The Molecular Structures and Expression Patterns of Two Distinct zebrafish

Dickkopf 3

Chuan-Yang Fu and Huai-Jen Tsai*

Institute of Molecular and Cellular Biology, National Taiwan University,

No. 1, Roosevelt Road, Sec. 4, Taipei 106, Taiwan

*Corresponding Author:

Huai-Jen Tsai,

Room 307, Fisheries Science Building, No. 1, Section 4, Roosevelt Road, Taipei,

Taiwan 106

e-mail: [email protected]

Tel: 886-2-3366-2487

Fax: 886-2-2363-8483

Key Words:

Zebrafish, , muscle, expression pattern, molecular structure

1 The Molecular Structures and Expression Patterns of Two Distinct zebrafish

Dickkopf 3 Genes

Chuan-Yang Fu and Huai-Jen Tsai Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan The Wnt signaling pathway is a cellular communication pathway that plays

critical roles in development and disease. A major class of Wnt signaling regulators is

the Dickkopf (Dkk) family, which is a secreted glycoprotein. The Dkk family has

been identified in birds and mammals, and known it consists of dkk1, 2, 3, 4 and a

dkk3-related (soggy). However, in low vertebrates, only dkk1 has been defined,

the others are still unknown. Here, we cloned two zebrafish dkk3 genes, which were

dkk3 and dkk3-related gene (dkk3r, also named the long-isoform dkk3). Based on the unrooted radial gene tree analysis of the Dkk genes among vertebrates, the zebrafish dkk3 and dkk3r we cloned were homologous of the dkk3 of other higher vertebrates.

The deduced amino acid residues of two zebrafish Dkk3 were 283 and 293, respectively, sharing 45% identity. Compared to the Dkk3 of other vertebrates, zebrafish Dkk3 shared two conserved Cysteine-rich domain 1 and 2, but difference in

N- and C-terminal amino acid sequence. Using reverse transcription-polymerase chain reaction and whole-mount in situ hybridization, we demonstrated that both dkk3 and dkk3r were maternally expressed. In addition, dkk3 and dkk3r were ubiquitously expressed during 16 hours post-fertilization (hpf). However, they were expressed in head, somite and neuron tube at 24 hpf. Interestingly, while dkk3r was particularly detected in craniofacial neuron tissue after 24 hpf, dkk3 was restricted in craniofacial arch muscles and pancreas. These evidences suggested that dkk3 and dkk3r shared the same expression patterns before 24 hpf, but displayed different patterns after 24 hpf.

Thus, using zebrafish as our system model, it is suggested that the results, as noted

2 above, may provide more insight into the molecular structures and expression patterns of the lower vertebrate dkk3 genes.

3 1. Introduction

The Dickkopf (Dkk) family consists of four genes (dkk1–4) and a dkk3-related

gene—soggy (sgy) or dkk1L (Krupnik et al., 1999). Dkks are secreted that

contain a signal sequence and two conserved cysteine-rich domains: an N-terminal

cysteine-rich domain unique to Dkks, and a C-terminal cysteine-rich domain related

to that of the colipase fold. The five Dkk proteins share 37–50% identity and

contain two conserved cysteine-rich regions separated by a variable linker region

(Krupnik et al., 1999).

Dkk1, Dkk2 and Dkk4 inhibit Wnt signaling by binding to LRP5/6 and the

transmembrane protein Kremen which results in LRP5/6 internalization and prevents

Wnt and Frizzled from forming an active complex with LRP5/6 (Mao et al., 2002;

Semënov et al., 2001). Dkk2 can also activate the Wnt pathway in certain situations,

depending on the cell type, the presence of Wnt ligands and levels of LRP5/6 (Brott

and SoKol 2002; Wu et al., 2000; Li et al., 2002). Dkk3 is the most divergent member

of the Dkk family by DNA sequence, function, and evolution (Niehrs., 2006). Unlike

the other Dkk members, Dkk3 does not regulate Wnt signaling in various activity assays, including Wnt-dependent secondary axis induction in Xenopus embryos and

