Zebrafish glce-A and glce-B genes both encode proteins of 585 amino acids. The gene products are homologous to the human protein sequence (67% and 73% respectively) (fig. 1a). Compared to the human, mouse and bovine sequences, the zebrafish proteins lack part of the N-terminus. The C-terminal domain is the most conserved region of Glce. Analysis of the hydrophobicity index determined utilizing the SOSUI 23 and the TMPRED 24 algorithms, reveals the presence in both zebrafish proteins of a conserved hydrophobic domain of ~20 amino acids located between residue 10 and 30 at the N-terminus of the proteins (fig. 1b). As the mammalian enzyme, zebrafish epimerase is a "type-two" transmembrane protein with predicted localization in the Golgi apparatus 4. The glce-A and the glce-B locus mapped to linkage group LN25 between markers Z24369 and Z20832 and to linkage group 7 between markers Z21519 and Z9521, respectively (fig. 1c). Based on the mapping of neighbor genes, both chromosomal regions are synthenic with human chromosome 15q22, i.e. the region harboring the epimerase gene.

glce transcript level was examined at different developmental stages and compared to that of ext2-A, an HS polymerase acting upstream the epimerization step 3. The expression of shh which is activated during gastrulation, and that of ef-1 which is expressed at similar level throughout development, were also monitored. glce-A, glce-B and ext2-A transcripts were present in fertilized embryos at developmental stages prior to the onset of zygotic transcription, indicating that these messages are maternally derived. The expression of HS biosynthesis enzymes reached a peak at the onset of gastrulation following midblastula transition (fig. 2a). Assay of epimerase activity in embryonic extracts at different developmental stages, was consistent with the level of mRNA encoding these proteins. In particular epimerase activity at 10 hpf was twice that observed at the 64-cells stage (fig. 2b).

glce transcripts were localized in embryos at different developmental stages by in-situ hybridization using gene-specific antisense riboprobes (fig. 2c–l). During the early stages a diffuse staining was observed throughout the blastoderm. At the beginning of segmentation, staining was detected along the entire dorsal axis (fig. 2i,j). At 24 hpf, however, glce expression was higher in the newly forming brain (fig. 2k). At this site epimerase transcripts were mostly detected at the perimeter of the forth ventricle (fig. 2l). The staining specificitity was confirmed through the use of sense control riboprobes in which case only background staining was seen. No significant differences were detected in the expression pattern of the two glce genes.

To investigate the functional significance of the epimerase during zebrafish development the protein was ectopically expressed by injecting embryos (1–2 cell stage) with capped in vitro-transcribed mRNA. The majority of injected embryos displayed a ventralizing phenotype whose severity correlated with the dose of mRNA injected (250 to 1000 pg) (table 1). The affected embryos had smaller head size, expanded blood islands, and abnormal tail somites (fig. 3b,c,f). More strongly ventralized embryos also lacked a notochord and developed somites that were not chevron-shaped and were fused in the midline below the neural tube (fig. 3g). Overexpression of glce-B produced an identical spectrum of phenotypes as the overexpression of glce-A. However, the highest frequency of severely affected embryos was observed when 250 or 500 pg of glce-A and glce-B mRNA were administered together in which case most of the animals failed to form an anterior axis (fig. 3d). In this treatment group, epimerase enzymatic activity at 10 hpf was 73% higher the level detected in uninjected embryos (fig 3h).

In order to better characterize the phenotype of embryos overexpressing Glce, the expression of dorsal and ventral markers were analyzed by in situ hybridization 2526. The expression domain of eve1, a marker of ventral and lateral regions at early gastrula stages, was expanded at the shield stage (fig. 3i,j). Likewise the range of expression of bmp2b was greatly enlarged in embryos at the 70% epiboly stage (fig. 3k,l). In contrast expression of fkd3, a marker of the presumptive neuroectoderm, was reduced by Glce overexpression (fig. 3m,n). Similarly the expression domain of chordin, a marker of the dorsal organizer, was reduced (fig. 3o,p). The fact that overexpression of the epimerase alters the pattern but does not prevent the expression of dorsal determinants, is consistent with the idea that Glce acts on D/V axis formation downstream the Wnt/β-catenin pathway that regulates chordin gene expression 27. glce is also a target of the Wnt/β-catenin transactivation pathway 28 raising the possibility that zygotic glce expression is coordinated with that of other D/V determinants.

Because head size is affected following ectopic expression of glce (fig 3b–d) the expression pattern of the neuroectoderm-specific markers krox 20 29 and opl (zic1) 30 was determined during somitogenesis. The expression of myoD, a transcript specifically localized to somitic mesoderm was also examined 31. During somitogenesis, krox-20 is normally expressed in hindbrain rhombomeres R3 and R5 both of which are dorsal ecdoderm derivatives. A reduced area of krox-20 expression was detected at the 5 somite stage in most of the embryos injected with 250 pg each of glce-A and glce-B mRNA whereas opl expression was undetectable (fig. 3q,r). myoD expression in the cells adjacent to the axial mesoderm and in the somitic furrows was also reduced implying a role of Glce in establishing mesodermal cell fate (fig 3s,t).

