Sonic hedgehog signaling directly targets Hyaluronic Acid Synthase 2, an essential regulator of phalangeal joint patterning

Dev Biol. 2013 Mar 15;375(2):160-71. doi: 10.1016/j.ydbio.2012.12.018. Epub 2013 Jan 8.

Abstract

Sonic hedgehog (Shh) signal, mediated by the Gli family of transcription factors, plays an essential role in the growth and patterning of the limb. Through analysis of the early limb bud transcriptome, we identified a posteriorly-enriched gene, Hyaluronic Acid Synthase 2 (Has2), which encodes a key enzyme for the synthesis of hyaluronan (HA), as a direct target of Gli transcriptional regulation during early mouse limb development. Has2 expression in the limb bud is lost in Shh null and expanded anteriorly in Gli3 mutants. We identified an ∼3kb Has2 promoter fragment that contains two strong Gli-binding consensus sequences, and mutation of either site abrogated the ability of Gli1 to activate Has2 promoter in a cell-based assay. Additionally, this promoter fragment is sufficient to direct expression of a reporter gene in the posterior limb mesenchyme. Chromatin immunoprecipitation of DNA-Gli3 protein complexes from limb buds indicated that Gli3 strongly binds to the Has2 promoter region, suggesting that Has2 is a direct transcriptional target of the Shh signaling pathway. We also showed that Has2 conditional mutant (Has2cko) hindlimbs display digit-specific patterning defects with longitudinally shifted phalangeal joints and impaired chondrogenesis. Has2cko limbs show less capacity for mesenchymal condensation with mislocalized distributions of chondroitin sulfate proteoglycans (CSPGs), aggrecan and link protein. Has2cko limb phenotype displays striking resemblance to mutants with defective chondroitin sulfation suggesting tight developmental control of HA on CSPG function. Together, our study identifies Has2 as a novel downstream target of Shh signaling required for joint patterning and chondrogenesis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aggrecans / metabolism
  • Animals
  • Base Sequence
  • Body Patterning* / genetics
  • Chondrogenesis / genetics
  • Extracellular Matrix Proteins / metabolism
  • Extremities / embryology*
  • Gene Expression Regulation, Developmental
  • Glucuronosyltransferase / deficiency
  • Glucuronosyltransferase / genetics
  • Glucuronosyltransferase / metabolism*
  • Hedgehog Proteins / metabolism*
  • Hyaluronan Synthases
  • Hyaluronic Acid / metabolism
  • Joints / embryology*
  • Joints / enzymology*
  • Kruppel-Like Transcription Factors / metabolism
  • Limb Buds / embryology
  • Limb Buds / metabolism
  • Mesoderm / embryology
  • Mesoderm / metabolism
  • Mice
  • Molecular Sequence Data
  • Mutation / genetics
  • Nerve Tissue Proteins / metabolism
  • Promoter Regions, Genetic / genetics
  • Proteoglycans / metabolism
  • Signal Transduction* / genetics
  • Zinc Finger Protein Gli3

Substances

  • Aggrecans
  • Extracellular Matrix Proteins
  • Gli3 protein, mouse
  • Hedgehog Proteins
  • Kruppel-Like Transcription Factors
  • Nerve Tissue Proteins
  • Proteoglycans
  • Shh protein, mouse
  • Zinc Finger Protein Gli3
  • link protein
  • Hyaluronic Acid
  • Glucuronosyltransferase
  • Has2 protein, mouse
  • Hyaluronan Synthases