Figure 1.
The phenotype of the s1pr2as10 mutant.
(A-H) Lateral views of wild-type (WT) (A, C, E and G) and s1pr2as10 mutants (B, D, F and H). Pericardial edema (B’), small eyes, and tail blisters (B’) were observed in 72-hpf s1pr2as10 mutant embryos (B). Two separate hearts (white arrows, D) were detected at 24 hpf, whereas a fused heart (white arrow, F) was observed at 48 hpf in s1pr2as10 mutant embryos. Paraffin sectioning with haematoxylin and eosin staining revealed the presence of two atria and one ventricle in s1pr2as10 mutant embryos at 72 hpf (H). (I-M) Ventral views of wild-type (WT) (I), s1pr2as10 (J), mil (K), and the intercross mutant progeny of s1pr2as10 and mil mutants (L and M) at 24 hpf are shown. The arrows indicate the positions of the hearts. (N) Cardiomyocytes were labeled via cmlc2 staining. Two representative s1pr2as10 mutant embryos are shown. Both contacting and separated cardiomyocytes were detected in the s1pr2as10 mutant embryos from the 22 ss to 28 hpf. (O) Percentages of WT and s1pr2as10 mutants containing contacting myocardia at different developmental stages. Scale bar = 100 µm. The error bars indicate the standard error.
Figure 2.
Development at low temperature rescues the s1pr2as10 cardiac phenotype.
(A) s1pr2as10 mutants raised at 28.5°C contained hearts with two atria (dark purple, amhc staining) and one ventricle (red, cmlc2 staining), and presented with tail blisters at 72 hpf. (B) s1pr2as10 mutant embryos raised at 22.5°C contained hearts with a single atrium and ventricle, and exhibited blood circulation at 72 hpf (arrow). (C) Percentages of s1pr2as10 mutant embryos raised at either 28.5 or 22.5°C containing two atria and one ventricle (2a1v) or one atrium and one ventricle (1a1v) at 72 hpf. (D) Percentages of s1pr2as10 mutant embryos raised at either 28.5 or 22.5°C displaying blood circulation at 72 hpf. The error bars indicate the standard error. Statistical significance was determined using Student’s t-test. *** indicates p<0.001.
Figure 3.
Low temperature shifts the timing of formation of the cardiac cone.
(A) Lateral views of 22-ss wild-type (WT) and s1pr2as10 mutant embryos raised at 28.5 or 22.5°C. (B) Time-lapse analyses of the medial migration of bilateral cardiomyocytes during the ∼17–30-ss in Tg(cmlc2:EGFP, cmlc2:H2AFZmCherry)cy13 transgenic fish (WT) or s1pr2as10 mutant embryos raised at 28.5 or 22.5°C. Five WT or s1pr2as10 mutant embryos were analyzed for each stage. The images outlined in red indicate the stage at which cardiac cone formation took place under each condition. (C) In situ hybridization of 19-ss wild-type (WT) or 26-ss s1pr2as10 mutant embryos raised at 28.5°C or 22.5°C with a cmlc2 RNA probe. The number of embryos displaying cmlc2 staining/the total number of embryos analyzed is shown for each panel. (D) Different degrees of myocardial migration defects were observed in gata5 and bon morphants at 24 hpf. Class I (single heart tube), Class II (cardiomyocytes in close proximity but not in contact), and Class III (two separate hearts) defects are shown. (E) Percentages of each class of myocardial migration defects in 10 ng gata5-MO or bon-MO-injected embryos raised at 28.5 or 22.5°C. Scale bar = 100 µm. Statistical significance was determined using Student’s t-test. * indicates p<0.05.
Figure 4.
Gene ontology (GO) and pathway analyses for differentially expressed genes identified by DNA microarray analysis.
GO and pathway analyses were conducted on different groups of genes using two strategies. (A) Genes with a greater than 1.3-fold change in expression between embryos raised at 28.5 and 22.5°C were used in strategy 1. GO analysis was performed using the zebrafish database. (B) Genes with a greater than 1.5-fold change in expression between embryos raised at 28.5 and 22.5°C were used in strategy 2. GO analysis was performed using the human database. The GO and pathway analyses were performed on genes from both Groups I and II or on genes from Groups III, IV and V. Box and Whisker plots for each group indicate the median (line within the box), 25th and 75th percentiles (top and bottom of the box), and 10th and 90th percentiles (Whiskers/error bars). The P-value from GO and pathway analyses are smaller than 0.1.
Figure 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 s1pr2as10 (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 s1pr2as10 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 s1pr2as10 mutant embryos raised at 22.5°C. The error bars indicate the standard error. (J) Knockdown of fn1 expression in s1pr2as10 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 s1pr2as10 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.
Figure 6.
Expression of two tenascin genes is affected by temperature.
In situ hybridization against tenascin-c (tnc) (A-H) and tenascin-w (tnw) (L-O) in 22-ss wild-type (WT) and s1pr2as10 (as10) mutant embryos raised at 28.5°C or 22.5°C. Expression of tnc increased in the pharyngeal arches (white arrows in B and D) and the cells between the brain and eyes of embryos raised at 22.5°C, but was unaffected in the trunk somite region (E-H). (I) qRT-PCR revealed a trend towards increased expression of tnc mRNA in s1pr2as10 mutant embryos raised at 22.5°C. (J) Knockdown of tnc expression with tnc-MO1 in s1pr2as10 mutants increased the percentage of 26-ss embryos raised at 22.5°C with the Class III cardia bifida phenotype. (K) Injection of s1pr2as10 mutant embryos raised at 28.5°C with human TNC protein partially rescued cardia bifida phenotypes at 24 hpf. (L-O) Decreased tnw expression in all tissues, including scattered epidermal cells in the head (black arrows in M and O) was observed in both 22-ss WT and s1pr2as10 mutants raised at 22.5°C. (P) qRT-PCR was used to confirm that expression of tnw mRNA is significantly reduced in 22-ss WT and s1pr2as10 mutant embryos raised at 22.5°C. (Q) Knockdown of tnw with tnw-MO1 in s1pr2as10 mutant embryos raised at 28.5°C partially rescued cardia bifida phenotypes at 24 hpf. (R) Injection of s1pr2as10 mutant embryos raised at 22.5°C with 100 pg tnw mRNA increased the percentage of 26-ss embryos with the Class III cardia bifida phenotype. Scale bars = 100 µm. The error bars indicate the standard error. Statistical significance was determined using Student’s t-test. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.
Figure 7.
Reactive oxygen species (ROS) mediate mitigation of cardia bifida in zebrafish embryos incubated at low temperature.
(A) s1pr2as10 mutant embryos raised at 22.5°C were treated with 50 or 150 µM N-acetyl cysteine (NAC) at the tailbud stage, and were then incubated at 22.5°C. Different degrees of myocardial migration defects were observed at the 26 ss. Class I (single heart tube), Class II (cardiomyocytes in close proximity but not in contact), and Class III (two separate hearts). (B) A proposed model showing how low temperature mitigates cardia bifida in zebrafish embryos. Statistical significance was determined using Student’s t-test. * indicates p<0.05.