Figure 7.

Altering normal HCN4 expression results in aberrant Lefty expression. (A) As visualized via in situ hybridization analysis, Lefty is normally expressed unilaterally in the left lateral plate mesoderm in NF stage 27 embryos (i). Altering HCN4 expression induced both full or partial bilateral and abnormal Lefty expression but did not result in a complete reversal of expression (solely right-sided). One of the most frequently observed bilateral phenotypes was a partial secondary field on the right side of embryos with the midline maintained by an absence of Lefty expression (ii). In the other commonly seen phenotype, Lefty transcripts were expressed across the anterior ventral surface of animals in an undefined shape with no visible midline gap (iii). Images are ventral views with anterior at top. (B) Overexpressing HCN4 and HCN4-DN(AAA) injection led to significant percentages of abnormal embryos compared with uninjected embryos (χ² = 19.0086, df = 4, *p < 0.05). There was no statistical difference between overexpression and dominant-negative phenotypes. n = 56–71 embryos per condition across 3 biological replicatesOE = HCN4-OE, DN = HCN4-DN(AAA). Scale bars = 200 μm.