Curran KL and Grainger RM (2000), Expression of activated MAP kinase in Xenopus l...

XB-ART-9939

Dev Biol 2000 Dec 01;2281:41-56. doi: 10.1006/dbio.2000.9917.

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Expression of activated MAP kinase in Xenopus laevis embryos: evaluating the roles of FGF and other signaling pathways in early induction and patterning.

Curran KL , Grainger RM .

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FGF signaling has been implicated in germ layer formation and axial determination. An antibody specific for the activated form of mitogen-activated protein kinase (MAPK) was used to monitor FGF signaling in vivo during early Xenopus development. Activation of MAPK in young embryos is abolished by injection of a dominant negative FGF receptor (XFD) RNA, suggesting that MAPK is activated primarily by FGF in this context. A transition from cytoplasmic to nuclear localization of activated MAPK occurs in morula/blastula stage embryo animal and marginal zones coinciding with the proposed onset of mesodermal competence. Activated MAPK delineates the region of the dorsal marginal zone before blastopore formation and persists in this region during gastrulation, indicating an early role for FGF signaling in dorsal mesoderm. Activated MAPK was also found in posterior neural tissue from late gastrulation onward. Inhibition of FGF signaling does not block posterior neural gene expression (HoxB9) or activation of MAPK; however, inhibition of FGF signaling does cause a statistically significant decrease in the level of activated MAPK. These results point toward the involvement of other receptor tyrosine kinase signaling pathways in posterior neural patterning.

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Species referenced: Xenopus laevis
Genes referenced: gal.2 hoxb9 mapk1 tbxt

