample concentration showed decreased specific lysis as lower E:T ratios. While most HAM/TSP PBMC derived exosomes were able to sensitize targets for CTL response, the magnitude of this response varied among patients  were used and failed to isolate exosomes that could sensitize targets for CTL lysis , a subset known to be dysfunctional in HAM/TSP [While HTLV-1 proviral DNA levels are significantly increased in T cells in the CSF of HAM/TSP patients compared to peripheral blood , 76, no lls Fig. h and CD8 HAM/TSP .Fig. 7Hy+ exosome detection was apparent in HAM/TSP CSF (12/20), we could not find evidence of Tax+ exosomes in a small group of asymptomatic carrier (AC) CSF samples (data not shown) . Collectively, these observations suggest that the detection of Tax in exosomes isolated from the CSF could serve as an additional biomarker of progression to HAM/TSP. The lack of Tax protein present in the exosomes from all HAM/TSP CSF samples probably reflects the diversity in the HAM/TSP patient population and the biology of the disease.While Tax+ exosomes can be detected in the CSF of HAM/TSP patients suggests exosomal transference of HTLV-1 antigens to antigen presenting cells (APCs) may also occur in the CNS, and could play a role in perpetuating neuroinflammation in HAM/TSP through damage to various CNS resident cells such as astrocytes, microglia or oligodendrocytes  for whom there is no detectable virus [The observations in this study on the detection of HTLV-1 viral antigens in exosomes found in virus-free CSF supernatant has implications for other virally associated neuroinflammatory diseases. For example, Ebola virus has been shown to be associated with long-term neurologic sequelae even though the virus is thought to have been cleared , 82, andle virus , indicatle virus , 85. Thele virus , 87. ColThe understanding of exosomes and their contribution to human disease is a quickly expanding field. Evidence suggests that exosomes play a large role in immunity and infection. Some viruses have been shown to alter exosome cargo with viral components in another mechanism of immune evasion. Here we show that HTLV-1 virus incorporates HTLV-1 Tax, an important viral regulatory protein, into exosomes found in the CSF and produced by HTLV-1 infected PBMCs. These exosomes could sensitize cells for lysis by HTLV-1 specific CTLs which are indicated in HAM/TSP neuropathology. This suggests that exosomes may have a functional role in CNS damage in HAM/TSP patients.g for 2 h and filtered through a 0.22 μm MCE filter , after which it was considered exosome-free. Exo-free CRPMI consisted of RPMI 1640 supplemented with 10% Exo-free FBS, 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, and 2 mM l-glutamine.HTLV-1 infected cell lines HUT102, MT-2 and C8166 and the uninfected Jurkat cell line were maintained in exosome-free (Exo-free) complete RPMI. Specifically, FBS was spun at > 90,000×6 cells per well in a 12-well plate and incubated at 37 °C for 5 days. ND PBMCs were maintained in culture with the addition of 100 IU/mL recombinant human (rh) IL-2. Culture supernatant was collected and then spun at 1300 rpm for 10 min to remove cells. Spun supernatant was then pushed through a 0.22 μm MCE filter (EMD Millipore) to remove large debris, apoptotic bodies and extracellular vesicles > 220 nm. Cerebrospinal fluid (CSF) was obtained through lumbar puncture of study participants by neurologist and nurse practitioners of the Neuroimmunology Clinal group. CSF was spun at 1300 rpm for 10 min to remove cells and supernatant was stored in 1 mL aliquots at − 80 °C prior to use.A total of 36 HAM/TSP, 10 ND, and 2 HTLV-II patient PBMC samples were used for ex vivo incubation and later exosome isolation. PBMCs were isolated by Ficoll-Hypaque  centrifugation, and were cryopreserved in liquid nitrogen prior to use. All NDs were noted to be healthy and HTLV-1 negative. Briefly, PBMCs were placed in Exo-free CRPMI at 8 × 10® particles NT80 (Ceres #CN1030) and NT82 (Ceres #CN2010) were provided by Ceres Nanosciences, Inc.  and have previously been shown to concentrate exosomes from media [+ or Mg+) for a final 30% slurry . Thirty microliters of 30% NT slurry was then added to 1 mL of ex vivo PBMC supernatant and rotated overnight at 4 °C or at room temperature for 1 h. After trapping, samples were spun at 13,000 rpm for 5 min and then washed with 500 μL DEPC H20. Exosome control NT86 (ctrl NT) was similarly placed in PBS (w/o Ca+ or Mg+) for a final 30% slurry. When working with CSF, samples were first diluted 1:1 in PBS (w/o Ca+ or Mg+) prior to nanotrapping.Nanotrapom media . Each waHTLV-1 Tax, Alix, CD63, and Actin were detected from nanotrapped exosomes by Western blot. Briefly, after washing, samples were resuspended in 100 μM Tris–HCl, 8 μM urea, 1% triton. After the addition of 4× LDS and DTT, samples were heated at 95 °C for 6 min, with a vigorous vortex for 5–10 s every 2 min. After heating, samples were run on a 4–12% Novex Bis–Tris 1.0 mm gel  and then transferred in an X-blot module onto a 0.45 μm nitrocellulose membrane. After blocking with 3% BSA in TBS, membranes were probed with anti-Tax , anti-CD63 or anti-Alix  overnight at 4 °C. For imaging, anti-ms HRP secondary antibody  was applied at 1:5000 or goat anti-ms IR 680RD secondary  was used for CSF samples. For detection of Actin, blots were stripped for 5–10 min with Restore PLUS Western Blot Stripping Buffer (ThemoFisher Scientific), reblocked and reprobed with Actin (Santa Cruz Biotech) at 1:100 overnight at 4 °C.® Acetylcholine/Acetylcholine Esterase Activity Assay Kit  was used following the manufacturer’s instructions. Briefly, a 1× running buffer negative control (20 mL of H2O and 5 mL of 5× reaction buffer) and two positive controls, one consisting of acetylcholinesterase and one consisting of hydrogen peroxide, were made and plated on a 96-well plate. Exosomes were treated and fluorescence of acetylcholinesterase activity was measured with a GLOMAX multidetection system  every 15 min for 1 h to find optimal activity. Changes in absorption were monitored at 412 nm during incubation period at 37 °C [AchE activity of exosomes after nanotrapping was carried out as described in detail previously  with briat 37 °C .For CD81 and HTLV-1 Tax, nanotrapped exosomes were resuspended in 1× TBS for assessment by MESO Enzyme Linked Immunosorbent Assay (ELISA) . Briefly, samples were freeze-thawed for 4 cycles of 5 min each and then blotted onto a MESO Sector Imager 96-well high-bind plate (MSD Technologies) in duplicate wells overnight. The plate was then blocked with blocker A for 2 h, washed with PBS ×2 and then probed with anti-CD81 (Santa Cruz Biotech) or anti-HTLV-1 Tax (Lt4) for 1 h, shaking. After a repeat wash, the plate was probed with goat anti-ms sulfa secondary Ab . After a final wash, MSD Read buffer was added at 1:2 dilution in Ultra Pure Water and the plate read using an MSD Sector Imager (courtesy of Dr. Avi Nath).5 cells/well in 10% Exo-Free CRPMI and allowed to proliferate for 3–5 days in culture at 37 °C. At 3, 4, or 5 days, plates were removed from culture and 50 μL of tritium solution was added to each well. Cells were incubated for 4 h at 37 °C after which they were washed and harvested onto a film mat. Five milliliters of scintillation fluid was added after which thymidine incorporation was measured using a β plate counter .Thawed ND or HAM/TSP PBMCs were placed on 96-well plate in triplicate at 3 × 107 PBMCs were stained in FACS buffer  with 10 μL/1 × 107 cells of anti-CD4 APC, anti-CD8 FITC and anti-CD25 PE . Cells were then sorted into CD4+CD25−, CD4+CD25+, CD8+CD25−, CD8+CD25+ T cell subsets on a BD FACS Aria flow cytometer. After sorting, cells were briefly placed in 20% FBS, 2× antimycobacterial, antibacterial (ThermoFisher Scientific) RPMI 2 mM l-glutamine. Cells were then spun at 1300 rpm for 10 min and resuspended in Exo-free CRPMI supplemented with 100 IU/mL hrIL-2 and plated at 7.5 × 105 cells per well in a 12-well plate for 5 days at 37 °C. For HAM/TSP PBMCs, no exogenous stimulation was provided. Endogenous activation of HAM/TSP PBMC was achieved by incubation of PBMCs for 18–24 h prior to FACS sorting as described above.To understand which T cell subsets were responsible for exosomes found in PBMC culture, sorting was performed on thawed or previously activated ND PBMCs. Activation occurred with addition of 100 ng/mL anti-CD3  and 300 IU/mL rhIL-2 24 h prior to sorting. At least 5 × 10l-glutamine) for up to 1 month. Clones were tested for efficiency of target killing prior to use. Targets B cells were pulsed for 1 h at 37 °C with Tax peptide 11-19 or nanotrapped exosomes from tissue culture supernatant of 11 separate HAM/TSP PBMC ex vivo cultures and 4 ND PBMC cultures. One HAM/TSP supernatant was nanotrapped with NT86 as a negative control. Anti-HLA Class I ab , Oxford UK) was added to CTLs after co-culture with targets at 5 μg/mL. The CTL Europium cytotoxity release assay was performed as