The expression of stc1a, but not other stc genes, is regulated by Ca²⁺ levels. (A–D) Wild-type zebrafish embryos were raised in embryo media containing the indicated Ca²⁺ concentration until 3 days post fertilization (dpf). The mRNA expression levels of stc1a(A), stc1b(B), stc2a(C), and stc2b(D) were determined by qRT-PCR and normalized by β-actin mRNA levels. Data shown are from 3 independent experiments, each containing 10–15 embryos/group. In this and all subsequent figures, data shown are Mean ± SEM unless stated otherwise. Different letters indicate significant differences between groups by one-way ANOVA followed by Tukey’s multiple comparison test (P < 0.05) unless stated otherwise. (E–H) Zebrafish of the indicated genotypes were raised in E3 embryo medium until 5 dpf. The mRNA levels of stc1a(E), stc1b(F), stc2a(G), and stc2b(H) were measured, normalized, and shown. Data shown are from 3 independent experiments, each containing 10–15 larvae/group. ***P < 0.001 by unpaired two-tailed t-test.

Genetic deletion of stc1a results in cardiac edema, body swelling, and premature death. (A) Schematic diagram of Stc1a protein and the indicated mutants. The N-linked glycosylation site and conserved cysteine residues are shown by red and black bars, respectively. (B) Morphology of fish of the indicated genotypes at the indicated stages. Lateral views with anterior to the left and dorsal up. Scale bar = 0.5 mm. (C–F) Body length, somite number, and head-trunk angle of the indicated genotypes at 24 hpf (C,D) and 48 hpf (E,F). n = 15–42 fish/group. (G) Survival curves. Progenies of stc1a (+17)^+/– intercrosses were raised in E3 embryo medium. Dead embryos were collected daily and genotyped individually. The survival curves of indicated genotypes and the total fish numbers are shown. P < 0.0001 by log-rank test.

Genetic deletion of stc1a results in elevated body Ca²⁺ content and increased NaR cell proliferation. (A) Total body Ca²⁺ content in 5 dpf zebrafish larvae of the indicated genotypes. Data shown are from 3 independent experiments, each containing 35 larvae/group. **P < 0.01 by unpaired two-tailed t-test. (B–D) Progenies of stc1a (+17)^+/– intercrosses (C) or progenies of stc1a (Δ18 + 1)^+/– intercrosses (D) were raised in the E3 embryo medium to 5 dpf. NaR cells were detected by in situ hybridization using an igfbp5a cRNA probe. After NaR cells were visualized and quantified in each fish, fish were genotyped individually. Representative images are shown in (B) and quantified data in (C,D). Scale bar = 0.2 mm. n = 33–70 larvae/group (C) and 19–46 larvae/group (D). (E,F) NaR cells in 5 dpf larvae of the indicated genotypes were scored following a published proliferation scoring index (Liu et al., 2018). Cells that divided 0, 1, or 2 times were scored as -, +, and ++. *P < 0.05 and ***P < 0.001 by Chi-square test. Total number of cells is shown above the bar. (G,H) Progenies of stc1a (+17)^+/–; Tg (igfbp5a: GFP) intercrosses were raised in E3 embryo medium until 5 dpf. GFP-expressing NaR cells were scored as described in (E). Representative images are shown in (G) and quantified results in (H). Scale bar = 0.2 mm. ***P < 0.001 by Chi-square test.

Papp-aa and Igfbp5a are indispensible for Stc1a action in NaR cells. (A) Embryos of the indicated genotypes were raised in E3 embryo medium until 5 dpf. The mRNA levels of papp-aa were measured and normalized. n = 15–17 larvae/group. (B)Tg(igfbp5a: GFP) embryos were injected with stc1a targeting gRNAs and Cas9 mRNA at the 1-cell stage. Embryos were raised in E3 embryo medium. The injected embryos were treated with BMS-754807 (BMS, 0.3 μM), ZnCl₂ (8 μM), or Bastimastat (200 μM) from 3 to 5 dpf. NaR cells were quantified and shown. n = 20–39 larvae/group. (C,D) Progeny of papp-aa^+/–; Tg (igfbp5a: GFP) intercrosses (C) or igfbp5a^{– /–}; Tg (igfbp5a: GFP) intercrosses (D) were injected with stc1a targeting gRNAs and Cas9 mRNA. The injected embryos were raised and NaR cells were quantified at 5 dpf. Each larva was genotyped afterward. n = 5–19 larvae/group (C) and n = 39–43 larvae/group (D). (E,F) Larvae of the indicated genotypes were raised in E3 embryo medium and NaR cells were quantified at 5 dpf. n = 9∼78 larvae/group (E) and n = 4∼34 larvae/group (F).

Stc1a promotes NaR cell quiescence by suppressing IGF-PI3 kinase-Akt-Tor signaling in NaR cells. (A) Progenies of stc1a (+ 17)^+/–; Tg (igfbp5a: GFP) intercrosses were raised in E3 embryo medium to 3 dpf and treated with DMSO or 0.3 μM BMS-754807 (BMS). Two days later, fish were fixed and phospho-Akt positive cells in the yolk sac region were detected by immunostaining. These fish were genotyped individually afterward. n = 14–47 larvae/group. (B,C) Progeny of stc1a (+ 17)^+/–; Tg (igfbp5a: GFP) intercrosses were raised in E3 embryo medium and treated with DMSO, 0.3 μM BMS-754807 (BMS), 0.06 μM Wortmannin (Wort), 8 μM MK2206 (MK), 5 μM Rapamycin (Rapa), or 10 μM U0126 from 3 to 5 dpf. After NaR cells were quantified, these fish were genotyped individually. Representative images (B) and quantified data are shown (C). n = 7–35 larvae/group. Scale bar = 0.2 mm. (D) Progeny of stc1a (Δ18 + 1)^+/–; Tg (igfbp5a: GFP) intercrosses were raised in E3 embryo medium to 3 dpf and treated with DMSO, 0.3 μM BMS-754807 (BMS), 5 μM Rapamycin (Rapa), or 10 μM U0126 from 3 to 5 dpf. NaR cells were quantified as described above. n = 4–28 larvae/group.

Perturbation of the Papp-aa-Igfbp-a-IGF signaling loop rescues the body edema and premature death in stc1a^{– /–} fish. (A,C) Gross morphology of fish of the indicated genotypes at the indicated stages. Lateral views with anterior to the left and dorsal up. Scale bar = 0.5 mm. (B,D) Survival curves. Embryos were raised in E3 embryo medium. Dead embryos were collected daily and genotyped individually. The survival curves of indicated genotypes and the total fish numbers are shown. P < 0.0001 by log-rank test. (E–J) Progeny of stc1a (+ 17)^+/– intercrosses were raised in E3 embryo media containing DMSO (E,F), 0.3 μM BMS-754807 (G,H) or 5 μM Rapamycin (I,J) starting from 3 dpf until the indicated time. Representative morphological views of the indicated genotypes at the indicated stages are shown in (E,G,I). Scale bar = 0.5 mm. P < 0.0001 by log-rank test. Dead fish were collected daily and genotyped individually. The survival curves of the indicated genotypes and the total fish numbers are shown in (F,H,J).

Proposed model of Stc1a function as a [Ca²⁺] state-regulated regulator of local IGF signaling and its role in epithelial cell quiescence-proliferation balance.