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Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts

  1. Amrutha Kizhedathu
  2. Rose Sebastian Kunnappallil
  3. Archit V Bagul
  4. Puja Verma
  5. Arjun Guha  Is a corresponding author
  1. Institute for Stem Cell Science and Regenerative Medicine (inStem), GKVK Campus, India
  2. SASTRA University, India
Research Advance
Cite this article as: eLife 2020;9:e57056 doi: 10.7554/eLife.57056
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
Wnt signaling is required for G2 arrest in Tr2 tracheoblasts.

(A) Cartoon representing the cell cycle phasing of cells in wild type Tr2 DT at different larval stages based on FUCCI. (B-C) Impact of knockdown of Chk1 and components of the Wnt signaling pathway on cell numbers in Tr2 DT. (B) Graph shows cells numbers in wild type (btl-GAL4), Btl-Chk1RNAi (btl-GAL4/UAS-Chk1RNAi), and Btl-Wnt pathway components RNAi (btl-GAL4/+; UAS-TCFRNAi/+, btl-GAL4/UAS-DshRNAi, btl-GAL4/UAS-Fz2RNAi) at L2 and 16–24 h L3. (C) Graph shows cell numbers in wild type (tub-GAL80ts/+; UAS-ArmRNAi/Tb) and Armadillo mutants (tub-GAL80ts/+; btl-GAL4/UAS-ArmRNAi) at 24–26 h L3. Larvae were grown at 18°C and transferred to 29°C at 0–2 hr into L3. (D-E) Impact of reduction in levels of Chk1 or in levels of different components of the Wnt signaling pathway on mitotic indices in Tr2 DT. (D) Graph shows frequency of pH3+ nuclei at 16–24 h L3 in wild type (btl-GAL4), Chk1 mutant (btl-GAL4/UAS-Chk1RNAi), and Wnt pathway mutants (btl-GAL4/+; UAS-TCFRNAi/+, btl-GAL4/UAS-DshRNAi, btl-GAL4/UAS-Fz2RNAi) (E) Graph shows the frequency of pH3+ nuclei in wild type (tub-GAL80ts/+; UAS-ArmRNAi/Tb) and Armadillo mutants (tub-GAL80ts/+; btl-GAL4/UAS-ArmRNAi) at 24–26 h L3. Larvae were grown as stated above. (F) Expression pattern of Wnt reporters Fz3-GFP (fz3-GFP) and Nkd- LacZ (nkd-lacZ) in Tr2 DT at different larval stages. Panels show immunostaining for GFP in Fz3-GFP (fz3-GFP) (top panel) and β-Gal in Nkd- LacZ (nkd-lacZ) (Bottom panel) and their respective secondary controls (G) Quantitative PCR analysis of Fz3 mRNA levels in micro-dissected Tr2 DT fragments at different stages. Graph shows fold change in mRNA levels with respect to L2 (n = 3 experiments, n ≥ 15 Tr2 DT fragments/condition/experiment, mean ± standard deviation). DT = Dorsal Trunk, DB = Dorsal Branch, TC = Transverse Connective. Scale bar = 20 µm (mean ± standard deviation, n ≥ 7 tracheae) Student's paired t-test: *p<0.05.

Figure 1—source data 1

Figure 1B Numerical data for number of cells in Tr2 DT of wild type, Btl-Chk1RNAi and Btl-Wnt pathway RNAi larvae at different stages.

Figure 1C Numerical data for number of cells in Tr2 DT of wild type and Btl-ArmRNAi larvae at 24–26 h L3.

https://cdn.elifesciences.org/articles/57056/elife-57056-fig1-data1-v4.xlsx
Figure 1—figure supplement 1
List of developmental signaling pathway knockdowns not showing proliferation at 16–24 h L3.

(A) Impact of reduction of components of various developmental signaling pathways on cell numbers in Tr2 DT. Table shows cells numbers in wild type (btl-GAL4), Btl-hopRNAi (btl-GAL4/UAS-hopRNAi), Btl-CiRNAi (btl-GAL4/UAS-CiRNAi), Btl-pi3kRNAi (btl-GAL4/UAS-pi3kRNAi), Btl-BtlDN (btl-GAL4/+, UAS-BtlDN/+), Btl-TkvRNAi (btl-GAL4/UAS-TkvRNAi), Btl-YkiRNAi (btl-GAL4/+; UAS-YkiRNAi/+), Btl-BskRNAi (btl-GAL4/+; UAS-BskRNAi/+), Btl-EcRRNAi (btl-GAL4/+; UAS-EcRRNAi/+) and Btl-EGFRRNAi (btl-GAL4/+; UAS-EGFRRNAi/+) at 16–24 h L3 (mean values ± standard deviation, n ≥ 7 tracheae).

