Expression of metallothionein gene during embryonic and early larval development in zebrafish

Aquatic Toxicology 69 (2004) 215–227

Expression of metallothionein gene during embryonic and early larval development in zebrafish Wen-Ya Chen b , Joseph Abraham Christopher John a , Chih-Hung Lin a,b , Hui-Fen Lin a , Shao-Chun Wu a , Cheng-Hui Lin b , Chi-Yao Chang a,∗ a

Molecular Genetics Laboratory, Institute of Zoology, Academia Sinica, 128, Academia Road, Section 2, NanKang, Taipei 11529, Taiwan, ROC b Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan Received 9 January 2004; received in revised form 18 April 2004; accepted 16 May 2004

Abstract Metallothionein (Mt) has been considered as a molecular marker of metal pollution in aquatic ecosystems. Less is known about the expression of mt gene during embryogenesis. Here, we report the cloning, sequencing, and the expression pattern of mt gene during developmental stages in zebrafish. The zebrafish embryogenesis when takes place in a medium containing a dosage of 1000 ␮M zinc resulted in high mortality, indicating the deleterious effect of zinc on development. The zebrafish mt gene consists of three exons encoding 60 amino acids with 20 conserved cysteine residues. RT-PCR result indicates the maternal contribution of Mt transcripts. Using digoxigenin (DIG)-labeled anti-sense RNA probe, whole-mount in situ hybridization was performed to observe the expression pattern of zebrafish mt gene during embryonic and early larval stages. Stronger as well as ubiquitous expression of mt gene during early embryonic stages narrowed to specific expression after hatching. The mt promoter region contains seven copies of putative metal-responsive elements (MREs), which are shown to be important for the high level activity by deletion analysis. The expression of mt gene during embryogenesis implies its significant role on development. © 2004 Elsevier B.V. All rights reserved. Keywords: Zebrafish; Danio rerio; Metallothionein; Zinc; Metal-responsive element (MRE); Whole-mount in situ hybridization; Embryonic development; Promoter

1. Introduction Metallothionein (Mt) has been shown to be a potential biomarker for metal contamination in aquatic environment (Benson et al., 1990; Couillard, 1997; AETE, 1999; Langston et al., 2002). As vertebrates are being ∗ Corresponding author. Tel.: +886 2 2789 9570; fax: +886 2 2653 8842. E-mail address: [email protected] (C.-Y. Chang).

the target species, they have served as prime context for the study of fish Mts. On the other hand, zinc (Zn), is considered as one of the major trace elements indispensable for multiple biological functions and embryonic development (Rubin, 1972; Keen and Hurley, 1989). Zn deprivation induces congenital malformations (Falchuk, 1998a,b; MacDonald, 2000; Black, 2001). However, excess Zn can be highly toxic to embryos (Domingo, 1994; Erbach et al., 1995). But it was demonstrated that Mt provides protection

0166-445X/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2004.05.004

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against the lethal toxic dosage of Zn through sequestration (Liu et al., 1991) and also plays an important role in Zn regulation during development (Olsson et al., 1987, 1989, 1990). Mts are a class of low molecular weight, cysteine-rich, heavy metal binding proteins, shown to be involved in heavy metal ion homeostasis and detoxification (Kägi et al., 1984; Hamer, 1986; Kägi and Schaffer, 1988; Schroeder and Cousins, 1990; Suzuki et al., 1993). Mt transcription is regulated by short cis-acting elements, termed metal-responsive elements (MREs), which are generally present in multiple copies (Stuart et al., 1985). A wealth of information has been acquired on the structure and functional analysis of fish mt promoters (Samson and Gedamu, 1995, 1998; Ren et al., 2000; Lin et al., 2004). A zebrafish mt promoter containing four MREs was shown to be susceptible to Zn induction (Yan and Chan, 2002). Moreover, fish mt promoters are functional in heterologous system (Zafarullah et al., 1988; Olsson et al., 1995). Metal-responsive transcription factor (MTF-1) is responsible for Mt gene expression (Heuchel et al., 1994). Role of MTF-1 on embryogenesis (Günes et al., 1998), embryo mt expression (Andrews et al., 2001), and its pattern of expression in zebrafish embryos (Chen et al., 2002) are documented. More recently, Riggio et al. (2003) revealed that Mt is accumulated in oocyte and after fertilization, the large pool of maternal Mt decreases during early stages of embryogenesis. Ultimately, therefore, the presence of Mt has a possible role on the normal development of embryos. Although the pattern of Mt gene expression during development was studied extensively in sea urchin (Angerer et al., 1986; Cserjesi et al., 1997), no valid information available on the expression pattern of mt during embryogenesis and hatching in fish. Zebrafish is considered as an important vertebrate animal model, particularly useful for studying gene expression pattern during developmental stages because of the wealth of classic embryological studies on this organism. Furthermore, zebrafish embryo is capable of exchanging metals with the environment, as in open embryonic system of mammals (Davidson, 1990; Riggio et al., 2003). This deserves importance in eco-toxicological view for further studies and tempted us to elucidate the pattern of mt expression during fish embryogenesis.

