Control of limb initiation and antero-posterior patterning by cooperation between Meis and Tbx transcription factors [RNA-seq]

Meis1 and Meis2 are highly similar homeodomain transcription factors that regulate organogenesis through cooperation with Hox proteins. Overexpression experiments have suggested an essential role for Meis factors in limb proximo-distal patterning; however, loss-of-function experiments supporting this notion have not been reported. Meis1 and Meis2 are coexpressed during limb development, first in the lateral plate mesoderm, before limb induction, and then they become restricted to a proximal domain of the growing limb bud. Here, we report that complete double conditional Meis1/2 inactivation in the lateral plate mesoderm leads to limb agenesis. Meis factors cooperate with Tbx factors in this function, extensively co-binding with Tbx to genomic sites and co-regulating enhancers of fgf10, a critical factor in limb initiation. Limbs with three deleted Meis alleles develop a complete PD set of limb skeletal elements, but show proximal-specific skeletal hypoplasia and agenesis of posterior skeletal elements. This failure in posterior skeletal element specification reveals an early role of Meis factors in establishing the limb AP prepattern required for Shh activation at later stages. Our results uncover novel roles for Meis transcription factors in early limb development and identify their involvement in new molecular interaction networks that regulate organogenesis. Overall design: Transcriptomic analysis comparing Meis1Ko;Meis2KO mutant limbs and WT limbs. RNA was isolated from the two hindlimbs E10 (30-31 somites approximately) of individual embryos. Six mutant and five WT embryos were used for analysis.

Meis1与Meis2是高度相似的同源盒结构域转录因子（homeodomain transcription factors），可通过与Hox蛋白（Hox proteins）协同调控器官发生过程。过表达实验已提示Meis家族因子在肢体近-远轴模式形成中发挥关键作用，但支持该结论的功能丧失实验尚未见报道。Meis1与Meis2在肢体发育过程中存在共表达现象：首先于肢体诱导前的侧板中胚层（lateral plate mesoderm）中表达，随后被局限于生长中肢芽（limb bud）的近端区域。本研究报道，在侧板中胚层中完全条件性失活Meis1/2会导致肢体发育不全（limb agenesis）。在此功能中，Meis家族因子与Tbx家族因子（Tbx factors）协同发挥作用：二者可广泛共结合于基因组位点，并共同调控成纤维细胞生长因子10（fgf10）的增强子（enhancers）——fgf10是肢体起始过程中的关键调控因子。携带3个Meis等位基因缺失的肢体可发育出完整的近-远轴骨骼全套结构，但呈现近端特异性骨骼发育不全以及后部骨骼元素发育不全。这种后部骨骼元素特化障碍，揭示了Meis家族因子在建立肢体前后轴预模式中的早期作用，而该预模式是后续阶段音猬因子（Shh）激活所必需的。本研究结果揭示了Meis转录因子在肢体早期发育中的全新功能，并确定其参与调控器官发生的新型分子互作网络。整体实验设计：转录组分析对比Meis1敲除；Meis2敲除（Meis1Ko;Meis2KO）突变肢体与野生型（WT）肢体。RNA提取自单个胚胎胚胎期第10天（约30-31体节（somites））的双侧后肢。本实验共纳入6个突变胚胎与5个野生型胚胎用于分析。