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doi: 10.1242/10.1242/dev.00286

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Requirements for transcriptional repression and activation by Engrailed in Drosophila embryos

National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

View larger version (90K): [in a new window]   Fig. 1. Effect of paired-Gal4-driven En on the expression of negative and positive targets. In this and all subsequent figures the domain of paired-Gal4 expression is marked by a blue rectangle. Embryos are all at st11 and the identity of segments is indicated (T, thoracic; A, Abdominal). Whole embryos are shown lying on their side, whereas higher magnifications show ventral views with the ventral midline running in the middle from left to right. (A,B) Expression of ci in a wild-type embryo (A) and in an embryo expressing ectopic En under the control of paired-Gal4 (prd-G4 UAS-en) (B). In addition to repression in the normal domain of En expression, ectopic repression of ci expression is seen in alternate segments, within the paired domain. (C,D) Expression of hh in a wild-type (C) and paired-Gal4 UAS-en (D) embryo. Here, ectopic expansion of hh expression is seen in the paired-domain (D). (E,F) Expression of endogenous en in a wild-type (E) and paired-Gal4 UAS-en (F) embryo. A probe from the 3' UTR that is not present in the UAS-en construct was used to detect endogenous expression. Endogenous en expression is activated by ectopic En. Note that repression and activation occur both in the thorax and abdomen.

View larger version (30K): [in a new window]   Fig. 2. Structure and possible effects of VP16En. (A) In VP16En, the repressor domain of En (as defined by Han and Manley, 1993) has been removed and replaced by the VP16 transactivating domain. Note that eh2, the domain of En implicated in cooperative binding with Exd, is still present in VP16En. (B) Two possible modes of activation by En. If En activates hh directly or by activating the expression of an intermediate activator (A), VP16En is expected to activate hh expression too. By contrast, if En activates through repression of a repressor, VP16En should activate the expression of this repressor and the net result would be repression of hh expression. (C) Expected effect of VP16En on negative targets of En (such as ci). The expected outcome is the same whether En represses ci expression directly or by repressing an intermediate activator (A').

View larger version (73K): [in a new window]   Fig. 3. VP16En activates genes that are normally repressed by En. Embryos placed side by side are of the same genotype except that embryos on the right-hand side express VP16En under the control of paired-Gal4. (A,B) Expression of ci in the abdominal region of wild-type (A) and paired-Gal4 UAS-VP16En (B) embryos. In the wild-type (A), ci is repressed by En in every segment. paired-Gal4-driven VP16En leads to ectopic activation of ci expression thus blotting out repression in alternate segments (B). (C,D) Expression of wg in the abdominal region of wild-type (C) and paired-Gal4 UAS-VP16En (D) embryos. Expression of wg is broadened in response to VP16En expression in alternate segments (D). (E) Expression of wg in a ciCE embryo. No expression is detectable in the segmented ectoderm. This is expected because Hh signaling (hence Ci) is required for continued wg expression (Tabata et al., 1992). (F) Expression of wg in a ciCE embryo that expresses VP16En under the control of paired-Gal4. Ectopic expression of wg is induced even though Ci is absent, consistent with the possibility that VP16En could activate wg expression directly.

View larger version (113K): [in a new window]   Fig. 4. VP16En represses the expression of en and hh, two positive targets of En. Wild-type embryos are shown on the left and paired-Gal4 UAS-VP16En embryos on the right. (A,B) Expression of hh in wild type (A) and in an embryo expressing VP16En (B). (C,D) Expression of en in a wild-type embryo (C) and in an embryo expressing VP16En (D). A specific probe that does not recognize VP16En was used to detect en mRNA. In B and D, expression of the target gene (hh and en, in purple) is clearly repressed in the cells that express VP16En (labelled in ochre).

