Angiopoietin-1 (Angpt1) is a glycoprotein ligand important for maintaining the vascular system. It signals via a receptor tyrosine kinase expressed on the surface on endothelial cells, Tie2. This receptor can undergo regulated ectodomain cleavage that releases the ligand-binding domain (sTie2) into the circulation. The concentration of sTie2 is increased in a range of conditions, including peripheral arterial disease and myocardial infarction, where it has been suggested to bind and block Angpt1 resulting in vascular dysfunction. Here we use a joint mathematical modelling and experimental approach to assess the potential impact of sTie2 on the ability of Angpt1 to signal. We find that the concentrations of sTie2 relative to Angpt1 required to suppress signalling by the ligand are more than ten–fold higher than those ever seen in normal or disease conditions. In contrast to the endogenous sTie2, an engineered form of sTie2, which presents dimeric ligand binding sites, inhibits Angpt1 signalling at seventy-fold lower concentrations. While loss of Tie2 ectodomain can suppress Angpt1 signalling locally in the cells in which the receptor is lost, our study shows that the resulting increase in circulating sTie2 is unlikely to affect Angpt1 activity elsewhere in the body. In humans there are three angiopoietins, angiopoietin-1 (Angpt1), Angpt2 and Angpt44. Angpt1 is constitutively secreted under normal conditions by perivascular cells4 and has crucial vascular protective effects, acting to maintain endothelial quiescence, suppress vessel inflammation, prevent microvessel regression and inhibit vascular leakage5.The angiopoietins are a group of secreted glycoprotein ligands that act mainly on the vasculature4. This transmembrane protein is expressed on endothelial cells7. Tie2 is activated by Angpt1, stimulating receptor phosphorylation, intracellular signalling and functional effects5. Endothelial cell effects of Angpt1 include suppression of apoptosis, enhancement of cell:cell junction integrity and inhibiting expression of inflammation-associated genes5.The primary receptor for angiopoietins is the receptor tyrosine kinase Tie29. Soluble Tie2 (sTie2) originates from endothelial cells where it is generated by proteolytic cleavage and release of the ectodomain from full-length receptor located at the cell surface9. sTie2 contains the ligand binding domain of the receptor and can bind angiopoietins, preventing them from interacting with cellular Tie2 and thereby inhibiting angiopoietin action9. sTie2 is increased in a number of diseases14. The possibility that increased sTie2 could contribute to vascular pathology by inhibiting Angpt1 protective signalling has been highlighted in peripheral arterial disease, myocardial infarction, vessel permeability in inflammation, organ failure in post cardiac-arrest syndrome and trauma, pulmonary hypertension, and neoangiogenesis19. sTie2 therefore may be an important target for therapeutic inhibition.In addition to cellular Tie2, a soluble form of the angiopoietin receptor has been detected in the circulationsTie2 has the potential to have profound effects on regulating angiopoietin availability and contribute to vascular pathology in diseases in which it is elevated. Despite this we know relatively little about how sTie2 affects the ability of angiopoietins to act on endothelial cells. In this study, therefore, we use computational modelling and experiment to assess the effects of sTie2 on the ability of Angpt1 to bind and activate its cellular receptor.20 for Angpt1 binding in the absence and presence of sTie2  on endothelial cells. To test this prediction we directly examined the effects of sTie2 on the ability of Angpt1 to activate cellular Tie2. Human endothelial cells were challenged with Angpt1, or control vehicle, in the absence or presence of sTie2 Fig. , and recie2 Fig. . This acie2 Fig. . Additioie2 Fig. , confirm28. Examples of such ligand-traps include TNF-α receptor ectodomain, Etanercept, used in treatment of rheumatoid arthritis29, and the VEGF ligand-trap, Aflibercept, used in a number of pathologies including diabetic macular oedema30. In contrast to physiological soluble receptors, ligand-traps are usually fusion proteins in which the ligand-binding domain is engineered to be presented in a dimeric format28. We were interested to investigate how a dimeric form of sTie2, similar to that which would be used for a ligand trap, compares with monomeric sTie2 in their ability to affect Angpt1 action. As shown in Fig. Soluble receptor ectodomains are used as therapeutic ligand-traps to block effects of pathological or inappropriately elevated concentrations of ligand31 and vary little with disease, though a decrease to 14pM has been reported in sepsis17. Circulating sTie2 concentrations in humans have been reported to be in the region of 100–200 pM in normal conditions31, rising as high as 350 pM in severe sepsis17. The molar ratio of sTie2:Angpt1 found in vivo therefore is normally around 3–6 rising to 25 in severe sepsis. Such relative concentrations are much lower than those we found to affect the ability of Angpt1 to assemble tetrameric Tie2 complexes in our model and suppress Tie2 activation in our experiments. This suggests that circulating sTie2 is unlikely to influence the ability of circulating Angpt1 to activate cellular Tie2 at the concentrations found in normal and disease states in vivo.Our results show that Angpt1 activation of cellular Tie2 is poorly responsive to sTie2. We found that in order to suppress signalling-competent Tie2 complex formation by 50% sTie2 is required at concentrations in the range of 350–200 fold that of Angpt1 in our model and experiments. Concentrations of Angpt1 in humans under normal conditions are around 30–70pMin vivo and not incorporated in our model and experiments could enhance the ability of sTie2 to modify Angpt1 action, although there is no evidence for such factors to date. In fact, in vivo sTie2 is likely to be less effective at suppressing Angpt1 action on endothelium than our data suggest. This is because in vivo Angpt1 is released by pericytes and smooth muscle cells on the abluminal surface of the endothelium, and the ligand is also present in the extracellular matrix around vessels4. Therefore, to access this pool of Angpt1 circulating sTie2 would have to cross the endothelial permeability barrier to access the ligand. In a number of diseases where sTie2 is increased this is accompanied by an increase in Angpt213. As Angpt2 is able to bind Tie23 it is conceivable that where Angpt2 concentrations are increased sufficiently this ligand could compete with Angpt1 for binding to sTie2 and further decrease the ability of sTie2 to modulate Angpt1.It is possible that additional factors present 26. There have also been reports of Tie2 levels varying across different vascular beds32, although others report uniformity in Tie2 expression33. Our simulations show that sTie2 remains relatively ineffective at modulating Angpt1-activation of cellular Tie2 tetramerization even when cellular Tie2 levels are varied over a 100-fold range. Thus we found that increasing cellular Tie2 levels ten-fold further decreased the effectiveness of sTie2 to affect Angpt1 action. Decreasing cellular Tie2 levels in our simulations lowered the concentration of sTie2 required to cause 50% suppression of Angpt1-induced cellular Tie2 activation to 57 nM. Even this lowered concentration of sTie2 corresponds to a molar ratio of more than 300 fold sTie2:Angpt1. Further decreases in cellular Tie2 are likely to increase sensitivity to sTie2, though at such low levels of cellular Tie2 the signal from the cellular receptor will be negligible irrespective of sTie2.Cellular levels Tie2 are known to be increased at sites of angiogenesisin vivo based on the lack of association between circulating sTie2 and disease severity in septic and non-septic critically ill patients34.Our data do not discount Tie2 shedding as an important mechanism regulating endothelial responses to Angpt1. Clearly, proteolytic shedding of cell surface Tie2 would suppress the ability of Angpt1 to signal through Tie2 in cells in which shedding has occurred. Furthermore, it is possible that local very high concentrations of released sTie2 could accumulate abluminally or be present near the surface of endothelial cells undergoing proteolytic Tie2 shedding. In both of these situations Angpt1 actions could be inhibited in the locality of the cells undergoing shedding, although the ability of sTie2 to affect Angpt1 even locally could be blunted where Angpt2 is also locally released and able to compete for sTie2 binding. Furthermore, as discussed, the released ectodomain is unlikely to affect circulating or abluminal Angpt1 action elsewhere in the vasculature, minimizing systemic effects. Others have also questioned whether sTie2 has any systemic effect on Angpt1 action In contrast to natural circulating sTie2, a ligand trap consisting of a dimeric Tie2 ectodomain fusion protein was far more effective at blocking Angpt1-induced cellular Tie2 activation. This likely reflects the increased avidity of the dimeric trap providing it with a significant kinetic advantage relative to monomeric sTie2.In conclusion, this study shows that normal and pathological levels of sTie2 have little effect on Angpt1-induced Tie2 clustering and activation in endothelial cells. These data suggest that therapeutic targeting sTie2 in conditions where the ectodomain is elevated would have little if any effect. However, dimeric sTie2 is able to inhibit Angpt1 activation of endothelial Tie2 at much lower concentrations. Thus in contrast to endogenous monomeric sTie2, the dimeric engineered ectodomain could provide a route for suppression of angiopoietin action.992-Tie2 antibody were obtained from R&D Systems. Human umbilical vein endothelial cells (HUVEC) were maintained in Medium 200 supplemented with Low Serum Growth Supplement and 10% (v/v) foetal bovine serum. Monomeric sTie2 and dimeric sTie2 were purified from medium of transfected HEK-293 cells using the transfection and purification methods we previously described for dimeric sTie235. For monomeric sTie2, cDNA encoding Tie2 ectodomain residues 1–442 (including the secretory leader sequence) was obtained as previously described35 and cloned into the expression vector in frame with a C-terminal hexahistidine tag. cDNA encoding dimeric sTie2 was as described previously35. Following purification protein was dialysed against phosphate buffered saline (PBS) with 10% glycerol and protein concentration determined. Purity was assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining and was routinely better than 99%. All other materials were as previously described36.Recombinant human Angpt1, antibodies recognizing Tie2 extracellular domain and anti-phosphoY20 for Angpt1-Tie2 binding and corresponding numerical simulations were performed with Matlab (R2015b) SimBiology package and described in the Supplementary Information.The mathematical model based on biochemical kinetics37. Western blots were quantified by densitometric analysis of images. Cellular Tie2 was quantified by comparison of band density with the standard curve constructed from known amounts of recombinant Tie2 blotted and probed on the same membranes. The number of Tie2 molecules at the cell surface was derived from the percentage of total cellular Tie2 that is surface localized and total number of Tie2 receptors in each cell. The percentage of total Tie2 at the cell surface was determined by surface biotinylation as we previously described38 and quantification of biotinylated and non-biotinylated Tie2 by immunoblotting following separation with streptavidin-agarose.The number of Tie2 receptors in HUVEC was determined by immunoblotting. Cells were counted and then lysed in Laemmli sample buffer containing 100 mM dithiothreitol Cleared lysates were heated to 100 °C for 5 min before resolving proteins by SDS-PAGE. In parallel known amounts of recombinant Tie2 were also resolved by SDS-PAGE. Proteins were transferred to PVDF or nitrocellulose membranes and probed with Tie2 antibody. Immunoreactive proteins were detected with peroxidase conjugated secondary antibody and chemiluminescent detection992-Tie2 antibody. Immunoreactive proteins were detected with peroxidase conjugated secondary antibody and chemiluminescent detection37. Blots were stripped and re-probed with anti-Tie2 or anti-vinculin to assess loading. Bands on Western blots were quantified by densitometric analysis.Prior to ligand treatment HUVEC were incubated in serum-free medium for one hour. As appropriate, and indicated in Results, cells were treated with 0.18 nM Angpt1 in the absence and presence of sTie2 for 15 min. In preliminary experiments we confirmed that at 15 min Angpt1-induced Tie2 phosphorylation had plateaued Supplementary Fig. Supplementary Information


