1. Volume 41, Issue 2, Pages 157-169.e5 (April 2017)
Aubergine Controls Germline Stem Cell Self-Renewal and Progeny Differentiation via Distinct Mechanisms 
Xing Ma, Xiujuan Zhu, Yingying Han, Benjamin Story, Trieu Do, Xiaoqing Song, Su Wang, Ying Zhang, Marco Blanchette, Madelaine Gogol, Kate Hall, Allison Peak, Perera Anoja, Ting Xie 
Developmental Cell 
Volume 41, Issue 2, Pages e5 (April 2017)
DOI: /j.devcel
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2. Developmental Cell 2017 41, 157-169. e5DOI: (10. 1016/j. devcel. 2017
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
Aub Is Required for GSC Maintenance and Early Germ Cell Differentiation
(A–E and H) Cap cells are highlighted by broken ovals, whereas arrows and arrowheads indicate spectrosomes in GSCs and CBs, respectively. Hts labels spectrosomes in GSCs and CBs and branched fusomes in cysts.
(A and B) Three-day-old (A) and 20-day-old (B) wild-type (WT) ovaries contain three and two GSCs, respectively.
(C–E) Three-day-old (C), 10-day-old (D), and 20-day-old (E) aub mutant ovaries carry two GSCs, one GSC, and no GSC, respectively. The aub mutant germaria in (C) and (D) also contain excess CBs, whereas that in (E) does not contain any germ cells.
(F and G) Quantification results show that aub mutant germaria lose GSCs significantly faster with age (F) and accumulate significantly more CBs (G) than wild-type germaria.
(H) The aubQC/Df mutant germarium contains excess CBs.
(I) Quantification results show that aubQC/Df and aubHN/Df mutant germaria contain significantly more CBs than aubHN/aubQC mutant germaria.
All error bars represent SE in this and the following figures, and all p values herein are determined by Student's t test. Scale bar, 10 μm.
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4. Figure 2
Aub Controls GSC Self-Renewal by Preventing DNA-Damage-Induced Checkpoint Activation
(A–D) LacZ-negative marked GSCs and marked cysts are indicated by broken circles and arrows, respectively; LacZ-positive unmarked GSCs are highlighted by circles. Nuclear membrane protein Lamin C (LamC) labels terminal filaments and cap cells.
(A and B) The marked control GSCs remain for 10 days (A) and 21 days (B) ACI.
(C and D) The marked aub mutant GSC remains 10 days ACI (C), but is lost from the niche 21 days ACI, which is evidenced by the presence of a marked aub mutant cyst (arrow) (D).
(E) Quantification shows that marked aub mutant GSCs are lost faster than marked control GSCs.
(F and G) Germaria carrying only marked mutant GSCs are filled with LacZ-negative marked CBs (arrowheads) 21 days ACI.
(H–J) GSCs are negative for ApopTag labeling in the aub mutant germarium (I) as in the control germarium (H) (J shows quantification results). ApopTag-positive germ cells (arrows) away from the germarial tip are observed in both control and aub mutant germaria. Ovals highlight cap cells and GSCs.
(K and L) nos-gal4-driven expression of FLAG-tagged p35 in germ cells, including GSCs (circles) (K), and a p35-expressing germarium contains three GSCs (circles) (L).
(M–O′) The p35-expressing aub mutant germarium contains two GSCs (circles, N) in contrast to the aub mutant germarium containing one GSC (circle, M). (O) and (O′) show GSC and CB quantification results, respectively. In (O′), the germaria without any GSCs are excluded.
WT, wild-type. Scale bar, 10 μm. See also Figure S1.
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5. Figure 3
Aub Is Required Intrinsically to Maintain GSCs by Preventing Chk2-Dependent Checkpoint Activation
(A and B) The control (A) and lok mutant (B) germaria contain two GSCs (ovals), which are negative for γ-H2AvD.
(C–E) The aub mutant (D) and lok aub double mutant (E) germaria contain one and two GSCs (circles), respectively, which are positive for γ-H2AvD (C shows results of GSC quantification).
WT, wild-type. Scale bar, 10 μm. See also Figure S2.
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6. Figure 4
Aub Promotes Early Germ Cell Differentiation by Enhancing Bam Function
(A and B) Both wild-type (WT) (A) and aub heterozygous mutant (B) germaria carry two GSCs (ovals) and one CB (arrow).
(C) Quantification results show that double aub and bam heterozygous mutant germaria contain significantly more CBs than single bam or aub heterozygous mutant germaria.
(D and E) In contrast to the bam heterozygous mutant germarium carrying two CBs (arrows, D), the bam and aub transheterozygous germarium contains excess CBs (arrows, E).
(F–I) Two days after heats-hock treatment (2d AHS), the bam-expressing aub mutant germarium (H) contains only differentiated cysts (arrow) but no GSCs or CBs in comparison with the wild-type germarium (F) carrying two GSCs (circles) and one CB (arrow) and the aub mutant germarium (G) carrying one GSC (circle) and many CBs (arrow). (I) CB quantification results.
Scale bars, 10 μm.
