Skip to main content
. 2021 Oct 13;10:e69288. doi: 10.7554/eLife.69288

Figure 2. Tbx5 maintains aldh1a2 expression in Xenopus foregut lpm.

Figure 2—source data 1. Summary of in-situ hybridization results from Xenopus CAS9/CRISPR injection experiments in F0 embryos.
The tbx5 exon5 sgRNA (targeting the DNA binding domain; Steimle et al., 2018) causes approximately 40% of injected embryos to have a molecular phenotype as revealed by in-situ hybridization. 2 nl of a mixture containing 50 pg/nl sgRNA with 0.5 ng/nl Cas9 protein (PNA Bio CP01-20) was injected on either side of the sperm entry point at the 1 cell stage (total of 200 pg sgRNA and 2 ng Cas9 protein per embryo), embryos were cultured to NF34 and assayed by in-situ hybridization for the indicated genes.

Figure 2.

Figure 2—figure supplement 1. Tbx5 LOF phenotype at stage NF43/44 and quantification of reduced Aldh1a2 immunostaining.

Figure 2—figure supplement 1.

(A) Immunostaining of stage NF43 transgenic Xenopus laevis Tg(nkx2-5:GFP) reporter embryos injected with either mismatch-MO (control) or Tbx5-MO. Confocal optical sections through the cardiopulmonary-foregut region showing Fibronectin+ tissue boundaries (purple), GFP in the ventral pharynx, trachea, and ventricle; and Sox2 (red) in the pharyngeal/esophageal endoderm. Tbx5-depleted embryos exhibit severe cardiac hypoplasia, a lack of ventricular trabeculae, and a single, undivided foregut tube lacking Nkx2-1 respiratory progenitors. (B) Immunostaining of NF44 X. tropicalis embryos for Fibronectin (purple), Sox2 (red), and FoxF1 (green) shows that tbx5 mutants, have a single Sox2+ foregut tube and severe cardiac hypoplasia phenocopying X. laevis Tbx5 morphants. Injection of human TBX5 RNA rescues trachea-esophageal morphogenesis and cardiac chamber development. (C) Representative images for quantitation of Aldh1a2 immunofluorescence signal in Tbx5 LOF embryos at NF34. Nikon Elements Analysis AR software was used to determine the average volume pixel intensity of the fg lpm region (dotted yellow lines). Each dot in the graphs represents a distinct fg lpm/pSHF region from N=3 total embryos, all imaged with identical confocal laser settings. *p<0.05, parametric two-tailed paired t-test. at, atrium; eso, esophagus; lb, lung bud; LOF, loss-of- function; lpm, lateral plate mesoderm; MO, morpholino; pha, pharynx; tra, trachea; vent, ventricle.
Figure 2—figure supplement 1—source data 1. Quantitation of Xenopus NF34 fg lpm/pSHF Aldh1a2 immunostaining volume pixel intensity.