Cohesins are vital for chromosome organisation during meiosis and mitosis. In addition to the important function in sister chromatid cohesion, these complexes play key roles in meiotic recombination, DSB repair, homologous chromosome pairing and segregation. Egg-laying mammals (monotremes) feature an unusually complex sex chromosome system, which raises fundamental questions about organisation and segregation during meiosis. We discovered a dynamic and differential accumulation of cohesins on sex chromosomes during platypus prophase I and specific reorganisation of the sex chromosome complex around a large nucleolar body. Detailed analysis revealed a differential loading of SMC3 on the chromatin and chromosomal axis of XY shared regions compared with the chromatin and chromosomal axes of asynapsed X and Y regions during prophase I. At late prophase I, SMC3 accumulation is lost from both the chromatin and chromosome axes of the asynaptic regions of the chain and resolves into subnuclear compartments. This is the first report detailing unpaired DNA specific SMC3 accumulation during meiosis in any species and allows speculation on roles for cohesin in monotreme sex chromosome organisation and segregation. Cohesins have also been implicated in promoting nucleolar structure and function16.Cohesins are non-histone proteins of the structural maintenance of chromosomes (SMC) group. Traditionally they are described as the molecular glue that keeps sister chromatids physically attached after DNA replication during both mitosis and meiosis. More recently it has been discovered that cohesins are also central to a number of other fundamental mechanisms in chromosome biology  is the inclusion of the SMC3 subunit.Mitotic cohesin is a multiprotein complex comprising four subunits: SMC3, SMC1α, an α-kleisin subunit (Rad21), and a sister chromatid cohesin component . There are also meiosis specific isoforms including SMC1β, Rad21L and Rec8 (α-kleisin subunit isoforms), and the sister chromatid cohesin subunit isoform Stromal Antigen 3 (SA3/STAG3). Although many of the meiosis specific functions remain unclear it is known that some meiotic cohesin subunits appear to act in functionally specific combinations15. In mouse, RAD21L is recruited to the chromatin loops of the XY sex body, a pattern similar to the ɣH2AX repressive histone variant which marks silenced sex chromatin17. Another aspect of meiotic sex chromosome regulation involves an association with nucleoli which has been implicated in their organisation and transcriptional silencing due to the close association at pachytene18. Proteins that localise to the sex chromosomes during pachytene have also been observed to accumulate in nucleoli which may provide some insights into meiotic nucleolar function20. During mouse zygotene, nucleoli form on nucleolar organising regions (NORs) however at pachytene these nucleoli disassociate from the NORs, coalesce and migrate to the sex body until diplotene21. While similar nucleolar-sex chromosome organisation has been reported in multiple eutherian species24, metatherians25 and even invertebrates26, the purpose of this association remains unclear.In meiotic cells, immunostaining for cohesin complexes shows the same localisation as the SC to the extent that some investigations now report the CC to be part of the SC28 with seven known pseudoautosomal regions  that mediate pairing to form an alternating XY 10 sex chromosome chain during meiosis I30. The complexity of monotreme sex chromosomes and their homology to the avian Z raises fundamental questions about the mechanisms underlying meiotic pairing, recombination, evolution of meiotic sex chromosome inactivation (MSCI) and sex chromosome segregation. Our recently published findings indicate that the platypus SC may be different to other mammalian SCs with 3 significantly divergent copies of SYCP3 which are expressed at high levels in platypus testis31. However, there is currently no information about the role of cohesin proteins in platypus meiosis.The platypus carries a highly unusual sex chromosome system comprising 5X and 5Y chromosomesIn the present study we examine cohesin and synaptonemal complex dynamics during meiotic prophase I in platypus, with particular focus on the sex chromosome chain. We discovered a highly dynamic and specific reorganisation of accumulated cohesin associated with the formation of the sex chromosome complex and to a large nucleolar structure. Our finding of temporal and differential cohesin accumulation specifically on asynapsed sex chromosome chromatin loops implicates a possible role for cohesin in sex chromosome organisation at prophase I. Specifically we speculate roles in unpaired DNA-specific functions and/or establishment of configurations or markers for faithful alternate segregation of the 10 sex chromosome elements at anaphase I.In order to investigate the dynamics of SC formation in monotreme prophase I, we used antibodies raised against the central element protein SYCP1 and the cohesin subunit SMC3 to visualise chromosome axial cores. SMC3 is a component of all meiotic cohesin complexes discovered to date and platypus SMC3 is highly conserved with over 99% pairwise identity with human SMC3 at the amino acid level Figure . Western32 suggests that this is a large nucleolus.SYCP1 immunostaining showed an expected SC staining pattern in platypus prophase I cells, however the SMC3 antibody detected remarkably strong localised accumulations unlike mouse cells at the same stage Fig. . As this5 (Oa_Bb-152P15) and Y2 (CH236-145P9). Y5 and Y2 consistently reside within the regions of SMC3 accumulation, demonstrating that the platypus sex chromosomes experience significantly more cohesin loading in prophase I relative to the autosomes  which also lacks a central element signal (SYCP1). This is because the bulk of the sex chromosome DNA is not homologous and therefore doesn’t undergo SC-mediated synapsis involving SYCP1. As the intense SMC3 accumulations in platypus prophase I nuclei lacked SYCP1 this represented the location of sex chromosomes. This was confirmed by immuno-FISH on meiotic spreads using anti-SMC3 and anti-SYCP1 antibodies followed by two Y chromosome specific BAC probes targeting Y which al34  regions. After SMC3 immunostaining, imaged cell coordinates were recorded prior to a series of DNA FISH experiments using combinations of Xes Table . At mid es Table . By contes Table .Table 1P5 BACs and four X1 BACs, b) X1Y1 [PAR1] and Y4X5 [PAR8], c) X3 and Y3, d) Y3X4 [PAR6] and X2Y2 [PAR3], e) Y2 and Y5. This revealed that the X and Y specific sequences  consistently colocalised with the intense SMC3 domain which lacked central element staining  were positioned either outside or on the periphery of the SMC3 domain and in all cases had associated central element staining  five Xing Fig. . In conting Fig. .Figure 831 and the absence of platypus MSCI32 currently remain unclear in terms of pairing checkpoints or preparations for metaphase and segregation of the 10 sex chromosomes.This is the first detailed analysis of the organisation at male meiotic prophase I of the most complex known mammalian sex chromosome system. We observed massive and differential recruitment of cohesin specifically to unpaired parts of the sex chromosome chain and this specific and transient cohesin accumulation to the sex chromosome complex coincided with nucleolar association and progressive condensation. There were clear differences between platypus and mouse in the temporal and spatial cohesin accumulation dynamics including the rapid loss of SMC3 from specific regions of sex chromosomes. The relationship between these differences and our recent reports on the highly diverged platypus SYCP338. In some cases the former may simply be due to the fact there is only one chromosomal axis on the sex chromosomes whereas the signal observed between autosomal homologues is created from the close proximity of two axial cores41. Such a phenomenon was also observed for grasshopper B chromosomes42. In addition, generalisations are confounded by differing cohesin subunit loading dynamics occurring on AEs following pachytene38. Interestingly, in grasshopper preleptotene cells the X chromosome lacks SMC3, however by leptotene the levels on the X are similar to the autosomes and by pachytene the loading appears weaker than that observed on the synapsed autosomes39. In contrast, other mammals such as the armadillo have SMC3 signals on the unpaired sex chromosome axial cores which is indistinguishable in intensity from autosomal SMC343. In horse this is more pronounced, where the X shows greater SMC3 recruitment at axial cores than that seen on the autosomes44. Contrasting this are observations in rat where cohesins form scattered foci along the axial core length41. Despite the evolution of species specific variations in cohesin loading, platypus sex chromosomes stand out by the sheer magnitude of axial core SMC3 accumulation relative to autosomes.In platypus we observed a remarkably different XY cohesin recruitment programme in terms of the extent and specificity to unpaired regions which deviates significantly from that described for other species. Although there are exceptions, the majority of studied species show cohesin sex chromosome loading at pachytene as weaker, as for REC8 in mouse, or comparable to autosomes, such as STAG3 or SMC332. The association of the condensed sex chromosome chain with the nucleolus in combination with heavy cohesin loading specifically on the unpaired DNA invites the idea that MSCI may not be required to avoid checkpoint arrest. We are unable to test this hypothesis by inducing autosomal inversions or depleting SMC3 in platypus meiosis therefore rendering these ideas untestable. However, the identification of additional antibodies recognising DDR pathway components and detailed epigenetic profiles on the sex chromosomes in platypus may aid in understanding the observed cohesin dynamics in respect to meiotic progression and possibly segregation. It has been shown that NOR chromosomal positioning dictates pachytene nucleolar localisation during spermatogenesis45 