Liu et al., 2020 - The metalloproteinase Papp-aa controls epithelial cell quiescence-proliferation transition. eLIFE   9 Full text @ Elife

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 isolated by FACS. The levels of papp-aa, papp-ab, and papp-a2 mRNA were measured and shown. Data shown are Mean ± SEM, n = 4. (B–C) NaR cells and other cells in four dpf Tg(igfbp5a:GFP) larvae were separated by FACS. The levels of papp-aa (B) and papp-ab (C) mRNA were measured and shown. *, p<0.05 by unpaired two-tailed t test. n = 3. (D) Whole mount in situ hybridization analysis of papp-aa mRNA in three dpf larvae. HB, hindbrain. pLL, posterior lateral line ganglion. Arrowheads indicate papp-aa mRNA signal in the yolk sac region. A sense cRNA probe was used as a negative control. Scale bar = 0.2 mm. (E) Tg(igfbp5a:GFP) fish of the indicated stages were analyzed by double label staining. Scale bar = 20 µm.

Excel spreadsheet containing quantitative data for <xref rid='fig1' ref-type='fig'>Figure 1</xref>.

Figure 2

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 embryos. They were transferred to the low [Ca2+] (0.001 mM) or normal [Ca2+] (0.2 mM) embryo medium at three dpf. Two days later, NaR cells in each fish were detected by igfbp5a mRNA expression and quantified. These fish were genotyped individually afterwards. (B–C) Progeny of papp-aa+/- intercrosses were treated as described in (A). Representative images are shown in (B) and quantified data in (C). Scale bar = 0.1 mm. n = 10 ~ 30 fish/group. In this and all subsequent figures, data shown are Mean ± SEM. Different letters indicate significant differences among groups by one-way ANOVA followed by Tukey’s multiple comparison test (p<0.05). (D–E) Progeny of papp-aa+/-;Tg(igfbp5a:GFP) fish intercrosses were raised in E3 medium to three dpf and transferred to the low [Ca2+] (0.001 mM) or normal [Ca2+] (0.2 mM) embryo medium. Two days later, the number of GFP-expressing NaR cells in each larva was quantified. The larvae were genotyped individually subsequently. Representative images are shown in (D) and quantified data in (E). Scale bar = 0.1 mm. n = 16 ~ 82 fish/group. (F–G) Fish of the indicated genotypes were raised in E3 embryo medium to 7 dpf and 12 dpf and stained with Alizarin Red. Scale bar = 0.2 mm.



Figure 2-S1 Genetic deletion of <italic>papp-aa</italic> 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+/- intercrosses were treated as described in (A). NCC cells (B) and HR cells (C) were labeled by slc12a10.2 mRNA and atp6v1al mRNA in situ hybridization and quantified. Each larva was genotyped afterwards. n = 7 ~ 31 fish/group.

Excel spreadsheet containing quantitative data for <xref rid='fig2s1' ref-type='fig'>Figure 2—figure supplement 1</xref>.

Figure 2-S3 Global and local effects of genetic deletion of <italic>papp-aa.</italic>

(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), and head-trunk angle (D) were measured at 24 hpf and head-trunk angle (E) at three dpf. Data shown are means ± SEM. n = 6 ~ 24 fish/group. No statistical significance was found using one-way ANOVA followed by Tukey’s multiple comparison test. (F) Fish of the indicated genotypes were raised in E3 medium to three dpf. NaR cells were imaged before and 8 hr after the low [Ca2+] treatment. The apical opening was marked by dotted lines. Scale bar = 10 µm.

Excel spreadsheet containing quantitative data for <xref rid='fig2s3' ref-type='fig'>Figure 2—figure supplement 3</xref>.

Figure 2-S4 Genetic deletion of <italic>papp-aa</italic> 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 mm. The number of calcified vertebral columns among the first ten columns were quantified and shown. n = 6 ~ 26 fish/group.

Excel spreadsheet containing quantitative data for <xref rid='fig2s4' ref-type='fig'>Figure 2—figure supplement 4</xref>.

