Combinatorial expression patterns of heparan sulfate sulfotransferases in zebrafish: II. The 6-O-sulfotransferase family.

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 6-O-sulfotransferases (6-OST) catalyze the transfer of sulfate groups to the 6-O position of glucosamine residues of HS. We report here the characterization and developmental expression analysis of the 6-OST gene family in the zebrafish. The zebrafish 6-OST gene family consists of four conserved vertebrate orthologues, including a gene duplication specific to zebrafish. We examined the mRNA expression patterns in several tissues/organs throughout early zebrafish development, including early cleavage stages, eyes, somites, brain, internal organ primordial, and pectoral fin development. Members of the 6-OST gene family have spatially and temporally distinct restricted expression, suggesting in vivo functional differences exist between members of this family.     

Combinatorial expression patterns of heparan sulfate sulfotransferases in zebrafish: III. 2-O-sulfotransferase and C5-epimerases.

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-O-sulfotransferase (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.     

Heparin sulphate d-glucosaminyl 3-O-sulfotransferase 3B1 plays a role in HBV replication

Hepatitis B virus infection is a worldwide epidemic and is closely associated with the development of hepatocellular carcinoma. Nevertheless, the molecular mechanisms of HBV infection and carcinogenesis remain elusive. Using a hepatocyte model of HBV infection and comparing the gene expression profiling analysis we found that heparan sulfate d-glucosaminyl 3-O-sulfotransferase 3 B1 (HS3ST3B1,3-OST3-B) is down-regulated in the hepatocytes of chronic HBV infection model. HS3ST3B1 showed potent inhibitory effect on HBV replication. The inhibitory effect of HS3ST3B1 overexpression was lost upon gene silencing of HS3ST3B1 or when a catalytic inactive mutant of HS3ST3B1 was expressed. Our study revealed the anti-viral activity of HS3ST3B1 on HBV replication. It is conceivable that possible therapeutic applications of HBV infection could be devised by manipulating HS3ST3B1 activity.     

Expression of sox11 gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development.

To investigate the role of sox genes in vertebrate development, we have isolated sox11 from zebrafish (Danio rerio). Two distinct classes of sox11-related cDNAs were identified, sox11a and sox11b. The predicted protein sequences shared 75% identity. In a gene phylogeny, both sox11a and sox11b cluster with human, mouse, chick, and Xenopus Sox11, indicating that zebrafish, like Xenopus, has two orthologues of tetrapod Sox11. The work reported here investigates the evolutionary origin of these two gene duplicates and the consequences of their duplication for development. The sox11a and sox11b genes map to linkage groups 17 and 20, respectively, together with other loci whose orthologues are syntenic with human SOX11, suggesting that during the fish lineage, a large chromosome region sharing conserved syntenies with mammals has become duplicated. Studies in mouse and chick have shown that Sox11 is expressed in the central nervous system during development. Expression patterns of zebrafish sox11a and sox11b confirm that they are expressed in the developing nervous system, including the forebrain, midbrain, hindbrain, eyes, and ears from an early stage. Other sites of expression include the fin buds and somites. The two sox genes, sox11a and sox11b, are expressed in both overlapping and distinct sites. Their expression patterns suggest that sox11a and sox11b may share the developmental domains of the single Sox11 gene present in mouse and chick. For example, zebrafish sox11a is expressed in the anterior somites, and zebrafish sox11b is expressed in the posterior somites, but the single Sox11 gene of mouse is expressed in all the somites. Thus, the zebrafish duplicate genes appear to have reciprocally lost expression domains present in the sox11 gene of the last common ancestor of tetrapods and zebrafish. This splitting of the roles of Sox11 between two paralogues suggests that regulatory elements governing the expression of the sox11 gene in the common ancestor of zebrafish and tetrapods may have been reciprocally mutated in the zebrafish gene duplicates. This is consistent with duplicate gene evolution via a duplication-degeneration-complementation process.     

Human gamma- to beta-globin gene switching in transgenic mice.

Previous studies demonstrated correct tissue- and temporal-specific expression of human gamma- and beta-globin genes in transgenic mice; however, expression was extremely low. When the erythroid-specific DNase I super-hypersensitive (HS) sites that are normally located upstream of the human beta-globin locus were fused individually to gamma- or beta-globin genes, expression increased to endogenous mouse globin levels but temporal specificity was lost. In contrast, when the HS sequences were combined with fragments containing both gamma- and beta-globin genes, correct developmental regulation was restored. We suggest that human gamma- to beta-globin gene switching during development results from competition of individual globin gene family members for interaction with the HS sequences and that factors influencing these competitive interactions determine temporal specificity.     

