The transcriptional co-factor Chip acts with LIM-homeodomain proteins to set the boundary of the eye field in Drosophila

Abstract

Development involves the establishment of boundaries between fields specified to differentiate into distinct tissues. The Drosophila larval eye-antennal imaginal disc must be subdivided into regions that differentiate into the adult eye, antenna and head cuticle. We have found that the transcriptional co-factor Chip is required for cells at the ventral eye-antennal disc border to take on a head cuticle fate; clones of Chip mutant cells in this region instead form outgrowths that differentiate into ectopic eye tissue. Chip acts independently of the transcription factor Homothorax, which was previously shown to promote head cuticle development in the same region. Chip and its vertebrate CLIM homologues have been shown to form complexes with LIM-homeodomain transcription factors, and the domain of Chip that mediates these interactions is required for its ability to suppress the eye fate. We show that two LIM-homeodomain proteins, Arrowhead and Lim1, are expressed in the region of the eye-antennal disc affected in Chip mutants, and that both require Chip for their ability to suppress photoreceptor differentiation when misexpressed in the eye field. Loss-of-function studies support the model that Arrowhead and Lim1 act redundantly, using Chip as a co-factor, to prevent retinal differentiation in regions of the eye disc destined to become ventral head tissue.

Fig. 1.

Chip inhibits retinal differentiation at the ventral eye-antennal disc boundary. (A) A w^– Chip mutant clone in the adult head autonomously forms an ectopic ventral eye (arrow). (B,C) Late third instar eye-antennal discs with Chip mutant clones identified by the absence of blue X-gal staining. Photoreceptors are stained with anti-Elav (brown). Anterior is to the left in this and all subsequent figures. Small ventral clones (B) and large clones generated in a Minute background (C) differentiate ectopic photoreceptors (arrows). (D-F) Early third instar discs stained with anti-Ey (D,E, green in F). (D) Wild type. (E,F) Chip mutant clones generated in a Minute background marked by the absence of GFP (blue in F). Ey is misexpressed in the antennal disc in the absence of Chip. (G-J) Chip mutant clones generated in a Minute background in late third instar eye-antennal discs are marked by the absence of GFP (blue in H,J). Photoreceptors are stained with anti-Elav (red in H,J). Outgrowths from the ventral eye-antennal disc show strong Ey expression (G, green in H) and a stripe of Ato (I, green in J) proximal to the region of Elav expression, indicating that a morphogenetic furrow initiated at the distal tip of the outgrowth.

Fig. 2.

Chip does not regulate dorsoventral polarity. (A,B) Wild-type eye discs stained with X-gal to reveal mirr-lacZ expression (A) or fng-lacZ expression (B). (C-F) Eye discs with Chip mutant clones generated in a Minute background (C,D) and eye discs with hth^B2 mutant clones generated in a Minute background (E,F), marked by the absence of GFP (blue in C,E). Staining with anti-β-galactosidase (green) shows the expression pattern of mirr-lacZ (C,E) or fng-lacZ (D,F). Photoreceptors are stained with anti-Elav (brown in A, red in C-F). Ventral outgrowths within Chip mutant clones (arrows in C,D) express fng in all cells and do not express mirr. hth mutant clones also show normal mirr and fng expression, but usually do not produce outgrowths. (G-I) Eye discs expressing E(spl)mβ-lacZ, stained with anti-β-galactosidase (red). (G) Wild type. (H,I) Chip mutant clones generated in a Minute background, marked by the absence of GFP (green). E(spl)mβ-lacZ marks the equator in wild-type discs, but is not expressed in Chip mutant outgrowths (arrows) in late third instar (H) or early third instar (I).

Fig. 3.

Hth represses photoreceptor differentiation independently of Chip. (A-C) Early third instar discs stained with anti-Hth (A,B, green in C). (A) Wild type. (B,C) Chip mutant clones generated in a Minute background marked by the absence of GFP (blue in C). Loss of Chip does not affect Hth expression. (D-G) Chip mutant clones generated in a Minute background in late third instar discs are marked by the absence of GFP (blue in E,G). Photoreceptors are stained with anti-Elav (red in E,G). Hth (D, green in E) and wg-lacZ (F, green in G) are still expressed in the anterior eye disc and show new expression domains within the outgrowth in Chip mutant clones (arrows). Hth is thus not downstream of Chip and does not require Chip as a co-factor to control wg expression. (H-K) Eye discs misexpressing Hth (blue in I,K) in wild-type or Chip mutant cells marked by co-expression of GFP (green in I,K). Both types of clones failed to express Elav (H, J, red in I,K) posterior to the morphogenetic furrow. Hth does not require Chip to prevent photoreceptor differentiation.

