Fig. 3

Loss of tet2/3 disrupts pharyngeal cartilage development by impairing chondrocyte differentiation (A) Re-clustering analysis showing six sub-clusters of EMCs indicated by different colors. Top, wild-type EMCs. Bottom, tet2/3-DKO EMCs. (B) Pseudotime-ordered analysis of EMCs. The black lines highlight the branching of the two differentiation trajectories in pseudotime. (C) Four modules of transcription factors identified with distinct expression features. Feature plots showing expression scores for each module. (D) Split bars showing the cellular composition of each replicate for different samples. (E) Violin plots showing the expression level of indicated genes in each EMC sub-cluster of wild-type (blue) and tet2/3-DKO (red) samples. (F–H) In situ hybridization assays showing the transcripts of lin28a, nrp2a, and col2a1a in embryos injected with indicated morpholinos at 48 hpf. Upper, lateral views, anterior toward the left. Lower, dorsal (F and G), or ventral (H) views, anterior toward the left. (I) Tg(col2a1a: EGFP) transgene embryos injected with control morpholino (left) and tet2/3 DMOs (right). Top, schematic of pharyngeal cartilage in embryos with different phenotypes. Bottom, the cartilage is shown by col2a1a signals labeled with green fluorescence. Scale bars, as indicated. (J–L) Alcian blue staining showing the morphology of craniofacial cartilage in embryos injected with indicated morpholinos and mRNAs at 5 dpf. Embryos were injected with tet2/3 DMOs (J), with tet2/3 DMOs and tet2/3 mutant mRNAs (K), and with tet2/3 DMOs and wild-type tet2/3 mRNAs (L). Scale bars, as indicated. See also Figure S4.