molecular cloning and developmental expression of zinc finger transcription factor mtf-1 gene in...

Biochemical and Biophysical Research Communications 291, 798–805 (2002)

Molecular Cloning and Developmental Expressionof Zinc Finger Transcription Factor MTF-1 Genein Zebrafish, Danio rerio

Wen-Ya Chen,*,† Joseph Abraham Christopher John,* Chen-Hui Lin,† and Chi-Yao Chang*,1

*Institute of Zoology, Academia Sinica, NanKang, Taipei, Taiwan, Republic of China; and †Department of Aquaculture,National Taiwan Ocean University, Keelung, Taiwan, Republic of China

Received January 28, 2002

Metal-responsive transcription factor, MTF-1 is azinc finger protein, shown to be essential for embry-onic development. Homozygous knockout mouse em-bryos for MTF-1 die in utero at day 14 of gestation, dueto liver decay. In the present study, we report thecomplete nucleotide sequence of cDNA encoding ze-brafish MTF-1 and the amino acid sequence similaritywith that of mouse, human, fish and Drosophila. Thesize of the zebrafish MTF-1 cDNA is 3,379 bp and thecoding region (1,779 bp) encodes a polypeptide of 593amino acids. The putative zinc finger and transactiva-tion domains comprised by zebrafish MTF-1 were alsodetermined. The zebrafish MTF-1 shows high identityof 97, 93, 93 and 67% in the DNA binding zinc fingerdomain and 51, 44, 48 and 20% overall identity withfugu, human, mouse and Drosophila, respectively. RT-PCR results show the maternal expression of MTF-1transcripts. The pattern of MTF-1 gene expressionduring embryonic and early larval development wasstudied by whole-mount in situ hybridization usingDIG-labeled anti-sense RNA probe. Stronger and ubiq-uitous expression was observed during the embryonicstages whereas, specific expression, especially in theneural parts, was observed throughout the stagesstudied after hatching. © 2002 Elsevier Science (USA)

Key Words: MTF-1; zinc finger protein; expression;zebrafish; RT–PCR; whole mount in situ hybridization;embryonic development; hatching gland; eye; brain.

Metal-responsive transcription factor (MTF-1) is ahighly conserved protein, involved in metal-inducedtranscriptional activation (1–4). It contains a DNAbinding domain of six zinc fingers of the C2H2 type at

1 To whom correspondence should be addressed at 336, Institute ofZoology (44), Academia Sinica, 128, Academia Road, Section 2, Nan-Kang, Taipei 11529, Taiwan, Republic of China. Fax: 886-2-26538842. E-mail: [email protected].

tinct transcriptional activation domains, which arecharacterized by a series of high density acidic, prolineand serine/threonine residues (5). MTF-1 exerts itsactivity by binding to the cis regulatory sequences,called metal-responsive elements (MREs) (6–8). TheseMREs are short sequence motifs of 9–12 conservedbase pairs (bps), contain a well-defined 7 bp core se-quence (TGCRCNC) (2, 3, 9–11). Besides metal-induced expression (3), it also plays a role in cellularresponses to oxidative stress and hypoxia (12, 13).Most importantly, the elimination of MTF-1 gene bytargeted gene disruption in the embryonic mouse leadto death, due to liver degeneration (14). The mRNA forheavy-chain subunit of �-glutamylcysteine synthetase(�-GCS), an essential enzyme for glutathione (GSH)biosynthesis and shares similar function as metallo-thionein (MT) in cellular homeostasis and in detoxi-fication processes, was also found to be reduced inMTF-1 knockout embryos (14). Furthermore, MTF-1 isexpressed in embryonic stem cells, throughout mousegestation and ubiquitously as well as constitutivelyexpressed in adult mice and in general, it seems toinfluence gene expression in many cells (14, 15). How-ever, the expression pattern of MTF-1 during embry-onic stage is still unknown.

Zebrafish, Danio rerio, is a small tropical freshwaterteleost, has high fecundity and short generation time(3–4 months). Its rapid development of the externallyfertilized, translucent embryos makes it an excellentunique vertebrate embryologic and genetic model sys-tem (16, 17). This vertebrate organism bridges thegap between Drosophila/Caenhorhabditis elegans andmouse/human genetics (18). Currently, MTF-1 genehas been isolated and characterized from mouse (2, 15),human (19), fugu (20) and Drosophila (21). Consider-ing the above background information and the impor-tance of zebrafish as a model system, in the present

the N-terminal region, followed by at least three dis-

doi:10.1006/bbrc.2002.6517, available online at http://www.idealibrary.com on

7980006-291X/02 $35.00© 2002 Elsevier Science (USA)All rights reserved.

study, as a first report, we provide the structure andamino acid sequence comparison analysis of the MTF-1along with the pattern of expression of MTF-1 duringdevelopmental stages.

