The position and function of the Notch-mediated eye growth organizer: the roles of JAK/STAT and four-jointed

Abstract

In many animal systems, the local activation of patterning signals in spatially confined regions (organizers) is crucial for promoting the growth of developing organs. Nevertheless, how organizers are set up and how their activity influences global organ growth remains poorly understood. In the Drosophila eye, local Notch activation establishes a conserved dorsal-ventral organizer that promotes growth. The dorsal selector Iroquois complex defines the position of the organizer at the mid-first instar, and through its ligand, unpaired, the Janus kinase (JAK)/signal transducers and activator of transcription (STAT) pathway is thought to mediate global growth downstream of the organizer. However, here we show that the unpaired/JAK/STAT pathway is actually a fundamental element in the spatial control of the organizer, upstream from Notch activation. Furthermore, we identify four-jointed, a target of the Fat and Hippo tumour-suppressor pathways, as a mediator of the growth controlled by the organizer. These findings redefine the process of organizer formation and function, and they identify four-jointed as a regulatory node, integrating multiple growth-control pathways.

Figure 1

General upd/JAK/STAT activity drives dorsal overgrowth that is linked to ectopic organizer formation. (A) Control wild-type (wt) adult eye. (B) Ubiquitous overexpression of upd drives a dorsal protrusion (arrow). (C–H) Eye discs stained for β-galactosidase (lacZ) and wg, or the neuronal marker Elav as indicated. Expression of the mirr–lacZ, Ser–lacZ, and slp–lacZ enhancer trap lines in wild type discs (C,E,G) and ey–Gal4>upd discs (D,F,H). (I,J) Schematics of the changes in gene expression. Double arrows represent signalling from the organizer to the adjacent cells. Red arrowhead points to the organizer position (C,D); the small red arrowhead points to the dorsal–ventral axis in the head, and the white arrowhead points to the eye (E). an, anterior; D, dorsal; ey, eyeless; Iro-C, Iroquois complex; JAK, Janus kinase; mirr, mirror; Ser, Serrate; slp, sloppy pair; STAT, signal transducers and activator of transcription; upd, unpaired; V, ventral; wg, wingless.

Figure 2

Transient ventral expression and the activity of the upd/JAK/STAT pathway. (A–H) Late first-instar eye discs stained for wg protein (blue), in which GFP represents the JAK/STAT reporter (10XSTAT92E–GFP) and β-galactosidase (lacZ, red) monitors upd–lacZ (A–D) or mirr–lacZ (E–H) expression. (I) Control GFP⁺ clones and (J–L) upd⁺ clones induced at 0–24 h AEL. Arrow in the inset (J) indicates the repressed mirr–lacZ. (K) Histogram of the area of upd⁺ clones in D and V regions. Only discs with no mirr repression (n=18) were scored. Data are means±s.e.m. P-value of 0.0348 was derived from Student's t-test. (L) Mosaic upd⁺ eye discs stained for pH3 (purple) and cut (red). AEL, after egg laying; D, dorsal; GFP, green fluorescent protein; JAK, Janus kinase; mirr, mirror; pH3, phosphorylated histone H3; STAT, signal transducers and activator of transcription; upd, unpaired; V, ventral; wg, wingless.

Figure 3

Inhibition of the JAK/STAT pathway alters the DV Iro-C expression. (A, G–I) ey–FLP-induced clones of JAK/STAT pathway genes marked by loss of GFP (green), with mirr–lacZ expression (red) and wg protein (blue). (B,F) Expression of mirr–lacZ in the larval and adult eye in wt (B,D) and ey–Gal4>PIAS (C,E,F) grown at 26.5°C (B–E) or 25°C (F). (G–I) Examples of eye discs with mutant clones running along the DV boundary and marked by loss of GFP (green). Note the altered border of mirr–lacZ expression (red). an, anterior; DV, dorsal–ventral; ey, eyeless; flp, flip; GFP, green fluorescent protein; Iro-C, Iroquois complex; JAK, Janus kinase; mirr, mirror; PIAS, protein inhibitor of activated STAT; STAT, signal transducers and activator of transcription; upd, unpaired; wg, wingless; wt, wild type.

Figure 4

upd drives overgrowth upstream from the Notch pathway. (A) There is no eye overgrowth induced by upd when the mirr border is eliminated by ubiquitous expression of mirr (ey–Gal4>upd>mirr). (B) Small eye size owing to ubiquitous fng expression is not altered by removing upd (os¹; C) or by adding hop^Tum-l (D) or a upd+ transgene (E; 100% penetrance). (F) Small eye size in ubiquitous PIAS (51 out of 63 flies with small eyes) or the eye loss (12 out of 63) is rescued by (G) N^ICD (100%, n=32). (H) Gain of upd in eyg⁻ (eyg^XE119/eyg^M3–12), a pharate adult is shown. ey, eyeless; eyg, eyegone; fng, fringe; mirr, mirror; N^ICD, Notch intracellular domain; PIAS, protein inhibitor of activated STAT; upd, unpaired.

Figure 5

Fj mediates growth by the organizer. (A) The expression of fj (visualized by fj–lacZ, white arrows in A–C) diminishes when Notch activity is compromised (B). Atonal (red) marks the position of the morphogenetic furrow (A,B). (C) Ectopic dorsal fj expression (arrowhead) in discs overexpressing upd. wg (pink) unambiguously defines the DV axis in the discs. (D) The growth defect associated with diminished Notch activity is partly rescued by (E) the gain of fj expression. (F) Map of GS-58F and GS-D67 within the fj locus. (G) New model of the sequential roles of upd/JAK/STAT, Notch and fj in normal eye growth. Dl, Delta; DV, dorsal–ventral; ey, eyeless; eyg, eyegone; fj, four-jointed; fng, fringe; GS, Gene Search; hh, hedgehog; Iro-C, Iroquois complex; JAK, Janus kinase; N, Notch; Ser, Serrate; slp, sloppy pair; STAT, signal transducers and activator of transcription; upd, unpaired; wg, wingless; wt, wild type.