Combinatorial Expression Patterns of Heparan Sulfate Sulfotransferases in Zebrafish: I. The 3-O-Sulfotransferase Family

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1 DEVELOPMENTAL DYNAMICS 235: , 2006 PATTERNS & PHENOTYPES Combinatorial Expression Patterns of Heparan Sulfate Sulfotransferases in Zebrafish: I. The 3-O-Sulfotransferase Family Adam B. Cadwallader and H. Joseph Yost* Heparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains termed the HS fine structure, which gives HS specific binding affinities for extracellular ligands. HS 3-Osulfotransferases (3-OST) catalyze the transfer of sulfate groups to the 3-O position of glucosamine residues of HS, a rare, but essential HS chain modification required for HS fine structure. We report here the first characterization and developmental expression analysis of the 3-OST gene family in a vertebrate. There are eight 3-OST genes in zebrafish: seven genes with homology to known 3-OST genes in mouse and human, as well as a novel, 3-OST-7. A phylogenetic comparison of human, mouse, and zebrafish indicates the 3-OST family can be subdivided into two distinct subgroups. We examined the mrna expression patterns in several tissues/organs throughout early zebrafish development, including early cleavage stages, somites, brain, internal body organ primordial, and pectoral fin development. The 3-OST gene family has both specifically expressed and ubiquitously expressed genes, suggesting in vivo functional differences exist between members of this family. Developmental Dynamics 235: , Wiley-Liss, Inc. Key words: zebrafish; heparan sulfate; 3-O-sulfotransferase; proteoglycan; 3-OST Accepted 19 September 2006 INTRODUCTION Heparan sulfate proteoglycans (HSPGs) are present on the cell surface or secreted extracellularly where they play a variety of biological roles, including cell adhesion, cell signaling during development, blood coagulation, wound healing, and growth factor-mediated proliferation (Rosenberg et al., 1997; Bernfield et al., 1999; Rapraeger, 2001; Esko and Selleck, 2002; Kramer and Yost, 2003; Hacker et al., 2005). HSPGs are composed of a core protein to which heparan sulfate (HS) chains, alternating glucuronic acid and N-acetyl glucosamine resides, are attached. The sugar chains undergo a series of extensive modifications beginning with N-deacetylation and N- sulfation of a subset of the glucosamine residues. Subsequently, a subset of glucuronic acid residues is converted to iduronic acid by HS C5- epimerase. After epimerization, the chains are further modified by means of the O-sulfation pathway. Sulfates are added to the 2-O position of uronic acid residues and at the 6-O and 3-O positions of glucosamine residues to create unique, sulfated domains termed the HS fine structure. The placement and spacing of sulfates in the HS fine structure confer the distinct, specific binding properties of HS (Nakato and Kimata, 2002). Modification of HS at the 3-O position of glucosamine residues is catalyzed by the HS 3-O-sulfotransferase (3-OST) family. The 3-O sulfation of glucosamine residues is the rarest modification of HS, accounting for 0.5% of the total sulfate in an HS chain (Colliec-Jouault et al., 1994; Shworak et al., 1994). The Caenorhabditis elegans genome encodes one putative 3-OST gene (hst-3) (Turnbull et al., 2003), whereas the human and mouse genomes contain at least seven Huntsman Cancer Institute, Center for Children, Department of Oncological Sciences, University of Utah, Salt Lake City, Utah Grant sponsor: NHLBI; Grant sponsor: The Huntsman Cancer Foundation. *Correspondence to: Joseph Yost, Huntsman Cancer Institute, Center for Children, University of Utah, 2000 Circle of Hope #4210, Salt Lake City, UT joseph.yost@hci.utah.edu DOI /dvdy Published online 31 October 2006 in Wiley InterScience ( Wiley-Liss, Inc.