2. Materials and methods 2.1. Fish and embryo Fish were reared under standard laboratory conditions, at 28 ◦ C (Brand et al., 1995). Embryos were raised in the presence of 0.003% 1-phenyl-2-thiourea (PTU; Sigma), to prevent the pigment formation. The stages of embryo were determined by following Kimmel et al. (1995). 2.2. Hatching of zebrafish eggs in a medium with Zn Each 50 number of zebrafish eggs were collected and allowed to hatch in 1000 ml of deionic water containing ZnCl2 at 10, 100, or 1000 ␮M. One set was kept as control, without ZnCl2 . The mortality was observed and recorded at every 12 h. 2.3. Probe preparation Degenerated primers were designed, based on the highly conserved regions of fish mt gene sequences, and used for the probe preparation, by RT-PCR. The primers are 5 -CGG GAT CCA TGG AYC CYT GYG ADT GCK CYA A-3 (forward); 5 -GGA ATT CTT RCA CAC RCA GCC WCA RGC RCA-3 (reverse); where Y = C or T; D = A or G or T; K = G or T; R = A or G; W = A or T. The RT-PCR was performed as described by Lin et al. (2004). Briefly, total RNA was extracted from the viscera of zebrafish using the RNAzol method (RNAzolTM B REAGENT, TEL-TEST “B”, INC.). For reverse transcription, a reaction mixture containing 10 ␮g of total RNA, 1 mM methyl mercury hydroxide (CH3 HgOH), 5 mM ␤-mercaptoethanol, 400 ␮M dNTPs, 30 ng/␮l oligo-dT, 0.5 U/␮l RNAsin, and 1 U/␮l moloney murine leukemia virus reverse transcriptase (MMLV-RT, Stratagene, La Jolla, CA, USA) in 70 ␮l of 1× first strand buffer was incubated at 37 ◦ C for 1 h. The resulting products were amplified by adding 0.05 U/␮l Taq DNA polymerase (Viogene, Shijr, Taipei, Taiwan) and 0.3 ␮g/␮l each of the above forward and reverse primers with a Perkin-Elmer Cetus DNA thermal cycler 480, using a program consisting of 1 cycle of 5 min at 95 ◦ C, 1 min at 80 ◦ C (hot start); 35 cycles of 2 min at 95 ◦ C (denaturation),

