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Figure 1. Manipulation of Rab11 function at the one- or four-cell stage leads to indistinguishable and weak effects on Pitx2c expression in the lateral plate mesoderm. (a-f) Effectiveness of constructs. Both Xenopus and human dnRab11 constructs interfere with integration of multiciliated cells (MCC) into the tadpole epidermis. Four-cell embryos were injected with mRNA encoding nuclear β-galactosidase (nuclear β-Gal) alone (a, d) or in combination with Xenopus (b, c) or human (e, f) wildtype (b, e) or dominant-negative (c, f) Rab11 mRNAs. Embryos were cultured to tadpoles stages and processed for scanning electron microscopy. Note that wildtype Rab11 did not impact on MCC migration while MCCs were virtually absent from dnRab11-injected specimens, as described (Kim et al., ). Scale bar represents 10 µm. (g-l) Low-frequency laterality defects in Rab11-manipulated Xenopus tadpoles. Embryos were injected at the one- or four-cell stage into the marginal zone with 2.4 (low) or 3.2 (high) ng of Xenopus or 9.6 ng of human dnRab11, cultured to Stage 30 and processed for Pitx2c ISH. Specimens were scored for wildtype (g), bilateral (h), inverted (i), or absent (j) Pitx2c expression in the LPM. (k, l) Deviations from normal left-sided patterns were pooled as LR-defects in bar graphs representing statistical evaluation of Xenopus (k) and human (l) dnRab11 injections. Note that differences between one- and four-cell injections were not significant (n.s.) in most cases, and that in the two cases where significances were recorded, the four-cell injections were more efficient. Note also that effects were overall low except for cases when very high doses of human dnRab11 were injected (l). n, number of embryos analyzed. |
