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First published online March 1, 2007
doi: 10.1242/10.1242/dev.02800

Development 134, 1161-1169 (2007)
Published by The Company of Biologists 2007
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Drosophila follicle cells are patterned by multiple levels of Notch signaling and antagonism between the Notch and JAK/STAT pathways

Efrat Assa-Kunik1,*, Isabel L. Torres2,*, Eyal D. Schejter1,{dagger}, Daniel St Johnston2 and Ben-Zion Shilo1,{dagger}

1 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
2 The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.


Figure 1
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  		Fig. 1. Early oogenesis in Drosophila. A diagram of the anterior portion of a wild-type ovariole, including the germarium and four young egg chambers (stages 1-4). Polar cells (green) and stalk cells (red) are shown both at the precursor stage (near the germarium), and following their differentiation. Oocytes are marked in blue throughout. fc, follicle cells; post., posterior; ant., anterior.

 

Figure 2
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  		Fig. 2. Delta is required in anterior follicle cells for stalk formation. Dl homozygous clones were induced in FRT82B DlM1/FRT82B GFP females, and are marked by the loss of GFP (green). (A,A') Germline cysts completely surrounded by Dl-mutant follicle cells fuse with the neighboring anterior cyst, but have a normal posterior stalk (arrowhead in A'). Fas3 (red) marks the follicle cell membranes. (B) Both the anterior (arrowhead) and posterior (arrow) pairs of polar cells (marked with PZ80, blue) are specified in Dl follicle cell clones. (C,C') Two confocal planes of a pair of fused egg chambers show that the anterior polar cells (C', arrowhead) of the older cyst are mutant for Dl. Polar cells are labeled with neurA101 (blue) and Fas3 (red). (D,D') In rare cases, an anterior stalk (arrowhead in D) forms even when both anterior polar cells (Fas3, red) are mutant for Dl. (E) Dl stalk cell clones appear as wild type. neurA101 (blue) and Fas3 (red).

 

Figure 3
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  		Fig. 3. Activation profile of Notch during early stages of follicle cell patterning. (A-A'') During the earliest stages of oogenesis, Notch activation in follicle cells (monitored by anti-ß-gal staining of the Su(H)m8-lacZ reporter, red) is restricted to the polar cells (marked by A101-GAL4/UAS-mCD8::GFP, green), and cannot be observed in stalks. The prominent signal in follicle cells of later egg chambers corresponds to the differentiation of main-body follicle cells and their switch from mitosis to endoreplication, following germline Dl induction of Notch signaling at stages 5-6 of oogenesis (Lopez-Schier and St Johnston, 2001Go; Ruohola et al., 1991Go). (B) Anti-ß-gal staining of the Notch reporter m7-lacZ identifies essentially the same pattern as the Su(H)m8-lacZ reporter. (C) However, with the m7-lacZ reporter, low levels of Notch activation could also be observed in the stalk cells at early stages (arrow). (D) Immunolocalization of Dl (red) reveals relatively higher levels of Dl protein in the germ line than in the follicle cells in early egg chambers.

 

Figure 4
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  		Fig. 4. The levels of Dl from follicle cells determine stalk size. (A,B) Precursor-stage polar cells (green, marked by A101-GAL4/UAS-mCD8::GFP) and stalk cells (red, marked by the how93F reporter) initially align as broad neighboring bands in region 3 of the germarium. The polar cell precursors are always positioned posteriorly. Nuclei are stained with DAPI (blue). (C,D) In situ hybridization using a fluorescent Kul RNA probe. In surface (C) and cross-section (D) views, Kul RNA (red) is detected in all follicle cells at early stages, including the stalk (arrow), but hardly in the germ line itself (arrowhead). (E,F) Notch activation during early oogenesis, monitored by X-Gal staining of the Su(H)m8-lacZ reporter. Arrows point to the germarium and arrowheads to stage 2 polar cells. Notch activation in A101-GAL4/UAS-dskul-ovarioles (F) is elevated as compared with wild type (E). (G) Expression of UAS-dskul in polar cells by A101-GAL4 (green) leads to an increase in stalk-cell number. The stalk is marked by the 93F lacZ reporter (red). (H) Quantification of stalk size in various genetic backgrounds. Stalk-cell numbers are increased following expression of dskul in either polar (dark red columns) or stalk cells (orange columns), and reduced in Dl heterozygous ovaries (green columns). (I,I') Expression of Notch dsRNA in the stalk cells (green) using the 24B-GAL4 driver leads to loss of the stalk marker Bib (red). (J) Expression of p35 in both polar and stalk cells using the 7025-GAL4 driver results in an abnormal stalk containing an excess of cells (arrows).