2 3424 CADWALLADER AND YOST Fig. 1. Phylogenetic analysis of the predicted zebrafish 3-O-sulfotransferases (3-OST) genes were conducted using MEGA version 3.1 (Kumar et al., 2004). Scale indicates the number of amino acid substitutions per site adjusted by the equal input model. zf, zebrafish; m, mouse; h, human; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster. 3-OST genes (Shworak et al., 1997, 1999; Xia et al., 2002, 2005). The extensive number of 3-OST genes in vertebrate species and the rarity of 3-OST modifications suggest that individual members of the 3-OST multigene family might regulate the production of functionally distinct HS fine structures. The importance of 3-O sulfation in fine structure formation is not well understood. While the requirement for a 3-OST modification in the HS/ antithrombin interaction has been well characterized, knockout of the 3-OST-1 gene in mouse, the only 3-OST knockout currently available, failed to provide supporting in vivo data (Shworak et al., 2002; HajMohammadi et al., 2003). Although the 3-OST-1 knockout mouse did exhibit other phenotypes, including intrauteral growth retardation, unperturbed HS/antithrombin interaction suggests that other 3-OSTs may have overlapping functions (Shworak et al., 2002; HajMohammadi et al., 2003). Two 3-OST genes, HS3st-A and HS3st-B, were recently characterized in Drosophila (Kamimura et al., 2004). RNAi-mediated disruption of HS3st-B results in the reduction of Notch signaling and neurological defects. The 3-OST-2 modification of HS has been linked to light-induced changes in the pineal gland in the rat (Borjigin et al., 2003; Kuberan et al., 2004). Although the biological significance of this is unknown, it is an intriguing finding given the rarity of HS 3-O sulfation. The large number of genes in the vertebrate 3-OST family suggests functional heterogeneity, such that the ability of a cell type to generate specific fine structures on HSPGs, and thus to modulate cell signaling pathways, might depend on the expression of specific OSTs. However, there has not been a systemic analysis of the expression of the 3-O-sulfotransferase gene family in any organism. Here we report the molecular identification and categorization of eight genes that appear to encode the 3-OST family in the zebrafish, Danio rerio, and examine the expression of each gene in early development by whole-mount in situ RNA hybridization. The unique expression of each 3-OST, with striking differences in tissue distribution in many developing structures, provides a foundation for future studies into how the HS 3-O-sulfotransferase family functions during early development. RESULTS Cloning of the Zebrafish 3-O- Sulfotransferase Family All previously characterized HS 3-OST family members have a type II transmembrane structure, composed of two distinct regions (Shworak et al., 1997, 1999; Xia et al., 2002, 2005). The first region, composed of the first third of the gene, is highly divergent among members of the family, both within and among species. The second region,

3 3-O-SULFOTRANSFERASE FAMILY IN ZEBRAFISH 3425 Fig. 2. Amino acid alignment of 3-O-sulfotransferases (3-OST) family member catalytic domains. The putative 5 PAPS binding site (5 -PBS) and the 3 PAPS binding site (3 PBS) were based upon the consensus sequence (Shworak et al., 1999; Negishi et al., 2001) A: Alignment of subgroup 1 3-OST sequences. Human 3-OST-3A, a subgroup 2 member, was added for reference. B: Alignment of subgroup 2 3-OST sequences. Human 3-OST-1, a subgroup 1 member, was added for reference. H, human; zf, zebrafish. the C-terminal two thirds of the gene, comprises the highly conserved catalytic domain (Shworak et al., 1999). Using the human 3-OST catalytic domains as a template, we initially identified members of the zebrafish 3-OST family by performing a BLAST search of early versions of the publicly available zebrafish databases. This search identified eight unique sequences representing the catalytic domains of the zebrafish 3-OST gene family. The fragments were amplified by polymerase chain reaction (PCR), and subsequently, 5 and 3 rapid amplification of cdna ends (RACE) were used to obtain full-length open-reading