Two-kidney one-clip (2K1C) hypertensive rats were treated with lisinopril (10 mg/kg/day for four weeks) or with pyrvinium pamoate . The treatment with lisinopril reduced the systolic blood pressure from 220 ± 4 in 2K1C rats to 112 ± 5 mmHg (P < 0.05), whereas the reduction in blood pressure with pyrvinium pamoate was not significant . The levels of collagen types I and III, osteopontin, and fibronectin decreased in the unclipped kidney in both treatments compared with 2K1C rats. The expressions of β-catenin, p-Ser9-GSK-3beta, and the β-catenin target genes cyclin D1, c-myc, and bcl-2 significantly decreased in unclipped kidney in both treatments (P < 0.05). In this study we provided evidence that β-catenin-dependent signaling pathway participates in the renal fibrosis induced in 2K1C rats.The mechanism of hypertension-induced renal fibrosis is not well understood, although it is established that high levels of angiotensin II contribute to the effect. Since  Hypertension is a major risk factor for development and progression of chronic kidney disease . The mai in vitro studies have shown that Ang II stimulates fibronectin, TGF-β, CTGF, and PAI-1 synthesis [Ang II works as a systemic vasoconstrictor, as a modulator of renal microcirculation, and as a regulator of sodium tubular transport . Howeverynthesis . Severalynthesis . In factynthesis .β-catenin signaling pathway participates in organogenesis and tissue homeostasis, and its deregulation has been linked in the pathogenesis of human diseases, including cancers and degenerative diseases [β-catenin. The nuclear translocation of β-catenin allows its association with T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors initiating the transcription of β-catenin-dependent target genes [β is assembled to form the destruction complex of β-catenin. Once the complex is assembled, the phosphorylation of β-catenin by glycogen synthase kinase-3β (GSK-3β) is the signal for ubiquitination and degradation by the proteasome system [β-catenin-dependent signaling, is altered and might have a role in fibrotic kidneys after obstructive injury  [β-catenin target genes in different cellular contexts [β-catenin signaling participates in the renal interstitial fibrosis induced by Ang II remains to be investigated. Recently, it was reported that β-catenin signaling was potently targeted by pyrvinium pamoate [β-catenin-dependent signaling pathway contributes to renal fibrosis induced in this model.The Wnt/diseases . In the et genes . In the e system . There aruction) . In addicontexts –19. Howe pamoate , an anthAll animal studies were performed in accordance with the Guiding Principles in the Care and Use of the Laboratory Animals for the American Physiological Society and were approved by the Ethics Committee of Animal Care of the Pontificia Universidad Católica de Chile. Animals were maintained at constant room temperature with a 12 h light/dark cycle in the institutional animal care facilities  with free access to food and water.μg/kg, single dose every 3 days) by oral gavage for 2 weeks starting from sixth week after surgery. The experiments with pyrvinium pamoate were subsequently performed to lisinopril experiments; therefore, each setting has its own sham control and 2K1C group. Pyrvinium pamoate was purchased from Sigma-Aldrich Co.  and lisinopril was obtained from Recalcine CFR Pharmaceuticals. Systolic blood pressure (SBP) was measured at the end of experiments in conscious rats before the sacrifice using the tail-cuff plethysmography with a Grass polygraph. The rats were sacrificed at the end of 8th week after surgery by overdose of ketamine : xylazine. An independent group of 2K1C and sham rats (n = 8 per group) was formed with the purpose of monitoring the level of SBP throughout the experimental period (0–8 weeks). The unclipped kidney was immediately excised and frozen at −80°C or fixed in Bouin's solution and processed for conventional histology, immunofluorescence, and immunohistochemistry.Male Sprague-Dawley rats of 100–125 g were anesthetized with ketamine : xylazine i.p. (25 : 2.5 mg/kg) and a silver clip (0.20 mm) was placed around the left renal artery through a left flank incision. Animals were randomly divided into four groups  according to the treatment: sham operated control, 2K1C rats without treatment, 2K1C rats treated with lisinopril (10 mg/kg/day) by oral gavage for four weeks starting from fourth week after surgery, and 2K1C rats treated with pyrvinium pamoate , active β-catenin clone 8E7 , total GSK-3β , p-Ser9-GSK-3β , and the target genes cyclin D1, c-myc, and bcl-2  or fibronectin  and incubated overnight at 4°C. Proteins were detected using enhanced chemiluminescence techniques. The β-actin levels  were used as a load control and the densitometric analysis was performed using ImageJ .Proteins were extracted from whole kidneys sections and were homogenized in RIPA buffer  supplemented with protease and phosphatase inhibitors mixture. Proteins were separated by 10–12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked using 5% skim milk or BSA in 0.1% PBS-Tween for 1 hour at room temperature. The immunoblotting was performed using primary antibody against total μm thick sections as previously done [Kidney tissue sections were fixed in Bouin's solution and embedded in Paraplast Plus. The immunohistochemistry and immunofluorescence were carried out in 5 sly done , 22. BriP < 0.05) was considered to be significant.The results are expressed as mean ± standard error (SEM). Differences between groups were assessed by a nonparametric Kruskal-Wallis test followed by Dunn's multiple range test. Statistical tests were performed using the GraphPad Prism software v 5.0 . A probability of 95% (P < 0.05) compared to sham rats at the same time . SBP of 2K1C rats continues to increase until fourth week (209 ± 4 mmHg) and remains high until eighth week (216 ± 8 mmHg) .Previously, the SBP through all experimental period was weekly evaluated in an independent group of 2K1C rats. SBP was significantly high in the third week after surgery   .Renal fibrosis was evaluated by immunohistochemical staining for collagen type I, collagen type III, and OPN and the level of fibronectin protein by Western blot in the unclipped kidney. The unclipped kidneys from hypertensive rats showed a significant increased fibrosis assessed by deposition of collagen types I and III Figures  and it wβ-catenin signaling in 2K1C hypertensive rats and the effect of ACE inhibition, we evaluated the protein level of several components of the β-catenin signaling pathway by Western blot analysis in the unclipped kidney from 2K1C Goldblatt rats without treatment or treated with lisinopril. The protein levels of the β-catenin were significantly increased in unclipped kidney from hypertensive rats when compared to sham . Moreover, we evaluated the levels of the classic β-catenin-dependent target genes such as cyclin D1, c-myc, and bcl-2. Our results indicated an increase in the level of cyclin D1, c-myc, and bcl-2 in the unclipped kidney from 2K1C hypertensive rats compared to sham  [β-catenin target genes; therefore the upregulation of all these genes might account for the important role of β-catenin-dependent signaling in the control of adult tissue renewal and fibrosis. Despite that, we do not show regulation of the transcription factor TCF/LEF in our model; the upregulation of LEF-1 and TCF transcription factor has been demonstrated in the UUO rats [β-catenin with the transcription factor LEF-1 in the nuclei of podocytes has been shown in rats with focal glomerulosclerosis [β-catenin with TCF/LEF transcription factors could mediate the upregulation of its target genes.Several pathways have been described as contributors in this process, including TGF-ignaling  as a maiisorders . In agrel injury , our datel (UUO) , 31 and el (UUO) , collageel (UUO) , 32, andel (UUO)  are all UUO rats , 34 and clerosis ; therefoβ is one of the main effectors of the Ang II-induced renal damage [β in the nonclipped kidney after the treatment with enalapril. Coincidentally, the cross talk and cooperation between the Wnt/β-catenin signaling and TGF-β signaling pathways in the fibrotic processes have been showed [β-catenin signaling is a common effector of different pathways activated by Ang II to promote fibrosis in the kidney.On the other hand, as we mentioned previously, there is wide evidence suggesting that TGF-l damage . Using 2l damage  showed an showed , 25, 36.β-catenin signaling, suggesting a modulator role for RAS in this pathway. The levels of β-catenin, p-Ser9-GSK-3β, and the β-catenin-dependent gene products decreased after the treatment with lisinopril, suggesting that Ang II can stimulate the β-catenin signaling to promote the fibrosis in 2K1C hypertensive rats. A cross talk between the Ang II signaling and the β-catenin pathway appears to occur, since Ang II modulates GSK-3β phosphorylation inducing fibrosis in the heart [β-catenin signaling pathway in the kidney. Recently, Zhou et al. (2015) [β-catenin signaling pathway activation; it could suggest that the activation of this pathway contributes to maintaining the overactivation of RAS in chronic kidney disease.It is worth noting that our data showed that the ACE inhibition in 2K1C hypertensive rats was associated with an inhibition of the he heart . However. (2015)  reportedβ-catenin signaling reduced renal β-catenin accumulation and decreased fibrosis [β-catenin signaling pathway reduces renal fibrosis in the unclipped kidney from 2K1C hypertensive rats. Furthermore, a novel finding in our study was the effect of pyrvinium pamoate (an FDA-approved drug) on reducing renal fibrosis in the unclipped kidneys. Recently, it was shown that pyrvinium pamoate may have therapeutic benefit in two different models of myocardial remodeling [β-catenin signaling was potently targeted by pyrvinium pamoate through the activation of casein kinase 1, novel mechanisms of action of pyrvinium pamoate have been suggested in different cancer cells lines, identifying pyrvinium pamoate as a novel anticancer drug able to target mitochondrial respiration in hypoglycemic/hypoxic conditions [β-catenin-dependent signaling pathway in 2K1C rats, the possibility that pyrvinium pamoate could also affect other