Combinatorial expression patterns of heparan sulfate ...

DEVELOPMENTAL DYNAMICS 236:581–586, 2007

PATTERNS & PHENOTYPES

Combinatorial Expression Patterns of Heparan Sulfate Sulfotransferases in Zebrafish: III. 2-OSulfotransferase and C5-Epimerases 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 give HS specific binding affinities for extracellular ligands. HS 2-Osulfotransferase (2-OST) catalyzes the transfer of sulfate groups to the 2-O position of uronic acid residues of HS. We report here the characterization and developmental expression patterns of 2-OST in several tissues/organs throughout early zebrafish development, including early cleavage stages, eyes, somites, brain, internal organ primordial, and pectoral fin. The 2-OST gene has spatially and temporally distinct expression, which is a surprise given the essential role of 2-OST in HS fine structure formation. Furthermore, although 2-OST and C5-epimerase are predicted to be interdependent for protein translocation from the endoplasmic reticulum to the Golgi, their expression is not coordinately regulated during zebrafish development. Developmental Dynamics 236:581–586, 2007. © 2006 Wiley-Liss, Inc. Key words: zebrafish; heparan sulfate; 2-O-sulfotransferase; proteoglycan; 2-OST; C5-epimerase; Glce; GlceA; GlceB Accepted 20 November 2006

INTRODUCTION Heparan sulfate proteoglycans (HSPGs) are present on the cell surface or secreted extracellularly where they play a variety of roles in 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 sugar chains of alternating glucuronic acid and N-acetyl glucosamine resides are attached. The sugar chains undergo a series of extensive modifications beginning with N-deacetylation and Nsulfation of a subset of the glu-

cosamine residues. Subsequently, a subset of glucuronic acid residues is converted to iduronic acid by heparan sulfate (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 confers the distinct, specific binding properties of HS (Nakato and Kimata, 2002). The HS 2-O-sulfotransferase (2-OST) catalyzes the addition of sulfates to urinoic acid residues. Most invertebrates

and vertebrates contain one 2-OST gene (Kobayashi et al., 1996, 1997; Bullock et al., 1998; Turnbull et al., 2003; Nogami et al., 2004). Drosophila contains two genes, which show high homology to other 2-OSTs, HS2ST, and pipe (Sergeev et al., 2001). Although pipe resembles an HS 2-OST, recent evidence suggests Pipe functions as a sulfotransferase for a substrate other than HS (Zhu et al., 2005). The importance of the 2-O-sulfation in fine structure formation has been examined in several species. RNAi of the Caenorhabditis elegans hst-2 gene, the C. elegans 2-OST orthologue, resulted in multiple phenotypic effects, including aberrant morphology and defects in egg laying (Turnbull et

Huntsman Cancer Institute, Center for Children, Department of Oncological Sciences, University of Utah, Salt Lake City, Utah *Correspondence to: H. Joseph Yost, Huntsman Cancer Institute, Center for Children, University of Utah, 2000 Circle of Hope #4280, Salt Lake City, UT 84112. E-mail: [email protected] DOI 10.1002/dvdy.21051 Published online 28 December 2006 in Wiley InterScience (www.interscience.wiley.com).

© 2006 Wiley-Liss, Inc.

582 CADWALLADER AND YOST

al., 2003). Several groups have begun to examine neuronal defects in hst-2 mutant worms (Bulow and Hobert, 2004; Kinnunen et al., 2005). Recently, a 2-OST in chicken was identified and found to be ubiquitously expressed throughout the chicken limb bud (Nogami et al., 2004). The 2-OST gene has been extensively studied in the mouse, for which a gene-trap knockout mouse has been created. HS 2-OST⫺/⫺ mice die neonatally from bilateral renal agenesis (Bullock et al., 1998). The knockout mice also have eye and skeleton defects, as well as less penetrant cleft palate and polydactyly (Merry and Wilson, 2002; Princivalle and de Agostini, 2002; Wilson et al., 2002; Turnbull et al., 2003). HS from HS 2-OST⫺/⫺ mice has a novel composition and structure but lacks detectable 2-O sulfation, suggesting there is only one 2-OST in the mouse (Merry et al., 2001). The single 2-OST gene in nearly all species suggests the 2-OST plays a major role in fine structure formation of HS. Here, we report the molecular identification and categorization of the zebrafish HS 2-O-sulfotransferse and examine the expression in early development by whole-mount in situ RNA hybridization. The unique expression of the 2-OST gene provides a foundation for future studies into how the HS 2-OST is regulated during early development. We also examine, for the first time in any organism, the colocalization of the HS 2-OST and C5-epimerase genes.