frames (ORFs) for each 3-OST gene. We propose nomenclature for each zebrafish 3-OST family member based on phylogenetic analysis of the gene family in zebrafish, human, and mouse (Fig. 1). Zebrafish contain orthologues of all previously identified vertebrate 3-OST genes, as well as an additional, novel family member, which we named 3-OST-7 (Fig. 1). Members of the zebrafish 3-OST family share at least 63% similarity within the catalytic domain to the corresponding human isoform, with the exception of zebrafish 3-OST-5, which shows only 53% similarity to human 3-OST-5. Zebrafish 3-OST-7, which shows 66% identity to human 3-OST-1 and 58% identity to human 3-OST-5, was named because alignment between human and zebrafish using ensembl ( suggests that 3-OST-7 is syntenic with human 3-OST-5. One nomenclature issue that cannot be resolved by sequence comparisons is distinguishing between two zebrafish genes that are similar to human 3-OST-3A and 3-OST-3B, because the two human genes share 100% identity throughout the catalytic domain, while the corresponding zebrafish genes are only 84% similar, precluding further distinction within the 3-OST-3 group. Therefore, to avoid confusion, we have named the zebrafish genes 3-OST-3X and 3-OST-3Z. 3-O-Sulfotransferase Subgroups In addition to providing for a rational nomenclature for zebrafish 3-OST family members that will allow comparisons to mammalian genes, phylogenetic analysis also revealed the existence of two distinct 3-OST family subgroups. These subgroups, which we termed subgroup 1 and subgroup 2 (Fig. 1), can be defined on the basis of sequence similarity, genomic structure, and previously characterized in vitro biochemical function. 3-OST subgroup 1 includes the human, mouse, and zebrafish 3-OST-1, -5, and -7 genes, as well as C. elegans hst-3 and Drosophila HS3st-A. Human and mouse members of this family have previously been characterized by their ability to create the HS fine structure domain that binds antithrombin (Shworak et al., 1997; Xia et al., 2002; Duncan et al., 2004). The zebrafish members of this subgroup all contain their entire ORF within one exon and lack transmembrane domains. Previously, only human and mouse 3-OST-1 had been shown to lack a transmembrane domain (Shworak et al., 1997). Members of this family share a common sequence similarity in the catalytic domain, when compared with members of subgroup 2 (Fig. 2A).

4 3426 CADWALLADER AND YOST TABLE 1. Expression of Zebrafish 3-O-Sulfotransferase Genes During Early Development a Subgroup 1 Subgroup 2 3-OST-1 3-OST-5 3-OST-7 3-OST-2 3-OST-3X 3-OST-3Z 3-OST-4 3-OST-6 Maternal Polster (w) (w) Somites (u/a) (p) (pv) (w) (we) Notochord (u) (w) (w) (w) Floorplate (u) (w) (w) (w) Hypochord (u) (w) (w) (w) Tail Bud (w) (w) (w) (w) Kupffer s vesicle Eye (we) (w) (w) (we) Lateral Line Primordia CNS Telencephalon (u) (w) Diencephalon (w) Midbrain (u) (d) (w) (e) Hindbrain Spinal Cord (n) (n) (n) Olfactory system (w) (w) (w) Heart Gut (a) Proctodeum Pancreas Liver Pronephric duct/kidney Branchial Arches Otic Vesicle Pectoral Fin Muscle Pectoral Fin AER a Embryonic axis (a, anterior; p, posterior; d, dorsal; v, ventral); e, early somitigenesis; n, neurons; u, ubiquitous; w, weak. The 3-OST subgroup 2 includes the human, mouse, and zebrafish 3-OST-2, -3(A, B, X, Z), -4, and -6 genes, as well as Drosophila HS3st-B. Human 3-OST-2 (O Donnell et al., 2006), 3-OST-3A and -3B (Shukla et al., 1999; Tiwari et al., 2004), 3-OST-4 (Tiwari et al., 2005), and mouse 3-OST-3B (Shukla et al., 1999), 3-OST-6 (Xu et al., 2005) have previously been characterized by their ability to create the 3-O sulfation necessary for herpes simplex virus-1 (HSV-1) entry. Zebrafish members of this subgroup all have their ORF split between two exons. As opposed to subgroup 1, all members of subgroup 2 contain an N-terminal domain, a transmembrane domain and a SPLAG domain (Shworak et al., 1999). Alignment of the catalytic domain of this subgroup reveals a common similarity (Fig. 2B). Expression of Zebrafish 3-OST Genes The rarity of HS 3-O sulfation, along with the large number of 3-OST genes in vertebrates, suggests expression of each gene may indicate potential