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Figure 2. Laterality Defects in Genome-Edited F0 myo1d Mutant Tadpoles
(A) Schematic depicting Myo1d protein structure (sgRNA sites indicated).
(B and C) Appearance and pitx2 gene expression in WT (B) and F0 myo1d mutant (C) tadpoles.
(D) Compilation of pitx2 expression patterns. BCD, blastopore closure defect; NTD, neural tube closure defect. Note that mutant embryos with WT appearance showed WT pitx2 expression in the left LPM, while stunted specimens with a convergent extension (CE) phenotype lacked expression or displayed mRNA expression on both sides. |
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Figure 4. Functional Interaction between myo1d and PCP
(A and B) Morphant specimens displayed enhanced apical surfaces of GRP cells at stage 18 (A) and delayed neural tube closure at stage 18 (B).
(C) Convergent extension defects in Keller open-face explants of myo1d morphants at stage 22.
(D) Co-injection of myo1d AUG-MO with two antisense MOs directed against vangl2 (at sub-phenotypic doses each) disrupted LR axis formation, as determined by expression of pitx2 in the LPM. Numbers represent analyzed specimens, which were derived from 3 independent experiments for apical constriction defects of GRP cells, 7 experiments for neural tube closure delay, and 4 experiments for myo1d and vangl2 interaction during LR axis formation. To determine the cell surface area, 25 cells from a central part of the GRP were analyzed in each case.
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Figure S1. Myo1d is required for LR axis formation, Related to Figure 1.
(A-H) nodal1 (A-D) and pitx2 (E-H) expression in wildtype (A, E) and myo1d morphant (B-D, F-H) embryos, as
determined by WM-ISH with antisense probes for nodal1 (A-D) and pitx2 (E-H).
(I, J) Downregulation of Myo1d protein in myo1d morphants. (I) Tadpole lysates were probed with Mab4E12 before
(left) and after (right) affinity purification (ap). (J) Embryos were injected at the 2-4 cell stage with 1 ng of AUG-MO or
an antisense MO containing 5 mismatches (MM-MO). Affinity purified Mab4E12 was used to probe western blots
containing lysates from stage 28 embryos.
(K, L) Lefty1 mRNA expression in wildtype (K) and myo1d morphant (L) specimen, as shown by transversal
histological sections of WM-ISH stained embryos at stage 24. fp, floor plate; hc, hypochord; MM-MO, mismatch MO. |
