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Originally published In Press as doi:10.1074/jbc.M311416200 on March 15, 2004

J. Biol. Chem., Vol. 279, Issue 20, 21406-21414, May 14, 2004
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Xenopus laevis Macrophage Migration Inhibitory Factor Is Essential for Axis Formation and Neural Development*

Masaki Suzuki{ddagger}§, Yumi Takamura§, Mitsugu Maéno¶, Shin Tochinai||, Daisuke Iyaguchi||, Isao Tanaka||, Jun Nishihira{ddagger}**, and Teruo Ishibashi{ddagger}

From the {ddagger}Department of Molecular Biochemistry, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, the ||Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, and the Department of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan

Received for publication, October 17, 2003 , and in revised form, March 9, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophage migration inhibitory factor (MIF)1 was originally discovered as a lymphokine derived from activated T cells that inhibited the random migration of macrophages (1, 2). 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 (3). MIF was also found to be essential for T cell activation (4). In addition, MIF has isomerase activity that catalyzes the tautomerization of D-dopachrome and phenylpyruvate (5, 6). Oxidoreductase activity has been implicated in the regulation of oxidative cell stress (7, 8). MIF is involved in the control of cell proliferation (9, 10), including the suppression of p53-mediated growth arrest (11) and the regulation of cell growth mediated by binding to c-Jun activation domain-binding protein 1 (Jab1) (12). Moreover, MIF regulates innate immunity through the modulation of Toll-like receptor 4 (13).

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 (14). 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 (15-17). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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-{beta}-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 (18). 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 x 0.1 x 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.


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  		TABLE I
Diffraction data and final refinement statistics

 
Structure Determination and Refinement—The structure was determined by molecular replacement with the human MIF structure (Protein Data Bank code 1MIF [PDB] ) 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 (19). 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 (20). 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 (21). Total RNA was extracted from embryos and adult organs by the AGPC method (22). 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-1{alpha} 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-1{alpha}) 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-1{alpha} (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-1{alpha} 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-1{alpha} 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 (23) using the expanded ClustalW program.2 Positions with gaps were excluded from the analysis. The degree of support for internal branches of the tree was assessed by 1000 bootstrap replicates (24).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (GenBankTM number BE681403 [GenBank] ). 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 GenBankTM/EMBL/DDBJ data base under accession number AB111063 [GenBank] .

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 (25). The N-terminal proline residue, which is the catalytic center of the isomerase activity of mammalian MIF (26), is conserved in XMIF. In mammalian MIF, a number of aromatic residues are clustered around this proline (27, 28). 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 (29, 30), is conserved in XMIF. Two cysteine residues that form the catalytic center of oxidoreductase activity (7) 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 (31). Cys59 is conserved in chicken MIF and in P. chilotes MIF as well, but not in those of jawless fishes (25). 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).



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  		FIG. 1.
Amino acid sequence alignment of MIF and DDT proteins from craniate species. The numbering system is for mammalian MIF. Hyphens denote gaps, which were inserted to optimize the alignment according to a previous report (25). Residues conserved in all the proteins listed are boxed, including the N-terminal proline that is the catalytic center of the isomerase activity of MIF (26), and those conserved in mammalian MIF and XMIF are marked by a plus sign (+). The asterisks represent the residues clustered around the N-terminal proline (27, 28). The dollar sign ($) denotes the lysine residue, which has also been implicated in the enzymatic activity (29, 30). The number sign (#) indicates the cysteine residues that form the catalytic center of the oxidoreductase activity of MIF (7, 8). The secondary structure elements shown below are those of human MIF (27). Species: Bota, Bos taurus (cattle); Gaga, Gallus gallus (chicken); Hosa, Homo sapiens (human); Meun, Meriones unguiculatus (Mongolian gerbil); Mumu, Mus musculus (mouse); Mygl, M. glutinosa (hagfish); Pach, P. chilotes (cichlid); Pema, P. marinus (lamprey); Rano, Rattus norvegicus (rat); Xela, X. laevis. The GenBankTM accession number for each protein is indicated to the right of the C terminus, except in the case of bovine MIF (SWISS-PROT accession number P80177 [GenBank] ).

