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• W2061404401 abstract "Macrophage migration inhibitory factor (MIF) is an immunoregulatory cytokine involved in both acquired and innate immunity. MIF also has many functions outside the immune system, such as isomerase and oxidoreductase activities and control of cell proliferation. Considering the involvement of MIF in various intra- and extracellular events, we expected that MIF might also be important in vertebrate development. To elucidate the possible role of MIF in developmental processes, we knocked down MIF in embryos of the African clawed frog Xenopus laevis, using MIF-specific morpholino oligomers (MOs). For the synthesis of the MOs, we cloned a cDNA for a Xenopus homolog of MIF. Sequence analysis, determination of the isomerase activity, and x-ray crystallographic analysis revealed that the protein encoded by the cDNA was the ortholog of mammalian MIF. We carried out whole mount in situ hybridization of MIF mRNA and found that MIF was expressed at high levels in the neural tissues of normal embryos. Although early embryogenesis of MO-injected embryos proceeded normally until the gastrula stage, their neurulation was completely inhibited. At the tailbud stage, the MO-injected embryos lacked neural and mesodermal tissues, and also showed severe defects in their head and tail structures. Thus, MIF was found to be essential for axis formation and neural development of Xenopus embryos. Macrophage migration inhibitory factor (MIF) is an immunoregulatory cytokine involved in both acquired and innate immunity. MIF also has many functions outside the immune system, such as isomerase and oxidoreductase activities and control of cell proliferation. Considering the involvement of MIF in various intra- and extracellular events, we expected that MIF might also be important in vertebrate development. To elucidate the possible role of MIF in developmental processes, we knocked down MIF in embryos of the African clawed frog Xenopus laevis, using MIF-specific morpholino oligomers (MOs). For the synthesis of the MOs, we cloned a cDNA for a Xenopus homolog of MIF. Sequence analysis, determination of the isomerase activity, and x-ray crystallographic analysis revealed that the protein encoded by the cDNA was the ortholog of mammalian MIF. We carried out whole mount in situ hybridization of MIF mRNA and found that MIF was expressed at high levels in the neural tissues of normal embryos. Although early embryogenesis of MO-injected embryos proceeded normally until the gastrula stage, their neurulation was completely inhibited. At the tailbud stage, the MO-injected embryos lacked neural and mesodermal tissues, and also showed severe defects in their head and tail structures. Thus, MIF was found to be essential for axis formation and neural development of Xenopus embryos. Macrophage migration inhibitory factor (MIF) 1The abbreviations used are: MIF, macrophage migration inhibitory factor; XMIF, Xenopus macrophage migration inhibitory factor; MO, morpholino oligomer; HPP, p-hydroxyphenylpyruvate; EST, expressed sequence tag; DDT, d-dopachrome tautomerase; RACE, rapid amplification of cDNA ends; UTR, untranslated region; RT, reverse transcription; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. was originally discovered as a lymphokine derived from activated T cells that inhibited the random migration of macrophages (1Bloom B.R. Bennett B. Science. 1966; 153: 80-82Crossref PubMed Scopus (1277) Google Scholar, 2David J.R. Proc. Natl. Acad. Sci. U. S. A. 1966; 56: 72-77Crossref PubMed Scopus (1095) Google Scholar). Later studies have revealed that this protein has various functions. For example, MIF is released from the anterior pituitary gland of lipopolysaccharide-challenged mice and potentiates lethal endotoxemia (3Bernhagen J. Calandra T. Mitchell R.A. Martin S.B. Tracey K.J. Voelter W. Manogue K.R. Cerami A. Bucala R. Nature. 1993; 365: 756-759Crossref PubMed Scopus (934) Google Scholar). MIF was also found to be essential for T cell activation (4Bacher M. Metz C.N. Calandra T. Mayer K. Chesney J. Lohoff M. Gemsa D. Donnelly T. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7849-7854Crossref PubMed Scopus (619) Google Scholar). In addition, MIF has isomerase activity that catalyzes the tautomerization of d-dopachrome and phenylpyruvate (5Rosengren E. Bucala R. Åman P. Jacobsson L. Odh G. Metz C.N. Rorsman H. Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar, 6Rosengren E. Åman P. Thelin S. Hansson C. Ahlfors S. Björk P. Jacobsson L. Rorsman H. FEBS Lett. 1997; 417: 85-88Crossref PubMed Scopus (214) Google Scholar). Oxidoreductase activity has been implicated in the regulation of oxidative cell stress (7Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Jüttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (270) Google Scholar, 8Kleemann R. Mischke R. Kapurniotu A. Brunner H. Bernhagen J. FEBS Lett. 1998; 430: 191-196Crossref PubMed Scopus (61) Google Scholar). MIF is involved in the control of cell proliferation (9Sasaki Y. Kasuya K. Nishihira J. Magami Y. Tsuchida A. Aoki T. Koyanagi Y. Int. J. Mol. Med. 2002; 10: 579-583PubMed Google Scholar, 10Yasuda Y. Kasuya K. Nishihira J. Sasaki Y. Tsuchida A. Aoki T. Koyanagi Y. Int. J. Mol. Med. 2002; 10: 463-467PubMed Google Scholar), including the suppression of p53-mediated growth arrest (11Hudson J.D. Shoaibi M.A. Maestro R. Carnero A. Hannon G.J. Beach D.H. J. Exp. Med. 1999; 10: 1375-1382Crossref Scopus (571) Google Scholar) and the regulation of cell growth mediated by binding to c-Jun activation domain-binding protein 1 (Jab1) (12Kleemann R. Hausser A. Geiger G. Mischke R. Burger-Kentischer A. Flieger O. Johannes F.-J. Roger T. Calandra T. Kapurniotu A. Grell M. Finkelmeier D. Bunner H. Bernhagen J. Nature. 2000; 408: 211-216Crossref PubMed Scopus (505) Google Scholar). Moreover, MIF regulates innate immunity through the modulation of Toll-like receptor 4 (13Roger T. David J. Glauser M.P. Calandra T. Nature. 2001; 414: 920-924Crossref PubMed Scopus (495) Google Scholar). The involvement of MIF in a variety of intracellular and extracellular events has led us to speculate that MIF might also have important functions in the development of mammals and other vertebrates. Little is known about the involvement of MIF in vertebrate development, except that the expression of chicken MIF in the developing eye lens is correlated with cell differentiation (14Wistow G.J. Shaughnessy M.P. Lee D.C. Hodin J. Zelenka P.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1272-1275Crossref PubMed Scopus (181) Google Scholar). To investigate the possible functions of MIF in developmental processes, we carried out loss-of-function experiments in embryos of the African clawed frog Xenopus laevis. For this purpose, we cloned a cDNA for Xenopus MIF (XMIF), and synthesized XMIF-specific antisense morpholino oligomers (MOs). MOs are gaining wide use in developmental biology for blocking the translation of mRNA because of their high efficacy and specificity (15Summerton J. Biochim. Biophys. Acta. 1999; 1489: 141-158Crossref PubMed Scopus (565) Google Scholar, 16Heasman J. Kofron M. Wylie C. Dev. Biol. 2000; 222: 124-134Crossref PubMed Scopus (464) Google Scholar, 17Heasman J. Dev. Biol. 2002; 243: 209-214Crossref PubMed Scopus (463) Google Scholar). In this study, the MO-mediated knockdown of MIF caused a severely altered phenotype, which demonstrated that MIF was an essential factor in Xenopus embryogenesis, and which, furthermore, suggested the importance of mammalian MIF in the development of mammals. Cloning of XMIF cDNA—We prepared total RNA from X. laevis liver and carried out rapid amplification of cDNA ends (RACE) reactions using the SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA). The primers used for 5′- and 3′-RACE were 5′-TGCACACGGCACAAGGATCAGTTGA-3′ and 5′-CTGATCCTTGTGCCGTGTGCAGTCTG-3′, respectively. The 5′ and 3′ primers used for the amplification of the full-length cDNA were 5′-AGTGTGCGACCCGTTCTCCATCTTT-3′ and 5′-AATTGCTGTACTTTGTTTATTCAGAAGGGAATG-3′, respectively. The product of this end-to-end PCR was cloned into pBluescript KS+. We sequenced four clones and found that all of them were identical. Preparation of Recombinant MIF Proteins—Escherichia coli BL21(DE3)pLysS, transformed with pET-3a containing Xenopus or rat MIF cDNA, was cultured, and MIF expression was induced by isopropyl-β-d-1-thiogalactopyranoside. The cells were disrupted with a French pressure cell disrupter. From the clarified homogenate, MIF was purified by S-hexylglutathione affinity column chromatography. Determination of Isomerase Activity—The rates of the keto-enol tautomerization of p-hydroxyphenylpyruvate (HPP) catalyzed by MIF were determined as previously described (18Knox W.E. Colowick S.P. Kaplan N.O. Methods in Enzymology. 