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

Marie Kmita,Rachel Paul,Ines Desanlis

doi:10.3390/jdb8040031

Abstract

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.

Highlights

Limb development in mouse initiates around E9.5, when the forelimb bud emerges from the lateral plate mesoderm
To gain a better understanding of the developmental program underlying limb patterning, we analyzed the transcriptome at single-cell resolution from mouse forelimb buds isolated from embryos at gestational day 10.5 (E10.5), embryonic day 11 (E11).5 and E12.5 (Figure 1a)
The dimensional reduction procedure applied to our single-cell RNA-seq dataset was based on the identification of the 3000 most variable expressed genes, from which the lowest and highest expressed genes were removed

Summary

Introduction

Limb development in mouse initiates around E9.5, when the forelimb bud emerges from the lateral plate mesoderm. At this stage, undifferentiated mesenchymal cells receive signals from a group of ectodermal cells at the distal tip of the limb bud, referred to as the Apical Ectodermal Ridge (AER). Factors (Fgfs), notably Fgf, whose function promotes limb bud growth and provides distal positional information to the underlying mesenchyme [1]. Cells at the distal-posterior margin of the limb bud express Sonic Hedgehog (Shh), establishing a gradient of SHH signaling along the anterior–posterior (A–P) axis, which underlies the A–P patterning of the developing limb [3].

Methods

Results

Discussion

Conclusion