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Fig.1. Heart defect in a tadpole after sustained spib morpholino inhibition of myeloid cell differentiation. (A–E) X. laevis Tg[lurp1:egfp] tadpole that was injected with Xtspib-e1i1MO into dorsal blastomeres, 40 ng dose. Tadpole was photographed at stage 40 (A and B) and stage 41 (C–E), with fluorescence from eGFP shown (B and D). The complete inhibition of myeloid cell differentiation caused by loss of spib has been sustained to stage 40. A large edema formed during this time interval, indicative of cardiac dysfunction (A, C, E). (F–J) Control sibling tadpole that was injected with the same morpholino into ventral blastomeres, photographed at stage 40 (F and G) and stage 41 (H–J). Fluorescent embryonic macrophages can be seen distributed throughout the tadpole (G and I). Hematopoiesis also observed in pronephros and liver (G and I). No edema developed and heart morphology appeared normal. Anterior is to the left in lateral (A–D, F–I) and ventral (E and J) views. Ed, edema; M, myeloid/macrophage; PN, pronephros; Lv, liver; MB and HB, additional domains of Tg[lurp1:egfp] fluorescence in mid and hindbrain.
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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.3. Spib morpholino affects myeloid cell production and also cardiac muscle morphogenesis. (A–J) Stage 31 tadpoles injected with Xtspib-e1i1MO into dorsal blastomeres (A–F), 40 ng dose, or control ventral blastomeres (G and H), or non-injected sibling (I and J), hybridized with probes for mpo and mlc2 (myocardium). Two tadpoles with severe macrophage deficit are shown (A–D) and one with a milder reduction (E and F). Myocardial phenotype severity appears linked to the number of functional macrophages produced. Anterior is to left in lateral views and ventral view of heart-forming regions. (K and L) Transverse section through the myocardium of the mild phenotype tadpole (E, F, K) compared with non-injected control (I, J, L), Nuclear-Fast Red counterstained (NFR). Section plane indicated (Sp). Scale bar = 100 μm. En, endoderm; Mc, myocardium; Ec, endocardium. (M–T) Stage 31 tadpoles injected with the spib morpholino, 40 ng dose and with same sequence of blastomere injections presented. Tadpoles hybridized to mpo and tnni3 (myocardium). One tadpole is shown with severe macrophage deficit (M and N) and one with a milder reduction (O and P).
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Fig.4. Spiba mRNA expression within the heart-forming region. (A–C) Wholemount in situ hybridization for myeloid spiba, ventral views of heart-forming region, at stage 27 (A), stage 29 (B) and stage 31 (C). Red triangle depicts position of forming myocardium (B and C). (D–F) Transverse sections through heart region of same embryos, with section planes depicted (A–C). Stage 27 (D), stage 29 (E) and stage 31 sections (F). To ease identification, the approximate position of the (myocardial) mesoderm has been artificially-darkened (E and F). NFR (D–F) nuclear counterstained. Scale bar = 100 μm. Sp, section plane; M, myeloid/macrophage; HF, heart field; Mc, myocardium; Ec, endocardium; VM, ventral midline; En, endoderm.
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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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Fig.6. Lurp1 morpholino disrupts myocardial morphogenesis prior to heart tube formation. (A–D) Tadpoles injected with the lurp1-MO into dorsal blastomeres, 12 ng dose, at stage 31 (A) and stage 34 (C), compared with non-injected sibling embryos (B and D). Tadpoles hybridized to mhcα (myocardium). Anterior is to left in ventral view of heart-forming regions. (E and F) Heart section of the stage 34 tadpole with dorsal blastomere morpholino and flat, bilateral myocardium (C and E), compared with control stage 34 tadpole (D and F). No counterstain. Scale bars = 100 μm. Sp, section plane; L, left; R, right; Mc, myocardium; Ec, endocardium; Lv, liver.
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Fig.7. Endocardial smad3 expression is detected in macrophage-morpholino defective embryos. (A–F) Tadpoles injected into dorsal blastomeres with spib morpholino, 40 ng dose (A and B), or lurp1 morpholino, 12 ng dose (C and D), or non-injected sibling (E and F). The spib morpholino caused a mild macrophage deficit phenotype (A and B) while the lurp1 morpholino induced abnormal, broadened morphology at the ventral midline (D). Tadpoles carry the Tg[smad3-egfp] reporter and were hybridized with probes for mpo and eGFP. At stage 32, the transgenic line gives eGFP expression within head, eye, pronephros, somite and notochord domains (A, C, E), while strong expression occurs in the forming endocardium, but not in myocardium at these stages, nor in macrophages. Anterior is to left in lateral views and ventral view of heart-forming regions. (G–I) Transverse heart sections of the tadpoles. Cells underlying the malformed myocardium gave endocardium-type smad3 reporter expression in morpholino tadpoles (G and H) while the forming endocardium stained strongly positive in the control (I). The right-sided myocardial region only, was presented for the morpholino injected tadpoles due to their broader ventral surface and to allow the necessary image magnification. NFR-counterstained. Scale bars = 100 μm. Sp, section plane; R, right; Mc, myocardium; Ec, endocardial cell activity.