function. On one extreme, ubiquitous expression of all 3-OST family members would suggest either that other posttranslational mechanisms are necessary to ensure regulated 3-O sulfation of HS or that family members are functionally equivalent. In contrast, distinct, nonoverlapping expression would suggest that cells regulate 3-OST gene expression, which in turn may regulate the range of possible HS fine structures the cell can create. To analyze the expression differences between members of the HS 3-OST family, we performed mrna in situ hybridization with riboprobes specific to each member. In situ hybridization of embryos from early cleavage stages through 48 hr postfertilization (hpf) was performed for each 3-OST gene, and the results of these analyses are summarized in Table 1. Table 1 is not a complete list of all structures showing expression, but gives representative examples of differential expression. Most 3-OST family members show extensive brain expression, but in most cases, expression is often restricted to very specific brain subdivisions (Fig. 5). 3-OST-1 had the most widespread expression of the entire family. 3-OST-1 was expressed intensely during early cleavage stages (Fig. 3A), continuing through early (Fig. 3B) and mid- (Fig. 3C) somitogenesis in a variety of structures (Table 1). At 24 hpf (Fig. 4A), expression becomes restricted to head and anterior somites. Expression remained in head and anterior somites through 36 hpf, when expression appeared in developing gut and liver. By 48 hpf, expression was restricted to head (Fig. 5A,B), gut and pectoral fin (Fig. 6A). Expression of 3-OST-2 was both spatially and temporally restricted to developing brain, otic vesicle, and olfactory areas during early zebrafish development (Table 1). No expression of 3-OST-2 was detected until 24 hpf,

5 3-O-SULFOTRANSFERASE FAMILY IN ZEBRAFISH 3427 when expression was seen in otic vesicle and developing brain (Fig. 4B). The 3-OST-2 was expressed in all major divisions of developing brain (Table 1), yet was spatially and temporally restricted to specific cell populations in each brain division (Fig. 5C,D). Expression of 3-OST-3X (Table 1) was observed throughout early cleavage stages (Fig. 3D) and became distinct during early somitigenesis, when expression was seen in neural tube and lateral plate mesoderm (Fig. 3E). This expression continued through mid-somitigenesis, when expression began in midbrain (Fig. 3F). At 24 hpf (Fig. 4C), expression continued in spinal cord and began in hindbrain, brachial arches, pectoral fin, and lateral line primordia. Expression at 36 hpf was seen in olfactory areas, hindbrain, and midbrain (Fig. 5E,F). At 48 hpf, expression in spinal cord had receded anteriorly, while expression continued in pectoral fin apical ectodermal ridge (AER) (Fig. 6B). Expression of 3-OST-3Z was not detected in early cleavage stage embryos (Fig. 3G), but began in bud stage embryos in polster and forebrain regions. During early (Fig. 3H) and mid- (Fig. 3I) somitigenesis, expression extended throughout brain and spinal cord. During mid-somitigenesis, expression was detected in ventral regions of posterior somites, as seen in Figure 3I. At 24 hpf (Fig. 4D), expression continued throughout brain and spinal cord, as well as pectoral fin, lateral line primordium, proctodeum, and dorsal fin fold. Expression in brain at 36 hpf became restricted to Fig. 3. Fig. 3. Expression of heparan sulfate (HS) 3-Osulfotransferases (3-OST) genes during early zebrafish development. A C: Expression of 3-OST-1 is ubiquitous in 2 cell (A), 7 somite (B), and 18 somite (C) embryos. D F: Expression of 3-OST-3X in 2 cell (D), 13 somite (E), and 18 somite (F) embryos. G I: Expression of 3-OST-3Z in 2 cell (G), 7 somite (H), and 18 somite (I) embryos. J L: Expression of 3-OST-4 in 2 cell (J), 5 somite (K), and 18 somite (L) embryos. M O: Expression of 3-OST-5 in 2 cell (M), 7 somite (N), and 18 somite (O) embryos. P R: Expression of 3-OST-6 in 2 cell (P), 13 somite (Q), and 18 somite (R) embryos. FB, forebrain; HB, hindbrain; LPM, lateral plate mesoderm; MB, midbrain; NT, neural tube; PND, pronephric duct; S, somites; SC, spinal cord; SCN, spinal cord neurons; TB, tail bud.

