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First published online 24 September 2003
doi: 10.1242/dev.00760

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Tgfß signaling acts on a Hox response element to confer specificity and diversity to Hox protein function

Aurélie Grienenberger¹, Samir Merabet^1,*, John Manak^2,3, Isabelle Iltis¹, Aurélie Fabre¹, Hélène Bérenger¹, Matthew P. Scott^2,3, Jacques Pradel¹ and Yacine Graba^1,

¹ Laboratoire de Génétique et Biologie du Développement, IBDM, CNRS, Université de la méditerranée, Parc Scientifique de Luminy, Case 907, 13288 Marseille Cedex 9, France
² Department of Developmental Biology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305-5427, USA
³ Department of Genetics, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305-5427, USA

View larger version (70K): [in a new window]   Fig. 1. Identification and regulation of a wg midgut enhancer. (A) wg upstream regulatory elements that drive (red bars) or do not drive (black bars) expression in the midgut when fused to a lacZ reporter. (B-G) Comparison of wg transcription and XC enhancer activity. wg trancripts (B,D,F) and lacZ (C,E,G) transcripts were detected by in situ hybridization. Arrows indicate PS8, the site of wg midgut expression. At all stages examined, shown here as lateral views of stage 11 (B,C) and stage 13 (D,E) embryos, XC enhancer activity exclusively mimics wg expression in the embryonic midgut. (F,G) Magnified ventral views of stage 16 embryos. The arrowheads indicate the central midgut constriction. (H-K) XC enhancer regulation recapitulates wg regulation. The activity of the XC enhancer visualized by lacZ transcripts is lost in abdAJX2 homozygous-mutant embryos (H) and diminished in dpps4 homozygous mutants (I). wg transcripts (J) and XC enhancer activity (K) are no longer detected in the central midgut following mesodermal expression of the Hth-En fusion protein in 24B-Gal4/UAS-hth-en embryos.

View larger version (82K): [in a new window]   Fig. 2. wg expression and XC enhancer activity depends on Dpp and Wg signaling. (A,B) wg transcripts revealed by in situ hybridization. Arrowheads indicate PS8, the site of wg midgut expression in wild-type embryos (A). wg expression is completely abolished in the midgut of dpps13 homozygous-mutant embryos (B). (C-L) Regulation of XC or XC([Hox/Pbx2-3-4]) enhancer activity by AbdA, Dpp and Wg visualized by in situ hybridization to lacZ transcripts. All panels show magnifications of lateral views of the midgut of stage 14 embryos, except panel F, which is from a stage 15 embryo. Embryos in C,J,K and L have been processed in the same conditions and the length of staining time was identical. Arrows indicate sites of ectopic enhancer activity. The embryo in C bears a wild-type copy of the XC enhancer and serves as a reference for the activity of XC variants. With respect to wg expression, XC enhancer activity in the wild type (C) is lost in dpps13 homozygous-mutant embryos (D). The embryo in D has been overstained to ensure the absence of lacZ staining. Mesodermal ubiquitous expression of Dpp in 24B-Gal4/UAS-dpp embryos induces ectopic activity of the XC enhancer posterior to PS8 (E) in cells where AbdA is present. At stage 15, ectopic sites of XC activity coincide with the sites of extra constrictions that form in this genotype (F). Mesodermal ubiquitous expression of AbdA in 24B-Gal4/UAS-abdA embryos weakly induces ectopic activity of the XC enhancer anterior to PS8 (G), in close proximity to the PS7 Dpp source. In 24B-Gal4/UAS-abdA embryos, ectopic XC([Hox/Pbx2-3-4]) enhancer activity is stronger than XC enhancer activity, and is also detected more anteriorly, close the source of Dpp in VM cells close to PS3-4 (H). Simultaneous expression of AbdA and Dpp in 24B-Gal4/UAS-dpp/UAS-abdA embryos induces ectopic XC enhancer activity anterior and posterior to PS8 (I). XC activity is diminished in wgIL114 homozygous embryos shifted to restrictive temperature at 7 hours of development at 25°C (J) or in 24B-Gal4/UAS-Tcf(DN) embryos expressing the dominant-negative form of the Wg transcriptional effector Drosophila Tcf (K). In wgILL114 homozygous mutants grown at 29°C, ectopic Dpp signaling provided by 24B-Gal4/UAS-dpp still induces, although at lower levels, ectopic XC enhancer activity (L).

View larger version (63K): [in a new window]   Fig. 4. Two Hox binding sites within Box2 are required for XC enhancer activity. Functional requirement of Hox6/7 sites for XC or XC([Hox/Pbx2-3-4]) enhancer activity visualized by in situ hybridization to lacZ transcripts. All embryos have been processed under the same conditions, with identical staining times. All panels show magnifications of lateral views of the midgut of stage 14 embryos. The embryo in A bears a wild-type copy of the XC enhancer and serves as a reference for the activity of XC variants. The deletion of Box2 (B), or the mutation of the two Hox binding sites (Hox6/7) found in Box2 (C), results in a strong diminution of XC enhancer activity. The activity of the XC([Hox/Pbx2-3-4]) enhancer (D) is stronger than that of the full-length XC (A), and is also significantly diminished upon mutation of the Hox6/7 sites (E).

View larger version (65K): [in a new window]   Fig. 3. Evolutionary conservation of the wg midgut enhancer. (A,B) Enhancer activities visualized by in situ hybridization to lacZ transcripts. Embryos are shown in a lateral view. The D. virilis counterpart of the XC enhancer drives expression in the central part of the midgut (B), as does the D. melanogaster XC enhancer (A), although at a reduced level. (C) Alignment of the D. melanogaster (mel), D. pseudoobscura (pseu) and D. virilis (vir) sequences. Sequences in red emphasize sequence identity. Two long stretches of sequence conservation are boxed in yellow. Consensus binding sites are indicated in blue for Hox and Hox/Pbx complexes, and in green for Mad/Medea (referred to as DRS) and Creb proteins. Sequences after the arrow are deleted in XC([Hox/Pbx2-3-4]).