cellular processes cannot be ruled out and future studies using a different β-catenin inhibitor may answer these questions.Our results are in agreement with previous studies in UUO model of renal damage showing that inhibition of the Wnt/fibrosis , 39. In modeling , 41 and modeling  showed tnditions  and to inditions  and a noβ-catenin signaling pathway is active in tubular cells as early as day 3 after UUO, and the activation of Wnt/β-catenin signaling pathway occurs independently of the increased blood pressure. Additional studies are required to better understand the contribution of the β-catenin signaling in the fibrotic lesions of unclipped kidney, in particular a Wnt/β-catenin signaling inhibitor without effect on blood pressure. However, we have recently provided evidence that in vitro Ang II induced the expression of profibrotic factors through the β-catenin-dependent signaling in mouse collecting duct cells. Ang II upregulated the β-catenin protein levels together with GSK-3β phosphorylation and β-catenin target genes. Interestingly, all these effects were prevented by pyrvinium pamoate, indicating that, in M-1 collecting duct cells, the β-catenin signaling pathway mediates the stimulation of fibrotic factors in response to AT1 receptor activation [Interestingly, our data showed that the treatment of hypertensive rats with pyrvinium pamoate treatment tends to decrease SBP; however, this decrease was not statistically significant; therefore, the animals remain exposed to high blood pressure levels. It is known that Ang II contributes to fibrotic lesions by the direct activation of profibrotic pathways in the kidney . Furthertivation  independβ-catenin signaling pathway is active in fibrotic process in the unclipped kidney from 2K1C hypertensive rats. The inhibition of β-catenin signaling pathway by pyrvinium pamoate and lisinopril decreases renal fibrosis in 2K1C hypertensive rats. These findings provide a better understanding of the mechanisms involved in renal damage in hypertension and open new therapeutic approaches to control the fibrosis induced by hypertension.In summary, our findings suggest that With the purpose of monitoring the level of systolic blood pressure (SBP) throughout the all experimental period (0-8 weeks) we performed an independent group of 2K1C and sham rats (n=8 rats per group) and the SBP was measured at 0, 3, 4 and 8 weeks after surgery. A significant increase on SBP was observed from the 3rd week after surgery. (p<0.05)


However, it has been difficult to study its pleiotropic effects, because of the dual capacity of β-catenin to coordinate cadherin-dependent cell adhesion and to act as a component of Wnt signal transduction. To distinguish between the divergent functions of β-catenin during peripheral nervous system development, we made use of a mutant allele of β-catenin that can mediate adhesion but not Wnt-induced TCF transcriptional activation. This allele was combined with various conditional inactivation approaches.We show that of all peripheral nervous system structures, only sensory dorsal root ganglia require β-catenin for proper formation and growth. Surprisingly, however, dorsal root ganglia development is independent of cadherin-mediated cell adhesion. Rather, both progenitor cell proliferation and fate specification are controlled by β-catenin signaling. These can be divided into temporally sequential processes, each of which depends on a different function of β-catenin.While early stage proliferation and specific Neurog2- and Krox20-dependent waves of neuronal subtype specification involve activation of TCF transcription, late stage progenitor proliferation and Neurog1-marked sensory neurogenesis are regulated by a function of β-catenin independent of TCF activation and adhesion. Thus, switching modes of β-catenin function are associated with consecutive cell fate specification and stage-specific progenitor proliferation.The online version of this article (doi:10.1186/s12915-015-0134-4) contains supplementary material, which is available to authorized users. Canonical Wnt signaling is one of the most important evolutionarily conserved signaling pathways in embryonic development -3. Therein vivo. The β-catenin protein is composed of 12 Armadillo repeats and is flanked by an amino-terminal domain and a conserved helix-C next to a carboxy-terminal domain. Nuclear β-catenin interacting with TCF/Lef recruits transcriptional co-activators to the transcription complex by means of its N- and C-terminal, initiating TCF/Lef-mediated transcription [Ctnnb1) manipulation to either or both functions of β-catenin. To distinguish between signaling and adhesion functions of β-catenin in the skin, a transgene has previously been used [Ctnnb1dm) that results in expression of a mutated form of the protein from its endogenous locus. The protein expressed from this allele (β-catenin-dm) is degradable in the absence of Wnt, is fully functional in adherens junctions, and lacks known signaling properties as a transducer of Wnt-dependent transcriptional activation [Ctnnb1dm display no phenotype [β-catenin is the bottleneck of canonical Wnt signaling, as it is the central, non-redundant component of the pathway. This trait has made β-catenin especially attractive as a target for genetic manipulation to elucidate the impact of Wnt signaling cription   [Wnt1-Cre Ctnnb1flox/flox embryos resulted in a drastic reduction of sensory neuronal, and complete absence of glial, lineages in the dorsal root ganglia (DRG), whereas other neural derivatives, such as sympathetic ganglia and the enteric nervous system, appeared to develop normally [Ctnnb1dm, together with β-catenin null and other conditional knockout alleles, and reveal distinct requirements of β-catenin functions for sensory neurogenesis. Surprisingly, while β-catenin/α-catenin-mediated cellular adhesion plays a negligible role in sensory neuron formation, several steps in sensory neurogenesis require β-catenin, but only some of these are dependent on β-catenin-mediated transcriptional activation.Neural crest cells (NCCs) are a population of multipotent cells that delaminate from the dorsal part of the neural tube during neurulation of vertebrate embryos . Upon dent1-Cre) ,25. Inacnormally . Sensorynormally . HoweverWnt1-Cre Ctnnb1flox/dm (βcat-Sig) animals. In these mice, Cre-mediated conditional deletion of β-catenin normally expressed from the Ctnnb1flox allele leads to complete replacement of the endogenous β-catenin protein by the signaling mutant form derived from the Ctnnb1dm allele  embryos  [β-gal) upon Cre-mediated recombination.To investigate the role of β-catenin signaling during neural crest development independently of β-catenin-mediated cell adhesion, we generated e (R26R) , which eR26R revealed similar, but less severe, phenotypes in βcat-Sig R26R embryos when compared to βcat-Null R26R embryos  expression was used to be able to trace recombined BAT-gal positive cells [BAT-gal reporter.The lineage-specific phenotypes observed in  BAT-gal . In thesve cells . Double βcat-Null animals and substitution of wild type β-catenin by the double mutated form with a truncated C-terminus present in recombined cells of βcat-Sig mutants  lacks the ability to attract transcriptional co-activators to TCF/Lef transcription factors [βcat-Sig R26R animals suggests that transcription of Neurog1 is not connected with the function of β-catenin as an activator of TCF/Lef-dependent transcription.Expression of Neurog1 was lost in expected . Interess Figure G-H, O. C factors , the expBAT-gal Wnt-reporter. Quantifications revealed that at least 40% of Neurog2- and Sox10-positive cells were positive for β-galactosidase at E9 . Triple staining for Krox20, neurofilament and the transcription factors Islet1/2, which mark motor and sensory neurons, showed a loss of dorsal as well as ventral Krox20-expressing BCCs at E12.5 in both β-catenin mutant embryos Figure , J-L. KrIn summary, the expression of the transcription factors Neurog2 and Krox20 depends on β-catenin as co-transcription factor for TCF/Lef-mediated transcription. In contrast, Neurog1 expression involves a co-transcription-independent function of β-catenin, indicating that sensory neuronal subtypes in the DRG are specified by distinct mechanisms.βcat-Null animals exhibited more severe phenotypes than βcat-Sig mutants, with first phenotypic variations manifested at E10.5 when the developing DRG coalesce laterally of the neural tube. The presence of N-cadherin and α-catenin at E12.5  mice, in which α-catenin is conditionally deleted in NCCs [βcat-Null and αcat-Adh embryos, the epithelial integrity of the Wnt1-Cre-expressing dorsal neural tube was disturbed [As shown above,  in NCCs ,41. α-Caisturbed   double mutant embryos, which mimics βcat-Null embryos with respect to loss of adhesion and TCF/Lef transcription. If expression of Neurog1 depends on additive effects of these two functions, αcat-Adh βcat-Sig double mutants should display the same phenotype as the βcat-Null. However, transverse sections through DRG of E10.5 αcat-Adh βcat-Sig R26R embryos revealed expression of Neurog1 comparable to control animals .Intriguingly, unlike in 5 Figure H, DRG prβcat-Sig R26R or βcat-Null R26R embryos at E11.5 . In contrast, the expression of the essential desmosome component junction plakoglobin (JUP) was absent in migrating NCCs at E9, but became evident from E10.5 onwards and was readily detectable in DRG progenitors by E11.5   is vital for the secretion of all Wnt proteins -46, evenral tube ,47. Use Taken together, our data and previous reports -24 indicβcat-Null, βcat-Sig, and αcat-Adh embryos. β-catenin signaling, on the other hand, is essential for proper DRG development. However, β-catenin exploits both TCF activation-dependent and -independent signaling to regulate proliferation at different developmental stages as well as distinct cell fates associated with consecutive waves of sensory neurogenesis.