2. Academic Press, New York1955: 289-292Google Scholar). Reaction mixtures contained 0.5 m sodium borate (pH 6.2), 0.5 mm p-hydroxyphenylpyruvic acid (Sigma), and 0.25 μg/ml MIF. Crystallization and Data Collection—Crystals of XMIF were obtained by the hanging-drop vapor diffusion method with a reservoir solution containing 0.1 m ammonium sulfate and 20% PEG 4000. The reservoir solution was mixed with XMIF at 7.5 mg/ml in a 1:1 volume ratio. After 1 week, the crystals had grown to a size of 0.1 × 0.1 × 0.1 mm3. The space group of the crystal was R3 with cell dimensions of a = b = 94.41 Å and c = 115.58 Å. A complete x-ray diffraction data set was collected in-house using an R-AXIS IV++ imaging plate at 100 K and the data were processed by the program d*trek. The statistics used for the data collection are summarized in Table I.Table IDiffraction data and final refinement statisticsResolution (Å)aValues in parentheses are for the outermost resolution shell.30-2.5(2.59-2.5)Number of observed reflections21,734Unique reflections13,284Completeness (%)97.0 (97.0)Averaged redundancy1.67 (1.62)Averaged I/σ(I)5.1 (1.7)Rmerge (%)bRmerge=∑h∑i|⟨Ih⟩−Ih,i|/∑h∑i⟨Ih⟩, where Ih,i is the ith used observation for unique reflections and ⟨Ih⟩ is the mean intensity for unique reflections.8.7 (26.3)Total number of non-hydrogen atomsProtein875 × 4Solvent52Resolution range for refinement (Å)10-2.5R-factor (%)cR−factor=∑|Fobs−Fcal|/∑Fobs, where Fobs and Fcal are observed and calculated structure factor amplitudes.24.1R-free-factor (%)dCalculated using a random subset of the data (10%) not included in the refinement.26.6Average B-factor (Å2)Protein50.0Solvent40.6a Values in parentheses are for the outermost resolution shell.b Rmerge=∑h∑i|⟨Ih⟩−Ih,i|/∑h∑i⟨Ih⟩, where Ih,i is the ith used observation for unique reflections and ⟨Ih⟩ is the mean intensity for unique reflections.c R−factor=∑|Fobs−Fcal|/∑Fobs, where Fobs and Fcal are observed and calculated structure factor amplitudes.d Calculated using a random subset of the data (10%) not included in the refinement. Open table in a new tab Structure Determination and Refinement—The structure was determined by molecular replacement with the human MIF structure (Protein Data Bank code 1MIF) using the program AMoRe (CCP4 suite), a molecular replacement package, which includes routines to run a complete molecular replacement. Structure refinement was performed using the program CNS, a flexible macromolecular structure determination software suite for x-ray crystallography and solution NMR spectroscopy, with manual interventions of the model modification using O, a general purpose macromolecular modeling program. After locating 52 water molecules, we refined the model of XMIF to an R-factor of 24.1% and an R-free factor of 26.6% for the region of 10-2.5-Å resolution. Non-crystallographic symmetry (NCS) restraints were applied throughout the refinement. Embryos and Injection of Reagents—Adult frogs were maintained in dechlorinated tap water in our laboratory at 20-23 °C. Ovulation was induced by the injection of human chorionic gonadotropin (250 units) into females. Fertilized eggs were obtained by artificial insemination using a sperm suspension prepared by mincing testes that were removed from a properly anesthetized male frog. Developmental stages were designated according to the system of Nieuwkoop and Faber (19Nieukoop P.D. Faber J. Normal Table of Xenopus laevis, North-Holland, Daudin, Amsterdam1967Google Scholar). Fertilized embryos were dejellied with 2.5% thioglycolic acid (pH 8.3) and were washed several times in Steinberg's solution (58 mm NaCl, 0.7 mm KCl, 0.5 mm Ca(NO3)2, 4.6 mm Tris-HCl, 8 mg/liter phenol red, pH 7.4). At the 4-cell stage, embryos dipped in 3% Ficoll solution were injected with MO (Gene Tools, Philomath, OR) and/or synthesized mRNA using a Nanoject microinjector (Drummond, Broomall, PA) (18.4 nl/embryo). The resultant embryos were cultured until the blastula stage in the same solution and were then allowed to develop further in Steinberg's solution. Whole Mount in Situ Hybridization—Whole mount in situ hybridization was performed as described previously (20Shain D.H. Zuber M.X. J. Biochem. Biophys. Methods. 