Notch-dependent expression of the archipelago ubiquitin ligase subunit in the Drosophila eye

Abstract

archipelago (ago)/Fbw7 encodes a conserved protein that functions as the substrate-receptor component of a polyubiquitin ligase that suppresses tissue growth in flies and tumorigenesis in vertebrates. Ago/Fbw7 targets multiple proteins for degradation, including the G1-S regulator Cyclin E and the oncoprotein dMyc/c-Myc. Despite prominent roles in growth control, little is known about the signals that regulate Ago/Fbw7 abundance in developing tissues. Here we use the Drosophila eye as a model to identify developmental signals that regulate ago expression. We find that expression of ago mRNA and protein is induced by passage of the morphogenetic furrow (MF) and identify the hedgehog (hh) and Notch (N) pathways as elements of this inductive mechanism. Cells mutant for N pathway components, or hh-defective cells that express reduced levels of the Notch ligand Delta, fail to upregulate ago transcription in the region of the MF; reciprocally, ectopic N activation in eye discs induces expression of ago mRNA. A fragment of the ago promoter that contains consensus binding sites for the N pathway transcription factor Su(H) is bound by Su(H) and confers N-inducibility in cultured cells. The failure to upregulate ago in N pathway mutant cells correlates with accumulation of the SCF-Ago target Cyclin E in the area of the MF, and this is rescued by re-expression of ago. These data suggest a model in which N acts through ago to restrict levels of the pro-mitotic factor Cyclin E. This N-Ago-Cyclin E link represents a significant new cell cycle regulatory mechanism in the developing eye.

Fig. 1.

Ago accumulates at the MF in a subset of Ato-positive and Elav-positive cells. Drosophila ago promoter activity is detected several rows of cells anterior to the wave of Elav-positive clusters. (A-A′) Merged confocal sections of a wild-type (wt) Drosophila larval eye disc stained for Ago (red) and Ato (blue). Arrows mark overlapping `holes' in Ato and Ago expression immediately posterior to the MF. Arrowhead indicates the position of the morphogenetic furrow (MF). In these and all following images, posterior is to the left. (B-B′) Merged confocal sections of a wild-type larval eye disc stained for Ago (blue) and Elav (red). Arrows in mark Ago-positive cells that reside anteriorly within the Elav-positive clusters. (C-C′) Merged confocal sections of UAS:R-DHFR_ts-EGFP; ago>Gal4 larval eye discs stained for GFP (blue) and Elav (red). Arrow indicates GFP expression extending into the MF anterior to the first row of Elav-positive nuclei.

Fig. 2.

hh and N pathway mutants cause a decrease in Ago levels at the MF. (A-B′) Ago (red) fails to accumulate at the MF in smo³ clones marked by the absence of GFP (A-A′). Higher magnification (B-B′) shows rescue of Ago accumulation at clonal boundaries that cross the MF (arrows). (C-C′) Ago (red) fails to accumulate at the MF in Psn²²⁷ clones (arrow in C) marked by the absence of GFP (arrow in C′). Insets show higher magnification views of clones (outlined by dotted lines). (D-D′) Ago (red) fails to accumulate at the MF in Dl^RevF10,Ser^RX82 clones marked by the absence of GFP. Arrowheads indicate the position of the MF.

Fig. 3.

N is necessary for elevated Ago expression and patterning at the MF. (A) Confocal section of a heat-shocked control FRT80B eye disc stained for Ago. (A′) Confocal section of an N^ts3 eye disc stained for Ago after a 2-hour temperature shift to 31°C. Arrow indicates residual Ago expression at the MF. Insets show higher magnification views of an N^ts3 eye disc at t=0 hours (left) and t=6 hours (right) of heat shock. Note the overall drop in Ago levels and the appearance of disordered groups of Ago-positive nuclei posterior to the MF.

Fig. 4.

Elevated Dl restores Ago in smo mutant cells. (A-A′) Confocal sections of smo³ clones marked by the absence of GFP (green) generated in the presence of Hsp70>Dl and stained for Ago (red). Arrows denote a delayed rescue of Ago levels in mutant clones. (B-B′) High-magnification view of a confocal section of smo³ clones marked by the absence of GFP (green) generated in the presence of Hsp70>Dl and stained for Ago (red). Arrows denote the delayed Ago expression in the mutant clone. Arrowheads denote the location of the MF.

Fig. 5.

