Drosophila p53 isoforms have overlapping and distinct functions in germline genome integrity and oocyte quality control
- Department of Biology, Indiana University, United States
Figures
Figure 1 with 2 supplements
The p53B protein isoform is expressed in the germline where it colocalizes with p53A in nuclear bodies.
(A) Drosophila p53 mRNA and protein isoforms. Left: The four p53 mRNA isoforms with introns as lines, translated regions of exons as orange boxes, and 5’ and 3’ untranslated regions as black boxes. …
Figure 1—figure supplement 1
Rare mCh-p53B expression in somatic follicle cells.
(A, B) mCh-p53B in nuclear bodies in a small group of follicle cells in a mCh-p53B female (A), and colocalization with GFP-p53A in follicle cells in a mCh-p53B / GFP-p53A female (B). White arrows …
Figure 1—figure supplement 2
p53A and p53B are expressed in the male germline.
A testis expressing GFP-p53A (A, A’) or mCh-p53B (B, B’), with labeling with anti-Hts (germline fusomes) and DAPI (blue) shown in A’, B’. Scale bar is 20 μm.
Figure 2 with 1 supplement
p53A is necessary and sufficient for IR-induced apoptosis in the soma.
(A–B) The p53 isoform-specific mutants created at the endogenous p53 locus with CRISPR / Cas9. Each allele is a small deletion (red asterisk) in the unique 5’ coding exon of p53A (A) and p53B (B) …
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Figure 2—source data 1
Counts of TUNEL-positive follicle cells for Figure 2.
- https://cdn.elifesciences.org/articles/61389/elife-61389-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
p53 isoform-specific alleles.
(A) Left: Shown are the location of p53A2.3 and p53B41.5 deletion alleles and the coordinates of primers (red font) used for RT-PCR. Expanded maps below indicate the coordinates of the deletion …
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Figure 2—figure supplement 1—source data 1
Counts of TUNEL-positive follicle cells for Figure 2.
- https://cdn.elifesciences.org/articles/61389/elife-61389-fig2-figsupp1-data1-v2.zip
Figure 3
p53A and p53B are expressed in the early female germline, but only p53A is required for IR-induced germline apoptosis.
(A) Illustration of three regions of the germarium: germline stem cells (GSC), their primary daughter cystoblasts (CB), dividing cystocytes (CC), GSC spherical cytoskeletal spectrosome (S), branched …
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Figure 3—source data 1
Counts of Tunel-positive germline cells for Figure 3.
- https://cdn.elifesciences.org/articles/61389/elife-61389-fig3-data1-v2.xlsx
Figure 4 with 1 supplement
p53A is necessary and sufficient for IR-induced expression of proapoptotic genes in the germline.
(A-H’) Confocal micrographs of the expression of the p53 activity reporter, hid-GFP, in germaria of females with the indicated p53 allele genotypes (rows), without IR (left two columns) or with IR …
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Figure 4—source data 1
Quantification of hid-GFP intensity for Figure 4.
- https://cdn.elifesciences.org/articles/61389/elife-61389-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
hid-GFP expression in region 2a responds to meiotic DNA breaks.
Hid-GFP expression in wild type (A) and mei-W681/ Df(2R)BSC782 mutants (B). Scale bars are 25 μm.
Figure 5 with 1 supplement
p53B protein levels fluctuate in p53 bodies during early meiosis.
(A-C’) Micrographs p53 bodies in germaria from GFP-p53A (A-A’, green) and mCh-p53B (B-B’, red), and GFP-p53A / mCh-p53B females (C-C’). (D–F) Representative quantification of GFP-p53A and mCh-p53B …
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Figure 5—source data 1
Quantification of GFP-p53A and mCh-p53B intensity in nuclear bodies for Figure 5.
- https://cdn.elifesciences.org/articles/61389/elife-61389-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
mCh-p53B levels fluctuate in p53 bodies during early meiosis.
(A, A’) A germarium from a GFP-p53A / mCh-p53B female showing localization of p53A and p53B proteins to p53 nuclear bodies in germarium regions 1, 2, and 3, with DAPI labeling of DNA (blue) shown in …
Figure 6 with 3 supplements
p53A and p53B mutants have persistent germline DNA damage.
(A-D’) Images of ovarioles of indicated genotypes immunolabeled with anti-γ-H2Av (red) (A–D) to detect DNA breaks, and counterstained with the DNA dye DAPI (blue) (A’-D’). The ovarioles are oriented …
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Figure 6—source data 1
Quantification of persistent DNA breaks by stage for Figure 6.
