<i>Gata6</i>-Dependent GLI3 Repressor Function is Essential in Anterior Limb Progenitor Cells for Proper Limb Development

<div><p><i>Gli3</i> is a major regulator of Hedgehog signaling during limb development. In the anterior mesenchyme, GLI3 is proteolytically processed into GLI3R, a truncated repressor form that inhibits Hedgehog signaling. Although numerous studies have identified mechanisms that regulate <i>Gli3</i> function in vitro, it is not completely understood how <i>Gli3</i> function is regulated in vivo. In this study, we show a novel mechanism of regulation of GLI3R activities in limb buds by <i>Gata6</i>, a member of the GATA transcription factor family. We show that conditional inactivation of <i>Gata6</i> prior to limb outgrowth by the <i>Tcre</i> deleter causes preaxial polydactyly, the formation of an anterior extra digit, in hindlimbs. A recent study suggested that <i>Gata6</i> represses <i>Shh</i> transcription in hindlimb buds. However, we found that ectopic Hedgehog signaling precedes ectopic <i>Shh</i> expression. In conjunction, we observed <i>Gata6</i> and <i>Gli3</i> genetically interact, and compound heterozygous mutants develop preaxial polydactyly without ectopic <i>Shh</i> expression, indicating an additional prior mechanism to prevent polydactyly. These results support the idea that <i>Gata6</i> possesses dual roles during limb development: enhancement of <i>Gli3</i> repressor function to repress Hedgehog signaling in the anterior limb bud, and negative regulation of <i>Shh</i> expression. Our in vitro and in vivo studies identified that GATA6 physically interacts with GLI3R to facilitate nuclear localization of GLI3R and repressor activities of GLI3R. Both the genetic and biochemical data elucidates a novel mechanism by <i>Gata6</i> to regulate GLI3R activities in the anterior limb progenitor cells to prevent polydactyly and attain proper development of the mammalian autopod.</p></div>