Retinal ganglion cell (RGC) axons are topographically ordered in the optic tract according to their retinal origin. In zebrafish dackel (dak) and boxer (box) mutants, some dorsal RGC axons missort in the optic tract but innervate the tectum topographically. Molecular cloning reveals that dak and box encode ext2 and extl3, glycosyltransferases implicated in heparan sulfate (HS) biosynthesis. Both genes are required for HS synthesis, as shown by biochemical and immunohistochemical analysis, and are expressed maternally and then ubiquitously, likely playing permissive roles. Missorting in box can be rescued by overexpression of extl3. dak;box double mutants show synthetic pathfinding phenotypes that phenocopy robo2 mutants, suggesting that Robo2 function requires HS in vivo; however, tract sorting does not require Robo function, since it is normal in robo2 null mutants. This genetic evidence that heparan sulfate proteoglycan function is required for optic tract sorting provides clues to begin understanding the underlying molecular mechanisms.

Mutations in human Exostosin genes (EXTs) confer a disease called Hereditary Multiple Exostoses (HME) that affects 1 in 50,000 among the general population. Patients with HME have a short stature and develop osteochondromas during childhood. Here we show that two zebrafish mutants, dackel (dak) and pinscher (pic), have cartilage defects that strongly resemble those seen in HME patients. We have previously determined that dak encodes zebrafish Ext2. Positional cloning of pic reveals that it encodes a sulphate transporter required for sulphation of glycans (Papst1). We show that although both dak and pic are required during cartilage morphogenesis, they are dispensable for chondrocyte and perichondral cell differentiation. They are also required for hypertrophic chondrocyte differentiation and osteoblast differentiation. Transplantation analysis indicates that dak−/− cells are usually rescued by neighbouring wild-type chondrocytes. In contrast, pic−/− chondrocytes always act autonomously and can disrupt the morphology of neighbouring wild-type cells. These findings lead to the development of a new model to explain the aetiology of HME.

Heparan sulfate proteoglycans are important modulators of growth factor signaling in a variety of patterning processes. Secreted growth factors that play critical roles in angiogenesis bind to heparan sulfate, and this association is affected by 6-O-sulfation of the heparan sulfate chains. Addition of 6-O-sulfate is catalyzed by a family of sulfotransferases (HS6STs), and genetic manipulation of their function permits an assessment of their contribution to vascular assembly. We report on the biochemical activity and expression patterns of two zebrafish HS6ST genes. In situ hybridization reveals dynamic and distinct expression patterns of these two genes during development. Structural analysis of heparan sulfate from wild-type and morpholino antisense 'knockdown' embryos suggests that HS6ST-1 and HS6ST-2 have similar biochemical activity. HS6ST-2, but not HS6ST-1, morphants exhibit abnormalities in the branching morphogenesis of the caudal vein during embryonic development of the zebrafish. Our finding that HS6ST-2 is required for the branching morphogenesis of the caudal vein is the first in vivo evidence for an essential role of a gene encoding a heparan sulfate modifying enzyme in vertebrate angiogenesis. Our analysis of two zebrafish HS6ST genes suggests that a wide range of biological processes may be regulated by an array of sulfation-modifying enzymes in the vertebrate genome.