 



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  		FIG. 2.
Phylogenetic 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 (32). 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.

 
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).



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  		FIG. 3.
HPP tautomerase activities of rat MIF and XMIF. Plotted values of absorbance at 330 nm (A330) represent the MIF-catalyzed increase in enol HPP. , rat MIF; {blacksquare}, XIF.

 
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 (27, 33). Each monomer contains two {alpha}-helices and a four-stranded {beta}-sheet. Two additional {beta}-strands interact with the {beta}-sheets of adjacent subunits. The barrel structure, which contains three {beta}-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.



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  		FIG. 4.
Structural comparison of XMIF and human MIF. The main chains of XMIF (red) and human MIF (blue) are superimposed. The atomic coordinates for XMIF protein have been deposited in the Protein Data Bank under code 1UIZ.

 
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 (34).



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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-1{alpha} served as the RNA loading controls.

 
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).



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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.

 
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).



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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-1{alpha} 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).

 
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.



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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.

 
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 amount of MO1 used in the experiment described above) was injected into the embryos, only a slight morphological effect was observed (data not shown). We hypothesized that the reduced effect was because of the lower targeting efficiency of MO2 than that of MO1. However, injecting the embryos with 36.8 pmol of MO2 caused essentially the same phenotypic changes observed in MO1-injected embryos, although the effects were slightly less prominent (Fig. 9, B, E, and G). When MIF RNA (2 ng per embryo) was co-injected with MO2, we observed a partial phenotype recovery (Fig. 9, C, F, and H). However, no specific change of phenotype was observed by injecting MIF RNA alone (data not shown), suggesting that the constitutively expressed MIF level might be sufficient to maintain physiological functions, and, moreover, that extra expressed MIF minimally affected the phenotypes. The average ± S.D. of the body length of the control embryos (n = 20) was 4.52 ± 0.13 mm and that of MO2-injected embryos (n = 17) was 2.11 ± 0.30 mm, whereas that of co-injected embryos (n = 16) was 3.00 ± 0.70 mm. In addition to the recovery of body length, the head and tail structures were morphologically restored in many of the co-injected embryos (Fig. 9, E and F). A higher dose of MIF RNA (4 ng per embryo) did not help to improve the restoration of the phenotype, suggesting the saturation of MIF RNA.



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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.

 
Histological observation revealed that, in the rescued embryos, the neural tube at the trunk level had formed (Fig. 9H). However, the anterior neural structures, including the brain, eyes, and ears were still disorganized in the co-injected embryos. Table II summarizes the results of the rescue experiment. When the embryos were injected with MO2, we found severely defective head structures in all 17 viable embryos, and no eye capsules had formed (Grade 2). On the other hand, when MIF RNA was co-injected with MO2, 12 of 16 viable embryos showed only moderate defects (Grade 1), i.e. the eye capsules were partially formed and the head structure was less disordered than in the previous case. These data suggested that the disappearance of neural tissues and defective head and tail structures in MIF MO-injected embryos were specific effects of the knockdown of MIF. Thus, we concluded that MIF was essential for axis formation and neural development in Xenopus embryogenesis.


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  		TABLE II
Summary of the rescue experiment by co-injection of MO and MIF RNA

Two dorsal blastomeres of the 4-cell stage embryos were injected with MO1 (9.2 pmol), MO2 (36.8 pmol), MO2 (36.8 pmol) and MIF RNA (2 ng), MIF RNA (2 ng), or 10% Steinberg's (solvent). Morphological phenotypes of the resultant embryos at stage 32 were classified into 4 categories.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
MIF was initially identified as a lymphokine, and today it is regarded as an important immunoregulatory protein and as a key component of the cytokine network that plays a number of roles in both acquired and innate immunity (3, 4, 13, 35, 36). The significance of MIF in the immune system is being recognized to an increasing extent. Clinically, MIF is emerging as a prospective target of therapy for human diseases primarily affecting immunity (35, 37-40). On the other hand, a number of findings have revealed many functions of MIF that are not restricted to those carried out as part of the immune system (9-12, 39, 41-44). Considering the various functions of MIF, it seemed possible that MIF might have essential roles in vertebrate development.

