Figure 2. MEK1 depletion affects cell competence to exit pluripotency and enter into differentiation.

(A) Sixteen-cell embryos were injected in one animal blastomere with 25 ng Mk-MO ATG and 2.5 ng FLDX. Next, these embryos were injected at blastula stage 8.5 with recombinant BMP4 (2 ng), NODAL (10 ng), or NOGGIN (36 ng) proteins into the blastocoele, collected at early gastrula stage 10.5, and processed for WISH with xk81a1 (epidermis, animal view), t/bra (mesoderm, lateral view), and sox2 (neural tissue, dorsal view). FLDX (orange staining) was used to trace Mk-MO injected cells (white arrows). (B) Four-cell embryos were injected with 25 ng Mk-MO per blastomere, animal caps were explanted at blastula stage 8.5 and cultured in the presence of 20 ng/ml BMP4, 100 ng/ml NOGGIN, 20 ng/ml NODAL (low), or 200 ng/ml NODAL (high) until late gastrula stage 13, and processed for RT-qPCR. (C) Four-cell embryos were injected with 25 ng Mk-MO per blastomere, collected at stage 10.5 and processed for RT-qPCR. (D) Embryos injected as in (C) were processed for WISH analysis at late gastrula stage 13 with pou5f3.2 (oct25) and ventx2 probes. a: animal view; v: ventral view; l: lateral view; d: dorsal view. For all qPCR graphs, error bars represent s.e.m. values of four independent experiments with two technical duplicates. For statistical analyses, samples were compared with the respective control by Unpaired Student’s t-test. *p<0.05, **p<0.005. ***p<0.0001. In A and D, the number of embryos exemplified by the photograph over the total number of embryos analyzed is indicated.

DOI: http://dx.doi.org/10.7554/eLife.21526.006

Figure 2—figure supplement 1. Neural induction in vivo depends on MEK1 activity.

Sixteen-cell embryos were injected in one ventral-animal blastomere with 3 ng of dominant-negative Smad5 (Smad5sbn) mRNA and 25 ng Mk-MO ATG, as indicated. Embryos were fixed at late gastrula stage 13, and processed for WISH with the indicated probes. The number of embryos exemplified by the photograph over the total number of embryos analyzed is indicated. Lateral views.

DOI: http://dx.doi.org/10.7554/eLife.21526.007

Figure 2—figure supplement 2. MEK1 is required to inhibit the expression of the pluripotency genes pou5f3.2 and ventx2.

(A) Embryos injected with 25 ng Mk-MO at 16-cell stage in one animal dorsal blastomere were grown until late gastrulation stage 13 and processed for WISH with pou5f3.2 and ventx2 probes. (B) Embryos injected with 25 ng Mk-MO at 16 cell stage in one animal ventral blastomere were grown until mid-neurula stage 18 and processed for WISH with pou5f3.2 probe. (C) Four-cell embryos were injected in each blastomere with 25 ng Mk-MO and grown until blastula stage 9, when animal caps were isolated, cultured in vitro until late gastrula stage 13 and then processed for RT-qPCR. In A and B, the number of embryos exemplified by the photograph over the total number of embryos analyzed is indicated.

DOI: http://dx.doi.org/10.7554/eLife.21526.008