Download Argonaute 1 regulates germ cell division and oocyte determination

Argonaute 1 regulates germ cell division and oocyte determination in
Drosophila melanogaster
Mohd Ghows Mohd Azzam and Ji-Long Liu
MRC Functional Genomics Unit, University of Oxford
[email protected]
Summary
Ago1 affects germ cell division
Argonaute 1 (Ago1) is a member of the Argonaute/PIWI protein family that involves
in small RNA-mediated gene regulation. In Drosophila melanogaster, Ago1 plays a
B
A
B
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specific role in microRNA biogenesis. Previous studies have demonstrated that
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Ago1 regulates the fate of germline stem cells. However, the function of Ago1 in
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other aspect of oogenesis is still elusive. Here we report the distribution of Ago1
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protein in Drosophila egg chambers. We find that Ago1 protein is enriched in the
oocytes and also distributed in the cytoplasm of follicle cells. Using mitotic clonal
GFP
Hoechst
C
D
analysis, we analyze multiple ago1 mutant alleles. Here, we find that the oocyte do
not form in many mutant clones. Approximately 30% of the mutant clones have only
8 nurse cells and they do not develop an oocyte. These results indicate that the level
of Ago1 affects the number of cystoblast divisions and oocyte determination.
orb
Hoechst + GFP + orb
Figure 3: Loss of Ago1 results in fewer cyst cell divisions. (A-D) Projection of an egg
chamber clone (GFP –ve) with only 8 nurse cells and no oocyte. This phenotype is the most
common occurring in the mutants.
D. melanogaster microRNA biogenesis
Ago1 phenotypic severity increases with age
Clones with and without oocyte
100%
100%
95%
80%
% of egg chamber
80%
Figure 1: microRNA biogenesis in Drosophila melanogaster. Ago1 (highlighted in the red box)
plays an important role of binding to the miRNA and incorporating it into the RISC.
100%
98%
73%
62%
59%
60%
Control
41%
38%
40%
Ago1 k08121
Ago1 14
27%
Ago1 EMS
20%
20%
5%
0%
0%
Normal clones - 7 DAE
Normal clones - 14 DAE
2%
0%
Clones with no oocyte -7 DAE
Clones with no oocyte - 14
DAE
*DAE – Days after eclosion (hatching)
Ago1 affects oocyte determination
Comparison of phenotype between 7 DAE and 14 DAE
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65
60
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2 NCs - 14 DAE
4 NCs - 14 DAE
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No. of cells
8 NCs - 14 DAE
40
16 NCs - 14 DAE
35
32 NCs - 14 DAE
30
2 NCs - 7 DAE
Hoechst + Ago1 + Actin
B
20
D
C
4 NCs - 7 DAE
8 NCs - 7 DAE
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2
0
GFP
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Ago1
Merged
16 NCs - 7 DAE
8
0
1 0 0 2
4
7
1
0 1
Ago1 k08121
3
1 0 0 2 0 0
3 3
Ago1 14
32 NCs 7 DAE
0 0 0 0 1 0
Ago1 EMS
E
Conclusion
Hoechst + GFP + orb + Histone H4
• Ago1 affects cyst cell division in the germarium resulting in 8 germline cells per
egg chamber in the Ago1 mutants.
F
•Oocyte determination is affected in the absence of Ago1.
G
GFP
•Older flies have more severe phenotypes than younger flies.
Acknowledgements
Hoechst + GFP
Figure 2: Ago1 mutant clones have oocyte determination defect. (A) Ago1 protein trap line
showing the distribution of Ago1 (green) in egg chambers. Enrichment of Ago1 in the developing
oocyte is seen in the stage 7 egg chamber. Heat-shock induced mitotic recombination generates
Ago1 homozygous mutant clones (GFP -ve). (B-G) Mitotic clones. (B-D) Follicle cell clones. GFP
(green) and Ago1 (yellow). (H-I) ago1 severe phenotype where follicle cells in egg chambers
above stage 5 begin to deteriorate and there is no oocyte formation.
Malaysian Ministry of Higher Education
This work was funded by the UK Medical Research Council.