and we observe here a conserved central nucleolar location due to the ‘interstitial’ NOR on platypus chromosome 6. The fact that in other species such as mouse the nucleolar-sex chromosome association begins later during pachytene may imply a requirement for platypus sex chromosomes association with nucleoli for chaperoning through the critical period of homologue synapsis21. Such a requirement is supported by the observation of Daish and colleagues that Y5 closely and consistently associates with the nucleolus from the earliest pachytene stages32 prior to chain contraction (see below) however this association was highly likely but also showed exceptions at early stages of pachytene as seen in Fig. 46 and also at the mating-type heterochromatic region of fission yeast where it is recruited by Swi6(HP1)47.In platypus early pachytene, the cohesin subunits SMC3 and STAG3 colocalise with autosomal SCs similar to other species however cohesin is then massively recruited to the unpaired sex chromosome axial cores. Only in platypus is the increased level of cohesin enrichment restricted to unpaired regions of the sex chromosomes. This raises the question of what cohesin may be doing differently on the unpaired DNA in terms of function and/or interactions and invites speculation on roles in protecting unpaired DNA from checkpoint surveillance machinery which may otherwise halt meiotic progression or simply one of facilitating condensation of the sex chromatin. We recently showed that therian-like sex chromosome transcriptional silencing (MSCI) evolved after the divergence of monotremes due to absence of repressive epigenetic marks such as γH2AX and DNA damage repair pathway components on the sex chromosome chain during late pachytene49. Nucleolar association is required for establishment of X chromosome inactivation in somatic cells50 demonstrating strong functional links between heterochromatin, cohesins, the nucleolus, and sex chromosomes, however the significance of this in the context of platypus meiosis remains unclear and currently is functionally untestable.Another area which requires investigation is the mechanism underlying the alternate segregation of the 10 sex chromosomes, an event which may be contingent on the sex chromosome specific accumulation and/or modification of structural complexes incorporating elements of both cohesin and synaptonemal complex components. There are examples to support such speculation in species with achiasmate sex chromosomes which utilise SC structural components to mediate faithful segregation52 which possibly represents an ancestral mode of organising heteromorphic sex chromosomes that predates the advent of sex chromosome specific silencing (MSCI). Interestingly, while monotremes and birds have sex chromosomes that share significant homology53 the heteromorphic chromosomes experience meiosis in males and females respectively. An example where proteins normally function to mediate homologous synapsis having roles in heteromorphic sex chromosome associations is seen in marsupials where SC proteins form a structure called the dense plate. The dense plate appears to mediate and maintain sex chromosome associations in the absence of a PAR54 and therefore further highlights the dynamism and functional flexibility of the meiotic machinery following sex chromosome evolution. To our knowledge however this is the first report showing such a marked association of SMC3 on XY specific chromatin.Our results show that reorganisation of SMC3 distribution coincides with sex chromosome chain condensation during pachytene. In birds the ZW pair undergoes synaptic adjustment and equalisationIn summary, we have shown that organisation of the platypus sex chromosome complex during prophase shows distinct and dynamic cohesin loading that is differentially loaded between paired and unpaired parts of the sex chromosome chain, for which we offer a simplified and functionally speculative model Fig. . Key poiThe authors confirm that all experiments were performed in accordance with relevant guidelines and regulations at the University of Adelaide. All experimental approaches were done according to the University of Adelaide biosafety and ethics committee regulations . Platypus samples were collected in 2002  and 2008 (AEC permit no. S-49-2006) at the Upper Barnard River  during the breeding season. Mouse testis were obtained from three week old animals (Swiss white).et al.30. Briefly the testis were placed in a petri dish containing 1 x PBS with 1 x Protease Inhibitor (Roche). The surrounding tunica albuginea was punctured and the testis material was teased apart using needles resulting in the release of meiotic cells and sperm from the ruptured tubules. A pipette was used to break up and flush the remaining material to ensure maximal release of cells. The resulting cell suspension was mixed with DMSO , slow cooled and stored in liquid nitrogen for future use.The method of meiotic sample preparation and storage was the same as previously described by Daish et al.55. Briefly, a 75 µL aliquot of the testis 10% DMSO suspension was thawed. Either 75 µL 0.3 M sucrose or 75 µL of 75 µM KCl was added to the testis material and left to sit at room temperature for 5 minutes. Next 50 µL of 1% PFA/0.15% Triton X-100 pH 9.5 was dropped onto slides and spread with a pipette tip. 20 µL of the testis material/hypotonic solution was then dropped onto the PFA coated slides. Slides were kept in a moist chamber for 2 hours and then washed in 50 mL of 1 X PBS followed by a final wash with water and wetting agent. Slides were air dried and used immediately for immunostaining or stored at −20 °C.Meiotic spreads of testis material were prepared by a method based on that previously described by Peters et al.52. Briefly, slides were blocked in 1 X PBS with 0.5% w/v BSA and 0.5% w/v milk powder three times for five minutes and incubated in a humid chamber with a primary antibody (1:200 dilution) in 1 X PBS/10% w/v BSA for either 2 hours at 37 °C or overnight at room temperature. Slides were then washed in 1 X PBS three times for five minutes each and blocked in 10% v/v goat serum in blocking buffer for three times for five minutes each and incubated at 37 °C for 2 hours in a humid chamber with a secondary antibody (1:400 dilution) in 10% v/v goat serum in blocking buffer . Finally slides were washed three times for five minutes in 1 X PBS, dipped in DAPI (1:5000 dilution) for one minute, washed twice with MQ water and mounted with Vectashield (Vecta Laboratories).Immunostaining was carried out as per Schoenmakers 39.To observe the central element we used an antibody  raised against the central element protein; Synaptonemal Complex Protein 1 (SYCP1). We employed the use of an SMC3 antibody  to visualise the axial core in platypus since it has been previously shown that SMC3 recruitment follows synaptic progressionIn Situ Hybridisation (FISH) based on the original protocol described by McDougall et al. (1972) was optimised for PFA fixed meiotic material. Briefly, BAC DNA probes were directly labelled using Klenow and a random 9mer primer overnight with either SpectumOrange or SpectrumGreen 2′deoxyuridine-5′-trisphosphate (Abbott Molecular). To reduce the background signal on slides, labelled probes were subsequently co-precipitated with salmon sperm and sonicated platypus male genomic DNA. Pellets were dissolved in deionised formamide and 2 X hybridisation buffer containing dextran sulfate. Slides containing the PFA fixed meiotic material were dehydrated in an alcohol series (70–100% ethanol) for 5 minutes each, treated with RNase A (100 µg/mL) for 30 minutes at 37 °C, pepsin  for 10 minutes at 37 °C, fixed in formaldehyde  in PBS/50 mM MgCl2, dehydrated in an alcohol series (70–100% ethanol) for 5 minutes each, denatured in 70% formamide/2 X SSC at 70 °C for 7 minutes for the first FISH and 3 minutes for each subsequent FISH on the same slide, dehydrated (70–100% ethanol) for 5 minutes each and air dried ready for hybridisation with the labelled probe. The probes were applied to the dry slide and covered with a coverslip, sealed with rubber cement and incubated overnight at 37 °C in a moist chamber. The coverslip was removed and slides were washed three times in 50% formamide/2 x SSC followed by 2 x SSC, once in 2 X SSC at 42 oC for 5 minutes, once at 60 oC in 0.1 X SSC for 5 minutes and finally once at 42 oC in 2 X SSC for 5 minutes. Slides were then stained with DAPI for one minute, washed twice with MQ water and mounted with Vectashield (Vecta Laboratories).A method of Fluorescence http://www.gimp.org/) and ImageJ .Slides were visualised using a Zeiss AxioImager 2.1 microscope equipped with a 10x ocular and 10x, 20x, 63x and 100x objective lenses. Fluorescent tags were visualised using 3 filters: DAPI, for DAPI stained DNA; GFP, for SpectrumGreen and Alexa 488 and; DS red, for SpectrumOrange and CY3. Images were recorded via an Axiocam CCD-camera and Zeiss Axiovision software. Unless otherwise stated, images were captured using the 100x objective lens, appearing as 1000x with the 10x ocular. Images were processed using Axiovision (Zeiss), GIMP 2.8 (Supplementary Data


We previously demonstrated the effects of Ankaferd hemostat (AH) on human umbilical vein endothelial cells (HUVECs) in Turkish Journal of Hematology [1]. Endothelial cells adhered to each other within seconds and critical intracellular transcription factors were activated just after the application of AH (5 µL) to the HUVECs . Rapid vWe further determined that the cellular effects of AH on HUVECs are clinically important in pharmacobiological hemostasis ,2,3. End


HOXA10 and HOXB13 revealed association with Epithelial-Mesenchymal Transition (EMT). While HOXA10 expression correlated positively with E-Cadherin and negatively with Vimentin expression, HOXB13 showed the reverse trend. Chromatin immunoprecipitation study in vitro revealed the ability of E7 to increase HOX gene expression by epigenetic regulation, affecting the H3K4me3 and H3K27me3 status of their promoters, resulting from a loss of PRC2-LSD1 complex activity. Thus, besides identifying the deregulated expression of HOX cluster members in HPV16 positive cervical cancer and their association with EMT, our study highlighted the mechanism of HPV16 E7-mediated epigenetic regulation of HOX genes in such cancers, potentially serving as bedrock for functional studies in the future.The Homeobox (HOX) genes encode important transcription factors showing deregulated expression in several cancers. However, their role in cervical cancer pathogenesis, remains largely unexplored. Herein, we studied their association with Human Papillomavirus type 16 (HPV16) mediated cervical cancers. Our previously published gene expression microarray data revealed a significant alteration of 12 out of 39 HOX cluster members among cervical cancer cases, in comparison to the histopathologically normal controls. Of these, we validated seven  by quantitative real-time PCR. We identified decreased HOXA10 expression as opposed to the increased expression of the rest. Such decrease was independent of the integration status of HPV16 genome, but correlated negatively with E7 expression in clinical samples, that was confirmed  Cervical cancer is the fourth leading cancer among women globally with HPV16 infection alone associating with ~50% of the cases –8. Such HOX genes are a family of transcription factors that show deregulated expression in a variety of cancers and known as targets of PRC2-complex