FGF mediated MAPK and PI3K/Akt Signals make distinct contributions to pluripotency and the establishment of Neural Crest
- Northwestern University, United States
Figures
Figure 1 with 1 supplement
FGF signaling is required for proper blastula stage gene expression.
(A) In situ hybridization examining FGFR4 expression in wildtype Xenopus embryos collected at blastula (stage 9, lateral view, animal pole up), late gastrula (stage 12, dorsal view, anterior up), …
Figure 1—figure supplement 1
Blocking FGF signaling using dnFGFR4, but not dnFGFR1, leads to a loss in neural crest gene expression at mid-neurula stages.
(A–B). In situ hybridization examining FoxD3, Sox9, and Snail2 expression in mid-neurula stage (stage 16) embryos injected with dnFGFR4 (A) or dnFGFR1 (B). Asterisk denotes injected side, marked by …
Figure 2
Blocking FGF signaling in pluripotent blastula cells interferes with adoption of an epidermal state and neuralizes cells.
(A–D) In situ hybridization examining expression of Epidermal Keratin (EPK) (A) Oct60 (B) Sox3 (C) or Nrp1 (D) in animal pole explants injected with dnFGFR4 or chordin for phenotypic comparison. …
Figure 3 with 1 supplement
FGF signaling directs the transit from a pluripotent to a lineage restricted state through regulation of Erk and Akt activation.
(A) Schematic representation of the FGF receptor and select signaling cascades activated downstream, highlighting the Ras/MAPK (Erk) and PI3K/Akt cascades. (B) Western blot of lysates from animal …
Figure 3—figure supplement 1
Meki (RDEA119) and PI3Ki (LY294) block activation of the MAPK and Akt cascades respectively.
(A–B) Western blot of lysates from animal pole explants treated with DMSO, Meki, or PI3Ki. Stage 9 (A) or Stage 13 (B) explants were cultured in vehicle or inhibitor treated media and collected …
Figure 4 with 1 supplement
PI3K/Akt signaling but not MAPK signaling is required for pluripotent blastula cells to transit to a neural progenitor state.
(A–B) Animal pole explant assay examining Sox2, Sox3, and Nrp1 expression in Chordin (Chd) induced animal cap explants treated with Meki (RDEA119) (A) or injected with dnPI3K (B). Explants were …
Figure 4—figure supplement 1
Blocking PI3K/Akt activation using PI3Ki (L) (LY294) or PI3Ki (W) (Wortmannin) phenocopies the effects of dnPI3K on Chordin-mediated neural induction.
(A–C) Animal pole explant assay examining Sox2, Sox3, and Nrp1 expression in Chordin (Chd) induced animal cap explants injected with dnRaf (A) or treated with PI3Ki (L) (B) or PI3Ki (W) (C). …
Figure 5 with 2 supplements
MAPK and PI3K/Akt differentially alter the transit of pluripotent cells to restricted cell states.
(A–B) Animal pole explant assay examining expression of Xbra and MyoD (A) or Endodermin and Sox17 (B) in explants cultured with or without activin after treatment with Meki (RDEA119) or injection …
Figure 5—figure supplement 1
MAPK and PI3K/Akt signaling are differentially required for Endodermin expression.
(A) Animal pole explant assay examining expression of Endodermin in explants cultured with or without activin after injection with dnRaf or treatment with PI3Ki (L) (LY294) or PI3Ki (W) …
Figure 5—figure supplement 2
Blocking MAPK or PI3K activation differentially alters animal pole explant gene expression (A–B) qRT-PCR analysis of animal cap explants treated with Meki (RDEA119) and cultured alongside sibling embryos collected at blastula stages (stage 9) (A) or treated with PI3Ki (LY294) and cultured alongside sibling embryos collected at early neurula stages (stage 13) (B).
Blocking MAPK activation inhibits Snail1, FoxD3, Zic1, and Sox2 expression while blocking PI3K activation increases Zic2, Sox2, Sox3, Sox11, Sox17, and MyoD expression. (*p<0.05; **p<0.01).
Figure 6
Prolonged MAPK activation alters the timing of pluripotency gene expression.
(A–B) Animal pole explant assays examining Sox3 expression in animal cap explants injected with constitutively active Mek (Act-Mek). Explants were cultured alongside sibling embryos and collected at …
Figure 7 with 2 supplements
Reprograming cells to a Neural Crest state establishes and requires high MAPK and low PI3K/Akt activity.
(A) Western blot of lysates from Pax3-GR/Zic1-GR injected animal pole explants. Explants were cultured alongside sibling embryos and collected at blastula (stage 9) and early neurula (stage 13) …
Figure 7—figure supplement 1
Reprograming to a Neural Crest state sustains FGFR4 expression.
(A) Animal pole explant assay examining FGFR4 expression in animal cap explants injected with Pax3-GR/Zic1-GR. Explants were cultured alongside sibling embryos and collected at blastula (stage 9), …
Figure 7—figure supplement 2
MAPK activation is required for neural crest factor expression at mid-neurula stages.
(A–D). In situ hybridization examining FoxD3, Sox9, and Snail2 expression in mid-neurula stage (stage 15) embryos injected with dnRaf (A) or treated with the PI3Ki (Wortmannin) (C). Western Blot …
Figure 8
Summary of the effects of MAPK or PI3K/Akt inhibition.
(A) Schematic representation of MAPK and PI3K/Akt cascade activation in animal cap explants (staged by sibling embryos) at the blastula stage (stage 9), midgastrula stage (stage 11), and early …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.33845.017