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1 min at 55 ◦ C (annealing), and 2 min at 72 ◦ C (extension). After 35 cycles, the reaction mixtures were incubated at 72 ◦ C for an additional 10 min to allow complete synthesis. This PCR product was treated with BamHI and EcoRI, and sub-cloned into the same sites of pBluescript SK(−) vector (Stratagene, La Jolla, CA, USA) for sequencing. The Mt cDNA fragment of 141 bp was sequenced and used as a probe. 2.4. Screening mt gene from zebrafish genomic library The probe was labeled with [␣-32 P]dCTP and rediprime II kit (Amersham Biosciences, NJ, USA) and used for screening 1.2 × 106 plaques of ␭ Fix II zebrafish genomic library (Chen et al., 2001). After three times of screening, the zebrafish mt gene was isolated and sub-cloned into the HindIII site of pBluescript SK(−) vector for sequencing. Nucleotide sequencing was performed by the commercially available dideoxynucleotide chain-termination method (ABI PrismTM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase) using PRISM 377 DNA Sequencer (Perkin-Elmer, MA, USA). 2.5. Screening zebrafish Mt cDNA from zebrafish brain cDNA library To isolate the Mt cDNA, 1.2 × 106 plaques of ␭ zebrafish brain cDNA library prepared by using ␭ ZAP® II Predigested EcoRI/CIAP-Treated Vector Kit (Stratagene, La Jolla, CA, USA) (Tsai et al., 2001), was screened for three times using the above probe (141 bp). In vivo excision of the zebrafish Mt cDNA with pBluescript SK(−) was performed from the ␭ ZAP II vector. The Mt amino acid sequence alignment was performed using BioEdit program. 2.6. RT-PCR Total RNA was extracted from various developmental stages of zebrafish following RNAzol method (RNAzolTM B REAGENT, TEL-TEST “B”, INC.) and used for one-step RT-PCR (Life Technologies, Grand Island, NY, USA). ␤-Actin was used as an internal control. The forward and reverse primers for Mt were designed from the exon 1 and 3 regions of zebrafish mt

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gene. The following primers were used. Mt: 5 -CCC AAG CTT ATT TCT AAG GAA CTT TCA AGC-3 (forward), 5 -CCG CTC GAG TAA ATA CCA CCA TTT ATT TTA G-3 (reverse); ␤-actin: 5 -GTC CCT GTA CGC CTC TGG TCG-3 (forward), 5 -GCC GGA CTC ATC GTA CTC CTG-3 (reverse). The RT-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 ◦ C for 30 s, 72 ◦ C for 1 min, and a final extension of 1 cycle at 72 ◦ C for 7 min. The RT-PCR products, Mt and ␤-actin cDNAs with 540 and 678 bp, respectively were subjected to 3% agarose gel electrophoresis. 2.7. Whole-mount in situ hybridization Whole-mount in situ hybridization was performed with digoxigenin (DIG)-labeled RNA probes (Wilkinson, 1992). The probes were prepared by using a set of primers, designed based on the Mt cDNA. The primers were: 5 -CCC AAG CTT ATT TCT AAG GAA CTT TCA AGC-3 (forward) and 5 -CCG CTC GAG TAA ATA CCA CCA TTT ATT TTA G-3 (reverse). The forward and reverse primers contain HindIII and XhoI sites, respectively. The 540 bp fragment was PCR amplified and sub-cloned into pBluescript SK(−) vector, containing T7 and T3 promoters. Using T7 and T3 RNA polymerase, the digoxigenin-labeled anti-sense and sense RNA probes were synthesized. 2.8. Construction of recombinant plasmids Two plasmids, pGL3-1.4zMT and pGL3-0.2zMT containing 1.4 and 0.2 kb of the zebrafish promoter were constructed using PCR strategy (Fig. 5A). The primers were 1.4 kb (forward): 5 -CCG CTC GAG AGA CAC TGC ACA CG-3 ; 0.2 kb (forward): 5 -CCG CTC GAG GTG ATT GCT GAT TG-3 ; 1.4 and 0.2 kb (reverse): 5 -CCG AAG CTT TTC CAG AGA GTA TCC-3 . The inserts were sub-cloned into pGL3-Basic Luciferase Reporter Vector (Promega, Madison, WI, USA) at XhoI and HindIII sites. 2.9. Transfection and Zn induction Human hepatocellular carcinoma (Hep3B) cells were grown in Dulbecco’s modified Eagle’s medium

