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First published online November 11, 2004
doi: 10.1242/10.1242/dev.01442

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A role for extra macrochaetae downstream of Notch in follicle cell differentiation

Department of Biological Chemistry, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185, USA

View larger version (25K): [in a new window]   Fig. 1. An ovariole, and early acquisition of cell fates in the follicle cells. The germarium is at the anterior (left, upper bracket), and progressively older egg chamber stages (Spradling, 1993) develop to the posterior (right). Somatic stem cells (blue) produce follicle cell precursors (brown), which form main body precursors that express EYA (brown) and polar/stalk cell precursors (yellow) that ultimately eliminate EYA expression. Main body cells differentiate at stage 6 (light brown). Unpaired is emitted from the polar cells (red) and received by STAT in the stalk cells (green). Polar, stalk and main body cells are all thought to require the activity of Notch (N) for differentiation. NC, nurse cells; FC, follicle cells; O, oocyte.

View larger version (66K): [in a new window]   Fig. 2. EMC overexpression phenotype. (A-C) Nuclei of ovarioles, stained with DAPI. The germarium is to the left. (A) A wild-type ovariole. (B,C) Individual ovarioles from females driving gene expression under the control of C306-Gal4: (B) EP3620; (C) UAS-emc5.3. Scale bar: 50 µm.

View larger version (105K): [in a new window]   Fig. 3. EMC expression pattern in ovaries. Anterior is to the left. (A,E) Females containing the emcP5C enhancer trap, stained with anti-ß-galactosidase (red) and with anti-Fas3 (green), showing the germarium and a stage-2 egg chamber (A) or a stage-3 egg chamber and a polar/stalk group (E). Arrows in A indicate terminal filament, cap and inner sheath cells. The bracket in A indicates follicle cells in a germarium. The bracket in E indicates polar and stalk cells. (B-D) The same germarium and early ovariole from a wild-type female stained with anti-EYA (B,C; red in C) and anti-EMC (C,D; green in C). The blue arrowheads indicate intercyst follicle cells in the germarium. The white arrowheads indicate stalk cells between the germarium and a stage-2 egg chamber. The red arrowheads indicate stalk cells between a stage-2 egg chamber and a stage-4 egg chamber. (F,G) The same stage-3 and stage-4 egg chamber and polar/stalk group from a wild-type female stained with anti-Fas3 (red in F) and anti-EMC (F,G; green in F). Arrowheads indicate stalk cells; arrows indicate polar cells. (H) Section of a wild-type stage-6 egg chamber stained with anti-EYA (left panel, and red in middle panel) and anti-EMC (right panel and green in middle panel). Arrowheads indicate a polar cell expressing EMC but not EYA. (I) Posterior of a stage-10 egg chamber possessing a clone of cells (dashed line) that are overexpressing EP3620 under the control of ayGal4. Overexpressing cells are marked with GFP (green). Red indicates EMC. Scale bars: in A, 25 µm in A-G, I; in H, 10 µm in H.

View larger version (127K): [in a new window]   Fig. 4. Loss-of-function phenotypes of emc (A,B,D-Q,S) compared with those of Notch (C,R,T). Mutant cells are marked by the absence of GFP (A-E,I-K,M,O,P; green in A-D,I,J,M,O,P). (A-C) clones of cells mutant for emc1 (A), emcAP6 (B) or N55E11 (C), stained with anti-Fas3 (red). (D-G) A portion of the same egg chamber showing a clone of emc1 mutant cells marked by the loss of GFP (D,E; green in D) expressing elevated Fas3 (D,F; red in D) but not extra PZ80 (D,G; blue in D). Inset in G: PZ80 expression in polar cells from the same experiment. The lines indicate emc mutant cells. (H,I) A brightfield (H) and a fluorescent section (I) of the same stage-8 egg chamber possessing a small clone of emcAP6 mutant cells (arrowhead) marked by the absence of GFP (I, green) and stained with anti-Cyclin B antibody (I, red). The arrow indicates yolk. The arrowhead indicates mutant cells magnified in J-L. (J-L) Magnified region of I, marked with GFP (J,K; green in J), and stained with anti-Cyclin B antibody (J,L; red in J). Arrowheads indicate an emc mutant cell expressing Cyclin B. (M,N) Posterior of the same stage-10 egg chamber, showing a clone of emcAP6 mutant cells (inside dashed line) and their heterozygous neighbors (green, outside dashed line) stained with DAPI (M,N; red in M). (O,P) Single sections (O) and a Z-stack (P) of different egg chambers from animals possessing emc mutant clones marked with GFP (green) and stained with anti-Fas3 antibody (red). Arrowheads indicate expected position of polar cells. Arrows indicate polar cells. (Q-T) Fused egg chambers from different females possessing clones of cells mutant for emc1 (Q,S) or N55E11 (R,T) stained with DAPI (Q,R) and with anti-Fas3 antibody (S,T). Arrows indicate oocyte nuclei; arrowheads indicate polar cells. Scale bars: in A, 10 µm in A-G,J-P; in H, 25 µm in H,I; in Q, 25 µm in Q-T.