during embryonic development –11. The Since HOX cluster members function as transcription factors, the common thought was that these show enhanced expression in cancers driving carcinogenesis by activating the downstream signalling cascades. However, several studies reported reduced expression of HOX cluster encoded molecules. Aberrant expression of HOX cluster genes is integral to a network of regulatory mechanisms involved in normal adult tissue homeostasis, and maintenance and activation of stem cell self-renewal process, crucial to malignant transformation –20. The Therefore, in this study, we focussed on elucidating the expression profile of HOX cluster member genes associated with cervical cancer pathogenesis, and their probable association with the Epithelial Mesenchymal Transition (EMT). We also aimed at identifying the association of HOX cluster expression with viral factors like HPV16 integration status and E7 expression among the cervical cancer cases. Finally, we elucidated the role of HPV16 E7 in epigenetically affecting the expression of HOX cluster genes through concomitant functional abrogation of PRC2-LSD1 complex in cervical cancer cell lines. Towards this, we estimated the H3K4me3 and H3K27me3 status of the promoters of the HOX genes with altered expression.To identify the HOX cluster members with deregulated expression among HPV16 positive cervical cancer cases in comparison to the histopathologically normal control samples (HPV negative and HPV16 positive), microarray data generated on 20 HPV16 positive cervical cancer cases, 11 HPV16 positive histopathologically normal samples and 11 HPV negative histopathologically normal samples (Accession No: GSE67522) was used. The histopathologically normal samples were grouped together for comparison, as the HPV negative and HPV16 positive normal samples did not reveal any differentially expressed genes as described in our previous study .Out of 39 HOX cluster genes, the microarray analysis revealed expression levels of 31 genes. Of these, 12 members of the HOX cluster portrayed significant differential expression among the cervical cancer cases, as compared to the histopathologically normal controls Figure ..We validated the expression profile of seven out of the twelve deregulated HOX cluster members, HOXA10, HOXA13, HOXB13, HOXC8, HOXC9, HOXC11 and HOXD10, using SYBR green based quantitative real-time PCR. For such analysis, we used a sample set of seventy cervical cancer cases  and thirty histopathologically normal cervical samples (11 HPV16 positive and 19 HPV negative). Such analysis, confirmed significant altered expression of all seven HOX cluster members among the cervical cancer cases, in comparison with the HPV negative control samples . Of thesDeregulated expression of several HOX genes in cervical cancer cases prompted us to interrogate the impact of such changes in cervical cancer pathogenesis. Existing reports –24, highE-Cadherin expression was significantly decreased among the cervical cancer cases  as compared to the HPV negative control samples. The cervical cancer samples with episomal HPV16 genome showed a reduction by 11.79-fold (p=0.03), while the cervical cancer samples with integrated HPV16 genomes showed a reduction by 6.02-fold (p=0.04) Figure . HoweverExpression of both the EMT markers, E-Cadherin and Vimentin, showed correlation with the expression of the HOX cluster members, HOXA10 and HOXB13 . While Hin vitro, where HOXA10 showed significantly reduced expression  in C33A cells transfected with E7 expressing plasmid, as compared to untransfected C33A cells . However, HPV16 E7 seemed to play an important role in the activation of HOX cluster genes despite the fact that some of the HOX cluster members failed to show any significant correlation with HPV16 E7.The HOX cluster members that revealed altered expression in cervical cancer cases in our study, also show deregulated expression in one or more of the other cancer types. Similar to our observations, HOXA13 shows enhanced expression in oesophageal squamous cell carcinoma, gastric cancer and hepatocellular carcinoma –35. HoweThe increased expression of majority of the HOX cluster members was also in line with our hypothesis of HPV16 E7 driven abrogation of PRC2-LSD1 complex activity leading to loss of gene silencing. A significant correlation of HOXA10 and HOXD10 expression with HPV16 E7 expression highlighted the impact of E7 on HOX gene expression. One of the possible mechanism of HOXA10 reduction could be through HPV16 E7-dependent activation of DNMT1 leading to HOXA10 promoter methylation , 41. On in vitro observations could be due to the heterogeneity of cancer tissues as opposed to the homogenous pool of cells in vitro. The gain of H3K4me3 and loss of H3K27me3 marks at the promoters of HOX cluster genes could explain the activated expression of such genes, under the influence of HPV16 E7 expression. McLaughlin-Drubin et al., [Although E7 expression did not show significant correlation with the expression of majority of the HOX cluster members among the cervical cancer samples, E7 overexpression in HPV negative cervical cancer cell line resulted in the activation of HOX cluster genes. Such differences in clinical and  et al.,  had earl et al., . In this et al., .In conclusion, along with identifying altered expression of HOX cluster genes and their association with metastasis in HPV16 related cervical cancers, our study also highlighted the master regulatory role of HPV16 E7 in modulating the expression of HOX cluster genes. Such expression alteration predominantly involved enrichment of H3K4me3 and loss of H3K27me3 marks at the gene promoters. Thus global studies employing ChIP-Seq for identifying the status of H3K4me3 and H3K27me3 marks at the gene promoters, along with RNA-Seq, seem to be important to identify the concomitant changes in the transcriptome brought about by HPV16 E7. This would further identify and confirm the role of HPV16 E7 in the epigenetic regulation of host cellular transcription. Such studies would also help in identifying molecular targets for therapy of HPV-active cervical cancer, which have distinctly different molecular phenotypes from HPV-inactive cervical cancer, as has been identified in a recent report studying TCGA cervical cancer data .The healthy control cervical biopsy samples were derived from married women aged 36 – 70 years (median: 52 years) without any prior history of cervical dysplasia/malignancy, from Calcutta Medical College and Hospital, Kolkata, West Bengal, India and were histopathologically confirmed as normal. The Cervical cancer samples used for this study were derived from married subjects aged 34 – 65 years (median: 52 years) attending a cancer referral hospital . The cervical cancer biopsy samples were histopathologically confirmed as invasive squamous cell carcinomas  categorised using the WHO classification and clinically diagnosed as stage III and above as per FIGO classification. Samples included in the different study groups did not differ significantly in terms of their median age.The samples were collected with informed consent from the participants, approved by the ethical committee for human experimentation of the National Institute of Biomedical Genomics (NIBMG) and frozen post collection. We performed all experiments in accordance with the approved ethical guidelines and regulations. All such clinical samples were tested for their HPV/HPV16 status by PCR based detection as described earlier . Confirm-12 v4 Expression Bead Chip array based platform generated by our laboratory [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67522). This data was used to identify the distinct differences of HOX cluster members at the gene expression level, between the HPV16 positive cervical cancer cases and the histopathologically normal control samples.The microarray data was generated using Illumina HumanHTboratory  and submRNA isolation, reverse transcription and quantitative real-time PCRs were all performed, as described previously . The pri2. C33A cells were transfected with1 μg of plasmid pcDNA3.1-HPV16 E7 vector generated in our laboratory [HPV negative cell line C33A and HPV16 positive cell line SiHa were cultured in DMEM supplemented with 10% FBS, 50 Units/ml of penicillin and 50 μg/ml of streptomycin at 37°C and 5% COboratory , using L3) and incubated at room temperature for 30 mins with agitation. The DNA was reverse cross-linked by adding 12μl of 5M NaCl per 100μl elute and incubated at 65°C overnight, followed by RNase A treatment and purified using PCR purification kit.SiHa and C33A cells were cultured as described above. For the analysis of H3K4me3 and H3K27me3 marks, we used SiHa cells and C33A cells, untransfected or transfected (HPV16 E7 expressing). The cells were cross-linked using formaldehyde at 1% final concentration and incubated at room temperature for 10 mins. The cross-linking was quenched using glycine at a final concentration of 0.125M and incubated at room temperature for 5 mins, followed by washing twice with 1X PBS. The cells were harvested by scraping in 1X PBS (with Protease Inhibitor cocktail) and pelleting the cells by centrifugation at 300xg for 5 mins, at 4°C. The cells were re-suspended in 1ml of SDS lysis buffer  and incubated on ice for 15 mins. The lysate was sonicated at 30% amplitude, 14 × 10 secs pulses, 20 secs pauses, followed by centrifugation at 14,000 rpm at 4°C. The lysate was pre-cleared with 80μl of 50% slurry of Protein A Agarose beads for 60 mins at 4°C, followed by centrifugation at 4,000 rpm for 1 min at 4°C. Specific antibody (5μg) was added to the supernatant and precipitated overnight at 4°C with rotation. After overnight rotation, 60μl of blocked Protein A Agarose beads  were added to the antibody/protein complex, followed by rotation for 60 mins at 4°C. The beads were pelleted by centrifugation at 1,000 rpm for 1 min at 4°C, followed by sequential washing with 1X Low Salt Immune Complex wash buffer , 1X High Salt Immune Complex wash Buffer , 1X LiCl Immune Complex wash Buffer  and 1X TE . The beads were eluted with freshly prepared elution buffer  was performed to check for statistical significance of the changes in expression levels, as the ΔCt values did not follow normal distribution. Multiple sets of experiments using cell line model were compared using ANOVA for statistically significant differences in expression levels between the experimental categories. All statistical analyses were done using SPSS v16.0. The p values were reported after performing Bonferroni multiple testing corrections.


The images for S1 FigDkk3 cds. (B) Validation of the Dkk3 exon 2 and exon 3 primer sets using increasing concentrations of 1st strand cDNA primed total RNA isolated from two independent mouse astrocyte preparations. Each data point determined in triplicate. (C) Dkk3 mRNA levels were normalized to GAPDH mRNA. Data shown as (mean ± SE) from 3 independent experiments.(A) The position of the PCR primer pairs used for amplification of exon 2 and exon 3 shown on the (TIF)Click here for additional data file.S2 FigDkk3 gene. TSS2 is positioned upstream of the forward LoxP site (black box) of the gene and the downstream LoxP site (in black) is positions 35 nt upstream of exon 3. Position of the CFP234 5’RACE primer indicated by arrow. (B) Sequence of the 5’UTR of the Cfp mRNA captured by 5’RACE highlighted in yellow.(A) Map of the insertion of the CFP promoter trap in gene-edited intron 2 of the (TIF)Click here for additional data file.


