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First published online August 24, 2007
doi: 10.1242/10.1242/dev.010140

Development 134, 3297-3305 (2007)
Published by The Company of Biologists 2007
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FoxF is essential for FGF-induced migration of heart progenitor cells in the ascidian Ciona intestinalis

Jeni Beh, Weiyang Shi, Mike Levine, Brad Davidson*,{dagger} and Lionel Christiaen{dagger}

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


Figure 1
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  		Fig. 1. FoxF expression in anterior B7.5 cells requires Ets1/2 activity. (A-C) In situ hybridization on neurula (A,B) and tailbud (C) embryos. Arrowheads (A,B) indicate the anterior B7.5 cells, which migrate and form the trunk ventral cells (TVCs; white arrowheads in C). Notice the FoxF expression in trunk epidermal cells. (D-G'') The B7.5 lineage cells were visualized with an anti-ß-galactosidase antibody (red), and FoxF expression was revealed by fluorescent in situ hybridization (green). (D-D'') Embryo electroporated with the Mesp>lacZ control. Within the B7.5 lineage, FoxF is only expressed in the anterior TVCs and not in the posterior tail muscles. (E-E'') Embryo co-electroporated with Mesp>lacZ and Mesp>Ets:VP16. Ets:VP16 induces all four B7.5 lineage cells to migrate and express FoxF. (F-F'') Embryo co-electroporated with Mesp>lacZ and Mesp>Ets:WRPW. Ets:WRPW inhibits both the migration of B7.5 cells and FoxF expression. (G-G'') Embryo co-electroporated with Mesp>lacZ and Mesp>Mesp:VP16. All B7.5 cells remain in the tail; anterior cells fail to express FoxF.

 

Figure 2
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  		Fig. 2. FoxF is an immediate target of the FGF/MAPK/Ets pathway. (A, top) Map of the FoxF gene (red) and 5' upstream region on chromosome arm 3q. (A, middle) VISTAplot showing sequence conservation (50-100%) between two Ciona species (http://genome.lbl.gov/vista/index.shtml). (A, bottom) Summary diagram of trunk ventral cell (TVC) and epidermis expression with various 5' enhancers fused to the lacZ reporter. (B) Embryo expressing the -3052 to +1 enhancer attached to the lacZ reporter, showing TVC (arrow) and epidermal expression. (C) The FoxF minimal TVC enhancer sequence (-1135 to -840) is highly conserved between C. intestinalis and C. savignyi. Boxes indicate the E-box (red) and the three Ets1/2-binding sites (green). (D) A 295 bp (-1135 to -840 bp) genomic DNA fragment, fused to the Forkhead (FoxA-a) basal promoter, drives lacZ expression specifically in the TVCs (arrow). (E) Mutational analysis. The histogram displays the proportions of embryos showing TVC staining (n=total embryos). Diagrams depict the wild-type -1135 to -840 fragment and the indicated mutations. (F) The -3052 to +1 cis-regulatory region with deleted E-box motif drives expression in epidermis only. (G) The -1135 to -840 ({Delta}E-box) does not drive lacZ expression in TVCs (arrow). (H) Cis-trans complementation test. Ets:VP16 can restore enhancer activity of the -1135 to -840 ({Delta}E-box) construct. The cis-trans complementation is abolished when the three Ets1/2 sites are mutated (errors bars indicate standard deviation). (I) The -1135 to -840 ({Delta}E-box) enhancer co-electroporated with Mesp>Ets:VP16. Ets:VP16 causes all four B7.5 cells to migrate into the trunk and causes the mutated enhancer to drive lacZ expression in all cells. Arrows in B,D,F,G,I point to the TVCs (not stained in F and G).

