Transcriptional Trajectories in Mouse Limb Buds Reveal the Transition from Anterior-Posterior to Proximal-Distal Patterning at Early Limb Bud Stage.

Limb patterning relies in 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 A-P to P-D 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 that revealed a general switch from A-P to P-D genetic program between E10.5 and E11.5. Interestingly, all the transcriptional trajectories at E10.5 end with cells expressing either proximal or distal markers suggesting a progressive acquisition of P-D identity. Moreover, we identified three categories of genes expressed in the distal limb mesenchyme characterized by distinct temporal expression dynamics. Among these are Hoxa13 and Hoxd13 (Hox13 hereafter), which start to be expressed around E10.5, and importantly the binding of the HOX13 factors was observed within or in the neighborhood of several of the distal limb genes. Our data are 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. Based on these results and the evidence that HOX13 factors restrict Hoxa11 expression to the proximal limb, in progenitor cells of the zeugopod, we propose that HOX13 act as a key determinant of P-D patterning.