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First published online 13 August 2008
doi: 10.1242/dev.021923

Development 135, 3021-3030 (2008)
Published by The Company of Biologists 2008
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Negative-feedback regulation of proneural proteins controls the timing of neural precursor division

Pao-Ju Chang1,*, Yun-Ling Hsiao1,*, An-Chi Tien2, Yi-Chen Li1 and Haiwei Pi1,{dagger}

1 Department of Life Science, Chang-Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan.
2 Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.


Figure 1
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  		Fig. 1. SOP is arrested at G2 phase in phyl mutants. (A-K) SOPs were labeled by anti-Sens staining (green). (A,B) Wild-type pupal thoraces at 14 hours (A) and 16 hours (B) APF. Almost all wild-type SOPs have divided within these 2 hours. (C,D) SOP division is delayed in phyl2 mutant clones (GFP negative). Arrows indicate phyl2-mutant SOPs, which remain as single cells at 22-24 hours APF (C) and do not divide until 24-28 hours APF (D). (E,G,I) Wild-type pupal thoraces. (F,H,K) phyl2 mutant clones. (J) phyl2/phyl4 hypomorphic mutant. (E-F') Thoraces stained with anti-PH3 antibody in red. Yellow and white arrows indicate the wild-type mitotic and phyl2 mutant SOPs, respectively. (G-H') Thoraces labeled for CycB expression in red. Yellow and white arrows indicate the wild-type and mutant SOPs, respectively. All SOPs accumulate high levels of CycB. (I-J') Thoraces labeled for BrdU incorporation in red. BrdU signals are observed in the two sister cells resulting from the first division of the SOP (indicated by arrows), but are never detected in wild-type SOPs (I) and in phyl2/phyl4 hypomorphic mutant SOPs (J). (K,K') BrdU incorporation assay in phyl2 null mutant clones. SOP and its lineage cells were labeled by anti-Sens (green), and BrdU signals are shown in red. The broken lines mark the boundary of phyl2 mutant clone (GFP negative). Although both anti-Sens staining and GFP signals are shown in green, anti-Sens staining signals are stronger than GFP and are arranged in regular arrays. The uniform GFP signals are weaker due to HCl hydrolysis used in the BrdU labeling procedures (see Materials and methods). BrdU signals can be easily detected in neighboring wild-type SOP-lineage cells (indicated by yellow arrows), but not in phyl2 mutant SOPs (indicated by white arrows).

 

Figure 2
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  		Fig. 2. phyl mediates G2-M transition by positively regulating stg mRNA expression. (A,B) stg mRNA expression pattern at the AWM of wild-type (A) and phyl2/phyl4 (B) wing disks at 2-4 hours APF. Arrows indicate the stg-positive SOPs. In phyl mutants, stg expression is significantly reduced in AWM SOPs (B). (C-F) Ectopic expression of stg rescues the defects in G2-M transition. (C) Percentage of cell division of thoracic SOPs at 22-24 hours (column 1 and 2) and 24-26 hours (column 3-4) APF. SOP division defect is strongly rescued when stg was added back transiently to phyl mutant pupae by heat-shock treatment (lanes 2 and 4). *P<0.0005, n=4-9 thoraces. The numbers in the parenthesis are the number of SOP scored. (D-F) Images of pupal thoraces stained with anti-Sens antibody (green). (D) Wild-type. (E) phyl2/phyl4; hs-Gal4/+ pupa with two pulses of heat-shock treatment. (F) phyl2/phyl4; hs-Gal4 UAS-stg/+ pupa with two pulses of heat-shock treatment.

 

Figure 3
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  		Fig. 3. phyl negatively regulates Ac protein levels in SOPs. (A-F) Ac expression is shown in red and Sens expression is shown in green. (A-A'') In wild-type pupal thorax at 12-13 hours APF, Ac is expressed at high levels in newly specified SOPs (arrowheads). Ac levels are diminishing in more mature SOPs which express higher levels of Sens (arrows). (B-B'') Thoracic region of Eq-Gal4/UAS-myc-ac wing disk. Although Myc-Ac (red) is clearly detected in most non-SOP disk cells and in newly specified ectopic SOPs that express Sens at low levels (arrowheads), Myc-Ac levels are strongly downregulated in late-stage ectopic SOPs (arrows). (C) Endogenous Ac protein disappears normally from the ap-Gal4>UAS-lacZ control thorax at 14-16 hours APF after a 10-hour incubation at 29°C. (D) When proteasome activity in ap-Gal4>UAS-DTS5 pupal thorax was disrupted under the same incubation condition as C, highly elevated levels of Ac are detected in SOPs. (E-E'') Ac protein levels are maintained in phyl2-mutant SOPs (GFP-negative) at 13-14 hours APF whereas it has disappeared from the neighboring wild-type SOPs. (F,F') Ac protein levels are strongly downregulated within phyl-misexpression clones (GFP positive).