outlined in the DELFIA protocol . Specific lysis was calculated as a percentage:HTLV-1 Tax specific CTL line and target B cell line were generated from an HLA A0201 HAM/TSP patient and cryopreserved prior to use. As described previously , CTLs weTissue culture supernatant or resuspended nanotrapped exosomes were pushed through a syringe at a total volume of 300 μL into the chamber of the NanoSight LM10 instrument  for extracellular vesicle visualization and quantification. Briefly, a laser beam is passed through the chamber containing the sample and light scattering and Brownian motion is quantified as a vesicle size distribution and quantity. For exosome visualization by flow cytometry (nanoFACS), samples were loaded into a Beckman Coulter MoFlo XDP cell sorter  as described extensively by Danielson et al. .t-tests were run to establish difference between groups and significance was considered anything with a p-value < 0.05. The two-tailed Fisher exact test was used to assess differences between groups on parametric questions (Tax+ or Tax− exosomes) using GraphPad QuickCalcs software. Statistical analyses of 2 × 2 contingency tables were performed using GraphPad QuickCalcs software with two-tailed Fisher exact tests. p-values < 0.05 were considered significant.Bar graphs and scatter plots were generated in GraphPad Prism 7.0 . Two-tailed unpaired Additional file 1: Figure S1. Characterization of Nanotrapped (NT80 + 82) exosomes by Nanosight. Tissue culture supernatants from HTLV-1 infected cells prior to nanotrapping (Pre-NT) and after nanotrapping (+NT) were analyzed for size and concentration by Nanosight. Nanotrapped exosomes (+NT) were resuspended in 300 μL for imaging. NT80 + 82 particles alone (NT Alone) were also analyzed. Pre-nanotrapped (Pre-NT) and post-nanotrapped (post NT) vesicle distributions were likewise analyzed according to size and shown in the right panel inset.


The host-pathogen interface is a crucial battleground during bacterial infection in which host defenses are met with an array of bacterial counter-mechanisms whereby the invader aims to make the host environment more favorable to survival and dissemination. Interestingly, the eukaryotic Wnt signaling pathway has emerged as a key player in the host and pathogen tug-of-war. Although studied for decades as a regulator of embryogenesis, stem cell maintenance, bone formation, and organogenesis, Wnt signaling has recently been shown to control processes related to bacterial infection in the human host. Wnt signaling pathways contribute to cell cycle control, cytoskeleton reorganization during phagocytosis and cell migration, autophagy, apoptosis, and a number of inflammation-related events. Unsurprisingly, bacterial pathogens have evolved strategies to manipulate these Wnt-associated processes in order to enhance infection and survival within the human host. In this review, we examine the different ways human bacterial pathogens with distinct host cell tropisms and lifestyles exploit Wnt signaling for infection and address the potential of harnessing Wnt-related mechanisms to combat infectious disease. The innate immune response is the first, and in many successful cases, the primary barrier between bacterial invader and human host. Fine-tuned through co-evolution with microbial insults and largely evolutionarily conserved across the Metazoa, the innate immune system specializes in both pathogen recognition and the formation of a rapid response . At the Ehrlichia chaffeensis utilizes a type 1 secretion system (T1SS) effector to activate Notch signaling which indirectly downregulates PU.1, a transcriptional activator of TLR2 and 4  and cell proliferation .Mounting research within the last two decades has demonstrated that the conserved eukaryotic signaling pathway Wnt is a significant part of the interplay between the human host and both extracellular and intracellular bacterial pathogens. The discovery of the Wnt pathway began with the identification of the murine oncogene elopment , 11. Latelopment . Wnt proelopment . Canonicelopment  14). In. Inint-12+ pathway and the planar cell polarity (Wnt/PCP) pathway. In the Wnt/Ca2+ pathway, Wnt ligands signal through Fzd and the coreceptor receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2) to induce Dvl-dependent phospholipase C (PLC) cleavage of phosphatidylinositol 4,5-biphosphate (PIP2), producing inositol triphosphate (IP3) and diacyl glycerol (DAG) (3 acts on Ca2+ channels at the endoplasmic reticulum resulting in a wave of cytosolic Ca2+ that drives protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CAMKII) activity. This controls nuclear translocation of nuclear factor of activated T cells (NFAT) for target gene transcription and actin polymerization through the Rho GTPase CDC42. NFAT target genes have been most thoroughly researched in the context of osteoclast formation and T cell regulation. In the transcription-independent Wnt/PCP pathway, cell polarity and migration are regulated through the direct interaction of Dvl and Dvl-associated activator of morphogenesis (DAAM1), G protein activation of the small GTPase Rac, and Dvl activation of phosphoinositide 3-kinase (PI3K)   15). IP. IP2+ pae (PI3K)  16). No. No2+ paPathogen manipulation of Wnt signaling as a mechanism of innate immune subversion takes advantage of three main outcomes of the signal cascade: cell fate determination (including maintenance of epithelia and endothelia), anti-inflammatory effects, and phagolysosome formation. In this review, we highlight mechanisms of Wnt pathway manipulation by several extracellular, and obligate intracellular pathogens, with a specific focus on the virulence factors involved and the consequences on innate immune subversion. Salmonella enterica causes typhoid fever or non-typhoidal salmonellosis in humans. The bacteria are commonly foodborne pathogens but can also be transmitted fecal-orally  that together encode the T3SS. A strain lacking Spi2 is unable to induce vascular permeability, indicating this pathogenic mechanism is Spi2-dependent. This model demonstrates how salmonellae permeabilize endothelial layers utilizing T3SS factors without directly manipulating cell-cell contacts, a mechanism that is employed by other endothelial pathogens such as Rickettsia. Whether this implicates activation of Wnt signaling as a potential therapeutic for S. enterica infection, and whether specific S. enterica pathogenicity island effectors are responsible for Wnt pathway inhibition remains to be investigated.Recently, another pathogenic mechanism was discovered by which salmonellae inhibit canonical Wnt signaling at the gut-vascular barrier (GVB), a system of tight and adherens junctions in the capillaries underlying the gut epithelium that functions as a size-selective barrier to molecules traversing the gut barrier . Similarmination . In the Salmonella colitis mouse model have demonstrated that S. Typhimurium is able to stimulate Wnt signaling in intestinal stem cells in contrast to the inhibitory mechanism deployed in the epithelium and endothelium. The T3SS effector AvrA, a deubiquitinase, has been shown to activate canonical Wnt signaling in intestinal stem cells through deubiquitination and subsequent stabilization of β-catenin . It. ItR. cothe cell . A correR. conorii infection relative to uninfected controls from 5 to 180 hpi . The pathogen is an obligately intracellular bacterium and infects mononuclear phagocytes including monocytes. E. chaffeensis enters the host cell through phagocytosis and replicates within a membrane-bound vacuole to form a microcolony known as a morula , 32E. che morula , 89, 90. control –94. E. cis genes . Accordi vacuole . β-caten vacuole .E. chaffeensis maintains pathway activity throughout infection of the monocyte , a severe inflammatory disease of the colon that arises from opportunistic infection by C. difficile typically following antibiotic-induced disturbances in the normal flora population proportions (C. difficile pathogenesis is highly dependent on the expression of a family of exotoxins that includes toxin A (TcdA) and toxin B (TcdB)  . The toxal cells . These tal cells . Howevern humans .C. difficile is known to inhibit cell proliferation within the intestinal epithelium, a process typically regulated by canonical Wnt signaling, through the direct inhibitory effect of TcdA and TcdB on the Wnt pathway . T. TC. dife arrest . TcdA-mee arrest .C. difficile infection . T. TH. pyl strains . CagA innfection . In a manfection . While tnfection . This coH. pylori infection in addition to manipulation of the signaling pathway through interference with adherens junctions. LRP5/6 and Dvl are both phosphorylated in a Cag-independent, T4SS-dependent manner in a gastric epithelial infection model  systems to sense bacterial population density and regulate biofilm development, mammalian host defense, host-microbe interactions, virulence and metabolite acquisition  was shown to promote bacterial