Wnt1/Fz8 signaling in cultured cells (Krupnik et al., 1999; Brott and SoKol 2002;

Wu et al., 2000). Dkk3 also does not physicallyinteract with LRP5/6 or Kremen (Mao et al., 2002; Mao and Niehrs 2002). However, Caricasole et al. (2003) demonstrated that Dkk3 is a weak inhibitor of Wnt7A signaling in PC12 cells although co-expression of LRP5 or LRP6 is required to uncover this activity Dkk3 displays

Wnt inhibitor activity in the osteocarcinoma Saos-2 cell line, measured by decreased cytoplasmic levels of β-catenin (Hoang et al., 2004), but does not inhibit Wnt reporter

Tcf/Lef luciferase activity assays in a prostate line (Kawano et al., 2006).

Therefore, the relationship between Dkk3 and Wnt signaling is unclear despite its

4 sequence similarity to the other Dkk genes.

dkk3 is expressed during embryonic development in many organs, including neural epithelium, limb bud, bone and heart, particularly in regions of epithelial-mesenchyme transformation (Monaghan et al., 1999). dkk3 is also widely expressed in adult tissues, with the highest levels found in the heart and brain

(Krupnik et al., 1999). Especially, dkk3 was differentially expression during mouse and chick development (Monaghan et al., 1999; Diep et al., 2004; Fjeld et al., 2005;

Nie et al., 2005). The lines of evidences reveal that the expressed of dkk3 gene among different species may exhibit a different and complicate manner. Therefore, the first step, it is important to elucidate the molecular structure and expression pattern of the dkk3 gene in the fish.

In this study, we report the cloning and bioinformatic analysis of two zebrafish dkk3 genes, dkk3 and dkk3-related gene (dkk3r, also named the long-isoform dkk3). In addition, we presented the developmental expression patterns of these two dkk3 genes. dkk3 and dkk3r were ubiquitously expressed during 16 hours post-fertilization (hpf).

However, they were expressed in head, somite and spinal cord at 24 hpf. Interestingly, while dkk3 was particularly detected in craniofacial neuron tissue after 24 hpf, dkk3r was restrictedly detected in craniofacial arch muscles and pancreas.

5 2. Results and Discussion

2.1. Identification and characterization of zebrafish dkk3 and dkk3r gene

BLASTing the zebrafish EST database with Xenopus laevis DKK3 sequence

(GenBank Accession No. NP_001116948) using genome database (Ensembl, http://www.ensembl.org/), we found a candidate gene (NP_001083014) of zebrafish

dkk3. Employing RT-PCR strategy, we cloned a cDNA coding sequence of zebrafish

dkk3 with the length of 855 base pairs (bp) long, predicted to encode a protein

comprising 283 amino acids (Fig. 1). BLASTing GenBank protein database, we found

that the dkk3 is mapped in the same contig of zebrafish 25, and it is

encoded by 7 exons. In mammalian animals, The Dkk family consists of four genes

(dkk1–4) and a dkk3-related gene—soggy (sgy) or Dkk1L (Krupnik et al., 1999). Up to

now, dkk3 isoform was not discovered in vertebrate. Base on the zebrafish dkk3

sequence, we blast the database and found that the dkk3r encoded by NP_001152755

shares 48% amino acid identity with zebrafish dkk3. This protein comprises 293

amino acids (Fig. 1). According to the sequence, we cloned a cDNA coding sequence

of zebrafish dkk3r with the length of 882 base pairs. Based on BLAST GenBank

protein database, we found that the dkk3r was mapped in the same contig of zebrafish

chromosome 3, and it is encoded by 7 exons.