β-catenin is a multifaceted regulatory protein that apart from mediating β-catenin-dependent cellular adhesion elicits various transcriptional responses in a context-specific manner . These sβcat-Null but much less so in βcat-Sig mutant animals. In the dorsal neural tube used as a control tissue, the deletion of either β-catenin or α-catenin resulted in loss of cell-cell adhesion and, thus, in the disruption of the epithelial integrity. In contrast, deletion of α-catenin did not affect DRG formation or growth, unlike loss of β-catenin in βcat-Null embryos. Crossing αcat-Adh with βcat-Sig animals also did not produce the phenotype of βcat-Null mutant DRG, excluding any compensatory mechanisms between TCF/Lef transcription and down-stream factors of stabilized adhesion. Additionally, we showed that DRG cells and their projecting axons commence to express plakoglobin as the DRG coalesces and maintain this expression with further development. Taken together, these findings substantiate that cadherin adhesion junctions are negligible for sensory neurogenesis and that cell-cell adhesion in the developing DRG might be mediated via desmosomes. Moreover, the differences in DRG formation observed in βcat-Sig versus βcat-Null embryos can apparently not be attributed to an adhesion phenotype. This, in turn, suggests that the mutated form of β-catenin in βcat-Sig mice not only maintains the ability to preserve cadherin-mediated adhesion, but also rescues a function of β-catenin independent of its function as an activator of TCF/Lef transcription.Cadherin adherens junctions associate transmembrane cadherin proteins with the actin cytoskeleton via a β-catenin - α-catenin complex. Cellular adhesion mediated by this process might conceivably be important for DRG formation, because DRG size and cellular composition were severely affected in conditional βcat-Sig nor in βcat-Null embryos, consistent with earlier reports that canonical Wnt signaling does not regulate proliferation of early NCCs [βcat-Sig and βcat-Null mutant embryos failed to incorporate EdU after a one hour pulse even though they expressed KI67. These results indicate that DRG progenitors in both mutant animals are stuck in cell cycle, most probably in G1 phase. Wnt signaling has been shown to regulate cell proliferation by promoting G1 progression [βcat-Sig and βcat-Null mutants was observed at E10 and E11.5. Therefore, at a stage during development when NCCs coalesce to form DRG, cell cycle progression of DRG progenitors is controlled by Wnt/β-catenin-dependent transcriptional activation, possibly mediated by Lef1  and Sox10-Cre Wlsflox/flox animals , revealin vivo [β-catenin-deficient DRG. Surprisingly, however, our study reveals that several processes underlying DRG formation do neither depend on a structural role of β-catenin in adherens junctions nor solely to attract transcriptional co-activators for initiating TCF/Lef mediated transcription. Recently it has been shown that some TCFs also act as repressors for other modes of transcription [βcat-Sig embryos is capable of maintaining the capacity to de-repress the TCF/Lef Groucho repression complexes, as its TCF/Lef-binding domain is preserved. This theory is supported by the fact that Neurog1 is expressed in the DRG progenitors of βcat-Sig animals, even though consensus sequences for TCF binding are located close to the transcription start site of the mouse Neurog1 gene [βcat-Sig mice, although defective in TCF/Lef activation, preserves a specific signaling function of β-catenin, next to its ability to maintain proper formation of the cadherin adhesion complex. This made it possible to demonstrate that distinct β-catenin signaling functions regulate DRG size and cellular composition in a spatiotemporal manner. The interaction partners and targets mediating these stage-specific functions remain to be identified.Although canonical Wnt signaling has previously been implicated in neural crest development and, in particular, sensory neurogenesis ,20,21,55in vivo . To deficription -10,12,58og1 gene  and β-caog1 gene . In any Wnt1-Cre mice [Sox10-Cre mice [Ctnnb1tm2Kem (Ctnnb1flox) mice [Ctnnb1dm mice [Ctnna1tm1Efu (Ctnna1flox) mice [In vivo fate mapping was performed utilizing either the ROSA26 reporter expressing β-galactosidase (R26R) [ROSA26 reporter expressing EGFP [R26R or EGFP. To monitor Wnt/β-catenin transcription, animals were crossed with the BAT-gal reporter line [Mouse experiments were performed in accordance with Swiss guidelines and approved by the Veterinarian Office of the Kanton of Zürich, Switzerland. The Cre-loxP system was used to conditionally knockout various genes. The generation of mutant mice has been reported previously: Cre mice , Sox10-CCre mice , Ctnnb1tox) mice , Ctnnb1d1dm mice , and Ctnox) mice . In vivoe (R26R)  or the Ring EGFP  mouse liter line .Wls (Wlsflox) was generated . The targeting approach was to flank the first exon with loxP sites. The first exon encodes the transcriptional start and the complete signal sequence. Excision of the ‘floxed’ region, therefore, leads to a Wls null-allele. Bl6 was used as the background strain. The targeting vector was generated by assembling the 5’ and 3’ homology arms with the loxP site flanked first exon and the FRT site flanked neomycin selection cassette  and Wls-lo (5'- GGCGGCATGGAAGCCAAGGGC -3') and 34 cycles of 94°C for 30 seconds, 57°C for 30 seconds and 72°C for 1 minute, to amplify a wild type fragment of 267bp length and, respectively, a mutant fragment 353bp in length.Genotyping for the floxed LacZ reporter gene expression was detected using X-Gal staining [staining . For immstaining .All quantifications were performed on 12 μm transverse sections. Three control and three mutant embryos were quantified at each stage for each type of mutant. To determine rostral-caudal differences quantifications were performed in each embryo on levels of the upper and lower limbs as well as on an intermediate level between the two, collecting two to three sections for each level. As relative proportions were consistent on all three levels in all animal types, we averaged the output of eight sections per embryo. Statistical analyses were performed using the two-tailed unpaired Student’s t-test between control and mutant animals with Microsoft Excel. All results are shown as mean ± standard deviation. Pictures in figures were chosen from the intermediate sections between the upper and lower limbs.


Despite the critical role of endothelial Wnt/β-catenin signaling during central nervous system (CNS) vascularization, how endothelial cells sense and respond to specific Wnt ligands and what aspects of the multistep process of intra-cerebral blood vessel morphogenesis are controlled by these angiogenic signals remain poorly understood. We addressed these questions at single-cell resolution in zebrafish embryos. We identify the GPI-anchored MMP inhibitor Reck and the adhesion GPCR Gpr124 as integral components of a Wnt7a/Wnt7b-specific signaling complex required for brain angiogenesis and dorsal root ganglia neurogenesis. We further show that this atypical Wnt/β-catenin signaling pathway selectively controls endothelial tip cell function and hence, that mosaic restoration of single wild-type tip cells in Wnt/β-catenin-deficient perineural vessels is sufficient to initiate the formation of CNS vessels. Our results identify molecular determinants of ligand specificity of Wnt/β-catenin signaling and provide evidence for organ-specific control of vascular invasion through tight modulation of tip cell function.DOI:http://dx.doi.org/10.7554/eLife.06489.001 Organs develop alongside the network of blood vessels that supply them with oxygen and nutrients. One way that new blood vessels grow is by sprouting out of the side of an existing vessel, via a process called angiogenesis. This process relies on signals that are received by the endothelial cells that line the inner wall of blood vessels, and that direct the cells to form a new ‘sprout’, consisting of tip and stalk cells.In the developing brain, the Wnt/β-catenin signaling pathway helps direct the formation of blood vessels. In this pathway, a member of a protein family called Wnt signals to specific proteins on the surface of the cells lining the blood vessels. Much effort has gone into uncovering the identity of these proteins, with many studies looking at blood vessel development in the brain of mouse embryos.In this study, Vanhollebeke et al. turned to zebrafish embryos to uncover new regulators of angiogenesis and define their roles during the multi-step process of blood vessel development in the brain. A variety of experimental techniques were used to alter and study the activity of different Wnt signaling pathway components. These experiments revealed that the Wnt7a and Wnt7b proteins signal to an endothelial cell membrane protein complex containing the proteins Gpr124 and Reck.Vanhollebeke et al. then created ‘mosaic’ zebrafish embryos, which contained two genetically distinct types of cells—cells that were missing one of the components of Wnt/β-catenin signaling pathway, and wild-type cells. Visualizing the growth of the vessels showed that all the new blood vessels that sprouted had normal tip cells. However, the cells in the stalk of the sprout could be either normal or missing a signaling protein.These findings demonstrate that Wnt/β-catenin signaling controls the pattern of blood vessel development in the brain by acting specifically on the invasive behaviors of the tip cells of new sprouts, a cellular mechanism that allows efficient organ-specific control of vascularization.DOI:http://dx.doi.org/10.7554/eLife.06489.002 Endothelial cells (ECs) acquire organ-specific characteristics to adapt to the requirements of their host tissues. The central nervous system (CNS) vascular microenvironment serves as a paradigm for blood vessel specialization because CNS ECs develop a set of junctional, cellular trafficking, and metabolic properties, collectively called the blood–brain barrier (BBB), that protect the CNS from blood-borne toxins and pathogens. Brain angiogenesis and barriergenesis are temporally coupled through a distinct, and tissue-specific, developmental program . The besIn addition to classical components of the Wnt/β-catenin pathway, such as Frizzled receptors and Lrp5/Lrp6 co-receptors , recent At present, the cellular mechanisms by which Wnt/β-catenin signaling controls the complex and multistep process of CNS blood vessel formation remain largely unexplored. Here, we leverage the zebrafish model to study the molecular machinery governing