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First published online 24 January 2007
doi: 10.1242/dev.02788

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Combinatorial signaling in the specification of primary pigment cells in the Drosophila eye

Department of Molecular, Cell and Developmental Biology, Department of Biological Chemistry, Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.

View larger version (152K): [in this window] [in a new window]   Fig. 1. EGFR activation and Delta expression in pupal cone cells. (A) Summary of events in the larval third instar eye disc based on Flores et al. (Flores et al., 2000) and Tsuda et al. (Tsuda et al., 2002). Activation of EGFR in R cells (green) causes the derepression of Delta. This process also requires the function of two novel nuclear proteins (Ebi and Sno) and the proteosome complex (Tsuda et al., 2002). Sequential and combinatorial integration of EGFR, Notch and Lz in cone-cell precursors (yellow) causes the expression of D-Pax2 and other genes involved in the specification of cone-cell fate. (B,C) Wild-type mid-pupal eye disc. Delta protein (green) is expressed in apical tips of pupal cone cells (B). The corresponding nuclei are marked by the expression of D-Pax2 lacZ (red, C). (D-F) MAPK activation in cone cells of a mid-pupal eye discs. Eye discs were stained for activated MAPK (red, D) and also for D-Pax2 lacZ (green, E). These two signals co-localize in the pupal cone-cell nuclei (yellow, F). (G-L) Delta is transcribed in pupal but not in larval cone cells. (G-I) In third instar eye discs, Delta-lacZ (Dl-lacZ; green, G) is not expressed in cone cells, which are marked with Cut (red, H). Residual lacZ expression is in R cells (notice the lack of overlap in I). (J-L) In pupal eye discs, Dl-lacZ (green, J) is expressed in cone cells, which are marked with Cut (red, K). The overlap is evident in the merged panel (yellow, L).

View larger version (131K): [in this window] [in a new window]   Fig. 2. EGFR is required for Delta expression in pupal cone cells. (A-C) Delta expression in EGFRts1-mutant clones subjected to a nonpermissive temperature during pupal development. (A) GFP expression marks wild-type cells (green); non-GFP cells are mutant for EGFRts1. (B) The same disc as in A was co-stained for Delta expression (red). (C) The merged panel shows that the Delta protein is restricted to wild-type cells. (D-F) Expression of the cone-cell marker, Cut, in EGFRts1 clones transferred to non-permissive conditions during the mid-pupal stages. (D) GFP marks wild-type tissue; non-GFP cells are mutant for EGFR. (E) The same disc as in D was co-stained for Cut (red). (F) The merged panel shows that the loss of EGFR function in pupal stages does not compromise cone-cell fate. (G-J) Expression of a dominant-negative version of EGFR (EGFRDN) in cone cells blocks Delta expression. (G,H) Wild-type (control) pupal eye discs stained for Delta showed its expression in cone cells (red, G); the spa-Gal4 driver was also expressed in cone cells (red, H). spa-Gal4, UAS-EGFRDN pupal eye discs stained for Delta showed loss of Delta expression in the cone cells (I), whereas spa-Gal4, UAS-EGFRDN pupal eye discs stained for Cut showed that a loss of EGFR function during pupal stages does not disrupt cone-cell fate specification (J). (K-N) Ectopic activation of EGFR in cone cells promotes Delta expression. MAPK activation in wild-type third instar eye disc (K) is seen in cells at the furrow followed by low levels of activation in the differentiated R cells behind the furrow (K). (L) In spa-Gal4, UASEGFRact third instar eye disc, MAPK is activated at high levels at later stages in the developing cone cells, which express spa-Gal4. (M) Wild-type expression of Delta (green) in the third instar eye disc is limited to R cells and is not expressed in cone cells (red). (N) In spa-Gal4, UAS-EGFRact third instar eye disc, ectopic activation of Delta (green) is seen in cone cells (red, arrow).

View larger version (151K): [in this window] [in a new window]   Fig. 3. Delta expression in the pupal cone cells requires the function of ebi and sno. (A-C) Delta protein expression in observed in pupal cone cells in wild type (red, A) and is reduced in pupal cone cells of snoE1/sno93i (D-F) and spa-Gal4, UAS-ebiDN (G-I) genotypes. Cone cells are marked with Cut (green; B,E,H) and the merged panels are shown in C,F and I.

View larger version (127K): [in this window] [in a new window]   Fig. 4. Requirement of Notch signaling for primary pigment-cell specification. (A-C) Pupal eye discs stained for Bar. Bar expression in seen in the primary pigment cells (A) and is lost in Nts pupal eye discs shifted to a non-permissive temperature 10 hours after pupation (B) or when UAS-Su(H)DN is expressed in the pigment cells using the pigment-cell-specific driver 54C-Gal4 in pupal eye discs (C). (D-F) Wild-type cut expression in pupal cone cells (D) remains unchanged in Nts when shifted to a nonpermissive temperature 10 hours after pupation (E) or in an 54C-Gal4 UAS-Su(H)DN background (F). (G-J) Overexpression of Delta in pupal cone cells using the spa-Gal4, UAS-Delta (UAS-Dl) combination causes ectopic primary pigment-cell specification; a high level of expression of the Delta protein is shown (G, compare with Fig. 1B). (H) Wild-type pupal eye disc stained for Dlg to mark the membranes shows four cone cells and a single row of pigment cells between ommatidia. (I) spa-Gal4, UAS-Dl pupal eye disc stained for Dlg show multiple rows of cells between ommatidia (arrow). (J) spa-Gal4, UAS-Dl pupal eye disc stained for Bar show the over specification of Bar-positive primary pigment cells.

View larger version (50K): [in this window] [in a new window]   Fig. 5. Requirement for Lz and Notch, but not EGFR, in primary pigment-cell specification. (A-F) Pupal discs stained for Bar (red). (A) Wild-type mid-pupal eye disc showed Bar expression in two primary pigment cells per cluster. (B) hsp70-Gal4-UAS-EGFRDN pupal eye disc subjected to heat shock at 29°C at the mid-pupal stage showed no defects in Bar expression or primary pigment-cell specification. (C) hsp70-Gal4, UAS-NDN pupal eye discs subjected to heat shock at 29°C at the mid-pupal stage showed a loss of Bar expression. (D) Primary pigment-cell specification in lzts114 at a permissive (25°C) temperature showed a wild-type pattern of primary pigment-cell specification. (E,F) Dosage-sensitive interaction between Notch and lz during primary pigment-cell fate specification. (E) lzts114/Y, Delta/+ (Dl/+) combination incubated at 25°C showed dosage-sensitive interactions causing a loss of Bar-expressing primary pigment cells. (F) lzts114, EGFRnull/+ pupal eye disc from flies incubated at 25°C showed no disruption in the specification of primary pigment-cell fate. (G) Signal integration during primary pigment-cell specification. In the larval cone cells, a low level of EGFR activation is required for the establishment of their fate but is insufficient to promote transcriptional activation of Delta. Once cone-cell fate is established, EGFR activation continued to rise and, in the pupal stages, caused the transcription of Delta. Activation of the Notch pathway in the adjacent undifferentiated cells in combination with Lz promoted the specification of primary pigment-cell fate. This process did not require input from the EGFR pathway. (H) Summary of cellautonomous combinations of Notch and EGFR inputs in the specification of neuronal (R1, R6 and R7) and non-neuronal cell types from undifferentiated cells behind the furrow. The fate specification of these cells also requires a transcriptional input from Lz.