View larger version (101K): [in a new window]   Fig. 5. Expression of slp is repressed by En. (A) Expression of slp in a wild-type st11 embryo. (B) Expression of slp in an embryo that expresses both ectopic en and lacZ under the control of paired-Gal4 [prdG4(Z) UAS-En]. slp transcripts (black) disappear where En is ectopically expressed (here the domain of ectopic expression is recognized with a lacZ probe in red). (C) Abdominal region (A5-A8) of a wild-type embryo showing the expression of slp at high magnification. (D) Similar view of an st11 embryo expressing VP16En under the control of paired-Gal4. Notice the broadening of the slp stripes in odd-numbered segments, where paired-Gal4 is active.

View larger version (98K): [in a new window]   Fig. 6. Evidence for bona fide activation by En. (A,B) Expression of hh (A) and en (B) in the abdominal region of slp (34B) mutant embryos. The expression patterns are identical. Note that expression widens in even-numbered segments, whereas it is decaying in oddnumbered segment (see Cadigan, 1994). (C,D) Expression of hh in slp (34B) mutant embryos that express either En (C) or VP16En (D) in the paired domain. Expression of hh is in purple and that of VP16En is in ochre. In both cases, hh expression is activated, especially in the ventral region (compare with homologous segments in plain slp-, panel B). Thus, in the absence of the repressor Slp, VP16En and En have the same effect on expression of hh (and also of en; not shown). By contrast, in the presence of slp, expression of hh is activated by En (Fig. 1D), whereas it is repressed by UAS-VP16En (Fig. 4B). Note that, in slp-1, the effect of VP16En is stronger than that of En.

View larger version (106K): [in a new window]   Fig. 7. Wg signaling contributes to the activation of the positive targets of En. (A) Expression of Wg with paired-Gal4 in the absence of en and slp leads to weak, but significant expression of hh in oddnumbered segments (where hh decays in slp-). Panels (B) and (C) are repeated from Fig. 6A,C to allow comparison with the effect of Wg. As seen, Wg and En have a similarly weak positive impact on hh expression. Together, Wg signaling (here induced with activated Armadillo, A*) and En cause strong activation of hh expression (panel D). (E) hh transcription in a wgCX4 embryo overexpressing En under the control of paired-Gal4. The histochemical reaction was allowed to proceed for a long time to reveal the weak signal. Expression is weak. Therefore, En can partially activate hh transcription in the absence of Wg signaling but Wg signaling is required for full activation. Indeed, as shown in (F), co-expression of En and activated Armadillo leads to strong hh expression. Remember that VP16En is a stronger activator than En in slp mutants. This is probably because VP16En activates Wg expression (Fig. 3D). Therefore, in the absence of slp, expression of VP16En leads to expression of both wg and en, thus causing maximal activation of hh expression.

View larger version (103K): [in a new window]   Fig. 8. Exd and Hth are required for En to activate genes in the abdomen. In all panels, abdominal segments A4 to A8 of es11 embryos are shown. (A) Expression of En protein and hh RNA in an exd deficient embryo. Expression of both En and hh has begun to decay. (B) Transcription of hh in an exd- embryo expressing exogenous En under the control of the paired-Gal4 driver. No ectopic activation is seen (most ochre cells are not black). There may be slightly increased residual expression of hh (compare black signal in A and B) but this could be in underlying neuroblasts. (C) In the presence of exd+, paired-Gal4-driven En activates hh expression through the domain of ectopic expression (see the widening of hh expression in alternate segment as shown for anterior segments in Fig. 1D). (D) Ectopic En does not activate hh expression in a zygotic hth mutant embryo (hth- paired-Gal4, UAS-en). This shows that a mutation in hth has the same effect as removal of exd (compare with panel B).