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(DMEM, Gibco BRL, Life Technologies, Grand Island, NY, USA) supplemented with 100 IU/ml penicillin, 100 ␮g/ml streptomycin, 10% fetal bovine serum (FBS), 10 mM HEPES, and 1 mM MEM non-essential amino acid solution at 5% CO2 and 37 ◦ C humidity incubator and seeded at a density of 5 × 105 cells per well in a 6-well plate. On the following day, the cells were transiently co-transfected with 0.1 ␮g of pRL-CMV (internal control) and 1 ␮g each of pGL3-1.4zMT, pGL3-0.2zMT, pGL3-Basic (negative control) using LIPOFECTAMINE PLUSTM reagent kit (Gibco BRL, Life Technologies, Grand Island, NY, USA). After 27 h of co-transfection, the culture medium was replaced with a medium containing 200 ␮M ZnSO4 (or without 200 ␮M ZnSO4 for control) in serum-free medium for 7 h. The cells were then harvested and assayed for luciferase activity using Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA). The luciferase activity was normalized with Renilla Luciferase activity, which was expressed from the co-transfected pRL-CMV vector.

3. Results 3.1. Effect of zinc on zebrafish embryo To understand the effect of Zn on zebrafish development, we hatched the eggs in deionic water containing ZnCl2 at different concentrations. The mortality was recorded at every 12 h. In control, 11% mortality was observed as early as shield stage (6 h post fertilization (hpf)), which increased to 21% at prim-5 stage (24 hpf) and maintained throughout the experimental period. Only 3% mortality was recorded at shield stage with 10 ␮M Zn and no more mortality was noted again. Hundred micromolars Zn caused no mortality at shield stage, but some mortality was recorded as development continued with a high level of 41% at 5 days post hatching (dph). At shield stage, the highest level of tested Zn concentration (1000 ␮M) produced just 5% mortality. This result, compared to the control indicates that Zn improves the survival rate of embryos and the larvae when added to deionized water. However, 1000 ␮M Zn caused >40% mortality at prim-5 stage, which increased to 100% on 4 dph (Fig. 1).

3.2. Sequence analysis of zebrafish mt gene The complete nucleotide sequence of zebrafish mt gene spanning 3293 bp (accession number: AY514791) and the deduced amino acid sequence are presented in Fig. 2A. Screening of 1.2 × 106 plaques of zebrafish brain cDNA library resulted in the identification of a Mt cDNA with the size of 522 bp (accession number: AY514790). Sequence analyses of zebrafish mt gene and its cDNA revealed that the coding region is interrupted by two introns of 94 and 664 nucleotides at nucleotide positions 26 and 186, respectively. Intron 1 is 68% AT-rich and intron 2 is 72% AT-rich. The open reading frame (ORF) encodes 60 amino acids. The upstream promoter region of the zebrafish mt gene contains a typical TATA box. Seven putative MREs, with five MREs (1, 2, 4, 6, and 7) having the motif TGCRCNC consensus core sequence in forward orientation, and two MREs (3 and 5) with the GNGYGCA sequence in reverse orientation were observed. Three possible polyadenylation signals with the AATAAA sequence were identified in the down stream region (Fig. 2A). The deduced amino acid sequence of zebrafish Mt was compared with that of vertebrates of various Classes including, mouse (Mammalia), snake (Reptilia), chicken (Aves), and xenopus (Amphibia) revealed high homology (>60%; except xenopus, which had 32% homology) (Fig. 2B). However, the ␣ and ␤ domains are highly conserved. Moreover, the total number of cysteine residues (20; 33%) and their locations are highly conserved (Fig. 2B). 3.3. Expression of mt in zebrafish during development The RT-PCR for zebrafish mt and ␤-actin cDNAs with 540 and 678 bp, respectively, demonstrated the expression of zebrafish mt at one-cell stage onwards, indicating the maternal contribution. The Mt transcripts could be observed through all the tested stages of zebrafish embryogenesis and early hatching period (Fig. 3). Whole-mount in situ hybridization was performed to observe the expression pattern of Mt transcripts in zebrafish embryos using DIG-labeled sense (data not shown) and anti-sense RNA probes. Strong signals are observed in the non-yolk cytoplasm of one-cell

