HEPARAN SULFATE PROTEOGLYCANS IN ZEBRAFISH DEVELOPMENT. Amanda Adeleye1, Beth Viviano2, Penny Jensen2, and Scott Saunders2,3, Biology Department, Washington University, St. Louis, M1, Dept. of Pediatrics2 and Dept. of Mol. Biol. & Pharm.3, Washington University School of Medicine, St. Louis, MO2,3
INTRODUCTION: Heparan sulfate proteoglycans are glycoproteins that bind and modulate the function of a number of ligands in vivo. These molecules are found ubiquitously in complex multicellular organisms and their functions are especially crucial during embryonic development. [1] Heparan sulfate chains are synthesized initially as repeating disaccharides of N-acetyl-glucosamine and glucuronic acid, which are then further modified by the processes of epimerization and sulfation.[2] This resulting heparan sulfate chain has immense structural diversity, and it is this diversity that is believed to give rise to the specificity of proteoglycan functions.[3] There are several enzymes in the heparan sulfate biosynthetic pathway that contribute to this structural diversity. One of these is the heparan sulfate 6-O-sulfotransferase-2 gene (HS6ST-2), which has previously been reported to be involved in angiogenesis and somite development in vivo.[4] In this study, we have sought to identify other significant phenotypes associated with the loss of HS6ST-2 function in zebrafish.
METHODS: Zebrafish embryos were injected with morpholinos known to disrupt function of the HS6ST-2 transcript. The zebrafish were grown to 24, 48 or 72 hours past fertilization (hpf). At the designated time point zebrafish were stained with CellTrace Bodipy TR methyl ester (MP 34556; Molecular Probes), washed 3 times in egg water and fixed in 4% paraformaldehyde. Afterwards Zebrafish were maintained in PBS. Zebrafish images were taken using an Olympus FV5-PSU confocal and analyzed using Olympus 1X71 microimaging suite and Adobe Photoshop
RESULTS: Morpholino injected zebrafish had phenotypes including abnormalities in both head and tail development with varying penetrance. Zebrafish injected with higher concentrations of HS6ST-2 morpholinos showed more severe and distinct phenotypes, when compared with zebrafish injected with a concentration of morpholinos previously reported
DISCUSSION: The HS6ST-2 morpholinos induced phenotypes confirming an essential role for 6-O-sulfation of heparan sulfate in zebrafish development. The more severe phenotypes seen following injection of slightly higher concentrations of morpholinos imply novel requirements for 6-O-sulfation in zebrafish patterning, which have not been previously reported. Further studies will be required to characterize the molecular basis of these defects.
[1] Viviano et al., 2005 Altered hematopoiesis in glypican-3-deficient mice results in decreased osteoclast differentiation and a delay in endochondral ossification, Devlomental Biology. 282 (2005), pp 152-162
[2] Bink et al., 2003 Heparan Sulfate 6-O-Sulfotransferase Is Essential for Muscle Development in Zebrafish, J. Biol. Chem. 278, pp 31118-31127
[3] Bernfield et al., 1999 Functions of Cell Surface Heparan Sulfate Proteoglycans, Annual Review of Biochemistry. 68, pp 729-777
[4] Ibid 2