Fig 7 contains an error on the x-axis. Fecal testosterone metabolites (fTM) and fecal glucocorticoid metabolites (fGM) values that are plotted on day 2 after an ACTH injection should be plotted on day 1, and the values that are plotted on day 1 should be plotted on day 2. Please see the corrected


C9orf72) is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). While functional haploinsufficiency of C9orf72 resulting from the mutation may play a role in ALS/FTD, the actual cellular role of the protein has been unclear. Recent findings have now shown that C9orf72 physically and functionally interacts with multiple members of the Rab small GTPases family, consequently exerting important influences on cellular membrane traffic and the process of autophagy. Loss of C9orf72 impairs endocytosis in neuronal cell lines, and attenuated autophagosome formation. Interestingly, C9orf72 could influence autophagy both as part of a Guanine nucleotide exchange factor (GEF) complex, or as a Rab effector that facilitates transport of the Unc-51-like Autophagy Activating Kinase 1 (Ulk1) autophagy initiation complex. The cellular function of C9orf72 is discussed in the light of these recent findings.Hexanucleotide repeat expansion in an intron of Chromosome 9 open reading frame 72 ( C9orf72; DeJesus-Hernandez et al., Amyotrophic Lateral Sclerosis (ALS) is a progressive, fatal neurodegenerative disease affecting motor neurons . The most prominent feature of the protein is the presence of a “Differentially expressed in normal and neoplastic cells” (DENN) domain  translation of the hexanucleotide expansion  should be pursued. Understanding of how the mutant hexanucleotide expansion of C9orf72 affect neuronal membrane traffic and autophagy would benefit from analyses using appropriate cellular and While the manuscript was in its final stage of review, Chen and colleagues reported a C9ORF72/SMCR8/WDR41-containing complex that also contain ATG101 . A C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy. Sci. Adv. 2(9): e1601167). The C9orf72 complex has Rab39B GEF activity, and also regulates Ulk1 levels as well as autophagic activity.BLT conceived and wrote the article.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We discover that two different HS modifying enzymes, Hs2st and Hs6st1, indeed differentially modulate the properties of emerging Fgf8 protein concentration gradients and the Erk signalling output in response to Fgf8 in living tissue in ex vivo cultures. Both Hs2st and Hs6st1 are required for stable Fgf8 gradients to form as rapidly as they do in wild-type tissue while only Hs6st1 has a significant effect on suppressing the levels of Fgf8 protein in the gradient compared to wild type. Next we show that Hs2st and Hs6st1 act to antagonise and agonise the Erk signalling in response to Fgf8 protein, respectively, in ex vivo cultures of living tissue. Examination of endogenous Fgf8 protein and Erk signalling outputs in −/−Hs2st and −/−Hs6st1 embryos suggests that our ex vivo findings have physiological relevance in vivo. Our discovery identifies a new class of mechanism to tune Fgf8 function by regulated expression of Hs2st and Hs6st1 that is likely to have broader application to the >200 other signalling proteins that interact with HS and their function in neural development and disease.Fibroblast growth factor (FGF) morphogen signalling through the evolutionarily ancient extracellular signalling-regulated kinase/mitogen activated protein kinase (ERK/MAPK) pathway recurs in many neural and non-neural developmental contexts, and understanding the mechanisms that regulate FGF/ERK function are correspondingly important. The glycosaminoglycan heparan sulphate (HS) binds to FGFs and exists in an enormous number of differentially sulphated forms produced by the action of HS modifying enzymes, and so has the potential to present an extremely large amount of information in FGF/ERK signalling. Although there have been many studies demonstrating that HS is an important regulator of FGF function, experimental evidence on the role of the different HS modifying enzymes on FGF gradient formation has been lacking until now. We challenged  Summary: Regulating the fine structure of cell surface carbohydrates provides a mechanism for fine tuning the formation of morphogen gradients in the embryonic mouse fore-brain. Fibroblast growth factors (FGFs) are secreted signalling proteins that function as morphogens by forming protein concentration gradients emanating from focal sources to elicit dose-dependent outcomes . The bron HS polymer is synthesised by EXT enzymes and modified by enzymatic addition and removal of sulphate groups by heparan sulphotransferases (HSTs) and sulphatases (SULFs), respectively. HS modifying enzymes underwent evolutionary expansion from the origin of multicellular animals suggesting a role in evolving tissue complexity. Mammals have 17 HS modifying enzymes classed into five categories  according to exactly where on the uronic acid–glucosamine disaccharide residue they add or remove sulphate groups. Many HS modifying enzymes are expressed in developing brain, pointing to functional importance in neural development , while Hs6st1 sulphates position 6 of glucosamine (6-O HS sulphation). We turned to the tractable system provided by Fgf8 signalling in the ventricular zone (VZ) of the cortico-septal boundary (CSB) at the developing mouse embryonic day (E)14.5 telencephalic midline to investigate the function of Hs2st and Hs6st1 in Fgf8 gradient formation, and the Erk response to Fgf8 in living tissue. Severe developmental phenotypes map to the CSB when FGF8/Fgf8 or HS are disrupted in humans or mice, indicating their functional importance in this region  from the tissue. According to the model, reaching a steady-state protein concentration gradient is key for the interpretation of the gradient by cells . FGF proelopment . Furtherll lines . Althougs region . Hs2st−/ embryos . This woHs2st and Hs6st1 in Fgf8 gradient formation modulation in vivo is difficult as there are many variables that are not tenable to experimental control, such as the amount of Fgf8 protein emanating from the source. Therefore, we designed an ex vivo bead assay in which we challenged slices of embryonic forebrain in which HS was enzymatically removed (Heparanase treatment) or lacking functional HS modifying enzymes Hs2st (Hs2st−/− tissue) or Hs6st1 (−/−Hs6st1 tissue) with a constant amount of Fgf8 to probe the formation of a Fgf8 protein gradient through time. The ex vivo assay allowed us to probe Fgf8 gradient emergence in much smaller timescales, that is impossible to assay in vivo, providing a higher temporal resolution of Fgf8 gradient formation.Fgf8 gradient formation involves the production of Fgf8 at the source, followed by Fgf8 transport through the extracellular matrix in the tissue, activation of the signalling pathway through receptor binding of responding cells, and finally Fgf8 protein clearance. The emergence of the Fgf8 protein gradient could be modulated by HS modifying enzymes at any step of the pathway. Dissecting the role of Fgf8 mRNA expression is restricted to the angle between the cerebral cortex and the septum at the CSB, identifying this region as the source of Fgf8 protein in vivo (ex vivo by embedding beads infused with recombinant Fgf8 protein (or BSA control) into the VZ at the CSB angle of telencephalic midline tissue explants (dissected region indicated by red dashed line in [Fgf8]) varies with distance (d) from the Fgf8-bead edge .In E14.5 mouse telencephalon, WT) tissue was challenged with an Fgf8-soaked bead, the concentration of Fgf8 protein ([Fgf8]) is highest closest to the Fgf8-bead and decays with increasing distance from the bead edge ([Fgf8] [expressed as arbitrary units (AU)] in the tissue against distance from the bead at each time-point produces classic decay curves with [Fgf8] falling non-linearly with increasing distance from the bead. Comparison of these curves shows no change over the 1-4 h period, indicating that a steady state gradient is achieved <1 h after introducing the Fgf8-bead into the tissue  gradient over time by removing HS (pre-treating WT tissue with heparanase) or using tissue from −/−Hs2st or −/−Hs6st1 embryos. Representative images of Fgf8 immunofluorescence show that these three conditions resulted in an unstable [Fgf8] gradient that does not achieve the WT steady state between 1 and 4 h  indicating impaired ability for forming a steady state [Fgf8] gradient. There were also significant deviations from the WT [Fgf8] gradient, although here, heparanase-treated, −/−Hs2st, and −/−Hs6st1 experiments showed different effects. Following heparanase treatment, the spatial concentration curve was higher than WT after 1 h and then dropped rapidly, becoming lower than WT by 2 h and barely detectable after 4 h, all of these differences were significant. Both −/−Hs2st and −/−Hs6st1 tissue also formed non-steady-state [Fgf8] gradients. However it is important to note that the [Fgf8] gradient in −/−Hs2st tissue was not significantly different to the WT [Fgf8] gradient after 4 h in culture, although the [Fgf8] gradient fluctuated in the earlier time-points. The spatial concentration curve formed in −/−Hs2st tissue was significantly higher than WT after 1 h, lower than WT after 2 h, and not significantly different to WT after 4 h  (−/−Hs2st and −/−Hs6st1 experiments) (These data demonstrate that the formation of a steady-state eriment) E,I,M,Q, [Fgf8] gradient is determined by three independent factors: [Fgf8] at the source; the amount of Fgf8 protein in the gradient (the amplitude); and the rate at which [Fgf8] decays with increasing distance from the source (the decay). In these experiments [Fgf8] at the source is constant (see below). We next asked whether the HS manipulations contribute to changing the [Fgf8] gradient by modulating its amplitude, decay, or both.The [Fgf8] gradient from the area under the curves in WT . Comparing [Fgf8] amplitude between time-points of each condition, the amplitude of WT tissue remained constant over time with no significant differences, indicating a steady-state amplitude was achieved as early as 1 h of culture . Heparanase treatment resulted in amplitude not significantly different to WT for the first 2 h followed by a significant drop to very close to detection threshold ; the more negative the slope the more rapid the [Fgf8] decay as a function of distance (WT and any HS condition at any time-point (Table S1C), indicating that none of the HS manipulations described here have a significant impact on [Fgf8] decay as a function of distance from the source. Interestingly, although the gradient of −/−Hs6st1 was significantly different from WT at the 4 h time-point, neither the [Fgf8] amplitude or [Fgf8] decay , gives [Fgf8] at the source which is the surface of the Fgf8 bead (<1 µm from bead edge), and there were no significant differences  except between WT and heparanase treated tissue at the 4 h time-point. This indicates the bead [Fgf8] was constant in all conditions and time-points (except heparanase treated cultures after 4 h). This was confirmed by direct quantification of bead [Fgf8] , so bead [Fgf8] was not a variable contributing to differences observed between other conditions and time-points.The decay of a protein gradient is described by different equations depending on both diffusion kinetics and whether the degradation rate, for example clearance of Fgf8 by RME, is linear or non-linear as a function of distance from the source , as opposed to modulating the decay of Fgf8 as a function of distance from the source ([Fgf8] decay). Critically, our analysis found that Fgf8 behaved differently in living −/−Hs2st and −/−Hs6st1 tissue with a much stronger effect in −/−Hs6st1 tissue, indicating that the Hs6st1 plays the dominant role in supressing Fgf8 levels.To conclude, the rapid (<1 h) formation of a steady state WT cultures, pErk forms a gradient with the highest concentration of pErk ([pErk]) closest to the bead ([pErk] gradient after 4 h (red=black>>blue lines in Table S2). Our analysis quantifies both Fgf8 and pErk signals at each distance from the Fgf8-bead, and these values were combined in a [pErk] (y-axis) vs [Fgf8] (x-axis) plot to generate a dose response curve showing the pErk response elicited by different [Fgf8] in the tissue , and the [Fgf8]/[pErk] dose response shows strong de-sensitisation to Fgf8 . The [Fgf8]/[pErk] dose response shows that −/−Hs2st tissue becomes progressively more sensitive to Fgf8 over time  significantly reduces the  to Fgf8 J. In cone points C and thecultures D where t compare I to L. Wex vivo experiments showed us how living tissue reacts when challenged with an implanted bead acting as an experimentally introduced source of Fgf8, likely at non-physiological levels. HS was found to be critical for [Fgf8] gradient formation and the Erk response to Fgf8 as the loss of HS resulted in no detectable [Fgf8] gradient after 4 h in culture and loss of Erk response to Fgf8. Hs2st was