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First published online 20 October 2004
doi: 10.1242/dev.01426

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Induction and autoregulation of the anti-proneural gene Bar during retinal neurogenesis in Drosophila

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 (117K): [in a new window]   Fig. 1. Bar expression in the basal undifferentiated cells and R1/6 photoreceptor cells is independently regulated. Antibodies used for staining are as indicated in each panel with matched colors in this and all subsequent figures. Posterior is towards the left and dorsal is upwards in all discs unless mentioned otherwise. (A) Two tier nuclei layers in the longitudinally sectioned eye disc. Photoreceptor nuclei stained by Elav (red) localize apically, whereas undifferentiated cell nuclei stained by BarH1 (green) localize in the basal region of the disc. R1/6 photoreceptors (yellow arrow) are labeled by both Elav and BarH1 antibodies. Bar is also expressed in peripodial cells (small white arrow) and basal undifferentiated cells (double arrowheads) behind the furrow (marked by large white arrow). (B) Complementary expression patterns of Bar and Ato in the basal undifferentiated cells along the furrow (marked by arrow). Two confocal images are combined to illustrate the Bar-Ato pattern. (C) Co-expression of Bar and Lz in a majority of basal undifferentiated cells (marked by white arrow) except in some Bar-expressing cells immediately posterior to the furrow (marked by yellow arrow). An eye disc is longitudinally sectioned. (D-I) Lz is not required for Bar expression in the basal undifferentiated cells. A lzr15 LOF clone is identified by the absence of Lz staining (D,E,G,H; red) and marked with broken lines in F,I. Bar expression is downregulated in R1/6 cells (D-F, arrows), but not in the basal undifferentiated cells (G-I) within the lzr15 LOF clone. (J) Bar is expressed in R1/6 cells in the apical photoreceptor level. An arrow indicates Bar expression in R1/6 cells of wild-type eye disc. (K,L) Gl is not required for Bar expression in the basal undifferentiated cells. A gl1 mutant eye disc stained with antibodies for Elav (red) and BarH1 (green) is shown at the apical photoreceptor nuclei level (K) and the basal undifferentiated cell level (L). Bar expression in most R1/6 photoreceptors is strongly downregulated or absent, except a few cells marked by an arrow (K), while its expression in the basal undifferentiated cells is not much changed (L). Broken lines in K,L mark the position of morphogenetic furrow; Bar is expressed only behind the furrow.

View larger version (87K): [in a new window]   Fig. 2. Bar expression in the basal undifferentiated cells depends on a posterior secreted signal. (A-C) Morphogenetic furrow can be formed and progresses in ato1 mutant eye disc. The furrow is shown by apical membrane constriction intensely stained by Dlg membrane marker (B, arrow) and by Ato expression (C, arrow) in the middle of ato1 mutant eye disc. (D-F) A graded Bar expression in ato1 mutant eye disc. Bar is expressed posterior to the furrow in ato1 mutant eye disc. Bar expression level is high near the posterior margin but decreases significantly near the furrow, suggesting that Bar expression may depend on a secreted factor(s) from the posterior margin (F). An area marked with a rectangle in E is magnified in F. MF, morphogenetic furrow.

View larger version (74K): [in a new window]   Fig. 3. Hh signaling is required for Bar expression in the basal undifferentiated cells. Eye discs containing smo3 LOF clones at different positions, close to the posterior margin (A-D) and near the furrow (E-H), are stained with antibodies for lacZ (clone marker; red), BarH1 (green) and Ato (blue). Bar expression is lost in smo3 LOF clones located close to the posterior margin (C, white arrow) or near the furrow (G, white arrow). Some Bar expression remains in part within the smo3 LOF clone near wild-type cells (C,G; yellow arrows), and ectopic Ato expression is observed in the region of Bar loss within the clones (D,H; red arrows). Apical through basal sections of confocal images are combined in order to see the BarH1 expression in single image.