Figure 1—figure supplement 2
Loss of Chk1 or Wnt signaling leads to continuous proliferation of tracheoblasts through L3.

(A) Impact of knockdown of Chk1 and TCF on cell numbers in Tr2 DT. Graph shows cells numbers in wild type (btl-GAL4), Btl-Chk1RNAi (btl-GAL4/UAS-Chk1RNAi), and Btl-TCFRNAi (btl-GAL4/+; UAS-TCFRNAi/+) at L2, 0–8 h L3, 16–24 h L3 and 32–40 h L3. (L2 and 16–24 h L3 data same as Figure 1B, mean values ± standard deviation, n ≥ 7 tracheae) Student's paired t-test: *p<0.05.

Phosphorylated Chk1 levels are diminished in the absence of Wnt signaling.

(A) Activated Chk1 (phospho-Chk1Ser345, pChk1) immunostaining (white) in Tr2 DT in Chk1 mutants and Wnt pathway mutants. pChk1 immunostaining in Tr2 DT in (i) wild type (btl-GAL4), (ii) Btl-Chk1RNAi (btl-GAL4/UAS-Chk1RNAi) (iii) Btl-TCFRNAi (btl-GAL4/+; UAS-TCFRNAi/+) at L2 and (iv) wild type treated with secondary antibody alone. Scale bar = 20 µm.

Wnt signaling regulates Chk1 transcription.

(A) Quantitative PCR analysis of Chk1 mRNA levels in micro-dissected Tr2 DT fragments at different stages. Graph shows fold change in Chk1 mRNA in Tr2 DT fragments from wild type (btl-GAL4), Wnt pathway loss-of-function (btl-GAL4/+; UAS-TCFRNAi/+) and Wnt pathway gain-of-function (UAS-ArmS10/+; btl-GAL4/+) larvae. Fold change has been represented with respect to L2 (n = 3 experiments, n ≥ 15 Tr2 DT fragments/condition/experiment, mean ± standard deviation). (B) Analysis of Chk1-lacZ expression in Tr2 DT in wild type and Wnt pathway mutants. β-Gal immunostaining in larvae expressing Chk1-lacZ at L2, 0–8 h L3 and 32–40 h L3 and in Btl-TCFRNAi (btl-GAL4/Chk1 lacZ; UAS-TCFRNAi/+) at L2. (C) Impact of overexpression of Chk1 and ATR in TCF mutants. Graph shows cell numbers in wild type (btl-GAL4, UAS-FUCCI/Cyo GFP; +/Tb), Btl-TCFRNAi (btl-GAL4, UAS-FUCCI/+; UAS-TCFRNAi/+), Btl-TCFRNAi, Chk1 (btl-GAL4, UAS-FUCCI/+; UAS-TCFRNAi/UAS-Chk1) and Btl-TCFRNAi, ATR (btl-GAL4, UAS-FUCCI/+; UAS-TCFRNAi/UAS ATR) at 16–24 h L3. (D) Impact of reduction of TCF and overexpression of Chk1 on levels of pChk1 in Tr2 DT. pChk1 immunostaining (white) in Btl-TCFRNAi, UAS-Chk1 (btl-GAL4, UAS-FUCCI/+; UAS-TCFRNAi/UAS-Chk1) animals at L2 (This image has been acquired at a lower laser power and gain setting compared to Figure 2A to prevent saturation of white pixels). (E) Impact of reduction of TCF and overexpression of ATR on levels of pChk1 in Tr2 DT. pChk1 immunostaining (white) in Btl-TCFRNAi, UAS-ATR (btl-GAL4, UAS-FUCCI/+; UAS-TCFRNAi/UAS-ATR) animals at L2 (mean values ± standard deviation, n ≥ 7 tracheae) Scale bar = 20 µm. Student's paired t-test: *p<0.05.

Figure 3—source data 1

Figure 3C Numerical data for number of cells in Tr2 DT of wild type, Btl-TCFRNAi, Btl-TCFRNAi, Chk1 and Btl-TCFRNAi, ATR expressing larvae at 16–24 h L3.

https://cdn.elifesciences.org/articles/57056/elife-57056-fig3-data1-v4.xlsx
Figure 4 with 2 supplements
Wg, Wnt5, Wnt6 and Wnt10 are required to maintain Chk1 expression.