found to play a minor role in the maintaining a stable Fgf8 gradient as removal of Hs2st resulted in [Fgf8] fluctuations during [Fgf8] gradient formation before eventually reaching WT [Fgf8] levels, albeit taking a longer time than WT tissue. Meanwhile, Hs6st1 was found to be needed to suppress [Fgf8] during the [Fgf8] gradient formation. The ex vivo experiments also showed that Hs2st antagonises the Erk response to Fgf8, while Hs6st1 is needed to promote Erk response to Fgf8.The ex vivo for the differential control exerted by Hs2st and Hs6st1 on the Fgf8 protein concentration gradient and the pErk response it elicits apply in vivo? In order to address this, we examined the expression of Hs2st, Hs6st1, Fgf8, Fgfr1, and pErk in the CSB region of E14.5 WT, −/−Hs2st, and −/−Hs6st1 embryos. Hs2st and Hs6st1 are expressed at relatively high levels in the VZ at the CSB angle  provided a constant [Fgf8], so the effects we describe can be attributed to perturbations on Fgf8 diffusion and/or degradation. [Fgf8] varies with distance from the source as a power law gradient, a model describing the distribution of a protein diffusing from a source and being cleared from the tissue in a concentration-dependent manner, for example higher Fgf8 concentrations causing increased Fgf8 degradation through an Fgf8-sensing feedback mechanism  or leaving HS intact but losing the function of different HS modifying enzymes (−/−Hs2st and −/−Hs6st1 experiments) all caused significant fluctuations in the gradient amplitude (amount of Fgf8 in the gradient) through time that were not observed in WT cultures. Although the fluctuations in Fgf8 gradient amplitude observed in both −/−Hs2st and −/−Hs6st1 tissue were significant, we observed an initial surge in amplitude at the 1 h time-point in −/−Hs2st, while the surge was only observed in the 2 h time-point in −/−Hs6st1. The Fgf8 amplitude of both mutant tissues reached WT levels after 4 h in culture. This prompted the question of whether the differences we observed in the Fgf8 gradient formation and the Erk response to the Fgf8 gradient due to the loss of Hs2st and Hs6st1 was due to a timing issue of when steady state is achieved, or that Hs2st and Hs6st1 have fundamentally different roles in the process of establishing the Fgf8 gradient and the Erk response. Analyses of the Fgf8 bead cultures for a longer time period would give further insight into this question, however culturing explants with Fgf8 beads for periods longer than our current study is not viable as Fgf molecules have a relatively short half-life. For example, Fgf10  has a half-life of ∼5 h. This is supported by other studies which performed similar Fgf bead assays using similar time-frames as this study . In contrast to amplitude, the [Fgf8] decay, revealed by the slope of log [Fgf8] vs log distance power function plots, was not significantly altered by removing HS or losing the function of Hs2st or Hs6st1. This indicates that the mechanism regulating the decay of Fgf8 as a function of distance from the source, including feedback mechanisms regulating decay, were not significantly affected by loss of Hs2st or Hs6st1 function. Net [Fgf8] decay is determined both by rates of diffusion and degradation and dissecting the regulation of each component, particularly by Hs6st1 which has the greater effect on Fgf8 concentration, is a topic for future research.The formation of a steady state protein concentration gradient requires the balancing of three factors: concentration at the source; diffusion through the tissue; and degradation in the tissue. In our echanism . We findis study . TherefoWT tissues form a steady-state [Fgf8] gradient in <1 h when challenged with an exogenous Fgf8 bead. While the loss of Hs2st affected [Fgf8] amplitude stability  CSB in addition to higher Erk activation when compared to WT. However, this higher Erk activation was not much higher than the Erk activation in −/−Hs2st tissue. We reasoned that the increase in Erk activation found in −/−Hs6st1 tissue in vivo was due to the increased levels of Fgf8 (∼threefold increase) found in vivo  supports the hypothesis that regulating specific HS sulphation via expression of HS modifying enzymes provides a more nuanced mechanism to control Fgf8 morphogen function than globally regulating HS synthesis. Removing HS effectively shuts down Fgf8 function while removing Hs2st or Hs6st1 play more modulatory roles. Furthermore, in contrast to the promiscuous interaction of HS with all paracrine FGFs, we find that Hs2st and Hs6st1 each have distinct effects on Fgf8 and may well have similarly specific relationships with other FGFs, and some of the >200 HS interacting signalling molecules comprising the heparanome in mammals  as opposed to its modification  and LacZ-IRES-hPLAPHs6st1 (Hs6st1−) mutant alleles were maintained on a CBA background ; goat anti rabbit Alexa Fluor 488 (1/200) (Invitrogen); and Fgf8 staining was detected via TSA Plus Fluorescence Systems (Perkin-Elmer). LacZ staining performed as described previously  and immunofluorescence  were performed on escribed . Primaryeviously . N≥3 forWT, −/−Hs2st, or −/−Hs6st1 explants, some of which were pre-treated with Heparanase (Sigma) for 2 h at 37°C, were cultured for 1, 2, or 4 h prior to fixation, sectioning (10 µm frozen sections) and processing for Fgf8 and pErk immunofluorescence and DAPI counterstaining.Modification of methods described by Fig. S1 for illustration of ROI placement) and used to quantify signal intensity at increasing distances from the edge of the bead into the tissue (for Fgf8 and pErk gradient quantification) and in the outer edge of the bead (for Fgf8 only). Data were then combined and graphed using GraphPad Prism 6 (GraphPad). Images from all genotypes of each time point experiment were imaged, processed and analysed in parallel to minimise technical variation for comparison between genotypes of the same time point. Values from BSA control cultures were subtracted to remove background fluorescence. Fgf8 fluorescence in in vivo sections were quantified using IMAGEJ. Quantification of Fgf8 in vivo: mean fluorescence for boxes of 100×180 µm drawn at the CSB were obtained for at least 2 sections per embryo with N being number of embryos analysed. WT, N=4; −/−Hs2st, N=3; −/−Hs6st1, N=3.Fluorescent sections were imaged using Leica AF6000 epifluorescence microscope connected to a DFC360 camera on constant exposure settings for three channels, DAPI, Fgf8, and pErk, and analysed using IMAGEJ. IMAGEJ was used to draw 10-µm wide regions of interest (ROI) on each image  and culture durations  using Kolmogorov–Smirnov (KS) test with Bonferroni correction to compare [Fgf8] and [pErk] gradients and ANOVA followed by Holm-Sidak post hoc test to compare [Fgf8] amplitude, [Fgf8] at the bead and slopes of Fgf8 distribution. The Fgf8 and pErk fluorescence intensity were normalised to the value at the point closest to the bead of the 1 h time point in WT tissue, which was designated as 100%. Statistical comparisons were made between WT, −/−Hs2st and −/−Hs6st1 for Fgf8 fluorescence intensity of in vivo sections using one-way ANOVA followed by Dunnett's post hoc test.Statistical comparisons were made between different tissue conditions (


ZEB1 coding region mutations.To identify the genetic basis of posterior polymorphous corneal dystrophy (PPCD) in families mapped to the PPCD1 locus and in affected individuals without OVOL2 was screened in the PPCD family in which linkage analysis established the PPCD1 locus and in 26 PPCD probands who did not harbor a ZEB1 mutation. Copy number variation (CNV) analysis in the PPCD1 and PPCD3 intervals was performed on DNA samples from eight probands using aCGH. Luciferase reporter assays were performed in human corneal endothelial cells to determine the impact of the identified potentially pathogenic variants on OVOL2 promoter activity.The promoter, 5’ UTR, and coding regions of OVOL2 mutation analysis in the first PPCD1-linked family demonstrated segregation of the c.-307T>C variant with the affected phenotype. In the other 26 probands screened, one heterozygous coding region variant and five promoter region heterozygous variants were identified, though none are likely pathogenic based on allele frequency. Array CGH in the PPCD1 and PPCD3 loci excluded the presence of CNV involving either OVOL2 or ZEB1, respectively. The c.-307T>C variant demonstrated increased promoter activity in corneal endothelial cells when compared to the wild-type sequence as has been demonstrated previously in another cell type.OVOL2 promoter variant c.-307T>C was herein identified in the original family that established the PPCD1 locus. However, the failure to identify presumed pathogenic coding or non-coding OVOL2 or ZEB1 variants, or CNV involving the PPCD1 and PPCD3 loci in 26 other PPCD probands suggests that other genetic loci may be involved in the pathogenesis of PPCD.Previously identified as the cause of PPCD1, the  ZEB1) on chromosome 10 that likely result in ZEB1 haploinsufficiency. Bas BasOVOL2ZEB1 mutation was not identified were submitted to the UCLA Clinical Microarray Core for array comparative genome hybridization (aCGH) to identify copy number variation (CNV) using a custom Agilent 8x60K array . The custom array was designed for the interrogation of: a 16.7 Mb region (hg19: 17.3 Mb– 34.0 Mb) on chromosome 20 encompassing the refined PPCD1 interval and approximately 2.7Mb (0.54 Mb 5’ and 2.17 Mb 3’) of flanking sequence; and a 5.2 Mb region (hg19: 29.1Mb– 34.3 Mb) on chromosome 10 containing the ZEB1 gene (31.6 Mb– 31.8 Mb) and 5 Mb (2.5 Mb 5’ and 2.5 Mb 3’) of flanking sequence.[Genomic DNA samples from eight PPCD probands in whom a sequence. The arra® LTX (Life Technologies) according to the manufacturer’s recommendations. To test the impact of the OVOL2 c.-307T>C variant on promoter activity, HCEnC-21T cells were transfected with 250 ng of either a OVOL2 PWT, OVOL2 Pc.-307T>C, or empty (pGL3-Basic) promoter luciferase construct (generously provided by Dr. Alison J. Hardcastle of the University College London).[™ BCA Protein Assay Kit (Life Technologies) and FilterMax F5 microplate reader . Promoter activity was measured using the Dual-luciferase® Reporter Assay System (Promega) according to manufacturer’s instructions. Each trial was normalized to Renilla firefly luminescence in order to account for variability in transfection efficiency.Immortalized human corneal endothelial cells (HCEnC-21T) were seeded at 50% confluency into 24-well plates. The following day, the cells were transfected using LipofectamineThe probability and corresponding 95% confidence interval of the identified variant, c.-1409G>C, found within the screened probands was estimated based on the observed frequency in the probands and from the observed frequency of same variant in the population assuming binomial distribution of allele frequencies.ZEB1 translational start site in 31 PPCD probands without a ZEB1 coding region mutation.[ZEB1 coding region mutation were identified, we screened the 1 kb upstream of the initiation methionine in these six individuals. The known single nucleotide polymorphism (SNP) c.-803G>C  0.316) was identified in the heterozygous state in two individuals, one of whom also demonstrated a second known SNP ) in the heterozygous state. Of the 37 DNA samples from these individuals without a ZEB1 promoter, 5’ UTR, or coding mutation, 27 had sufficient DNA to analyze the 1.8kb region upstream of the OVOL2 translational start site.We have previously reported the absence of presumed pathogenic variants in the non-coding 1 kb region (containing both the 5’ UTR and promoter) upstream of the mutation. After siOVOL2, one proband (P1) is a member of the family that we mapped to the PPCD1 locus and in which we identified a OVOL2 promoter variant (c.-307T>C) that segregated with the affected phenotype.[OVOL2 in the remaining 26 PPCD probands in whom a ZEB1 truncating mutation was not identified revealed five different heterozygous variants in eight probands, while the remaining 18 probands did not demonstrate any heterozygous variants within either the OVOL2 promoter or 5’ UTR (OVOL2 promoter (6/54 chromosomes corresponding to an observed MAF of 0.111) was double the global MAF of 0.0541, the difference was not statistically significant (p = 0.064). Given this, and as the global minor allele frequency is much higher than the population prevalence of PPCD, the c.