1996; 31: 185-188Crossref PubMed Scopus (64) Google Scholar). Briefly, embryos were fixed in MEMFA (0.1 m MOPS, pH 7.4, 2 mm EGTA, 1 mm MgSO4, 3.7% formaldehyde) for 2 h at room temperature. Hybridization was performed with a digoxigenin-labeled antisense or sense RNA probe for XMIF that was synthesized from a 403-bp insert of XMIF cDNA, including the whole coding region, which had been cloned in pBluescript. The enzymes used for the synthesis of antisense and sense probes were T7 and T3 RNA polymerases, respectively. An antisense probe of zic-2, a pan-neural marker, was synthesized using a 2.9-kb full-length insert that had been cloned in pBluescript. The positive signals were visualized using BM purple (Roche Diagnostics). Embryos were fixed again in MEMFA solution, dehydrated, and replaced with benzyl benzoate/benzyl alcohol (2:1) to enhance the transparency. RT-PCR Analysis—The reverse transcription (RT)-PCR analysis was carried out according to the methods described in a previous report (21Braun M. Wunderlin M. Spieth K. Knöchel W. Gierschik P. Moepps B. J. Immunol. 2002; 168: 2340-2347Crossref PubMed Scopus (34) Google Scholar). Total RNA was extracted from embryos and adult organs by the AGPC method (22Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). cDNA was synthesized using a SuperScript II preamplification system (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. DNA was amplified by Ex Taq polymerase (Takara, Otsu, Japan) using XMIF primers (5′-CCGATACCCTTCTGTCCGAT-3′ and 5′-CATTCCAGCCAACGTTTGCA-3′, 265 bp), EF-1α primers (5′-CCTGAATCACCCAGGCCAGATTGGTG-3′ and 5′-GAGGGTAGTCTGAGAAGCTCTCCACG-3′, 221 bp), and histone H4 primers (5′-CGGGATAACATCCAGGGCATC-3′ and 5′-CATAGCGGTAACGGTCTTCCT-3′, 186 bp). The amplification reactions consisted of 28 (XMIF), 24 (H4), and 21 (EF-1α) cycles. In each case, denaturing, annealing, and extension steps (1 min at 94 °C, 1.5 min at 55 °C, 1 min at 72 °C, respectively) were carried out. After a single extension step (5 min at 72 °C), products were separated by 1% agarose gel electrophoresis and transferred to a nylon membrane (Biodyne B; Pall, Ann Arbor, MI). The membrane was then hybridized with a radiolabeled DNA probe for XMIF (403 bp), EF-1α (PstI/SacI fragment, 378 bp), or histone H4 (PCR-amplified fragment, 186 bp). Synthesis of Capped RNA for in Vitro Translation and Injection—For the synthesis of XMIF RNA, including its 5′-untranslated region (UTR), the pBluescript plasmid containing the full-length XMIF cDNA was linearized with HindIII. From this template, capped RNA was synthesized using the mMessage mMachine T7 Kit (Ambion, Austin, TX). Xenopus EF-1α RNA was synthesized from the control template provided with the kit. For the synthesis of XMIF RNA without its 5′ UTR, the coding sequence was amplified and subcloned. The primers used were 5′-CCGCTCGAGCCGCCACCATGCCTGTCTTCACCAT-3′ and 5′-GCCTCTAGATTAGGCAAAGGTAGATCC-3′. A 9-nucleotide Kozak consensus sequence for translation initiation (underlined) was included in the 5′ primer. The PCR product was inserted into the XhoI-XbaI site of pCS2+. The plasmid was linearized with NotI, and capped RNA was synthesized using the mMessage mMachine SP6 Kit. In Vitro Translation—XMIF or EF-1α protein was synthesized using nuclease-treated rabbit reticulocyte lysate (Promega, Madison, WI). The translation reaction mixture (20 μl) contained 14 μl of reticulocyte lysate, 20 μm methionine-free amino acid mixture, 5 μCi of l-[35S]methionine (Amersham Biosciences), and 0.5 μg of capped RNA. Translation was carried out at 30 °C for 90 min, followed by addition of RNase A to a concentration of 0.2 mg/ml and incubation at room temperature for 5 min. Translated products were analyzed by 15% Tricine-SDS-PAGE and visualized by autoradiography. Histological Observation—Embryos were fixed in 2% paraformaldehyde in Steinberg's solution, dehydrated, embedded in paraffin (Paraplast; Structure Probe, West Chester, PA), sectioned at 7 μm, and stained with hematoxylin and eosin. Phylogenetic Analysis of MIF Sequences—The phylogenetic tree was constructed with the aligned amino acid sequences of MIF homologs by the neighbor-joining method (23Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar) using the expanded ClustalW program. 