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First published online 30 January 2008
doi: 10.1242/dev.011940

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Ephrin signaling establishes asymmetric cell fates in an endomesoderm lineage of the Ciona embryo

Department of Molecular and Cell Biology, Division of Genetics, Genomics and Development, Center for Integrative Genomics, University of California, Berkeley, CA 94720, USA.

View larger version (58K): [in this window] [in a new window]   Fig. 1. A7.6 group genes mark mesoderm fate and TTF-1 marks endoderm fate in A6.3 lineages. (A) A7.6-specific marker genes Hand-like, FGF8, Delta-like and MyTF are specifically expressed in A7.6 at late 110-cell stage. Delta-like is also expressed in animal blastomeres b7.9, b7.10 next to A7.6. (B) TTF-1 is expressed in endoderm blastomeres. (C,D) Schematic of 32-, 64- and 110-cell embryos showing the mixed lineage of the A6.3 blastomere. At the 32-cell stage, A6.3 is the only endomesoderm blastomere in the Ciona embryo that will give rise to mesoderm (red) and endoderm (yellow) progenies.

View larger version (54K): [in this window] [in a new window]   Fig. 2. Inhibition of MAPK signal causes ectopic expression of A7.6 group genes in the anterior endoderm. (A-E) 110-cell Ciona embryos treated with 2 µM U0126 at late 32-cell stage. (A-D) A7.6 group genes are ectopically expressed in the anterior endoderm blastomeres upon MEK inhibition, whereas A7.6 expression is unaffected (numbers indicate affected embryos/total embryos). Conversely, the endoderm-specific marker TTF-1 is completely lost in the endoderm when MAPK signaling is inhibited (E). (F,G) Endogenous pattern of MAPK activation in the wild-type embryo. At the 64-cell stage (F), dpERK staining is observed in the nuclei of notochord precursors (A7.3, A7.7), all endoderm blastomeres (A7.1, A7.2, A7.5, B7.1, B7.2) and mesenchyme cells (B7.3, B7.7), but is strikingly absent from A7.6 mesoderm. This MAPK dichotomy in A7.5/A7.6 siblings persists to the 110-cell stage (G). (H) MAPK activation visualized by dpERK staining is completely lost in embryos treated with U0126.

View larger version (57K): [in this window] [in a new window]   Fig. 3. Ci-ephrin-Ad is broadly expressed in the animal hemisphere. (A) Ciona embryos at progressively older stages hybridized for Ci-ephrin-Ad. (B) Schematic representation of Ci-ephrin-Ad-expressing cells in the animal hemisphere. The black dots indicate blastomeres that express the gene. The Ci-ephrin-Ad transcript is present in all animal blastomeres at the 16-cell stage, but diminishes in two of the progenies, b6.5 and b6.7, at the 32-cell stage. Zygotic expression further diminishes at the 64-cell stage. The relative positioning of the b6.5 and A6.3 blastomeres is shown in the vegetal view of the 32-cell embryo.

View larger version (83K): [in this window] [in a new window]   Fig. 4. Ci-ephrin-Ad establishes asymmetric fates in A6.3 lineages. (A-D) Upon Ci-ephrin-Ad MO injection, A7.6 group genes are severely downregulated in Ciona embryos. The numbers of embryos showing a complete loss of marker gene expression/total embryos are indicated. Whereas Hand-like, FGF8 and MyTF display an almost complete loss of expression, Delta-like seems to be the most resilient to MO inhibition, with only 40% loss of expression. The remaining 60% embryos show normal or slightly reduced expression in A7.6. (E) When Ci-ephrin-Ad function is inhibited, ectopic dpERK staining is observed in the A7.6 blastomeres of all injected embryos. (F) Brachyury expression is expanded to the A-line neural tissues in MO embryos, as previously shown (Picco et al., 2007). (G) Ectopic expression of the endoderm marker TTF-1 in A7.6 blastomeres of MO-injected embryos. The ectopic expression is variable and is seen on only one side of the embryo shown here. (H) Schematic illustrating that TTF-1 expression (yellow) is expanded into A7.6 of MO-injected embryos. (I,J) Inhibition of Ci-ephrin-Ad function results in transformation of A7.6 mesoderm fate to endoderm fate. In wild-type arrested embryos (J), alkaline phosphatase (AP) activity is observed in endoderm blastomeres. For an unknown reason, the staining is consistently stronger in the anterior endoderm. (I) In Ci-ephrin-Ad MO-injected embryos, AP staining is also observed in A7.6 blastomeres, suggesting a fate transformation from mesoderm to endoderm.

