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Fig. 3. Ectopic expression of HCN2 rescues the BR− muscle phenotype. a Embryos were microinjected (Inj) with HCN2 mRNA (wild-type channel, WT) either in the two cells (HCN2 WT-group, turquoise arrows) or in one cell (1/2 HCN2 WT, see b blue arrow) at the two-cell stage. Brain was removed at stage 25, and animals with and without brain (Ctrl and BR−, respectively) were analyzed for muscle structure and patterning at early- (stages 30–41) and late-stage (stages 42–48). b 1/2 HCN2-WT injection: embryos were microinjected with HCN2 and lacZ mRNA in one of the cells at two-cell stage (blue arrow). The injection side was confirmed by enzymatic detection of β-galactosidase, β-gal (dorsal view of one Ctrl animal is showed on the right). Rostral is up. Scale bar, 1 mm. c Quantification of the mean percentage of abnormal embryos (macroscopic phenotype) and statistical comparisons between uninjected BR− embryos (No Inj) and the different injected-BR− populations (Water, black arrows: water-injection in the two cells; HCN2: HCN2-WT mRNA injection in the two cells; 1/2 HCN2 + β-gal: co-injection of HCN2-WT and lacZ gene reporter in one LR side). Values are plotted as mean % ± s.d. (no-pooled data from two different replicates). d, e Typical muscle phenotype for uninjected BR− (d) and HCN2-WT injected BR− (e), as seen under polarized light. Rostral is upper right and dorsal is up. Scale bar, 100 μm. f, g Quantification of the mean length of myotome fibers and statistical comparisons among uninjected Ctrl and uninjected BR− (BR−), HCN2-WT injected BR- and 1/2 HCN2-WT (measured on uninjected contralateral side) at early- (f one-way ANOVA, P < 0.01) and late- (g one-way ANOVA, P < 0.01) stages after brain removal. No significant differences after a posteriori analysis were detected among the different Ctrl groups. Data represent the mean and s.d. of two independent replicates. For all panels, number in bars indicates n or number of animals for each group. P values after after z-test c or post-hoc Bonferroni’s test f, g are indicated as **P < 0.01, ns P > 0.05 Image published in: Herrera-Rincon C et al. (2017) © The Author(s) 2017. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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