The amino acid sequence of XMIF is 67-71% identical to that of mammalian MIF, and the crucial residues required for some MIF functions are conserved in XMIF. Of all the MIF homologs with known amino acid sequences, the most similar to XMIF is chicken MIF, the amino acid sequence of which is 77% identical to that of XMIF. In addition to the EST that was used to clone the XMIF cDNA, an EST that is similar to mammalian DDT has also been isolated from Xenopus (GenBankTM accession number BC043871 [GenBank] ). This EST contains an entire open reading frame for a possible product of 117 amino acids, which is 66% identical to human DDT and only 27% identical to XMIF. The presence of the open reading frame is supporting evidence for our initial assumption that the functions of XMIF correspond to those of mammalian MIF.

The three-dimensional structure of XMIF (Fig. 4) is very similar to that of mammalian MIF. In addition, the aromatic residues that form the catalytic pocket around the N-terminal proline of MIF are all conserved in XMIF, whereas DDT has a totally different set of residues clustered around the proline (28). Moreover, the HPP tautomerase activity of XMIF was found to be almost equal to that of rat MIF. If we take into account all of these findings, it is clear that XMIF is the ortholog of mammalian MIF. Therefore we consider the results presented here to be useful for understanding the functions of mammalian MIF. We also consider it reasonable to assume that MIF is important in mammalian development.

Targeted disruption of the MIF gene in mice causes no detectable developmental abnormalities (45-47). However, some compensation mechanism that rescues the developing embryos from the loss of MIF may be activated during the generation of knockout mice. By applying the MO-mediated knockdown strategy, we observed an altered phenotype of MIF-targeted Xenopus embryos. In the MO-injected Xenopus embryos, the assumed compensation mechanism may not have been fully activated. Alternatively, the compensation may be less efficient in Xenopus than in mice.

In the RT-PCR analysis, a large amount of MIF mRNA was detected at the blastula stage (stage 9). Because the MIF message was also detected abundantly in early cleaving embryos and the level of expression declined after the blastula stage, the transcript detected at stage 9 was presumed to be of maternal origin. In the whole mount in situ hybridization analysis, MIF mRNA was detected in the animal pole area at stage 9, which is likely to reflect the distribution of MIF mRNA in the oocyte. The accumulation of MIF mRNA near the animal pole correlates with the region of the early embryo in which cell division is most accelerated. Thus, MIF might be important for the promotion of cell division in the animal hemisphere. However, the functions of XMIF in very early embryos cannot be examined by MO-mediated targeting because MO injection does not deplete the preformed proteins in the oocyte. The role of XMIF in the early processes of embryogenesis before gastrulation remains to be investigated using a different strategy.

The injection of MIF MO at the 4-cell stage had no detectable effect on subsequent processes up to gastrulation. This may indicate that the MIF protein stored in the oocyte is required for early developmental processes, and that the MIF synthesized from the maternal mRNA after the 4-cell stage has no additional functions. Alternatively, the MIF mRNA in the early embryo may be the source of MIF protein that functions in the gastrula embryo. Although the transcription of MIF mRNA from the zygotic genes may have started at the gastrula stage, the weak MIF signal detected at stage 11 (Fig. 6B) might also have been derived from undegraded maternal mRNA. Even the region in which no MIF signal was detected may have contained MIF protein synthesized from the maternal mRNA, which might have important roles in that region.