(A) Quantitative PCR analysis for levels of Wg, Wnt5, Wnt6 and Wnt10 mRNA in micro-dissected Tr2 DT fragments at different stages. Graph shows fold change in Wg, Wnt5, Wnt6 and Wnt10 mRNA in Tr2 DT fragments from wild type (btl-GAL4) larvae at L2, 0–8 h L3 and 32–40 h L3. Fold change has been represented with respect to L2 (n = 3 experiments, n ≥ 15 Tr2 DT fragments/condition/experiment, mean ± standard deviation). (B) smFISH for Wg, Wnt5, Wnt6 and Wnt10 mRNA in Tr2 DT at L2 and 32–40 h L3. Arrowheads point to the sites of mRNA accumulation. (Scale bar = 5 µm) (C) Impact of knockdown of components of the Wnt signaling pathway on cell numbers in Tr2 DT. Graph shows cells numbers in wild type (btl-GAL4, Same as Figure 1B) and Btl-Wnt pathway componentsRNAi (btl-GAL4/+; UAS-WgRNAi/+, btl-GAL4/+; UAS-Wnt5RNAi/+ btl-GAL4/+; UAS-Wnt6RNAi/+, btl-GAL4/+; UAS-Wnt10RNAi/+) at L2 and 16–24 h L3. (D) Impact of reduction in levels of different components of the Wnt signaling pathway on mitotic indices in Tr2 DT. Graph shows frequency of pH3+ nuclei at 16–24 h L3 in wild type (btl-GAL4, Same as Figure 1D) and Wnt pathway mutants (btl-GAL4/+; UAS-WgRNAi/+, btl-GAL4/+; UAS-Wnt5RNAi/+, btl-GAL4/+; UAS-Wnt6RNAi/+, btl-GAL4/+; UAS-Wnt10RNAi/+) (E) Impact of expression of mutant Wg, Wnt5 and Wnt6 on cell numbers in Tr2 DT. Graphs show cell numbers in wild type and Wg mutants (Wgts) at 24–26 h L3, (Larvae were grown at 18°C and transferred to 29°C at 0–2 hr into L3) Wnt5 mutant (Wnt5[400] FRT19A) and Wnt6 (Wnt6 KO) on cell numbers in Tr2 DT. (F) Activated Chk1 (phospho-Chk1Ser345, pChk1) immunostaining (white) in Tr2 DT in Wnt pathway mutants. pChk1 immunostaining in Tr2 DT in (i) Btl-WgRNAi(btl-GAL4/+; UAS-WgRNAi/+), (ii) Btl-Wnt5RNAi(btl-GAL4/+; UAS-Wnt5RNAi/+), (iii) Btl-Wnt6RNAi(btl-GAL4/+; UAS-Wnt6RNAi/+) and Btl-Wnt10RNAi(btl-GAL4/+; UAS-Wnt10RNAi/+) at L2. (G) Quantitative PCR analysis of Chk1 and Fz3 mRNA levels in micro-dissected Tr2 DT fragments at L2. Graphs show fold change in Chk1 and Fz3 mRNA in Wild type, Btl-TCFRNAi(btl-GAL4/+; UAS-TCFRNAi/+), Btl-WgRNAi (btl-GAL4/+; UAS-WgRNAi/+), Btl-Wnt5RNAi(btl-GAL4/+; UAS-Wnt5RNAi/+), Btl-Wnt6RNAi(btl-GAL4/+; UAS-Wnt6RNAi/+) and Btl-Wnt10RNAi(btl-GAL4/+; UAS-Wnt10RNAi/+) in Tr2 DT fragments. Fold change has been represented with respect to Wild type (n = 3 experiments, n ≥ 15 Tr2 DT fragments/condition/experiment, mean ± standard deviation).(mean values ± standard deviation, n ≥ 7 tracheae) Scale bar = 20 µm. Student's paired t-test: *p<0.05.

Figure 4—source data 1

Figure 4C Numerical data for number of cells in Tr2 DT of wild type, Btl-WgRNAi, Btl-Wnt5RNAi, Btl-Wnt6RNAi and Btl-Wnt10RNAi larvae at different stages.

Figure 4E Numerical data for number of cells in Tr2 DT of wild type, Wgts, Wnt5[400] and Wnt6[KO] larvae at different stages.

https://cdn.elifesciences.org/articles/57056/elife-57056-fig4-data1-v4.xlsx
Figure 4—figure supplement 1
Wnt ligands do not affect each others expression.