The deduced amino acid sequence of zebrafish Dkk3 and Dkk3r were compared

with other vertebrates Dkk3, the alignment data showed that Dkk3 and Dkk3r had the

same conserved cysteine-rich domains: an N-terminal cysteine-rich domain unique to

Dkks(Cys-1), and a C-terminal cysteine-rich domain related to that of the colipase

fold(Cys-2) as they were described by Krupnik et al. (1999). The deduced amino acid

sequence of zebrafish Dkk3 shared 14–15%, 14–17%, 27–43%, 19%, 12% and 8%

identities with that vertebrates Dkk1, Dkk2, Dkk3, Dkk4, Soggy, and Ciona Dkk,

respectively (Table 1). Meanwhile, the deduced amino acid sequence of zebrafish

6 Dkk3r shared 13–14%, 15%, 27–36%, 18%, 5-8% and 6% identities that of with

vertebrates Dkk1, Dkk2, Dkk3, Dkk4, Soggy, and Ciona Dkk, respectively (Table 1).

To examine the evolutionary relationship between teleost and other species Dkk, we

constructed a phylogenetic tree based on the deduced amino acid residues of zebrafish

Dkk (Fig. 2). Results showed that zebrafish Dkk3 and Dkk3r were clustered into

DKK3 subfamily. Taken together, these results suggest that the Dkk3 and Dkk3r are zebrafish ortholog of mammalian DKK3.

2.2. Temporal expression of dkk3 and dkk3r in zebrafish

We used specific primers to detect the temporal expressions of zebrafish dkk3 and dkk3r gene transcripts by RT-PCR. Total RNAs were extracted from zebrafish embryos at 10 stages, ranging from 1-, 4-, 8-, 16-, 24- and 48-hpf. As shown in Figure

3, we were able to detect zebrafish dkk3 and dkk3r gene at 1-cell stage through 48 hpf

(Fig. 3), indicating that zebrafish dkk3 and dkk3r gene transcripts were both maternally expressed, a very early embryonic marker.

2.3. Spatial Expression of dkk3 in Zebrafish Embryos

dkk3 is expressed during embryonic development in many organs, including neural epithelium, limb bud, bone and heart, particularly in regions of epithelial-mesenchyme transformation (Monaghan et al., 1999). dkk3 is also widely expressed in adult tissues, with the highest levels found in the heart and brain

(Krupnik et al., 1999). Especially, dkk3 was differentially expressed during mouse

and chick development (Monaghan et al., 1999 and Nie et al., 2005). To examine the

spatial expression of zebrafish dkk3, whole-mount in situ hybridization (WISH) was

performed on zebrafish embryos collected from 4 to 72 hpf. The data showed that

zebrafish dkk3 transcripts were detected were ubiquitously expressed during 16hpf.

7 (Figs. 4B, 4C, 4D). After 16 hpf, dkk3 were detected in head and truck (Fig. 4D, 4E).

Histochemical section of trunk was also performed to confirm the expression of

zebrafish dkk3 at cell level. This analysis showed that zebrafish dkk3 is expressed in

somite and neuron tube (Fig. 4E’). This early expression patterns were quite different

to the patterns of other vertebrates reported by Monaghan et al. (1999), Diep et

al.(2004), Fjeld et al. (2005), and Nie et al., (2005). We also noticed that dkk3 was detected in craniofacial arch muscles such as adductor mandibulae, levator arcus palatine, adductor operculi, adductor hyoideus, lateral rectus, and other tissues such as nucleus, fin bud and pancreas (Figs. 4G, 4H, 4I, 4J, 4H’ 4J’). These data suggested that the spatial expression of zebrafish dkk3 is different to that of mammals and other vertebrates.

2.4. Spatial expression of dkk3r in zebrafish embryos

In part, dkk3 isoform was never discovered in vertebrate. We had cloned the zebrafish dkk3r. In order to study the spatial expression pattern of zebrafish dkk3r,

WISH was performed on zebrafish embryos collected from 4 to 72 hpf. Similar to the expression patterns of zebrafish dkk3 during early embryogenesis, the data showed that zebrafish dkk3r transcripts were detected were ubiquitously expressed during hpf.

(Figs. 5B, 5C, 5D). After 16 hpf, dkk3r were detected in head and truck (Figs. 5D, 5E).