View larger version (70K): [in a new window]   Fig. 9. VP16En requires hth throughout the germ band to activate positive targets of En and only in anterior thoracic segments to activate negative targets. All panels show anterior segments (T1, T2, T3, A1, in register) of st11 embryos that express VP16En under the control of the paired-Gal4 driver. (A) In the absence of slp, VP16En (in ochre, seen with anti-HA) activates expression of hh (blue), especially in the midventral region. This point is already made in Fig. 6D for abdominal segments. The same effect is seen on en expression (not shown). (B) In the absence of hth (and also slp), VP16En no longer activates ectopic hh expression. This panel shows a slp hth double mutant with VP16En in brown and hh in blue. Almost all endogenous hh has disappeared and no ectopic expression is seen. (C) VP16En activates ectopic expression of wg in T2 and A1 (where paired-Gal4 is active) in an otherwise wild-type embryo. (D) In a hth mutant, ectopic wg expression is still activated in A1 but not in T2. (E,F) A similar effect is seen on the expression of slp, another negative targets of En.

View larger version (47K): [in a new window]   Fig. 10. En requires exd/hth to repress endogenous targets, but only at the anterior of the germ band. Embryos shown at the top (A, B, C) are all deficient for exd (or hth) and, in all panels, En is expressed under the control of paired-Gal4 (e.g. in T2 and A1). (A) In hth-, expression of ci is repressed by En in A1 but not in T2. In this embryo, all En originates from the transgene since endogenous expression decays in hth mutants. (A') For comparison, the effect of ectopic En on ci expression in hth+ is reproduced from Fig. 1B (showing only the left side of the embryo). (B) As with ci, expression of slp is repressed in A1 but not in T2 in exd- embryos. Here ectopic En is shown in ochre (as detected with an antibody) and slp expression is in blue. Note that in A1, En is present but not slp. (B') Again, for comparison, we show the effect of En expression on slp expression in an otherwise wild type embryo (as in Fig. 5B). (C) In exd mutants, activation of hh expression by En is abolished in both T2 and A1. Here, En protein is shown in ochre and expression of hh is shown in brown. Residual expression (a probable remnant of previous activation by pair-rule gene products) is seen in the normal domain, but not ectopically as happens if exd+ is present (shown in C'). (C') Expression of hh is activated in T2 and A1 of otherwise wild type embryos (as shown previously in Fig. 1D).

View larger version (67K): [in a new window]   Fig. 11. Role of Ubx or Abd-A in repression by En. (A) Coexpression of En and Abd-A (ochre) repress ci expression at the anterior of the germ band (as well as in the abdomen) of a hth mutant embryo. This includes T2 and also head segments (not shown). Remember that expression of En alone in a hth mutant does not lead to repression there (Fig. 10A). (B) Ectopic expression of Abd-A alone in a wild-type embryo (or in a hth mutant; not shown) has no effect on ci expression. Normal repression in the domain of en expression is seen but no ectopic repression is seen in the paired domain (marked with an anti-Abd-A antibody in ochre). (C) Coexpression of En and Antp does not lead to ci repression in T2 of a hth mutant. Expression of ci is the same as if En alone were expressed (as in Fig. 10A). In this picture, expression of Antp (ochre) is partially masked by the signal coming from ci transcripts (blue). (D) As expected, paired-Gal4 driven Antp alone has no effect on ci expression whether in a wild type or a hth mutant (not shown). (E-F) Co-expression of Ubx allows VP16En to activate wg expression in T2 of a hth mutant embryo (E), whereas VP16En is unable to activate there on its own (F; also shown in Fig. 9D). (G) Distribution of ci transcripts (blue) and En protein (ochre) in a Ubx, abd-A double mutant [Df(3R)Ubx109] overexpressing En with paired-Gal4. In such embryos, repression of ci occurs throughout the domain of ectopic En expression. There is no overlap between En (ochre) and ci (blue). Thus, removal of Ubx and Abd-A does not prevent En from repressing in the abdomen (compare to Fig. 1B). (H) Expression of ci (blue) and En (ochre) in a Ubx, abd-A, hth64-1 triple mutant. Here, no repression of ci expression is seen despite the strong ectopic expression of En (see panel I). Thus in the absence of Ubx, Abd-A and Hth, the repressive activity of En is abolished. (I) Embryos of the same genotype as in H stained for En alone. This panel shows that the paired-Gal4 driver is not affected in the triple mutant.

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