Organ LR patterning defects in cdon−/−embryos (A) CRISPR/Cas9 target was showed. The sequence in the exon 3 of cdon gene was chosen as the target (yellow highlighted). (B) Sequencing results for the genomic DNA in WT embryos, cdon+/− embryos and cdon−/− embryos. (C) Nucleotide sequences highlighted in yellow are CRISPR/Cas9 targets. In the cdon mutant, the sequence “AAGGGC” was changed to “TTGATGAATGGGG”. Cdon mutant harbors a frameshift mutation that is predicted to result in the production of truncated Cdon protein (104 amino acids). (D) Cdon−/− embryos were found to display liver LR defects using WISH. d1, left-sided liver in controls (95.5%, n = 111); d2, left-sided liver in cdon−/− embryos (71.2%, n = 118); d3, liver bifida in cdon−/− embryos (17.8%, n = 118); d4, right-sided liver in cdon−/− embryos (11.0%, n = 118). (E) Percentages of left-sided liver, liver bifida, and right-sided liver in cdon−/− embryos (n = 118), controls (n = 111) and cdon−/− embryos injected with cdon mRNA (n = 89), cdon−/− embryos show a statistically significant difference compared to controls, and cdon−/− embryos injected with cdon mRNA show a statistically significant difference compared to cdon−/− embryos. (F) Cdon−/− embryos displayed heart LR defects. f1, normal-loop heart in controls (97.5%, n = 122); f2, normal-loop heart in cdon−/− embryos (72.7%, n = 128); f3, no loop heart in cdon−/−embryos (16.4%, n = 128); f4, reversed loop heart in cdon−/− embryos (10.9%, n = 128). The blue dashed line indicates the atrial edge. (G) Percentages of normal looping, no looping, and reversed looping of the heart in cdon−/− embryos (n = 128), controls (n = 122) and cdon−/− embryos injected with cdon mRNA (n = 90), cdon−/− embryos show a statistically significant difference compared to controls, and cdon−/− embryos injected with cdon mRNA show a statistically significant difference compared to cdon−/− embryos. Statistical analysis was performed using Student’s t-test. “*” p < 0.05, “**” p < 0.01, “***” p < 0.001. Notice: “control” refers to wild-type embryos.
|