MATERIALS AND METHODS

Fish and embryo maintenance. Fish were raised and maintainedunder standard laboratory conditions, at 28°C, as described byBrand et al. (22). Embryos were raised in 0.003% PTU (Sigma), toprevent pigment formation. The age of the embryos was determinedby morphological feature (23).

Cloning and sequence analysis. The MTF-1 cDNA was preparedas described previously (24), from lambda zebrafish embryonic cDNAlibrary (Stratagene, #937457). Degenerated primers were designed,based on the highly conserved regions of mouse, human and fuguMTF-1 gene sequences, and used for the probe preparation. Theforward and reverse primers are, 5�-ACC TAC AGC ACR GCA GGSAAC CTG-3� and 5�-ATG TTG AAV GCC TTC TCA CAG CCK-3�,respectively. Database homology searches were made using the Na-tional Center for Biotechnology Information BLAST server (25). TheMTF-1 sequence alignment was performed using BioEdit program. Aphylogenetic radial gene tree was constructed by implementing theFitch program of the PHYLIP package.

RT–PCR. One-step RT-PCR (Life Technologies) was performed,using total RNA from various developmental stages. �-actin wasused as an internal control. The primers used were, MTF-1: 5�-GCTCTA GAA TGA AGA GAT ACC AGT GTC TGT TC-3� (forward),5�-CCG CTC GAG TTT GTC GTG ACC CCG GAT GTG GCT-3�(reverse); �-actin: 5�-GTC CCT GTA CGC CTC TGG TCG-3� (for-ward), 5�-GCC GGA CTC ATC GTA CTC CTG-3� (reverse). TheRT-PCR program was 1 cycle of 50°C for 30 min and 94°C for 2 min,followed by a PCR amplification with 40 cycles of 94°C for 30 s, 56°Cfor 30 s, 72°C for 1 min and a final extension of 1 cycle at 72°C for7 min. The RT-PCR products were subjected to 3% agarose gelelectrophoresis.

Whole-mount in situ hybridization. Whole-mount in situ hybrid-ization was performed with digoxigenin-labeled RNA probes (26).The probes were prepared by using a set of primers, designed basedon the MTF-1 cDNA coding region. The primers were: 5�-GCT CTAGAA TGG GAG AGA ATG GCC CTC TCT CG-3� (forward) and5�-CCG CTC GAG ATA TCC AAT CAT ACC TGA ATT T CC-3�(reverse). The Xba I and XhoI sites in the forward and reverseprimers, respectively are underlined. The 1,779 bp fragment wasPCR amplified and sub-cloned into pBluescript SK(�) vector, con-taining T7 and T3 promoters. Using T7 and T3 RNA polymerase,the digoxigenin labeled sense and antisense RNA probes weresynthesized.

RESULTS AND DISCUSSION

Cloning and sequencing of zebrafish MTF-1 cDNA.MTF-1 is a highly conserved, ubiquitously expressedzinc finger transcriptional activator, and has beenstudied in mouse (2, 15), human (19), fugu (20) andDrosophila (21). We have cloned and sequenced thezebrafish MTF-1 cDNA from lambda zebrafish embry-onic cDNA library, by screening 1.2 � 106 plaques. Thesize of the cDNA was 3,379 bp (Fig. 1A). The zebrafishMTF-1 cDNA contains a 5� untranslated region (UTR),a long 3� UTR and a coding region spanning 59, 1,541[including poly(A) tail with 18 ‘A’ residues] and 1779

bp, respectively. The length of 3� UTR is so long, acommon feature among mammalian and fish MTF-1genes (20). Zebrafish MTF-1 consists of 593 amino ac-ids, translated from ATG to TAG of the coding region.The size of the amino acid sequence is smaller thanthat of other species studied so far.

Amino acid analysis of zebrafish MTF-1. Theunique N-terminal region of zebrafish MTF-1, is fol-lowed by a DNA binding site, containing six zinc fin-gers of C2H2 type (Fig. 1B). The C terminal region ofthe protein contains a transactivation domain com-prised of a series of three putative distinct activationdomains, including an acidic-, a proline- and a serine/threonine-rich regions. Figure 2A highlights the con-servation of the functional domains, especially theDNA-binding domain of zebrafish MTF-1 with that ofother species analyzed. The entire amino acid sequence(593 aa) shows an identity of 51, 44, 48 and 20% withthat of fugu, human, mouse and Drosophila, respec-tively. The sequence of DNA binding domain with sixzinc fingers shows 97, 93, 93 and 67% homology withthat of fugu, human, mouse and Drosophila, respec-tively. The amino acid sequence in the acidic regionshows 73, 58 and 54% homology and serine/threonine-rich region shows 65, 54 and 49% homology with thoseof fugu, human and mouse, respectively. The zinc fin-ger DNA binding domain, involved in specific DNArecognition is found to be the most conserved part,followed by the transactivation domain.