2www.ddbj.nig.ac.jp/E-mail/homology.html. Positions with gaps were excluded from the analysis. The degree of support for internal branches of the tree was assessed by 1000 bootstrap replicates (24Felsenstein J. Evolution. 1985; 39: 783-791Crossref PubMed Google Scholar). cDNA Cloning of XMIF—Although the cloning of full-length cDNA for MIF homologs from Xenopus has not been previously described, an expressed sequence tag (EST) that is similar to mammalian MIF cDNA has been isolated from Xenopus embryos (GenBank™ number BE681403). We assumed that this EST was derived from a gene encoding a protein that was the functional counterpart (ortholog) of mammalian MIF. Thus, we refer to the product of the gene as XMIF (we also refer to it simply as MIF when there is no need to distinguish it from mammalian MIF), and we cloned its full-length cDNA from Xenopus liver by the RACE method. The primers used for the RACE reactions were selected from the EST. Sequencing of 17 clones obtained from 5′ RACE identified two major transcription start sites at -59 and -47, relative to the start codon. Using the terminal sequences of the 5′- and 3′-RACE products, we amplified and cloned a full-length cDNA for XMIF. The resultant 527-bp cDNA started at -59, and contained an open reading frame, the length of which was identical to that of mammalian MIF. The sequence of this cDNA has been deposited in the GenBank™/EMBL/DDBJ data base under accession number AB111063. Comparison of XMIF and Mammalian MIF—Fig. 1 shows the deduced amino acid sequence of XMIF compared with those of MIF homologs from different craniate (vertebrate and hagfish) species, including a jawed fish (Paralabidochromis chilotes) and two jawless fishes (Petromyzon marinus and Myxine glutinosa) of distant taxa (25Sato A. Uinuk-ool T.S. Kuroda N. Mayer W.E. Takezaki N. Dongak R. Figueroa F. Cooper M.D. Klein J. Dev. Comp. Immunol. 2003; 27: 401-412Crossref PubMed Scopus (35) Google Scholar). The N-terminal proline residue, which is the catalytic center of the isomerase activity of mammalian MIF (26Bendrat K. Al-Abed Y. Callaway D.J. Peng T. Calandra T. Metz C.N. Bucala R. Biochemistry. 1997; 36: 15356-15362Crossref PubMed Scopus (144) Google Scholar), is conserved in XMIF. In mammalian MIF, a number of aromatic residues are clustered around this proline (27Sugimoto H. Suzuki M. Nakagawa A. Tanaka I. Nishihira J. FEBS Lett. 1996; 389: 145-148Crossref PubMed Scopus (64) Google Scholar, 28Sugimoto H. Taniguchi M. Nakagawa A. Tanaka I. Suzuki M. Nishihira J. Biochemistry. 1999; 38: 3268-3279Crossref PubMed Scopus (106) Google Scholar). These surrounding residues are all conserved in XMIF, suggesting their importance in the function or formation of the structure of MIF. In addition, the invariant lysine residue at position 32, which contributes to the isomerase activity of the protein (29Johnson W.H. Czerwinski R.M. Stamps S.L. Whitman C.P. Biochemistry. 1999; 38: 16024-16033Crossref PubMed Scopus (14) Google Scholar, 30Soares T.A. Goodsell D.S. Ferreira R. Olson A.J. Briggs J.M. J. Mol. Recognit. 2000; 13: 146-156Crossref PubMed Scopus (31) Google Scholar), is conserved in XMIF. Two cysteine residues that form the catalytic center of oxidoreductase activity (7Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Jüttner S. Brunner H. Bernhagen J. J. Mol. Biol. 1998; 280: 85-102Crossref PubMed Scopus (270) Google Scholar) are also conserved. One of these cysteines (Cys56) is conserved in all known MIF homologs from craniate species, including d-dopachrome tautomerase (DDT), and also in some homologs from nematodes. The other cysteine (Cys59) residue of the catalytic center undergoes cysteinylation in human suppressor T hybridoma cells. This modification is essential for the immunosuppressive activity of MIF as a glycosylation-inhibiting factor (31Watarai H. Nozawa R. Tokunaga A. Yuyama N. Tomas M. Hinohara A. Ishizaka K. Ishii Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13251-13256Crossref PubMed Scopus (50) Google Scholar). Cys59 is conserved in chicken MIF and in P. chilotes MIF as well, but not in those of jawless fishes (25Sato A. Uinuk-ool T.S. Kuroda N. Mayer W.E. Takezaki N. Dongak R. Figueroa F. Cooper M.D. Klein J. Dev. Comp. Immunol. 