Lineage commitment of embryonic cells involves MEK1-dependent clearance of pluripotency regulator Ventx2
- Aix Marseille Univ, CNRS, France
Figures
Figure 1 with 2 supplements
MEK1 depletion impairs embryonic development.
(A) Mk-MO and Mk-MO ATG were designed to target MEK1 translation. Western blot analysis of blastula stage nine embryos injected with 25 ng per blastomere of either MO at the 4 cell stage revealed …
Figure 1—figure supplement 1
MEK1 depletion by morpholinos.
(A) Four-cell embryos were injected in each blastomere with 50 pg GFP‐CAAX mRNA with or without 25 ng Mk-MO, fixed at blastula stage 9, cryosectioned and stained with anti-phospho-MEK1 antibody. The …
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Figure 1—figure supplement 1—source data 1
Values of blastopore closure ratios.
Details are shown in Figure 1—figure supplement 1 and Materials and methods.
- https://doi.org/10.7554/eLife.21526.004
Figure 1—figure supplement 2
Gene expression analysis of MEK1-depleted gastrula embryos.
(A–D) Four-cell embryos were injected in each blastomere with 25 ng Mk-MO, collected at early gastrula stage 10.5 and processed for RT-qPCR to quantify changes in the expression levels of …
Figure 2 with 2 supplements
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 …
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 …
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 …
Figure 3 with 1 supplement
MEK1 is required for Ventx2 clearance and asymmetric distribution during cell division.
(A,B) Four-cell embryos were injected in each cell with 50 pg GFP-CAAX, 50 pg Ventx2-Myc, 50 pg 2SAVentx2-Myc RNAs, and 25 ng Mk-MO, as indicated. Embryos were fixed at blastula stage 9, or gastrula …
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Figure 3—source data 1
Myc signal intensity ratios between daughter nuclei.
Each value corresponds to the ratio calculated from one individual confocal slice between α and β daughter nuclei (see legend to Figure 3 and Materials and methods for further details).
- https://doi.org/10.7554/eLife.21526.010
Figure 3—figure supplement 1
Ventx2 degradation and asymmetric distribution require MEK1 activity.
(A) In silico analysis of phosphorylation sites in the Ventx2 protein and prediction of kinases involved, with Kinasephos2 software. (B) Schematic representation of the Ventx2 protein. HD indicates …
Figure 4 with 1 supplement
Ventx2 knockdown rescues the competence of MEK1-deficient cells to differentiate.
(A) 4 cell embryos were injected in each cell with 50 pg Ventx2-Myc or 50 pg 2SAVentx2-Myc RNAs, and processed for WISH analysis at late gastrula stage 13 with pou5f3.2 probe. Stabilized 2SAVentx2 …
Figure 4—figure supplement 1
Ventx2 knockdown restores germ-layer formation in MEK1-deficient embryos.
(A) Four-cell embryos were injected with 50 pg 2SAVentx2-Myc RNA per cell, fixed at tailbud stage 25 and processed for WISH with pou5f3.2 probe. (B) Four-cell embryos were injected with 30 ng …
Additional files
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Supplementary file 1
GSK3 is not a negative regulator of the pluripotency gene network and is not required for MEK1-dependent Ventx2 clearance in vivo.
(A) Four-cell embryos were injected with 300 pg DN-GSK3 RNA per blastomere. Embryos were collected at stage 25 and processed for WISH analysis with sox2 probe. DN-GSK3 efficiently induced secondary body axes, indicating that the dose used was functional. (B) Embryos injected as in (A), were collected at stage 10.5 and processed for RT-qPCR. (C-D) Embryos injected as in (A) were processed for WISH analysis at early gastrula stage 10.5 with gsc (C, vegetal view) and ventx2 (D, top: vegetal view, bottom: animal view) probes. (E) Embryos injected at the 8 cell stage with 300 pg DN-GSK3 RNA in one dorsal animal blastomere were processed for WISH analysis at late gastrula stage 13 with pou5f3.2 and ventx2 probes (anterior view). (F-G) Four-cell embryos were injected in each cell with 50 pg GFP-CAAX, 50 pg Ventx2-Myc, and 25 ng Mk-MO (F), or 50 pg GFP-CAAX, 50 pg Ventx2-Myc and 300 pg of DN-GSK3 (G) Animal caps were explanted at blastula stage nine and cultured until gastrula stage 11, fixed and processed for anti-Myc (red), and anti-GFP (green) immunostaining, and DNA was stained with DAPI (blue). Note that Ventx2-Myc is detectable only in MEK1 depleted caps. For the qPCR graph, error bars represent s.e.m. values of three 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. In C, D and E, the number of embryos exemplified by the photograph over the total number of embryos analyzed is indicated. In F and G scale bar is 20 μm.
- https://doi.org/10.7554/eLife.21526.014
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Supplementary file 2
Phylogenetic tree of Ventx deuterostome genes.
Boxes indicate members with orthology relationship, like coelacanth Ventx, Xenopus Ventx2 and human VENTX (blue arrows). Sequences were collected from ENSEMBL, JGI, A-STAR and NCBI public databases (see Supplementary file 4). Ventx homeodomain sequences were aligned using Jalview software (RRID:SCR_006459) and the phylogenetic tree was obtained by Neighbor Joining analysis of percentage identity.
- https://doi.org/10.7554/eLife.21526.015
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Supplementary file 3
The Evolutionary history of Ventx family genes.
(A) Synteny of the Ventx genomic region in gnathostomes. Blue dotted boxes indicate species-specific gene duplication events. Note that a triplication event, giving rise to Ventx1, Ventx2 and Ventx3, occurred in the last common ancestor of tetrapods. One or more Ventx paralogs was subsequently lost during squamata, archosaura and testudina evolution. Mammals lost both Ventx1 and Ventx3 paralogs and exclusively kept Ventx2. Mouse represents an extreme case with a total loss of Ventx genes. (B) Simplified tree of vertebrates, which displays typical situations regarding the number of Ventx genes in main evolutionary branches.
- https://doi.org/10.7554/eLife.21526.016
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Supplementary file 4
EMBOSS prediction of PEST destruction motifs in Ventx orthologs.
- https://doi.org/10.7554/eLife.21526.017
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Supplementary file 5
Probes used for WISH.
- https://doi.org/10.7554/eLife.21526.018
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Supplementary file 6
Primers used for RT-QPCR.
- https://doi.org/10.7554/eLife.21526.019
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Source data 1
Related to Supplementary file 2.
VENTX homeodomain sequences.
- https://doi.org/10.7554/eLife.21526.020