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stage embryo indicates the presence of high level Mt transcripts, suggesting that Mt mRNA is supplied maternally. This non-yolk cytoplasm is squeezed out and streams to the animal pole to form a cytoplasm cap (blastodisc) and produces an intensive signal (Fig. 4A). This segregation process continues through the cleavage period (Fig. 4B–D). The heavy signal observed at the end of gastrula period (Fig. 4E), becomes light with a uniform distribution at the 5-somite stage (Fig. 4F). As segmentation period proceeds, the ubiquitous expression becomes restricted to the tail region (Fig. 4G), which further intensifies at the prim-5 stage (Fig. 4H). After hatching, no significant mt signal could be observed in the tail region. On 1 dph, clear as well as distinct mt expression signals are seen in common cardinal vein and chloride cells (Fig. 4I). The dorsal view shows a clear pattern of expression with stronger stain in pronephric region and light stain in retina (Fig. 4J). The signals observed on 1 dph at lateral

and dorsal views become stronger on 2 dph (Fig. 4K and L). The intensity of signals at common cardinal vein, pronephric region, and retina almost vanished on 3 dph. However, a mosaic pattern of expression with a specific signal at olfactory pit is observed (Fig. 4M and N). The intensity of staining due to mt expression at cerebellum and pronephric region on 4 dph becomes increased and maintains until 12 dph (Fig. 4O–R). An intensive signal could be noticed in the hyosymplectic on 4 dph (Fig. 4O). On 12 dph, the mt expression is confined to the cerebellum, lens, pronephric region, pronephric duct, ceratobranchials, and pancreas (Fig. 4Q and R). These data clearly show that mt expression plays a role on zebrafish embryogenesis and early larval development. 3.4. Promoter activity of zebrafish mt promoter The promoter region of the zebrafish mt gene, spanning 1.4 kb and its deletion containing 0.2 kb

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Fig. 3. RT-PCR for mt gene expression at various developmental stages. C indicates the negative control (without template). ␤-Actin was used as an internal control.

proximal region were ligated into pGL3-Basic vector (Fig. 5A) and transfected into Hep3B cells. The plasmid constructs, pGL3-1.4zMT and pGL3-0.2zMT containing seven and four MREs, respectively showed no significant luciferase activity and the activity was comparable with control. However, addition of ZnSO4 increased the expression of plasmids, pGL3-1.4zMT and pGL3-0.2zMT to 34- and 3-fold, respectively (Fig. 5B). The results show that zebrafish mt promoter comprising all the seven MREs can provide high level promoter activity and this promoter is functional in a heterologous system.

4. Discussion Zn is one of the important essential metals for the embryonic development (Watanabe et al., 1997; Falchuk, 1998a). However, deprivation as well as high level of Zn causes abnormality (Falchuk, 1998a) or toxic to the embryos (Domingo, 1994; Erbach et al., 1995), suggesting a requirement of Zn at a particular level. It has been shown that 70% of fish hepatic Zn is bound to Mt, which is expressed at extreme high levels (Thompson et al., 2002). Mts are efficient

sequestering molecules that can maintain intracellular ‘free’ Zn2+ at femtomolar range (Outten and O’Halloran, 2001). Mt proteins can sequestrate, up to seven Zn atoms in its ␤ and ␣ fragments, four and three atoms, respectively (Winge and Miklossy, 1982; Nielson and Winge, 1983; Kurasaki et al., 1999) and release as required for the biological activities. Mts act as Zn donors to apoenzymes (Jacob et al., 1998) and zinc-finger transcription factors requiring Zn for their activity. Thus, Zn and Mts are considered to have roles on normal embryogenesis and early larval development. Here, as a first step, we investigated the effect of Zn on zebrafish development. The mortality, 11–21% from shield to prim-5 stages observed during the zebrafish development in a medium without Zn indicates the requirement of Zn for development (Fig. 1). The supply of 10 ␮M Zn, decreased the mortality of embryos. However, excess of Zn caused 100% mortality on 4 dph, showing the embryotoxicity due to Zn. Sequence analysis of zebrafish mt revealed that it has a tripartite exon–intron similar to that of other fish Mt genes (Hamer, 1986). The splicing junctions follow the GT-AG rule (Breathnach and Chambon, 1981; Hamer, 1986). Intron 1 of zebrafish mt is 68% AT-rich,

