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FIG. 5.
Expression of XMIF in developing embryos and organs of an adult frog. The RT-PCR/Southern blot analysis was performed as described under “Experimental Procedures.†A, MIF was expressed maternally; levels decreased at the gastrula and neurula stages (stages 11 and 15, respectively), and increased again at the tailbud stage (stage 20). B, MIF was ubiquitously expressed in various organs in the adult frog. Histone 4 (H4) and EF-1α served as the RNA loading controls.
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FIG. 6.
XMIF is expressed in the developing central nervous system. Whole mount in situ hybridization analysis was performed for the blastula (stage 9), gastrula (stage 11), early neurula (stage 13), neurula (stage 15), early tailbud (stage 26), and tailbud (stage 34) embryos. A, lateral view of a stage 9 embryo hybridized with the MIF probe. No signal was detected in control hybridization using a sense probe (not shown). B and C, lateral views of stage 11 embryos hybridized with antisense (B) and sense (C) probes. The arrow in B indicates the signal in the dorsal marginal zone. D-G, lateral (D and E) and dorsal (F and G) views of stage 13 embryos hybridized with antisense (D and F) and sense (E and G) probes. The arrows in D and F indicate the expression in the anterior region of the neural plate. H-J, lateral (H) and dorsal (I and J) views of stage 15 embryos hybridized with antisense (H and I) and sense (J) probes. K, lateral view of a stage 26 embryo. L and M, lateral views of stage 34 embryos hybridized with antisense (L) and sense (M) probes. The bar shown in A indicates 1 mm. ap, animal pole; vp, vegetal pole; a, anterior; p, posterior.
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FIG. 7.
MIF MOs inhibit translation of MIF RNA. Synthesized RNA of MIF, including its 5′ UTR (+U), MIF without 5′ UTR (-U), or EF-1α was translated in vitro in the presence of MO1 (1), MO2 (2), or the standard control MO (C) at the indicated concentrations. MO1 efficiently inhibited the translation of MIF, whereas the control MO had no effect on the translation (lanes 1-3 and 6). MO2 also inhibited the translation of the same RNA, with reduced efficiency (lanes 4 and 5). MO2 did not inhibit the translation of MIF RNA lacking an MIF-derived 5′ leader sequence (lanes 7 and 8). MO1 and MO2 had no effect on the translation of an RNA unrelated to MIF (lanes 9-11).
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FIG. 8.
MIF MO disturbs the early differentiation of neural tissue as well as notochord formation. Two dorsal blastomeres of 4-cell stage embryos were injected with standard control MO (9.2 pmol) (B, E, H, K, and M), or with MIF MO (MO1, 9.2 pmol) (C, F, I, L, and N). These embryos were cultured until stage 32 (tailbud stage) for histological or in situ hybridization analyses. A, D, G, and J are uninjected control embryos. D-L, transverse sections at the level of the eye (D-F), at the level of the ear (G-I), and at the level of the trunk (J-L). Note that MIF MO-injected embryos lack axial structures such as the notochord, somites, and neural tissues. M and N, the expression of zic-2, a pan-neural marker, revealed that MIF MO inhibited the expression of neural differentiation markers, although some neural cell-like cells were observed histologically (see the arrow in F). The bars shown in A, D, and M indicate 1 mm, 100 μm, and 1 mm, respectively. l, lens; r, retina; m, midbrain; p, pharyngeal cavity; h, hindbrain; n, notochord; o, otic vesicle; sc, spinal cord; so, somite; g, gut.
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FIG. 9.
The effect of MIF MO is partially recovered by simultaneous injection of wild-type MIF RNA. Two dorsal blastomeres of the 4-cell stage embryos were injected with MO or MIF RNA, and these injected embryos were cultured until stage 32. Fixed embryos were subjected to morphological and histological analyses. A, uninjected control embryos. B and C, embryos were injected with MO2 (36.8 pmol) (B) or MO2 (36.8 pmol) and MIF RNA (2 ng) together (C). D-F, high magnification views of an uninjected embryo (D), an MO2-injected embryo (E), and an MO2- and MIF RNA-injected embryo (F). G and H, transverse sections at the trunk level of an MO2-injected embryo (G) and an MO2- and MIF-injected embryo (H). Note that MO2 disturbed the differentiation of the neural tube, but MIF RNA was able to rescue neural tube formation. The bars shown in A, D, and G indicate 1 mm, 1 mm, and 100 μm, respectively.
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mif (macrophage migration inhibitory factor (glycosylation-inhibiting factor) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 9, lateral view, animal up.
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mif (macrophage migration inhibitory factor (glycosylation-inhibiting factor) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, dorsal view, anterior left.
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mif (macrophage migration inhibitory factor (glycosylation-inhibiting factor) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
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mif (macrophage migration inhibitory factor (glycosylation-inhibiting factor) ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 34, lateral view, anterior left, dorsal up.
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