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 2A. Papp-aa-IRES-mCherry, Papp-aa E501A-IRES-mCherry, or mCherry expressing NaR cells were detected by GFP and mCherry expression. NaR cells expressing mCherry or Papp-aa-mCherry (yellow, double labeled by GFP and mCherry) were scored following a published scoring system (Liu et al., 2018). Representative images are shown in (A) and quantified data in (B). Scale bar = 50 µm. +, one cell division, -, no division. ****, p<0.0001 by Chi-square test. The total cell number is shown above the bar. (C) Conditioned media collected from HEK293 cells co-transfected with Igfbp5a and the indicated plasmid were analyzed by western blotting. Intact and cleaved Igfbp5a bands were indicated. (D–E) Tg(igfbp5a:GFP) fish were transferred to the low [Ca2+] medium containing 0–8 µM ZnCl2 (D) or 200 µM Batimastat at three dpf (E). After two days of treatment, NaR cells were quantified and shown. n = 18 ~ 25 fish/group. **, p<0.001 by unpaired two-tailed t test. (F–H) Tg(igfbp5a:GFP) embryos were injected with BAC(igfbp5a:mCherry, BAC(igfbp5a:hSTC1-IRES-mCherry) (G) or BAC(igfbp5a:hSTC2-IRES-mCherry) (H). They were raised and subjected to the low [Ca2+] stress test described in Figure 2A. NaR cells expressing mCherry or human STC (yellow, double labeled by GFP and mCherry) were scored following a published scoring system (Liu et al., 2018). Representative images are shown in (F) and quantified results in (G and H). ++, two cell division, +, one cell division, -, no division during the experiment. ****, p<0.0001, Chi-square test. Total cell number is shown above the bar.

Excel spreadsheet containing quantitative data for <xref rid='fig3' ref-type='fig'>Figure 3</xref>.

Figure 5

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 in (A) and quantitative results in (B). Scale bar = 0.1 mm. n = 25 ~ 58 fish/group. (C–D) Tg(igfbp5a:GFP) fish were transferred to the low [Ca2+] medium 0–8 µM ZnCl2 (C) or 200 µM batimastat (D) at three dpf. After one day treatment, they were analyzed by immunostaining for phospho-Akt. n = 18 ~ 24 fish/group. ****, p<0.0001, unpaired two-tailed t test. (E and F) Progeny of papp-aa+/-;Tg(igfbp5a:GFP) intercrosses were injected with BAC(igfbp5a:mCherry) or BAC(igfbp5a:myr-Akt-mCherry). They were raised and subjected to the low [Ca2+] stress test described in Figure 2A. NaR cells expressing mCherry or myr-Akt were scored as described in Figure 3. Representative images are shown in (E) and quantified data in (F). ++, two cell division, +, one cell division, -, no division during the experiment. Scale bar = 20 µm. ****, p<0.0001 by Chi-square test. Total number of cells is shown above the bar.

Figure 6

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 shown in (A) and quantified data in (B). Scale bar = 0.2 mm. n = 25 ~ 27 fish/group. (C) Wild-type fish were treated with 90 µM NBI-31772 with or without 0.3 µM BMS-754807 from 3 to 4 dpf. The number of cells positive for phosphorylated Akt staining were quantified and shown. n = 19 ~ 23 fish/group. (D) Larvae treated as described in (C) were stained for phosph-S6 and quantified. n = 14 ~ 15 fish/group. (E) Tg(igfbp5a:GFP) fish were treated with NBI-31772 (90 µM) together with BMS-754807 (0.3 µM), MK2206 (8 µM), or Rapamycin (5 µM) from 3 to 5 dpf. NaR cell number was quantified and shown. n = 10 ~ 24 fish/group. (F) Tg(igfbp5a:GFP) fish were treated with human IGF1 (150 ng/ml) in E3 embryo medium from 3 to 5 dpf. NaR cells were quantified and shown. n = 35 ~ 36 fish/group. **p<0.01, unpaired t-test. (G) Proposed model of Papp-aa function as a [Ca2+]-regulated molecular switch of IGF signaling in epithelial cells. Left panel: under normal [Ca2+] conditions, Papp-aa proteolysis activity is suppressed. Igfbp5a is intact and it inhibits IGF signaling by binding to IGFs and prevents their binding to the IGF1 receptor. The IGF- PI3 kinase-Akt-Tor signaling is inhibited in NaR cells. Right panel: under low [Ca2+] conditions, Papp-aa activity is increased. This increases Igfbp5a proteolytic cleavage and releases IGFs from the Igfbp5a/IGF complex. Bioavailable IGFs binds to IGF1 receptor and activates PI3 kinase-Akt-Tor signaling in NaR cells and promotes their reactivation and proliferate.


Acknowledgments:
ZFIN wishes to thank the journal eLIFE for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Elife