Histochemical section of trunk was also performed to confirm the expression of zebrafish dkk3r at cell level. This analysis showed that zebrafish dkk3r was expressed in somite and neuron tube (Fig. 5E’). This evidence indicates that the expression of zebrafish dkk3r and dkk3 were similar before 16 hpf. However, unlike dkk3 express in craniofacial arch muscles and pancreas, dkk3r was detected in craniofacial neuron tissue, such as anterior lateral line ganglia, octaval ganglia, posterior lateral line ganglia, cerebellum, epiphysis, forebrain ganglia and fin bud(Figs. 5G, 5H, 5I, 5J, 5H’

8 5J’). These data suggested that the spatial expression of zebrafish dkk3r was different

to that of zebrafish dkk3.

2.5. Conclusion

In this study, we cloned and characterized the molecular structures and

expression patterns of zebrafish dkk3 and dkk3r. In comparing our zebrafish system model to higher vertebrates, we demonstrated that zebrafish Dkk3 and Dkk3r were zebrafish ortholog of mammalian Dkk3, and also find a novel dkk3 isoform, dkk3r which is a unique isoform that has not been reported in mammals, amphibians, and avian homologues. However, expression patterns data indicated that there is a highly difference between zebrafish two dkk3 and vertebrate Dkk3. This finding is particularly important to understand Dkk3 function during embryogenesis in lower vertebrates because the Dkk3 knock-out mice develops normally (Barrantes et al.,

2006). Therefore, the present study displays the empirical groundwork for further study required to understand the Dkk3 biological function and the regulatory mechanism in lower vertebrates.

9 3. Experimental & procedures

3.1.Nucleic acids extraction and cDNA library synthesis

To obtain total RNA, zebrafish embryos aged 48-72 hpf were collected and

immediately stored in liquid nitrogen. The frozen embryos were homogenized with

TRIzol reagent (Biorad) and their total RNAs were extracted according to the manufacturer’s instructions. First strand cDNA was synthesized from 3 ng of total

RNA using a SuperScript II (Invitrogen).

3.2. Molecular cloning of dkk3 and dkk3r genes

A cDNA fragment was amplified using the polymerase chain reaction from cDNA

library with the following primers: dkk3F: ATGTTTCTGCTCGGATTCAG; dkk3R:

TCAGACGATGTAGTCGATCT. dkk3rF: ATGCTGAAATCGATGATATTGTG;

dkk3rR: TCAGTCCTCATTTCCTTCACCGG. Thirty-five cycles of PCR

amplification were performed by Phusion DNA polymerase (FINNZYMES). Each cycle consisted of denaturation for 40 sec at 94°C, 1 min of annealing at 57°C, and 30 sec of extension at 72°C. The last extension step was extended for 10 min at 72°C.

The PCR fragment was then cloned into the pGEM T-Easy vector (Promega) and transformed into Escherichia coli DH5α and sequenced. dkk3 and dkk3r Genbank

nucleotide accession number: NM_001089545 and NP_001152755.

3.3. Bioinformatics analysis of dkk3 and dkk3r sequences

The cDNA database from NCBI (http://ncbi.nlm.nih.org) was used to search for

sequence annotations indicative of possible homology to zebrafish DKK3. Nucleotide

sequences were translated by using the sequence available through the BCM Search

Launcher interface (http://searchlauncher.bcm.tmc.edu). Multiple sequence alignment

10 of the deduced amino acid sequences of DKK3 was performed using ClustalW

(Thompson et al., 1994), and phylogenetic trees were constructed by using the neighbor-joining method (Pearson et al., 1999) through the EMBL-EBI interface

(http://www.ebi.ac.uk/Tools/clustalw/). The accession numbers of sequences used in

