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Differential requirement for STAT by gain-of-function and wild-type receptor tyrosine kinase Torso in Drosophila

¹ Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
² Department of Biomedical Genetics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 633, Rochester, NY 14642, USA
³ Dana-Farber Cancer Institute, M649B, 44 Binney Street, Boston, MA 02115, USA
⁴ Howard Hughes Medical Institute, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA

View larger version (86K): [in a new window]   Fig. 1. The torGOF mutant phenotype is suppressed by loss of mrl activity. (A,C,E) tll mRNA expression patterns. (B,D,F) Larval cuticles. (A) In wild-type embryos, tll expression is restricted to the anterior and posterior poles. The posterior tll expression domain occupies 15% of egg length (EL). The anterior tll expression is controlled by both the Tor and Bicoid pathways (Pignoni et al., 1992) and is not discussed in this paper. (B) A wild-type larva has eight ventral abdominal denticle bands (A1 to A8), and posterior spiracles containing Filzkörper materials (arrowhead). (C) Embryos laid by females heterozygous for torY9, a gain-of-function allele, show expansion of tll expression domains that causes enlargement of the terminal cell fates at the expense of the central cell fates (Klingler et al., 1988). As a consequence, (D) most denticle bands are frequently deleted, and occasionally ectopic Filzkörper material can be observed (arrowhead). (E) Removal of the maternal mrl gene product from torY9 embryos resulted in a reduction of the ectopic tll expression associated with torY9 to levels similar to those of wild-type embryos (see Materials and Methods). (F) The ventral denticle bands were mostly restored in these embryos and they exhibit phenotypes that are similar to mrl embryos derived from homozygous germline clones (GLC embryos). Similar results were obtained when a second gain-of-function tor mutation, torRL3, was used (data not shown).

View larger version (117K): [in a new window]   Fig. 2. Mutations in hop or mrl do not enhance Draf or Ras1 mutant phenotypes. (A) In mrl GLC embryos, the size of the posterior tll expression domain appears similar to wild type. (B) hop or mrl GLC embryos have identical phenotypes (mrl6346 mutant is shown) with a characteristic deletion of A5. (C) The tll expression pattern in hopC111 DrafC110 GLC embryos is indistinguishable from that in DrafC110 GLC embryos. (D) These embryos exhibit cuticle phenotypes that resemble those of hopC111 embryos. (E) The size of the posterior tll expression domain in hopC111 DrafPB26 double GLC embryos is similar to that of DrafPB26 GLC embryos. (F) The cuticles of these embryos exhibit defects of both hopC111 and DrafPB26 mutants. (G) 19% of the Ras1C40B mrl6346 double mutant embryos (n=18/97) have residual posterior tll expression that is similar to Ras1C40B GLC embryos. (H) The cuticle defects of the Ras1C40B mrl6346 double GLC embryos are a combination of those associated with Ras1C40B and mrl6346 mutants, respectively, i.e., they show characteristic deletion of A4 and A5 due to the mrl6346 mutation and defects in posterior structures similar to Ras1C40B and mrl6346 GLC embryos.

View larger version (36K): [in a new window]   Fig. 3. TorGOF activates and associates with Mrl. (A) Mrl activity was measured by a gel mobility shift assay in S2 cell extracts using an oligonucleotide (top strand sequence: GGATTTTTCCCGGAAATG. Bottom strand sequence: GACCATTTCCGGGAAAAA) optimal for Mrl binding (Yan et al., 1996). Control (lane 1) shows basal levels of Mrl activity in S2 cells. Transfection of Hop (lane 4; 10 µg) resulted in a significant increase in Mrl DNA-binding activity. Transfection of DNA encoding wild-type Tor (lane 2; 10 µg) or TorGOF (lane 3; 10 µg) significantly increased the DNA-binding activity of Mrl to levels similar to those observed following Hop transfection. Cells treated with the vanadate-H2O2 (100 µM sodium orthovanadate, 1 mM hydrogen peroxide) (see Sweitzer et al., 1995) strongly activate Mrl (lane 5) and result in similar gel-shift bands. Addition of an anti-Mrl antibody caused a supershift of the protein-DNA complex (lane 6), suggesting it is due to Mrl-oligonucleotide association. (B) Tor protein was precipitated with anti-Tor antibody (Cleghon et al., 1996) from wild-type and torGOF embryo extracts, respectively. Note Mrl (80 kDa) was co-precipitated with Tor (135 kDa) from both wild-type and torGOF embryos in the presence of vanadate.

View larger version (22K): [in a new window]   Fig. 4. Mrl-binding sites in the tll promoter. (A) The binding of Mrl to sites 1 and 2 was assayed by a gel mobility shift assay using synthetic oligonucleotides corresponding to the two sites and surrounding sequences. The Hop/Mrl pathway was activated by treating S2 cells with vanadate-H2O2 (Sweitzer et al., 1995). We find that site 1 binds strongly to Mrl, while the affinity of site 2 is lower. Addition of anti-Mrl antibody produced a supershift for each protein-oligo complex, which is consistent with the binding of Mrl to these sequences. (B) The positions of the two Mrl-binding sites are shown relative to the tll transcription start, and their sequences are shown and compared with STAT-binding consensus and optimal Mrl-binding sequences. (C) The 5.9 kb regulatory fragment upstream from the tll transcription start site is sufficient to drive lacZ expression in a pattern similar to that of endogenous tll in wild-type embryos. (D) In torGOF embryos, this promoter fragment drives lacZ expression in expanded domains. (E) A mutant 5.9 kb fragment was generated by disrupting both Mrl-binding sites (see Materials and Methods). In wild-type embryos, the expression pattern of lacZ driven by this mutated 5.9 kb fragment was not affected. (F) However, in torGOF embryos, the expansion of lacZ expression pattern was reduced.

View larger version (18K): [in a new window]   Fig. 5. Differential requirement of STAT for RTK signaling. The RTK Tor induces the expression of its target gene tll by derepression via activating the Ras-MAPK signaling pathway. Additional yet unidentified activators (A) and repressors (R2), which may or may not be controlled by Tor, determine the transcription levels of tll in a combinatorial manner. The activators (A) and repressors (R2) are unevenly distributed in cells along the anteroposterior axis of the embryo. In the central region of the embryo, there are higher levels of the repressors or lower levels of activators than the posterior region (only one possibility is shown). Mrl (STAT) is not essential for tll expression under wild-type conditions. However, TorGOF activates Mrl. Activated Mrl is required to overcome the higher levels of repressors (R2) in tissues where tll is not normally expressed, resulting in developmental abnormalities. Arrow and bar indicate activation and repression, respectively. Dotted lines represent undermined events.