Nie S and Chang C (2007), PI3K and Erk MAPK mediate ErbB signaling in Xen...

XB-ART-36374

Mech Dev 2007 Jan 01;1249-10:657-67. doi: 10.1016/j.mod.2007.07.005.

Show Gene links Show Anatomy links

PI3K and Erk MAPK mediate ErbB signaling in Xenopus gastrulation.

Nie S , Chang C .

???displayArticle.abstract???
ErbB signaling regulates cell adhesion and movements during Xenopus gastrulation, but the downstream pathways involved have not been elucidated. In this study, we show that phosphatidylinositol-3 kinase (PI3K) and Erk mitogen-activated protein kinase (MAPK) mediate ErbB signaling to regulate gastrulation. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways to control cell behaviors. Activation of either PI3K or Erk MAPK rescues gastrulation defects in ErbB4 morphant embryos, and restores convergent extension in the trunk mesoderm as well as coherent cell migration in the head mesoderm. The two signals preferentially regulate different aspects of cell behaviors, with PI3K more efficient in rescuing cell adhesion and spreading and MAPK more effective in stimulating the formation of filopodia. PI3K and MAPK also weakly activate each other, and together they modulate gastrulation movements. Our results reveal that PI3K and Erk MAPK, which have previously been considered as mesodermal inducing signals, also act downstream of ErbB signaling to participate in regulation of gastrulation morphogenesis.

???displayArticle.pubmedLink??? 17716876
???displayArticle.pmcLink??? PMC2098746
???displayArticle.link??? Mech Dev
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: egfr erbb4 fn1 mapk1 pik3ca pik3cb pik3cg
???displayArticle.morpholinos??? erbb4 MO1

???attribute.lit??? ???displayArticles.show???