2003; 27: 401-412Crossref PubMed Scopus (35) Google Scholar). We carried out a phylogenetic analysis of XMIF and the other MIF homologs listed in Fig. 1, and found that the branching pattern of the resultant phylogenetic tree corresponded essentially with the evolutionary relationships among the species (Fig. 2).Fig. 2Phylogenetic tree of MIF and DDT sequences (Fig. 1) constructed by the neighbor-joining method. The length of the branches is proportional to the phylogenetic distances estimated using the empirical method for protein distances of Kimura (32Kimura M. The Neutral Theory of Molecular Evolution, Cambridge University Press, Cambridge1983Crossref Google Scholar). A plant MIF homolog from Arabidopsis thaliana (TrEMBL accession number Q9LU69) has been included as an outgroup. The numbers indicate the percentages of 1000 bootstrap replicates in which the same internal branch was recovered. The scale bar indicates an evolutionary distance of 0.1 amino acid substitution per position in the sequence. The species abbreviations are the same as those used in Fig. 1.View Large Image Figure ViewerDownload (PPT) Because crucial amino acid residues for the isomerase activity of mammalian MIF were shared with XMIF, we expected XMIF to have the same activity as mammalian MIF. The determined rate of keto-enol tautomerization of HPP catalyzed by XMIF was comparable with that catalyzed by rat MIF (Fig. 3). We carried out x-ray crystallographic analysis to compare the structure of XMIF with that of mammalian MIF. In the crystal structure of XMIF (Fig. 4), one trimer and one monomer were contained in an asymmetric unit, in which MIF formed a trimer around the crystallographic 3-fold axis. Mammalian MIF proteins also exist as a trimer (27Sugimoto H. Suzuki M. Nakagawa A. Tanaka I. Nishihira J. FEBS Lett. 1996; 389: 145-148Crossref PubMed Scopus (64) Google Scholar, 33Suzuki M. Sugimoto H. Nakagawa A. Tanaka I. Nishihira J. Sakai M. Nat. Struct. Biol. 1996; 3: 259-266Crossref PubMed Scopus (188) Google Scholar). Each monomer contains two α-helices and a four-stranded β-sheet. Two additional β-strands interact with the β-sheets of adjacent subunits. The barrel structure, which contains three β-sheets (one from each monomer), forms the solvent-accessible channel positioned at the center of the trimer. These structural features are conserved in mammalian MIF. The root mean square deviation for main chain atoms between XMIF and human MIF and between XMIF and rat MIF are 0.55 and 1.30 Å, respectively. Expression of XMIF mRNA—RNAs from Xenopus embryos and those from organs of an adult frog were subjected to RT-PCR followed by Southern blot analysis to determine the expression levels of MIF mRNA. Fig. 5A shows the expression of MIF mRNA in embryos at different stages of development. Early embryos contained a substantial amount of maternally expressed MIF mRNA. The mRNA level decreased in the gastrula stage (stage 11) and recovered in the early tailbud stage (stage 20). A high expression level continued through the tail-bud stage. Fig. 5B shows the expression of MIF mRNA in organs of the adult frog. As has been observed in the case of mammalian MIF, XMIF was ubiquitously expressed. Moreover, the expression pattern was similar to that of rat MIF, which is expressed at high levels in the brain and kidney, and at lower levels in the heart, lung, spleen, and liver (34Sakai M. Nishihira J. Hibiya Y. Koyama Y. Nishi S. Biochem. Mol. Biol. Int. 1994; 33: 439-446PubMed Google Scholar). Using whole mount in situ hybridization, MIF mRNA was detected in the animal pole area at the blastula stage (stage 9) (Fig. 6A). At the gastrula stage (stage 11), expression of MIF mRNA decreased once but a very small amount of the transcript was detected in the deep layer of the dorsal marginal zone (Fig. 6B). When observing the stained embryos without clearing, MIF mRNA was not detected in the superficial layer of the dorsal region at stage 11 (data not shown). At the early neurula stage (stage 13), MIF was obviously detected in the neural plate. The expression level was relatively high in the anterior region (Fig. 6, D and F). At stage 15, MIF was detected in the anterior and posterior