Able 1. Analysis of cartilage phenotype by Alcian staining.Table 2. Results of morpholino microinjection experiments (2 experiments for each combination).Phenotype ( ) Samples Std CO-Mo I exp II exp Moxat1 I exp II exp Moxat3 I exp II exp Moxat1+3 I expn56 37 76 46 50 38StrongWeak 7 5 8 11 16No effect 93 95 92 89 84 87 35 MoXat3 MoXat1 Std CO O Otx2 Nrp1 Twist Otx2 Nrp1 Twist 81 75 91 68 72 74 89 79 80 94 93 SampleExpression level alteration ( )nStrong Slight Increase 57773-63-4 reduction reduction 14 4 3 1 1 9 8 9 11 31 29 12 12 19 25 28 24 18 18 32 42 60 8No effect86 84 85 78 74 54 68 73 71 37 29doi:10.1371/journal.pone.0069866.tOtx2 Nrp1 TwistD, E). Furthermore, consistent with the pharyngeal skeleton phenotype, a clear reduction in the expression of Twist (Fig. 3 H, I), a key gene expressed in NCC and promoting epithelial mesenchymal transition and migration [26,32], was get SPDB observed in 26 of embryos. This percentage is in good 16574785 agreement with that of tadpole larvae showing a strong phenotype in the pharyngeal arches; another 60 of embryos showed a weak reduction of Twist expression (Table 2). On the other hand, injection of single MOs had a weak effect on these molecular markers: a strong reduction was observed in less than 10 of cases, and a weak reduction in about 18?8 of embryos (depending on the marker) (Fig. S2; Table 2). As a control, around 95 of embryos injected with a standard control MO (8 ng) had no skeletal phenotype, and only a few had a weak reduction in pharyngeal arches (Fig. S3I; Table 1); whenMoXat1+Otx2 Nrp1 Twist117doi:10.1371/journal.pone.0069866.tsimilarly injected embryos were scored for molecular marker expression, about 85 of them showed no alteration, 12?4 displayed a weak reduction and very few a strong reduction (Fig. S3A ; Table 1). The distributions of the diverse skeletal phenotypes obtained in these experiments were significantly different in combinedFigure 3. Results of combined antisense MoXat1 and MoXat3 injections in Xenopus embryos. Reduction of Xotx2 (A or J , respectively for strong or slight reduction), nrp-1 (D , strong; M , slight) and Twist (G , strong; P , slight) expression is observed on the injected side of embryos (inj), compared to uninjected side (un). Strong or weak reduction (I, R respectively) of pharyngeal skeleton is observed on the injected side of antisense MO treated swimming tadpoles compared to control side. Beta-gal red staining traces injected side of embryos. doi:10.1371/journal.pone.0069866.gMulti-AT-Hook Factors in XenopusMoxat1+Moxat3 injected embryos compared to embryos injected with either standard or Moxat1 or Moxat2 morpholinos (Table S1); similar statistical support to our conclusions was observed also for the effects on molecular markers (Table S2). Finally, although we did not detect Xhmg-at-hook2 mRNA in our RT-PCR experiments, we have also designed and injected a MO (MoXat2) targeting this mRNA. Either when injected alone or when injected in combination with MoXat1 or MoXat3, MoXat2 did not elicit any phenotype or increased the effects of the other two MOs, in agreement with Xhmg-at-hook2 negligible level of expression (data not shown) and further strengthening the specificity of the effects obtained with MoXat1 and MoXat3.XHMG-AT-hook1 Biochemical Properties are Distinct from Those of Xenopus XLHMGA2ba and Human HMGAThe newly described Xhmg-at-hook transcripts code for noncanonical HMGA proteins since they have multiple AT-hooks and no C-terminal acidic tail. To ch.Able 1. Analysis of cartilage phenotype by Alcian staining.Table 2. Results of morpholino microinjection experiments (2 experiments for each combination).Phenotype ( ) Samples Std CO-Mo I exp II exp Moxat1 I exp II exp Moxat3 I exp II exp Moxat1+3 I expn56 37 76 46 50 38StrongWeak 7 5 8 11 16No effect 93 95 92 89 84 87 35 MoXat3 MoXat1 Std CO O Otx2 Nrp1 Twist Otx2 Nrp1 Twist 81 75 91 68 72 74 89 79 80 94 93 SampleExpression level alteration ( )nStrong Slight Increase reduction reduction 14 4 3 1 1 9 8 9 11 31 29 12 12 19 25 28 24 18 18 32 42 60 8No effect86 84 85 78 74 54 68 73 71 37 29doi:10.1371/journal.pone.0069866.tOtx2 Nrp1 TwistD, E). Furthermore, consistent with the pharyngeal skeleton phenotype, a clear reduction in the expression of Twist (Fig. 3 H, I), a key gene expressed in NCC and promoting epithelial mesenchymal transition and migration [26,32], was observed in 26 of embryos. This percentage is in good 16574785 agreement with that of tadpole larvae showing a strong phenotype in the pharyngeal arches; another 60 of embryos showed a weak reduction of Twist expression (Table 2). On the other hand, injection of single MOs had a weak effect on these molecular markers: a strong reduction was observed in less than 10 of cases, and a weak reduction in about 18?8 of embryos (depending on the marker) (Fig. S2; Table 2). As a control, around 95 of embryos injected with a standard control MO (8 ng) had no skeletal phenotype, and only a few had a weak reduction in pharyngeal arches (Fig. S3I; Table 1); whenMoXat1+Otx2 Nrp1 Twist117doi:10.1371/journal.pone.0069866.tsimilarly injected embryos were scored for molecular marker expression, about 85 of them showed no alteration, 12?4 displayed a weak reduction and very few a strong reduction (Fig. S3A ; Table 1). The distributions of the diverse skeletal phenotypes obtained in these experiments were significantly different in combinedFigure 3. Results of combined antisense MoXat1 and MoXat3 injections in Xenopus embryos. Reduction of Xotx2 (A or J , respectively for strong or slight reduction), nrp-1 (D , strong; M , slight) and Twist (G , strong; P , slight) expression is observed on the injected side of embryos (inj), compared to uninjected side (un). Strong or weak reduction (I, R respectively) of pharyngeal skeleton is observed on the injected side of antisense MO treated swimming tadpoles compared to control side. Beta-gal red staining traces injected side of embryos. doi:10.1371/journal.pone.0069866.gMulti-AT-Hook Factors in XenopusMoxat1+Moxat3 injected embryos compared to embryos injected with either standard or Moxat1 or Moxat2 morpholinos (Table S1); similar statistical support to our conclusions was observed also for the effects on molecular markers (Table S2). Finally, although we did not detect Xhmg-at-hook2 mRNA in our RT-PCR experiments, we have also designed and injected a MO (MoXat2) targeting this mRNA. Either when injected alone or when injected in combination with MoXat1 or MoXat3, MoXat2 did not elicit any phenotype or increased the effects of the other two MOs, in agreement with Xhmg-at-hook2 negligible level of expression (data not shown) and further strengthening the specificity of the effects obtained with MoXat1 and MoXat3.XHMG-AT-hook1 Biochemical Properties are Distinct from Those of Xenopus XLHMGA2ba and Human HMGAThe newly described Xhmg-at-hook transcripts code for noncanonical HMGA proteins since they have multiple AT-hooks and no C-terminal acidic tail. To ch.