Fig. 4.

Transcriptomic analyses reveal loss of TFAP2 genes compromises midface Alx1, Alx3 and Alx4 gene expression. (A) Bulk RNA-seq volcano plot of frontonasal and maxillary prominence (FNP/MxP) tissues isolated from E10.5 control and Tfap2^Δ/NCKO littermates (cartoon workflow on the left). Downregulated (purple) and upregulated (gold) genes are shown. (B) Enrichr (Kuleshov et al., 2016) terms based on the RNA-seq downregulated genes. (C) Overview of the scRNA-seq experiment performed on E11.5 Tfap2^HET controls and Tfap2^NCKO mutants. The top box displays the workflow of sorting GFP-positive head CNCCs from pharyngeal arch 2 (PA2) and rostral of, and including, PA2. Created using Biorender.com. Below is the resulting uniform manifold approximation and projection (UMAP) plot of the three major groupings and genes enriched in each. The mesenchyme population is outlined and is further subset and re-clustered (D) using MAGIC imputation (van Dijk et al., 2018). Dashed lines on the mesenchyme UMAP indicate the positional identities, with their respective gene signatures. These include clusters for the FNP (two generated), MxP, mandibular prominence (MdP) and PA2. (E) Violin plots of Alx1, Alx3 and Alx4 gene expression between conditions and divided based on clusters. The FNP clusters are outlined. (F) Frontal view of whole-mount in situ hybridizations for Alx3 and Alx4 between E10.5 controls and Tfap2^Δ/NCKO mutants. Scale bars: 500 µm. Dashed lines outline the FNP. (G,H) Real-time PCR analysis for Alx1, Alx3 and Alx4 gene expression based on cDNA generated from E11.5 FNP/MxP CNCCs sorted from Tfap2^HET-Tfap2^NCKO pairs (G) or E10.5 medial and lateral FNP (mFNP, lFNP) bulk tissue isolated from control-Tfap2^Δ/NCKO pairs (H).