Fig. 5

hspb12 knockdown randomized spaw, pitx2, lefty2 and lefty1. (A) 0.5 pmol hspb12 MO1 increased bilateral spaw expression compared to control MO (Χ² test; p<0.001). (B, C) 14-18 somite embryos. (B) Control MO injected embryo with left sided spaw and midline lefty1 expression. (C) hspb12MO1-injected embryo with normal spaw and lefty1 expression. (D) Hspb12MO1 randomized pitx2 expression (Χ² test; p<0.001). (E) hspb12 MO1 injection did not induce ectopic lefty2, but increased the number of embryos lacking lefty2 expression (Χ² test; p<0.001). (F-H) lefty1 and lefty2 in situ hybridization at 22-24 somites. (F) Control MO with normal lefty1 and lefty2 in left brain and heart field respectively. (G) In this embryo, Hspb12MO1 suppressed normal lefty1 and lefty2. (H) In another embryo, Hspb12MO1 induced bilateral lefty1 and lefty2. lefty1 and lefty2 were never observed exclusively on the right. (I) Hspb12MO1 injection caused bilateral or absent lefty1 expression in the brain (Χ² test; p<0.001). (J) Injection of 0.25 pmole of hspb7MO1 or hspb12MO1 individually did not affect left-sided spaw expression in the LPM, but injection of both MO together induced bilateral and right-sided spaw. (K) A dose of 0.25 pmole of hspb12MO1 or hspb7MO1 individually perturbed expression of lefty2 in heart, while the combination of 0.25 pmole each of hspb7MO1 and hspb12MO1 induced an even greater degree of abnormal expression. (L) Similarly, 0.25 pmole of hspb7MO1 or hspb12 MO1 induce a similar alteration in the expression of lefty1 in the brain, while the combination induces further abnormal expression.