생쥐의 사지발생 과정에서 SWI/SNF 염색사 리모델링 복합체의 기능에 관한 연구

Abstract

During development and differentiation, alterations in gene expression require modification of chromatin status, which allows or restrains the access of transcription factors to the regulatory elements. For developmental progression, the function of context-dependent transcription factors responding to developmental signals needs the recruitment of chromatin remodelers. Recent studies have identified the interlinked transcriptional networks by the enrichment of these factors in the genomic loci by using a limb bud, which is an excellent model to understand tissue patterning. Thus, the molecular circuits of regulators orchestrating limb development involve distinct functions of chromatin remodelers. The graded Sonic hedgehog (Shh) signaling governs the vertebrate limb skeletal patterning along anteroposterior (AP) axis by regulating the activity of bifunctional Gli transcriptional regulators acting as activator or repressor. Gli target genes are activated, repressed or derepressed in response to a gradient of Hedgehog (Hh) signaling, but the mechanisms by which bifunctional Gli proteins collaborate to regulate target genes are poorly understood. Furthermore, the genetic networks involved in limb patterning are well defined, whereas the epigenetic control of the process by chromatin remodelers remains unclear. Here, I found that Srg3/mBaf155, a core subunit of SWI/SNF chromatin remodeling complex, is essential for Shh/Gli-driven limb AP skeletal patterning. Genetic analysis and spatiotemporal distributions of gene expression analyzed by whole-mount in situ hybridization have uncovered the dual requirements of SWI/SNF complex in Hh pathway during limb development. Specific inactivation of Srg3 in the limb bud mesenchyme hampered the transcriptional upregulation of both Shh receptor Ptch1 and its downstream effector Gli1 upon morphogen Shh stimulation and induced ectopic activation of Hh pathway in Shh-free region. In limb buds lacking Srg3, the attenuated sensing of Shh caused the redistribution of Shh-descendants as well as the downregulation of target genes. Without severe defects in the formation of signaling centers such as the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER), Srg3-deficient limb buds established the intact AP axis but progressively lost their anterior and posterior identities. Analysis of these progressive phenotypes provided genetic evidence how modulation of Shh responsiveness drives the fate of posterior limb skeletal progenitors and activation of ectopic Hh pathway is detrimental to the formation of anterior skeletal elements. Bifurcating role of Srg3 in Hh pathway distalized the distributions of epithelial-mesenchymal signaling components such as Shh, BMP antagonist Gremlin1 (Grem1) and FGF. Subsequently, Srg3 deficiency led to aberrant BMP activity and disruption of chondrogenic differentiation in zeugopod and autopod primordia. Notably, I found that Shh responsiveness and Gli repressor activity are collaboratively required for the spatiotemporal regulation of Grem1 expression, which affects the onset of digit chondrogenesis. My study uncovers the bifurcating function of SWI/SNF complex in Hh pathway to determine the fate of AP skeletal progenitors.