Transcriptome of Shox2+ cells in the developing limb

Vertebrate appendage patterning is programmed by Hox-TALE factors-bound regulatory elements. However, it remains enigmatic which cell lineages are commissioned by Hox-TALE factors to generate regional specific pattern and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis, and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis. RNA sequencing profiling the transcriptome of Shox2+ in the developing limb脊椎动物附肢模式形成由结合了Hox-TALE因子（Hox-TALE factors）的调控元件所编程。然而，目前仍不清楚哪些细胞谱系受Hox-TALE因子调控以产生区域特异性模式，以及是否存在其他Hox-TALE辅因子。本研究探究了Shox2转录调控因子在肢体模式形成过程中所调控的转录机制。我们利用成骨谱系特异性Shox2失活方法，研究发现尽管Shox2在多种细胞谱系中广泛表达，但Shox2缺陷时观察到的肢近节缺失现象，是成骨谱系中Shox2功能丧失的特异性结果。染色质免疫沉淀测序（ChIP-Seq）分析显示，Shox2与围绕骨骼发生基因的顺式调控增强子存在强相互作用，这些增强子同时也被Hox-TALE因子结合，这支持了Shox2在成骨谱系命运决定与骨骼模式形成中具有谱系自主性功能。Pbx染色质免疫沉淀测序（Pbx ChIP-Seq）进一步实现了全基因组范围内对同时被Pbx、Meis及Shox2转录因子共占据的顺式调控模块的鉴定。对ChIP-Seq与RNA测序（RNA-Seq）数据的整合分析以及转基因增强子实验表明，Shox2作为阻遏因子，通过与近侧肢间充质中活跃的增强子相互作用来塑造肢近节，并拮抗TALE因子在成骨过程中的阻遏功能。本研究通过RNA测序对发育中肢体内Shox2阳性细胞的转录组进行了表征。