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

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.

SUBMITTER: Liu J

PROVIDER: S-EPMC3820152 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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

Liu Jiang J Li Qiang Q Kuehn Michael R MR Litingtung Ying Y Vokes Steven A SA Chiang Chin C

Developmental biology 20130108 2

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 ...[more]

PMID: 23313125

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Project description:Sonic hedgehog (Shh) is a key signal in establishing different digit fates along the anterior-posterior axis of the vertebrate limb bud. Although the anterior digits appear to be specified by differential concentrations of Shh in a traditional, morphogen-like response, recent studies have suggested that posterior digits are specified by an extended time of exposure to Shh rather than, or in addition to, a threshold concentration of Shh. This model for digit patterning depends upon continued Shh signaling in the posterior limb through mid-to-late bud stages. We find that cyclopamine, a potent antagonist of Shh signaling, can down-regulate hedgehog target genes in the posterior limb throughout the time Shh is expressed, indicating that continued active Shh signaling indeed takes place. To further explore the relative roles of time and concentration of Shh during limb development, we carried out two additional series of experiments. To test the effect of limiting the time, but not the amount of Shh produced, we treated chick embryos with the hedgehog antagonist cyclopamine at various stages of limb development. We find that short exposures to Shh result in specification of only the most anterior digits and that more posterior digits are specified sequentially with increasing times of uninterrupted Shh activity. To test the effect of limiting the level of Shh produced, but not the time of exposure, we genetically modified Shh production in mice. As previously shown, reducing both the concentration of Shh produced and the duration of Shh exposure results in a loss of posterior digits. We find that maintaining a low level of Shh production throughout the normal time frame of ZPA signaling results in a near complete restoration of the posterior-most digits. These data are consistent with, and lend additional support to, the model that concentration of Shh seen and duration of exposure both contribute to the dose-dependent specification of digit identities, but for the posterior-most digits the temporal component is the more critical parameter.

Dataset Information

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

Publications

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

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