6 3428 CADWALLADER AND YOST telencephalon, tectal regions, and hindbrain, as well as spinal cord. Expression was also seen in lateral line primordia in posterior tail and several internal organ primordia. These staining patterns were constant through 48 hpf in brain (Fig. 5G,H) and pectoral fin muscle and AER (Fig. 6C). The 3-OST-4 was detected as a maternal transcript (Fig. 3J). During early (Fig. 3K) and mid- (Fig. 3L) somitigenesis, expression was ubiquitous. During mid-somitigenesis (Fig. 3L) more intense expression was seen in hindbrain, which became apparent at 24 hpf (Fig. 4E) along with punctate expression throughout the spinal cord. At 36 hpf, expression was regionalized in brain (Fig. 5I,J). Although a low level of staining was seen throughout the head, specific expression was observed in olfactory epithelium, as well as forebrain and hindbrain. At 48 hpf, brain expression continued and otic vesicle expression began. 3-OST-5 expression changed dynamically during early zebrafish development. Expression was observed in early cleavage stages (Fig. 3M) and ubiquitous expression continued through early somitigenesis (Fig. 3N). During mid-somitigenesis (Fig. 3O), expression was confined to forebrain, midbrain and spinal cord neurons. Expression shifted to olfactory epithelium and a subset of spinal cord neurons at 24 hpf (Fig. 4F) and restricted to the head region at 36 hpf. Expression in 36 hpf embryos was seen in olfactory epithelium, telencephalon, and tectum (Fig. 5K M). At 48 hpf, expression again changed; forebrain and midbrain ventricle regions expressed 3-OST-5, while olfactory epithelium expression persisted (Fig. 5N P). 3-OST-6 was detected as a maternal transcript (Fig. 3P). During early somitigenesis, predominant expression was seen in hindbrain and spinal cord neurons (Fig. 3Q). This expression continued through mid-somitigenesis when spinal cord neurons became more prevalent (Fig. 3R). By 24 hpf (Fig. 4G), expression occurred throughout the brain, with intense staining seen in hindbrain and no expression in spinal cord. This expression became more intense by 36 hpf (Fig. 5Q,R) and began to weaken at 48 hpf. 3-OST-7 was expressed in early cleavage embryos and remained expressed through mid-somitigenesis. By 24 hpf (Fig. 4H), strong expression was seen throughout the brain and weaker expression seen in posterior somites. Distinct expression of 3-OST-7 was first seen in 36 hpf brain (Fig. 5S,T), pancreas, and weakly in pectoral fin muscle (Fig. 6D). This expression continued through 48 hpf. DISCUSSION While in vitro characterization of human and mouse 3-OST family members has focused on potential roles of each gene in creating an antithrombin binding site or entry of HSV-1 in cultured cells, relatively few studies have focused on characterization of 3-OST family members in vivo. Here, we show zebrafish contain orthologues of all seven of the higher vertebrate 3-OSTs, as well as an additional, novel 3-OST family member, 3-OST-7. We propose that 3-OSTs in vertebrates are divided into two subgroups. Each 3-OST family member showed specific expression patterns, even among very closely related family members. Both 3-OST-3X and 3-OST- 3Z, the closest related family members with 71% identity, displayed large differences in expression, indicating the in situ hybridization probes did not