Loss of Mcm2 disrupts left-right asymmetry development. (A) Heart looping is randomized upon Mcm2 knockdown. Upper panel shows representative images of correct (D-loop) and aberrant heart looping (no loop, L-loop) at 48 hpf after in situ hybridization (ISH) for cmlc2. A, atrium; V, ventricle. Scale bar: 100 μm. Stacked bar graph summarizes heart looping experiments. D, D-loop; N, no loop; L, L-loop. P = 0.0003 (CTRL vs Mcm2 MO), P < 0.0001 (splCTRL vs Mcm2 splMO). n = 5–10 experiments. Number of embryos: NI = 309; CTRL = 166; Mcm2 MO = 128; splCTRL = 113; Mcm2 splMO = 194. (B) Example images of insulin (ins) ISH labelling of the endocrine pancreas (arrows). R, right (correct) or L, left (wrong position) of the embryonic midline. Scale bar: 100 μm.Graph displays quantification of aberrant pancreas position upon Mcm2 knockdown. P = 0.0052 (CTRL vs Mcm2 MO), P < 0.0001 (splCTRL vs Mcm2 splMO). n = 4–9 experiments. Number of embryos: NI = 299; CTRL = 146; Mcm2 MO = 109; splCTRL = 113; Mcm2 splMO = 192. (C) Loss of Mcm2 results in ambiguous southpaw (spaw) expression at 22 ss, which can be partially rescued by co-injection of mcm2 RNA. Upper panel shows examples of correct and aberrant spaw distribution. P < 0.0001 (splCTRL vs Mcm2 splMO), P = 0.0029 (splMO vs Mcm2 splMO + mcm2 RNA). n = 3 experiments. Number of embryos: NI = 156; splCTRL = 166; Mcm2 splMO = 152, mcm2 RNA = 142; Mcm2 splMO + mcm2 RNA = 181. (D) ML216-mediated inhibition of Bloom and Werner helicases from tailbud stage until 22ss does not affect asymmetry development. n = 3 experiments. Number of embryos: DMSO = 56; 1 μM ML216 = 61; 5 μM ML216 = 60; 10 μM ML216 = 57; 25 μM ML216 = 54; 50 μM ML216 = 53. All data analysed using two-tailed Fisher's exact test.
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