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First published online October 26, 2007
doi: 10.1242/10.1242/dev.010850

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A combinatorial code of maternal GATA, Ets and ß-catenin-TCF transcription factors specifies and patterns the early ascidian ectoderm

Ute Rothbächer^*,, Vincent Bertrand^*,, Clement Lamy and Patrick Lemaire

Institut de Biologie du Développement de Marseille Luminy (IBDML), CNRS-UMR6216/Université de la Méditerranée Aix-Marseille, F-13288 Marseille Cedex 9, France.

View larger version (60K): [in this window] [in a new window]   Fig. 1. Isolation of a cis-regulatory module driving early animal expression. (A) Ci-fog expression at the 16-cell stage. Anterior is to the top for animal (an) and vegetal (veg) views, to the left for the lateral (lat) view. (B) lacZ reporter activity (X-Gal) at the 110-cell stage following electroporation of the pfog-2000::NLS-lacZ construct. Lateral view, as in A. (C) Timing of lacZ reporter activation in animal blastomeres after electroporation of the pfog-2000 construct. Animal views of representative embryos. (D) Phylogenetic footprinting between C. intestinalis and C. savignyi sequences covering 2 kb upstream of the ATG of Ci-fog. The conserved region is shown in pink, the heterologous Ci-bra basal promoter in green. The level of lacZ reporter activity is indicated for each electroporated construct (n80), from very strong (++++) to absent (-); with more than half of embryos counted having all animal cells stained (++++), 50-90% of animal cells stained (+++), 25-50% (++) or 1-25% (+) of animal cells stained.

View larger version (65K): [in this window] [in a new window]   Fig. 2. GATAa is necessary and sufficient for FGF-independent animal-wide activity. (A) Deletion constructs of the conserved pfog region identify two GATA sites required for lacZ reporter expression. The level of lacZ reporter activity in animal blastomeres at the 110-cell stage is quantified as in Fig. 1; n80 for each construct. The pfog-m314 construct has a point mutation in each of the two GATA binding site (red crosses). (B) Animal view of 110-cell stage embryos co-injected with pfog-314 and either control-MO or GATAa-MO, stained with X-Gal. Numbers indicate the proportion of positive embryos. (C) lacZ reporter activity at the 110-cell stage in embryos co-injected with G12 and either water, GATAa-MO, GATAa-MO with GATAa mRNA or GATAa-MO with GATAb mRNA. Numbers indicate ß-gal-positive embryos. (D) lacZ reporter activity at the 110-cell stage in embryos electroporated with G12 with or without U0126 treatment. The quantification of expression within the animal region is sorted by cell lineage derivatives of the 32-cell stage (bottom right corner). A representative untreated embryo is shown (110-cell stage, upper right corner, animal view, anterior is up).

View larger version (78K): [in this window] [in a new window]   Fig. 3. GATAa specifies epidermis. (A) Expression of the epidermis markers epiB and epi1 in control and EnR-GATAa mRNA-injected embryos. Embryos shown in the top and middle panels were cleavage-arrested at the 64-cell stage with cytochalasin and stained at the equivalent of the neurula stage for the presence of epi1 or epiB transcripts (animal view, anterior to the top). Embryos shown in the bottom panels were left to cleave normally and stained at the early tailbud stage for epi1 transcripts. (B) Expression of epiB at late gastrula stage in control and GATAa-MO, GATAb-MO or double-injected embryos. Numbers indicate the proportion of positive embryos. Percentages refer to embryos with strongly reduced expression.

View larger version (67K): [in this window] [in a new window]   Fig. 4. Timing of animal restriction. (A) Animal and vegetal reporter expression at the 16-cell stage as visualized by lacZ mRNA detection. Lateral views, anterior to the left, numbers indicate the proportion of positive embryos with restricted expression. (B) At the 8-cell stage, the mRNA expression of animal and vegetal markers is partially restricted. Representative embryos are shown across the top; diagram of experimental results at the bottom. Red indicates the percentage of embryos with stronger animal expression; white indicates equal animal and vegetal expression; yellow indicates stronger vegetal expression. Numbers of embryos counted are indicated on the colored bars.

View larger version (68K): [in this window] [in a new window]   Fig. 5. GATAa activity is blocked in the vegetal region by ß-catenin-TCF. (A) GATA transcriptional activity (G12 reporter mRNA, upper panel) and ß-catenin-TCF activity (Ci-foxD mRNA, lower panel) was visualized at the 16-cell stage in control, ß-catenin-MO- and TCF-MO-injected embryos. Lateral views, anterior to the left. Numbers indicate embryos with vegetal expression. (B) Bar chart of the mean number of positive cells per embryo in each condition in A (yellow is animal, blue is vegetal expression; error bars indicate confidence limits for P=0.05). (C) G12 reporter activity at the 110-cell stage in control, 100 mM and 200 mM LiCl-treated embryos. The x-axis represents the percentage of positive embryos; the y-axis indicates values at increasing concentrations of LiCl. The total numbers of embryos counted (n) are indicated at the bottom for each experimental condition. (D)Effect of GATAa-MO or Ci-GATAa mRNA injection on pFoxD activity at the 16-cell stage. Animal to the top, anterior to the left, numbers indicate the proportion of embryos with restricted vegetal expression.

View larger version (94K): [in this window] [in a new window]   Fig. 6. Mechanism of differential readout of GATAa activity in pan-animal versus neural territories. Top: Schematic of the pfog-314 construct, showing the nucleotide position of the longest available EST, RACE-PCR fragments (results not shown) and position of an intron; green indicates sequences with basal promoter activity (-290). The putative GATA-binding sites of pfog are indicated in blue. Representative embryos electroporated with the indicated construct were stained for lacZ reporter activity at the 110-cell stage. A,B,D,E are animal views and C is a vegetal view; anterior to the top; n35. (A) Eliminating the intron maintains animal expression. (B) GATA sites from the otx neural a-element (red) maintain animal-wide expression. (C) -290 sequences in association with the ebra enhancer drive expression in notochord precursors and (D) in association with the otx neural a-element show neural and sporadic animal expression. (E) Ets site deletion can derepress animal-wide expression. (F) Quantification of constructs showing animal-wide expression. 90-100% indicates the percentage of embryos with all animal cells stained; 50-90% indicates the percentage of embryos with more than half of animal cells stained; <50% indicates the percentage of embryos with fewer than half of animal cells stained.

View larger version (26K): [in this window] [in a new window]   Fig. 7. Model for early animal patterning in ascidians. Schematic views of the different events taking place in the early Ciona egg, 8-cell and 32-cell embryo and the corresponding molecular mechanisms defining the ectodermal territories.