-1409G>C variant is very unlikely to be pathogenic. Screening of each of the four OVOL2 coding exons in the 27 probands revealed only one heterozygous variant (c.327C>A), which was identified in three probands. As the c.327C>A variant has a MAF of 0.0403, much higher than the population prevalence of PPCD, this variant is also very unlikely to be pathogenic.Of the 27 probands who had sufficient DNA to perform screening of the henotype., 24 Screr 5’ UTR . Of the OVOL2 was screened in affected and unaffected members of the family with PPCD that was the first to be mapped to the PPCD1 locus on chromosome 20  were identified in five of the eight probands, ranging in size from 134 to 14,007 base pairs  proband, and the second gain present in three (3/8) probands; and a loss in intron 2 of PIGU, which was identified in three (3/8) probands. There were no identified CNVs within OVOL2. In the PPCD3 locus, CNV were identified in eight genomic regions, although none involved ZEB1.CNV analysis within the PPCD1 and PPCD3 loci was performed in eight of the 27 PPCD probands without a se pairs . Three oOVOL2 promoter activity in human corneal endothelial cells. Immortalized human corneal endothelial cells, HCEnC21T, were transfected with a luciferase reporter construct containing either no promoter (empty), an OVOL2 wild-type promoter (OVOL2 PWT), or an OVOL2 promoter harboring the c.-307T>C mutation (OVOL2 Pc.-307T>C). While both OVOL2 PWT and OVOL2 Pc.-307T>C were able to drive the expression of luciferase, OVOL2 Pc.-307T>C produced significantly higher levels of luciferase expression when compared to the OVOL2 PWT  and one family initially diagnosed with autosomal dominant CHED that was mapped to the region on chromosome 20 containing OVOL2, promoter mutations in OVOL2 have been convincingly associated with PPCD1.[The recent identification of entified., 25 We ientified., 18 Althentified. Given thentified. This varth PPCD1., 25OVOL2 or ZEB1 coding region or promoter mutation or CNV involving either gene in 26 PPCD probands indicates that other genes may be associated with PPCD. In the PPCD mouse, a chromosomal inversion flanked by deletions involving Csrp2bp and Dzank1 has been identified in the portion of chromosome 2 that is syntenic to the human PPCD1 locus.[Csrp2bp fusion transcripts and Ovol2, which is located 37 kb from Csrp2bp and 66 kb from the breakpoint of the chromosomal inversion. While it is possible that the identified genetic rearrangement leads to PPCD1 in the mouse through upregulation of Ovol2 expression, as is the case in humans, the authors provide compelling evidence for the disruption of Csrp2bp as being causative for PPCD1 in the mouse.[CSRP2BP in any of the 8 probands analyzed for CNV. Although identification of other affected pedigrees of sufficient size to perform whole exome sequencing and CNV analysis to identify additional genetic loci should be performed, we also plan to perform and encourage other investigators to perform sequencing of presumed OVOL2 and ZEB1 regulatory sequences to identify other potentially pathogenic sequence variants.Our failure to identify a presumed pathogenic D1 locus., 29 The he mouse., 29 HoweZEB1, as it does in corneal epithelial cells. OVOL2 maintains the epithelial cell state by repressing mesenchymal genes such as ZEB1.[PAX6, OVOL2 induces the transcriptional profile of corneal epithelial cells in fibroblasts, suggesting that OVOL2 may serve as a master regulator of the epithelial cell state.[OVOL2 results in enhanced promoter activity in corneal endothelial cells compared to the wild-type sequence. As it has been previously reported that OVOL2 represses ZEB1 expression, our promoter activity results are consistent with the hypothesis that PPCD-associated OVOL2 promoter mutations lead to PPCD by causing OVOL2 overexpression and subsequent ZEB1 insufficiency.[In silico analyses have predicted PPCD-associated OVOL2 promoter mutations cause a gain or loss of putative transcription factor binding sites, potentially leading to OVOL2 dysregulation.[OVOL2 and ZEB1 mutations on the expression of these transcription factors, the endothelial transcriptome and corneal endothelial morphology and function are ongoing, and are anticipated to clarify whether both PPCD1 and PPCD3 share a common molecular pathway.Additional studies will also need to be performed to elucidate the role of OVOL2 in the corneal endothelium, and to determine whether OVOL2 acts to repress the expression of  as ZEB1. When cotll state. In contrficiency., 27 In sgulation., 25 FurtS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


CNTNAP2). In addition, we demonstrate that FoxP1/4 bind to the regulatory region of very low density lipoprotein receptor (VLDLR), as has been shown for FoxP2 previously. Interestingly, FoxP1/2/4 individually or in combinations regulate the promoters for SV40, zebra finch VLDLR and CNTNAP2 differentially. These data exemplify the potential for complex transcriptional regulation of FoxP1/2/4, highlighting the need for future functional studies dissecting their differential regulation in the brain.The Forkhead transcription factor FOXP2 is implicated in speech perception and production. The avian homolog, FoxP2 Fox genes perform tissue specific functions during development and mutations can cause cancer and other diseases  transcription factors comprise 19 highly evolutionary conserved, structurally related families, FoxA to FoxS. The defining feature of these genes is the Fox domain which binds to regulatory regions of target genes. Many FoxP subfamily members in vertebrates, FoxP1 to FoxP4 , encoding one of the reelin receptors, and Contactin-associated protein-like 2 (CNTNAP2) gene which codes for a neurexin called CASPR2  were bred in our colony at the Freie Universität Berlin. All procedures were performed according to the guidelines of the governmental law (TierSchG), under permits granted by the local Berlin authorities governing research involving animals. All birds were sacrificed with an Isoflurane overdose.Male zebra finches (Co-Immunoprecipitation (Co-IP) was done using Dynabeads® Protein G  following the manufacturer’s protocol with a few changes as follows. We first incubated the antibody-Dynabeads mixture for 15 min at room temperature  lysis medium for 15 min on ice. Extracts were centrifuged for 10 min at 1500 g and supernatant was stored at −80°C until used. Cell extracts and co-IP’ed proteins were separated by SDS PAGE (8%–10%), transferred to a polyvinylidene fluoride membrane , and blocked with Roti-Immunoblock for 2 h or overnight at 4°C. The membranes were then incubated with the desired antibody  25 μl of OptiMEM (GIBCO) containing 30 ng of pGL4.13 (Luciferase gene driven by the SV40 promoter which is known to be regulated by FoxP subfamily members) and 30 ng of pGL4.75  and 250 ng total vector over-expressing the different FoxPs for each well; and (b) 1 μl of Lipofectamine™ 2000 (Invitrogen) in 25 μl OptiMEM that was premixed for 5 min at RT. After combining (a) and (b) the mix was incubated for 20 min at RT, then it was added to the cells. After 4–6 h of incubation we changed the medium to 75 μl of antibiotic containing medium and incubated for further 48 h at 37°C in a CO2 incubator.For SV40 we seeded ~30000 HeLa cells in 200 μl DMEM medium (GIBCO) containing Penicillin-Streptomycin  per well of a 96-well white flat bottom plate . Transfection was performed using Lipofectamine™ 2000 (Invitrogen) following manufacturer’s protocol. Briefly, after seeding plates were incubated for 24 h at 37°C at 5% COVLDLR and CNTNAP2 we used HeK293 cells, and luciferase assays were done as described previously  following manufacturer’s protocol in an Elisa plate reader . Mean background from untransfected wells was subtracted from all other wells. We present Luciferase results as mean Relative Light Units (Luciferase RLU/Renilla RLU) calculated from the normalized values of 4–7 independent assays.The pGL4-VLDLR promoter was cloned as described .Proteins for electrophoretic mobility shift assays (EMSAs) were purified using the Protino Ni-NTA Agarose  according to manufacturer’s protocol. Protein was quantified using BCA1 (Sigma). EMSA assays were carried out as published previously, using the Oligo described for VLDLR , and therefore we used FoxP2 and FoxP4 polyclonal antibodies to pull down protein complexes, and all three antibodies for Western blot detection. As a negative control we used IgG antibodies of the same species in which the specific FoxP antibodies were raised. After IP with FoxP2 or FoxP4 antibodies, we detected co-IP’ed FoxP1 Figures . After ISince the majority of FoxP expressing cells in Area X and the surrounding striatum express FoxP1/2/4 . The SV40 promoter has a putative core consensus sequence for binding (TATTTRT) human and murine Foxp1/2/4 , in the same range as reported for mice and human FOXP1/2/4. Also, like its mammalian homologs, zebra finch FoxP4 repressed transcription stronger than FoxP1 or FoxP2 did, and FoxP2 was a weak repressor of the SV40 promoter . There were no significant differences between cells expressing FoxP1 or FoxP4 alone or in any of the combinations tested. We did observe a stronger transcriptional repression in cells expressing FoxP2 in combination with another FoxP subfamily member than when FoxP2 was expressed alone . However, we cannot rule out the formation and action of homodimers of FoxPs in those cells. Taken together, FoxP target genes in cells that express only FoxP2 may be less strongly regulated than those that are co-expressed with FoxP1 and/or FoxP4.We assessed whether the homotypic and heterotypic interaction of zebra finch FoxP proteins lead to differential regulatory activity. To do so we used the SV40 promoter driving Luciferase (VLDLR for a number of reasons: (a) it is a direct target of FoxP2 in humans, mice, and zebra finches . However, FoxP2 and FoxP4 were less effective and not significantly different from each other in their ability to activate the VLDLR promoter (p = 0.999). Combinations of FoxP1/2/4 did activate the VLDLR promoter to a similar degree . Comparing the activation of FoxP2 alone to the FoxP1/2 and FoxP1/2/4 combination (p = 0.0078 and p = 0.012 respectively) we found a significant difference on the VLDLR promoter but not with the FoxP2/4 combination (p = 0.88). There were no differences between the individual activation by FoxP4 and the combinations with FoxP1 (p = 0.10) or FoxP2 (p = 0.98), but there was a significant difference to the FoxP1/2/4 combination (p = 0.034). Taken together FoxP1/2/4 are not only repressors, but are able to bind to the VLDLR promoter and activate it differently depending on the co-expression with other FoxPs.To test whether binding of FoxP1/2/4 to the CNTNAP2 because: (a) CNTNAP2 is associated with speech disorders, as well as ASD, dyslexia and intellectual disability .To further test the regulatory ability of zebra finch FoxPs on a target gene relevant in Area X, we chose CNTNAP2 promoter contained a sequence gap located ~0.75 kb upstream of the start codon of the first exon of CNTNAP2 . FoxP1 was able to bind to the CNTNAP2 oligo and resulted in two shifted bands, consistent with the possibility that it binds in two of the three FoxP2 binding sites . Moreover, in combination, the different FoxPs also failed to regulate expression driven by the CNTNAP2 promoter, . Interestingly, combinations of FoxP4 with either FoxP1 or FoxP2 did not result in repression or activation, in contrast to FoxP1 or FoxP2 alone, suggesting that hetero-dimerization might affect the regulation of FoxPs. Taken together we show that FoxP1 and FoxP2 can bind to and regulate the CNTNAP2 promoter, with opposing activities. CNTNAP2 is an example of how FoxPs may tune the regulation of different targets genes via homo- and hetero-dimerization.To investigate whether binding of FoxP1 and FoxP2 to the r Figure . When teo Figure  FoxP4 faSV40, VLDLR and CNTNAP2 and regulate their transcriptional activity. Zebra finch FoxP1/2/4 repressed the SV40 promoter activity, as reported for mouse and human , which is in the opposite direction of the present findings with HeK293 cells. This highlights the plasticity with which FoxP proteins can regulate target genes in different cellular contexts, depending on different binding of co-factors that change the regulation of the same gene  two ways to associate or interact: the leucine zipper motif and domain swapping, e.g., the exchange of identical structural elements involving the Forkhead domain (Stroud et al., VLDLR and CNTNAP2. All zebra finch FoxP proteins studied bound to the VLDLR oligonucleotide, which was previously shown to bind to FoxP2 (Adam et al., VLDLR oligonucleotide contains a partial FOXP core sequence, ATTT (Stroud et al., ATTTAT (Wang et al., RTTTAY (Pierrou et al., CNTNAP2 oligonucleotide contains three putative FoxP2 binding sites. One TATTTAT (Enard et al., Finally, our data are the first to address neural targets regulated by FoxP1 and FoxP4. We tested the binding of two known targets of FoxP2 in zebra finches, VLDLR and CNTNAP2. Importantly, different FoxP combinations resulted in specific, differential transcriptional regulation. Together, our data demonstrate how versatile and variable FoxP regulation can be in the neural context.In summary, we show that zebra finch FoxP proteins can interact with each other in all combinations in the songbird brain. We show that all neurally expressed FoxPs have the capacity to bind to and regulate the target genes EM and CS: experiment design, analysis of data and wrote the manuscript. EM did all experiments.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.


