FIGURE SUMMARY
Title

Cell-autonomous regulation of epithelial cell quiescence by calcium channel Trpv6

Authors
Xin, Y., Malick, A., Hu, M., Liu, C., Batah, H., Xu, H., Duan, C.
Source
Full text @ Elife
Fig. 3 supplemental 1

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

EXPRESSION / LABELING:
Genes:
Fish:
Condition:
Anatomical Term:
Stage: Day 5
PHENOTYPE:
Fish:
Condition:
Observed In:
Stage: Day 5
Fig. 5

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

EXPRESSION / LABELING:
Antibody:
Fish:
Conditions:
Knockdown Reagents:
Anatomical Term:
Stage: Day 4
PHENOTYPE:
Fish:
Conditions:
Knockdown Reagents:
Observed In:
Stage Range: Day 4 to Day 5
Fig. 5 supplement 1

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Figure 1

Morphology of <italic>trpv6</italic> mutant fish.

(A–B) Gross morphology (A) and body size (B) of trpv6Δ8-/-; Tg(igfbp5a:GFP) fish and siblings. Values are Mean ± SEM, n = 7–23 fish. No statistical significance was found.
Figure 1 supplement 1

Morphology of <italic>trpv6</italic> mutant fish.

(A–B) Gross morphology (A) and body size (B) of trpv6Δ8-/-; Tg(igfbp5a:GFP) fish and siblings. Values are Mean ± SEM, n = 7–23 fish. No statistical significance was found.

PHENOTYPE:
Fish:
Observed In:
Stage Range: Prim-5 to Days 7-13
Figure 2

Genetic deletion of Trpv6 increases epithelial cell apical opening.

(A–B) Progenies of a trpv6Δ8+/-;Tg(igfbp5a:GFP) intercross were injected with or without the BAC(igfbp5a:Trpv6-mCherry) DNA. At 3 dpf, larvae were photographed, followed by individual genotyping. Representative images are shown in (A). The apical opening of NaR cells were quantified and shown in (B). Data are Mean ± SEM, n = 8–42. Different letters indicate significant difference at p<0.05, one-way ANOVA followed by Tukey’s multiple comparison test.
Figure 2—figure supplement 1.

Genetic deletion of Trpv6 increases epithelial cell apical opening.

(A–B) Progenies of a trpv6Δ8+/-;Tg(igfbp5a:GFP) intercross were injected with or without the BAC(igfbp5a:Trpv6-mCherry) DNA. At 3 dpf, larvae were photographed, followed by individual genotyping. Representative images are shown in (A). The apical opening of NaR cells were quantified and shown in (B). Data are Mean ± SEM, n = 8–42. Different letters indicate significant difference at p<0.05, one-way ANOVA followed by Tukey’s multiple comparison test.
Figure 2—figure supplement 2.

Inhibition of Trpv6 increases epithelial cell proliferation.

(A–B) Tg(igfbp5a:GFP) fish were treated with Ruthenium Red (2 µM) or GdCl3 (100 µM) from 3 to 5 dpf. Representative images are shown in (A). NaR cell numbers were quantified and shown in (B). **, **** indicate p<0.01 and 0.0001 by unpaired t-test.
Figure 3

<italic>trpv6</italic>, <italic>igf1ra</italic>, <italic>igf1rb</italic> expression in <italic>trpv6</italic><sup>-/-</sup>.

(A–C) trpv6Δ7+/-; Tg(igfbp5a:GFP) intercrosses were raised in normal or low [Ca2+] solutions from 3 to 5 dpf. Caudal fin was clipped for genotyping and 4–6 fish from the same genotype group were pooled for RNA extraction and RT-qPCR. Gene expression level was normalized to 18 s. Values shown are Mean ± SEM, n = 3.
Figure 3—figure supplement 2.

Akt-Tor pathway activation in <italic>trpv6</italic><sup>-/-</sup>.

(A–B) trpv6Δ8+/-; Tg(igfbp5a:GFP) intercrosses were treated with 0.3 µM BMS-754807 or DMSO. At 4 dpf, the treated fish were subjected to immunostaining using an anti-phospho-Akt antibody (A) or an anti-phospho-S6 antibody (B).
Figure 3—figure supplement 3.

Inhibition of Trpv6 increases Akt signaling.

(A–B) Wild type fish were treated with Ruthenium Red (30 µM) or GdCl3 (100 µM) from 3 to 4 dpf and analyzed by immunostaining using an anti-phospho-Akt antibody. Representative images are shown in (A) and quantified results shown in (B). **, **** indicate p<0.01 and 0.0001 by unpaired t-test.
Figure 4

Schematic diagram showing the <italic>BAC(igfbp5a:GCaMP7a)</italic> construct.

Filled boxes indicate igfbp5a ORF and open boxes indicate its UTRs. The iTol2 cassette and GCaMP7a reporter cassette were introduced into DKEYP-56B7 by homologous recombination. The igfbp5a sequence from the start codon to the end of first exon was replaced by the GCaMP7a cassette.
Figure 4—figure supplement 2.

Validation of <italic>Tg(igfbp5a:GCaMP7a)</italic> fish.

(A–B) Embryos injected with BAC(igfbp5a:GCaMP7a) DNA were raised in E3 embryo solution and imaged at 3 dpf before and after the addition of the indicated chemicals (Ionomycin: 5 µM, CaCl2: 10 mM, EGTA: 10 mM).
Figure 4—figure supplement 3.

Inhibition of Trpv6 decreases [Ca<sup>2+</sup>]<sub>i</sub> in NaR cells.

(A–C) Time-lapse images of 3 dpf larvae were taken after the addition (A) and removal (B) of 100 µM Ruthenium red (RR) at the indicated time points. Fluorescence change of GCaMP7a (green) and mCherry (red) were quantified and shown in (C). Mean ± SEM, n = 8. **,***, and **** indicate p<0.01, p<0.001, p<0.0001 by multiple t-tests.
Figure 4—figure supplement 4.

Inhibition of IGF1 receptor does not change [Ca<sup>2+</sup>]<sub>i</sub> in NaR cells.

Time-lapse images of 3 dpf Tg (igfbp5a:GCaMP7a) larvae at the indicated time points after adding 0.3 µM BMS-754807. Changes in GCaMP7a (green) and mCherry (red) signal intensity were quantified. Representative images are shown in (A) and quantified results are shown in (B). Mean ± SEM, n = 3. No significance was found by two-way ANOVA followed by Dunnett's multiple comparison test.
Figure 5—figure supplement 2.

Inhibition of PP2A does not change [Ca<sup>2+</sup>]<sub>i</sub> in NaR cells.

Time-lapse images of 3 dpf Tg (igfbp5a:GCaMP7a) larvae at the indicated time points after adding 1 µM Okadaic acid (OA) (A, B) or 0.1 µM Calyculin A (C, D). Changes in GCaMP7a (green) and mCherry (red) signal intensity were quantified. Representative images are shown in (A, C) and quantified results are shown in (B, D). Mean ± SEM, n = 3. No significance was found by two-way ANOVA followed by Dunnett's multiple comparison test.
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Elife
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