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First published online 22 October 2003
doi: 10.1242/dev.00818

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Bar homeodomain proteins are anti-proneural in the Drosophila eye: transcriptional repression of atonal by Bar prevents ectopic retinal neurogenesis

¹ Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
² Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
³ Department of Ophthalmology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

View larger version (92K): [in a new window]   Fig. 1. Complementary expression pattern of Bar and Ato in basal undifferentiated cells. Posterior is towards the left and dorsal is upwards in all figures unless mentioned otherwise. (A) Scanning electron micrograph of adult compound eye. (B) Third instar eye disc stained with antibodies against Ato, Elav and Sens. The arrow indicates the position of the morphogenetic furrow. (C) Magnified view of B near the furrow showing four different stages of Ato expression (1-4). The 3'- and 5'-regulatory elements of ato specify the stage 1 and stages 2-4 of ato expression, respectively. (D,E) Complementary expression patterns of Bar and Ato. Longitudinal (D) and tangential (E) sections of the eye disc. Note that Bar and Ato expression is complementary in the basal cells along the furrow (D, arrow). (F) Two tier layers of eye disc. Photoreceptor and basal cell layers were stained with antibodies for Elav (red) and BarH1 (green), respectively. White and yellow arrows indicate the morphogenetic furrow and the R1/6 cells, respectively. R1 and R6 photoreceptors are labeled by both antibodies.

View larger version (118K): [in a new window]   Fig. 2. Bar is required for downregulation of Ato expression. (A-C) Ato expression is strongly elevated within Bar loss-of-function clones (A). Bar mutant cells are marked by the loss of GFP staining (B). Bar mutant cells showed ubiquitous upregulation of Ato expression (C, white arrow) and individually singled-out Ato+ cells (C, yellow arrow). (D) dpp-Gal4 drives lacZ expression in the dorsoventral lateral margins of the eye disc and in the ventral region of the antennal disc (arrow). (E,F) Misexpression of BarH1 by dpp-Gal4 strongly downregulates Ato expression. The furrow progression was strongly decreased in the ventral margin region of the eye disc where dpp drives BarH1 expression (E, arrowhead). Ato expression in the ventral region of antennal disc was dramatically downregulated (E,F, arrows).

View larger version (93K): [in a new window]   Fig. 3. Ectopic Ato+ cells form photoreceptor clusters. Bar loss-of-function clones were identified by the absence of GFP (A-I, green) or BarH1 (J-O, green) staining and marked with broken lines in C,F,I,L,O. (A-I) Ectopic Ato+ cells further differentiate into mature R8 photoreceptor cells within Bar loss-of-function clones. The number of cells expressing Sca (A-C; early R8 marker), Sens (D-F; early R8 marker) and Boss (G-I; late R8 marker) was increased within the clones. (J-O) Excess numbers of ommatidial clusters are formed in Bar loss-of-function clones. Photoreceptor clusters were detected by Elav (J-L) or Arm (M-O) staining. The size of some clusters within the Bar mutant clone was smaller than that of the wild-type ommatidia clusters, suggesting that photoreceptor recruitment in these ectopic clusters is not yet complete. White and yellow arrows in O indicate normal or ectopic photoreceptor clusters, respectively.

View larger version (115K): [in a new window]   Fig. 4. Lateral inhibition and interommatidial spacing function in Bar loss-of-function clones. Bar loss-of-function clones were identified by the absence of anti-GFP staining and marked with broken lines in C,F. Sens (A-C) and dpERK (D-F) staining within the furrow were normal with regular spacing in Bar mutant regions. White and yellow arrows mark R8 equivalence groups in the wild-type and the Bar mutant regions, respectively.

View larger version (130K): [in a new window]   Fig. 5. Bar represses the expression of ato at the level of transcription. (A) The expression of 3'F:5.8 ato-lacZ in eye-antennal disc of wild-type fly. ß-gal is detected in a few columns posterior to the furrow where endogenous Ato is normally absent. This is probably due to the perdurance of ß-gal protein, because ß-gal mRNA expression of 3'F:5.8 ato-lacZ line faithfully mimics the expression pattern of endogenous ato mRNA (Sun et al., 1998). (B-D) Bar represses ato expression at the transcription level through 3'-regulatory element of ato. Bar mutant cells were marked by the loss of GFP staining (B,C) and showed dramatically increased expression of ß-gal within the clone (D, arrows). (E) The expression of 5'F:9.3 ato-lacZ in the eye disc of wild-type fly. (F-H) Bar mutant cells showed a dramatically elevated expression of 5'F:9.3 ato-lacZ within the clone. Bar mutant cells were marked by the loss of GFP staining (F,G). (I) The expression of 5'F:9.3 ato-lacZ in eye-antennal disc of wild-type fly. Note normal ato-lacZ expression in the antennal disc. (J-L) Misexpressed BarH1 by dpp-Gal4 strongly downregulates the expression of 5'F:9.3 ato-lacZ (J,K) and of 5'F:7.2 ato-lacZ (L). Ato expression in the ventral region of the antennal disc was strongly downregulated (J-L, arrows). (M-P) Bar represses ato expression at the transcription level through 5'-regulatory element of ato. Note that many cells within Bar loss-of-function clones were lacZ-positive but Sens negative (N,O, arrows).

View larger version (117K): [in a new window]   Fig. 6. Transcriptional repression of ato by Bar is CiFL-independent. (A-D) Third instar eye-antennal disc of hhP30 (enhancer trap) (Ma et al., 1993) was stained with antibodies for ß-gal (Hh-lacZ; A, blue), CiFL (A,B,D, red) and Ato (C; A,D, green). The expression domains of CiFL and Ato approximately overlap within the furrow (A,D). (E-H) Complementary expression pattern of CiFL and Bar. Wild-type third instar eye-antennal discs were stained with antibodies for CiFL (E,F,H, red) and BarH1 (E,G,H, green). (I-L) Bar loss-of-function clone (I,L, loss of GFP staining) shows no increase in CiFL expression (J). Note that the same clone shows elevated expression of Ato (K).

View larger version (51K): [in a new window]   Fig. 7. Model for ato repression by Bar in the developing eye disc. Ato expression within and anterior to the morphogenetic furrow (MF) is resolved into single R8 founder cells behind the furrow. The founder cells initiate the R8 cell differentiation and form photoreceptor clusters by sequentially recruiting other photoreceptors. The nuclei of photoreceptors are apically localized (orange region). Hh secreted from photoreceptor cells activates Ato expression via CiFL (gray region). Bar homeodomain proteins are expressed in basal undifferentiated cells behind the furrow (green region) and repress ato transcription, establishing the complementary anteroposterior pattern of Ato and Bar zones.