| |
FIGURE 1: FoxA4 depletion altered the expression of mesodermal markers, impaired axis elongation, expanded the prechordal mesoderm and reduced the notochord.
(A–J) Expression of mesodermal markers at stage 13, dorsal views. (A, F) myod. (B, G) bra. (C, H) gsc. (D, I) frzb1. (E, J) chd. Embryos were injected into one dorsal cell at the 4-cell stage with 40 ng of CtrlMO (A) or of FoxA4MO (B), or with 20 ng of each MO into both dorsal cells at the 4-cell stage, (CtrlMO: B,C,E; FoxA4MO: G, H, J) or at the 1-cell stage (CtrlMO: D; FoxA4MO: I). The myoD domain was shortened on the injected side (is) by FoxA4MO (58%, n = 12) (F). The bra domain was split by FoxA4MO (G) (79%, n = 24). The gsc domain was posteriorly expanded by FoxA4MO (100%, n = 15) (H). The frzb1 domain was posteriorly expanded by FoxA4MO (100%, n = 14) (I). The chd domain was affected in 100% of the injected embryos (n = 14) (J). Among these, 71% showed an expansion of the anterior chd domain, corresponding to the PM (green arrow), while the posterior domain (corresponding to the notochord) was severely reduced (red arrow). In the remaining 29%, the entire axial mesoderm appeared much thinner and shorter than in CtrlMO-injected siblings (inset in J). Similar results were obtained after injection of 20 ng of FoxA4MO into 1-cell stage embryos (Fig. S1B). (K) The body length between the cement gland and the tip of the tailbud was measured at tailbud stage in embryos injected before the first cleavage with 20 ng of CtrlMO (green bar, n = 25) or of FoxA4MO (red bar, n = 27). P<0.0001, two-tailed t-test. (L) Notochord/embryo length ratio measured in the group of embryos corresponding to those shown in (B, G). Green bar, CtrlMO (n = 18); red bar, FoxA4MO (n = 24). P<0.0001, two-tailed t-test. (M–R) Expression of axial mesodermal markers at stage 10.25. (M, P) gsc. (N, Q) frzb1. (O, R) chd. Embryos were injected with 20 ng of CtrlMO (M–O) or of FoxA4MO (P–R). Injections were performed unilaterally in 1 cell at the 2 or 4-cell stage or at the 1-cell stage, giving similar results. Unilateral injections are shown here. The injected side was identified by DOG fluorescence and is oriented to the right (insets in M–R). In gastrulae, we were able to observe an expansion of gsc (58%, n = 40; P) and of frzb1 domains (88%, n = 33; Q). chd expression was reduced in the less involuted, superficial cells on the injected side (red arrows), but a cloud of deep cells expressing chd persisted (green arrows), and even appeared to be expanded on the injected side in some embryos, like the one shown below (50%, n = 10) (R).
doi:10.1371/journal.pone.0110559.g001 |
