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First published online 23 January 2008
doi: 10.1242/dev.015206

Development 135, 829-837 (2008)
Published by The Company of Biologists 2008
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Multiple RTK pathways downregulate Groucho-mediated repression in Drosophila embryogenesis

Einat Cinnamon1, Aharon Helman1, Rachel Ben-Haroush Schyr2, Amir Orian3, Gerardo Jiménez4 and Ze'ev Paroush1,*

1 Department of Biochemistry, Faculty of Medicine, The Hebrew University, PO Box 12272, Jerusalem 91120, Israel.
2 Department of Cellular Biochemistry and Human Genetics, Faculty of Medicine, The Hebrew University, PO Box 12272, Jerusalem 91120, Israel.
3 The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 31096, Israel.
4 Institut de Biologia Molecular de Barcelona-CSIC and Institució Catalana de Recerca i Estudis Avançats, Parc Científic de Barcelona, 08028-Barcelona, Spain.


Figure 1
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  		Fig. 1. Phosphorylation of Groucho in the ventral neuroectoderm depends on EGFR signalling. (A-I) Ventral views of stage 10 embryos; anterior is towards the left. (A-C) Wild-type embryo stained with both {alpha}pGro (A; red) and {alpha}dpERK (B; green) antibodies. (C) Merge. There is a significant overlap between the staining in cells that border the midline, whereas only pGro, but not dpERK, is detected in more lateral cells. (D-F) Wild-type embryo double-stained with {alpha}pGro (D; red) and {alpha}Gro (E; green) antibodies. (F) Merge. The staining is largely mutually exclusive, indicating that Gro is phosphorylated in cells straddling the ventral midline, whereas in more lateral ectodermal regions it is mostly in its unphosphorylated state. (G-I) Homozygous Egfrf2 mutant embryos stained for pGro (G; red) and Gro (H; green). (I) Merge. pGro staining is decreased in the ventral neuroectoderm (compare with A,D), and is replaced by {alpha}Gro staining (compare with E). (F,I) There is complementarity between the {alpha}pGro and {alpha}Gro staining, attesting to the specificity of our {alpha}pGro antibodies (see Fig. 2D below). (C,F,I) Arrowheads indicate the ventral midline. (J) {alpha}pGro and {alpha}Gro antibodies differentially recognise the phosphorylated and nonphosphorylated forms of Gro, respectively, in western blot analysis, using a denaturing gel. Bacterially expressed GST-Gro fusion protein is recognised mainly by {alpha}Gro antibodies (lane 1). Phosphorylation of Gro by ERK2 in vitro leads to its detection primarily by {alpha}pGro, and prevents its recognition by {alpha}Gro, antibodies (lane 2). Incubation of phosphorylated Gro with a nonspecific phosphatase (2.5u CIP, lane 3; 5u CIP, lane 4) reverses the recognition by the antibodies, suggesting that phosphorylation itself is enough to cause the differential recognition by the antibodies.

 

Figure 2
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  		Fig. 2. Groucho is phosphorylated in cellular blastoderm embryos at the termini and in seven central transverse stripes. (A-C) Lateral view of a stage 5 blastoderm embryo, double-stained with {alpha}pGro (A; red) and {alpha}Gro (B; green) antibodies. (C) Merge. Phosphorylated Gro is detected at both poles, and in seven stripes in the trunk region of the embryo (A). {alpha}Gro staining is complementary to that of {alpha}pGro (C). (D) The pGro signal is significantly reduced in embryos devoid of maternally contributed Gro (germline clone; GLC), stained by {alpha}pGro antibodies. (E-G) Confocal optical cross-sections of a stage 4 syncytial blastoderm embryo, stained for {alpha}pGro (E; red) and {alpha}dpERK (F; green). (G) Merge. {alpha}dpERK and {alpha}pGro staining colocalises at the anterior and posterior termini. dpERK is mostly cytoplasmic, whereas pGro is predominantly nuclear. In this and subsequent figures, embryos are oriented with the anterior towards the left and dorsal side upwards.