	Fig. 1. MAPK and PI3K regulate mesoderm formation and gastrulation at different developmental stages in Xenopus. Embryos were incubated with the MAPK inhibitor U0126 or the PI3K inhibitor LY294002 at 20 μM (a) or 50 μM (b) from stage 4 or stage 10 onward. DMZ explants were dissected at stage 10, mesodermal markers were analyzed by RT-PCR at stage 11, and the morphology of the explants was examined at stage 16. While both inhibitors reduced the elongation of the DMZ explants in a dose-dependent manner when applied from either early or late stages, only inhibitors applied from stage 4 could efficiently block expression of a panel of mesodermal markers.
	Fig. 2. ErbB signaling regulates activation of PI3K and Erk MAPK pathways in Xenopus DMZ explants. DMZ explants from embryos overexpressing ErbB4 (1 ng mRNA/embryo), ErbB4-MO (20 ng MO/embryo), p110caax (1 ng mRNA/embryo), Δp85 (1 ng mRNA/embryo) or MEK∗(1 ng mRNA/embryo) were examined at gastrula stages for activation of PI3K (pAkt) or Erk MAPK (dp-Erk) pathways. Overexpression of ErbB4 enhanced, while depletion of ErbB4 reduced, the levels of both pAkt and dp-Erk. p110caax and MEK∗ stimulated pAkt and dpErk, respectively; but they also weakly increased phosphorylation of the other pathway component, suggesting a crosstalk between the two pathways.
	Fig. 3. Activation of PI3K or Erk MAPK rescued gastrulation defects in ErbB4 morphant embryos. (a) Coinjection of 5–10 pg of p110caax or MEK∗ RNA rescued the open blastopore and axial defects induced by depletion of ErbB4 (20 ng ErbB4-MO/embryo). (b) Embryos with different phenotypes were counted and summarized in the bar graph.
	Fig. 4. Activation of PI3K or Erk MAPK rescued convergent extension of DMZ explants from ErbB4 morphant embryos. Coexpression of 5–10 pg mRNA/embryo of p110caax or MEK∗ rescued elongation of the DMZ explants from ErbB4 morphant embryos (20 ng ErbB4-MO/embryo) at mid-neurula (stage 18) and tailbud (stage 24) stages. At tailbud stages, explants from ErbB4-MO injected embryos started to dissociate, and p110caax prevented cell shedding more efficiently than MEK∗ did.
	Fig. 5. Both PI3K and MAPK are required for optimal rescue of convergent extension in ErbB4-depleted explants. (a) 20 μM of U0126 or LY294002 were applied at stage 10 to DMZ explants from ErbB4-depleted (20 ng ErbB4-MO/embryo) and p110caax or MEK∗ (5–10 pg mRNA/embryo) rescued embryos, respectively. While both p110caax and MEK∗ rescued convergent extension of the explants, treatment with either U0126 or LY294002 reduced the elongation of the explants. (b) Morphometric analyses of the elongation of the DMZ explants (15 explants for each sample). The length-to-width (L/W) ratios of the DMZ explants were measured and calculated, and the statistical significance of the differences was analyzed using Student’s t-test. The difference between control and ErbB4-morphant was significant (p-value less than 0.0005), but the control and the rescued samples were not significantly different (p-values of 0.81 and 0.37 for p110caax and MEK∗ rescued samples, respectively, compared with the control explants). Treatment of the rescued samples with the chemical inhibitors significantly decreased the L/W ratios of the explants (p-values less than 0.003 compared with the control or the rescued explants).
	Fig. 6. Activation of PI3K and Erk MAPK rescued mediolateral intercalation of dorsal mesodermal cells. Membrane-tethered fluorescent proteins (EGFP and Cherry) were injected separately into two dorsal blastomeres of 4-cell embryos, together with ErbB4-MO (20 ng/embryo) and RNAs encoding p110caax or MEK∗ (5–10 pg/embryo). Cells from ErbB4 morphant embryos respected the midline and did not intermingle, but cells co-expressing the MO and p110caax or MEK∗ intercalated with each other, so that cells with different fluorescent signals mixed across the midline.
	Fig. 7. PI3K and MAPK rescued head mesoderm migration on fibronectin. (a) Migration of head mesoderm was impaired in explants from ErbB4 morphant embryos (20 ng ErbB4-MO), and both p110caax and MEK∗ (5–10 pg mRNA) rescued the cell migration. The distance of migration was enhanced, and the cell–cell contact between the migratory cells was restored. (b) Activation of either p110caax or MEK∗ rescued the distance of head mesoderm migration partially but significantly (with p-values of 0.03 from Student’s t-test). The percentage of the explants that maintained cell–cell adhesion was also greatly improved, with p110caax rescued the adhesion more efficiently to a level with no statistical difference from that of control (p = 0.16; for MEK∗, p = 0.03).
	Fig. 8. PI3K rescued cell–cell adhesion more efficiently than Erk MAPK. Dorsal mesodermal cells were dissociated for an hour before reaggregated on agarose-coated dish for 3 h. Cells from ErbB4 morphant embryos (20 ng ErbB4-MO) did not reaggregate well, so that loose clusters formed. Coexpression with p110caax (5–10 pg mRNA) effectively rescued cell adhesion to a level similar to that of control, while MEK∗ (5–10 pg mRNA) was less efficient in the rescue, so that aggregates were looser in structure.
	Fig. 9. PI3K rescued mesodermal cell spreading on fibronectin more efficiently than MAPK. The morphology of the mesodermal cells attached to fibronectin was examined and the number of the spreading cells was quantified in the bar graph. While ErbB4-MO (20 ng/embryo) reduced cell spreading on FN matrix, coinjection of p110caax or MEK∗ (5–10 pg mRNA/embryo) increased the percentage of the spreading cells significantly (p < 0.0001 compared with ErbB4-MO alone). Expression of p110caax, but not MEK∗, rescued cell spreading to a level comparable to that of the control cells (p = 0.14 and 3E-05 for p110caax and MEK∗, respectively, compared with the control).
	Fig. 10. Activation of PI3K and MAPK restored the formation of membrane protrusions in cells from ErbB4 morphant embryos. Membrane-tethered EGFP was used to label cell membrane, and formation of the cell protrusions in dissociated DMZ cells on FN matrix were observed by time lapse movies. (a) Cells from ErbB4 morphant embryos (20 ng ErbB4-MO/embryo) showed reduced filopodia and lamellipodia, but with increased membrane blebs. Coexpression of p110caax or MEK∗ (5–10 pg mRNA/embryo) restored the formation of lamellipodia and filopodia. (b) Quantification of the membrane protrusions. Lamellipodia, filopodia and membrane blebs were counted separately and summarized in the bar graph. ErbB4-MO reduced the average lamellipodia and filopodia from 1.7 and 3.7 to 0.3 and 0.7 per cell per minute, respectively, while increased the formation of the membrane blebs from 0.2 to 1.4 per cell per minute (p-values less than E-10 for all type of protrusions, as assayed by Student’s t-test). Coexpression of p110caax and MEK∗ restored the frequencies of lamellipodia and filopodia (p > 0.05 for all protrusions compared with the control cells), with MEK∗ stimulating filopodia more efficiently. The total numbers of the cells counted were given under the bar graph.

References [+] :

Adam, Heregulin regulates cytoskeletal reorganization and cell migration through the p21-activated kinase-1 via phosphatidylinositol-3 kinase. 1998, Pubmed