Research ArticlePhysiology

Ca2+ concentration–dependent premature death of igfbp5a−/− fish reveals a critical role of IGF signaling in adaptive epithelial growth

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Science Signaling  18 Sep 2018:
Vol. 11, Issue 548, eaat2231
DOI: 10.1126/scisignal.aat2231
  • Fig. 1 Deletion of Igfbp5a leads to premature death.

    (A) Structure of wild-type (WT) Igfbp5a, Igfbp5aΔ4, and Igfbp5aΔ11. SP, signal peptide. N, L, and C indicate the N-, L-, and C-domain of Igfbp5a. Asterisk (*) indicates a stop codon. The underlined amino acids were introduced by a frameshift. aa, amino acids. (B and C) F2 progeny of igfbp5aΔ4+/− intercrosses (left panel) and igfbp5aΔ11+/− (right panel) intercrosses were raised in E3 solution until 3 or 5 dpf and transferred into either fish system water (B) or 5PPT high [Ca2+] solution (C). The fish were sampled randomly and genotyped at the indicated stages. The genotype distributions are shown. ****P < 0.0001 by χ2 test. ns, not statistically significant. Total number of fish in each group is shown above the bar.

  • Fig. 2 igfbp5a−/− mutant fish develop and grow normally in Ca2+-rich solutions.

    (A) Progeny of igfbp5a+/− intercrosses were raised in E3 solution until 5 dpf and transferred into high [Ca2+] 5PPT solution. The vertebrate columns of 12 dpf larvae were visualized by calcein staining and quantified. Data shown are means ± SEM. n = 18 to 19 fish per group. No statistically significant differences were found among the groups using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. (B to D) Embryos of the indicated genotypes were raised in E3 solution. Their body length (B) and somite number (C) were measured at 24 hours postfertilization (hpf), and head-trunk angle (D) was measured at 72 hpf. Data shown are means ± SEM. n = 12 to 73 fish per group. No statistically significant differences were found using one-way ANOVA, followed by Tukey’s multiple comparison test. (E) Progeny of igfbp5a+/− intercrosses were sampled at the indicated stages. After NaR cells were visualized by in situ hybridization using a trpv5/6 complementary RNA (cRNA) probe and quantified, the fish were genotyped individually. Data shown are means ± SEM. n = 6 to 14 fish per group. No statistically significant differences were found by one-way ANOVA, followed by Tukey’s multiple comparison test.

  • Fig. 3 Deletion of Igfbp5a increases mortality and impairs adaptive NaR cell proliferation.

    (A) Progeny of igfbp5a+/− intercrosses were raised in E3 solution until 5 dpf and transferred into low-[Ca2+] solution. Dead larvae were collected daily and genotyped. The survival curve is shown, and the numbers of fish are indicated. P < 0.0001 by log-rank test. (B and C) Fish of the indicated genotypes were raised in solutions containing the indicated Ca2+ concentration to 11 dpf and stained by Alizarin red. Representative images are shown in (B), and the average number of calcified vertebral columns is shown in (C). n = 19 to 29 fish per group. ***P < 0.001, ****P < 0.0001, unpaired two-tailed t test. Scale bar, 0.5 mm. (D) Embryos of the indicated genotypes were raised in E3 solution until 72 hpf and transferred to solutions containing the indicated Ca2+ concentration. The expression of trpv5/6 mRNA was measured by quantitative polymerase chain reaction (qPCR) and normalized to the NaR cell number for the group. Values are means ± SEM of three independent experiments, each containing RNA isolated from a pool of 20 larvae of the indicated stages. Different letters indicate statistically significant differences (P < 0.05 by one-way ANOVA, followed by Tukey’s multiple comparison test). (E) Progeny of igfbp5a+/− intercrosses were raised in E3 solution to 72 hpf and transferred to solutions containing the indicated Ca2+ concentration. The fish were sampled at 120 hpf. NaR cells were visualized by in situ hybridization using the trpv5/6 cRNA probe and quantified. Each larva was genotyped afterward. Data shown are means ± SEM. n = 13 to 40 fish per group. Different letters indicate statistically significant differences (P < 0.05 by one-way ANOVA, followed by Tukey’s multiple comparison test). (F) Tg(igfbp5a:GFP) fish of the indicated genotypes were raised in E3 solution until 72 hpf and transferred to low-[Ca2+] solution. The number of GFP-labeled NaR cells was quantified at 72, 96, 104, and 120 hpf. Data shown are means ± SEM. n = 7 to 36 fish per group. Different letters indicate statistically significant differences (P < 0.05 by one-way ANOVA, followed by Tukey’s multiple comparison test).

  • Fig. 4 Deletion of Igfbp5a but not its paralog Igfbp5b impairs low-[Ca2+] stress–induced Akt-Tor signaling in NaR cells.