Expression analysis of chick Wnt and frizzled genes and selected inhibitors in early chick patterning.

Wnt signaling is an important component in patterning the early embryo and specifically the neural plate. Studies in Xenopus, mouse, and zebrafish have shown that signaling by members of the Wnt family of secreted signaling factors, their Frizzled receptors and several inhibitors (sFRP1, sFRP2, sFRP3/Frzb1, Crescent/Frzb2, Dkk1, and Cerberus) are involved. However, very little is known about the expression of genes in the Wnt signaling pathway during early anterior neural patterning in chick. We have performed an expression analysis at neural plate stages of several Wnts, Frizzled genes, and Wnt signaling pathway inhibitors using in situ hybridization. The gene expression patterns of these markers are extremely dynamic. We have identified two candidate molecules for anterior patterning of the neural plate, Wnt1 and Wnt8b, which are expressed in the rostral ectoderm at these stages. Further functional studies on the roles of these markers are underway.     

Skin-specific expression of ictacalcin, a homolog of the S100 genes, during zebrafish embryogenesis.

Full-length cDNA coding for the ictacalcin gene, a homolog of the S100 genes, was isolated in zebrafish and mapped on linkage group 16 using the LN54 radiation hybrid panel. The homology and phylogenetic analyses, based on the deduced amino acid sequences, showed the orthologous relationship of ictacalcin genes between zebrafish and other fish species. However, ictacalcin genes constitute an out-group with respect to other members of the S100 gene family. This result supports the findings that fish ictacalcin genes are new members of the S100 gene family and may have evolved after the divergence of teleosts and tetrapods. The zebrafish ictacalcin gene was zygotically transcribed from 12 hours postfertilization onward and was stably expressed throughout adulthood. During zebrafish embryogenesis, the ictacalcin gene was specifically expressed in striated epidermal cells covering the entire embryo. The ictacalcin staining in keratinocytes of striated epithelia was absent in the cytoplasm surrounding the nuclei, but it was highly concentrated in the peripheral margin. Tissues enriched with epithelia folds, such as olfactory epithelium, hatching gland, pectoral fin buds, urogenital opening, and pharynx, showed a robust ictacalcin expression. The strikingly heavy staining of ictacalcin in the pharyngeal region provides us with an early marker to follow the pharynx formation in zebrafish embryos.     

Isolation of the human testatin gene and analysis in patients with abnormal gonadal development.

We have previously isolated the testatin gene using a modified mRNA differential display method on RNA from developing male and female mouse gonads. This gene is specifically expressed during early testis development, immediately after the onset of the testis-determining gene SRY: The protein encoded by testatin has features that are characteristic for type 2 cystatins, a family of small inhibitors of cystein proteases such as the cathepsins. We have now isolated the human orthologue of this gene. We describe here the sequence, genomic structure, chromosomal location, and expression pattern of the human testatin gene. Like mouse testatin, human testatin is specifically expressed in the testis, suggesting that it has a function in reproduction. We have therefore also investigated whether the human testatin gene plays a role in disorders of gonadal development, by sequencing the gene in patients with gonadal dysgenesis, with true hermaphroditism, and in children with less well-defined intersex conditions. We found no sequence aberrations in these patients apart from an H109P polymorphism which was also found in fertile controls. This is the first genetic analysis of testatin in humans.     

Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish.

We mined the zebrafish genomic sequence database and identified contigs containing segments of several dopamine receptor genes. By using a polymerase chain reaction amplification strategy, we generated full-length cDNAs encoding a single dopamine D3 receptor and three distinct D2 receptor subtypes. Zebrafish dopamine receptor genes were mapped by using the T51 radiation hybrid panel. The D3 receptor gene (drd3) mapped to linkage group (LG) 24. The three D2 receptor genes were localized to LG 15 (drd2a), LG 16, (drd2b), and LG 5 (drd2c). With the exception of the drd2b gene, each of these map positions was syntenic with regions of human chromosomes containing orthologs of the zebrafish dopamine receptor genes. Whole-mount in situ hybridization was used to investigate expression of the D2 and D3 receptor genes. Expression of the drd3 gene was first detected at mid-somitogenesis and was particularly prominent in somites. Thereafter, the drd3 gene was expressed diffusely throughout the brain and spinal cord. The three D2 receptor genes were expressed throughout the central nervous system (CNS) in distinct but overlapping patterns. In early embryos, the drd2a gene was expressed exclusively in the epiphysis, whereas the drd2c gene was localized to the notochord. After 24 hpf, the drd2a, drd2b, and drd2c genes were differentially expressed throughout the CNS. The identification of dopamine receptor genes in zebrafish should allow us to use the power of zebrafish genetics to analyze the functional properties of this important class of neurotransmitter receptors.     

