Relief of gene repression by torso RTK signaling: role of capicua in Drosophila terminal and dorsoventral patterning

Abstract

Differentiation of the embryonic termini in Drosophila depends on signaling by the Tor RTK, which induces terminal gene expression by inactivating at the embryonic poles a uniformly distributed repressor activity that involves the Gro corepressor. Here, we identify a new gene, cic, that acts as a repressor of terminal genes regulated by the Tor pathway. cic also mediates repression along the dorsoventral axis, a process that requires the Dorsal morphogen and Gro, and which is also inhibited by Tor signaling at the termini. cic encodes an HMG-box transcription factor that interacts with Gro in vitro. We present evidence that Tor signaling regulates terminal patterning by inactivating Cic at the embryo poles. cic has been evolutionarily conserved, suggesting that Cic-like proteins may act as repressors regulated by RTK signaling in other organisms.

Figure 1

cic is required for repression of tll and hkb during terminal development. Cuticle phenotypes of embryos derived from wild-type (A) and homozygous cic¹ (B) females. Note the strong suppression of trunk segmentation in the cic¹ mutant embryo. Embryos from females carrying the cic¹ allele in trans with a deficiency of the cic region show a slightly more severe phenotype (not shown). (C–H) RNA expression patterns of tll (C,D), hkb (E,F), and tor-RE-lacZ (G,H) in embryos derived from wild-type (C,E,G) and homozygous cic¹ (D,F,H) females. Derepression is observed in all cases in cic¹ mutant embryos. In this and following figures, anterior is to the left and dorsal is up.

Figure 2

cic acts downstream of the Tor pathway and is not required for Gro activity. (A) Diagram of the Tor cascade. (B,C) Cuticle phenotypes of embryos derived from doubly homozygous tor; cic females (B) and from cic¹ females carrying germ-line clones of Draf (C); the embryos display a cic-like phenotype, suggesting that cic acts downstream of Draf. The roles of cic, tor, and Draf in terminal patterning are strictly maternal and the mutations do not show paternal rescue. (D,E) Antibody staining for activated Erk protein in wild-type (D) and cic¹ (E) embryos; the similar staining indicates that the domain of Tor signaling is unaffected in cic¹ mutant embryos. (F–H) Effects on Sex-lethal (Sxl) expression of ectopic hairy (h) activity driven by the hunchback (hb) promoter in otherwise wild-type (F), gro^E48 (G), and cic¹ (H) embryos. Embryos were stained with an antibody against the active Sxl protein. Repression of Sxl by Hairy requires gro, however the Hairy/Gro complex does not require cic function.

Figure 3

cic is required for repression of zen and acts through the VRE. RNA expression patterns of zen (A,B), twi (C,D), and St.2-lacZ-VRE (E,F) in embryos derived from wild-type (A,C,E) and homozygous cic¹ (B,D,F) females. Derepression of zen and lacZ expression driven by the eve stripe 2 enhancer (arrowhead) is observed in cic¹ embryos. In contrast, the similar pattern of twi expression in wild-type and cic¹ embryos indicates that activation of twi by Dorsal is independent of cic.

Figure 4

cic encodes an evolutionarily conserved HMG box transcription factor. (A) Diagram of the cic genomic region showing the insertion sites of the hobo element causing the cic¹ mutation and the nearby P(PZ) bwk⁸⁴⁸² P element (transposons are not represented at scale). The cic transcription unit spans ∼8 kb and includes at least two introns. We have mapped in detail only one intron in the 5′ region of the gene (not shown). The diagram shows the region deleted in a small deficiency caused by imprecise excision of P(PZ) bwk⁸⁴⁸², bwk^Δ14 (see Materials and Methods), which does not complement cic¹. The genomic construct used to rescue the cic phenotype and a rescued embryo are also shown. (E) EcoRI; (N) NotI; (X) XbaI. (B) Maternal expression of cic. cic transcripts accumulate in early blastoderm embryos (stages 1–3) but become undetectable after the onset of gastrulation (stage 5). In situ hybridizations were carried out with an anti-sense cic RNA probe. (C) Amino acid sequence of the deduced Cic protein. The HMG box domain is shown in bold. Possible MAPK phosphorylation sites (P-X-S/T-P) are underlined. Note the presence of multiple homopolymeric stretches in the protein, particularly in the amino-terminal domain. (D) Sequence alignment of HMG box domains from Drosophila Cic (D) and two related proteins from humans (H; GenBank accession no. AB002304) and C. elegans (C; GenBank accession no. Z50797). Periods indicate identical residues. (E) Sequence alignment of the Cic carboxy-terminal domain (amino acids 1308–1355) with the corresponding region of the human and C. elegans proteins; the strong conservation indicates that these proteins are true orthologs. We have not detected significant similarities outside of the HMG box and carboxy-terminal domains.

Figure 5

Cic binds to Gro in vitro. (A) Diagram of the Cic protein and three fragments tested for interaction with Gro. (B) ³⁵S-labeled Gro was incubated with similar amounts of the indicated GST fusions bound to glutathione-Sepharose beads. After washing the beads, the retained Gro protein was detected by autoradiography. The Cic^834–1403 domain binds to Gro, whereas the other two domains show little or no binding. GST fusions of Hairy and Dorsal^1–378 were used as positive controls (Paroush et al. 1994; Dubnicoff et al. 1997), whereas GST–Hairy^1–286 (a mutant lacking the carboxy-terminal Gro-binding motif) was the negative control (Jiménez et al. 1997). Binding of Gro to Dorsal^1–378 appears very weak in our assay. We have not observed significant similarities between the Cic^834–1403 domain and other Gro-binding motifs.

Figure 6

Cic is negatively regulated by the Tor pathway. (A) Pattern of Cic protein distribution in wild-type embryos. The protein localizes predominantly in the nucleus (inset; surface view of blastoderm embryo) and is present in medial but not terminal regions of the embryo. (B,C) Pattern of Cic protein in cic¹ (B) and tor mutant embryos (C). Note the lack of staining in the cic¹ background, and the accumulation of Cic protein at the poles of tor mutant embryos (arrowheads). (D) Model for Cic function in terminal and dorsoventral patterning. Cic associates with Gro to form a protein complex that represses tll and hkb expression in the central region of the embryo. The Tor RTK pathway inactivates Cic at the embryo poles, thus allowing activation of tll and hkb by other maternal factors. A similar mechanism operates in the regulation of zen, except that repression in middle regions of the embryo is only ventral and also requires Dorsal and other corepressors such as Dri and Cut. In both cases, repression could involve additional factors.