 

Figure 3
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  		Fig. 3. FoxF function is necessary for TVC migration. (A-F) Embryos electroporated (A,C-F) or injected (B) with Mesp>GFP to mark the B7.5 lineage (green). The red channel detects the fluorescent background of the Ciona embryo. (A) Wild-type embryo with normal anterior TVC and posterior tail muscle positions (lateral view). (B) Embryo co-injected with Mesp>GFP and FoxF morpholino. Anterior B7.5 cells fail to detach from their sister muscle cells and to migrate into the trunk (ventral view). (C) Mesp>GFP co-electroporated with Mesp>FoxF:VP16. All B7.5 lineage cells have migrated into the trunk (ventral view). (D) Mesp>GFP co-electroporated with Mesp>FoxF:WRPW. All B7.5 cells remain in the tail. (E) Mesp>GFP co-electroporated with Mesp>Ets:VP16. All B7.5 cells migrate into the trunk (lateral view). (F) Mesp>GFP co-electroporated with Mesp>Ets:VP16 and Mesp>FoxF:WRPW. Inhibited TVC migration occurs that is comparable to that observed with FoxF:WRPW alone. (G) The five distinct classes of migration phenotypes. (H,I) Proportions of embryos distributed among the five phenotypic classes in each condition, including EtsVP/FoxFW and EtsW/FoxVP epistasis tests; color coding is as in G.

 

Figure 4
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  		Fig. 4. FoxF function is not required for Hand-like expression in the TVCs. Electroporated Ciona embryos hybridized with the Hand-like probe (green) and the B7.5 lineage cells visualized with an anti-ß-galactosidase antibody (red). (A-A'') Wild-type embryo electroporated with Mesp>lacZ alone. Hand-like is expressed in anterior trunk ventral cells (TVCs), but not in posterior tail muscles. (B-B'') Embryo co-injected with Mesp>lacZ and FoxF morpholino. TVC migration is inhibited, but Hand-like expression persists in the anterior cells. (C-C'') Embryo co-electroporated with Mesp>lacZ and Mesp>FoxF:WRPW. TVC migration is inhibited, but Hand-like exhibits normal expression in two anterior B7.5 cells. (D-D'') Embryo co-electroporated with Mesp>lacZ, Mesp>Ets:VP16 and Mesp>FoxF:WRPW. Hand-like expression is present in all B7.5 cells even though migration is inhibited by FoxF:WRPW.

 

Figure 5
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  		Fig. 5. The effects of FoxF:WRPW on GATA-a and NK-4 expression are variable. Embryos electroporated with Mesp>lacZ and the indicated constructs (left) were stained for ß-galactosidase (red) and either GATA-a (A,C,E) or NK-4 (B,D,F) mRNAs (green). (A,B) Wild-type embryos. (C-F) Embryos electroporated with Mesp>FoxF:WRPW. GATA-a and NK-4 expression persists (C,D) or is abolished (E,F) in non-migrating trunk ventral cells (TVCs). Arrowheads indicate anterior B7.5 cells.

 

Figure 6
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  		Fig. 6. Co-expression of Ets:VP16 and FoxF:WRPW results in a mis-positioned beating heart. (A,C) Wild-type heart (arrowhead) located between the endostyle (e) and the stomach (s). (B,D) Juvenile co-electroporated with Mesp>Ets:VP16 and Mesp>FoxF:WRPW. Beating heart tissue (arrow) is located where the tail is being reabsorbed above the stomach (s). Heart is missing from the wild-type location (*).

 

Figure 7
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  		Fig. 7. The early heart gene network regulates heart differentiation and migration. Mesp expression initiates heart specification in the B7.5 lineage. Mesp is thought to upregulate Ets1/2 expression in the whole B7.5 lineage. An FGF signal activates Ets1/2 in the anterior B7.5 cells, thus inducing both heart muscle specification and cell migration. Activated Ets1/2 activates process-specific genes for both heart differentiation (the heart-kernel genes) and cell migration (FoxF) in parallel pathways. Mesp presumably functions in parallel to Ets1/2 to regulate GATA-a, NK-4 and FoxF expression. FoxF and activated Ets1/2 function in parallel to control heart cell migration.

 

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© The Company of Biologists Ltd 2007