Cloning of the Zebrafish 2-OST

Fig. 1. Analysis of the heparan sulfate 2-O-sulfotransferase (2-OST) gene from multiple species. A: Amino acid alignment of 2-OST genes from several vertebrate species. B: Phylogenetic analysis of 2-OST genes from several species were conducted using MEGA version 3.1 (Kumar et al., 2004). The scale indicates the number of amino acid substitutions per site adjusted by the equal input model.

Using the human and mouse 2-OST genes as templates, we initially identified several exons showing high homology in the zebrafish by performing a BLAST search of early versions of the publicly available zebrafish databases. Further refinement of the sequences, using polymerase chain reaction (PCR), 5⬘ and 3⬘ rapid amplification of cDNA ends (RACE), and sequencing indicated that zebrafish contain one 2-OST gene. Analysis of the 2-OST gene locus in the Ensembl Vega Danio rerio genome assembly version v21 (http://www.

vega.sanger.ac.uk) suggests a syntenic relationship has been conserved between human, mouse, and zebrafish. The 2-OST gene is located between the genes gtf2b and sep15 on human chromosome 1 and mouse chromosome 3. The zebrafish 2-OST gene, located in an unannotated region of chromosome 2, maps between annotated sequences for gtf2b and sep15. Structurally, the HS 2-OST locus in human and mouse consists of seven exons, with the open reading frame

RESULTS AND DISCUSSION

(ORF) encompassing the 3⬘ end of exon 1 through the 5⬘ end of exon 7. Zebrafish was found to have a similar seven-exon structure and identical placement of the ORF. The ORF of the zebrafish 2-OST gene shows 85% identity to human and 84% identity to both mouse and Xenopus 2-OST genes (Fig. 1A). Phylogenetic analysis of the 2-OST family comparing zebrafish with other species indicates that the zebrafish 2-OST gene shows high conservation (Fig. 1B).

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Expression of Zebrafish 2-OST To analyze the expression of the HS 2-OST, we performed mRNA in situ hybridization with antisense riboprobes specific to the HS 2-OST gene. In situ hybridization of embryos from early cleavage stages through 48 hr postfertilization (hpf) was performed, and the results of these analyses are summarized in Table 1, which lists representative examples of differential expression. The 2-OST was expressed maternally (Fig. 2A) and during early cleavage stages. 2-OST was expressed ubiquitously during gastrulation (Fig. 2B). During early somitogenesis, expression became restricted to the somites and presomitic mesoderm (Fig. 2C). This expression continued through mid-somitigenesis (Fig. 2D,E), when expression was also seen weakly in the eye. During late somitogenesis (Fig. 2F), expression was seen in eye, weakly in hindbrain and in somites, with the strongest expression in caudal somites. At 24 hpf, expression was seen weakly throughout the brain (Fig. 3A) and in the eye and somites (Fig. 2G). Expression at 36 hpf becomes widespread throughout the embryo, with strongest expression in the brain, otic vesicle (Fig. 3B), and pectoral fin (Fig. 2H). At 48 hpf, expression remains widespread throughout the embryo (Fig. 2O), with the strongest staining seen in the head and pectoral fin (Fig. 3C).

