Excess Gpr125 causes C&E movement defects. (A-E) Lateral views of uninjected or gpr125 RNA injected embryos at 1 day post-fertilization (dpf). Anterior is leftwards. AP axis length phenotypic categories. Blue, greater than 95%; green, 80-95%; yellow, 40-79%; red, smaller than 40% of the average AP axis length of control embryos. Arrowheads in D and E indicate the cyclopic eyes and change of head position. (F-J) Ventral views of uninjected or gpr125 RNA-injected embryos at 3 dpf. Anterior is leftwards. Eye fusion defects were categorized into five groups (I-V) according to Marlow et al. (Marlow et al., 1998). (K) Quantification of AP axis shortening and eye fusion phenotypes. The colored bars correspond to the AP axis length phenotypic categories shown in B-E. Eye fusion phenotypes were quantified by cyclopia index (CI) according to Marlow et al. (Marlow et al., 1998). CI values are above the bars and numbers of embryos analyzed inside the bars. (L-O) Whole-mount in situ hybridization analyses of marker gene expression in uninjected or 200 pg gpr125 RNA-injected embryos at the two-somite stage. (L,M) Animal pole views, ventral upwards. (N,O) Dorsal views, anterior upwards. n, notochord; ne, neural ectoderm border; pm, prechordal mesoderm; s, somites. Red line indicates the width of the notochord at the first somites. (P,Q) mEGFP-labeled notochord (n) of control or 200 pg gpr125-Cherry RNA-injected embryos at the one-somite stage. Anterior is upwards. All measured notochord cells are outlined and the notochord boundary of the gpr125-injected embryo is marked with dashed lines. (R-U) Analyses of LWR and mediolateral (ML) alignment in the ectoderm or notochord of control (n=3 embryos, 158 and 131 cells, respectively) or 200 pg gpr125-Cherry RNA-injected embryos (n=6 embryos, 266 and 220 cells, respectively) at the one-somite stage. Rose diagrams depict cell orientation relative to the AP axis (vertical dashed line). Corresponding LWRs are expressed as mean±s.e.m. in the lower right corners. (V,W) Punctate and cytosolic distribution of Pk-GFP in control or gpr125 RNA-injected embryos. (X) Classes of Pk-GFP distribution in control or gpr125 RNA-injected embryos (155 or 183 cells, respectively). (Y,Z) ML alignment and LWR of ectodermal cells analyzed for Pk-GFP localization in control or gpr125 RNA-injected gastrulae.
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