Xenbase Image ID: 159722

Fig. 7. Knockdown of casp9 depletes the expression of early blood marker genes. (A) X. tropicalis embryos were injected in two CD4 blastomeres at 16-cell stage with 2.5 ng C9MOatg1, 4 ng CoMO or a combination of 2 ng C3MOatg and 2 ng C7MOatg in each blastomere. β-Gal RNA (10 pg) was coinjected as lineage tracer. WISH analysis of T3 globin and gata1 expression in the posterior part of the VBI is shown. Both T3 globin and gata1 expression were affected upon depletion of casp9, but not upon the combined depletion of casp3 and casp7. (B) Graphs representing the percentage of embryos injected in CD4 blastomeres at 16-cell stage, exposing the defects in marker expression. The embryos were scored using the phenotype in A as a reference (based on six independent experiments). (C) Depletion of T3 globin expression could be rescued by X. laevis casp9 RNA. X. tropicalis embryos were injected in two CD4 blastomeres at 16-cell stage with 2.5 ng CoMO, 2.5 ng C9MOatg1 or 2.5 ng C9MOatg1, combined with 100 pg X. laevis casp9 RNA in each blastomere. β-Gal RNA was coinjected as a lineage tracer. WISH analysis shows T3 globin expression in the posterior part of the VBI. The overexpression of Xlcasp9 could rescue the defect in T3 globin expression induced by casp9 depletion. (D) The percentages of embryos injected in CD4 blastomeres at 16-cell stage showing the defect in T3 globin marker expression (based on four independent experiments). (E) For quantitative RT-PCR analysis, X. tropicalis embryos were injected in two CD4 blastomeres at 16-cell stage with 4 ng CoMO, 2.5 ng C9MOatg1 with or without 100 pg X. laevis casp9 RNA, or a combination of 2 ng C3MOatg and 2 ng C7MOatg in each blastomere. RT-PCR analysis of T3 globin expression shows significant reduction upon injection of C9MOatg1, which is rescued upon coinjection of casp9 RNA. T3 globin expression is not significantly affected in C3/C7 double morphants. The graph shows the combination of three biological replicates. ns, not significant; *P<0.05; **P<0.005 (Student’s t-test). Image published in: Tran HT et al. (2017) Copyright © 2017. Image reproduced with permission of the Publisher, The Company of Biologists Ltd. Larger Image Printer Friendly View

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