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 (somatic LPM) 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 LPM 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. Overall design: Two 15 somite-stage Fgf10+/+ and two 16 somite-stage Fgf10-/- embryos (E8.75) were dissected in cold 1X DPBS . The heart and tissues anterior to somite 7 and posterior to somite 13 were dissected away. The resulting core of embryonic tissue included the forelimb field as well as other embryonic tissues present at this axial level. Single embryos were trypsinized and then filtered through a cell strainer, transferred into a 1% BSA coated tube and centrifuged for 10 minutes at 500g at 4°C. Cells were fixed as described using (Tran et al., 2022) and ~58-73k cells per embryo were stored in -80?. scRNASeq libraries were prepared using the Parse Evercode WT Mini v2 kit according to the manufacturers protocol. The libraries were sequenced on an Illumina NovaSeq X Plus.