Figure.2. and Table.1 are GenBank Human-DKK1：NP_036374, Mus-DKK1：

NP_034181, Chicken-DKK1 ： NP_034181, Xenopus-DKK1 ： (NP_001016283,

Human-DKK2：NM_014421, Mus-DKK2：NP_064661, Chicken-DKK2：XP_420494,

Xenopus-DKK2：NM_001082615, Human-DKK3：NM_001018057, Mus-DKK3：

NP_056629, Chicken-DKK3 ： NP_990456, Xenopus-DKK3 ： NP_001116948,

Human-DKK4：NP_055235, Mus-DKK4：NP_663567, Human-Soggy：AB047818,

Mus-Soggy ： AB051203, Ciona-DKKA ： NM_001078563, Ciona-DKKB ：

NM_001078211,

3.4. RT-PCR analysis

To detect the spatial and temporal expressions of zebrafish dkk3 and dkk3r, RT-PCR was performed by using the different specific primers as described in section 2.2 above. The total RNAs were extracted from embryos at 1, 4, 8, 16, 24 and 48 hpf. The primer pairs of dkk3F and dkk3R, dkk3rF and dkk3rR, were used to amplify cDNA fragments of 852 and 882, respectively. We used the amplification of zebrafish

β-actin (Kelly and Reversade, 1997) to serve as an RNA quality control in each tissue sample.

3.5. WISH

WISH of whole embryos was performed by using digoxigenin (DIG)-labeled riboprobes of dkk3 and dkk3r. We followed the procedures as described by Thisse and

Thisse (2008). Stained embryos were placed in 100% glycerol and evaluated with a

11 differential interference contrast microscope (DMR, Leica) with a color digital camera (COOLPIX 996, Nikon) attached. For histological examination, some stained embryos were embedded in cutting temperature -25℃ compound and sectioned at

10-nm intervals.

12 4. References Barrantes Idel, B., Montero-Pedrazuela, A., Guadano-Ferraz, A., Obregon, M.J., Martinez de Mena, R., Gailus-Durner, V., Fuchs, H., Franz, T.J., Kalaydjiev, S., and Klempt, M. (2006). Generation and characterization of dickkopf3 mutant mice. Molecular and Cellular Biology 26, 2317-2326.

Brott, B.K., and Sokol, S.Y. (2002). Regulation of Wnt/LRP signaling by distinct domains of Dickkopf proteins. Molecular and Cellular Biology 22, 6100-6110.

Caricasole, A., Ferraro, T., Iacovelli, L., Barletta, E., Caruso, A., Melchiorri, D., Terstappen, G.C., and Nicoletti, F. (2003). Functional characterization of WNT7A signaling in PC12 cells: interaction with A FZD5 x LRP6 receptor complex and modulation by Dickkopf proteins. The Journal of Biological Chemistry 278, 37024-37031.

Diep, D.B., Hoen, N., Backman, M., Machon, O., and Krauss, S. (2004). Characterisation of the Wnt antagonists and their response to conditionally activated Wnt signalling in the developing mouse forebrain. Brain Research 153, 261-270.

Fjeld, K., Kettunen, P., Furmanek, T., Kvinnsland, I.H., and Luukko, K. (2005). Dynamic expression of Wnt signaling-related Dickkopf1, -2, and -3 mRNAs in the developing mouse tooth. Developmental Dynamics 233, 161-166.

Hoang, B.H., Kubo, T., Healey, J.H., Yang, R., Nathan, S.S., Kolb, E.A., Mazza, B., Meyers, P.A., and Gorlick, R. (2004). Dickkopf 3 inhibits invasion and motility of Saos-2 osteosarcoma cells by modulating the Wnt-beta-catenin pathway. Cancer Research 64, 2734-2739.

Kawano, Y., Kitaoka, M., Hamada, Y., Walker, M.M., Waxman, J., and Kypta, R.M. (2006). Regulation of prostate cell growth and morphogenesis by Dickkopf-3. Oncogene 25, 6528-6537.

Krupnik, V.E., Sharp, J.D., Jiang, C., Robison, K., Chickering, T.W., Amaravadi, L., Brown, D.E., Guyot, D., Mays, G., and Leiby, K. (1999). Functional and structural diversity of the human Dickkopf gene family. Gene 238, 301-313.

13 Li, L., Mao, J., Sun, L., Liu, W., and Wu, D. (2002). Second cysteine-rich domain of Dickkopf-2 activates canonical Wnt signaling pathway via LRP-6 independently of dishevelled. The Journal of Biological Chemistry 277, 5977-5981.

Mao, B., and Niehrs, C. (2003). Kremen2 modulates Dickkopf2 activity during Wnt/LRP6 signaling. Gene 302, 179-183.