regions of the developing neural tissue (Fig. 6, H and I). In the early and late tailbud embryos (stages 26 and 34), intense expression was observed in the head region, including the brain and eye capsules (Fig. 6, K and L). The expression in the posterior region, which was observed at stage 15, was also detected at stage 26, and was not detectable at stage 34. In the late tailbud embryo, a moderate level of expression was observed in the dorsal region (Fig. 6L). Sections of stained embryos at the tailbud stage showed that mesodermal tissues such as head mesenchyme and somites, in addition to the brain, exhibited diffuse MIF signals (data not shown). Knockdown of XMIF Inhibits Neurulation—Because MIF mRNA was detected at high levels in neural tissues, we expected that MIF might play important roles in neural development. Thus, we injected antisense MOs for MIF into two dorsal blastomeres of 4-cell stage embryos, from which neural tissues are derived. We used two MOs for targeting MIF. MO1, which hybridizes to the area including the start codon (-1 to 24), was predicted by the manufacturer to be optimal for blocking the translation of MIF. MO2, which was complementary to a portion of the 5′ UTR (-28 to -4), was used for a rescue experiment, because it hybridizes only to endogenous MIF mRNA, not to co-injected MIF RNA lacking an MIF-derived 5′ leader sequence. We examined the effect of the MIF MOs in an in vitro translation system (Fig. 7). As expected, both MO1 and MO2 inhibited the translation of the full-length MIF RNA. MO1 almost completely blocked the translation at a concentration of 1 μm (Fig. 7, lane 2). In this system, MO2 had a lower targeting efficiency than MO1. With 1 μm MO2, an appreciable amount of MIF protein was synthesized. At 10 μm, MO2 was as effective as 1 μm MO1 (Fig. 7, lanes 4 and 5). Thus these MOs were likely to inhibit the translation of endogenous MIF mRNA in injected embryos, at different efficiencies. MO2, which was designed for co-injection with MIF RNA without its 5′ leader sequence, was shown to have no effect on the translation of that RNA (Fig. 7, lanes 7 and 8). In addition, MO1 and MO2 had no detectable effect on the translation of an RNA unrelated to MIF (EF-1a) (Fig. 7, lanes 9-11). Fig. 8 shows the results of the injection of MO1. In the embryos injected with MO1, early embryogenesis up to gastrulation proceeded normally, but neurulation was completely inhibited, i.e. embryos that had received the injection did not form any trace of a neural plate. These embryos also had severe defects in the head and tail structures, and histological examination showed that they were devoid of neural tissues, resulting in the absence of the brain, spinal cord, eye capsules, and otic vesicles. Although a mass of cells in the anterior dorsal region showed staining similar to that of neural cells (Fig. 8F), they did not form any structure found in normally developed neural tissues. More surprisingly, mesodermal tissues such as the notochord and somites did not form. The disappearance of these structures indicated that both anteroposterior and dorsoventral axis formation were entirely disordered in these embryos. We further confirmed the absence of neural tissues by in situ hybridization of two pan-neural markers, zic-2 and nrp-1. As shown in Fig. 8N, no expression of zic-2 was observed in the MO-injected embryos. Similarly, nrp-1 was not detected (data not shown). These results indicated that the injection of MIF MO inhibited the differentiation of neural precursor cells; in addition, it was found that even the anterior dorsal cells showing neural cell-like staining had not differentiated normally. To examine the specificity of the effect of MIF MO on embryogenesis, we used MO2 and MIF RNA in a rescue experiment. When 9.2 pmol of MO2 (equal to the am" @default.
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• W2061404401 date "2004-05-01" @default.
• W2061404401 modified "2023-10-12" @default.
• W2061404401 title "Xenopus laevis Macrophage Migration Inhibitory Factor Is Essential for Axis Formation and Neural Development" @default.
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• W2061404401 doi "https://doi.org/10.1074/jbc.m311416200" @default.
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