Wnt-dependent brain angiogenesis using a combination of targeted mutagenesis, morpholino knock-downs, RNA injections, genetic mosaics, and single-cell resolution real-time imaging. We find that EC-specific and Gpr124-dependent Wnt/β-catenin signaling is required for angiogenic sprouting throughout the zebrafish brain. In addition, we identify Reck , a GPI-anchored MMP inhibitor and angiogenic modulator , as a nogpr124 mutant alleles using TAL effector nucleases  sprouting from the dorsal aorta appears to be unaffected  begin to form by angiogenic sprouting from the dorsal wall of the PHBCs. These sprouts progress dorso-medially into the neural tissues to connect with the basilar artery and by 36 hpf, an average of four sprouts per hindbrain hemisphere have formed . In cont vessels . Defectsaffected . Injectihenotype  and bothhenotype . Asymmethenotype , possiblhenotype .Video 1.gpr124 mutant zebrafish can proceed through organogenesis in the complete absence of intracerebral blood vessels and approximately half of them reach adulthood , Pgp and by permeability assays . In contrast to the regular pattern of alternating longitudinal melanophore-rich dark stripes and light interstripes found in wild-types, gpr124 mutants exhibit discontinuous stripes formed by clusters of melanophores bordered by xanthophores and iridophores, most prominently in the dorsal aspect of the trunk  expression in a subset of wild-type ECs of the PHBCs starting at 26 hpf. Gradually, both the intensity and the number of GFP+ cells increased and these cells contributed to the PHBC, BA, and CtAs of 48 hpf wild-type embryos. In contrast, no GFP+ ECs could be detected in the PHBC or BA after gpr124 or reck knock-down  expression in the forming DRGs at 54 hpf  and thaTo test whether the combination of Reck and Gpr124 can co-activate signaling by Wnts other than Wnt7a and Wnt7b, we screened all 19 Wnts and Norrin for co-activation by Gpr124 alone, Reck alone, or Gpr124 plus Reck . This exIn earlier experiments, we observed that Gpr124 co-activation of Wnt7a- and Wnt7b-dependent signaling in STF cells could be stimulated by Lrp5 but not by Lrp6, and we postulated that one or more additional proteins might be required to enhance signaling in the presence of Lrp6 . The preTo examine whether the functional interactions between Gpr124, Reck and the Fz/Lrp5/6 complex reflect a multi-component membrane receptor complex involved in Wnt7 binding, their subcellular localization was investigated in 293 cells. We generated amino-terminal epitope-tagged version of Gpr124 (FLAG-Gpr124) and Reck (HA-Reck) and validated the biological activity of the fusion proteins in STF assays (data not shown) and brain vascular rescue experiments in zebrafish embryos . When exvegf genes were not increased in the absence of Gpr124 at 48 hpf   that labels perivascular cells presenting all characteristics of bona fide pericytes. We found extensive vascular wall coverage in both the presence and absence of Gpr124 function  , we detesiblings . As exped the BA . Mammalid the BA . To detefunction . We then embryos , but onl embryos  to emerg mutants . Similargpr124 mutant embryos using a novel Tg(kdrl:lifeact-EGFP) line that labels filamentous actin  ECs in the context of Tg(kdrl:ras-mCherry) gpr124, reck or Wnt/β-catenin deficient endothelial neighbors (Tg(kdrl:GFP) cells were transplanted into Tg(kdrl:ras-mCherry) wild-type hosts, tip cell genotypes were randomized  characterization of the pattern of Wnt/β-catenin signaling in the developing zebrafish brain vasculature and the consequences of reduced Wnt/β-catenin signaling on brain angiogenesis, (2) identification of Reck, together with Gpr124, as integral components of a novel Wnt7a/Wnt7b-specific receptor complex in both zebrafish and mammals, and (3) discovery of a distinctive requirement for Wnt/β-catenin signaling in tip cells during brain angiogenesis.Fz4−/− ECs to assemble into mosaic vessels, suggesting that Wnt/β-catenin signaling  (gpr124 and reck morphant vascular defects were not rescued by dll4 knock-down or gamma-secretase inhibitor (DAPT) treatment.Despite current evidence for the tight integration of multiple signaling systems in EC development, our data argue that the tip cell requirement for Wnt/β-catenin signaling operates through a mechanism that does not simply reflect a global modulation of VEGF and/or Notch signals. In hibition , appearsn (NICD) , gpr124 In our experiments, the Wnt reporter transgene labels both tip and stalk cells in nascent sprouts, as well as ECs in adult CNS vessels , reflectReck−/− mouse embryos die in utero at around E10.5 with prominent vascular defects including vessel dilation, hemorrhaging, and arrested development of the primary vascular plexus , in agreement with results from a previous study that examined Reck's role in DRG development s843, Tg(kdrl:ras-mCherry)s896, Tg(kdrl:NLS-GFP)zf109, Tg(hsp70l:dkk1-GFP)w32, Tg(hsp70l:Mmu.Axin1-YFP)w35, Tg(7xTCF-Xla.Siam:GFP)ia4, Tg(ngn1:gfp), Tg(UAS:EGFP), Tg(sox10:mRFP)vu234, Tg(kdrl:NLS-mCherry)is4 transgenic lines have been described elsewhere  (Tg(fli1:Myr-mCherry) was generated by fusing an oligonucleotide encoding the myristoylation (Myr) signal derived from Lyn to mCherry in the pTol2-fli1 vector, yielding the pTol2-fli1:Myr-mC plasmid. The Tg(kdrl:Lifeact-EGFP)s982 line was generated following cloning of the F-actin binding Lifeact-EGFP downstream of a 6.8 kb kdrl promoter in pTol2. The TgBAC(pdgfrb:citrine)s1010 line was generated using a BAC clone containing the start codon of pdgfrb (CH73-289D6) using a pRedET based protocol (Zebrafish were maintained and embryos were obtained and raised under standard conditions. Establishment and characterization of the lsewhere . The Tg(C-2A-mC)  was geneprotocol . Tol2 LTprotocol .All images were acquired using a Leica  M165 stereomicroscope or a Zeiss  LSM710 confocal microscope after embryo anesthesia with a low dose of tricaine and immobilization in 1% low-melting agarose in glass-bottom Petri dishes . Time-lapse images were recorded every 10 min for a total period of 16 hr. The microscope stage was enclosed in a temperature-controlled chamber, and samples were kept at 28.5°C. The Sulfo-NHS-Biotin leakage assay was performed by intra-ventricular injection of 5 nL of 0.5 mg/mL EZ-link Sulfo-NHS-Biotin  in D-PBS, with the help of a micromanipulator, in animals treated with a low dose of tricaine. After 60 min, animals were anaesthetized with an overdose of tricaine, and the brains and livers were dissected and fixed in 4% PFA overnight at 4°C before embedding in 4% agarose. Vibratome sections of 150 μm thickness were permeabilized in 0.4% PBT (Triton X-100) and blocked in 4% PBTB (BSA). Primary and secondary antibody staining was performed at 4°C overnight. The following primary antibodies were used: anti-GLUT1 , anti-P Glycoprotein [C219] . Biotin was detected with Alexa Fluor 647 streptavidin. In all confocal images and time points, brightness and contrast were adjusted linearly and equally. Three-dimensional reconstructions were done with the Imaris FilamentTracer software  in automatic detection mode before manual false-coloring and editing to highlight extra- and intra-cerebral vessels.gpr124 were designed and cloned using Golden Gate assembly  with the following primers: gpr124-Fo: CTTGTTCTTTTTGCAGGATCCGCCACCATGTCGGGACCCTGCGTC; gpr124-Re: CTATAGTTCTAGAGGCTCGAGTTATACAGTCGTCTCGCTCTTCC; gpr125-Fo: CTTGTTCTTTTTGCAGGATCCGCCACCATGTCGGTGCTTTGCGTCC; gpr125-Re: CTATAGTTCTAGAGGCTCGAGCTACACAGTAGTTTCATGCTTCCAC. Full-length zebrafish reck was amplified from 48 hpf cDNA and recombined into BamHI-XbaI digested pCS2 using In-fusion cloning  with the following primers: reck-Fo: CTTGTTCTTTTTGCAGGATCCACCATGAGCGGGTGTCTCCAGATC; reck-Re: ACGACTCACTATAGTTCTAGAGTCAGAGGTCAGAGGTCAGGG. gpr124/125 hybrids were generated by recombining two PCR fragments overlapping by 15 bp derived from the above mentioned constructs into BamHI-XhoI digested pCS2 using In-fusion cloning . In hybrids 1 and 2, the hinge corresponds to amino acid 742/743 of Gpr124 (VLHP742/V743IYT) and amino acid 739/740 of Gpr125 (PLHP739/V740IYA). In hybrids 3 and 4, the hinge corresponds to amino acid 1034/1035 of Gpr124 (HHCF1034/K1035RDL) and amino acid 1029/1030 of Gpr125 (HHCV1029/N1030RQD). Splice-blocking morpholinos against erbb3b (TGGGCTCGCAACTGGGTGGAAACAA) (sorbs3 (TTTCCGACAGGGAAAGCACATACCC) (reck (CAGGTAGCAGCCGTCACTCACTCTC) (gpr124 (ACTGATATTGATTTAACTCACCACA) were obtained from Gene Tools  and injected at the one-cell stage at 0.5, 4, 1, and 2 ng, respectively, unless otherwise stated. Injection of a standard control morpholino (up to 8 ng) did not affect brain vasculature or DRG formation.Capped messenger RNA was synthesized using the mMESSAGE mMACHINE kit . The following expression plasmids were generated and used in this study: full-length zebrafish GAAACAA) , sorbs3 CATACCC) , reck (CCACTCTC)  and gpr1Tg(kdrl:ras-mCherry)s896 embryos were injected with the indicated morpholinos. Donor Tg(kdrl:GFP)s843 and host embryos were dechorionated with pronase (1 mg/mL) for 5 min at 28°C in 1/3 Ringer solution supplemented with penicillin (50 U/mL)/streptomycin (50 µg/mL) before being incubated in agarose-coated dishes in the same medium. Twenty to 50 cells were removed from donor embryos at mid-blastula stages and transplanted along the blastoderm margin of age-matched host embryos which were subsequently grown at 28.5°C until the indicated stages. The contribution of GFP+ transplanted cells was assessed using a Leica M165 stereomicroscope and EC position within mosaic vessels was determined using confocal microscopy. Contribution of cells of defined genotype to the tip cell position was calculated as a percentage of the total number of mosaic CtAs or ISVs.Host one-cell stage fli1a gene was used to direct endothelial transgenic expression , 1-AKP , SB 216763 (50 μM), BIO (0.5 μM). LiCl was diluted in egg water, other drugs were prepared in 100% DMSO and diluted to the indicated concentration with egg water. As a control, volume-matched DMSO solutions in egg water were used.Tg(kdrl:GFP)s843gpr124 mutants and 20 wild-type siblings, cDNAs were amplified, labeled with Cy3 (gpr124 mutants) or Cy5 (wild-type siblings), and hybridized to the Agilent Zebrafish (V3), 4x44k