Furthermore, the deduced amino acid sequence ofzebrafish MTF-1 was analyzed phylogenetically, withrespect to the relatedness to the MTF-1 of other spe-cies. The radial gene tree shows the branching of ze-brafish MTF-1 together with fugu, indicates the simi-larity, whereas Drosophila is far away from thebranches of zebrafish/fugu and mouse/human, reveal-ing that Drosophila harbors the most distant MTF-1homologue (Fig. 2B).

Expression pattern of zebrafish MTF-1 transcriptsduring embryonic stage. MTF-1 plays an importantrole in the cellular stress response, mainly to heavymetals (3) and oxidative stress (12). The stress-inducedmetallothioneins MT-1 and MT-II and �-GCS (14) aretranscriptionally activated by MTF-1. It was shown byNorthern blot analysis, that MTF-1 is expressed inmost of the mouse tissues (heart, brain, spleen, lung,liver, skeletal muscle, kidney and testes) (15). Here wedemonstrated the expression of MTF-1 during embry-onic and early larval stages of zebrafish. RT-PCR wasperformed and the products, MTF-1 and �-actin cDNAswith 554 and 679 bp, respectively, were subjected to 3%agarose gel electrophoresis. MTF-1 transcripts, de-tected as early as 1-cell stage on wards, demonstratethe maternal expression (Fig. 3).

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FIG. 1. Nucleotide sequence and structure of zebrafish MTF-1. (A) Nucleotide sequence and the deduced amino acid of the zebrafishMTF-1 cDNA. Upper- and the lowercase letters in the nucleotide sequence denote the coding and non-coding regions, respectively. Asterisk(*) indicates the stop codon. (B) Schematic diagram showing the DNA binding site containing six zinc fingers of the TFIIIA type (C2H2) andputative transactivation domains of zebrafish MTF-1. Amino acid sequence of zinc fingers is also depicted, highlighting the C2H2 region. F1to F6 indicate the six zinc fingers.

FIG. 2. Sequence analysis of zebrafish MTF-1. (A) Amino acid sequence alignment of zebrafish, fugu, human, mouse, and DrosophilaMTF-1. Shaded black and gray colors show the complete sequence identity and similarity. Hyphens were introduced to fill the gaps foroptimal alignment. In addition, nuclear export signal (NES) and nuclear localization signal (NLS; which fits the consensus motif, KQREVKR)are also located. The putative domains are shown in boxes. GenBank accession numbers for the sequences are as follows: zebrafish,AF458116; fugu, AJ131393; human, X78710; mouse, X71327; and Drosophila, AJ271817. (B) Radial gene tree showing the MTF-1 inzebrafish, fugu, human, mouse, and Drosophila. The branch lengths are proportional to the difference between the amino acid sequences.

The expression pattern of MTF-1 gene was analyzedby whole-mount in situ hybridization of zebrafish em-bryos using DIG-labeled sense (data not shown) andanti-sense RNA probes. During zygote period, the non-yolk cytoplasm begins to stream towards the animalpole to form a cytoplasm cap, called blastodisc (27, 28)

and produces a strong staining due to the presence ofMTF-1 transcripts (Fig. 4A). The discrete staining inthe yolk at the lateral view shows the segregation ofblastodisc from the yolk still continues through thecleavage period (Figs. 4B and 4C). The expressionmaintains in the blastula period, which begins with theappearance of ball-like structure of blastoderm (Figs.4D and 4E). In the middle of gastrula period, 70%epiboly (8 h) shows a heavy signal in the head region(Fig. 4F). A uniform distribution of staining and for-mation of optic primordium are seen in the 5-somitestage (Fig. 4G). The 14-somite stage (Fig. 4H), is char-acterized by the separation of dorsal and ventral mus-cle masses. In 14- and 20-somites, the expression couldbe seen in the head and tail regions (Figs. 4I and 4J).The yolk was removed to observe the expression pat-tern more clear (Fig. 4N), which shows a ubiquitous

FIG. 3. RT–PCR analysis of MTF-1 gene expression at differentdevelopmental stages. �-actin was used as an internal control. Cindicates the negative control.