cross-hybridize with other family members. Expression of 3-OST-3Z is throughout the neural tube at 18 somites (Fig. 2F), whereas expression of 3-OST-3X is absent from the forebrain and expressed in only a subset of the midbrain (Fig. 2I). Additionally, brachial arch expression was seen for 3-OST-3X at 24 hpf (Fig. 2C), but was absent in 3-OST-3Z embryos (Fig. 2D). Analysis of 3-OST expression patterns suggests coordinated regulation exists between family subgroups (Fig. 1; Table 1). First, most tissues examined have at least one member of each subgroup expressed throughout development. Second, each subgroup contains at least one member with basal or ubiquitous expression and at least one member with specific expression. For example, 3-OST-1, a member of subgroup 1, is expressed ubiquitously, whereas 3-OST-5, another subgroup 1 member, is expressed in very distinct locations throughout the early zebrafish. In subgroup 2, 3-OST-6 is expressed ubiquitously throughout early zebrafish development, whereas 3-OST-2 has specific brain expression. These examples suggest expression is coordinated between subgroups and one member of each subgroup might be necessary for generation of specific HS fine structures in vivo. The diversity in expression of 3-OST family members suggests the previous biochemical studies need to be re-evaluated in the context of a biological system. Previous studies have Fig. 4. The 3-O-sulfotransferases (3-OST) family is spatially restricted at 24 hr postfertilization. A: 3-OST-1 is expressed throughout the head and in the anterior somites. B: 3-OST-2 is expressed basally in the brain and specifically in the otic vesicle. C: 3-OST-3X is expressed in the hindbrain, spinal cord, branchial arches, pectoral fin, and lateral line primordial. D: 3-OST-3Z is expressed in the brain, spinal cord, pectoral fin, lateral line primordial, proctodeum, and dorsal fin fold. E: 3-OST-4 is expressed specifically in spinal cord neurons. F: 3-OST-5 is expressed specifically in the olfactory region and in posterior spinal cord neurons. G: 3-OST-6 is expressed specifically in the hindbrain. H: 3-OST-7 is expressed basally throughout the embryo and specifically in the posterior somites. BA, branchial arches; DFF, dorsal fin fold; FB, forebrain; HB, hindbrain; LLP, lateral line primordial; MB, midbrain; OE, olfactory epithelium; OT, otic vesicle; PF, pectoral fin; S, somites; SC, spinal cord; SCN, spinal cord neurons. Fig. 5. Expression of 3-O-sulfotransferases (3-OST) family members in the brain. A,B: Lateral (A) and dorsal (B) expression of 3-OST-1 at 48 hours postfertilization (hpf). C,D: Lateral (C) and dorsal (D) expression of 3-OST-2 at 48 hpf. E,F: Lateral (E) and dorsal (F) expression of 3-OST-3X at 36 hpf. G,H: Lateral (G) and dorsal (H) expression of 3-OST-3Z at 48 hpf. I,J: Lateral (I) and dorsal (J) expression of 3-OST-4 in 36 hpf embryos. K P: Expression of 3-OST-5 in 36 hpf (K M; lateral [K], dorsal [L], and rostral [M]) and at 48 hpf (N P; rostral [N], lateral [O], and dorsal [P]). Q,R: Lateral (Q) and dorsal (R) expression of 3-OST-6 in 36 hpf embryos. S,T: Lateral (S) and dorsal (T) expression of 3-OST-7 in 36 hpf embryos. BA, branchial arches; DI, diencephalon; FB, forebrain; HB, hindbrain; MB, midbrain; OE, olfactory epithelium; OT, otic vesicle; RGC, retinal ganglion cells; TE, telencephalon.