 

Figure 5
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  		Fig. 5. Overexpression of Delta alters follicle cell fates and antagonizes Unpaired signaling. (A-B') Dl overexpression using the A101-GAL4 polar cell-specific driver induces extra polar cells (green). Some of these extra cells express the main-body follicle cell marker Eya (arrow, red in A,A') or the stalk marker Bib (arrowhead, red in B,B'). (C,C') Dl overexpression using the Upd-GAL4 polar cell-specific driver results in loss of the stalk and fusions between neighboring cysts. Two confocal planes of three germline cysts from a single ovariole are shown. Fas3 (red) marks polar cells in the oldest egg chamber (designated #1, arrowhead). (D) Dl overexpression using the 24B-GAL4 stalk cell-specific driver induces the expression of the polar cell marker, neurA101 (arrowhead, red).

 

Figure 6
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  		Fig. 6. Unpaired signaling from polar cells is limited in range. (A-B) Anti-GFP staining (green), monitoring JAK/STAT activation using the 2XSTAT92E-GFP reporter. Anti-ß-gal staining (A', red) of the Notch reporter m7-lacZ. During early stages of oogenesis, Upd signaling from the polar cells is capable of inducing strong STAT activation in stalk cells (arrows in A,A'), but fails to elicit activation in either the polar cells themselves, or in the neighboring main-body follicle cells. At later stages, follicle cell populations, including main-body and border cells, exhibit concomitant Notch and STAT activation (arrowheads in A,A',B). (C,C') Localization of STAT in early follicle cells. Ovarioles in which polar cells were visualized by A101-GAL4/UAS-GFP (green), were stained for STAT (red) and the nuclear marker DAPI (blue). Nuclear localization of STAT staining (boxes) shows the limited range of JAK/STAT activation by Upd emanating from polar cells. STAT staining alone (C) demonstrates the non-responsiveness of the polar cells to the Upd signal they produce, as STAT does not localize to polar cell nuclei (arrowhead in C, enlarged view). (D,D') Overexpression of Upd using the A101-GAL4 polar cell-specific driver. The range of JAK/STAT activation (monitored by nuclear localization of STAT, red) broadens exclusively towards the anterior. Polar cells remain refractory to the signal (arrowhead). (E) DAPI staining of egg chambers overexpressing Upd in polar cells reveals an abnormally long stalk (arrow). (F,F') Main-body follicle cell clones homozygous for N55e11 (marked by loss of GFP), display nuclear localization of STAT (arrow), demonstrating that Notch activation in these cells antagonizes the JAK/STAT pathway. Clones that were further than four cell-diameters from the polar cells did not display nuclear STAT, owing to the restricted diffusion of Upd (not shown).

 

Figure 7
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  		Fig. 7. Extra polar cells are formed in a hopscotch hypomorphic mutant. (A) At early stages of oogenesis, hopmv1/GA32 hypomorphs form a stalk, and the oocyte (marked by anti-BicD, green) moves to the posterior of the egg chamber as in wild type. At later stages, fusions occur and stalks are never seen. (B) Stalk cells migrate between two cysts to form a stalk with two layers connected by adherens junctions (labeled with E-Cadherin in red, arrow), but they never intercalate (B and D, arrow). (C) The stalk (labeled with E-Cadherin, red) collapses from stage 4/5. The oocyte is stained with anti-Orb (blue). (D) When the stalk collapses, a cluster of cells expressing high levels of Fas3 (red) accumulates adjacent to the older egg chamber (arrowhead). (E-F) The oocyte (labeled with anti-BicD, green) remains attached to the cells that express high levels of Fas3 (red, arrow in E'), which are identified as polar cells at later stages.

 

Figure 8
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  		Fig. 8. Scheme for the spacial and temporal order of determination of follicle cell fates by the Notch and JAK/STAT pathways. (A) At stage 1 of oogenesis, follicle cells encounter high levels of Dl from the germ line. Expression of Fng in the anterior polar/stalk precursors enhances Notch signaling, leading to the induction of polar cells (PC). The relatively lower levels of Dl expressed by follicle cells are further reduced by the metalloprotease Kul, thus leading to low levels of Notch activation in those follicle cells that do not contact the germ line. This low level of activation promotes cell viability and contributes to induction of the stalk cell fate. (B) The polar cells express the Domeless ligand Upd, which diffuses to neighboring follicle cells. The main-body follicle cells, which experience high or intermediate levels of Notch activation, are refractory to JAK/STAT signaling, and repress STAT nuclear localization. By contrast, the pre-stalk cells, which are subject to low levels of Notch activation, undergo JAK/STAT activation. The combined activity of the two signaling pathways thereby facilitates proper stalk cell-fate induction.

 

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© The Company of Biologists Ltd 2007