View larger version (100K): [in a new window]   Fig. 5. Molecular epistasis of Notch, EMC and EYA. (A-H) Loss-of-function clones of N55E11 (A,B,G,H,), emc1 (C,D) or eya54C2 (E,F) mutant cells marked with GFP (A,C,G), or anti-EYA (E, green) and stained with either anti-EMC (A,B,E,F; red in A,E) or anti-EYA (C,D,G,H; red in C,G). The dashed lines indicate clones of mutant cells. (I-L) FLP-OUT clones of cells expressing activated Notch (UAS-N34A: designated N-Act) in two separate stage-10 egg chambers, marked with GFP to show N34A-expressing cells in green (I,K) and stained with anti-EMC (I,J; red in I) or with anti-EYA (K,L; red in K). The arrows indicate cells overexpressing EMC (I,J) or EYA (K,L). The dashed lines (I,J) and arrows (K,L) indicate cells overexpressing UAS-N34A. Scale bar: 10 µm.

View larger version (123K): [in a new window]   Fig. 6. The effect of EMC overexpression on polar cell differentiation and on expression of EYA. (A-D) Two examples of fused egg chambers in which EP3620 is overexpressed using FLP-OUT Gal4, stained with anti-Fas3 antibody (A,C; red) and DAPI (A-D; blue in A,C), possessing three polar cell groups (arrows) rather than four, and having either a layer of cells between the fused egg chambers (B, arrowheads) or no such layer (D). (E-K) EP3620 (E,F,H-K) or UAS-LacZ (G) overexpressed using Upd-Gal4. (E,F) An ovariole (E) or two egg chambers in a different ovariole (F) stained with DAPI, showing an egg chamber with the oocyte positioned toward the anterior rather than the posterior of the ovariole (arrows). (G,H) Portions of the anterior end of an egg chamber (G) and the posterior end of one egg chamber (H, left) and anterior end of its neighbor (H, right) expressing UAS-LacZ (G) or EP3620 (H), and showing expression of EYA (blue), and the polar cell marker PZ80 (red). The arrowheads indicate the shape of the anterior end of the egg chamber and the position of the expected polar cell. The arrow indicates the region magnified in I-K. (I-K) EYA expression (I), polar cells (J) and Gal4-expressing cells (marked with GFP, K). The arrows indicate a polar cell expressing Gal4 (and presumably EMC) but not EYA. The arrowheads indicate a cell expressing Gal4 and EYA but not PZ80. Scale bars: in A, 25 µm in A-F; in G, 25 µm in G,H; in I, 10 µm in I-K.

View larger version (17K): [in a new window]   Fig. 7. Graphs showing the relationship between loss of polar cells, gain of polar cells and egg chamber stage in temperature-shift experiments. Upd-Gal4/+; P280/+; EP3620/+: dark gray; Upd-Gal4/+; P280/+; UAS-mCD8GFP/+: light gray. Egg chamber stage is plotted on the x-axis and percentage of expected polar cell groups in egg chambers of that stage that show loss of all polar cells (A) or that show four polar cells (B) is plotted on the y-axis. In each case, the value plotted is the average of three experiments. Error bars: standard deviation.

View larger version (8K): [in a new window]   Fig. 8. Model for the relationship between Notch, EMC and EYA during oogenesis. Left: in polar/stalk precursors, an unknown pathway (X) inhibits expression of EMC. In the absence of EMC, EYA is reduced. Right: in main body precursors, Notch promotes EMC expression, and EMC promotes EYA expression, leading to either the establishment or maintenance of main body cell identity. In addition, EMC activity promotes differentiation.