Pseudomonas putida P13 and Pantoea agglomerans P5 are well recognized for application as phosphate solubilizing bioinoculants and are used as solid carrier based. Liquid bioinoculants are preferred for economizing production process and longer shelf-life.Phosphorus is one of the low bioavailable macroelements. Use of microorganisms in biofertilizers could release phosphorus from insoluble compounds. 3 dissolved in phosphate buffer, respectively. Formulation 4 was nutrient broth containing 4% glycerol and formulation 5 was diluted nutrient broth containing 4% glycerol. Survival (cfu) and phosphate solubilization index (SI) were evaluated after 3 months.Five low cost liquid formulations were examined. Formulations 1, 2 and 3, were phosphate buffer, 0.2% and 0.5% KNO3 concentration decreased preserving ability. While using KNO3 at 0.2% was accompanied with reaching maximum SI level. Overall, less nutritious formulations (1 and 5) provided maximum preserving ability without bioactivity loss. In the case of strain P13, maximum survival obtained in formulations 2 and 3, whereas SI level decreased. Preserving ability in formulations 1, 4 and 5 was similar but less nutritious formulations (1 and 5), improved bioactivity.Considering strain P5, increase in KNOPseudomonas putida and Pantoea agglomerans.The results introduced two formulations of 1 and 5 as economically efficient liquid bioinoculants for  Nitrogen, phosphorus and potassium, are among the macronutrients which are required in large quantities for plants . Due to Pseudomonas putida strain P13 and Pantoea agglomerans strain P5 are well known as effective phosphate solubilizing bacteria of related bioinoculants in Iran , Tehran branch, Department of Applied Microbiology. Recovery of the two phosphate solubilizing bacteria (PSB) was made by sub-culturing few drops of stored bacterial stocks at −70°C onto nutrient agar medium. Plates were incubated at 30°C until growth was observed.2PO4 15.44 μM, NaCl 1.55 mM, Na2HPO4 27.09 μM . Cultures were then centrifuged at 5000 rpm for 15 minutes at 30°C and cell mass was collected and washed twice with equal volume of normal saline solution, subsequently. The cell mass was added to liquid formulations under sterile conditions .8 cfu/ml for strain P5 and 1 × 107 cfu/ml for strain P13 by measuring the 0.5 optical density at 540 nm wavelength OD(540nm). These values are within the range of bacterial cell number allowed for a biofertilizer according to the plant protection organization of Iran. The samples were perfectly sealed and stored at 25°C in dark conditions.The number of cells in each formulation was adjusted to 2 × 10The assessment of viable populations of the two strains was made at monthly interval for three months. Serial four-fold dilutions in normal saline solution were made from each sample. An aliquot of 100 μl from each dilution was dropped on nutrient agar medium as the spread plate technique. The number of colony forming units (cfu) was determined after 24 hours of incubation .(540nm) value of 0.5 was attained. 25 μl of each bioinoculant (at the OD(540nm) value of 0.5) was spotted onto plates of Sperber′s agar medium to evaluate hydrolysable phosphorus from tri-calcium phosphate qualitatively by forming the clear zone in the opaque medium (SI) was calculated as follows  value of 0.5 were calculated and used as control. The same incubation conditions were applied for preserved bacteria and control.Phosphate solubilization indexes of 24 hours cultures of strains P13 and P5 at ODThe assays were calculated as an average of three independent experiments. Data were analyzed using SPSS version 21. One-way analysis of variance (ANOVA) and Tukey test were used to examine values. P value <0.05 was considered significant.The ability of each formulation to preserve each single bacterial strain was evaluated over a three month period. Data related to population dynamics of strains P5 and P13, are presented in 8 cfu/ml, underwent a reduction in number in all formulations after one month of storage. However, there was no significant reduction in the number of bacteria afterwards. Formulations 2 and 3 induced a reduction in cell number higher than other formulations at the end of the storage period. The simple phosphate buffer tended to preserve better the number of cells in comparison to phosphate buffers containing potassium nitrate (5 × 106 cfu/ml). Furthermore, increasing the concentration of potassium nitrate in the formulation provoked a further reduction of bacterial population .Strain P5 with the initial population of 2×10Estimating the influence of nutritious formulations (formulations 4 and 5) on P5 viability, better maintaining ability by formulation of undiluted nutrient broth (formulation 4) was not longer than two months and overall better performance was provided by less nutritious formulation (formulation 5) after three months of storage.6 cfu/ml.The results of different formulations for preserving strain P5 indicated that both of the formulations 1 and 5 supported the highest maintaining ability and protected population maintenance of 5 × 107 cfu/ml), but with the limitation of cell proliferation for duration of one month (in formulation 2) and two months (in formulation 3) after storage. Statistically no significant difference was observed among formulations 1, 4 and 5 . Compared to the control, the addition of potassium nitrate to the phosphate buffer of formulations 2 (SI = 3.98) and formulation 3 (SI = 3.86) enhanced SI ability. Formulation 4, with undiluted nutrient broth, resulted into the lowest index of P solubilization (SI = 3.74).Bacteria of strain P5 with formulations 1 and 5 showed a SIcontrol= 3.5), formulations 1 and 5 enhanced the bacterial ability to solubilize phosphate. The highest SI for P13 was obtained with the simple phosphate buffer of formulations 1 (SI = 3.86) and diluted broth of formulation 5 (SI = 3.81). The presence of potassium nitrate in the phosphate buffer formulation reduced SI (contrary to the results observed for strain P5 in which maximum SI was obtained in the presence of 0.2% KNO3). Formulations 2, 3, and 4, induced a decline on phosphatase activity.In the case of strain P13 as compared to the control . In their study, it was evidenced that the majority of pathogenic bacteria were able to survive in either water or phosphate buffer after 30 weeks of preservation at room temperature, allowing to hypothesize that human and plant pathogenic bacteria can survive in water or phosphate buffer for several years.Considering both bacteria as Gram negative and facultative anaerobes, efficiency of each individual formulation was examined for each strain, separately. Maximum population of strain P5 and one of highest SI level and increasing cell number for at least about one month after storage of the strain P13. Formulation 2, contributed to maximum SI of strain P5 and formulation 3 decreased population level of the strain to its minimum. Our findings about the ability to sustain the Pseudomonas population size of the phosphate buffer based formulations are in line with findings of Vandenbergh and coworkers  were sufficient to maintain Pseudomonas for up to 160 days or more at room temperature.Maximum preserving ability during three months of storage for strain P13 was assured by phosphate buffers containing potassium nitrate. The formulations were confined by reducing oworkers , 10 who Pseudomonas (and other microorganisms meanwhile) at room temperature .The maintained population size for both strains was acceptable in formulation 4, but P. fluorescens preservation at room temperature. The reported potent bacterium maintained its antagonistic activity until 150 days without any remarkable reduction in bioactivity . Nutrient broth medium containing 2% glycerol amendment has been defined as an appropriate formulation for activity .The formulation 5 fitted with two main positive features; keeping both strains bioactive along with promotion of strain P5 to highest population density. However, the formulation was not so effective in maintaining the P13 strain population. Considering the direct relationship of plant growth promotion efficacy with the number of inoculated microorganisms, the population decline of strain P13 in formulation 5 could be counteracted with inoculation of more bacterial biomass.Pseudomonas. It is better to add extenders at on seed level or at sowing time  for on seed level use  can be added to water or microbial culture broth of inoculant formulation at on seed level. Sucrose is a common additive which can be used as an extender or an adhesive in liquid formulations of ing time –25. Pantevel use . Additioreported .In prior studies, it has been demonstrated that mixed inoculation of the two phosphate solubilizing bacterial strains P5 and P13, can be performed .In conclusion it would be possible to utilize formulations 1 and 5 for production of efficient liquid bioinoculants composed of single strain and both strains tested.


The data presented in this article are related to the research paper entitled “Anti-inflammatory effect of avenanthramides via NF-κB pathways in C2C12 skeletal muscle cells.”  [1] This article includes experimental procedures used to analyze the mode of binding between and IkB kinase (IKKβ) and avenanthramides which are a group of phenolic alkaloids found in oats. The protein-ligand docking and the computer simulation method of molecular dynamics (MD) for studying the physical interactions of molecules were performed. Specifications TableValue of the data•The molecule docking model first describes the mode of binding between Avns and IKKβ molecular domains.•Molecular dynamic (MD) simulations were carried out to establish the protein-ligand complex.•In summary, protein-ligand docking and molecular dynamics simulations methods were performed to understand the potential structure and the nature of molecular clusters with fine interactions with IKKβ.1The data presented in this article are supportive to the data presented in 22.1A standard protocol for protein-ligand docking studies was used. In short, all docking studies were performed using the Schrodinger modeling suite package . The crystallographic structure of the targeted IKKβ (PDB code: 3RZF) with bound inhibitors was used as a starting point to study the potential mode of Avns binding 2.2MD simulations were performed for IKKβ in complex with the structurally solved inhibitors. Each system was solvated in a cubic box with positive TIP3P water