Gene Expression Microarray.RNA was extracted from 20 FLAG-Gpr124 and HA-Reck were generated by recombining two PCR fragments overlapping by 15 bp derived from the above mentioned constructs into BamHI-XhoI digested pCS2 using In-fusion cloning . The FLAG peptide was inserted between the amino acid 48/49 of zebrafish Gpr124 and the HA peptide between the amino acid 23/24 of zebrafish Reck. The mouse Fz4-GFP and human FLAG-Dvl2 constructs were obtained from Addgene . HEK 293T cells were transfected with Lipofectamine 2000  1 day after seeding and were grown in IBIDI imaging chambers for 2 days before fixation for 15 min in 4% paraformaldehyde. Indirect immunofluorescence and proximity ligation assays were performed with the following antibodies: mouse monoclonal anti-FLAG M2  diluted at 1/2500, purified polyclonal rabbit anti-HA  diluted at 1/250 and Alexa-conjugated secondary antibodies  diluted at 1/5000. PLAs were performed following the manufacturer's instructions . DAPI nuclear counterstaining was performed for 2 min at 5 µg/mL.dll4 probe was generated with the following primers: dll4-ISH-Fo:GCAGCTTGGCTCACCTTTCTC and dll4-ISH-Re: TAATACGACTCACTATAGGGAGTCCTTTCTCCTGATGCCTGC and T7 was used for transcription and digoxigenin labelling. For whole-mount in situ hybridization, embryos were fixed in 4% paraformaldehyde overnight at 4°C and processed as described previously  1 day after seeding in a 96-well tray and were harvested 2 days later. Assays were performed in triplicate, and the ratio of activities of firefly luciferase (expressed from a stably integrated reporter with seven tandem Lef/Tcf binding sites) to Renilla luciferase was determined for each well. Wnt signaling components were expressed from a CMV promoter. The following amounts of plasmid DNA were transfected per well. Statistical analysis was performed using GraphPad software. Data presented in bar graphs represent mean ± SD. p-values were calculated by the one-way ANOVA (post hoc Tukey's test) and Student's t test for multiple and single comparisons of normally distributed data , respectively and by the Kruskal–Wallis (post hoc Dunn's test) and Mann–Whitney test for multiple and single comparisons of non-normally distributed data, respectively. p-values of tip cell genotype in mosaic vessels  were det review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and two reviewers.Thank you for sending your work entitled “Tip cell specific requirement for an atypical Wnt/β-catenin pathway during CNS angiogenesis” for consideration at The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.The reviewers agreed that this is a highly interesting, innovative and well-presented manuscript, highlighting the heterogeneity of the molecular mechanisms that underlie angiogenesis in different vascular beds. It will have broad impact on the field of angiogenesis.The major points raised that need to be addressed are:dll4 knock down and γ-secretase inhibition by DAPT did not rescue the phenotype of Gpr124MO. Considering however the cited paper by Corada et al., conceptually these experiments are not suited to rescue the Gpr124MO phenotype, as Dll4 should be down-regulated in Gpr124MO due to the diminished β-catenin signaling. In this case the over-expression of Dll4, rather than its inhibition should rescue the phenotype. The authors would need to determine the level of Dll4 expression in the Gpr124 and Reck mutants, which would determine whether tip cell specific β-catenin signaling does regulate Dll4 and thereby contribute to the tip cell expression profile and function. Along the same line, the authors should comment on the arterial differentiation of vessels within the Gpr124 and Reck mutants.1) The authors nicely cite and describe the importance of Notch signaling for tip-stalk cell formation and the possible role of Gpr124/Reck in this scenario. In the subsection entitled “Wnt/β-catenin signaling in tip cells”, the authors claim that 2) The authors do not provide direct evidence that Gpr124 and Reck physically interact at the membrane in a Fzd receptor complex. This should be possible to investigate in STF cells by biochemical assays , at least in the over-expressing situation. Can colocalization be observed by immunofluorescence in the overexpressing cells? 1) The authors nicely cite and describe the importance of Notch signaling for tip-stalk cell formation and the possible role of Gpr124/Reck in this scenario. In the subsection entitled “Wnt/β-catenin signaling in tip cells”, the authors claim that dll4 knock down and γ-secretase inhibition by DAPT did not rescue the phenotype of Gpr124MO. Considering however the cited paper by Corada et al., conceptually these experiments are not suited to rescue the Gpr124MO phenotype, as Dll4 should be down-regulated in Gpr124MO due to the diminished β-catenin signaling. In this case the over-expression of Dll4, rather than its inhibition should rescue the phenotype. The authors would need to determine the level of Dll4 expression in the Gpr124 and Reck mutants, which would determine whether tip cell specific β-catenin signaling does regulate Dll4 and thereby contribute to the tip cell expression profile and function. Along the same line, the authors should comment on the arterial differentiation of vessels within the Gpr124 and Reck mutants.dll4 transcriptional dysregulation in the cerebrovascular defects observed in the absence of the Gpr124/Reck-dependent Wnt/β-catenin signaling. In particular, we tested, using an enhancer from the fli1a gene, whether mosaic transgenic endothelial expression of dll4 in gpr124- and reck-deficient embryos was sufficient to overcome the absence of endothelial Wnt/β-catenin signaling in the PHBCs. To track expressing cells, dll4 and gpr124 coding sequences were fused to EGFP by a viral 2A peptide sequence and both sequential orientations were tested. While EGFP-positive cells were detected at equal frequencies in different segments of the vasculature, including the PHBCs, with all tested transgenic constructs, only gpr124-expressing cells could restore brain angiogenesis by leading new sprouts at tip cell positions  The authors do not provide direct evidence that Gpr124 and Reck physically interact at the membrane in a Fzd receptor complex. This should be possible to investigate in STF cells by biochemical assays , at least in the over-expressing situation. Can colocalization be observed by immunofluorescence in the overexpressing cells?As requested, we added new data showing that Gpr124, Reck and Frizzled co-localize in cell culture after over-expression . We addi


We previously demonstrated that 6-benzylthioinosine (6-BT) could induce the differentiation of a subset of acute myeloid leukemia (AML) cell lines and primary AML cells regardless of their cytogenetics. In this study we investigated whether Wnt signaling pathways played roles in 6-BT-induced differentiation of AML cells.We induced differentiation of HL-60 leukemic cells and primary AML cells in vitro using 6-BT. Real-time PCR (qPCR), western blot, and luciferase assays were used to examine the molecules’ expression and biological activity in canonical and noncanonical Wnt signaling pathways. AML cell differentiation was measured by the Nitroblue tetrozolium (NBT) reduction assay.2+ signaling in HL-60 cells. Pre-treatment of HL-60 cells with an inhibitor of glycogen synthase kinase-3β (GSK-3β), which activated canonical Wnt signaling, partly abolished the differentiation of HL-60 cells induced by 6-BT. Pre-treatment of HL-60 cells with an inhibitor of protein kinase C (PKC), resulting in inactivation of non-canonical Wnt/Ca2+ signaling, abolished 6-BT-induced differentiation of HL-60 cells. Several molecules in the non-canonical Wnt/Ca2+ pathway were detected in bone marrow samples from AML patients, and the expression of FZD4, FZD5, Wnt5a and RHOU were significantly reduced in newly diagnosed AML samples compared with normal controls.6-BT regulated the expression of both canonical and non-canonical Wnt signaling molecules in HL-60 cells. Both 6-BT and all-trans-retinoic-acid (ATRA) reduced canonical Wnt signaling and activated noncanonical Wnt/CaBoth canonical and non-canonical Wnt signaling were involved in 6-BT-induced differentiation of HL-60 cells, and played opposite roles in this process. Wnt signaling could be involved in the pathogenesis of AML not only by regulating self-renewal of hematopoietic stem cells, but also by playing a role in the differentiation of AML cells.The online version of this article (doi:10.1186/1471-2407-14-886) contains supplementary material, which is available to authorized users. Wnt signaling pathways are highly conserved and regulate cell fate decision at all stages of development in multiple tissue types, including hematopoietic stem cells , 2. Dere+ cells [2+-releasing pathway that is activated by the Wnt-stimulated G proteins. Calcium/protein kinase C(PKC) and calmodulin-dependent kinase II (CaMKII) were considered to be primary mediators of this signaling pathway [In the canonical Wnt/β-catenin signaling pathway, engagement of a Wnt protein by a Frizzled (FZD) receptor leads to stabilization of β-catenin, which then translocates into the nucleus to initiate target gene expression through interaction with the TCF/LEF transcriptional complex . Deregul+ cells . In both+ cells , 10. In + cells . The non pathway , 13. Non pathway . Wnt5a w pathway .We have demonstrated that 6-BT induced differentiation of various AML cell lines and primary AML cells . Since tHL-60 cells were cultured in Iscove’s modified Dulbecco’s medium  supplemented with 10% fetal bovine serum  and 1% penicillin–streptomycin. ATRA, PMA and NBT were purchased from Sigma. 