We confirmed the specificity of the MOs by in vitro translation analysis and by co-injection of MO2 and MIF RNA. In the in vitro analysis, MO1 and MO2 were both found to inhibit the translation of MIF RNA. Of the two MOs, MO1, which had been predicted to be most effective, inhibited the translation more efficiently than MO2. MO1 and MO2 had different sequences without any overlap and caused essentially the same morphological effect on injected embryos. The lower targeting efficiency of MO2 than of MO1, which was shown in the in vitro assay, was consistent with the observation that the MO2-injected embryos were less disordered than the MO1-injected embryos. Moreover, the co-injection of MIF RNA and MO2 partially rescued the altered phenotype caused by the MO2 injection. In addition to the restoration of the head structure (Table II), it was highly noticeable that the co-injected embryos formed the neural tube, which was absent in the embryos injected with MO2 alone. Taken together, these data strongly suggest that the phenotype of the MO-injected embryos, at least except for the disappearance of mesodermal tissues, was caused specifically by the knockdown of MIF.

We initially anticipated that MIF might be required for neural development. Indeed, the MO-mediated knockdown of MIF completely inhibited neurulation. However, it was unexpectedly found that the MIF MO-injected embryos also lacked mesodermal structures such as the notochord and somites. Although the mesodermal derivatives were not restored in the rescue experiment, it is still possible that the disappearance of mesodermal tissues in the MO-injected embryos was specifically caused by the knock-down of MIF, because injection of RNA does not always result in sufficient production of the encoded protein in the cells that require it, or because ectopic production of MIF protein in adjacent cells might have inhibited the normal differentiation of mesodermal cells. Therefore, the essentiality of MIF for developmental processes may not be neural tissue-specific in Xenopus. Because the notochord is formed in the dorsal mesoderm and it interacts with the ectoderm to induce neurulation, the initial effect of the MIF MO injection on neural development is thought to be the inhibition of notochord formation. By in situ hybridization at the gastrula stage, positive staining of MIF mRNA was hard to identify in the developing notochord. However, it is possible that a very small amount of MIF mRNA (below the limit of detection by in situ hybridization) was expressed in the dorsal mesoderm, and that this small amount was critical for the differentiation of the cells in this region to form the notochord. Alternatively, the effect of MO in the formation of the notochord occurred non-specifically. Histological observation in the restoration experiment showed that MO2-injected embryos lost the neural tube and notochord (6 of 6 examined embryos) but the embryos rescued by MIF RNA recovered only the neural tube (5 of 5 examined embryos) (Fig. 9, G and H). This suggests the possibility that MO2 happened to disturb the translation of (an) unknown gene(s) that is required for the development of the notochord but not for the neural induction from the dorsal mesoderm. Although it is difficult to address the exact reason for the incomplete restoration by MIF RNA, the present study at least demonstrates a significant role of MIF in differentiation of neural tissues and in the formation of the anteroposterior axis. In addition, dorsoventral axis formation was shown to be at least partly dependent on MIF, because the neural tube was formed in the embryos co-injected with MO2 and MIF RNA.

At the gastrula stage, MIF mRNA was detected in the deep layer of the dorsal marginal zone (Fig. 6B). Although the MIF signal was weak in the gastrula embryo, an increased signal was detected in the neural plate of the early neurula embryo (Fig. 6D), which indicated that zygotic expression had started. In the RT-PCR analysis (Fig. 5A), the level of MIF expression per whole embryo at the early tailbud stage (stage 20) was found to be much higher than that at the neurula stage (stage 15). We presume that the observed increase in the amount of MIF mRNA resulted partly or even primarily from proliferation of MIF-producing cells, in addition to elevated expression in those cells. Considering the abundant expression of MIF in the developing neural tissue, it is likely that MIF is required for the differentiation of neural precursor cells in a direct fashion. MIF may also be indirectly involved in neurulation, because notochord formation may require MIF. However, further investigation is necessary to confirm the direct or indirect involvement of MIF in neural development, and to explicate the molecular mechanism underlying the essentiality of MIF in embryogenesis.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1UIZ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by a grant-in-aid for National Project of Protein Structure and Function from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

** To whom correspondence should be addressed: Dept. of Molecular Biochemistry, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Sapporo 060-8638, Japan. E-mail: j_nisihi{at}med.hokudai.ac.jp.

1 The 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. Back

2 www.ddbj.nig.ac.jp/E-mail/homology.html. Back



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