Zygotic expression of Exostosin1 (Ext1) is required for BMP signaling and establishment of dorsal-ventral pattern in Xenopus

Abstract

Exostosin 1 (EXT1) is a glycosyltransferase that contributes to the biosynthesis of heparan sulfate proteoglycans (HSPG). Loss of ext1 function leads to the human genetic disorder hereditary multiple exostoses (HME) and inhibits development in mouse, zebrafish and Drosophila. In Xenopus, loss of maternal EXT1 leads to impaired wnt11 signaling, resulting in a loss of dorsal embryonic development (Tao et al., 2005), but the functions of zygotic ext1 have not been elucidated. In this study, morpholino oligonucleotides were used to generate a zygotic partial loss of function for ext1, in order to evaluate the requirements for ext1 function in gastrulation and paracrine signaling. Transcriptional profiling was carried out by microarray. Validation and subsequent analyses of gene expression were performed using Q-RT-PCR and in situ hybridization. Western blots were used to assess paracrine signaling pathway activity. Introduction of ext1 MO led to gastrulation defects, which were partially rescued by co-injection of ext1 mRNA. Microarray-based comparisons of gene expression in control vs. Ext1 MO embryos identified several developmentally significant genes that are dependent upon Ext1 function, including brachyury (Xbra). In addition, decreased Ext1 was shown to reduce the level of Wnt8 and BMP4 signaling and disrupt ventral-specific gene expression. Ext1 function is required for maintenance of normal levels of BMP and wnt, as well as their target genes. In addition, expression of xbra and the establishment of ventral mesoderm depend upon normal levels of Ext1. These findings suggest that ext1-dependent synthesis of HSPG is critical for wnt and BMP signaling, mesodermal identity, and ventral pattern.