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ZFIN ID: ZDB-FIG-180802-2
Klatt Shaw et al., 2018 - Intracellular Calcium Mobilization Is Required for Sonic Hedgehog Signaling. Developmental Cell   45(4):512-525.e5 Full text @ Dev. Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Gene:
Antibodies:
Fish:
Conditions:
Knockdown Reagents:
Anatomical Terms:
Stage: Prim-5
PHENOTYPE:
Fish:
Conditions:
Knockdown Reagents:
Observed In:
Stage: Prim-5

Fig. 1

Formation of Shh-Dependent Muscle Requires RyR Function

(A) Schematic illustration of the nuclei of Shh-dependent muscle cells in the somites of 26 hpf zebrafish embryos, presented relative to the notochord (ntc) in transverse section and in a superficial lateral view (anterior to left). Nuclei of different muscle types are color-coded: slow muscle pioneer cells ([MPs], gray), medial fast fibers ([MFFs], green), and superficial slow fibers ([SSFs], magenta).

(B, C, E, F, H, I, K, and L) Shh-dependent muscle fiber nuclei, marked by Prox1 (magenta) and En (green) expression, were visualized by immunostaining 26 hpf embryos: (B) wild-type (WT), (C) ryr1a and ryr3 MO-injected (ryr1a,3 MO), (E) vehicle-treated (0.5% DMSO) WT, (F) MZryr1a(−/−);ryr1b(−/−);MZryr3(−/−) mutant, (H and I) azumolene-treated WT, (K) azumolene-treated MZryr1a(−/−);ryr1b(−/−);MZryr3(−/−) mutant, and (L) 4-CmC-treated WT (supernumerary Prox1+ SSF nuclei are indicated with arrowheads). All nuclei co-expressing En and Prox1 were considered MPs. Scale bar, 25 μm.

(D and J) Quantification of distinct muscle cell types per somite. Data are represented as means ± SEM. As numbers of nuclei in WT and DMSO-treated embryos did not differ significantly, comparisons were made with control vehicle-treated embryos unless otherwise indicated. p < 0.01; ∗∗∗p < 0.0001; n.s., not significant.

(G) Traces of GCaMP fluorescence in electrically stimulated muscle of 24 hpf Tg(act2b:GCaMP6f) embryos incubated from 6 to 24 hpf in indicated solutions. Data are represented as means ± SEM. The maximum change in fluorescence from baseline, arrow in (G) is shown for each recorded embryo in (G′) with horizontal lines representing means. p < 0.01, ∗∗p < 0.001, ∗∗∗p < 0.0001. See also Figure S2.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
GCaMP sny210Tg chemical treatment by environment: 4-Chloro-m-cresol Prim-5 skeletal muscle cell IFL
sny210Tg chemical treatment by environment: azumolene sodium Prim-5 skeletal muscle cell IFL
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-eng IHC TU control Prim-5 fast muscle cell
IHC Prim-5 slow muscle cell muscle pioneer
IHC TU chemical treatment by environment: 4-Chloro-m-cresol Prim-5 fast muscle cell
IHC Prim-5 slow muscle cell muscle pioneer
IHC TU chemical treatment by environment: azumolene sodium Prim-5 fast muscle cell
IHC Prim-5 slow muscle cell muscle pioneer
IHC TU + MO1-ryr1a + MO1-ryr3 control Prim-5 fast muscle cell
IHC Prim-5 slow muscle cell muscle pioneer
IHC ryr1az42/z42 ; ryr1bz43/z43 ; ryr3z45/z45 control Prim-5 fast muscle cell
IHC Prim-5 slow muscle cell muscle pioneer
IHC ryr1az42/z42 ; ryr1bz43/z43 ; ryr3z45/z45 chemical treatment by environment: azumolene sodium Prim-5 fast muscle cell
IHC Prim-5 slow muscle cell muscle pioneer
Ab5-prox1 IHC TU control Prim-5 slow muscle cell
IHC Prim-5 slow muscle cell muscle pioneer
IHC TU chemical treatment by environment: 4-Chloro-m-cresol Prim-5 slow muscle cell
IHC Prim-5 slow muscle cell muscle pioneer
IHC TU chemical treatment by environment: azumolene sodium Prim-5 slow muscle cell
IHC Prim-5 slow muscle cell muscle pioneer
IHC TU + MO1-ryr1a + MO1-ryr3 control Prim-5 slow muscle cell
IHC Prim-5 slow muscle cell muscle pioneer
IHC ryr1az42/z42 ; ryr1bz43/z43 ; ryr3z45/z45 control Prim-5 slow muscle cell
IHC Prim-5 slow muscle cell muscle pioneer
IHC ryr1az42/z42 ; ryr1bz43/z43 ; ryr3z45/z45 chemical treatment by environment: azumolene sodium Prim-5 slow muscle cell
IHC Prim-5 slow muscle cell muscle pioneer
Phenotype Details
Fish Conditions Stage Phenotype
TU chemical treatment by environment: 4-Chloro-m-cresol Prim-5 slow muscle cell increased amount, abnormal
TU chemical treatment by environment: azumolene sodium Prim-5 fast muscle cell decreased amount, abnormal
Prim-5 slow muscle cell decreased amount, abnormal
Prim-5 slow muscle cell muscle pioneer decreased amount, abnormal
TU + MO1-ryr1a + MO1-ryr3 control Prim-5 fast muscle cell decreased amount, abnormal
Prim-5 slow muscle cell decreased amount, abnormal
Prim-5 slow muscle cell muscle pioneer decreased amount, abnormal
ryr1az42/z42 ; ryr1bz43/z43 ; ryr3z45/z45 control Prim-5 fast muscle cell decreased amount, abnormal
Prim-5 slow muscle cell decreased amount, abnormal
Prim-5 slow muscle cell muscle pioneer decreased amount, abnormal
ryr1az42/z42 ; ryr1bz43/z43 ; ryr3z45/z45 chemical treatment by environment: azumolene sodium Prim-5 fast muscle cell decreased amount, exacerbated
Prim-5 slow muscle cell decreased amount, exacerbated
Prim-5 slow muscle cell muscle pioneer decreased amount, exacerbated
sny210Tg chemical treatment by environment: 4-Chloro-m-cresol Prim-5 skeletal muscle cell GCaMP expression increased amount, abnormal
sny210Tg chemical treatment by environment: azumolene sodium Prim-5 skeletal muscle cell GCaMP expression decreased amount, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Developmental Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Cell, 45(4), Klatt Shaw, D., Gunther, D., Jurynec, M.J., Chagovetz, A.A., Ritchie, E., Grunwald, D.J., Intracellular Calcium Mobilization Is Required for Sonic Hedgehog Signaling, 512-525.e5, Copyright (2018) with permission from Elsevier. Full text @ Dev. Cell