Cell-specific alterations in the activation of the Pitx1 regulatory landscape caused by the loss of a single enhancer (RNA-seq)

Developmental genes often reply on multiple transcriptional enhancers to achieve correct expression during embryogenesis. These enhancers can have partially redundant activities, thus it is difficult to ascertain the direct impact of the loss of a single one of them as often only mild, if any, phenotypes are seen. In this work we use the testbed locus Pitx1 to characterise the regulatory and cell specific alterations following the loss of one of its enhancers Pen in vivo. We combine single cell transcriptomics and in embryo cell tracing and observe the global decrease previously observed in deletion of Pen hindlimbs results from an accumulation of low to non-expressing cells that affects more proximal rather than distal limb and leads to a clubfoot phenotype product of a localised heterochrony and loss of irregular connective tissue. We also analyse the contribution of the enhancer to the overall locus structure and activity and we suggest that in some cases redundant enhancers may locally enforce a robust activation of their gene's regulatory landscape. We analysed two biological replicates of Pitx1-GFP positive and negative cells and two biological replicates of Pitx1-GFP-deltaPen GFP postive and negative cells from hindlimb tissue to compare the expression differences between Pitx1 expressing and non-expressing cells in the two genetic backgrounds.发育基因通常依赖多个转录增强子（transcriptional enhancers），以在胚胎发生过程中实现精准的时空表达。此类增强子往往存在部分功能冗余，因此难以确定单个增强子缺失后的直接调控影响——即便能观测到表型，通常也仅为轻微表型。 本研究以模式研究位点Pitx1为实验对象，在体内对其增强子Pen缺失后的调控变化与细胞特异性改变进行系统表征。我们结合单细胞转录组学（single cell transcriptomics）与胚胎内细胞示踪技术，发现此前在Pen缺失的后肢样本中观察到的整体表达水平下降，实则源于低表达至不表达细胞的累积；这类细胞对肢体近端区域的影响更为显著，而非远端区域，并通过局部异时性改变与不规则结缔组织丢失，最终催生先天性马蹄内翻足表型。 此外，本研究还分析了该增强子对位点整体结构与基因活性的调控贡献，并提出：在部分场景下，功能冗余的增强子可在局部强化其靶基因调控区域的稳健激活。 我们从后肢组织中分别获取了两份Pitx1-GFP阳性与阴性细胞的生物学重复样本，以及两份Pitx1-GFP-ΔPen GFP阳性与阴性细胞的生物学重复样本，以此对比两种遗传背景下，表达与不表达Pitx1的细胞之间的转录表达差异。