clearance. LiCl promotes canonical Wnt pathway activity through the inhibition of GSK3β . In. InP. aeresponse . β-cateneratitis . Overexpof GSK3β . Variousof GSK3β . For exa tissues . During e cornea . In corrP. aeruginosa infection. P. aeruginosa contains two quorum-sensing systems which regulate specific signaling molecules that contribute to infection and survival (P. aeruginosa acyl-homoserine lactone (PAI) quorum-sensing molecule  is an extraintestinal pathogenic isolate of Escherichia coli and the causative agent of 40% of hospital- and 80% of community-acquired urinary tract infections  and enterohaemorrhagic E. coli (EHEC) (C. rodentium are members of the attaching and effacing (A/E) family of bacterial pathogens due to the destructive effect of colonization of the intestinal epithelium and enterocytes  –152. EPEerocytes , 151. ThIL6 expression . HlyA is inserted into epithelial and macrophage membranes and triggers rapid degradation of various host proteins involved in the proinflammatory response and cell-cell and cell-matrix interactions, including β-catenin . HlyA mepression . This stSeveral gaps in knowledge remain in reference to the role of Wnt signaling in subversion of innate immunity during UPEC infection. While exfoliation and proliferation are mechanisms of host response to UPEC infection, this response is beneficial for establishing a niche for persistent infection , 154. IdSalmonella, Chlamydia, Rickettsia, and Mycobacteria demonstrates canonical pathway activation contributes to an anti-inflammatory state which mediates immunosuppression and may prolong infection. The former two pathogens also appear to stimulate canonical Wnt signaling to prolong host cell survival by inducing either proliferation or differentiation of the host cell. Ehrlichia is unique in that studies have shown the bacterium activates non-canonical pathways which govern cytoskeletal reorganization and autophagy regulation, permitting phagocytosis of the bacterium without destruction by the lysosome. Inhibition of the canonical Wnt pathway by Salmonella and C. difficile occurs in intestinal epithelium and the intestinal capillary endothelium for the former and in the colonic epithelium for the latter, and contributes to pathogen manipulation of tissue barriers, facilitating dissemination into the bloodstream. In the case of Salmonella, inhibition of Wnt signaling may also lead to an inflammatory response that recruits target cells for Salmonella and promotes dissemination. Studies have shown that C. difficile and P. aeruginosa utilize multiple effector molecules to interfere with canonical Wnt signaling activity of the surrounding tissue during infection. In both cases, viability of the cell within the infected tissue is reduced which may contribute to nutrient accumulation or dissemination for the bacteria. E. coli and H. pylori, contrastingly, activate canonical and non-canonical Wnt signaling from their extracellular niches, resulting in increased proliferation or differentiation of cells in the surrounding epithelium. Although evidence is yet to demonstrate a clear phenotype, it is likely that pathway activation also contributes to an anti-inflammatory state in the bacterial replicative niche due to reciprocal regulation of the transcription factors NF-κB and β-catenin.Pathogens' respective niches and modes of dissemination shape how they avoid innate immune pressure during infection. Wnt signaling is a host cell pathway targeted by both obligately intracellular and extracellular bacterial pathogens alike, demonstrating the breadth of the pathway's control over eukaryotic cellular processes. A consistent theme among the intracellular pathogens discussed in this review is activation of the pathway in the host cell. Evidence from S. enterica, C. pneumoniae, E. chaffeensis, E. coli, and H. pylori; lectins and quorum-sensing molecules of P. aeruginosa; two-component regulatory systems of S. enterica; and likely many more unknown factors. Shared mechanisms involve disassembly of adherens junctions as is the case for C. trachomatis, R. rickettsii, H. pylori, and P. aeruginosa which may be a means for increasing the amount of cytosolic β-catenin available to activate the Wnt response element, or a means of inactivating the protein and facilitating its degradation. Cpn1027 of C. pneumoniae and TcdB of C. difficile both inactivate pathway components through direct binding. Such may be the case for E. chaffeensis TRP32, 47, and 120, as many host-bacterial interactions between negative regulators of Wnt signaling and the TRPs have been identified for which mechanisms have yet to be described. Pathogens also deploy enzymes during infection to manipulate Wnt signaling components at the post-translation modification level, as is the case with C. difficile TcdA and S. enterica AvrA and SopB. Research identifying Wnt pathway regulatory mechanisms and foreign molecules that can interfere with the pathway has applications ranging from research tools to potential therapeutics.Studies have implicated a range of bacterial effectors with the capability to manipulate canonical and non-canonical Wnt signaling, including T1,3,4SS effector proteins of As research detailing mechanisms of pathogenesis and bacteria-host interaction accumulates, our knowledge of how various cellular mechanisms participate in innate immune signaling expands. Wnt signaling regulates stem cell renewal, cell proliferation, and cellular morphology, but the ability of pathogens to usurp these processes to establish infection demonstrates that Wnt signaling is also a target for bacterial immunoevasion strategies. By drawing a parallel between pathway dysregulation during infection and dysregulation in “classical” diseases of Wnt signaling, we can identify targetable pathway components for drug intervention of infectious disease. One such strategy involves the use of the existing armaments of small molecule inhibitors and activators that target various components of canonical Wnt signaling thereby overriding the pathogen reprogramming strategy. Another approach is the use of antivirulence therapy to target the pathogen-host molecular interactions that are beneficial to bacterial virulence, thereby weakening the pathogen through impairing its ability to manipulate the Wnt pathway.Streptococcus and Porphyromonas gingivalis and demonstrate efficacy in reducing bacterial burden or excessive inflammation (Salmonella perturbation of the GVB represents a case in point—stimulation of Wnt signaling reverses infection-induced endothelial weakening (Multiple small molecules have been identified that modulate Wnt signaling activity through activation or inhibition of pathway components or regulators . Use of ammation –157. GSKammation . Modulatammation –161. Theeakening . The effeakening .C. difficile infection has been shown to be controlled by small molecules and monoclonal antibodies that target critical enzymatic or protein-protein interaction-mediating domains within TcdB (E. chaffeensis infection, as surface TRPs including TRP120 mediate phagocytosis of the bacterium through stimulation of Wnt pathway activity. As further research identifies the molecular determinants of such pathological events, interference with TRP-Wnt receptor complex could reduce the ability of E. chaffeensis to establish infection within monocytes. P. aeruginosa utilizes surface protein LecB to extracellularly modulate β-catenin activity. Antivirulence drugs in preclinical development that target LecB demonstrate how this protein can be manipulated to reduce infection, and suggest that the efficacy of these drugs is, at least in part, due to the disruption of P. aeruginosa modulation of Wnt signaling (Studies of how pathogens interact with the host signalosome allows us to exploit this knowledge to target crucial interactions between bacterial and host factors that enable bacterial virulence . Antivirhin TcdB , 165. Thignaling .The era of Wnt signaling research has shifted from understanding the basic molecular biology of the pathway to identifying how the pathway is manipulated by infectious agents. The ultimate goal is to use this knowledge to repurpose or develop novel therapeutics for infectious disease that are relevant in the age of antibiotic resistance. Pathogens have coevolved with the human host and are master manipulators of signaling pathways that have been in place throughout the evolution of metazoans. Understanding the interactions that dictate how pathogens can usurp the Wnt signal cascade to benefit their survival and dissemination facilitates the development of counter-defense strategies that enable the host innate immune system to control and clear infection.P. aeruginosa and E. coli which were contributed by LP. JW performed artwork. All authors participated in editing of the final draft.MR and JM conceived the work. MR gathered information and contributed all sections except for 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.