Hh and N pathway mutants cause a decrease in ago promoter activity at the MF. (A-A′) Confocal sections of smo³ clones marked by the absence of β-gal expression (red). ago promoter activity detected by the ago>Gal4,UAS:R-DHFR_ts-EGFP transgene (green) is much lower in the mutant clones (arrows). (B-B′) A higher magnification view of the disc in A-A′ shows rescued ago promoter activity in MF cells along clonal borders (arrowheads). (C-C′) Dl^RevF10,Ser^RX82 clones marked by the absence of β-gal (red) generated in ago>Gal4,UAS:R-DHFR_ts-EGFP (green) discs. ago promoter activity at the MF drops in the mutant clones (arrows) but is rescued in cells along clonal borders (arrowhead). (D-D′) Dl^RevF10,Ser^RX82 clones marked by the absence of β-gal (red) generated in ago>Gal4,UAS>EGFP (green) discs. ago>Gal4-driven expression of stable EGFP results in perdurance of GFP posterior to the MF only in wild-type cells. ago promoter activity is rescued in cells along clonal borders (arrowheads). (E-E′) Confocal sections of Su(H)^Δ47 clones marked by the absence of β-gal (red). ago promoter activity detected by the ago>Gal4,UAS:R-DHFR_ts-EGFP transgene (green) is reduced in the mutant clones (yellow arrow). The red arrow (E) indicates an example of elevation of ago>Gal4 activity in wild-type MF cells immediately adjacent to Su(H) mutant cells.

Fig. 6.

The ago promoter is responsive to changes in levels of N signaling. (A) Relative luciferase activity in Drosophila S2 cells transfected with ago2kb-luc or ago2kb6xmut-luc. Cells were co-transfected with either 300 ng of NICD plasmid, 50 ng of DaN plasmid, or both, as indicated. Error bars indicate mean ± s.d. (B) The ago locus. Three transcript variants, RA, RB and RC, have alternate first exons but share the same coding sequence (red bars, exons). Splicing patterns are indicated. Location of the P{GawB}ago[NP0850] element 53 bp upstream of the ago-RA start site is indicated. The 2 kb region cloned into the luciferase vector is indicated (black bar) as are the locations of putative transcription factor binding sites: six Su(H) binding sites (purple; arrows indicate orientation), two A sites (green), and the p53 binding sequence (blue). (C) Quantitative real-time PCR analysis of the expression of RA and RC mRNA levels in Hsp70>N^intra with (+) or without (–) a 1-hour heat shock as indicated. Error bars indicate mean ± s.d. (D-D′) Ago protein (red) fails to accumulate at the MF in da¹⁰ mutant clones marked by the absence of lacZ expression (magenta). Arrows indicate Ago protein in wild-type cells that flank a large da¹⁰ clone. (E) Gel shift analysis of an interaction between recombinant Su(H) protein [GST-Su(H)] and a radiolabeled fragment of the wild-type (wt) ago promoter containing two putative Su(H) sites closest to the ago-RA start site (see B). GST-Su(H) binds the wild-type probe (lane 2), and is efficiently competed by excess unlabeled wt DNA (lanes 3-5 contain 50, 100 and 500 ng DNA, respectively), but not by equivalent amounts of an unlabeled DNA containing mutations (altered bases indicated by dots) in the two consensus Su(H) sites (lanes 6-8). Free probe and shifted Su(H):DNA complexes are indicated.

Fig. 7.

The decrease in Ago levels in N pathway mutant clones results in a build-up of the SCF-Ago substrate Cyclin E. (A-A′) Confocal sections of an eye disc containing Dl^RevF10,Ser^RX82 double-mutant clones marked by the absence of GFP (green) stained for Cyclin E (blue). Note the elevated Cyclin E in Dl^RevF10,Ser^RX82 cells behind the MF. (B-B′) Merged confocal sections of Dl^RevF10,Ser^RX82 MARCM clones positively marked by GFP (green) and co-stained for Cyclin E (blue). Arrows highlight the fact that the effect of Dl^RevF10,Ser^RX82 on Cyclin E only occurs in cells within and behind the MF. (C-D′) Merged confocal sections of Dl^RevF10,Ser^RX82 MARCM clones (GFP positive; green) expressing an Ago:YFP fusion protein. Arrows indicate Dl,Ser mutant clones in which the Cyclin E levels are partially rescued by expression of Ago:YFP (compare the intensity of Cyclin E in C and D with that in A and B).