(A) Impact of knockdown of Wnt10 on cell numbers in Tr2 DT. Graph shows cells numbers in wild type (btl-GAL4, Same as Figure 1B) and Btl-Wnt10RNAi(btl-GAL4/UAS-Wnt10RNAi) at L2 and 16–24 h L3 (mean values ± standard deviation, n ≥ 7 tracheae). (B) smFISH for Wnt5 mRNA in Wnt5 mutant (Wnt5[400] FRT19A) Tr2 DT at L2. (Scale bar = 5 µm). (C) smFISH for GAPDH mRNA in Wild type (btl-GAL4) Tr2 DT at 32–40 h L3. Arrowheads point to the sites of mRNA accumulation. (Scale bar = 5 µm). (D) Quantitative PCR analysis of Wg, Wnt5, Wnt6 and Wnt10 mRNA levels in micro-dissected Tr2 DT fragments at L2. Graph shows fold change in Wg, Wnt5, Wnt6 and Wnt10 mRNA in Wild type (btl-GAL4), Btl-WgRNAi (btl-GAL4/+; UAS-WgRNAi/+), Btl-Wnt5RNAi(btl-GAL4/+; UAS-Wnt5RNAi/+), Btl-Wnt6RNAi(btl-GAL4/+; UAS-Wnt6RNAi/+) and Btl-Wnt10RNAi(btl-GAL4/+; UAS-Wnt10RNAi/+) in Tr2 DT fragments. Fold change has been represented with respect to Wild type (n = 3 experiments, n ≥ 15 Tr2 DT fragments/condition/experiment, mean ± standard deviation).

Figure 4—figure supplement 2
Loss of Wnt2, Wnt4 or WntD does not lead to precocious proliferation in Tr2 DT.

(A) Impact of reduction of Wnt2, Wnt4 and WntD on cell numbers in Tr2 DT. Table shows cells numbers in wild type (btl-GAL4), Btl-Wnt2RNAi (btl-GAL4/+; UAS-Wnt2RNAi/+), Btl-Wnt4RNAi (btl-GAL4/+; UAS-Wnt4RNAi/+) and Btl-WntDRNAi (btl-GAL4/+; UAS-WntDRNAi/+) at 16–24 h L3 (mean values ± standard deviation, n ≥ 7 tracheae).

Exit from G2 arrest is required for activation of Dpp signaling.

(A) phospho-Mad (pMad) immunostaining in wild type Tr2 DT at L2, 0–8 h L3, 16–24 h L3 and 32–40 h L3. (B) Impact of reduction of Chk1 or Wnt pathway components on pMad expression in Tr2 DT. pMad immunostaining in Tr2 DT from Btl-Chk1RNAi (btl-GAL4/UAS-Chk1RNAi) and Btl-Wnt6RNAi (btl-GAL4/+; UAS-Wnt6RNAi/+) animals at 16–24 h L3. (C) Impact of overexpression of activated form of Tkv (Btl-TkvQD) on pMad expression in Tr2 DT. pMad immunostaining in Tr2 DT from wild type and Btl-TkvQD (btl-GAL4/+; UAS- TkvQD /+) animals at L2. Scale bar = 20 µm.

Loss of Stg, Cdc2 or Cyclin B perturbs hypertrophic growth of Tr2 DT.

(A) Impact of reduction of Chk1 or different drivers of G2-M on the size of Tr2 DT at 32–40 h L3. Scatter plot shows length and width of Tr2 DT from Wild type, Btl-Chk1RNAi (btl-GAL4/UAS-Chk1RNAi), Btl-StgRNAi (btl-GAL4/+; UAS-StgRNAi/+), Btl-Cdc2RNAi(btl-GAL4/UAS-Cdc2RNAi) and Btl-CyclinBRNAi (btl-GAL4/+; UAS-CyclinBRNAi/+) at 32–40 h L3. Values shown in red are from data previously published in Kizhedathu et al., 2018. (n ≥ 6 tracheae). (B) Model for the regulation of G2 arrest by Wnt signaling. Wnt signaling negatively regulates G2-M by transcriptionally upregulating Chk1. Arrest in G2 negatively regulates Dpp signaling, preventing precocious proliferation and allowing for hypertrophic growth of Tr2 DT.

Author response image 1

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (Drosophila melanogaster)btl-GAL4Shiga et al., 1996FLYB: FBtp0001208This line was a gift from Dr.Shigeo Hayashi
Genetic reagent (Drosophila melanogaster)UAS-Chk1RNAiVDRC110076
Genetic reagent (Drosophila melanogaster)UAS-TCFRNAiVDRC3014
Genetic reagent (Drosophila melanogaster)UAS-Chk1Kizhedathu et al., 2018 and
This paper
FLYB: FBgn0261278Developed using clone from DGRC: UFO05423
AntibodyPhospho-Chk1 (Ser345)CST2348Rabbit monoclonal antibody (1:200)
Commercial assay or kitTyramide signal amplification systemThermo Fisher ScientificB40922

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