In vivo, an intra-articular injection of shRunx2 lentivirus followed with Jagged1/DAPT treatments was performed in rats. MMP-13 expression in articular cartilage was detected by immunohistochemistry. Finally, MMP-13 expression changes were analyzed in chondrocytes under IL-1β stimulation. Our findings showed that, CCK-8 staining and EdU labeling revealed suppression of cell proliferation by Notch signaling activation after Jagged-1 treatment in chondrocytes. Promoted differentiation was also observed, characterized by increased expressions of Col X, MMP-13 and Runx2. Meanwhile, Sox9, aggrecan and Col II expressions were down-regulated. The opposite results were observed in Notch signaling inhibited cells by DAPT treatment. In addition, Runx2 RNAi significantly attenuated the ‘regulatory sensitivity’ of Notch signaling on MMP-13 expression both in vitro and in vivo. However, we found there wasn’t significant changes of this ‘regulatory sensitivity’ of Notch signaling after Runx2 over-expression. Under IL-1β circumstance, MMP-13 expression could be reduced by both DAPT treatment and Runx2 RNAi, while Runx2 interference also attenuated the ‘regulatory sensitivity’ of Notch in MMP-13 under IL-1β stimulation. In conclusion, Notch signaling is an important regulator on rat chondrocyte proliferation and differentiation, and this regulatory effect was partially mediated by proper Runx2 expression under both normal and IL-1β circumstances. In the meanwhile, DAPT treatment could effectively suppress expression of MMP-13 stimulated by IL-1 β.Notch signaling is involved in the early onset of osteoarthritis. The aim of this study was to investigate the role of Notch signaling changes during proliferation and differentiation of chondrocyte, and to testify the mechanism of MMP-13 regulation by Notch and Runx2 expression changes during osteoarthritis. In this study, Chondrocytes were isolated from rat knee cartilages. Notch signaling was activated/inhibited by Jagged-1/DAPT. Proliferative capacity of Chondrocytes was analyzed by CCK-8 staining and EdU labeling. ColX, Runx2 and MMP-13 expressions were analyzed as cell differentiation makers. Then, Runx2 gene expression was interfered using lentivirus transfection (RNAi) and was over-expressed by plasmids transfected siRNA in chondrocytes, and MMP-13 expression was analyzed after Jagged-1/DAPT treatment.  Currently, there haven’t been effective interventions to prevent the initial onset or arrest the progression of OA, which is due to the limited understanding of molecular mechanisms in the progress of the disease.Osteoarthritis (OA) is the most common degenerative articular disorder nowadays2. Once OA is initiated, the tightly controlled metabolic balance of ECM will be disrupted by expression changes of certain genes in chondrocytes, leading to progressive loss of matrix proteins. Fundamentally, cleavage of Col II is considered as a critical early event of OA4.Healthy articular cartilage consists of chondrocytes and extracellular matrix (ECM). Under the physiological condition, chondrocytes produce both catabolic and anabolic proteins to keep the equilibrium of ECM, and ECM in turn functions to maintain the homeostasis of cellular environment and the structure of cartilage7. MMP-13 expression could be extraordinarily detected in the vicinity of early focal lesion in OA cartilage8. Therefore, characterizing the regulation of MMP-13 expression in chondrocytes is important to understanding molecular mechanisms of early OA.Initial cleavage of Col II is preferentially performed by Matrix metalloproteinase 13 (MMP-13), an interstitial collagenase expressed by articular chondrocytes9. In Runx2-haploinsufficient (Runx2+/−) mice, reduced MMP-13 was observed, and the pathology of instability-induced experimental OA was ameliorated by decreasing cartilage destruction10. Evidences above illustrate that Runx2 contributes substantially to pathogenesis of OA disease through its regulatory links to MMP-13.Runx2, which is known with a unique binding site in the MMP-13 promoter region, might be a focal point of abnormal signaling mechanisms that promote OA initiation12. Notch signaling is an evolutionarily conserved pathway that mediates cell-cell interaction required for cell fate determination13. This signaling is activated when a ligand-receptor interaction induces a series of receptor proteolytic cleavage to release Notch1 receptor intracellular domain  in cytoplasm. The NICD then translocated into nuclei and ultimately induces expression of downstream target genes14. Recent reports raised the issue of involvement of Notch signaling in the homeostasis of articular cartilage and pathogenesis of OA process17. Loss of RBPjκ-dependent Notch signaling in postnatal joint cartilage results in an early OA-like pathology18, while temporary suppression of Notch signaling in murine joints leads to delayed OA progression19. These data collectively indicate that there is a requisite role of Notch signaling in the onset and progression of OA. In addition, Shang et al.20 recently found that there is a significantly positive correlation between Runx2 expression, as well as MMP-13 expression, with Notch signaling in a chondrogenic cell line. All these studies above led us to hypothesize that MMP-13 expression was possibly regulated by Notch signaling through activation of Runx2 in chondrocytes, which regulatory mechanism might be required for the pathogenesis of early OA.Similarly, MMP-13 expression could also be stimulated by Notch signaling pathway in murine articular chondrocytes and rat OA modelTo test our hypothesis, we imitated alteration of Notch signaling in chondrocytes using exogenous recombinant rat Jagged-1 protein  and N-[N--L-alanyl]-S-phenylglycine t-butyl ester (DAPT)  respectively. Then proliferation and differentiation of mature chondrocytes were examined. Furthermore, Runx2 transcription was interfered by shRNA in Jagged-1/DAPT treated cells and animal models in order to clarify the regulatory mechanism of Notch signaling on MMP-13 through Runx2. Finally, we investigated whether the regulatory effect of Notch signaling on MMP-13 expression via Runx2 still exists under the stimulus of IL-1β.in vitro studies, qRT-PCR, western and staining experiments were performed on n = 3/group/time-point. In the in vivo studies, staining experiments on lentivirus/Jagged-1/DAPT injected cartilage samples were performed on n = 6 rats/group. The experimental protocol was approved by the Animal Ethics Committee of the university  and was carried out according to the Guidelines for the Care and Use of Laboratory Animals.14-day-old and 12-week-old male Sprague-Dawly (SD) rats were obtained from the Experimental Animal center of Sichuan University, China. Rats were randomly allocated to treatment groups/harvest time-points, fed with tap water and pellet food for easy access under standard temperature and humidity conditions, and killed by pulling their necks. In the 3) and were digested with 0.2% trypsin  and collagenase . Then a cell suspension was yielded. Isolated chondrocytes were suspended in DMEM medium supplemented with 10% FBS  and were counted in hemocytometer. The chondrocytes were seeded on a 90-mm petri dish at a density of 1.5 × 105/dish, and passaged approximately at a 7-day interval. Chondrocytes at P3 stage, as mature chondrocytes, were taken for subsequent experiments. Characterization of the chondrocytes in primary cultures was established by immunostaining of Col II and toluidine blue staining (data not shown).Chondrocytes were prepared from knee cartilage tissues of 14-day-old rats. Briefly, harvested cartilage tissues were cut into small pieces (<2 mmAscl and Pmel sites of the pGCSIL vector containing a CMV-driven GFP reporter (shRunx2). Scrambled shRNA unrelated to human gene sequences was used as a negative control (shControl).The lentivirus vector with Runx2 shRNA (ID NM_053470) was constructed by Genechem Co.,Ltd . The oligonucleotide sequences were designed and synthesized as follows: Runx2-shRNA-F: 5′-CCGGCAGCACGCTATTAAATCCAAACTCGAGTTTGGATTTAATAGCGTGCTGTTTTTg-3′, Runx2-shRNA-R: 5′-AATTCAAAAACAGCACGCTATTAAATCCAAACTCGAGTTTGGATTTAATAGCGTGCtg-3′. The combined sequences of the EGFP gene and Runx2 shRNA were cloned into the In vitro, the chondrocytes were seeded before transfection at 5 × 104 cells/well in 24-well plates and incubated in serum-free growth medium with polybrene (8 μg/ml). When cells reached 50% confluency, they were transfected with the shRunx2/shControl lentivirus at a multiplicity of infection of 100 (MOI = 100). Three days later, the transfection efficiency was measured by the percentage of GFP-positive cells/total cells, and the knocking-down efficiency was analyzed by qRT-PCR and western blot.In vivo, 20 μl carrier solution with shRunx2/shControl lentivirus (108tu/ml in the carrier solution) was slowly injected into the right articular cavity. 