View larger version (102K): [in a new window]   Fig. 4. Ato-EGFR signaling is required for the induction of Bar expression during furrow progression. (A-C) Bar expression level in the basal undifferentiated cells in wild-type eye disc is relatively even, although it appears to have a slightly higher level at the posterior margin and immediately posterior to the furrow (B, arrows). An area marked with a rectangle in A is magnified in B. (C) Predicted two activators for Bar expression in the basal undifferentiated cells: posterior margin signal (Hh) and furrow signal (blue arrow). P, posterior margin; MF, morphogenetic furrow. (D-F) Ato is required for Bar expression. Bar expression is absent (white arrow) immediately posterior to the furrow within ato1 LOF clone, marked by broken white line in F. A broken yellow line marks the normal anterior boundary of Bar expression right posterior to the furrow in wild type. Bar expression is rescued in the posterior part of the ato1 LOF clone (F, yellow arrow). Apical through basal sections of confocal images are combined in order to see Bar expression in single image. (G-I) EGFR is not activated within ato1 LOF clones. Yellow and white arrows mark the dpERK activation within the proneural clusters in the wild-type or ato1 mutant regions, respectively. The dpERK (dual phosphorylated extracellular signal regulated kinase) staining indicates the activation of EGFR signaling pathway. (J-L) egfrCO LOF clone shows no Bar expression immediately posterior to the furrow (white arrow in K). In K, the broken yellow line marks the normal anterior boundary of Bar expression right posterior to the furrow in wild type. White (K) and yellow (L) lines, respectively, mark the egfr LOF clone boundaries. Ectopic Ato expression (L) is observed in the posterior region of the eye disc where Bar expression is lost (lack of green in K).

View larger version (103K): [in a new window]   Fig. 5. Bar autoregulates its expression. (A-C) Bar expression in the basal undifferentiated cells is maintained after expression of its two activators, Hh and Ato, has ceased. A hh1 mutant eye disc stained with antibodies for Elav (red), BarH1 (green) and Ato (blue) is shown at the apical photoreceptor level (A) and the basal undifferentiated cell level (B). Ato expression is strongly downregulated but Bar expression is quite normal in hh1 mutant eye disc. In A, an arrow marks the arrested furrow. (C) A longitudinal section of hh1 mutant eye disc. (D) A longitudinal section of wild-type eye disc. (E-L) Bar positively autoregulates its transcription. Misexpression of BarH1 by dpp-GAL4 in the background of BarH2P058-lacZ ectopically induces lacZ expression (H,L; arrows) in the region of BarH1 misexpression shown by ectopic BarH1 protein in the ventral region of eye disc (E-H) and along the anteroposterior (AP) border of leg disc (I-L). The ventral region of eye disc marked by a rectangle in E is magnified in F-H. (I) The wild-type pattern of Bar expression (red) in a ring domain and the dpp driver pattern by GFP reporter expression (green) along the AP axis.

View larger version (52K): [in a new window]   Fig. 6. A model for Bar regulation during retinal neurogenesis. Ato expression (gray region) in the morphogenetic furrow (MF) is activated by Hh produced by photoreceptor cells (orange region) and initiates the generation of photoreceptor neurons. Bar homeodomain proteins, which are essential for transcriptional repression of ato (Lim and Choi, 2003), are expressed in basal undifferentiated cells behind the furrow (green region) by several mechanisms. Positive and inhibitory relationships labeled by arrows may be indirect. (i) Prior to photoreceptor differentiation at the time of furrow initiation, Bar expression in the basal undifferentiated cells is induced initially by a secreted signaling factor, Hh, from the posterior margin (yellow region). (ii) During furrow migration, Bar expression near the furrow is induced by Ato from the furrow. EGFR signaling may partially mediate nonautonomous effects of Ato on Bar expression. (iii) Hh produced in photoreceptor cells induces Dpp expression and may also contribute to Bar expression during furrow migration. Finally (iv), Bar is autoregulated to maintain its expression.