Fig. 2. Nucleotide sequence and amino acid comparison of zebrafish Mt. (A) Complete nucleotide sequence of zebrafish mt gene. Within the coding region, exons and introns are shown in upper- and lowercases, respectively. The introns contain the consensus GT-AG splicing signals. The decoded amino acid sequence is given in single letter code below the exons. The TATA box and the polyadenylation signal are boxed. The putative MREs are shown. The zebrafish mt gene sequence was deposited in the GenBank database (accession number: AY514791). (B) Alignment of zebrafish Mt amino acid sequences with mouse, snake, chicken and xenopus. Shaded black and gray colors indicate the complete sequence identity and similarity, respectively. Hyphens are introduced to fill-up the gaps for optimal alignment. Total number of amino acids is given at the right end of each sequence. GenBank accession numbers for the sequences are as follows: zebrafish: AY514790, mouse: MT-I: AAA39527, MT-II: AAA39528, MT-III: AAA39529, MT-IV: AAA20233, snake: BM401839, chicken: CAA29924, and xenopus: AAA28111.

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Fig. 4. Whole-mount in situ hybridization of zebrafish mt transcripts during embryonic and early larval stages. The embryo stages are given at the bottom of each panel. Lateral (A–F, H, I, K, M, O, Q) and dorsal (G, J, L, N, P, R) views are shown. Anterior is to the left in panels (F–R). Abbreviations: cb, cerebellum; cc, chloride cell; ccv, common cardinal vein; ce, ceratobranchials; hy, hyosymplectic; le, lens; op, olfactory pit; p, pancreas; pd, pronephric duct; pr, pronephric region; re, retina; and yse, yolk sac extension.

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Fig. 5. Functional analysis of zebrafish mt promoter. (A) Schematic representation of the structure of zebrafish mt promoter and the two promoter regions fused with the luciferase reporter. MREs are shown (1–7) with forward (solid black) and reverse (gray black) orientations. The constructs, pGL3-1.4zMT and pGL3-0.2zMT contain seven and four MREs, respectively. (B) Relative luciferase activity of the expression of various plasmid constructs, transfected into Hep3B cells, with or without induction of 200 ␮M ZnSO4 . Each experiment was performed in triplicate. The bar indicates the standard error of the measurements.

which is similar as that of rainbow trout MT-B (69%) and intron 2 of zebrafish mt and rainbow trout MT-B are 72 and 61% AT-rich, respectively (Zafarullah et al., 1988). Based on the sequence comparison of fish Mts, this zebrafish Mt gene (brain origin) cannot be clas-

sified accurately and at this time is referred to simply as Mt form. The zebrafish Mt cDNA encodes 20 cysteine residues (33%) organized into two distinct fragments, which is conserved through vertebrate Mt proteins