2 weeks later, rats were used for further injection treatment.21. Briefly, total RNA prepared from MC3T3-E1 cells was used to generate complementary DNAs for the Runx2 gene using PrimeScript™ Reverse Transcriptase reagent kit . cDNA was confirmed by DNA sequencing. After digestion with BglII and NotI , purification from agarose gel, PCR products was subcloned into pCMV-myc-C vector . The Runx2 plasmid DNA was transfected into cells (OE-Runx2) using Lipofectamine™ 2000 Transfection Reagent . Untreated Cells were used as control (OE-Control).Runx2 over-expression vector construction as previously describedIn vitro, untreated cells , Runx2 shRNA transfected cells (shRunx2) and scrambled shRNA transfected cells (shControl) were respectively divided into three groups for each test, including the group treated with exogenous recombinant rat protein Jagged-1 , DAPT  and DMSO . At first, the working density of Jagged-1 was confirmed by a gradient test. 6 hours after Jagged-1 treatment, chondrocytes were harvested to test Notch1 mRNA expression by qRT-PCR, showing 0.5 μg/ml the most effective density (p < 0.05)  Fig. . 100 μM In vivo, drug injection dosages were followed as previously described12 and according to our in vitro data. 2 weeks after shRunx2 lentivirus injection, animals were started to receive weekly intra-articular injection with DAPT , Jagged-1  or DMSO (0.3 ml) as control. After 4 injections of DAPT/Jagged-1, animals were sacrificed 24 h after the last injection, then the knee joints were carefully dissected and fixed in 3.7% PFA at 4 °C for 48 hours. Thereafter samples were embedded in paraffin. 5 µm sections were cut on an HM360 microtome . Each one of every 5 consecutive sections were stained with hematoxylin  and eosin  (H&E). Other sections were used for immunohistochemical staining.3 cells/well into 96-well plates and were allowed to adhere to the plates overnight. Cells were then divided and treated with different stimuluses  for 6, 12, 24 and 48 hours. 10 μl CCK-8 reagent was added to each well and incubated for another 4 hours. The absorbance was read (wavelength = 450 nm). Data of cell proliferation was assessed based on the average absorbance values of each group, according to the manufacturer’s protocol.The cell counting kit-8  was used for quantitatively evaluating the cell viability. Chondrocytes were seeded at 2 × 105/well. After Jagged-1/DAPT treatment, 10 μM EdU was added to the medium. Cells were then fixed with 3.7% formaldehyde, and were incubated with Click-iT reaction cocktail in dark. The nuclei were counterstained with DAPI . The stained cells were observed using a fluorescence microscope . Percentage of EdU-positive cells was calculated by number of red-fluorescent (Alexa 594-stained) cells/ number of DAPI-stained cells.A Click-iT® EdU imaging kits  was used for measuring proliferation capacity of chondrocytes. Chondrocytes were seeded in a 6-well plate at 10TMFirst Strand cDNA Synthesis Kit . qRT-PCR with cDNA was performed using 2xPCR Master Mix . Gene expression relative to GAPDH was calculated using the 2−ΔΔCT formula method. The sequences of primers are shown in Table Total RNA was isolated using TRIZOL reagent , chloroform and isopropanol. Reverse transcription of RNA was performed by RevertAidThe total protein was collected using a protein extraction kit . The protein concentrations were determined using the BCA assay . After denaturing at 95 °C for 5 mins, 5 mg aliquot of total protein of each sample was subjected to the 4–20% gradient SDS-PAGE, transferred to a PVDF membrane . The membrane was incubated in blocking solution with donkey serum  for 1 hour, and was incubated overnight at 4 °C with the primary antibodies against Col X , Col I , Col II , Sox 9 , Aggrecan , Notch1-ICD , MMP-13 , Runx2  and β-actin . One of the following secondary antibodies was used: HRP-labeled goat anti-rabbit IgG  and HRP-labeled goat anti-mouse IgG . Proteins in the blots were visualized by an ECL plus kit . The integrated optical density of bands was quantified with the ImageJ software. Each sample was normalized to β-actin.in vitro IFS, chondrocytes were fixed with 3.7% paraformaldehyde at first. Endogenous peroxidase activity was depleted with 3% hydrogen peroxide. then nonspecific antibody binding was blocked. The cells were incubated with a mouse monoclonal anti-MMP-13  as primary antibody, and a Cy3-labeled goat Anti-Mouse IgG  as secondary antibody. Nuclei was counterstained with DAPI . The fluorescence stained cells were observed under fluorescence microscope .For in vivo IFS and IHC, after dewaxing in xylene, sections were rehydrated in an alcohol series . Briefly, endogenous peroxidase activity was depleted with 3% hydrogen peroxide. then nonspecific antibody binding was blocked. The sections were respectively incubated with rabbit anti-GFP antibody  and mouse monoclonal anti-MMP-13  as the primary antibodies overnight at 4 °C. In immunofluorescent staining, sections were stained with a goat anti rabbit IgG H&L (FITC) secondary antibody . DAPI  was used for nuclear counterstaining. For immunohistochemistry staining, the tissue was stained with HRP labeled goat anti-mouse IgG (H + L)  followed by visualization of HRP activity by a DAB-HRP color development kit .For p value < 0.05 was considered to be the level of significance.All data are presented as mean ± standard deviation. Statistical significance among the groups with a single variable was assessed using one-way ANOVA analysis with Tukey’s test. The other statistical significance among groups was assessed using two-way ANOVA analysis with multiple comparisons. Analyses were performed using the SPSS 17.0 statistical software program. All experiments were performed at least thrice in triplicate. A To verify whether Notch signaling could be activated or suppressed in chondrocytes by Jagged-1/DAPT, expression of Notch1 was examined by qRT-PCR and western blot. After Jagged-1 treatment, Notch1 expression was increased at both mRNA Fig.  and protin vitro after Jagged-1 or DAPT treatment. The CCK-8 test showed that the viability of chondrocytes decreased in Jagged-1 treated group and increased in DAPT treated group. Compared with control, prominent changes were observed at 12 hours after both treatments (decreased by 70% in Jagged-1 group and increased by 1.7-fold in DAPT group)  Fig. . Consistup) Fig. .Figure 2p < 0.05), whereas DAPT treatment significantly reduced their expression   Fig. . On the 01) Fig. . These fp > 0.05)  Fig. . At the 05) Fig. .p < 0.01 in mRNA, p < 0.05 in protein, shRunx2 + jagged-1/shRunx2 vs. shControl + jagged-1/shControl). In contrast, the decrease rate of MMP13 expression treated by DAPT was also attenuated after Runx2 knocking-down. Especially when MMP13 expression was most dramatically changed at 12 hours after Jagged-1/DAPT treatment in the control group, the knocking-down of Runx2 remarkably abated the regulation effect of Jagged-1/DAPT treatment on MMP13   Fig. . Howeverol) Fig. . To direol) Fig. . Howeverol) Fig. . These fin vitro findings, we performed Jagged-1 and DAPT intra-articular injection in the knee joint of SD rats, and suppressed the Runx2 expression by local injection of shRunx2 lentivirus. After lentivirus injection, Runx2 was successfully knocked-down by approximately 75% in rat knee articular cartilage  and DAPT. These cells were further divided to shControl or shRunx2 group. Notch1, Runx2, and MMP-13 expression were examined within 48 h.p < 0.05). Such increased expression of MMP-13 was down-regulated by DAPT to about 50% at mRNA level and 60% at protein level respectively (p < 0.05)  Fig. . These rp < 0.05, shRunx2 + IL-1/shRunx2 vs. shControl + IL-1/shControl)  Fig. . Our res22. At the beginning of OA, the earliest degradation changes also occurred at the cartilage surface24. Accordingly, we speculated that the onset of OA is possibly associated with changes of Notch signaling pathway in chondrocytes, eventually resulting in an imbalance between production and degradation of ECM. To observe such early changes in OA, several time points within 48 h were chosen for observation after exogenous intervention of Notch signaling in mature chondrocytes.In normal articular cartilage surface, the immuno-activity of Notch receptors was undetectable. While in OA cartilage, Notch signaling is activated, and a much more abundant expression in the damaged areas was detected26. But how Notch signaling are involved in this phenotypic change of chondrocytes remains controversial30. Some studies showed that Notch signaling is associated with significantly increased cell proliferation30. These results could be an explanation for the chondrocyte cloning found primarily in the upper cartilage zone, that is, in the same location as the Notch-1 ligand positive cells31. Another theory is that surface damage to the ECM might contribute to the increased proliferation32. Such response together with increased synthesis of ECM is considered as an early attempt to repair in early OA. In this study, we found that enhanced Notch signaling resulted in a decrease of chondrocytes proliferation. Such inhibitory effect could disrupt the self-regeneration of chondrocytes. On the other hand, our data showed hypertrophic differentiation markers, Col X, MMP-13 and Runx2, were significantly increased by activating Notch signaling, demonstrating that articular chondrocytes might lose their differentiated phenotype and obtain a behavior with similarities to terminal differentiating