Fig. 4.

Chip requires its LIM interaction domain to repress photoreceptor differentiation. (A) Full-length Chip construct (ChipFL) and the Chip constructs with the LID deleted (ChipΔLID) or the dimerization domain deleted (ChipΔDD). The proline-rich (P-rich), dimerization (DD), other interaction (OID) and LIM interaction (LID) domains are indicated. The Chip^45F allele has a stop codon at position 111. (B) An eye-antennal disc with Chip^45F mutant clones expressing ChipΔLID marked by GFP co-expression (green). Photoreceptors are stained with anti-Elav (red). (C) The number of eye discs containing Chip mutant clones with no rescue construct, or with UAS-ChipFL, UAS-ChipΔLID or UAS-ChipΔDD, that show ectopic photoreceptor differentiation. All discs containing Chip mutant clones were counted, regardless of the position of the clones. The LID and DD domains are required for Chip to suppress eye differentiation.

Fig. 5.

Arrowhead (Awh) and Lim1 inhibit photoreceptor differentiation in a Chip-dependent manner. (A) Seven LIM-HD proteins are encoded in the Drosophila genome. The closest human homologue for each is given in parentheses. (B-G) Whole-mount in situ hybridization to eye-antennal discs with LIM-HD probes. (B) ap; (C) Awh; (D) CG32105; (E) tup; (F) Lim1; (G) Lim3. Only Awh, Lim1 and CG32105 are expressed in the ventral eye-antennal boundary region. (H-K) Eye discs that ectopically express Awh in clones of wild-type cells (H,I) or in Chip mutant clones (J,K) marked by co-expression of GFP (green in I,K). (L-O) Eye discs that ectopically express Lim1 in clones of wild-type cells (L,M) or in Chip mutant clones (N,O), marked by co-expression of GFP (green in M,O). Photoreceptors are stained with anti-Elav (H,J,L,N, magenta in I,K,M,O). Both Awh and Lim1 inhibit photoreceptor differentiation in wild-type, but not Chip mutant, cells.

Fig. 6.

Lim1 represses Ey in the antennal disc. (A-H) Eye discs that ectopically express Lim1 (A-D) or Awh (E-H) in clones of wild-type cells (A,B,E,F) or in Chip mutant clones (C,D,G,H), marked by co-expression of GFP (green in B,D,F,H), stained with anti-Ey (A,C,E,G, red in B,D,F,H). Lim1 and Awh can repress Ey expression in its normal anterior domain (stars), but inappropriately maintain a high level of Ey expression in posterior cells (arrow in A). Chip is required for full repression of Ey. (I) A wild-type eye-antennal disc stained with anti-Ey. (J,K) An eye-antennal disc with Lim1 mutant clones marked by the absence of GFP (green in K), stained with anti-Ey (J, red in K) and anti-Elav (blue in K). Ey expression expands into the ventral antennal disc in Lim1 mutant regions (arrow in K).

Fig. 7.

Lim1 and Awh redundantly contribute to limiting the eye field. (A-C) Lim1 mutant clones marked by the absence of GFP (green in C) in an Awh mutant eye-antennal disc, stained with anti-Eya (A, blue in C) and anti-Elav (B, red in C). A clone in the ventral antennal disc (arrow) misexpresses Eya and differentiates photoreceptors. (D) The numbers of discs with Lim1 clones in wild type and Awh mutant backgrounds that misexpress Eya and Elav and that form outgrowths. Removing Awh increases the frequency of all these phenotypes. (E) Schematic of an eye-antennal disc illustrating a model for the functions of Chip, Lim1 and Awh. Chip acts with Lim1 to prevent Ey expression in the anterior eye disc from spreading into regions fated to form the ventral head, and with Awh to regulate additional genes that inhibit photoreceptor differentiation. Yellow regions give rise to adult head cuticle, and the pink region to the maxillary palp.