A single erythroid-specific DNase I super-hypersensitive site activates high levels of human beta-globin gene expression in transgenic mice

Erythroid-specific DNase I super-hypersensitive (HS) sites that are normally located far upstream of the human beta-globin locus were inserted immediately upstream of a 4.1-kb fragment containing the human beta-globin gene. These constructs (HS beta) and a construct containing the beta-globin gene alone (beta) were microinjected into fertilized mouse eggs, and expression was analyzed in erythroid fetal liver and brain of day-16 embryos that developed. Only 7 of 23 animals that contained the beta gene alone expressed human beta-globin mRNA in erythroid tissue, and the average level of expression per gene copy was 0.3% of endogenous mouse beta-globin mRNA. In contrast, 50 of 51 transgenic mice that contained various HS beta constructs expressed the transgene specifically in erythroid tissue. The average level of expression per gene copy for constructs containing all five upstream HS sites was 109% of endogenous mouse beta-globin mRNA. Constructs that contained a single super-hypersensitive site (HS II beta) expressed 40% as much human beta-globin as mouse beta-globin mRNA per gene copy. These results demonstrate that the HS VI site, normally located downstream of the human beta-globin locus, is not required for high-level expression. Furthermore, the results demonstrate that high levels of human beta-globin gene expression can be obtained in transgenic mice even when a relatively small fragment of DNA (1.9 kb) containing erythroid-specific super-hypersensitive site II (HS II) is inserted upstream of the human beta-globin gene.     

Comparison of Iroquois gene expression in limbs/fins of vertebrate embryos

In Drosophila, Iroquois (Irx) genes have various functions including the specification of the identity of wing veins. Vertebrate Iroquois (Irx) genes have been reported to be expressed in the developing digits of mouse limbs. Here we carry out a phylogenetic analysis of vertebrate Irx genes and compare expression in developing limbs of mouse, chick and human embryos and in zebrafish pectoral fin buds. We confirm that the six Irx gene families in vertebrates are well defined and that Clusters A and B are duplicates; in contrast, Irx1 and 3, Irx2 and 5, and Irx4 and 6 are paralogs. All Irx genes in mouse and chick are expressed in developing limbs. Detailed comparison of the expression patterns in mouse and chick shows that expression patterns of genes in the same cluster are generally similar but paralogous genes have different expression patterns. Mouse and chick Irx1 are expressed in digit condensations, whereas mouse and chick Irx6 are expressed interdigitally. The timing of Irx1 expression in individual digits in mouse and chick is different. Irx1 is also expressed in digit condensations in developing human limbs, thus showing conservation of expression of this gene in higher vertebrates. In zebrafish, Irx genes of all but six of the families are expressed in early stage pectoral fin buds but not at later stages, suggesting that these genes are not involved in patterning distal structures in zebrafish fins.     

Molecular characterization of the zebrafish P2X receptor subunit gene family

P2X receptors are non-selective cation channels gated by extracellular ATP and are encoded by a family of seven subunit genes in mammals. These receptors exhibit high permeabilities to calcium and in the mammalian nervous system they have been linked to modulation of neurotransmitter release. Previously, three complementary DNAs (cDNAs) encoding members of the zebrafish gene family have been described. We report here the cloning and characterization of an additional six genes of this family. Sequence analysis of all nine genes suggests that six are orthologs of mammalian genes, two are paralogs of previously described zebrafish subunits, and one remains unclassified. All nine subunits were physically mapped onto the zebrafish genome using radiation hybrid analysis. Of the nine gene products, seven give functional homo-oligomeric receptors when recombinantly expressed in human embryonic kidney cell line 293 cells. In addition, these subunits can form hetero-oligomeric receptors with phenotypes distinct from the parent subunits. Analysis of gene expression patterns was carried out using in situ hybridization, and seven of the nine genes were found to be expressed in embryos at 24 and 48 h post-fertilization. Of the seven that were expressed, six were present in the nervous system and four of these demonstrated considerable overlap in cells present in the sensory nervous system. These results suggest that P2X receptors might play a role in the early development and/or function of the sensory nervous system in vertebrates.