chondrocytes. Such terminal differentiation is defined as hypertrophy and apoptosis33. This process leads to degradation of cell surroundings, as can be seen in OA cartilages and OA models. Evidences above suggested that early suppression of proliferation and promotion of differentiation by activating Notch signaling could aggravate early OA. It is worth mentioning that when and how Notch signaling was activated may be the pivotal determinations to diverse outcomes35. Our results came from a transient alteration of Notch signaling in normal and mature chondrocytes, and detections were completed in an initial and short term. Thus, the controversial results between our study and previous studies were possibly due to different experimental conditions30.In early OA, the normally quiescent chondrocytes undergo phenotypic shift, characterized by a transient proliferative response, a differentiation expressing hypertrophy-like changes and increased synthesis of cartilage-degrading enzymes, such as MMP-1336. During the degeneration process MMP-13 is known as the factor that preferentially cleaves Col II37. Our findings showed that when MMP-13 expression was up-regulated by Jagged-1 within 48 h, Col II expression was decreased and aggrecan was increased, which suggested that activity of Notch signaling was involved in the degradation of ECM during early OA by stimulating MMP-13 expression.The first sign of prominent degeneration in OA is the onset of fibrillation at the articular cartilage surface as collagen fibrils had been damaged. Progressive denaturation of Col II was found in early OA, and a net loss of this molecule is accompanied with the cartilage damage34. These evidences hinted that the role of Notch signaling in regulating MMP-13 expression depended on its dosage, as well as the duration and the context of the signaling activation. Interestingly, there is a putative CSL (Notch co-activator) binding site located in mouse and rats MMP-13 promoter sequence38. However, MMP-2 promoter area also has this binding site while MMP-2 was not regulated by Notch, suggesting that the regulation on MMP-13 by Notch signaling may achieved through a more circuitous mechanism11.Indeed, the regulation of MMP-13 by Notch signaling is highly complex. Generally, sustained overexpression of Notch signaling increased abundance of MMP-13. Conversely, MMP-13 abundance decreased in conditionally overexpression of Notch signaling, which could not repair the injury of induced OA but exacerbated disease progression instead9. In Runx2+/−mice, OA development was resisted with decreased MMP-13 expression10. In addition, overexpression of Notch signaling in chondrogenic cell line significantly induced expression of Runx220. In accordance with studies above, our data showed that transcriptional inhibition of Runx2 resulted in an attenuation of ‘regulation sensitivity’ of Notch signaling on MMP-13 expression. Our data, with other previous reports, suggested that early regulation of MMP-13 by Notch signaling could be partly mediated by Runx2 in mature chondrocytes.Runx2 is known as the direct upstream transcriptional regulator of MMP-13, and is regulated by Notch signaling in chondrocytes as well. In OA cartilage, Runx2 contributes to increase expression of MMP-1339. In a mouse surgical knee joints OA model, intra-articular injection of DAPT decreased expression of MMP-13, and the cartilage degradation was suppressed19. In our study, expression of MMP-13 induced by IL-1β was significantly suppressed by DAPT, highlighting that DAPT is a potential protector of Col II from degradation and might offer an optional therapy of clinical OA.DAPT is a small compound of Notch signaling inhibitor, which increases expression of Col II in the articular chondrocytesIn conclusion, our findings demonstrated that activation of Notch signaling was involved in initial and development of early OA damage by suppressing proliferation and increasing hypertrophic differentiation in mature articular chondrocytes. Moreover, our data suggested that the expression of MMP-13 might be partly regulated by Notch signaling though the activation of Runx2 in chondrocytes under both normal and IL-1β circumstances. However, the animal model we used in this study was rat with intact normal cartilage, while animals with OA cartilage were not used in the current studies. Further work using OA animal models is therefore needed to better understand how Runx2 is involved in the Notch signaling transmission, and how these effects function in the cartilage degradation during AO progression.Supplemental Figures


Ptychodera flava, an indirect-developing hemichordate that has larval morphology similar to echinoderms and adult body features that are similar to chordates.Mesoderm is generally considered to be a germ layer that is unique to Bilateria, and it develops into diverse tissues, including muscle, and in the case of vertebrates, the skeleton and notochord. Studies on various deuterostome animals have demonstrated that fibroblast growth factor (FGF) signaling is required for the formation of many mesodermal structures, such as vertebrate somites, from which muscles are differentiated, and muscles in sea urchin embryos, suggesting an ancient role of FGF signaling in muscle development. However, the formation of trunk muscles in invertebrate chordates is FGF-independent, leading to ambiguity about this ancient role in deuterostomes. To further understand the role of FGF signaling during deuterostome evolution, we investigated the development of mesodermal structures during embryogenesis and metamorphosis in P. flava. The mesoderm cells are then differentiated stepwise into the hydroporic canal, the pharyngeal muscle and the muscle string; formation of the last two muscular structures are controlled by FGF signaling. Moreover, augmentation of FGF signaling during metamorphosis accelerated the process, facilitating the transformation from cilia-driven swimming larvae into muscle-driven worm-like juveniles.Here we show that genes encoding FGF ligands, FGF receptors and transcription factors that are known to be involved in mesoderm formation and myogenesis are expressed dynamically during embryogenesis and metamorphosis. FGF signaling at the early gastrula stage is required for the specification of the mesodermal cell fate in P. flava embryo, and it is reiteratively used for the morphological transition during metamorphosis. The dependence of muscle development on FGF signaling in both planktonic larvae and sand-burrowing worms supports its ancestral role in deuterostomes.Our data show that FGF signaling is required for mesoderm induction and myogenesis in the The online version of this article (10.1186/s12862-018-1235-9) contains supplementary material, which is available to authorized users. Mesoderm is a unique feature of bilaterians and contributes to the increased complexity of body structures. During vertebrate development, mesoderm is subdivided into several regions, including axial, paraxial, intermediate and lateral plate mesoderm. Each of these regions gives rise to different tissues. For example, axial mesoderm forms the notochord, while paraxial mesoderm forms somites, which further differentiate into muscle and many types of connective tissue –5. In amXenopus, FGF signaling is required for the development of axial and paraxial mesoderm. Disruption of FGF signaling by expressing a dominant negative form of the FGF receptor resulted in the loss of both notochord and somites [no tail expression, which marks the dorsal axial mesoderm (putative notochord) [Studies in several vertebrate models have elucidated critical roles for fibroblast growth factor (FGF) signaling during mesoderm differentiation. For example, in  somites –8. In zetochord) –11. On tDrosophila melanogaster, the FGF receptor (heartless) null mutant embryos specified their primary mesoderm normally, but these tissues failed to go through its usual dorsolateral migration and thus resulted in the malformation of the body wall muscles [Caenorhabditis elegans, mutations in FGF ligand egl-17 and receptor egl-15 affect the specification and migration of the larval sex myoblasts, a small subset of body muscle structures [In order to decipher the ancestral role of FGF signaling in chordate mesoderm development, studies have also been carried out on invertebrate chordates, including amphioxus and ascidians. Intriguingly, FGF signaling was reported to only be required for the formation of anterior somites in amphioxus . In asci muscles , 16. In ructures . BecauseSaccoglossus kowalevskii, a direct-developing hemichordate acorn worm, it has been shown that inhibition of FGF signaling, by either downregulating fgfr-b or fgf8/17/18, resulted in the loss of the mesoderm and reduced the expression of several mesodermal and muscle marker genes [S. kowalevskii. However, it is unclear whether myogenesis is directly regulated by FGF signaling after mesoderm induction has completed. In the indirect-developing acorn worm, Ptychodera flava, it has been shown that U0126, an inhibitor against mitogen-activated protein kinase kinase (MEK), blocks mesoderm formation [To gain insight into the ancestral function of