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First published online July 11, 2006
doi: 10.1242/10.1242/dev.02448

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Ci-FoxA-a is the earliest zygotic determinant of the ascidian anterior ectoderm and directly activates Ci-sFRP1/5

Institut de Biologie du Développement de Marseille, UMR 6216, CNRS/Université de la Méditerranée, Parc Scientifique de Luminy, Case 907, F-13288 Marseille Cedex 9, France.

View larger version (47K): [in a new window]   Fig. 1. Ci-sFRP1/5 marks the difference between anterior and posterior ectoderm. In situ hybridization for Ci-sFRP1/5 at the 64-cell stage on whole embryos (A, animal view, anterior towards the left), whole animal explants (B), anterior animal a-line explants (C) and posterior animal b-line explants (D) explanted at the eight-cell stage. The scheme to the left illustrates an eight-cell stage embryo in lateral view. a- and b-line cells are shown in red and green, respectively.

View larger version (41K): [in a new window]   Fig. 2. Isolation of a minimum enhancer recapitulating Ci-sFRP1/5 expression. (A) Ci-sFRP1/5 locus in the Ciona intestinalis genome, showing the 5' flanking region and the first two exons. Coding/non coding conserved regions between C. intestinalis and C. savignyi are shown in white/pink. The scheme shows the position of the distal sFRP-1067 and proximal fragments tested in vivo. (B) Schematic view of the regulatory elements (with their length in brackets) tested at the 110-cell stage. With the exception of sFRP-1067, which includes the endogenous minimal promoter, all constructs were placed in front of the Cibrachyury minimal promoter. (a-c) Representative X-gal staining for embryos electroporated with the indicated DNA (percentages correspond to embryos electroporated with sFRP-640, sFRP-95 and sFRP-67 respectively). (a,c) Animal view, anterior towards the top. (b) Anterior view, animal to the top. (C) Alignment of the C. intestinalis and C. savignyi sequences of the sFRP-118 element, with putative Fox binding sites framed in red.

View larger version (20K): [in a new window]   Fig. 3. Consensus Fox-binding sites are necessary and sufficient for anterior ectodermal expression. (A) Activity of various endogenous and synthetic constructs containing Fox sites (red circles). Left: scheme of the constructs tested. Right: histogram representing the percentage of embryos with X-gal staining in different lineages. (B) Sequence of the 6-Fox element artificial construct tested in A, with the Fox sites boxed.

View larger version (48K): [in a new window]   Fig. 4. Ci-sFRP1/5 is a target of Ci-FoxA-a via the sFRP-118 element. (A) Weak expression of Ci-FoxA-a by whole-mount in situ hybridization at the eight-cell stage in the anterior animal (a4.2) and vegetal (A4.1) lineages. Lateral view, anterior towards the left, animal towards the top. The staining is detected in the nuclear region. (B-E) Effect of the down- or upregulation of Ci-FoxA-a on sFRP-118 activity at the 110-cell stage (histogram in centre is colour coded according to the lineage) and on endogenous Ci-sFRP1/5 expression at the 64-cell and neurula stages (right). (B,C) Embryos microinjected with control or Ci-FoxA-a morpholinos. The percentage of embryos with a-line staining is indicated. (D) Control and (E) embryos electroporated with the indicated Ci-FoxA-a overexpression construct. The percentage of embryos with ectopic b-line staining is indicated. Black arrows indicate ectopic posterior ectodermal expression. Anterior is towards the top, animal views are shown at the 64-cell stage, vegetal views are shown at the neural plate stage.

View larger version (66K): [in a new window]   Fig. 5. Ci-Fox-A-a activates anterior ectodermal markers. (A-F) Effect of pan-animal Ci-FoxA-a overexpression on the neural plate-stage activity of the sFRP-118 enhancer, visualized by X-gal staining. (A,B) Whole embryos; (C,D) anterior ectodermal explants cut at the eight-cell stage and cultured in isolation; (E,F) posterior ectodermal explants. The percentage indicates the proportion of stained explants. Whole embryos are shown in animal views at the neural plate stage. (G-O) Effect of pan-animal overexpression or knock down of Ci-FoxA-a on Ci-Otx (G-I), Ci-FoxC (J,K) and Ci-Ror-a (L-N). All embryos are shown in vegetal view at the neural plate stage, anterior towards the top. The percentages of embryos with ectopic expression in b-line cells are indicated. (O) Schematic vegetal view of the neural plate showing the position of anterior (a9.) and posterior (b9.) ectodermal lineages at neural plate stage. Anterior is towards the top.

View larger version (78K): [in a new window]   Fig. 6. Ci-FoxA-a suppresses posterior fates. Effect of morpholino knock-down (A,B) or pan-animal Ci-FoxA-a overexpression (C-F) on the expression of Ci-Delta2 (A,B, 64-cell stage; C,D, 110-cell stage) and Ci-Msxb (E,F, 110-cell stage). Expression is detected in the b7.9, 10 (Ci-Delta2) and b8.17, 18, 19, 20 (Ci-Delta2, Ci-Msxb) b-line neural precursors in wild-type embryos (A,C,E). The black arrow in B indicates ectopic expression in the anterior neural precursors (a7.9, 10) at the 64-cell stage. Percentages indicate the proportion of embryos with staining in the anterior (A,B) or posterior (C-F) ectoderm. All embryos are shown in vegetal view, anterior towards the top.

View larger version (30K): [in a new window]   Fig. 7. A model of anterior fate determination in the ascidian ectoderm at the eight-cell, 64-cell and neural plate stages. The anterior ectodermal lineage (red) gives rise to the trunk epidermis (not coloured), the palps (striped motif) and the sensory vesicle (red), while the posterior ectodermal lineage (green) gives rise to the tail epidermis (not coloured) and the dorsal row of the nerve cord (green) at the larval stage. At the eight-cell stage, Ci-FoxA-a expression is activated in the anterior animal cells. Presence of this factor leads to the activation of anterior ectodermal genes at the 64-cell and neural plate stages, and to the repression of posterior ectodermal genes. Vegetal expression of Ci-FoxA-a is not represented in this model.