View larger version (73K): [in this window] [in a new window]   Fig. 5. Ubiquitous activation of FGF signaling inhibits A7.6 group gene expression. (A) The constitutively active form of FGFR (torso-FGFR) is expressed under the ZicL enhancer. Brachyury is ectopically expressed in A-line neural blastomeres, consistent with ectopic activation of the MAPK pathway. Some ectopic Ci-Bra expression is also observed in the muscle and mesenchyme. (B) Ci-Bra expression in wild-type Ciona embryo. (C-F'') Embryos injected at the 1-cell stage with torso-FGFR mRNA. Expression of A7.6 group genes is modestly reduced. To demonstrate the variable degree of reduction, embryos with no expression (C,D,F), weak expression (C',D',E,F') and wild-type expression levels (C'',D'',E',F'') are shown together with the number of embryos affected. In addition to A7.6, there are three instead of two Delta-like-positive blastomere cells (E'') consistently observed in injected embryos [in wild-type embryos, Delta-like expression in A7.8 is highly variable and only seen in 20% of the embryos (Hudson and Yasuo, 2006), suggesting that MAPK usually activates Delta-like in these cells].

View larger version (72K): [in this window] [in a new window]   Fig. 6. Ephrin-Ad represses MAPK activity. (A) Delta-like expression is lost in both A7.6 (arrows, 80%) or one A7.6 (15%) of the Ciona embryos co-injected with ephrin-Ad MO and torso-FGFR mRNA, suggesting Ephrin-Ad and MAPK pathways function synergistically. Ectopic activation of Delta-like in b-line blastomeres is often observed (arrowheads). (B-E) Treating ephrin-Ad MO-injected embryos with the MEK inhibitor U0126 reverses the repression of A7.6 genes in A7.6 and ectopically activates them in the anterior endoderm. The number of affected embryos/total embryos is shown.

View larger version (81K): [in this window] [in a new window]   Fig. 7. Misexpression of Ci-ephrin-Ad in the endoderm activates mesoderm markers and represses endoderm markers. A FoxD enhancer was used to drive Ci-ephrin-Ad throughout the vegetal hemisphere at the 16-cell stage. (A-D) Ectopic expression of A7.6 group genes in the anterior endoderm (arrows) is observed in embryos misexpressing Ci-ephrin-Ad. The average number of endoderm cells misexpressing the mesoderm marker is shown. FGF8 appears to be most responsive to misexpression of Ci-ephrin-Ad; the others show an average of one or fewer ectopic expressing cell. (E) The endoderm marker TTF-1 is variably lost in the endoderm where Ci-ephrin-Ad is misexpressed, suggesting a partial transformation of endoderm to mesoderm fate.

View larger version (49K): [in this window] [in a new window]   Fig. 8. A7.6 marker gene expression does not require Nodal signaling from b6.5. (A,B) In late 32-cell stage wild-type Ciona embryos, Nodal is expressed in endoderm/endomesoderm blastomeres (A) and in b6.5 (B). (C,D) In embryos treated with U0126 at early 32-cell stage, vegetal expression of Nodal is unaffected (C), whereas b6.5 expression is completely blocked (D). (E-H) Embryos grown in U0126 beginning at the 16-cell stage display variable changes in A7.6 marker gene expression. (I-K) Hand-like, FGF8 and Delta-like expression in A7.6 is not affected by U0126 inhibition. (L) MyTF expression in A7.6 partially requires MAPK signaling at the 16-cell stage, but not at the 32- to 64-cell stage. (M,N) Ectopic expression of Nodal in the vegetal hemisphere induces ectopic Snail expression in the A-line neural lineage. (O-Q) Ectopic Nodal expression does not induce A7.6 gene expression in A7.5 or other endoderm blastomere, but is able to induce Hand-like and Delta-like expression in the A-line neural lineage (O, arrow; P, arrowheads) or to induce ectopic MyTF expression in mesenchyme lineages (Q, arrow).

View larger version (19K): [in this window] [in a new window]   Fig. 9. A model for A6.3 endomesoderm specification in Ciona. In A6.3, a combination of Nodal signaling and MAPK signaling maintains an endomesoderm state. Upon cell division, MAPK is inhibited in A7.6 by Ephrin-Ad from neighboring animal blastomeres, allowing endogenous Nodal to activate A7.6 mesoderm genes. In A7.5, Nodal is unable to activate such genes owing to inhibition by MAPK. The fates of blastomeres are indicated in different colors (red, mesoderm; yellow, endoderm).

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