FGF signaling for mesoderm, it is important to investigate Ambulacraria, which includes both echinoderms and hemichordates and is the closest group to the chordates –23. In cer genes . Therefoormation , althougP. flava. Unlike the direct-developing S. kowalevskii, which grows directly into a vermiform body [P. flava develops into a swimming larva before metamorphosing into a worm-like adult body plan [P. flava changes its body form and lifestyle dramatically, becoming a bottom-burrower with a muscular vermiform body [P. flava during different embryonic time windows. We also show that, in addition to environmental stimulations, FGF signaling is able to accelerate the progression of metamorphosis. These data suggest an ancient role for FGF signaling in muscle development in the common ancestor of deuterostomes.In this study, we investigated the roles of FGF signaling in mesoderm development during embryogenesis and metamorphosis of orm body , 29, P. ody plan –33. The ody plan , 35. Durorm body , 31. HerP. flava were collected from Chito Bay, Penghu Islands, Taiwan, as previously described [Rhodomonas lens at 5000 cells/ml. Under this condition, the cultures reached the Spengel stage within 3 months. Advanced Spengel larvae, with fully developed protocoel, mesocoels and metacoels were picked for metamorphosis experiments. Sea sand was collected from the adult habitat and used to induce metamorphosis. The sand was first filtered through a 300 μm mesh, washed with FSW three times, and soaked in antibiotic-FSW overnight at room temperature. After removing the antibiotic-FSW and washing three times with FSW, the sand was ready to be used for metamorphosis experiments. For the experiments using sterilized sea sand, sand was washed with reverse osmosis-purified water three times, autoclaved, and then washed three times with FSW.Mature escribed . Spawninescribed , 33, 36 escribed , 31 withP. flava that were derived from two or three separate breeding seasons. For each trial of the metamorphosis experiments, 10–15 Spengel larvae per condition were cultured in the presence of sand with or without the inhibitors or proteins. Each experiment was carried out over at least three replicate trials, unless otherwise indicated. The inhibitors and recombinant proteins used in this work include: SU5402 , PD173074 , U0126  and human basic-FGF . Inhibitors and recombinant protein were dissolved in DMSO or 0.1% BSA , respectively. The solvents were used to treat experimental control groups.Embryos were treated with inhibitors or recombinant proteins after fertilization or at the indicated developmental stages. Treatments were maintained until fixation. Experiments performed at specific embryonic stages were repeated at least three times using different batches of 3) after fixation. Following three washes with PBST, the samples were incubated in PBST with 0.1% Triton X-100 for 20 min, and then transferred to the blocking solution (PBST with 1% BSA) for at least 1 h at room temperature. Alexa Fluor 488 phalloidin  staining was performed at 4 °C overnight. Counterstaining was conducted using Hoechst 33342  for 1 h. Extensive washes were performed between every step. Specimens were mounted in PBST containing 70% glycerol and 0.1% NaN3, and imaged with either a Zeiss (Axio Imager A2) microscope (for embryos) or Leica (Z16APO) microscope (for specimens larger than 1 mm). All phalloidin stained samples were imaged on a Leica (TCS-SP5) confocal microscope.Digoxigenin-labeled RNA antisense probes were synthesized from cDNA clones using DIG RNA Labeling mix (Roche) and T7 or SP6 RNA polymerase (Promega). Fixation, dehydration and in situ hybridization were performed as described , and BM-S. kowalevskii were retrieved from GenBank and used as queries to search the P. flava embryonic transcriptome database [All genes investigated in this study were cloned by methods described previously , and thedatabase . SpecifiP. flava differentiates into several different structures during embryogenesis and metamorphosis  and three FGF receptors  identified previously [Fgf8/17/18 was the earliest fgf gene to be activated specifically in the apical ectoderm at the late blastula stage  or an inhibitor (U0126) against MEK, a downstream kinase of the FGF signaling. We also used exogenous recombinant FGF protein (bFGF) to enhance FGF signaling in the embryos. Two concentrations of each inhibitor and protein were used to determine optimal conditions for the following experiments  genes, striated muscle MHC (stMHC) and smooth muscle MHC (smMHC) , middle  and dorsal (foxf-positive) domains at the late gastrula stage. These domains represent different regulatory states, which possibly contribute to the formation of the three mesodermal structures, the pharyngeal muscle, the muscle string and the hydroporic canal.We then further examined the expression of the same four transcription factor genes in conjunction with other known myogenic genes in later stages of development. In these experiments, we tracked the differentiation of mesoderm into the three different structures, including the hydroporic canal, the pharyngeal muscle and the muscle string. In situ hybridization of the myogenic din gene –55 showenal Fig.  A4-5. Tofoxc shrank to the most ventral tip of the mesoderm . The morphology of the transforming larvae and juveniles upon bFGF treatments were also similar to the untreated individuals . These results suggest that FGF signaling plays a positive role in promoting sand-induced metamorphosis. However, due to the conflicting results from the three FGF inhibitor treatments, it remains uncertain whether FGF signaling is required to trigger metamorphosis.Given that FGF signaling was required for muscle development during embryogenesis, we set out to test the potential role of FGF signaling in the production of muscle during metamorphosis. We first analyzed the expression of genes encoding FGF ligands, FGF receptors, and two myogenic Fox factors, P. flava in the absence of environmental chemical/biological stimulating factors.In a previous study, we showed that autoclaved sand is less efficient at inducing metamorphosis, suggesting that some unknown, sand-associated, heat and pressure-labile factors are required to stimulate this morphological transition . To furtXenopus demonstrated that fgf8 is necessary for mesoderm formation [fgf8 and fgf24  in zebrafish blocks the formation of most posterior mesoderm [S. kowalevskii, in which fgf8/17/18 expressing cells are sufficient to induce mesoderm in adjacent cells [P. flava. The earliest FGF signaling in this species may also be mediated by FGF8/17/18, since it was one of the earliest FGF ligands to be produced (at the late blastula stage). The expression of fgf8/17/18 in the apical ectoderm also coincided with that of its ortholog in S. kowalevskii, suggesting the conserved role of FGF8/17/18 in mediating embryonic mesoderm induction in hemichordates. However, there are significant differences in how the mesoderm-derived coeloms are formed in these two species. In S. kowalevskii, the coeloms are formed from pouches evaginated from the archenteron through a process called enterocoely and the embryo directly develops into the juvenile form within days [P. flava, the protocoel, which is the anterior coelom, is also formed by enterocoely during embryogenesis. However, the formation of the paired posterior coeloms, the meso- and metacoels, is not by enterocoely but is through aggregation of the mesenchymal cells surrounding the digestive tract during the late larval stages, around two to 4 months after fertilization [FGF homologs are widely distributed in bilaterians and cnidarians –59. To dormation . Additiomesoderm . The connt cells . In thishin days . In the lization , 31. Ourfoxc, foxf, twist and myocardin, we discovered that the P. flava embryonic mesoderm is partitioned into at least three domains, and two of which, the ventral and middle domains, are involved in myogenesis. In addition, we found that FGF signaling is required for the maintenance of foxc and myocardin expression in the ventral and middle domains and the formation of the two muscular structures, the pharyngeal muscle and the muscle string. The role of FGF signaling in myogenesis has also been suggested in S. kowalevskii. When the translation of fgfr-b or fgf8/17/18 is inhibited by siRNAs, expression of muscle markers, such as muscle Lim protein and myosin, is greatly reduced [P. flava protocoel are thus similar, suggesting that these features are likely ancestral, at least in the common ancestor of the ambulacrarians.The vertebrate embryonic mesoderm is divided into the axial, paraxial, intermediate and lateral domains. These domains exhibit different regulatory states that govern the formation of diverse cell types during development. Similarly, based on the expression patterns of  reduced . In sea  reduced , 64. Mor reduced . The ontP. flava, metamorphosis takes only one to 2 days, but results in dramatic changes in both body plan and lifestyle. One intriguing finding in our study is that muscles are extensively generated during metamorphosis, coinciding with significant increases in the expression levels of the genes