Zebrafish Ext2 is necessary for Fgf and Wnt signaling, but not for Hh signaling

Sabine Fischer,Johan Ledin,Beata Filipek-Gorniok

doi:10.1186/1471-213x-11-53

Sabine Fischer, Johan Ledin + Show 1 more

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https://doi.org/10.1186/1471-213x-11-53

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Abstract

BackgroundHeparan sulfate (HS) biosynthesis is tightly regulated during vertebrate embryo development. However, potential roles for HS biosynthesis in regulating the function of paracrine signaling molecules that bind to HS are incompletely understood.ResultsIn this report we have studied Fgf, Wnt and Hedgehog (Hh) signaling in ext2 mutants, where heparan sulfate content is low. We found that Fgf targeted gene expression is reduced in ext2 mutants and that the remaining expression is readily inhibited by SU5402, an FGF receptor inhibitor. In the ext2 mutants, Fgf signaling is shown to be affected during nervous system development and reduction of Fgf ligands in the mutants affects tail development. Also, Wnt signaling is affected in the ext2 mutants, as shown by a stronger phenotype in ext2 mutants injected with morpholinos that partially block translation of Wnt11 or Wnt5b, compared to injected wild type embryos. In contrast, Hh dependent signaling is apparently unaffected in the ext2 mutants; Hh targeted gene expression is not reduced, the Hh inhibitor cyclopamine is not more affective in the mutants and Hh dependent cell differentiation in the retina and in the myotome are normal in ext2 mutants. In addition, no genetic interaction between ext2 and shha during development could be detected.ConclusionWe conclude that ext2 is involved in Fgf and Wnt signaling but not in Hh signaling, revealing an unexpected specificity for ext2 in signaling pathways during embryonic development. Thus, our results support the hypothesis that regulation of heparan sulfate biosynthesis has distinct instructive functions for different signaling factors.

Highlights

Heparan sulfate (HS) biosynthesis is tightly regulated during vertebrate embryo development
In ext2 and extl3 mutants, maternally deposited mRNA provides sufficient levels of Heparan Sulfate Proteoglycans (HSPG) to allow normal gastrulation of the embryo while HS polymerization is subsequently reduced in all tissues [46]. ext2 and extl3 mutants were originally isolated based on their defective limb development [47] and we have previously shown that Fgf10 signaling during limb development requires ext2 and extl3 [14]. ext2 and extl3 mutants show defects in cartilage and pharyngeal arch morphogenesis [48,49], development of the ear [50], and axon sorting in the optic tract [46,51]
Expression of the Fgf signaling target gene etv5b is reduced in ext2 mutants The zebrafish ext2 mutant only contains a fraction of the normal HS levels in its tissues at 24 hpf [46] but even though Fgf signaling is believed to depend on HSPGs, Fgf10 function in the developing limb is so far the only defective Fgf signaling activity observed in ext2 mutants [14]

Summary

Introduction

Heparan sulfate (HS) biosynthesis is tightly regulated during vertebrate embryo development. Heparan Sulfate Proteoglycans (HSPG) consist of proteoglycan core proteins to which long linear heparan sulfate (HS) chains carrying sulfate groups in different positions are attached [1,2,3]. They are ubiquitous components of cell surfaces and of the extracellular matrix. Fgf proteins are thoroughly studied HSPG binding proteins with important roles in cell migration, Wnt and Hh proteins are secreted signaling morphogens with functions in numerous developmental processes. Decreased HSPG sulfation reduces Shh signaling in mice [20] and decreased HS polymerization is suggested to interfere with the function of Indian hedgehog (Ihh) (discussed below)

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