6-BT was kindly provided by the National Cancer Institute Developmental Therapeutics Program. Bisindoylmaleimide (BIM) and BIO were purchased from CalBiochem. Primary antibodies for β-catenin, p-PKC (Thr638), PKC, p-CaMKII (Thr286), CaMKII and β-actin were purchased from Abcam. Primary antibodies for p-Rac (Ser71) and Rac were purchased from Cell Signaling Technology.Bone marrow mononuclear cells of patients treated at Qilu Hospital  were obtained between May 2008 and July 2009. Thirty patients had newly diagnosed AML, twelve had AML in complete remission, and ten were normal controls. Informed consent was obtained from each donor. Procedures for collecting samples were approved by the Drug and Clinical Investigations Ethics Committee of the Faculty of Medicine, Qilu Hospital, Shandong University.2 Profiler™ PCR array (PASH-043) was generously provided by SuperArray Bioscience Corporation . The PCR array was performed according to the manufacturer’s instructions. Briefly, total RNA was isolated from HL-60 cells after treatment with 6-BT (10 μM) or vehicle (0.01% DMSO) for 3 days. Reverse transcription was performed with M-MuLV reverse transcriptase (Fermentas) using an oligo(dT)18 primer. Genomic DNA contamination was eliminated by Dnase treatment using an RNeasy Micro Kit (Qiagen). Expression of Wnt molecules was tested by PCR on ABI Prism 7700 (Applied Biosystems). For data analysis, the ΔΔCt method was used. For each gene, fold changes were calculated as the difference in gene expression between 6-BT- or vehicle-treated cells; a positive value indicates gene up-regulation and a negative value indicates gene down-regulation.Human Wnt signal RTTotal RNA was isolated from HL-60 cells treated with 6-BT or vehicle for 1 day or 3 days, using TRIzol reagent (Invitrogen). RNA was transcribed into cDNA using the Enhanced Avian RT First Strand Synthesis kit (Sigma). RT-PCR was performed in triplicate using FastStart SYBR Green Master (Roche Diagnostics) on an Applied Biosystems 7500 Fast Real-Time PCR System. Primers used are available upon request.-ΔΔCT method with β-actin as an endogenous control.For patient samples, qRT-PCR was performed using SYBR Green PCR Master Mix (Toyobo) on an ABI Prism 7500 sequence detection system. All reactions were carried out in 20-μl reaction volume in triplicate. Fold changes in gene expression were determined using the 25 cells/mL) were cultured in 96-well plates. Cells were first treated with 10 μM of 6-BT or DMSO (0.01%) for 5 d, then with 20 uL of a solution of NBT (5 mg/mL) and PMA (100 ng/mL). Cells were incubated at 37°C for 30 min and at least 200 cells were counted for the positive percent.We used NBT reduction to evaluate differentiation of AML cells. To perform the NBT assay, 100 μL of HL-60 cells , then solubilized in radio immunoprecipitation assay (RIPA) lysis buffer containing 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 0.15 mol/l NaCl and 0.05 mol/l Tris–HCl, pH 7.2. Protein concentrations were determined with the bicinchoninic acid (BCA) assay protein reagent kit (Sangon) according to a standardized curve. Total proteins (30 ug/lane) were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes using standard procedures. Non-specific sites were blocked with 5% nonfat milk in PBS with 0.1% Tween-20. Primary antibodies were used according to the manufacturer’s instructions. The near-infrared fluorescence-labeled secondary antibodies detecting primary antibodies were IRDye 680 Goat Anti-Rabbit IgG and IRDye 800CW Goat Anti-Mouse IgG . Detection and quantification were performed with the Li-Cor Odyssey imaging system and its software.6 HL-60 cells were transfected with 2.5 μg of either TOPFLASH, FOPFLASH or NFAT luciferase along with 0.25 μg pRL-TK. Vehicle (0.01% DMSO), positive control (10 mM LiCL) or 6-BT (10 or 20 μM) were added 24 hours after transfection. After another 24 hours, cell lysates were prepared and reporter activity was measured using the Dual-Luciferase Reporter Assay System (Promega).The TOPFlASH is a luciferase reporter of β-catenin-mediated transcriptional activation. The backbone of TOPFlASH is the pTA-Luc vector of Clontech, which provides a minimal TA viral promoter driving expression of the firefly luciferase gene. 7 TCF/LEF binding sites were cloned into the Mlu1 site of this vector. The negative control FOPFLASH construct contains mutated TCF/LEF binding sites . NFAT-luCells were washed twice with PBS, then loaded with Fluo-3/AM (Molecular Probes) for 30 min, and warmed to 37°C before flow cytometry analysis using a FACScan (Becton Dickinson).Immunofluorescence was performed to identify subcellular localization of β-catenin. Three days after treatment with 6-BT (10 μM) or ATRA (1 μM), HL-60 cells were harvested by centrifugation. Drops of cells were plated on polylysine-coated slides and incubated at room temperature for 25 min, then fixed with 4% polyoxymethylene. Cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min, and then blocking was carried out with goat serum for 30 min to minimize nonspecific binding of the primary antibody. The β-catenin antibody  was applied at a 1:100 dilution overnight followed by three 5-min washes in PBS. FITC anti-rabit IgG (Jackson Lab) were used to detect β-catenin. Images were captured using a Zeiss Microscopy LSM 780 fluorescent microscope and analyzed with Image J software.Values are mean ± standard deviation (SD) from 3 independent experiments. Groups were compared using a Student’s two-tailed unpaired t test. For patient samples, the copy number of each gene is presented quantitatively as mean ± SD. The difference in copy number of each gene in the AML-ND, AML-CR, and CON groups was performed using a one-way ANOVA test. SPSS software (version 15.0) was used for all statistical analysis. Tests for statistical significance were two-sided. P values less than 0.05 were considered to indicate statistical significance.2 Profiler™ qPCR array. Twelve genes, Wnt5a, Wnt11, FZD2, FZD4, FZD5, FZD7, JUN, KREMEN1, RHOU, CCND1, PPC and B2M, were up-regulated more than 4-fold upon 6-BT treatment  are Wnt molecules or positive regulators, whereas most down-regulated genes  are in the canonical Wnt signaling pathway  or vehicle (0.01% DMSO) for 3 days. A total of 96 genes, including 5 housekeeping genes, were examined in the RT0 μM or vWnt5a, FZD4, FZD7, KREMEN1, RHOU, Wnt6, and DIXDC was compared in HL-60 cells treated with 6-BT or vehicle for 1 day or 3 days. We demonstrated that expression levels of Wnt5a, FZD4, FZD7, KREMEN1, and RHOU were significantly up-regulated after 6-BT treatment, whereas expression levels of Wnt6 and DIXDC were significantly down-regulated  that were treated with 6-BT (10 μM) for 24, 48 or 72 hours. Levels of β-catenin protein were measured by Western blot. β-catenin protein was significantly decreased upon treatment with 6-BT in all primary AML FAB M2 and M3 samples  for 4 hours and then tried to induce HL-60 differentiation by 6-BT (10 μM) or ATRA (1 μM) for 3 days, 6-BT and ATRA almost completely lost their capacity to induce differentiation of HL-60 cells , Wnt11 (~4.03 fold increase), FZD2 (~4.0 fold increase), FZD4 (~8.0 fold increase), FZD5 (~4.59 fold increase) and FZD7 (~7.46 fold increase) , AML patients in complete remission (AML-CR) and normal controls (CON). The mean expression level of FZD4, FZD5, Wnt5a and RHOU were significantly down-regulated in AML-ND samples compared with normal controls . Amanda et al. detected ability of Wnt5a protein to directly stimulate intracellular calcium flux and found Wnt5a protein treatment at high dose did not alter the intracellular concentration of Ca2+ in 293 cells [2+ signaling pathway. In our study, we found 6-BT up-regulated Wnt5a, as well as other noncanonical receptor molecules, such as FZD2, FZD4 and FZD5, indicating that receptor and/or co-receptor recruitment in Wnt/Ca2+/PKC activation is involved.On the other hand, non-canonical Wnt/Cam/CaMKII  and PKC m/CaMKII , which hm/CaMKII . Silencim/CaMKII , 39. Ectm/CaMKII . In our 93 cells . The act93 cells . Based o2+ influx and promoted degradation of β-catenin independent of GSK3β and β-TRCP [2+ from cytosolic stores resulted in calpain-mediated degradation of β-catenin [2+ pathway. In our study, we discovered 6-BT and ATRA increased Wnt5a level, induced Ca2+ influx and upregulation of PKC, which might be the possible mechanism of β-catenin degradation. However, our PCR array data suggested that up-regulation of noncanonical and down-regulation of canonical Wnt signaling pathway seemed to happen simultaneously, raising some questions of Wnt signaling pathway in leukemogenesis. Does activation by treatment by 6-BT and ATRA of noncanonical Wnt signaling lead to inactivation of canonical Wnt signaling, or is this a coincidence? The interrelationship between canonical and noncanonical Wnt/Ca2+ signaling pathways needs further exploration. The Wnt signaling pathway is context-dependent transduced to both canonical and noncanonical pathways based on the expression profile of Wnt, sFRP, WIF, DKK, and FZD co-receptors and the activity of intracellular Wnt signaling regulators [Previous studies reported the activation of noncanonical Wnt signaling lead to inactivation of canonical Wnt signaling. Topol and Ishitani et al. found activation of noncanonical Wnt signaling induced Cad β-TRCP , 43. Li -catenin . PKC-med-catenin , 46. Cho-catenin . Taken tgulators . BesidesFZD4, FZD5, Wnt5a and RHOU are significantly down-regulated in bone marrow samples from newly diagnosed AML patients compared to normal controls, suggesting a critical role of Wnt signaling pathway in the pathogenesis of AML.We demonstrated that both canonical and noncanonical Wnt signaling pathways were collectively involved in the 6-BT and ATRA induced leukemic cell differentiation. c-Myc. On the other hand, 6-BT treatment also increased in the transcriptional levels of the noncanonical Wnt ligands and receptors-Wnt5a, Wnt11, FZD2, FZD4, FZD5 and FZD7. The phosphorylation levels of CaMKII and PKC, effecors in noncanonical Wnt pathway, were upregulated, indicating the activation of noncanonical Wnt signaling pathway. Figure S2. 6-BT treatment had little effect on the mRNA level of β-catenin. Real-time RT-PCR detected β-catenin mRNA levels in HL-60 cells after being treated with DMSO (0.01%) or 6-BT for 3 days. β-actin was used as control. The values represent the means ± S.E. (n = 3). Figure S3. 6-BT substantially increased the number of cells in the G0/G1 phase while concomitantly reduced the number of cells in the S phase in HL-60 cells. After being treated with DMSO (0.01%) or 6-BT for 2 days, HL-60 cells were were harvested and washed twice in PBS, then fixed in 75% alcohol over night at 4°C. After washed in cold PBS thrice, cells were resuspended in 1 mL PBS with 40 μg PI and 100 μg RNase A  and incubated for 30 min at 37°C. Samples were then analyzed by FACS. (DOC 579 KB)Additional file 1: Figure S1: A proposed model of effect of 6-BT on both canonical and noncanonical Wnt signaling molecules in HL-60 cells. On the one hand, 6-BT treatment up-regulated level of KREMEN1 and down-regulated level of β-catenin, and concomitantly reduced the canonical Wnt signaling target gene


