Vg1-Nodal heterodimers are the endogenous inducers of mesendoderm
- Harvard University, United States
- Broad Institute of MIT and Harvard, United States
- Harvard Stem Cell Institute, United States
Figures
Figure 1 with 2 supplements
Maternal vg1 is required for mesendoderm formation.
(A) Zygotic and maternal vg1 (Zvg1 and Mvg1) mutants and wild-type (WT) embryo at 28 hours post-fertilization (hpf). See Figure 1—figure supplement 1 for information about the vg1 mutant alleles, …
Figure 1—figure supplement 1
vg1 mutant alleles.
(A) Single guide RNAs (sgRNAs) targeting the vg1/dvr1/gdf3 gene were designed using CHOPCHOP (Labun et al., 2016; Montague et al., 2014). Colored arrows indicate possible sgRNA targets, color-coded …
Figure 1—figure supplement 2
Left-right asymmetry in Zvg1 mutants.
(A) Heart positioning at 28 hpf in WT and Zvg1 embryos, detected by expression of cmlc2 by in situ hybridization. (B) Expression of southpaw (spaw), a Nodal ligand involved in left-right asymmetry, …
Figure 2
Endogenous Nodal signaling requires Vg1.
(A) Mvg1, maternal-zygotic vg1 (MZvg1) and maternal-zygotic oep (MZoep) mutants at 28 hpf. (B) Expression of Nodal target genes gsc, lft1 and ntl at 50% epiboly and sox32 at 90% epiboly in WT, Mvg1 …
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Figure 2—source data 1
Raw qPCR data for Figure 2E and F.
- https://doi.org/10.7554/eLife.28183.007
Figure 3 with 1 supplement
Vg1 processing requires Nodal.
(A) superfolderGFP (sfGFP) was inserted into vg1 downstream of the predicted basic cleavage site. (B) Mvg1 embryo injected with 50 pg of vg1-sfGFP mRNA, shown at 28 hpf. (C) Anti-GFP reducing …
Figure 3—figure supplement 1
Vg1 processing requires Nodal.
(A) Anti-GFP reducing immunoblot of WT and Mvg1 embryos injected with 50 pg of vg1-sfGFP or vg1-NC-sfGFP mRNA and 50 pg of cyc or sqt mRNA. Black arrowhead indicates full-length Vg1-sfGFP, open …
Figure 4 with 1 supplement
Vg1 secretion requires Nodal.
(A) Live imaging of Mvg1 embryo injected with 50 pg of vg1-sfGFP mRNA. Scale bar, 17 um. (B) Mvg1 embryos co-injected with 50 pg of vg1-sfGFP mRNA and 50 pg of cyc, sqt or bmp7a mRNA. Scale bar, 17 …
Figure 4—figure supplement 1
Vg1 secretion requires Nodal.
(A) Mvg1 embryos co-injected with 20 pg of vg1-sfGFP mRNA and 0.5 pg, 2 pg or 20 pg of cyc or sqt mRNA. These images were all captured under the same microscope settings. Arrowheads indicate small …
Figure 5 with 2 supplements
Vg1 and Nodal form heterodimers, and are only active when co-expressed.
(A) Anti-Flag reducing immunoblot (IB) of anti-HA immunoprecipitates (IP) from lysates of Mvg1 embryos injected with 50 pg of cyc-HA, sqt-HA and/or 50 pg of vg1-Flag mRNA. Black arrowhead indicates …
Figure 5—figure supplement 1
Vg1 and Nodal form heterodimers.
(A) Anti-Flag reducing immunoblot of anti-HA IP from lysates of WT embryos injected with 50 pg of vg1-HA mRNA and 50 pg of cyc-Flag or sqt-Flag mRNA. Black arrowhead indicates full-length protein; …
Figure 5—figure supplement 2
Vg1 and Nodal are only active when co-expressed.
(A) Transplantation experiments with DAB staining to show the location of the transplanted cells (orange) and lft1 expression (purple). (B) in situ hybridizations for a mesoderm marker (lft1) and an …
Figure 6
Vg1 can enable rapid response to low concentrations of Nodal.
Kinetic model comparing Nodal homodimer formation in the absence of Vg1 (red line) and Vg1-Nodal heterodimer formation in the presence of a maternal Vg1 pool (blue line). For both conditions Nodal …
Figure 7
Model for mesendoderm induction in zebrafish.
As described in the main text: (1) vg1 mRNA is inherited from the mother, and is ubiquitous in the early embryo; (2) Vg1 protein is synthesized ubiquitously and retained predominantly in the ER; (3) …
Tables
Table 1
Quantification of Vg1-sfGFP localization in Mvg1 embryos co-injected with 20 pg of vg1-sfGFP mRNA and 0.5–20 pg of cyc or sqt mRNA.
See Figure 4—figure supplement 1A for examples of Vg1-sfGFP extracellular localization.
| mRNA co-injected with 20 pg vg1-sfGFP | Intracellular | Extracellular puncta | Extracellular diffuse | Extracellular puncta + diffuse | |
|---|---|---|---|---|---|
| cyc (pg) | 0.5 | 2 | 3 | 0 | 0 |
| 1 | 5 | 5 | 0 | 0 | |
| 2 | 0 | 8 | 0 | 0 | |
| 5 | 0 | 7 | 0 | 0 | |
| 10 | 0 | 6 | 0 | 0 | |
| 20 | 0 | 9 | 0 | 0 | |
| sqt (pg) | 0.5 | 5 | 0 | 0 | 0 |
| 1 | 4 | 4 | 1 | 0 | |
| 2 | 0 | 3 | 2 | 4 | |
| 5 | 0 | 6 | 0 | 0 | |
| 10 | 2 | 0 | 3 | 5 | |
| 20 | 0 | 2 | 5 | 3 | |
Additional files
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Source code 1
Nodal homodimerization versus Vg1-Nodal heterodimerization simulation.
- https://doi.org/10.7554/eLife.28183.018
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Source code 2
Ordinary differential equations for Nodal homodimer simulation.
- https://doi.org/10.7554/eLife.28183.019
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Source code 3
Ordinary differential equations for Vg1-Nodal heterodimer simulation.
- https://doi.org/10.7554/eLife.28183.020
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Transparent reporting form
- https://doi.org/10.7554/eLife.28183.021
