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Figure 9. BMP-4 expression is reduced and incorrectly localized in response to altering HCN4 expression. (A) At NF stage 27 BMP-4 is expressed in 2 bilateral patches around the developing heart field (i). Altering HCN4 caused a loss of definition to the fields (ii) as well as an overall reduction in the size of the fields and intensity of expression (iii). (B) BMP-4 remains bilaterally expressed during heart looping (NF stage 32) with an additional asymmetric area of expression in the myocardium (yellow arrow, i). This left-sided myocardial expression helps to direct and orient heart looping. Overexpression of HCN4 and expression of HCN4-DN(AAA) not only disrupted the lateral BMP-4 fields of expression (ii), it also caused both a randomization and loss of myocardial expression (white arrow, iii). (C) By the end of looping at NF stage 35, BMP-4 is evenly expressed in the myocardium and retains its bilateral expression in the surrounding tissues (i). Similar to stage 27 and 32, disruption of HCN4 led to both a reduction in expression as well as a loss of field definition (ii, iii). Injected embryos frequently displayed malformed hearts who did not complete looping properly (yellow vs. white arrows). All abnormal images are from HCN4-DN(AAA) injected embryos but similar phenotypes were observed with HCN4-OE embryos. (A-C) Images are ventral views with anterior at top. (D) All stages and injection conditions induced significantly higher percentages of abnormal phenotypes compared with controls (Stage 27: χ2 = 32.4768, df = 4; Stage 32: χ2 = 82.2366, df = 4; Stage 35: χ2 = 36.0791, df = 4; All: *p < 0.05). There was no statistical difference between overexpression and dominant-negative phenotypes. n = 58–79 embryos per condition across 3 biological replicates. OE = HCN4-OE, DN = HCN4-DN(AAA). Scale bars = 200 μm. |