Burkholderia mallei is a host-adapted bacterium that does not persist outside of its equine reservoir. The organism causes the zoonosis glanders, which is endemic in Asia, Africa, the Middle East and South America. Infection by B. mallei typically occurs via the respiratory or percutaneous route, and the most common manifestations are life-threatening pneumonia and bacteremia. Glanders is difficult to diagnose and requires prolonged antibiotic therapy with low success rates. There is no vaccine to protect against B. mallei and there is concern regarding its use as a biothreat agent. Thus, experiments were performed to establish a non-human primate model of intranasal infection to study the organism and develop countermeasures. Groups of marmosets  were inoculated intranasally with B. mallei strain ATCC 23344 and monitored for clinical signs of illness for up to 13 days. We discovered that 83% of marmosets inoculated with doses of 2.5 X 104 to 2.5 X 105 bacteria developed acute lethal infection within 3–4 days. Signs of disease were severe and included lethargy, inappetence, conjunctivitis, mucopurulent and hemorrhagic nasal discharges, and increased respiratory effort with abdominal lifts. Burkholderia mallei was cultured from the lungs, spleen and liver of these animals, and pathologic examination of tissues revealed lesions characteristic of glanders. Challenge experiments also revealed that 91% of animals infected with doses ranging from 25 to 2.5 X 103 bacteria exhibited mild non-specific signs of illness and were culture negative. One marmoset inoculated with 2.5 X 103 organisms developed moderate signs of disease and reached humane end-points 8 days post-infection. The liver and spleen of this animal were colonized with the agent and pathological analysis of tissues showed nasal, splenic and hepatic lesions. Taken together, these data indicate that the marmoset is a suitable model to study respiratory infection by B. mallei. Burkholderia mallei is a non-motile Gram negative bacillus that does not persist in the environment outside of its natural equine reservoir. The organism causes the highly contagious and debilitating zoonotic disease glanders, which primarily affects horses, mules and donkeys. Glanders is an old disease, having first been described by Aristotle in 330 B.C. Other less commonly known names that have been used throughout history to describe the disease include equinia, malleus, droes and farcy. Though now considered exotic in North America and Europe, glanders once was endemic in these regions. Early settlers introduced the disease in the U.S. via the import of infected horses. By the 2nd half of the 19th century, glanders was widespread due to the Civil War, when it was readily transmitted to thousands of cavalry horses passing through remount stations. In response to this epidemic, industrial countries implemented vigorous eradication programs that entailed financial incentive to owners, destruction of infected animals, and disinfection of affected facilities. Great Britain was first to eradicate the disease in 1928, followed by the U.S. in 1934, Canada in 1938, and most European countries by 1965 [B. mallei bacilli disseminate to the liver, spleen and lymph nodes where it rapidly causes necrotizing abscesses [B. mallei from patients. The mortality rate is high (up to 50%) despite aggressive antimicrobial therapy. Burkholderia mallei is resistant to many antibiotics, which limits treatment options. The recommended two-phase treatment process involves the use of ceftazidime and meropenem (intensive phase), followed by TMP-SMX and co-amoxiclav (eradication phase) for several weeks [B. mallei.Humans, goats, dogs, cats, rabbits, and carnivorous predators living in the vicinity of equids with glanders, have been naturally infected. Camels have also been reported to harbor the disease and have been linked to zoonotic transmission –7, 10–13bscesses –18. The al weeks . ResponsB. mallei might be used as a bioweapon because it has been utilized in this manner on multiple occasions [B. mallei. German agents also infected thousands of mules in Mesopotamia with the organism, and a German agent was arrested in Russia with similar intentions in 1916. Between 1932 and 1945, the Japanese deliberately infected horses, civilians, and prisoners of war with glanders at the Pin Fan Institute in occupied Manchuria. More recently (1980s), the Soviet Union purportedly weaponized B. mallei and used it against opposition forces in Afghanistan [B. mallei as a Tier 1 agent, and the development of medical countermeasures (MCM) against the organism is highly desirable.Though human cases of glanders are rare, there is legitimate concern that ccasions , 20–25. hanistan . For theB. mallei and provide excellent platforms for developing MCM (i.e. identification and characterization of targets). In contrast, there have been very few reports describing the use of non-human primates as surrogates to study glanders and validate lead MCM candidates [B. mallei using the common marmoset. Our data indicate that this species is a suitable surrogate to study glanders.Several animal models have been developed to study glanders including hamsters, mice and horses –31. Thesndidates –34. To aB. mallei strain ATCC 23344 [-2 , 10-3 , 10-4 , 10-5 (250 CFU), and 10-6 (25 CFU) were used to infect animals.The CC 23344  was usedCallithrix jacchus) were obtained from Worldwide Primates Inc. The animals were >36 months old and weighed 220–430 grams at the time of infection, and were acclimatized to their new surroundings for at least two weeks. Prior to inoculation, the animals were anesthetized in their nest boxes with inhalant isoflurane gas until a respiratory rate of 10–12 breaths per minute was achieved. Once anesthetized, the animals were held in supine position and droplets of B. mallei suspension (5–10 μL) were delivered to the right nostril using a sterile filtered tip attached to a P200 Pipetman. The droplets were inhaled through the nasal passageways, and a total volume of 50 μL of bacterial suspension was administered.Both male and female, healthy, sexually mature common marmosets  supplemented with various food items  in order to recreate their previous diet at the Worldwide Primates Inc. Therapeutic intervention with antibiotics and/or analgesics was not provided as such treatments would have affected the experimental outcomes of the study. Infected animals were monitored every 8 hr.Marmosets were housed individually in stainless steel cages  positioned to allow visual and vocal communication. The animals were provided various forms of environmental enrichment including wooden perches, mirrors, teethable circular link baby toys, and Kong puzzle toys filled with forage items . Water and food were provided 3 points: complete anorexia and/or lack of water consumption for 24 hr; 3 points: non-responsiveness to external stimuli, recumbency, marked difficulty breathing (dyspnea); 2 points: a "dull" appearance, manifested in part by a reduced responsiveness to external stimuli or lethargic movements in the cage; 2 points: failure to enter the nestbox or climb onto a resting perch; 2 points: failure to consume water for 24 hr; 1 point: mild changes in respiration, such as slightly increased difficulty or an increased respiratory rate; 1 point: inappetence for 1 day. Any animal with a point score ≥ 3 was humanely euthanized. This was accomplished by anesthetizing marmosets in their nest boxes using inhalant isoflurane gas. Once anesthetized, the animals were administered a dose of 10–20 mg/kg ketamine intramuscularly and then euthanized by intracardiac injection of a pentobarbital dose of 240 mg/kg. This procedure is in accordance with the AVMA Guidelines for the Euthanasia of Animals. The median lethal dose (LD50) value was calculated according to Reed and Muench [Humane end-points were strictly observed. Marmosets exhibiting signs of moderate to severe discomfort were euthanized. The following scoring system was used to determine if euthanasia was appropriate: d Muench , and is A board-certified veterinary anatomic pathologist performed post-mortem examination of tissues and organs from all animals.B. mallei bacteria in tissues. To suppress growth of the marmoset normal flora, agar plates containing 8 μg/mL Polymixin B , 2 μg/mL Bacitracin  and 5 μg/mL Cyclohexamide  were utilized.Samples of lung, liver, spleen, and trachea were aseptically collected for bacterial culture prior to processing the tissues for cytology and histology. These samples were weighed, homogenized with 15-mL disposable tissue grinders (Fisherbrand), serially diluted, and plated on agar medium to calculate the number of viable Tissue imprints of lung, liver, and spleen were prepared on glass slides, air-dried for 10 min, fixed with 100% anhydrous methanol, and stained with modified Wright-Giemsa using an automated Aerospray Hematology Slide Stainer . Cytologic evaluation was performed by a board-certified veterinary clinical pathologist using an Olympus BX51 light microscope (Olympus Corporation). Images were captured using an Olympus DP71 camera and software (Olympus Corporation).B. mallei, the samples were taken out of the BSL3 laboratory, embedded in paraffin, cut into 3 μm-thick sections, and stained with hematoxylin and eosin (H&E). Histologic evaluations were performed by a board-certified veterinary anatomic pathologist using an Olympus BX41 microscope (Olympus Corporation). Images were captured using an Olympus DP71 camera and software (Olympus Corporation).Tissues selected for histology included sagittal sections of the right nasopharynx, sections of all lobes of the lungs, trachea, submandibular and tracheobronchial lymph nodes, liver, spleen, kidneys, adrenal glands, and representative sections of the gastrointestinal tract . The tissues were submersed in 10% buffered formalin  for 3 weeks, cut, placed into histology cassettes