Since both the phenotype and marker gene expression pattern following ectopic expression of Glce is reminiscent of that of chordino 30323334, ogon 333536 and radar 3738 mutants or of embryos misexpressing alk 8 39 in which the ventralizing activity of Bmps is enhanced, we examined whether Bmp2b activity is potentiated by Glce. For this purpose we titrated the dose of injected bmp2b mRNA to achieve a preponderance of partially ventralized embryos displaying V2 and V3 phenotype severity 4041 (table 1). This same dose (20 pg) was then administered together with glce-A and/or glce-B mRNA (250 pg). Following the treatment, the majority of embryos exhibited the most severe V4 class phenotype consistent with Glce activity potentiating the effect of Bmp2b.

The effect of Glce protein knockdown on D/V axis formation was next examined by administering antisense morpholino oligonucleotides (MO) targeting glce-A and glce-B transcripts. Most of the embryos after injection of 4 ng of antisense MO, displayed a mild dorsalized phenotype with reduced ventral tail fin (fig. 4b and table 2). At higher dose (8 ng) about half of the morphants showed kinked tail, enlarged heart cavity and in some animals the atrioventricular boundaries failed to form (fig. 4c). A dramatic shortening and reduction of the body mass with tail coiling similar to the phenotype associated with mutation of Bmp2b, and Bmp7 3441 was observed in the majority of the embryos receiving the highest dose (16 ng) of MO (fig. 4d). In this group of morphants the epimerase enzymatic activity was significantly decreased (34% of the control at 8 hpf) (fig. 4e). The inability of the MOs to completely abolish the Glce activity suggests that at this time residual maternally derived enzyme is still active. In spite of this, the effect of Glce knockdown on ventral cell fate was already detectable in the mild morphants as revealed by the faint staining of gata1 expressing blood islets (fig. 4g), a ventral tissue derivative 42. In stronger morphants, a broadening of the chordin expression domain wasobserved (fig. 4i–l). In addition, consistent with the axis dorsalization, the expression of bmp2b at shield stage was severely reduced as also evidenced by its undetectable expression in the tail during somitogenesis (fig. 4m–p). The administration of human or zebrafish glce mRNAs to embryos injected with MO rescued the enzymatic activity and prevented the development of the most severe dorsalized phenotypes (table 2).

In order to assess the dependency of Bmp activity on Glce level, embryos were injected with either 50 pg of bmp2b mRNA or 100 pg of bmp4 to generate a preponderance of V3-V4 ventralized embryos (table 3). Following randomization, half of the injected embryos received a mix of MOs targeting both glce-A and glce-B transcripts. About two-third of the embryos receiving the MOs displayed a normal-to-mild (V1-V2) ventralized phenotype whereas few developed the most severe V4 class phenotype. These results are in stark contrast to the embryos that had only received bmp2b and bmp4 mRNA supporting the concept that Glce is required for Bmp-mediated ventralization.

Embryos were obtained from natural mating of wild-type (Oregon, AB) fish and breed, raised and staged according to established criterias 70.

A query of the zebrafish dEST database identified a number of putative clones whose translated sequence matched the N- and C-terminus of the human protein. Analysis of the predicted protein sequence of these clones indicated that zebrafish have two highly homologous Glce proteins. Cloning of the putative glce cDNAs was performed by reverse transcription of adult male zebrafish mRNA (Qiagen Sensiscript) primed with oligo-dT. cDNAs were amplified by PCR by combining primers matching the different possible cDNA terminal sequences. Forward primers were 5'-ATGCGCTGTCTGGTGGCTCGAATCAATC ACAAGACT-3' and 5'-ATGCGTTGTCTGGCAGCCGGTGTTCACTACAAGACC-3'. Reverse primers were 5'-CTAGTTGTGCTTAGCCCGACCTCCTTTCAGGTAAGT-3' and 5'-TTAATTGTGCTTAGCCCTCCCTCCTTTCAGGTAGCT-3'. This strategy resulted in two products of the expected size (~1.8 kb) from two of the four possible primer combinations. The amplified products were named glce-A (GenBank AY388516) and glce-B (AY388517), cloned into pcDNA3.1-TOPO (Invitrogen) and sequenced.

The 5'-end translated region of the zebrafish homologue of human exostosin 2 (EXT2) gene, was cloned using a similar strategy. The existence of two zebrafish ext2 genes were predicted from alignments of published EST sequences. The 5'-end of the ext2-A coding sequence including part of the UT region was cloned by RT-PCR using forward and reverse primers of sequence 5'-CATTCAACTTAAATATTCACCATA-3' and 5'-GGCGCTCAGCAGGTCATTGTATTC-3' respectively. Sequencing of an expected 528 bp fragment confirmed the identity of the amplified cDNA.