encoding FGF ligands, FGF receptors and myogenesis factors. Indeed, the dual observations that FGF signaling is required for embryonic myogenesis and that exogenous bFGF protein accelerated the transformation support the notion that FGF signaling plays a positive role in myogenesis during this morphological transition. However, the issue of whether FGF signaling is indispensable to this morphological transition was not unequivocally resolved in our study. This uncertainty is due to our variable results using two different FGF receptor inhibitors, PD173074 and SU5402. The discrepant results may have been generated because of two potential reasons. First, the concentrations of the two drugs that were used for metamorphosis experiments were optimized in embryos (2.5 μM for PD173074 and 20 μM for SU5402). The penetration efficiency of the drugs may be lower in the Spengel larva, which has a bigger body cavity than the smaller embryos. A lower penetration efficiency would reduce the effective drug dose, leading to a lack of biological efficacy at low doses, and this dampening of efficacy may have been especially relevant for PD173074, which was used at a relatively low concentration. Second, although SU5402 is strongly selective for human FGFRs, cell-free kinase assays have revealed that it is also a potent inhibitor of other tyrosine kinase receptors  [P. flava is induced by multiple factors. Both physical contact with the sand and some as yet unidentified, sand-associated chemical or biological factor(s) are required to stimulate metamorphosis. It remains unclear whether these environmental factors are directly responsible for the observed upregulation of FGF ligand and receptor genes during metamorphosis, and if so, how the upregulation might be mediated. Regardless of the necessity for FGF signaling in this process, our data clearly show that FGF can accelerate metamorphosis, possibly by activating myogenic factors and to trigger the formation of the muscle fibers during this time. This massive generation of muscle fibers during metamorphosis is necessary for lifestyle changes, in which the cilia-driven planktonic larva transforms into a muscle-driven benthic worm.In nd JAK3) . TherefoP. flava. The extensive production of muscle and elevated expression of FGF ligand and receptor genes during metamorphosis strongly imply that FGF signaling is reiteratively used in mesoderm development during embryogenesis and metamorphosis in P. flava. Thus, the role of FGF signaling in muscle development is conserved at least in ambulacrarians and may have been present in the common ancestor of deuterostomes.In this study, we show that FGF signaling is required for mesoderm induction and muscle formation during embryogenesis of the hemichordate acorn worm Additional file 1:Table S1. Accession numbers for genes/proteins used in this study. Table S2. Primers used for cloning. (DOCX 25 kb)Additional file 2:Figure S1. Development of the mesodermal structures in P. flava. (A) The presumptive endomesoderm emerges as a thickened vegetal plate (yellow and red stripes) at the late blastula stage. (B) At the mid gastrula stage, the mesodermal cells (red) are specified at the tip of the archenteron (yellow). (C) At the late gastrula stage, the mesoderm develops into the protocoel that extends dorsally and forms a duct-like structure, the hydroporic canal (black arrow), which opens in the dorsal ectoderm to form a hydropore (black asterisk). (D) After hatching, the mesoderm of the tornaria larva further differentiates into the pharyngeal muscle (green arrow) and the muscle string (black arrowhead) that reaches to the anterior ectoderm. (E) At the Spengel larval stage, the protocoel is considerably enlarged, and two paired coeloms, the mesocoels (light purple) and metacoels (dark purple), form as two pairs of rings surrounding the stomach. (F) During metamorphosis, the protocoel forms a proboscis coelom at the Agassiz stage. (G) The transforming Agassiz has a more elongated posterior region, starts losing its cilia, and is incapable of swimming. (H) The juvenile has a typical tripartite body with an anterior proboscis, followed by a collar region and a trunk. The two black dots on the anterior ectoderm indicate the eye spots. Abbreviations: prob., proboscis; col., collar. (PNG 353 kb)Additional file 3:Perturbations of FGF signaling after fertilization. Phenotypes of embryos at 43 hpf (A1-F1) and 73 hpf (A2-F2) after treatment with FGF signaling inhibitors (B1-D2) or bFGF protein (F1-F2) upon fertilization. Control embryos were treated with DMSO or 0.1% BSA. The concentrations of each drug or protein are indicated in each panel. All embryos are shown from a lateral view with the mouth on the left. All panels are shown in the same scale, according to the scale bar in A1. Abbreviations: me, mesoderm; en, endoderm. (PNG 2340 kb)Additional file 4:P. flava that were treated with PD173074 (B1-B4), SU5402 (C1-C4) or U0126 (D1-D4) at different developmental stages (indicated by yellow circles on the left). (E1-H5) Tornaria larvae treated with SU5402 or U0126 at different developmental stages were observed and stained with Phalloidin (green). The larvae were counterstained with Hoechst 33,342 for nuclei (blue). The drugs and the concentrations used in the experiments are shown at the top of the panels, and the treatments were performed at the time points indicated by the yellow circles on the left. Abbreviations: ms, muscle string; pm, pharyngeal muscle; hc, hydroporic canal. (PNG 6751 kb)Inhibitions of FGF signaling at various developmental stages. (A1-A2) Phenotypes of the control embryos (DMSO-treated) at 43 hpf and 73 hpf. (B1-D4) Phenotypes of the late gastrula stage Additional file 5:snail, foxc, foxf, twist and myocardin genes during embryogenesis. In situ hybridization for snail (A1-A7), foxc (B1-B7), foxf (C1-C7), twist (D1-D7) and myocardin (E1-E7) at different developmental stages indicated on the left. Embryos at 43 hpf and 73 hpf were viewed from the lateral side with mouth to the left. Embryos in the inlays of panels B6 and B7 were observed from the ventral side to show the expression of foxc in the posterior ciliary band. The expression patterns of each gene at the late gastrula and tornaria stages are delineated schematically below the corresponding panels. All panels are shown in the same scale, according to the scale bar in A1. (PNG 8481 kb)Expression patterns of the Additional file 6:MHC genes identified in P. flava.(A) The amino acid sequences of the myosin head domains of the MHC proteins from 15 species were used to construct the phylogenetic tree. The three different types within the myosin superfamily, type I, II and V, were all well resolved. The two P. flava MHCs are grouped in type II (green branches), one within the skeletal/cardiac muscle MHC subgroup (Pf-stMHC) and the other in the smooth/non muscle subgroup (Pf-smMHC). Values at each node are Bootstrap inferences and the values lower than 50 are not shown. The scale bar indicates the substitutions per site. The abbreviations of the species names are: Amq, Amphimedon queenslandica; B, Bovine; Bb, Branchiostoma belcheri; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Dm, Drosophila melanogaster; Hs, Homo sapiens; Mm, Mus musculus; Pf, Ptychodera flava; Pl, Placopecten magellanicus; Rn, Rattus norvegicus; Sk, Saccoglossus kowalevskii; Sc, Saccharomyces cerevisiae; Sch, Schistosome mansoni; Sp, Strongylocentrotus purpuratus. (B1-C2) In situ hybridization analyses of P. flava stMHC (B1-B2) and smMHC (C1-C2) in 43 hpf and 73 hpf embryos. The embryo in the inlay of B2 was viewed from the dorsal side to show the expression of stMHC in the muscle string. The embryo in the inlay of C1 was observed from the apical side. All panels are shown in the same scale, according to the scale bar in B1. The green arrow indicates the pharyngeal muscle and the black arrowheads denote the muscle string in B2. (PNG 1292 kb)Phylogenetic and expression analyses of the two Additional file 7:Supplementary methods. (DOCX 77 kb)Additional file 8:foxc and foxf during metamorphosis. The mRNA expression levels of genes encoding FGF ligands and receptors (A) and two myogenic factors, foxc (B) and foxf (C), were measured at the Spengel (gray), Agassiz (green) and juvenile (blue) stages. The gene names are given on the X-axis, and the relative expression levels normalized to 18S rRNA are shown by the bars. The numbers above each bar indicate the fold differences between gene expression levels in the indicated stages relative to the Spengel stage. (PNG 123 kb)QPCR analyses of FGF ligands, FGF receptors, Additional file 9:Perturbations of FGF signaling during sand-induced metamorphosis. The morphology of a wild type Spengel larva (A1) and the Spengel larvae treated with 10 μM (A2) or 20 μM (A3) of SU5402. The images were taken 2 days after treatments. The morphology of individuals after cultured for 2 days with sand containing 0.1% BSA (B1–2), 100 ng/ml (B3–4) or 200 ng/ml bFGF protein (B5–6). The Spengel larvae transformed into either Agassiz  or juveniles . A1-A3 and B1-B6 are shown in two different scales, according to the scale bars in A1 and B1, respectively. (PNG 5404 kb)