Transcriptional trajectories highlight the transition from anterior-posterior to proximal-distal patterning in the early limb bud [scRNA-seq]

Limb patterning relies in a large part on the function of the Hox family of developmental genes. While the differential expression of Hox genes shifts from the anterior-posterior (A-P) to the proximal-distal (P-D) axis around embryonic day 11 (E11), whether this shift coincides with a more global change of P-D versus A-P patterning program remains unclear. By performing and analyzing the transcriptome of the developing limb bud from E10.5 to E12.5, at single cell resolution, we have uncovered transcriptional trajectories which revealed a general switch from A-P to P-D genetic program between E10.5 and E11.5. Interestingly, the transcriptional trajectories at E10.5 all end with cells expressing either proximal or distal markers suggesting a progressive acquisition of P-D identity. Moreover, we observed that distally expressed Hox genes, namely Hoxa13 and Hoxd13, act as a key determinant for P-D patterning as their transcriptional control results in their distal restricted expression, which in turn restricts Hoxa11 in the proximal limb bud domain, in progenitor cells of the zeugopod. Finally, we identified three categories of genes expressed in the distal limb mesenchyme characterized by distinct temporal expression dynamics. As anticipated from previous results, HOX13 binding was observed within or in the neighborhood of several of these genes consistent with previous evidence suggesting that the transition from the early/proximal to the late/distal transcriptome of the limb mesenchyme largely relies on HOX13 function. Single-cell RNA-seq analysis of mouse forelimb bud at E10.5, E11.5 and E12.5肢体模式形成在很大程度上依赖于发育基因Hox家族（Hox family）的功能。Hox基因的差异表达在胚胎第11天左右（E11）从前后轴（anterior-posterior, A-P）转向近远轴（proximal-distal, P-D），目前尚不清楚这一转变是否与近远轴相对于前后轴的模式形成程序的全局性变化相吻合。本研究通过对胚胎第10.5天（E10.5）至第12.5天（E12.5）发育中的肢芽进行单细胞分辨率的转录组分析，揭示了转录轨迹，发现在E10.5至E11.5期间存在从前后轴遗传程序向近远轴遗传程序的整体转换。有趣的是，E10.5时期的所有转录轨迹均以表达近端或远端标志物的细胞告终，这提示细胞的近远轴身份是逐步获得的。此外，本研究观察到远端表达的Hox基因——即Hoxa13与Hoxd13——是近远轴模式形成的关键调控因子：它们的转录调控使其表达局限于远端区域，进而将Hoxa11限制在肢芽近端的肢中段（zeugopod）祖细胞域中。最后，本研究鉴定出远端肢间充质中表达的三类基因，其特征为独特的时间表达动态模式。正如此前研究预期的那样，在其中多个基因的编码区或邻近区域检测到了HOX13结合位点，这与先前的研究证据相符——即肢间充质从早期/近端转录组向晚期/远端转录组的转变，在很大程度上依赖于HOX13的功能。本数据集源自小鼠胚胎第10.5天、11.5天及12.5天前肢芽的单细胞RNA测序（single-cell RNA-seq）分析。