In the human, the glce translational start site is located in a 602 bp exon. Assuming that zebrafish glce genes maintain the same genomic organization as the human, PCR primers were designed to amplify the exon containing the translation initiation site for each each zebrafish orthologue. PCR amplification with primers 5'-ATGCGCTGTCTGGTGGCTCGAATC-3' and 5'-AGATGAAGGGCAGATACACCTCGC-3' for glce-A and 5'-ATGCGTTGTCTGGCAGCCGGTGTTCACTACAAG-3' and 5'-GACCTTTAATGGTGGCATCGTCATTGATCAGGC-3' for glce-B using genomic male zebrafish DNA as template, gave products of the expected size (420 bp and 261 bp for glce-A and glce-B respectively) that were cloned in pGEM-T vector and sequenced to confirm their identity. These sets of primers were then used to determine the chromosomal location of each gene by radiation hybrid panel (LN54) mapping.

Antisense Morpholino oligonucleotides (MOs) (Gene Tools LLC) were designed to target the 5'-UT region of the genes of interest. glce-A and glce-B MOs had sequence 5'-AGCCATGAGGAACACGTCAGCAAAC-3' and 5'-TCCCTGCTTACCTGCAATGCAAACA-3', respectively. MOs were dissolved in water at a concentration of 4 mM and diluted in Danieu's buffer before injection.

Full-length glce cDNAs were subcloned in pT3TS vector at the BglII and EcoRV sites and in vitro-transcribed capped mRNAs were synthesized (T3 mMESSAGE mMACHINE kit, Ambion). mRNAs (1 μg) were tested prior to injection for protein expression in vitro using a rabbit reticulocyte lysate assay kit and ³⁵S-methionine. Labeled proteins were separated on 9% SDS-PAGE gel followed by autoradiography. Human glce mRNA was generated from a full length cDNA clone in pcDNA 3.1 vector using T7 RNA polymerase. The human clone (AY635582) was obtained by RT-PCR using primers of sequence 5'-CTGCATATGCTGTGCTTGGCA-3' and 5'-CTAGTTGTGCTTTGCCCTGCTGCCTTT-3' based on published coding sequences and on cDNA 5'-end extension experiments we have performed 28. bmp2b and bmp4 capped mRNA were kindly provided by Dr M. Mullins (U.Penn).

MOs and mRNAs were injected (1–3 nl) into the yolk of 1–2 cell embryos 71. Post-injection (6 h) embryos were sorted, the unfertile/damaged removed and the rest allowed to grow at 28°C for further observation.

Antisense digoxigenin-labeled riboprobes were generated using SP6 or T7 RNA polymerase-based labeling kit (Roche). cDNA clones for glce-a, glce-b, chordino, krox-20, opl, myoD, were generated by RT-PCR [see Additional file 1]. bmp2b, fkd3, and eve1 antisense riboprobes were generated by reverse transcription from cDNA clones (cb670, cb114, and cb872 respectively) obtained from the Zebrafish International Resource Center (University of Oregon). Whole embryo in situ hybridization was performed as previously described 72.

RNA was extracted and cDNA generated by reverse transcriptase using oligo-dT primers. An aliquot of the reaction was used as template for PCR amplification using gene-specific primers (Appendix I). Reactions were performed in duplicate and the product generated after 20 and 30 cycles analyzed by agarose electrophoresis to ensure that the products were quantitated during the exponential phase of the chain reaction.

Embryos were collected at the indicated times, dechorionated and washed in ice-cold 25 mM HEPES, pH 7.0 buffer containing 0.1% Triton X-100. Embryos were then homogenized in 200 μl of the same buffer with a pestle that fits tightly into an Eppendorf tube and stored at -70°C. The substrate for the epimerase enzymatic assay consisted of radiolabeled modified bacterial N-acetylheparosan prepared as described previously 28. The final N-sulfated heparosan product was purified by ion-exchange chromatography and eluted at higher ionic strength than the starting bacterial polysaccharide (0.66 M vs. 0.40 M NaCl). The epimerase enzymatic assay was performed as described by Crawford et al. 4. Briefly, reactions were set up by combining the homogenates from 20 embryos with labeled substrate (1 nmole ~30,000 dpm) followed by 2 h incubation at 28°C. Reactions were stopped by addition of DEAE-Sepharose (1 ml) equilibrated in 50 mM Na-acetate, 50 mM NaCl pH 4.0 buffer (1:1 volume) followed by 15 min incubation at 4°C. Tritiated water generated as a result of the epimerization of GlcA into IdoA was recovered in the supernatant following centrifugation at 10,000 rpm. The sample and a 800-μl rinse of the DEAE slurry with buffer, were counted for the associated radioactivity in a Wallac 1219 Rackbeta liquid scintillation counter. Values were corrected for a reaction blank obtained by adding the substrate to the embryo homogenate just prior to the addition of DEAE-Sepharose.