Seven-up, the Drosophila homolog of the COUP-TF orphan receptors, controls cell proliferation in the insect kidney

Abstract

Morphogenesis of the insect kidney, the Malpighian tubules, is controlled in Drosophila by a single large cell, the tip cell. It has been postulated that this cell sends out a mitogenic signal that induces the division of neighboring cells. The signal and the molecules that receive it have remained elusive. We show that the COUP-TF-related nuclear orphan receptor Seven-up is a key component that becomes induced in response to mitogenic EGF receptor signaling activity emanating from the tip cell. Seven-up in turn is capable of regulating the transcription of cell cycle regulators.

Figure 1

svp is required for MT growth. (A) Dorsolateral view of a stage 13 embryo visualizing two of the four everting tubules (inset, arrowheads) by mAb FascII labels tubule membranes. (B) mAb 22C10, mAb FascII double staining (both green) of a stage 16 tubule; 22C10 expression marks the neural tip cell. (C–E) Anti-Kr (red), mAb FascII (green) double stainings highlighting the tip mother cell in stage 10 (C, arrowhead), the two daughter cells shortly thereafter (D, arrowheads) and the tip cell in stage 14 (E, arrowhead). (F) Scheme of tip cell allocation. (G–I) svp expression monitored by in situ hybridization (G; stage 11 tubule) or via a svp lacZ line (H,I; late stage 11) showing the same expression pattern. Anti-β-gal (green), anti-Kr (red) double stainings. Yellow shows coexpression of svp and Kr in the tip mother cell (H; arrowhead) and the tip cell (I; arrowhead). (J–M,O) svp mutant embryos, (N) wild type. (J–L) Anti-Kr staining. The primordium (J), tip cell allocation (K,L), and tip cell differentiation (inset in L, mAb 22C10 stains) are normal. (M) Anti-Cut staining reveals a reduced tubule cell number in svp mutants (Table 1). (N,O) BrDU incorporation studies of stage 11 embryos. (tmc) Tip mother cell; (tc) tip cell.

Figure 2

Localized rho and S activity in the tip cells of the tubules. (A,B) rho expression in the tip mother cell (A, arrowhead) and the tip cell (B, arrowhead) as revealed by in situ hybridization. (C,D) S lacZ expression; double staining of anti-β-gal (green) and anti-Kr (red) revealing S expression in the tip mother cell (C, arrowhead), the tip cell (D, arrowhead), and its sibling cell (D, open arrowhead). (E) Summary of the rho (blue) and S (light blue) expression patterns in the everting tubules. (F) pnt–lacZ expression; double staining of anti-β-gal (red) and anti-Kr (green) revealing pnt expression in the tip mother cell (arrowhead) and its neighboring cells. (G,H) BrdU incorporation studies in EGFR mutants (flb^IK35; stage 12; G) and (stage 15; H).

Figure 3

svp expression is dependent on EGFR signaling. (A–D) svp lacZ expression shown by anti-β-gal antibody stainings (the same results were obtained monitoring svp expression by in situ hybridization). (A) svp expression in the tip region of a wild-type tubule (stage 15). (B) svp lacZ expression is abolished in mutants of the EGFR (flb^IK35). (C) Upon ectopic sSpi expression the svp domain is largely expanded (arrowheads point to cells that ectopically express svp; cf. A) and the cell number increases. (D) Ectopic expression of the dominant-negative Dras1^N17 allele using the XB2-3-Gal4, reduces svp expression (cf. A). (E,F) Anti-Cut stainings marking all of the tubule cells. (E) Reduced tubule cell number in flb^IK35 mutants. (F) Ectopic svp expression of rescues the tubules of flb^IK35 mutants. Approximately three times more cells were obtained compared to the mutant condition. (G) Summary of the results. Svp (red nuclei) is activated by EGFR signaling, which emanates from the tip cell (blue) and controls cell division.

Figure 4

EGFR signaling and Svp control cell cycle gene expression. BrDU incorporation in stage 10 wild-type (A) and G455.2–Gal4/UAS–Svp II tubules (mediating SvpII expression in all tubule cells) (B). Cell proliferation does not occur on one side of the outgrowing tubules as in wild type (A) but throughout the everting tubules (B, arrowheads). (C,D) Schematic representation of the BrdU incorporation studies in A and B. Red marks cell division. (E–I) RNA in situ hybridization experiments of wild-type (E–G) and (flb^IK35) mutant embryos (H–I). (E) stg expression in proliferating tubule cells of wild-type embryos occurs on one side of the outgrowing tubule (arrowhead) in early stage 10. (F) Upon ectopic expression of Svp, stg becomes ectopically expressed in cells that undergo extra divisions (arrowheads, cf. E and B). In wild type, cycE is also expressed asymmetrically in the outgrowing tubules in stage 10 (G; arrowhead) and in the distal proliferation zone (not shown). Localized transcription of stg (H; stage 11) and cycE (I; stage 10) is absent in flb^IK35 mutants. (J) Model of how tip cell (blue) signaling might control tubule growth. Dividing cells are red; See text for details.