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	FIG. 1. Embryos injected with 4 ng of an inactive form of the FGF receptor RNA (d50/Xss) (A and B) or 4 ng of the dominant negative form of the FGF receptor RNA (XFD) (Câ F). All embryos are oriented with the dorsal side of the blastopore toward the top. Immunostaining by anti-MAP kinase, activated, was observed to encircle the blastopore of stage 10.5/11 control embryos (A) but staining was absent (C) or greatly reduced (E) in embryos injected with XFD. Similar results were observed with Xbra in situ hybridization. Normal Xbra expression was observed in embryos injected with d50/Xss (B) but staining was either absent (D) or greatly reduced (F) in embryos injected with XFD. Sensorial ectoderm of animal caps was treated for 5 min with 50 ng/ml FGF. Uninjected ectoderm showed activated MAPK expression (G, see arrows) while those injected with XFD showed mosaic expression in which less then 20% of the cells were positive for activated MAPK (H, arrows) or showed no expression (H). Ectoderm not treated with FGF showed no expression as well (I). Scale bar, 225 mm.
	FIG. 2. Activated MAPK expression becomes localized to the nucleus of the animal cap during blastula stages and later is observed to increase dorsally. (Aâ C) Animal views of cleared embryos at stage 6 (A), stage 7 (B), and stage 8 (C) (magnified in insets). Nuclear staining was observed to increase between stages 6 and 7 (see insets). By stage 9 (D) staining was observed in the animal cap (ac) and marginal zone (mz), but not in the vegetal pole (vp). (E) A sagittal section through a stage 9 embryo. Equal amounts of ac, mz, and vp protein were analyzed by Western blot using the activated MAPK antibody (F). Activated MAPK was detected in the ac and mz, but not in the vp. An increase in activated MAPK expression was observed prior to dorsal lip formation (G, magnified in inset). (H) A sagittal section of the embryo pictured in G, dorsal staining is magnified in the inset. Scale bar, 350 mm.
	FIG. 3. Increased activated MAPK expression was observed to completely surround the blastopore at mid- to late gastrula stages (A, arrowhead indicates dorsal). (B) A sagittal section of staining in a late gastrula embryo (dorsal is on the right and the animal pole is up). Specifically, increased staining occurs in the presumptive mesoderm (pm) and posterior neural tissue (pn) but expression is lost in the involuted mesoderm (im) and is not present in the anterior neural tissue (b, brain). During neurula stages (posterior staining of stage 17 shown here in C and D) activated MAPK expression remains localized to posterior as well as anterior regions of the embryos. (C and D) Anterior on the right and posterior to the left. In a sagittal section through the midline of the embryo, posterior staining was specifically seen in the posterior mesoderm (pm, notochord) and neural (pn) tissue (D). Scale bar, 300 mm.
	FIG. 4. Activated MAPK expression is affected in the posterior mesoderm of XFD-injected embryos, but not in the posterior neural tissue. (Aâ D) Dorsal is up. A and B show activated MAPK around the blastopore in stage 11.5/12 uninjected embryos in whole mount and section, respectively. The arrowhead in A points to the approximate region pictured in B. The arrow in B points to activated MAPK present in posterior neural (pn) tissue. C and D show expression of activated MAPK in XFD-expressing stage 11.5/12 embryos in whole mount and section, respectively. The arrowhead in C shows the approximate region of section in D. The arrows in D indicate the approximate position of the pn and the absence of activated MAPK. (Eâ L) Anterior is to the right and dorsal is up unless otherwise noted. E shows the normal expression of activated MAPK in a stage 15/16 embryo from a dorsal view. The arrowheads denote the anteriorâ posterior extent of the expression of activated MAPK. F shows a sagittal section of a stage 15/16 embryo stained for activated MAPK. Note the presence of activated MAPK in the posterior mesoderm (m) and neural (n) tissues. G shows a dorsal view of a stage 15/16 embryo expressing XFD. The arrowhead denotes the presence of activated MAPK in a â tail-likeâ protrusion. The arrow shows the absence of activated MAPK in the posterior which does not form a tail. H shows a sagittal section of the posterior of a stage 15/16 embryo expressing XFD and b-gal, displaying the expression of activated MAPK in a tail-like protrusion. Note the expression of activated MAPK in the mesoderm is decreased or absent while the levels of activated MAPK in neural tissue is less affected. Mosaic expression of b-gal was also observed in both the mesoderm and the neural tissue. Colocalization of activated MAPK and b-gal is magnified in the inset (arrowhead). n is less affected. I shows a dorsal view of the normal in situ hybridization pattern for Xbra in an uncleared stage 15/16 embryo. The arrowheads denote the anteriorâ posterior extent of Xbra staining (not including the notochord which is not shown). J shows a sagittal section of Xbra expression in the posterior blastopore region (arrows). K shows a dorsal view of Xbra expression in a stage 15/16 embryo expressing XFD. The arrowhead shows the expression of Xbra in a tail-like protrusion, while the arrow denotes the lack of Xbra expression in the posterior of the embryo not forming a tail. L shows a sagittal section of a tail-like protrusion expressing Xbra in the posterior mesoderm (arrow). Scale bar, 150 mm.
	FIG. 5. Induction of HoxB9 RNA by dorsal mesoderm was not affected in animal caps expressing XFD. HoxB9 is present in the posterior neural tube of a stage 26 embryo by whole-mount in situ hybridization (between arrowheads in A). Some background staining was observed in the head. (B) Expression of HoxB9 in uninjected ectoderm recombined with dorsal mesoderm (arrowhead denotes condensed staining and arrow denotes diffuse staining). (C) Expression of HoxB9 RNA in XFD-expressing ectoderm recombined with dorsal mesoderm (arrowhead denotes condensed staining and arrow denotes diffuse staining). HoxB9 expression was not seen in untreated animal caps as seen in D (section). (E and F) The expression of HoxB9 (E, arrowhead) is not found in the RLDX-labeled dorsal mesoderm (F, arrowhead). (G) HoxB9 is present in the ectoderm (G, arrowhead) and not in the RLDX-labeled mesoderm (H, arrowhead). Scale bar, 150 mm.