for biopsy samples (Leica BIOSYSTEMS), and fixed in 10% buffered formalin for an additional 2 days. For nasopharyngeal tissues, the animals' heads were bisected and placed into decalcification solution (Leica BIOSYSTEMS) for 5 hr prior to cutting, placing into cassettes, and submersing into 10% buffered formalin. After verifying that fixed tissues no longer contained live B. mallei infection. The serum, which was obtained in the context of a previous study [Selected tissues were stained using a modified Gram stain method developed by the Histology Laboratory at The University of Georgia College of Veterinary Medicine, which identifies bacteria as Gram positive (purple-black) or Gram negative (dark pink-red). Selected tissues were also concurrently processed for examination by immunofluorescence microscopy. Briefly, tissue sections were incubated in a 10 mM sodium citrate buffer (pH 6.2) for 10 min at a temperature of 120°C to unmask antigens. Following this high-temperature antigen retrieval step, the tissues were washed, permeabilized with a solution containing 0.05% Triton X-100 for 20 min at room temperature, and probed for 1 hr with convalescent serum from mice that survived acute us study , was useB. mallei bacilli were performed inside a Class II Biosafety Cabinet (BSC) in a BSL3 laboratory. Animals infected with B. mallei were housed in wire front and top cages within an ABSL3-Ag room, which served as primary containment , at The University of Georgia Animal Health Research Center. The animal facility is part of the University of Georgia’s program that is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC-I). Post-mortem examination and tissue collection were performed in a BSL3 necropsy suite equipped with a Class II BSC. All experiments were performed in compliance with the rules and regulations of the U.S. Federal Select Agent Program and were approved by The University of Georgia Institutional Biosafety Committee (IBC) and Institutional Animal Care and Use Committee (IACUC). Animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All efforts were made to minimize animal suffering. Anesthesia and euthanasia procedures were performed by trained personnel including board-certified veterinarians, animal care staff, and research team members. The IACUC approval number covering this specific study is AUP A2013 01-018-Y2-A2.Preparation of the inoculum for infection and processing of tissues containing live Burkholderia mallei infection generally occurs via the respiratory or percutaneous route. The respiratory route of infection, and subsequent pulmonary disease, are of particular concern with respect to the use of B. mallei as a biothreat agent because the airways are the most likely route of entry for the bacterium during a biologic attack. For these reasons, we developed a marmoset model of intranasal infection to study the organism. Five groups of animals were inoculated with increasing doses of strain ATCC 23344 and monitored for clinical signs of illness for a period of up to 13 days. Two uninfected marmosets were included as controls and were housed in the same room with the infected animals. The results are summarized in 50 in mice has been reported to be 820 CFU [50 doses of 1,000–1,800 CFU [ 820 CFU . Nebuliz 820 CFU . The use,800 CFU , 38, 39.B. mallei, considerable effort was put into documenting the clinical presentation of disease. As expected, the severity and onset of clinical signs correlated with the bacterial dose administered. Marmosets in the low dose groups (25 and 250 CFU) displayed mild and non-specific signs of illness. At day 7 post-infection, the animals stopped grooming and by day 10, most exhibited withdrawn behavior , had decreased appetite and reduced activity, and developed dry and crusted nostrils. One marmoset from each group also displayed mild unilateral conjunctivitis at the end point of the study  displayed moderate symptoms of disease. By day 4, both reached humane experimental end points as they exhibited rough coats, unilateral conjunctivitis, clear nasal discharges, inappetence, lethargy, and increased respiratory rates  rapidly developed severe signs indicative of glanders. Two days post-infection, all three animals stopped grooming, were inappetent, and their water intake was reduced. One marmoset unexpectedly died ~60 hr post-challenge 544, . This anThe two uninfected control animals exhibited mild and non-specific signs of illness attributable, presumably, to the BSL3 environment. These included brief periods of decreased appetite and mildly withdrawn behavior , occasional soft stools, and rough unkempt coat at the end point of the study.B. mallei, we collected tissues from infected animals at the indicated end points and determined bacterial loads in the lungs, spleen, liver, and trachea. The results are shown in the last 4 columns of B. mallei in the spleen and liver, which correlates with the overt signs of illness displayed by the animal on day 8 post-infection.To examine replication and dissemination of B. mallei  were processed for culture, and the results show colonization of the spleen, liver and lungs  exhibiting moderate symptoms of infection were colonized with ngs F48, .B. mallei during infection, tissue imprints  were prepared at the indicated end points and examined by microscopy. The cytology findings are summarized in i.e. presence of erythroid, myeloid, and megakaryocytic precursor cells), and occasional segmented neutrophils with normal morphology (To investigate pathologic changes induced by rphology . The lunrphology .i.e. LPS), which perforates the nuclear and cellular membranes of the neutrophil and ultimately inhibits the cell’s ability to control water homeostasis. The splenic sample from marmoset D37 also contained numerous degenerate neutrophils. These findings are consistent with the moderate symptoms of glanders displayed by the animal on day 8 post-challenge and colonization of its spleen and liver with B. mallei  appeared normal, correlating with their unremarkable clinical presentation and culture negative status . Interes. mallei . The lunB. mallei  exhibited suppurative inflammation. Neutrophil morphology in these samples ranged from normal to vacuolated and degranulated. Neutrophil vacuolation and degranulation typically occur during inflammation and signify the release of bactericidal molecules . The samples also showed moderate lymphoid hyperplasia as well as moderate extramedullary hematopoiesis . These f. mallei . The livnegative . Conversnegative . The lunB. mallei  were examined and were clearly indicative of bacterial infection F48, . Represe. mallei . ConsistB. mallei, tissues were collected at the indicated end points, fixed with formalin, processed into thin sections, stained, and examined by microscopy. The histology findings are summarized in To further investigate pathologic changes induced by B. mallei  contained lesions indicative of infection. Marmoset D37 had severe bilateral mucopurulent and pyogranulomatous rhinitis, focally-extensive fibrinopurulent perihepatitis and hepatitis, and marked splenomegaly with microabscesses and pyogranulomas. All lesions contained numerous degenerate neutrophils, and the nasopharyngeal and liver specimens showed intralesional bacilli. These findings are consistent with the obvious symptoms of illness displayed by the animal on day 8 post-challenge , coloniz. mallei , and the. mallei . Interes. mallei , contain. mallei , culture. mallei , and the. mallei . The lunB. mallei  revealed lesions in the nasopharynx, lungs, spleen, and liver with intralesional bacilli . These f. mallei , and the. mallei . Both an. mallei . The nas. mallei ; immunof. mallei . Multinu5,000 CFU in vivo –42, wereB. mallei. Examination of liver samples from marmoset 544 revealed numerous, variably-sized, and scattered foci of necrosis composed of leukocyte nuclear debris, necrotic hepatocytes, and intralesional B. mallei bacilli  were examined and showed extensive pathology in their nasal cavity, lungs, liver and spleen with intralesional bacilli and multinucleated giant cells . The mosilli Fig –5H. The illi Fig . Grosslyilli Fig , large milli Fig , to multilli Fig –6H. Onlyilli Fig . The patilli Fig  and colo. mallei .B. mallei. We show that this non-human primate develops an illness similar to that described in other established models for the organism, and the disease onset and progression resemble that reported for humans with glanders. We also document the median lethal dose that causes acute disease by the intranasal route of inoculation and detail pathologic changes induced by B. mallei during infection.In this study, we demonstrate that the common marmoset is susceptible to intranasal infection with 3 bacteria exhibited mild and non-specific signs of illness and were culture negative for B. mallei. Only one marmoset inoculated with 2.5 X 103 organisms  and rapidly progressive lethal disease (3–8 days), the severity of which correlates with inoculum size. Symptoms in animals infected with medium, medium-high, and high doses consisted of subdued behavior , lethargy, inappetence, decreased water intake, decreased grooming behavior with piloerection (rough coats), ocular and nasal discharges, conjunctivitis, tachypnea (increased respiratory rate), and dyspnea . Natural cases of glanders in humans and other animals exhibit many of these signs, namely malaise, excessive lacrimation (tearing), nasal discharge, conjunctivitis, tachypnea, and dyspnea [We observed that marmosets infected with higher doses of  dyspnea , 45, 46. dyspnea , 27, 31.B. mallei were colonized with the organism . Additional findings in these samples consisted of highly vacuolated and degranulated neutrophils, band neutrophils, lymphoid hyperplasia, and increased extramedullary hematopoiesis  and histopathology  documented in other experimental models of glanders as well as in natural cases in equids and humans. Hence our findings complement those of Nelson and colleagues and underscore the usefulness and relevance of the marmoset model to study glanders and develop countermeasures.While preparing this manuscript, Nelson and colleagues published a report in which the common marmoset was used to develop a subcutaneous model of infection for . mallei . Marmose