- https://cdn.elifesciences.org/articles/61389/elife-61389-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Double labeling for oocyte marker Orb and γ-H2Av in stage 1.
(A-F’) Images of germarium and stage one labeled for γ-H2Av (red) and oocyte marker Orb (green) (A–F), and with DAPI DNA stain (blue) (A’-F’). (G) Quantification of percent stage one with γ-H2Av and …
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Figure 6—figure supplement 1—source data 1
Quantification of DNA breaks in orb-lableled ovaries of Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/61389/elife-61389-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
Quantification of γ-H2Av fluorescence intensity in oocytes (red) and nurse cells (blue) of stage one egg chambers.
Each dot represents a single oocyte or nurse cell. Horizontal lines represent mean and standard deviation. Asterisks represent adjusted p values of a two-way, pair-wise ANOVA computed through a …
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Figure 6—figure supplement 2—source data 1
Quantification of DNA breaks per cell for Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/61389/elife-61389-fig6-figsupp2-data1-v2.xlsx
Figure 6—figure supplement 3
DNA repair foci in p53 mutants depends on DNA meiotic breaks.
γ-H2Av labeling of wild type (A), mei-W681/ Df(2R)BSC782; p53A2.3 (B), and mei-681/Df(2R)BSC782; p53B41.5 (C).
Figure 7 with 3 supplements
p53A and p53B have overlapping and distinct functions in germline genome integrity and the meiotic pachytene checkpoint.
(A-E’) Drosophila ovarioles of indicated genotypes were immunolabeled for γ-H2Av (red A-E) to detect DNA breaks and counterstained with the DNA dye DAPI (blue A’-E’). The ovarioles are shown with …
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Figure 7—source data 1
Quantification of persistent DNA breaks by stage for Figure 7.
- https://cdn.elifesciences.org/articles/61389/elife-61389-fig7-data1-v2.xlsx
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Figure 7—source data 2
Quantification of karysome phenotype for Figure 7K.
- https://cdn.elifesciences.org/articles/61389/elife-61389-fig7-data2-v2.xlsx
Figure 7—figure supplement 1
Images of eggshell phenotype classes produced by p53 and okr single or double mutant mothers.
p53A+B+ = wild type. okr; p53A+B+ = okra single mutant. Class I- wild type; Class II: ventralized; Class III: short or missing dorsal appendages. Class IV: Small collapsed eggs with thin eggshells. …
Figure 7—figure supplement 2
Quantification of eggshell phenotype classes produced by p53 and okr single or double mutant mothers.
p53A+B+ = wild type. okr; p53A+B+ = okra single mutant. Class I- wild type; Class II: ventralized; Class III: short or missing dorsal appendages. Class IV: Small collapsed eggs with thin eggshells.
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Figure 7—figure supplement 2—source data 1
Quantification of egg phenotypes for Figure 7—figure supplement 2.
- https://cdn.elifesciences.org/articles/61389/elife-61389-fig7-figsupp2-data1-v2.xlsx
Figure 7—figure supplement 3
p53 mutant mothers have reduced fertility.
Hatch rates of eggs from p53 and okr single or double mutant mothers. The hatch rate for all okr single and double mutants was zero (complete female sterility). p53A+B+ = wild type. okr; p53A+B+ = ok…
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Figure 7—figure supplement 3—source data 1
Quantification of hatch rates for Figure 7—figure supplement 3.
- https://cdn.elifesciences.org/articles/61389/elife-61389-fig7-figsupp3-data1-v2.xlsx
Figure 8
Model: Drosophila p53 isoforms colocalize to nuclear bodies and have DNA lesion and cell type specific functions in the germline genotoxic stress response.
