Fig. 5

Expression of fibronectin 1 is affected by temperature.

(A-H) In situ hybridization against fibronectin 1 (fn1) in 22-ss wild-type (WT) (A, B, E, F) and s1pr2^as10 (as10) mutant (C, D, G, H) embryos raised at 28.5 or 22.5°C. Expression of fn1 mRNA increased at the midline region (white arrows in B and D) and bilateral LPM (black arrows in B and D) of both WT and s1pr2^as10 mutant embryos raised at 22.5°C, as compared to those raised at 28.5°C (A and C). Expression of fn1 at the tail bud or yolk regions was not affected (E-H). (I) qRT-PCR revealed a trend towards increased expression of fn1 mRNA in s1pr2^as10 mutant embryos raised at 22.5°C. The error bars indicate the standard error. (J) Knockdown of fn1 expression in s1pr2^as10 mutants increased the percentage of 26-ss embryos raised at 22.5°C with the Class III cardia bifida phenotype. (K) Injection of human fibronectin protein into s1pr2^as10 mutant embryos raised at 28.5°C partially rescued the cardia bifida phenotypes at 24 hpf. (K′) The red dye indicates the protein injection region at 14–16 hpf. Scale bars = 100 μm. Statistical significance was determined using Student’s t-test. * indicates p<0.05, *** indicates p<0.001.