 

Figure 4
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  		Fig. 4. phyl downregulates Sc but not Da protein levels in SOPs. (A-E) Anti-Sc or anti-Da staining is shown in red, and SOPs were labeled by anti-Sens staining (green). (A-A'') Sc expression pattern in the wild-type pupal thorax at 12-13 hours APF. Sc levels are high in newly specified SOPs (arrowheads), but gradually diminishing in more mature SOPs (arrows). (B-B'') Thoracic region of Eq-Gal4/UAS-myc-sc wing disk. Although Myc-Sc (red) is clearly detected in most non-SOP disk cells, its protein levels are strongly downregulated in late-stage single SOPs (arrows). (C-C'') Sc protein is accumulated in the phyl2 mutant SOPs (GFP-negative) at 14-15 hours APF. (D-E'') Da protein levels in AWM SOPs. Da protein (red) is expressed in both newly specified SOPs (arrowheads) at late third instar (D-D'') and in mature SOPs (arrows) at 2-4 hours APF (E-E'').

 

Figure 5
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  		Fig. 5. sina is required for phyl-mediated Ac downregulation. (A-D) Anti-Ac staining is shown in red. (A-C) Ac protein levels at the AWM of the third-instar wing disks. The dorsal and ventral Ac clusters are indicated by d and v, respectively. (A,A') ap-Gal4>UAS-GFP wing disk. Ac levels in the dorsal GFP-expressing clusters are comparable to that in the ventral clusters. GFP expression is shown in blue. (B,B') ap-Gal4>UAS-flag-phyl wing disk. Ac levels in the dorsal clusters are strongly reduced by misexpression of Flag-tagged Phyl (blue). (C,C') sina2/sina3 wing disk expressing Flag-Phyl in the dorsal clusters. Misexpression of Flag-Phyl fails to downregulate Ac protein levels in sina2/sina3 mutants. (D-D'') In the sina3 mutant clone (GFP negative), Ac accumulates in the SOPs (D') and these SOPs remained as single cells at 16-18 hours APF (D'').

 

Figure 6
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  		Fig. 6. Proneural proteins directly interact with Phyl. (A-C) Western blots of immunoprecipitates (IPs) or lysates from S2 cells expressing the indicated proteins. Immunoprecipitation was performed using anti-Flag antibody. (A) Phyl and Ac proteins interact in S2 cells. Myc-Ac protein was co-immunoprecipitated by anti-Flag antibody only when Flag-Phyl was co-expressed. (B) Myc-Sc protein could only be co-immunoprecipitated by anti-Flag antibody when Flag-Phyl was co-expressed. (C) Phyl and Da do not interact in S2 cells. HA-Da was not co-immunoprecipitated with Flag-Phyl. *Non-specific bands. (D,E) GST pull-down assay. (D) GST-Ac and GST-Sc specifically pulled down in vitro translated S35-labelled Phyl and Sina proteins, but not the control Luciferase (Luc) protein. GST protein did not pull down Phyl and Sina. (E) GST-Sina does not interact with Da. As positive controls, GST-Sina pulled down Phyl and Ac. (F) Phyl acts as an adaptor between Ac and Sina in yeast bridge assay. Interaction was scored by the growth in the -His -Ade (plate II) and -His -Ade -Met (plate III) selective plates. All yeast cells grow in the non-selective plates (plate I). Expression of the bridge protein was induced in the absence of 1 mM methionine (-Met).

 

Figure 7
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  		Fig. 7. Mutations in ac and sc rescue SOP division defect of phyl mutants. (A-F) Pupal thoraces stained with anti-Sens (green) and anti-Hnt (red) antibodies to label all SOP daughter cells. (A) Wild-type pupal thorax at 24-26 hours APF. All SOPs have divided into three-to five-cell clusters. (B) In female phyl2/phyl4 pupal thorax at 24-26 hours APF, 27% microchaetal SOPs have divided into two cells and almost no microchaetal SOP has divided into a three-to five-cell cluster. Arrowheads indicate SOP daughter cells of macrochaete (large bristle). Macrochaetal SOPs were not counted in this assay due to different division timing (Huang, 1991). (C) Female sc10-1/+; phyl2/phyl4 pupal thorax. Arrows indicate SOPs divided into three-to four-cell clusters. (D) Male phyl2/phyl4 pupal thorax at 22-24 hours APF. 27.8% SOPs have divided. Arrowheads, SOP daughter cells of macrochaetes. (E,F) accami/Y; phyl2/phyl4 (E) and scM6/Y; phyl2/phyl4 (F) pupal thoraces at 22-24 hours APF. Arrows indicate SOPs divided into three- or four-cell clusters. Arrowheads in E, SOP daughter cells of macrochaetes. (G) Percentage of SOP division in thoraces at 22-24 hours APF. **P<0.0005, *P<0.01, n=7-22 thoraces. The numbers in parentheses are the number of SOP scored. Mutant pupae with fewer than 10 SOPs were not scored.

 

Figure 8
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  		Fig. 8. Summary of G2-M transition in wild-type and phyl mutant SOPs. In wild-type pupae at 12 hours APF, Ac and Sc (shown in red) are enriched in newly specified SOPs. High levels of Ac and Sc proteins then further activate transcription of phyl, which in turn mediates Ac and Sc degradation between 12 and 14 hours APF. Depletion of Ac and Sc from SOPs at 14 hours APF leads to stg expression and the subsequent G2-M transition. Five daughter cells are generated by 24 hours APF. In phyl mutant SOPs, accumulation of Ac and Sc represses stg expression, leading to defects in G2-M transition. Most of the SOPs in phyl2 clones remain undivided between 12 and 24 hours APF. *In phyl2 mutant clones, Ac expression between 20 and 24 hours APF, and Sc expression between 16 and 24 hours APF are not determined.

 

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