FIG. 4. Whole-mount in situ hybridization of zebrafish MTF-1 transcripts during embryonic development. Anti-sense RNA for MTF-1coding region (1.7 kb), labeled with digoxigenin, was used as probe. The stages of the embryos are indicated at the bottom of each panel.Lateral (A, B, D–F, H, L), dorsal (C, G, I, K), and ventral (J, M) views are shown. Anterior is to the top in I–M. N–P are flat-mountedspecimens, excised from the yolk and viewed dorsally (anterior to the left). Abbreviations: cb, cerebellum; ey, eye; fb, forebrain; hb, hindbrain;hg, hatching gland; le, lens; mb, midbrain; me, mesencephalon; re, retina; nt, neural tube; op, optic primordium; tl, telencephalon.


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expression. During 26-somite stage, strong signalscould be observed in the cerebellum, neural tube, lensand retina regions (Figs. 4K and 4O). Figures 4L, 4Mand 4P show the expression pattern of MTF-1 gene atprim-5 stage (24 h). Expression in the eye and retinaregions maintains and the MTF-1 transcripts could beobserved in the hatching gland (Fig. 4L) also. Themorphogenesis of central nervous system (CNS), whichcontinues extensively from the segmentation period,becomes more prominent in the straightening period,especially during prim-5 stage. Brain morphogenesisadvances in this stage, showing fore-, mid- and hind-brain regions more prominently with extensive signalof MTF-1 gene expression (Fig. 4L). The pattern ofexpression is clearly seen when viewed dorsally, withthe distinction of telencephalon, mesencephalon andcerebellum (Fig. 4P).

Expression pattern of zebrafish MTF-1 transcriptsduring early larval stage. At 48 h, the MTF-1 geneexpression is restricted mostly towards the head partand slightly in pectoral fin buds (Fig. 5A). The intensityof bands seems to be high at some parts, especially incerebellum, diencephalon and retina on day 4 of post-hatching (Figs. 5B and 5C). On 10th day, the MTF-1expression is confined to the cerebellum, hyosymplec-tic, ceratohyal and ceratobranchials (Fig. 5D). The in-tensity of staining in the cerebellum region from the48 h stage becomes increased and maintains until 10th

day. Lichtlen et al. (29) revealed that MTF-1 is dispens-

able for neural differentiation, at least under non-stress conditions. However, it is obvious from this ex-pression pattern study that MTF-1 is expressingectopically in the nervous system, indicating a possibleessential role in the neural differentiation, at leastduring the early developmental stages.

A recent target gene search resulted in the finding ofa set of genes including, stress-responsive proteins (ex.MT-II, transforming growth factor �-1, tear lipocalinetc.), secretory liver proteins, liver-enriched transcrip-tional factors (ex. CCAAT/enhancer binding protein �),signal pathway factors etc. (30). The promoter of thesegenes contains the target sequence (MRE) for the bind-ing of MTF-1 and thus expanding its functional terri-tory. More excitingly, apart from DNA binding prop-erty, now it has been shown that MTF-1 binds withRNA, to participate in a p53-mediated apoptotic se-quence of events (31). However, the biochemical back-ground and the domains involved in the recognitionand binding are due for investigation. Moreover, thehigh expression of metallothioneins correlates with apoor prognosis and progressive disease in a number ofhuman tumors (32, 33). This leads to the speculationthat MTF-1 may play a role in tumor growth andtherapy resistance in vivo (30). The mechanisms in-volved in the degeneration of embryonic hepatocytes ofmouse remain to be understood. Similarly severalquestions about the role of MTF-1 are yet to be ad-dressed and deserve active investigation. Investigation

FIG. 5. Whole-mount in situ hybridization of zebrafish MTF-1 transcripts during early larval stages (48 h after fertilization and 4 and10 days after hatching). A and B and C and D are dorsal and lateral views, respectively, with anterior to the left. Abbreviations: cb,cerebellum; ce, ceratobranchials; cf, caudal fin; di, diencephalon; hy, hyosymplectic; pfb, pectoral fin bud; re, retina.


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on the role of MTF-1, in the development of tissuesother than the liver and later than E14 is not possibleusing the lethal embryonic phenotype of MTF-1 knock-out mice (34). But, the 1-ethyl-1-nitrosourea (ENU)mutagenesis facilitates zebrafish to become the verte-brate of choice for large-scale mutagenesis of genescrucial for development (35). Hence, induced mutagen-esis and knockdown experiments in zebrafish may elu-cidate and come out with essential information.

ACKNOWLEDGMENT

This work was supported by Institute of Zoology, Academia Sinica.

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molecular cloning and developmental expression of zinc finger transcription factor mtf-1 gene in...

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