Developmental and transcriptional programs that define the initiation of the forelimb.

The vertebrate forelimb initiates as a localized swelling in the somatic lateral plate mesoderm in response to TBX5-dependent transcription. The molecular pathways driving limb morphogenesis have been extensively studied but the steps directly preceding limb bud formation remain poorly characterized. To address this, we defined the temporal onset of forelimb initiation in mouse embryos using sequencing-based high-throughput approaches (RNA-seq, scRNA-seq, ChIP-seq and Ribo-ITP) benchmarked to known features in forelimb development, identifying four distinct stages. Using scRNA-seq at the onset of forelimb-specific transcription, we determined the transcriptional profile of the somatic lateral plate mesoderm and identified signature genes that distinguish the nascent forelimb from other cell types. This group includes multiple genes involved in neural projection as well as cell adhesion. Interestingly, these genes are highly enriched for TBX5-binding sites, suggesting they are candidate early transcriptional targets of TBX5. As TBX5 is essential for forelimb outgrowth, the identification of these genes suggests new mechanistic models for TBX5-driven limb initiation.