    (A to D) Fish of the indicated genotypes raised in E3 solution were transferred to solutions containing the indicated Ca2+ concentration at 72 hpf. After 24 hours, they were stained for phosphorylated Akt (pAkt) (A and B) or phosphorylated S6 (pS6) (C and D). Representative images are shown in (A) and (C). Scale bar, 0.1 mm. The numbers of cells positive for phosphorylated Akt or phosphorylated S6 in each fish were quantified, and the quantitative results are shown in (B) and (D). Values are means ± SEM. n = 6 to 12 fish per group. Different letters indicate statistically significant differences between groups (P < 0.05 by one-way ANOVA, followed by Tukey’s multiple comparison test). (E) Fish of the indicated genotypes were raised in E3 solution to 72 hpf and transferred to solutions containing the indicated [Ca2+] with or without U0126 (10 μM) or PD98059 (10 μM). After 8 hours, they were stained for phosphorylated Erk (pErk). The phosphorylated Erk signal was scored following a previously published scoring system (3). Total numbers of fish are shown above the bar. (F) Larvae of the indicated genotypes were raised in E3 solution to 72 hpf and transferred to solutions containing the indicated Ca2+ concentration. After 8 hours, they were stained for phosphorylated Akt. The number of cells positive for phosphorylated Akt was quantified. Values are means ± SEM. n = 13 to 18 fish per group. Different letters indicate statistically significant differences (P < 0.05 by one-way ANOVA, followed by Tukey’s multiple comparison test).

  • Fig. 5 Reintroduction of Igfbp5a or constitutively active Akt in NaR cells restores adaptive proliferation.

    (A) Structure of zebrafish Igfbp5a and its mutants. SP, N-, L-, and C-domains are indicated. Mutated residues are shown. (B and C) NaR division scoring system. Zebrafish igfbp5a−/−;Tg(igfbp5a:GFP) embryos injected with BAC(igfbp5a:Igfbp5a-IRESmCherry) constructs containing the indicated genes were raised in E3 solution until 72 hpf and transferred to low-[Ca2+] solutions. Igfbp5a-expressing NaR cells are labeled by both GFP and mCherry. The NaR division scoring system is shown in (B). During the 48-hour treatment period, NaR cells that divide 0, 1, or 2 times were scored as −, +, and ++. Scale bar, 0.05 mm. The quantified data are shown in (C). χ2 test was used for statistical analysis. **P < 0.01, ***P < 0.001. Total numbers of cells are shown above the bar. (D) The larvae described in (B) and (C) were collected after 24 hours in low-[Ca2+] solution and stained for phosphorylated Akt and mCherry. Scale bar, 0.02 mm. (E) BAC(igfbp5a:myr-Akt-mCherry) DNA was injected into igfbp5a−/−;Tg(igfbp5a:GFP) embryos. NaR division was scored as described in (B). χ2 test was used for statistical analysis. ****P < 0.0001. Total numbers of cells are shown above the bar.

  • Fig. 6 IGFBP5 but not two cancer-associated IGFBP5 mutants promotes the mitotic response of human epithelial cells to IGF stimulation.

    (A) Human LoVo epithelial cells were transfected with the indicated siRNA construct with or without pCMV-BP545. After 24 hours, they were treated with human IGF2 (400 ng/ml) for 3 days and analyzed by flow cytometry. The percentage of S-phase cells is shown. Values shown are means ± SEM. n = 3 independent experiments. Groups labeled with different letters are statistically significantly different from each other (P < 0.05 by one-way ANOVA, followed by Tukey’s multiple comparison test). (B) Structure of human IGFBP5 and its two cancer-associated mutants. SP, N-, L-, and C-domains are indicated. Mutated residues are shown. Asterisk (*) indicates a stop codon. (C) Zebrafish igfbp5a−/−;Tg(igfbp5a:GFP) embryos injected with the indicated human IGFBP5 construct were raised in E3 solution until 72 hpf and transferred to low-[Ca2+] solution. Forty-eight hours later, GFP and mCherry double-positive cells were quantified using the scoring system shown in Fig. 5B. χ2 test was used for statistical analysis. **P < 0.01, ***P < 0.001, ****P < 0.001. Total numbers of cells are shown above the bar.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/548/eaat2231/DC1

    Fig. S1. Generation of igfbp5a mutant fish.

    Fig. S2. igfbp5a mRNA levels in mutant fish.

    Fig. S3. Deletion of Igfbp5a does not alter igfbp5b expression.

    Fig. S4. igfbp5a Δ4 mutant fish are prone to dying under low Ca2+ stress.

    Fig. S5. CRISPR-Cas9–mediated transient deletion of Igfbp5a impairs adaptive NaR cell proliferation.

    Fig. S6. Expression of IGF and insulin ligand and receptor genes in NaR cells.

    Fig. S7. Deletion of Igfbp5a has no effect on phosphorylated Erk signaling.

    Table S1. Primers for cloning and sequencing of Igfbp5a, IGFBP5, and their mutants.

  • This PDF file includes:

    • Fig. S1. Generation of igfbp5a mutant fish.
    • Fig. S2. igfbp5a mRNA levels in mutant fish.
    • Fig. S3. Deletion of Igfbp5a does not alter igfbp5b expression.
    • Fig. S4. igfbp5a Δ4 mutant fish are prone to dying under low Ca2+ stress.
    • Fig. S5. CRISPR-Cas9–mediated transient deletion of Igfbp5a impairs adaptive NaR cell proliferation.
    • Fig. S6. Expression of IGF and insulin ligand and receptor genes in NaR cells.
    • Fig. S7. Deletion of Igfbp5a has no effect on phosphorylated Erk signaling.
    • Table S1. Primers for cloning and sequencing of Igfbp5a, IGFBP5, and their mutants.

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