View larger version (79K): [in a new window]   Fig. 5. Box2 binds in vitro to AbdA and is sufficient to drive an AbdA-dependent expression pattern in the embryonic midgut. (A-D) Box2 responds in vivo to AbdA. Enhancer activity is visualized by immunohistochemistry using an anti-ß-galactosidase antibody. All panels show magnifications of lateral views of the midgut of stage 14 embryos. Arrowheads indicate PS8, the site of wg midgut expression. Arrows indicate ectopic enhancer activity. An oligomer consisting of three copies of Box2 drives lacZ expression in the posterior midgut in the AbdA expression domain. Expression is first detected posterior to the normal site of wg expression (A) and later in a domain that includes the third, and part of the fourth, midgut chambers (B). Box2 enhancer activity no longer occurs in abdAJX2 homozygous mutants (C), and is induced ectopically in the entire midgut VM when AbdA is ubiquitously provided by a heat shock construct (D). (E) Gelshift experiments with AbdA, Exd and Hth proteins produced in vitro were performed on wild-type (lanes 1-10) and mutated (Box2m; lanes 11-14) forms of Box2. Box2m carries the same point mutations as those introduced in XC(Hox6/7). 3 µl of the programmed lysate were used for each protein and for the mock lysate (lanes 2 and 16). For the binding experiments combining Exd and Hth, the two proteins were simultaneously produced and 6 µl of the lysates were used. The anti-AbdA serum was used at a 1/20 dilution. The activity of the proteins were assayed on a DllR sequence (lanes 15-23), known to assemble an AbdA/Exd/Hth complex. The asterisk and the dot mark the position of the AbdA/Exd and AbdA/Exd/Hth complexes, respectively.

View larger version (115K): [in a new window]   Fig. 6. Requirement of Mad and Creb proteins for XC enhancer activity. Enhancer activity is visualized by in situ hybridization to lacZ transcripts. All panels show magnifications of lateral views of the midgut of stage 14 embryos. Arrowheads indicate the site of wg expression in PS8. Arrows indicate ectopic enhancer activity. All embryos have been processed under the same conditions and the staining times were identical. (A-C) Requirements in trans. (A) Wild-type embryo carrying the XC enhancer. The activity of the XC enhancer is lost in mad12-homozygous mutants (B) and is strongly reduced upon expression of a dominant-negative form of Drosophila CrebB in the mesoderm of 24B-Gal4/UAS-Creb(DN) embryos (C). (D-F) Requirements in cis. Mutation of the three DRS in XC(DRS1-2-3) results in the complete loss of enhancer activity in VM PS8 (D). It also induces ectopic activity (arrows) of the enhancer in endodermal cells from the central part of the midgut, and in a more anterior region close to the foregut/midgut boundary (D). XC(Creb1-2) that bears mutations in the two Creb binding sites shows a severely reduced enhancer activity (E). The mutation of the three DRSs and the two Creb binding sites results in a complete loss of enhancer activity (F), including in the territories where the enhancer is ectopically induced by XC(DRS1-2-3).

View larger version (49K): [in a new window]   Fig. 7. Mad and Drosophila CrebB proteins bind in vitro to the XC enhancer. (A) Gelshift experiments with Mad protein [50 ng (+) or 200 ng (++)] were performed on double-stranded oligonucleotides corresponding to wild-type or mutated (DRS2m) versions of DRS2, in the presence or absence of a 500-fold excess of cold DRS2 competitor. Lanes 1-3 show a dose-dependent binding of Mad to DRS2. Lanes 4-7 indicate that binding is competed by cold DRS2 (lanes 4 and 5), and that the integrity of the DRS2 site is required for Mad binding (lanes 6-7). (B) Similar experiments on double-stranded oligonucleotides corresponding to the wild-type or mutated (DRS1m) version of DRS1. Compared with the experiments in A, this gelshift shows that Mad protein binds DRS2 with a stronger affinity than DRS1. (C) Gelshift experiments with Drosophila CrebB protein [20 ng (+) or 100 ng (++)] were performed on double-stranded oligonucleotides corresponding to wild-type (Creb1-2) or mutated (Creb1-2m) versions of Creb-binding sites, in the presence or absence of a 500-fold excess of cold Creb1-2 competitor. Lanes 1-3 show a dose-dependent binding of Drosophila CrebB. Lanes 4-7 indicate that the binding of Drosophila CrebB is competed by the competitor, and that the integrity of the two Creb binding sites is required for binding to occur.

View larger version (24K): [in a new window]   Fig. 8. A model for the regionalization of AbdA activity: cis-regulatory readouts of a Hox/signaling combinatorial code subdivide the domain of AbdA protein function. Simultaneous requirement of Dpp signaling and AbdA results in localized wg transcriptional activation in PS8 VM cells. The direct convergence of AbdA and Dpp signaling on a wg cis-regulatory region has been established in this study. The model hypothesizes that the regulation of pnt and opa also results from direct cis-regulatory integration of Hox and signaling inputs. In the regulation of pnt, Wg-activated transcription factors synergise with AbdA, ultimately resulting in localized (PS8-10) transcription of pnt. In the regulation of opa that only occurs in the absence of Dpp signaling, Dpp-activated regulators inhibit opa activation through AbdA. According to this model, the cis-regulatory readout of a Hox/signaling combinatorial code is instructive in the regionalization of AbdA transcriptional activity, and thus confers functional diversity to AbdA. WRE, Wg response element; DRE, Dpp response element; HRE, Hox response element.

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