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Fig.2. Spib morpholino affects myeloid cell production but not cardiac specification. (A–D) Tailbud stage 23 embryos injected with the Xtspib-e1i1MO into dorsal blastomeres (A–D), 40 ng dose, or as a control in ventral blastomeres (E and F), or non-injected sibling (G and H). Embryos subjected to wholemount in situ hybridization for mpo (myeloid/macrophage) and nkx2-5 (heart field). Dorsal-injected embryos exhibited either a severe loss of the myeloid domain (A and B) or a milder reduction (C and D). Nkx2-5 mRNA expression detected in all examples. (I–P) Late tailbud stage 28 embryos injected with the spib morpholino, 40 ng dose and with the same sequence of blastomere injections presented. Embryos hybridized with probes for mpo and tbx5. Cardiac tbx5 mRNA detected in all examples. Anterior is to left, lateral views and ventral view of heart fields. |
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Fig.5. Lurp1 morpholino affects myeloid domain migration and cardiac muscle morphogenesis. (A–H) A stage 25 embryo injected with the lurp1-MO into both blastomeres at the two-cell stage (2/2) (A and B), 12 ng dose. Embryos similarly injected into dorsal blastomeres (C and D), or as a control in ventral blastomeres (E and F), or non-injected sibling (G and H) and hybridized with probes for mpo and nkx2-5. (I–P) Stage 35 tadpoles injected with the lurp1 morpholino, 12 ng dose, with the same sequence of blastomere injections presented and hybridized to mpo and mlc2. Anterior is to the left in lateral and detail ventral views. (Q–S) Transverse section through the heart region of tadpoles injected with lurp1 morpholino. Examples of a cardiac bifida phenotype (I, J, Q) and abnormal myocardial folding morphogenesis (K, L, R) were observed in both the two-cell stage and dorsal blastomere injection experiments but never after ventral blastomere injection (M, N, S). (T and U) Stage 25 heart field sections of embryo with dorsal blastomere morpholino (C, D, T) and non-injected sibling (G, H, U). Green arrowheads show dark mpo stain of macrophages. Asterisk (*) denotes mesodermal nkx2-5 stain. NFR-counterstained. Scale bar = 100 μm. Sp, section plane; M, myeloid/macrophage; En, endoderm; Mc, myocardium; Ec, endocardium. |
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Supplementary Fig. 3
Mesodermal nkx2-5 expression is detected in spib morpholino embryos. (A–D): Stage 25 embryo injected with the Xtspib-e1i1MO into dorsal blastomeres, 40 ng dose (A and B), or non-injected control (C and D). Embryos hybridized to mpo and nkx2-5. (E and F) Transverse sections through the heart field of the embryos. Inset image shows the whole section while main image depicts the region of the ventral midline. Arrowheads indicate dark mpo stain (E and F). NFR-counterstained. Scale bar = 100 μm. Sp, section plane; Me, mesoderm; En, endoderm. |