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(Kojima and Kägi, 1978; Kägi, 1993). The amino and carboxy terminals contain 9 and 11 cysteines, form ␤ and ␣ fragments, respectively (Kurasaki et al., 1999). The amino acid sequence coding these putative ␤ and ␣ fragments, which are shown to be essential for the conformation of the Zn capture domains, are present in zebrafish Mt protein. The amino acid sequence confirms the motif characteristics of Mt proteins. The zebrafish Mt amino acid identity was very high (>60%) with that of mouse, snake, and chicken but xenopus showed only 32% homology (Fig. 2B). Among fish Mts, zebrafish Mt shares 78–98% homology (data not shown). In vertebrates, all mt promoters contain highly conserved MRE sequences, responsible for the binding of MTF-1 (Cserjesi et al., 1992; Chen et al., 2002). Analysis of zebrafish mt promoter has indicated the presence of seven MREs organized into four proximal and three distal clusters. Usually, a pair of complementary MRE sequences are consistently located between 40 and 120 nucleotides prior to the transcription start point and are identified as the major promoter elements involved in mt gene expression (Stuart et al., 1985). But, zebrafish mt promoter contains four MREs within the proximal promoter region. Nomizu et al. (1993) revealed that Zn is acquired from maternal resources during organogenesis. Similarly, studies on sea urchin revealed that the eggs contain substantial levels of maternal Mt mRNA (Nemer et al., 1984). We also observed Mt transcripts in the oocytes (data not shown). Our RT-PCR data demonstrated the maternal supply of Mt mRNA and its constitutive expression through out the embryogenesis and hatching. Quantitatively, it was demonstrated that the pattern of Mt level accumulated during oogenesis slowly decreases during the early stages of development in zebrafish (Riggio et al., 2003). Our whole-mount in situ hybridization of zebrafish embryos demonstrated the gradual decrease in the level of Mt transcripts during the early embryonic stages, say upto 5-somite stage, and at 26-somite and prim-5 stages the signal due to the mt expression was highly concentrated at tail region. In our previously published report on the developmental expression of MTF-1, we observed a strong ubiquitous expression at 26-somite and prim-5 stages (Chen et al., 2002). Interestingly, the expression patterns of mt (Fig. 4H) and MTF-1 (Chen et al., 2002) at

prim-5 stage are in opposite manner. The former expressed well in the tail region while the latter in head region. Based on this, it can be speculated that Mt may be involved in urinary and digestive system whereas, MTF-1 not and the expression of mt in the tail region may be due to other regulatory factors. However, further study is deserved to elucidate more on these patterns of expression. Expression of mt in the hyosymplectic and ceratobranchials implies its role on jaw and gill development. The mt expression pattern is more comparable with MTF-1, during early hatching period. A great deal of interest has been shown on the induction of fish mt promoter activity in response to heavy metals (Bonham et al., 1987; Samson and Gedamu, 1995, 1998; Carvan et al., 2000; Yan and Chan, 2002; Lin et al., 2004). In our transfection experiments, the zebrafish mt promoter with all the seven MREs shows high susceptibility to metals, with higher level, say 34-fold induced activity at 200 ␮M ZnSO4 . But, the plasmid construct containing the proximal four MREs could produce only threefold induction (Fig. 5B). These results suggest that the distal promoter region can be expected to contain other regulatory elements and the requirement of distally located MREs of zebrafish mt promoter for maximal response to Zn. In fact, we expected an induction comparable with the plasmid containing seven MREs because it was demonstrated that two MREs are sufficient for substantial induction by heavy metals (example, Stuart et al., 1985; Searle et al., 1985, 1987). Moreover, in our laboratory, we observed the binding of a recombinant MTF-1 with the zebrafish proximal MREs one and two, through electrophoretic mobility shift assay (unpublished data). Even in the current study, the zebrafish mt proximal MREs do not fail to induce at all, suggesting that these MREs are influencing the induction process. On the other hand, Olsson and Kille (1997) showed that all the MREs in proximal and distal clusters are needed for maximal inducibility of stone loach mt gene. Moreover, Yan and Chan (2002) recently reported that a zebrafish mt promoter containing 835 bp showed significant induction in response to Zn, when transfected into a zebrafish caudal fin cell line. To conclude, in the present investigation, we have demonstrated the deleterious effect of excess Zn on zebrafish embryotoxicity, the structure of mt gene and its promoter and the expression pattern of mt gene

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during the developmental stages. Recently, Tan and Chan (1997) showed efficient gene transfer into zebrafish skeletal muscle, persistence, and strong expression of the injected plasmid constructs. So, this study paves the ways to go further to establish the use of this zebrafish Mt as a biomarker and facilitate to analyze the feasibility of developing transgenic zebrafish for toxicological studies.

Acknowledgements This study was supported by Grant No. NSC 92-2311-B-001-038 from National Science Council, Taiwan, Republic of China.

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