Journal: Mechanisms of Development

Article Title: Early cardiac morphogenesis defects caused by loss of embryonic macrophage function in Xenopus

doi: 10.1016/j.mod.2011.04.002

Figure Lengend Snippet: 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.

Article Snippet: Subsequently, 16 ng of lurp1-MO (plus 4 ng rhodamine-B dextran 10,000, Invitrogen) was injected into half of the embryos at the four-cell stage, into the same embryo region.

Techniques: Inhibition, Cell Differentiation, Injection, Fluorescence, Control

Journal: Mechanisms of Development

Article Title: Early cardiac morphogenesis defects caused by loss of embryonic macrophage function in Xenopus

doi: 10.1016/j.mod.2011.04.002

Figure Lengend Snippet: 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.

Article Snippet: Subsequently, 16 ng of lurp1-MO (plus 4 ng rhodamine-B dextran 10,000, Invitrogen) was injected into half of the embryos at the four-cell stage, into the same embryo region.

Techniques: Migration, Injection, Control, Sequencing, Staining

Journal: Mechanisms of Development

Article Title: Early cardiac morphogenesis defects caused by loss of embryonic macrophage function in Xenopus

doi: 10.1016/j.mod.2011.04.002

Figure Lengend Snippet: 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.

Article Snippet: Subsequently, 16 ng of lurp1-MO (plus 4 ng rhodamine-B dextran 10,000, Invitrogen) was injected into half of the embryos at the four-cell stage, into the same embryo region.

Techniques: Injection, Control

Journal: Mechanisms of Development

Article Title: Early cardiac morphogenesis defects caused by loss of embryonic macrophage function in Xenopus

doi: 10.1016/j.mod.2011.04.002

Figure Lengend Snippet: 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.

Article Snippet: Subsequently, 16 ng of lurp1-MO (plus 4 ng rhodamine-B dextran 10,000, Invitrogen) was injected into half of the embryos at the four-cell stage, into the same embryo region.

Techniques: Expressing, Injection, Transgenic Assay, Staining, Control, Activity Assay

Journal: Mechanisms of Development

Article Title: Early cardiac morphogenesis defects caused by loss of embryonic macrophage function in Xenopus

doi: 10.1016/j.mod.2011.04.002

Figure Lengend Snippet: 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 ). 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.

Article Snippet: Subsequently, 16 ng of lurp1-MO (plus 4 ng rhodamine-B dextran 10,000, Invitrogen) was injected into half of the embryos at the four-cell stage, into the same embryo region.

Techniques: Injection, Fluorescence