Comparison of HS 2-OST Expression From Various Organisms HS 2-OST has an essential role in HS fine structure formation, yet it is surprising that 2-OST had restricted expression patterns during early zebrafish development. While it is possible that an undiscovered 2-OST has complementary expression, a search of the latest zebrafish genome (build Zv6) has not uncovered a second 2-OST gene. This finding suggests some cells in the developing embryo use high levels of 2-OST, while others have a minimal or no requirement for 2-O-sulfation. In C. elegans, expression of hst-2 begins early in development, specifically

TABLE 1. Expression of Zebrafish 2-O-Sulfotransferase and C5-Epimerases During Early Developmenta

Maternal 60% epiboly Adaxial mesoderm Polster Somites Notochord Floorplate Hypochord Tail Bud Kupffer’s vesicle Eye Lateral Line Primordia CNS Telencephalon Diencephalon Midbrain Hindbrain Spinal Cord Olfactory system Heart Gut Proctodeum Pancreas Liver Pronephric duct/kidney Branchial Arches Otic Vesicle Pectoral Fin Muscle Pectoral Fin AER

2-OST

GlceA

GlceB

⫹ ⫹ (w) ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺

⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺

⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺

⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺

⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺

(l) (l) (l) (l)

(w)

(u)

(u) (u) (u) (u)

(u) (a) (u) (u) (u) (u)

⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺

a

a, anterior; l, ubiquitous late expression; u, ubiquitous; w, weak.

in the pharynx. During larval development, expression is seen in the pharynx, muscle, and a subset of neurons (Kinnunen et al., 2005). HS2ST expression in the chick is seen throughout the embryo at stages 17– 24, but expression is strongest in the pharyngeal arches and limb buds (Nogami et al., 2004). In the mouse, expression of Hs2st is widespread at 8.5 days postcoitum, with elevated expression in the hindbrain. Expression between 10.5 and 12.5 days postcoitum was most intense in the brain, particularly the midbrain– hindbrain boundary, sclerotome (somites), and mesenchyme of the limb (Bullock et al., 1998). Although it is challenging to correlate specific developmental stages among multiple species, expression of zebrafish Hs2st was expressed in developing structures in which expression was seen in other organisms (Table 1). Highest Hs2st expression in the

zebrafish was seen in the brain (Fig. 3A–C), somites (Fig. 2F–G), branchial arches (Fig. 2H), and pectoral fin (Fig. 3C). Similar to our previous analyses of the HS 3-OST (Cadwallader and Yost, 2006a) and 6-OST families (Cadwallader and Yost, 2006b), our analysis of zebrafish 2-OST expression in early embryos indicates that Hs2st is not expressed in every cell. This finding suggests some cells in the developing embryo use high levels of 2-OST, while others have a negligible requirement for 2-O-sulfation. The restricted expression pattern in early zebrafish development suggests that the relatively late neonatal death of the HS 2st⫺/⫺ mouse, with limited developmental defects (Bullock et al., 1998), reflects the possibility that 2-OST is not required in all cells during early development.


Comparison of Zebrafish 2-OST and C5-Epimerase Expression Recently, Pinhal et al. (2001) described a protein–protein interaction between C5-epimerase and HS 2-OST in cultured cells, suggesting that interaction between the two enzymes is required for translocation of both enzymes from the endoplasmic reticulum to the Golgi. If this protein interaction is obligatory for function, one would predict that 2-OST expression and C5-epimerase expression should be coordinated. Given our finding that 2-OST has dynamic expression patterns during development, we performed mRNA in situ hybridization with two recently characterized C5epimerases in zebrafish (Ghiselli and Farber, 2005; Table 1). Previous work reported that the two C5-epimerase genes were ubiquitously expressed during early zebrafish development (Ghiselli and

