Deletions in and1 and and2 lead to absent or reduced expression. (A) Schematic representation of deletions (red outlines) in and1 and and2. Black rectangles indicate the coding regions; white rectangles represent the untranslated exons of the genes. Numbers refer to base pairs in the gene sequence. The region around the deletion sites (red highlight) in wild-type and mutated sequences is shown below. (B) Whole-mount in situ hybridization of and1 and and2 antisense riboprobes of wild-type (nand1=10; nand2=10), and1−/−and2+/+ (nand1=4; nand2=5), and1+/+and2−/− (nand1=6; nand2=4) and and1−/−and2−/− (nand1=5; nand2=5) embryos at 2 dpf. Embryos were stained for 20 min. Black arrowheads indicate purple staining in median fin folds (mff) and pectoral fin folds (pff). Pectoral fin folds lacking expression are emphasized with a black dotted outline. Scale bars: 250 µm. (C) Relative fold change of and1, and2, and3, and4, col2a1a and col2a1b expression in wild-type sibling and double mutant tails at 5 dpf (n=30 tails per biological replicate). Wild-type expression level was set to 1. Fold change was determined by RT-qPCR using the ΔΔCq method. Error bars indicate standard error of ΔΔCq. *P<0.05 (unpaired two-tailed t-test).
|