Mao, B., Wu, W., Davidson, G., Marhold, J., Li, M., Mechler, B.M., Delius, H., Hoppe, D., Stannek, P., and Walter, C. (2002). Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling. Nature 417, 664-667.

Nie, X. (2005). Dkk1, -2, and -3 expression in mouse craniofacial development. Journal of Molecular Histology 36, 367-372.

Niehrs, C. (2006). Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene 25, 7469-7481.

Semenov, M.V., Tamai, K., Brott, B.K., Kuhl, M., Sokol, S., and He, X. (2001). Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6. Current Biology 11, 951-961.

Wu, W., Glinka, A., Delius, H., and Niehrs, C. (2000). Mutual antagonism between dickkopf1 and dickkopf2 regulates Wnt/beta-catenin signalling. Current Biology 10, 1611-1614.

14 Table 1. Identity of the deduced amino acid sequences of zebrafish DKK protein with other vertebrates DKK Species(GeneBank No.) Zebrafish DKK1 (NM_131003) Zebrafish DKK2 (NM_001111209) Zebrafish DKK3 (NM_001089545) Zebrafish related DKK3 (NP_001152755) (Identity％) (Identity％) (Identity％) (Identity％) DKK1 Homo sapiens (NP_036374) 45 41 15 14 Mus musculus (NP_034181) 49 39 14 13 Gallus gallus (NP_034181) 42 41 14 14 Xenopus laevis (NP_001016283) 44 38 15 14

DKK2 Homo sapiens (NM_014421) 36 58 14 15 Mus musculus (NP_064661) 36 57 14 15 Gallus gallus (XP_420494) 36 60 17 15 Xenopus laevis (NM_001082615) 37 58 14 15

DKK3 Homo sapiens (NM_001018057) 18 16 42 33 Mus musculus (NP_056629) 16 15 42 32 Gallus gallus (NP_990456) 15 16 43 36 Xenopus laevis (NP_001116948) 17 16 27 27

DKK4 Homo sapiens (NP_055235) 34 31 19 18 Mus musculus (NP_663567) 33 34 19 18

Soggy Homo sapiens (AB047818) 5 2 12 5 Mus musculus (AB051203) 3 4 12 8

DKKA Ciona intestinalis (NM_001078563) 18 21 8 6 DKKB Ciona intestinalis (NM_001078211) 12 21 8 6

15 hDKK3 MQRLGATLLCLLLAAAVPTAPAPAPTATSAPVKPGPALSYPQEEATLNEMFREVEELMED 60 mDKK3 MQRLGGILLCTLLAAAVPTAPAPSPTVTWTPAEPGPALNYPQEEATLNEMFREVEELMED 60 cDKK3 MRRGEGPAPRRRWLLLLAVLAALCCAAAGSGGR------RRAASLGEMLREVEALMED 52 xDKK3 ------zDKK3 -MLKSMILCLCVGLAVGSSVHRGAHLDISDTLEEH----VAHGQTTLNEMFREVEKLMED 55 zDKK3r MFLLGFSLCLAVVHGIVPEIPK-TDMDIIANMETN----AAQEQT-MSDVLKEVEELMED 54