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7. Figure 5
Aub Maintains Bam Protein Expression in Mitotic Cysts at the Transcriptional Level
(A–D′) In contrast to aub heterozygous 2-cell (A and A′) and 4-cell (B and B′) cysts (broken lines) expressing Bam protein, aub hemizygous mutant 2-cell (C and C′) and 4-cell (D and D′) cysts (broken lines) express less Bam protein.
(E and E′) Western blotting (E) shows that aub mutant ovaries express significantly less Bam protein than wild-type ovaries (asterisk denotes a non-specific protein recognized by the anti-Bam antibody). (E′) quantification results of three western blot experiments.
(F) qRT-PCR results show that bam mRNA expression levels are reduced in aub mutant ovaries in comparison with that in wild-type control ovaries.
(G–G″) Control germaria show bam-GFP expression in 2-cell (G), 4-cell (G′), and 8-cell (G″) cysts (broken lines).
(H–H″) aub mutant germaria exhibit reduced bam-GFP expression in 2-cell (H), 4-cell (H′), and 8-cell (H″) cysts (broken lines).
(I) Quantitative results show that bam-GFP increases its expression from control 2-cell to 8-cell cysts, which is significantly downregulated in aub mutant mitotic cysts.
(J and J′) Western blotting (J) shows that aub mutant ovaries express significantly less bam-GFP than control ovaries. (J′) Quantification results of three western blot experiments.
Scale bars, 10 μm.
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8. Figure 6
Aub Binds mRNAs Encoding Known GSC Self-Renewal and Differentiation Factors in Cultured GSCs
(A) Cultured bamΔ86 hs-bam GSCs express GFP-Aub fusion protein as single cells carrying a spectrosome (arrowhead) or dividing doublets carrying an elongated fusome (arrow). DNA-labeled nuclei (asterisk) are supporting somatic cells.
(B and C) One day after Bam induction, the cultured GSCs have differentiated into 4-cell (arrowheads, B), 8-cell (arrows, B), and 16-cell (arrow, C) cysts. DNA-labeled nuclei (asterisks) are supporting somatic cells.
(D) Autoradiographs show that GFP-tagged Aub can only be crosslinked to mRNAs in the presence of UV. Asterisk indicates the free GFP-Aub position, and the green box highlights the collected crosslinked GFP-Aub-RNA complexes.
(E) Aub binds to 5′ UTRs and -3′ UTRs but to a lesser extent to the coding regions (CDS).
(F) Five major Aub-binding motifs, and their appearance frequency and E values.
(G) Aub target mRNAs, including known self-renewal and differentiation genes.
(H) Aub binds its own mRNAs in the 5′ UTR, the coding region, and the 3′ UTR.
(I) Aub binds the 3′ UTR of bam.
(J) Aub binds to the 5′ UTR, intron, and 3′ UTR of sxl.
(K and L) Aub binds the 5′ UTRs and 3′ UTRs of brat and mei-p26.
(M and N) Aub binds the 5′ UTRs and 3′ UTRs of dnc (M) as well as an intron, exons and 3′ UTR of Rm62 (N).
See also Figure S3 and Table S1.
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9. Figure 7
Aub Controls Bam, Dnc, and Rm62 Expression at the Translation Level via Their 3′ UTRs in Germ Cells Possibly by Interacting with the eIF4 Complex and pAbp
(A–E) The expression of nos-gfp-bam3′UTR in aub mutant 4-cell and 8-cell cysts (arrowheads, C and D) decreases in comparison with their control counterparts (arrowheads, A and B). Circles indicate GSCs. (E) Quantification results. Scale bars, 10 μm.
(F) gfp mRNA levels from nos-gfp-bam3′UTR remain unchanged in the aub mutant ovaries in comparison with the control ovaries.
(G) nos-gfp-bam3′UTRΔ (deleting the Aub-binding site) significantly decreases GFP protein expression in 4-cell and 8-cell cysts in comparison with nos-gfp-bam3′UTR.
(H–J) nos-gfp-dnc3′UTRΔ (deleting the two Aub-binding sites, I) significantly decreases GFP protein expression in GSCs (circles) in comparison with nos-gfp-dnc3′UTR (H). (J) Quantification results.
(K–M) The 30-day-old dnc3′UTRΔ1 (deleting the first Aub-binding site in the 3′ UTR) mutant germarium carries one GSC (circle, L) in comparison with the 30 day-old control germarium carrying two GSCs (two circles, K). Arrowheads denote CBs. (M) GSC and CB quantification results show that GSCs in the dnc3′UTRΔ1mutant germaria are significantly fewer than the control germaria, but CBs remain unchanged.
(N) FLAG-eIF4A can pull down Aub-HA in S2 cells, whereas Aub-FLAG can pull down Myc-eIF4A.
(O) Myc-eIF4E can pull down Aub-HA in S2 cells, whereas Aub-FLAG can pull down Myc-eIF4E.
(P) Flag-eIF4G can pull down Aub-HA in S2 cells.
(Q) Aub-FLAG can pull down HA-pAbp in S2 cells, whereas FLAG-pAbp can pull down Aub-HA.
(R) A working model explaining the roles of Aub in maintaining GSC self-renewal and promoting GSC progeny differentiation, and proposing a potential strategy for Aub to control the translation of its target mRNAs.
See also Figure S4.
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