Fig. 2. Expression of heparan sulfate (HS) 2-Osulfotransferase (2-OST) and Glce-A and -B are not always coordinated during early zebrafish development. A–E: Early expression of HS 2-OST in two cell (A), 60% epiboly (B), 2 somite (C), 7 somite (D), and 13 somite (E) stages. F–N: Comparison of HS 2-OST (18 somites, F; 24 hr postfertilization [hpf], G; and 36 hpf, H), C5-epimerase-A (18 somites, I; 24 hpf, J; and 36 hpf, K), and C5-epimerase-B (18 somites, L; 24 hpf, M; and 36 hpf, N). Green arrows indicate areas where expression is stronger or unique to the C5-epimerase genes, black arrows represent areas where expression is similar between 2-OST and epimerase, and red arrows indicate areas where 2-OST is stronger. O: Expression of HS 2-OST at 48 hpf. BA, branchial arches; E, eye; EL, eye lens; FB, forebrain; HB, hindbrain; MB, midbrain; OT, otic vesicle; PF, pectoral fin; S, somites; TB, tail bud. Fig. 3. Heparan sulfate (HS) 2-O-sulfotransferase (2-OST) and C5-epimerase expression in brain and pectoral fin. A–C: Expression of HS 2-OST in a dorsal view of a 24 hr postfertilization (hpf) brain (A), a dorsal view of a 36 hpf brain (B), and a dorsal view of 48 hpf brain and pectoral fin (C). D–F: Expression of GlceA in a dorsal view of a 24 hpf brain (D), a dorsal view of a 36 hpf brain (E), and a dorsal view of a 48 hpf brain and pectoral fin (F). G–I: Expression of GlceB in a dorsal view of a 24 hpf brain (G), a dorsal view of a 36 hpf brain (H), and a dorsal view of 48 hpf brain and pectoral fin (I). The white arrowhead indicates expression in the pectoral fin; black arrowheads indicate lack of pectoral fin expression. E, eye; EL, eye lens; FB, forebrain; HB, hindbrain; MB, midbrain; MHB, midbrain– hindbrain boundary; OT, otic vesicle.

Fig. 2.

Fig. 3.

2-O-SULFOTRANSFERASE AND C5-EPIMERASES 585

Farber, 2005). Our results confirm that the C5-epimerase genes are expressed throughout the embryo during early development. Here, we show that, at later stages, expression begins to differ between the two epimerase genes. GlceA expression is confined to the brain from late somitigenesis through 48 hpf (Figs. 2I–K, 3D–F). GlceB expression is seen specifically in eye lens, midbrain, and hindbrain from 18 somites through 48 hpf (Figs. 2L–N, 3G–I) and in the somites at 24 hpf (Fig. 2M). The differences between GlceA and GlceB are best highlighted by the expression of GlceB in the eye lens (Fig. 2L,M, 3G,H), and GlceA expression in the forebrain at 24 hpf (Fig. 2J). Table 1 provides a comparison of the structures in which GlceA and GlceB are expressed. Comparison of 2-OST and Glce expression patterns reveals several tissues in which expression of the three genes is not coordinated (Table 1). In contrast, several tissues, for example, somites at 24 hpf (Fig. 2G,M), have high expression of both 2-OST and epimerase. Some tissues have high 2-OST expression and negligible Glce expression, for example, early somites (Fig. 2F,I,L) and pectoral fin (Figs. 2H,K,N, 3C,F,I). Conversely, other tissues have negligible 2-OST expression and high Glce expression, such as the eye lens (Fig. 3A,B,D,E,G,H). This finding indicates that 2-OST and C5epimerase expression patterns are not coordinately regulated during development, and some cell types might not use high levels of C5-epimerization or 2-O-sulfation for construction of HS fine structures unique to that cell type.

EXPERIMENTAL PROCEDURES Cloning of Zebrafish 2-OST Gene We identified and designed primers for one sequence in both the ensembl Danio rerio database (http://www.ensembl.org) and the NCBI zebrafish database (http://www.ncbi.nlm.nih.gov/ genome/seq/DrBlast.html) that had similarity to the human and mouse 2-OST genes. Where necessary, 5⬘ RACE and 3⬘ RACE were performed to identify complete ORFs according

to the manufacturer’s instructions (First Choice RLM-RACE, Ambion). Subsequently, the full-length ORF was amplified and cloned into pCR4Blunt TOPO (Invitrogen). Primer sets and sequences used are available upon request. The 2-OST sequence is available under GenBank accession numbers DQ812997.

Whole-Mount In Situ Hybridization Zebrafish embryos were fixed in sucrose buffered 4% paraformaldehyde, rinsed in phosphate-buffered saline (PBS), dehydrated in methanol, and stored at ⫺20°C. Riboprobes were synthesized from either the full-length ORF into pBlueScript or pCRscript (Stratagene). Two separate probe constructs were used for each HS2-OST and C5-epimerase genes. The linearized DNA templates were transcribed using 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 (Ho¨lle 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.

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