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Fig.8. Tissue replacement surgery recovers heart formation defects of macrophage-morpholinos. (A–D) Anterior-ventral tissue replacement surgery was performed at stage 16 between a donor embryo and a recipient embryo (A, Supplementary Movie 1, see Section 4). The donor carries the Tg[lurp1:egfp] transgene and had been injected with DsRed1 RNA into 2/2 blastomeres. The recipient embryo carries the Tg[mlc1v:egfp] transgene and was injected with the lurp1-MO into dorsal blastomeres, 16 ng dose. Graft recipient tadpole at stage 32 (B). White rectangle (B) shows position of fluorescence images (C-green, migratory macrophages; D-red, graft tissue). (F–H) The same tadpole now at stage 42 has a beating heart. White circle (F) shows position of fluorescence images, ventral view (G-green, donor-macrophages and recipient-heart; H-red, graft tissue). E, I, J: Sibling tadpole injected with lurp1-MO but was not operated on, shown at stage 32 (E) and stage 42 (I and J). The remnant of myocardial tissue does not form a lumen and does not beat (J). Anterior is to left in all views. Gr, graft; M, macrophage; IH, interhyoid facial muscle; H, heart.
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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.
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Supplementary Fig. 4
Target specificity of lurp1 translation-inhibiting morpholino. (A–D): Injection of 200 pg L1-eGFP-nuc RNA into one blastomere at the two-cell stage (1/2). Fluorescence viewed of same embryo at stage 10.5 (A and B) and stage 28 (C and D). (E–H): Similar injection of L1-eGFP-nuc RNA, followed by injection of 16 ng lurp1-MO (plus rhodamine-Dextran) to the same site at the four-cell stage. The lurp1-MO inhibits translation of the synthetic RNA in vivo. (I–L) Injection of 200 pg of control L2-eGFP-nuc RNA. (M–P) Similar injection of L2-eGFP-nuc RNA, followed by injection of 16 ng lurp1-MO. The lurp1-MO does not inhibit translation of the control RNA. Images of green fluorescence from eGFP and red fluorescence from rhodamine-Dextran are presented. Stage 10.5 embryos oriented with the blastopore at the bottom of each image while stage 28 embryos are lateral views.
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Supplementary Fig. 5
Lurp1 RNA splice-interfering morpholino similarly affects myocardial morphogenesis. (A and B) Stage 35 tadpole injected with Xllurp1-e2i2MO into dorsal blastomeres, 16 ng dose, hybridized to mpo and mlc2. The section plane (B) is indicated on the head (A). (C–E) Control tadpole with same morpholino in ventral blastomeres. Position of two sections (D andindicated (C). Scale bar = 100 μm. Sp, section plane; Mc, myocardium; Ec, endocardium; M, macrophage; Lv, liver.
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Supplementary Fig. 6
Spib mismatch morpholino does not affect myeloid cell production. (A–D) Stage 24 embryos injected with the mismatch control morpholino, Xlspiba-e1i1misMO, into both blastomeres at the two-cell stage (A and B), 40 ng dose, or into dorsal blastomeres (C and D). Embryos hybridized to mpo and nkx2-5. (E and F) Sibling embryo injected with the active spib morpholino, Xlspiba-e1i1MO, into dorsal blastomeres at 40 ng dose. (G–J) Stage 31 tadpoles with the same sequence of mismatch control spib morpholino and hybridized to mpo and mlc2. (K and L) Sibling tadpole injected with the active spib morpholino. Myeloid cell production and embryo development is unaffected by the mismatch control morpholino whereas the active morpholino inhibits myeloid development.
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Supl. Fig. 7.Lurp1 mismatch morpholino does not affect Xenopus development. (A–D) Stage 24 embryos injected with the mismatch control morpholino, lurp1-misMO, into both blastomeres at the two-cell stage (A and B), 12 ng dose, or into dorsal blastomeres (C and D). Embryos hybridized to mpo and nkx2-5. (E and F) Sibling embryo injected with the active lurp1 morpholino into dorsal blastomeres at 12 ng dose. (G–J) Stage 31 tadpoles with the same sequence of mismatch control lurp1 morpholino and hybridized to mpo and mlc2. (K and L) Sibling tadpole injected with the active lurp1 morpholino. Myeloid cell production and embryo development is unaffected by the mismatch control morpholino whereas the active morpholino causes macrophage migration from lateral positions and defective (broader) heart myocardium morphogenesis.
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Supplementary Fig. 2
Spib morpholino comparison of bilateral versus dorsal blastomere injection. (A): Tailbud stage 23 embryos injected with Xtspib-e1i1MO, 40 ng dose, into both blastomeres at the two-cell stage (2/2, A and B), into dorsal blastomeres (C and D), or non-injected control (E and F). Embryos hybridized with probes for mpo and nkx2-5. (G): Late tailbud stage 28 embryos, with the same sequence of spib morpholino blastomere injections presented and hybridized to mpo and tbx5. (M): Stage 31 tadpoles with the same sequence of spib morpholino blastomere injections and hybridized to mpo and tnni3. Different morpholino injected embryos were chosen for presentation compared to Fig. 2 and Fig. 3.
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