(A) The p53A (green) and p53B (red) isoforms are concentrated in p53 bodies of germline nuclei (blue). Trafficking of p53 isoforms through these bodies (double arrow) may mediate their functions in …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | w67c23 | Bloomington Drosophila Stock Center | FBal0095147 | |
| Gene (Drosophila melanogaster) | w1118 | Bloomington Drosophila Stock Center | FBal0018186RRID:BDSC_6598 | |
| Gene (Drosophila melanogaster) | p53 (p535A1-4) | Bloomington Drosophila Stock Center | FLYB:FBgn0039044; RRID:BDSC_6815 | FBal0138188 |
| Gene (Drosophila melanogaster) | p53 (p53A2.3) | Robin et al., 2019 | FLYB:FBgn0039044 | See Materials and Methods, Section 2 |
| Gene (Drosophila melanogaster) | p53 (p53B41.5) | this study | FLYB:FBgn0039044 | See Materials and Methods, Section 2 |
| Gene (Drosophila melanogaster) | okra (okraAA) | Ghabrial et al., 1998 | FLYB:FBgn0002989 | Obtained from T. Schupbach |
| Gene (Drosophila melanogaster) | okra (okraRU) | Bloomington Drosophila Stock Center | FLYB:FBgn0002989; RRID:BDSC_5098 | FBal0013236;Obtained from T. Schupbach |
| Gene (Drosophila melanogaster) | mei-W68 (Df(2 R)BSC782) | Bloomington Drosophila Stock Center | FLYB:FBgn0002716; RRID:BDSC_27354 | |
| Gene (Drosophila melanogaster) | mei-W68 (mei-W681) | Bloomington Drosophila Stock Center | FLYB:FBgn0002716; RRID:BDSC_4932 | FBal0012191 |
| Genetic reagent (Drosophila melanogaster) | hid-GFP | Tanaka-Matakatsu et al., 2009 | FLYB:FBgn0003997; RRID:BDSC_50751 | Obtained from W. Du |
| Genetic reagent (Drosophila melanogaster) | GFP-p53A | Zhang et al., 2015 | FLYB:FBtp0111619 | |
| Genetic reagent (Drosophila melanogaster) | mCh-p53B | Zhang et al., 2015 | FLYB:FBtp0098077 | |
| Sequence-based reagent | p53 gRNA | This study | 5’:CCTGGAGCA CGGAAGATTCTTG; 3’:GATCCACAG GCGTAGCCAGGTGG | |
| Sequence-based reagent | primer #501 | This study | PCR primer | CCAACAAGAT CGCTTGATCAGATA |
| Sequence-based reagent | primer #1,085 | This study | PCR primer | GGCCATGGG TTCCGTGGTCA |
| Sequence-based reagent | primer #1,061 | This study | PCR primer | GAGTCAGCAG TTCGGGTCTC |
| Antibody | Anti-GFP (Rabbit polyclonal) | Invitrogen | Cat# A11122 | IF(1:500) |
| Antibody | Anti-dsRed (Rabbit polyclonal) | Clontech | Cat# 632,496 | IF(1:200) |
| Antibody | Anti-dsRed (mouse polyclonal) | Clontech | Cat# 632,392 | IF(1:200) |
| Antibody | Anti-Hts 1B1 (mouse monoclonal) | Developmental Studies Hybridoma Bank | RRID:AB_528070 | IF(1:20) |
| Antibody | Anti-γH2Av (mouse monoclonal) | Developmental Studies Hybridoma Bank | RRID:AB_2618077 | IF(1:1000) |
| Antibody | Anti-orb 4H8 (mouse monoclonal) | Developmental Studies Hybridoma Bank | RRID:AB_528418 | IF(1:500) |
| Antibody | Anti-Vasa (rat monoclonal) | Developmental Studies Hybridoma Bank | RRID:AB_10571464 | IF(1:100) |
| Antibody | Alexa Fluor 488 anti-mouse (polyclonal) | Invitrogen | Cat# A11011 | IF(1:1000) |
| Antibody | Alexa Fluor 488 anti-rabbit (polyclonal) | Invitrogen | Cat# A11008 | IF(1:1000) |
| Antibody | Alexa Fluor 568 anti-mouse (polyclonal) | Invitrogen | Cat# A11004 | IF(1:1000) |
| Antibody | Alexa Fluor 568 anti-rabbit (polyclonal) | Invitrogen | Cat# A10042 | IF(1:1000) |
| Antibody | Alexa Fluor 488 anti-mouse IgG1 (polyclonal) | Invitrogen | Cat# A21121 | IF(1:1000) |
| Antibody | Alexa Fluor 568 anti-mouse IgG2b (polyclonal) | Invitrogen | Cat# A21144 | IF(1:1000) |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/61389/elife-61389-transrepform1-v2.docx
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Supplementary file 1
p values for frequency of nurse cells and oocytes with DNA breaks for Figures 6 and 7.
- https://cdn.elifesciences.org/articles/61389/elife-61389-supp1-v2.docx
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Supplementary file 2
ANOVA p value comparisons among genotypes for mean H2AV intensity in stage 1 oocytes and nurse cells for Figures 6 and 7.
- https://cdn.elifesciences.org/articles/61389/elife-61389-supp2-v2.docx
