The metalloproteinase Papp-aa controls epithelial cell quiescence-proliferation transition
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, United States
- College of Ocean and Earth Sciences, Xiamen University, China
- Department of Molecular Biology and Genetics, Aarhus University, Denmark
Figures
Figure 1
Papp-aa is highly expressed in NaR cells.
(A) Tg(igfbp5a:GFP) fish were raised in E3 embryo medium to 3 days post fertilization (dpf) and transferred to embryo media containing the indicated [Ca2+]. Eighteen hours later, NaR cells were …
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Figure 1—source data 1
Excel spreadsheet containing quantitative data for Figure 1.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig1-data1-v3.xlsx
Figure 2 with 4 supplements
Genetic deletion of papp-aa impairs NaR cell reactivation and bone calcification.
(A) Diagram of the experimental design. Progeny of papp-aa+/- fish intercrosses were raised in standard E3 embryo medium to three dpf. The progeny is a mixture of homo, hetero, and wild type …
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Figure 2—source data 1
Excel spreadsheet containing quantitative data for Figure 2.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Genetic deletion of papp-aa impairs NaR cell proliferation, but has no effect on HR and NCC cells.
(A) Progeny of papp-aa+/- intercrosses were treated as described in Figure 2A. NaR cells were visualized by trpv6 mRNA mRNA in situ hybridization. Scale bar = 0.1 mm. (B–C) Progeny of papp-aa+/- …
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Figure 2—figure supplement 1—source data 1
Excel spreadsheet containing quantitative data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig2-figsupp1-data1-v3.xlsx
Figure 2—figure supplement 2
Genetic deletion of papp-ab has no effect on NaR cell proliferation.
The experimental design is the same as described in Figure 2A. NaR cells were labeled by igfbp5a mRNA expression and quantified followed by individual genotyping afterwards. n = 10 ~ 26 fish/group.
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Figure 2—figure supplement 2—source data 1
Excel spreadsheet containing quantitative data for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig2-figsupp2-data1-v3.xlsx
Figure 2—figure supplement 3
Global and local effects of genetic deletion of papp-aa.
(A) Gross morphology of wild-type (WT), papp-aa+/-, and papp-aa-/- fish at the indicated stages. Lateral views with anterior to the left. Scale bar = 0.5 mm. (B–E) Body length (B), somite number (C),…
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Figure 2—figure supplement 3—source data 1
Excel spreadsheet containing quantitative data for Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig2-figsupp3-data1-v3.xlsx
Figure 2—figure supplement 4
Genetic deletion of papp-aa impairs bone calcification.
(A–C) Fish of the indicated genotypes were raised in E3 embryo medium to 10 dpf (B) and 12 dpf (C) and stained with Alizarin Red. Representative images of 10 dpf are shown in (A). Scale bar = 0.5 …
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Figure 2—figure supplement 4—source data 1
Excel spreadsheet containing quantitative data for Figure 2—figure supplement 4.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig2-figsupp4-data1-v3.xlsx
Figure 3
Papp-aa proteinase activity is in NaR cells critical.
(A–B) Progeny of papp-aa+/-;Tg(igfbp5a:GFP) intercrosses were injected with BAC(igfbp5a:mCherry) containing the indicated gene. They were subjected to the low [Ca2+] stress test described in Figure …
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Figure 3—source data 1
Excel spreadsheet containing quantitative data for Figure 3.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig3-data1-v3.xlsx
Figure 4 with 1 supplement
Papp-aa is an Igfbp5 proteinase and Papp-aa-mediated Igfbp5 proteolysis in NaR cells is critical.
(A) Purified human IGFBP1, 2, 3, 4, 5, and six was incubated for 2 hr at 28°C with conditioned media collected from HEK293 cells transfected with zebrafish Papp-aa and from Mock transfected cells. …
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Figure 4—source data 1
Excel spreadsheet containing quantitative data for Figure 4.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Conservation of Papp-aa cleavage site in Igfbp5a and 5b.
(A) L-domain sequence of human IGFBP5, zebrafish Igfbp5a, and Igfbp5b. K128 in human IGFBP5, K147 or K148 in zebrafish Igfbp5a and K144 or K145 in Igfbp5b are indicated in red. (B) Igfbp5a and the …
Figure 5 with 1 supplement
Papp-aa acts by regulating IGF-Akt-Tor signaling in NaR cells.
(A–B) Zebrafish embryos of the indicated genotypes were transferred to the low [Ca2+] medium at three dpf. One day later, they were fixed and stained for phospho-Akt. Representative images are shown …
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Figure 5—source data 1
Excel spreadsheet containing quantitative data for Figure 5.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Papp-aa acts via Igfbp5a and IGF signaling in NaR cells.
(A) Loss of Papp-aa abolishes Ca2+ deficiency-induced Tor activity. Zebrafish of the indicated genotypes were transferred to normal or low [Ca2+] medium at three dpf. One day later, they were fixed …
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Figure 5—figure supplement 1—source data 1
Excel spreadsheet containing quantitative data for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig5-figsupp1-data1-v3.xlsx
Figure 6 with 2 supplements
Disruption the IGF/Igfbp complex activates IGF-Akt-Tor signaling and promotes NaR cell reactivation.
(A–B) Tg(igfbp5a:GFP) fish were transferred to normal [Ca2+] medium containing the indicated doses of NBI-31772 at three dpf. Two days later, NaR cells were quantified. Representative images are …
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Figure 6—source data 1
Excel spreadsheet containing quantitative data for Figure 6.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
Disruption the IGF/Igfbp complex has no effect on pErk activity and Mek/Erk signaling is not critical.
(A). Wild-type embryos were transferred to normal [Ca2+] medium containing 90 µM NBI-31772 with or without 0.3 µM BMS-754807. The fish were sampled at four dpf and subjected to immunostaining using …
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Figure 6—figure supplement 1—source data 1
Excel spreadsheet containing quantitative data for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig6-figsupp1-data1-v3.xlsx
Figure 6—figure supplement 2
Treatment with fish IGF1 increases NaR cell reactivation and proliferation.
Tg(igfbp5a:GFP) fish were treated with salmon IGF1 (150 ng/ml) in E3 embryo medium from 3 to 5 dpf. NaR cells were quantified and shown. n = 17 ~ 24 fish/group. **p<0.01, unpaired t-test.
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Figure 6—figure supplement 2—source data 1
Excel spreadsheet containing quantitative data for Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/52322/elife-52322-fig6-figsupp2-data1-v3.xlsx
Additional files
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Supplementary file 1
PCR primers used in this study.
- https://cdn.elifesciences.org/articles/52322/elife-52322-supp1-v3.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52322/elife-52322-transrepform-v3.docx