hDKK3 TQHKLRSAVEEMEAEEAAAKASSEVNLANLPPSYHNETNTDTKVGNNTIHVHREIHKITN 120 mDKK3 TQHKLRSAVEEMEAEEAAAKTSSEVNLASLPPNYHNETSTETRVGNNTVHVHQEVHKITN 120 cDKK3 TQHKLRNAVQEMEAEEEGAKKLSEVNFENLPPTYHNESNTETRIGNKTVQTHQEIDKVTD 112 xDKK3 ------MEMGNETIHSQKEMTKNTD 19 zDKK3 TQHKLEEAVHQMENET----TNSLLNGRDFPDNFHDETTTEIKLGNRTIQLIERINKKTD 111 zDKK3r TQHKLEDAVHQMDNET----AKSSLHPQNVSSNLQNYSAIETIAGNQTISIGERINKTTD 110 **.*: ..: * *: hDKK3 NQTGQMVFSETVITSVGDEEGRRSHECIIDEDCGPSMYCQFASFQYTCQPCRGQRMLCTR 180 mDKK3 NQSGQVVFSETVITSVGDEEGKRSHECIIDEDCGPTRYCQFSSFKYTCQPCRDQQMLCTR 180 cDKK3 NRTGSTIFSETIITSIKGGENKRNHECIIDEDCETGKYCQFSTFEYKCQPCKTQHTHCSR 172 xDKK3 NHTGSTLYSETVITSLKNNSK-RHQECIVDEDCKSGNYCYFADSEYKCLPCKAT-EPCTR 77 zDKK3 NKTGKTHFSRTLIQNT-ERWNEVDHECMIDEDCGDGSFCLYEIVTSKCVPCQTTNMECTK 170 zDKK3r NSTEETN-NLSSIQPR-DKENIVDHECVIDEDCEKGKYCLYETHSSKCLPCKQLDASCTK 168 * : . . : * :**::**** :* : .* **: *::

Cys-1 domain hDKK3 DSECCGDQLCVWGHCTKMATRGSNGTICDNQRDCQPGLCCAFQRGLLFPVCTPLPVEGEL 240 mDKK3 DSECCGDQLCAWGHCTQKATKGGNGTICDNQRDCQPGLCCAFQRGLLFPVCTPLPVEGEL 240 cDKK3 DVECCGDQLCVWGECRKATSRGENGTICENQHDCNPGTCCAFQKELLFPVCTPLPEEGEP 232 xDKK3 DGECCEG-LCVWGQCAH-VTKGEGGTICESQEDCNPGFCCAVHSDLLFPVCTPLPGEGEP 135 zDKK3 DVECCGDQLCVWGVCAQNKTKGQSGTICQNQNDCSPQHCCAFHKALLFPVCRPKPQEGQG 230 zDKK3r DEECCAGQLCVWGQCTINITKGDAGTICQYQTDCKEDFCCAFHKALLFPVCITKPIERER 228 * *** . **.** * ::* ****: * **. ***.: ****** . * * :

Cys-2 domain hDKK3 CHDPASRLLDLITWELEPDGALDRCPCASGLLCQPHS-HSLVYVCKPTFVGSRDQDGE-- 297 mDKK3 CHDPTSQLLDLITWELEPEGALDRCPCASGLLCQPHS-HSLVYMCKPAFVGSHDHSEE-- 297 cDKK3 CHDPSNRLLNLITWELEPDGVLERCPCASGLICQPQSSHSTTSVCELSSNETRKNEKEDP 292 xDKK3 CLDPSNKLLDIMNWDVQPAGVLGRCPCSQGLVCQPQSHALVSTCQEPSPDDSKRSDLEVP 195 zDKK3 CEREGNQLMEVLLWED--EGPREHCPCAAGLLCQQIQ---KSSVCVDERHASGEGNED-- 283 zDKK3r CIISANHLMELLSWDMDGEGPQEHCPCAGELQCQHRG---RGALCLKSQNSSEEELTD-- 283 * .:*:::: *: * :***: * ** : : hDKK3 ------ILLPREVPDEYEVGSFMEEVRQELEDLERSLTEEMALREPAAAAAALLGGE 348 mDKK3 ------SQLPREAPDEYEDVGFIGEVRQELEDLERSLAQEMAFEGPAP-VESLGGEE 347 cDKK3 LNMDEMPFISLIPRDILSDYEESSVIQEVRKELESLE----DQAGVKSEHDPAHDLFLGD 348 xDKK3 ------EVIPPFIGIMPQEGQYYEDGTQLSDGPYASPSEESRSLESRYGDPLLAAAK 246 zDKK3 ------zDKK3r ------TLYSEIDYIV------293

hDKK3 EIDPCLRSSDCIDGFCCARHFWTKICKPVLHQGEVCTKQRKKGSHGLEIFQRCDCAKGLS 408 mDKK3 EI------349 cDKK3 EI------350 xDKK3 RVSVDVPEEVLPFVGIMEERDYEDSDVVPTSDSVPTSDGVDAGPLEETYETEPGLVEKSP 306 zDKK3 ------zDKK3r ------

hDKK3 CKVWKDATYSSKARLHVCQKI 429 mDKK3 ------cDKK3 ------xDKK3 FDDYK------311 zDKK3 ------zDKK3r ------:

Fig. 1. The deduced amino acid sequences of zebrafish DKK3 and DKKr3 compared with other DKK3 family from vertebrates. The alignment of amino acid sequences of three types TnnI by CLUSTALW(1.83). The conserved regions are boxed with color, including (1) red; Cys-1, Cysteine rich domain-1. (2) blue; Cys-2, Cysteine rich domain-2. Asterisks, two dot and one dot were indicated that amino acid residues were 100％, 75％, 50％ conserved among all species, respectively. ”-”, gaps created to maximize the degree of homology among all of the sequence compared.

16

Fig. 2. An unrooted radial gene tree of DKK among vertebrates. The gene tree was constructed with the neighbor-joining method (Pearson et al., 1999), using 1,000 bootstrap values. The marker length of 0.1 corresponds to 10% sequence difference. The DKK1, DKK2, DKK3, DKK4, Soggy clades, and Ciona were marked in red, green, blue, brown, purple and orange respectively. See the Experimental Procedure section for details on the sources of DKK. This phylogenic tree indicates that Zebrafish DKK1, DKK2, DKK3 and DKK3r clustered with their tetrapod counterparts into two distinct monophyletic groups. The Soggy and Ciona Dkk were unique monophyletic groups.

17

Fig. 3. Using RT-PCR to detect the temporal expression patterns of zebrafish dkk3 and dkk3r genes during the early development. Total RNAs were isolated from different developmental stages (hours of postfertilization; hpf) as indicated. Specific primers were designed for detecting the existence of dkk3 and dkk3r transcripts. The expected molecular size of each DNA fragment after RT-PCR amplification was indicated on the right. The two zebrafish dkk3 genes both started to be transcribed in one cell stage. The β-actin transcript was used as an internal control.

18

19 Fig. 4. The expression patterns of dkk3 transcript during the development of zebrafish embryos. Embryos at different stages as indicated were collected and hybridized with dkk3 using whole-mount in situ hybridization. Panels A, B, C, D, F, G and I were lateral views and anterior of embryo to the left; panel H and J were ventral view and anterior of embryo to the top. dkk3 was ubiquitously expressed during 16 hpf, but it was expressed in head, somite and neuron tube at 24 hpf. Panels F and F’ were whole-mount in situ hybridization and transverse section (red line) at 24 hpf embryos. Panel F’ was indicated that dkk3 gene expressed in somite and neuron tube. After 24 hpf, dkk3 was restrictedly expressed in craniofacial arch muscles, fin and pancreas. am：adductor mandibulae; lap： levator arcus palatini; ao：adductor operculi； ah：adductor hyoideus; lr：lateral rectus；N：neuron tube； n：nucleus；P：pancreas； s: somite； F：fin bud.

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21 Fig. 5. The expression patterns of dkk3r transcript during the development of zebrafish embryos. Embryos at different stages as indicated were collected and hybridized with dkk3r using whole-mount in situ hybridization. Panels A, B, C, D, F, G and I were lateral views and anterior of embryo to the left; panels H and J were dorsal view and anterior of embryo to the top. dkk3r was ubiquitously expressed during 16 hpf, but it was expressed in head, somite and neuron tube at 24 hpf. Panel F and F’ were whole-mount in situ hybridization and transverse section (red line) at 24 hpf embryos. Panel F’ was indicated that dkk3r gene expressed in somite and neuron tube. After 24 hpf, dkk3r was particularly detected in craniofacial neuron tissue and fin. alg：anterior lateral line ganglia; og： octaval ganglia; plg：posterior lateral line ganglia； c：cerebellum; e：epiphysis； fg：forebrain ganglia；N：neuron tube； s: somite； F：fin bud.

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