 

Figure 3
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  		Fig. 3. Groucho is phosphorylated by the Torso pathway. Wild-type (A) or mutant (B-F) stage 5 embryos, stained with {alpha}pGro (A,B,C,E) or {alpha}Gro (D,F) antibodies. The genetic inactivation of the Torso pathway, in tsl691 mutant embryos, leads to loss of pGro from the termini (B; compare with A). Reciprocally, pGro staining expands to more central regions in torY9 mutants, in which the Torso receptor is overactive, and a higher than normal uniform staining is observed throughout the embryo (C; compare with A). (D) A torY9 mutant embryo stained with the {alpha}Gro antibody, showing a larger posterior domain devoid of staining (compare with Fig. 2B). The central pGro stripes are not dramatically affected in either tsl691 or torY9 mutants. (E,F) A stage 5 DSor mutant embryo (GLC), stained for {alpha}pGro (E; red) and {alpha}Gro (F; green). pGro staining is absent from the termini (E; compare with A), and is replaced by {alpha}Gro staining, which is not detected normally in this region (compare with Fig. 2B). There are seven pGro stripes in the DSor mutant (E).

 

Figure 4
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  		Fig. 4. Phosphorylation of Groucho and downregulation of its repressor function are required for terminal gap gene expression. Expression of tll (A-D) and hkb (E-H) in stage 4 embryos maternally expressing Gro (B,F), GroAA (C,G) or GroDD (D,H). Embryos similarly expressing lacZ serve as controls (A,E). Expression of Gro leads to reduction in tll and hkb transcription at both termini (B,F), whereas that of GroAA almost completely eliminates tll and hkb expression (C,G). In the posterior of the embryo, tll and hkb expression remains only in a ventral-terminal domain (arrows in B,C,F,G). Expression of GroDD has no effect, indicating that it cannot repress tll and hkb (D,H).

 

Figure 5
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  		Fig. 5. Maternal expression of Groucho or its derivatives does not disrupt embryonic anteroposterior axis formation. The anterior localisation of hb (A-D) and posterior localisation of nos (E-H) RNA transcripts, as well as the degradation of the Cic protein at the termini (I-L) are indistinguishable in early stage 5 control embryos (A,E,I) and in embryos maternally expressing Gro (B,F,J) or its variants, GroAA (C,G,K) and GroDD (D,H,L). By contrast, the posterior zygotic hb stripe is derepressed posteriorly in Gro (B) and GroAA (C), but not in GroDD (D)-expressing embryos. hb is not derepressed in a ventroposterior domain in Gro (B) and GroAA (C) expressing embryos (white arrows), in accordance with the enduring hkb expression in these embryos (see Fig. 4F,G). (A-D,I-L) Black arrows indicate the same relative positions in all embryos.

 

Figure 6
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  		Fig. 6. Phosphorylated Groucho is a nuclear and stable protein that persists after MAPK activation has been extinguished. (A,A') Posterior terminus of stage 5 wild-type embryo, stained with {alpha}pGro antibodies (red) and {alpha}Lamin antibody (green) demarcating the nuclear membrane. Superficial (A) and transverse (A') confocal sections show the nuclear localisation of pGro, encircled by {alpha}Lamin staining. (B-D) Ventral view of wild-type stage 6 gastrulating embryo (note the invagination of the ventral furrow), stained for {alpha}pGro (B; red) and {alpha}dpERK (C; green). (D) Merge. pGro staining is detected at the termini even after MAPK activation has been turned off. Strong dpERK and pGro staining on both sides of the ventral furrow correlates with EGFR activation in this region. (E-E'') Model depicting possible implications of Gro phosphorylation to RTK target gene regulation. Prior to RTK activation (E), Gro is associated with its partner DNA-binding repressors (R), mediating repression of RTK target genes. Upon RTK pathway activation (E'), Gro is phosphorylated by MAPK. Modification of Gro downregulates its repressor activity, causing derepression of pathway target genes. MAPK is no longer active after RTK signalling has been turned off (E''), yet Gro remains stably phosphorylated and its activity attenuated, allowing for sustained RTK target gene expression.

 

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© The Company of Biologists Ltd 2008