7 3-O-SULFOTRANSFERASE FAMILY IN ZEBRAFISH 3429 Fig. 4. Fig. 6. Expression of 3-OST family members in the developing pectoral fin. A: Weak expression of 3-OST-1 is observed in the pectoral fin muscle (white arrowhead). B: Strong expression of 3-OST-3X is seen in the AER (black arrowhead). C: The 3-OST-3Z is strongly expressed in both the muscle and AER. D: The 3-OST-7 is expressed weakly in the fin muscle. examined the role of 3-O sulfation in CHO cells, which lack endogenous 3-O sulfation (Shukla et al., 1999; Yabe et al., 2001; Xia et al., 2002; Tiwari et al., 2004, 2005; Xu et al., 2005). The analysis presented here suggests each cell or tissue regulates 3-OST gene expression spatially and temporally, therefore the in vivo function of each 3-OST may be different than what has previously been characterized in vitro. Family members having ubiquitous expression may represent a general 3-O sulfation pathway, whereas family members with specific expression may represent specific developmental Fig. 5.

8 3430 CADWALLADER AND YOST or cell-signaling processes for which a specific 3-O sulfated fine structure is required. Comparisons of the expression patterns of 3-OST family subgroups suggest each subgroup contains members with general and specific expression. Future studies focusing on the in vivo differences between the subgroups in the biological context of a developing organism such as the zebrafish will be necessary to understand the interplay among 3-OST gene family members and subgroups. EXPERIMENTAL PROCEDURES Cloning of Zebrafish 3-OST Genes We retrieved eight sequences showing similarity to the human 3-OST gene family catalytic domains from the ensemble Danio rerio database ( as well as the NCBI zebrafish database ( ncbi.nlm.nih.gov/genome/seq/drblast. html). Primers were designed to amplify the eight sequences to confirm identity. Where necessary, 5 RACE and 3 RACE were performed to identify complete ORFs (First Choice RLM-RACE, Ambion), according to the manufacturer s instructions. Subsequently, the full-length ORF was amplified and cloned into pcr4-blunt TOPO (Invitrogen). At least 28 primer sets were used and the sequences are available upon request. The sequences are available under the following Gen- Bank accession numbers: 3-OST-1 (DQ812985), 3-OST-2 (DQ812986), 3-OST-3X (DQ812987), 3-OST-3Z (DQ812988), 3-OST-4 (DQ812989), 3-OST-5 (DQ812990), 3-OST-6 (DQ812991), and 3-OST-7 (DQ812992). WHOLE-MOUNT IN SITU HYBRIDIZATION Zebrafish embryos were fixed in sucrose buffered 4% paraformaldehyde, rinsed in phosphate-buffered saline, dehydrated in methanol, and stored at 20 C. Riboprobes were synthesized from the full-length ORF cloned into pbluescript (Stratagene). The linearized DNA templates were transcribed using either T3 or T7 polymerases and digoxigenin-labeling mixes (Roche). In situ hybridizations were carried out as previously described (Essner et al., 2000) using a Biolane HTI in situ machine (Huller and Huttner AG). Embryos were cleared in 70% glycerol in PBST and photographed using either a Leica MZ12 or a Nikon SMZ1000 dissecting microscope. Digital images were processed using Adobe Photoshop and ACD Systems Canvas. ACKNOWLEDGMENTS We thank J. Amack and J. Neugebauer for critical reading and B. Bisgrove and R. Dorsky for discussions. We also thank B. Demarest and N. Trede for technical assistance. NOTE ADDED IN PROOF In a companion paper, we have characterized the expression of the heparan sulfate 6-O-sulfotransferase family in zebrafish (Cadwallader and Yost, 2006). REFERENCES Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J, Zako M Functions of cell surface heparan sulfate proteoglycans. 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