ZFIN ID: ZDB-FIG-210715-1
Chambers et al., 2020 - Ppargc1a Controls Ciliated Cell Development by Regulating Prostaglandin Biosynthesis. Cell Reports   33:108370 Full text @ Cell Rep.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibodies:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage Range: 10-13 somites to Day 4
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage Range: 10-13 somites to Day 4

Fig. 1 ppargc1a-Deficient Zebrafish Display Phenotypes Commonly Associated with Cilia Defects

(A) WT sibling (top) and a ppargc1asa13186/sa13186 mutant (bottom) zebrafish with a curly tail, pronephric cyst (blue arrow, inset), and pericardial edema at 3 dpf. Heterozygous crosses resulted in the expected Mendelian ratio shown at the bottom right of the respective panels (WT group includes homozygous WT and ppargc1a+/sa13186 combined). Scale bar, 90 μm.

(B) JB4 transverse section of a 4 dpf WT sibling (left) and ppargc1asa13186/sa13186 mutant (right) zebrafish (n, notochord; tubule outlined in green, cysts outlined in red). Scale bar, 90 μm.

(C) Three days post-fertilization WT sibling (left) and ppargc1asa13186/sa13186 mutant (right) otoliths. Arrowheads indicate abnormal otoliths present in the mutant. Scale bar, 90 μm.

(D) Four days post-fertilization otic cilia (green, acetylated α-tubulin) of the cristae in WT (left) and ppargc1asa13186/sa13186 mutant (right) zebrafish. Scale bar, 10 μm.

(E) Dorsal view of 3 dpf WT sibling (top) and ppargc1asa13186/sa13186 mutant (bottom) approximately 24 h after being injected with 40 kDa dextran-FITC to assay for pronephric function. White arrowheads indicate PCT fluorescence in WT. White box highlights the approximate area of the PCT with no fluorescence. Scale bar, 90 μm.

(F) Three days post-fertilization WT sibling (top) and ppargc1asa13186/sa13186 mutant (bottom) stained via WISH for slc20a1a to mark PCT. Scale bar, 90 μm.

(G) Fifty-five hours post-fertilization WT and ppargc1a MO injected zebrafish stained via WISH for the heart marker myl7. Penetrance graph of atypical heart looping occurring in WT and ppargc1a morphants. Chi-square analysis indicates significance. n = 128 (WT), n = 103 (MO), p < 0.001. Scale bar, 90 μm.

(H) Whole-mount immunofluorescence of the Kupffer’s vesicle (KV) at the 10 ss stained for acetylated α-tubulin (cilia, green), anti-PKC (membrane boundary, red). Scale bar, 8 μm.

(I) Cilia length in micrometers of KV cilia.

(J) The number of cilia present in the KV.

(K) Twenty-four hours post-fertilization WT zebrafish stained via WISH to illustrate ppargc1a mRNA expression. Box is of approximate area of inset showing Ppargc1a (red) expression and the cilia marker acetylated α-tubulin (green). Scale bars, 65 μm for the whole embryo and 10 μm for the inset. Data are represented as mean ± SD; ∗∗p < 0.01 and ∗∗∗p < 0.001 (t test).

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
myl7 TU standard conditions Long-pec myocardium ISH
TU + MO1-ppargc1a standard conditions Long-pec myocardium ISH
ppargc1a TU standard conditions Prim-5 olfactory placode ISH
Prim-5 pronephric tubule ISH
Ab2-ppargc1a Prim-5 pronephric tubule apical part of cell IHC
slc20a1a TU standard conditions Protruding-mouth pronephric proximal convoluted tubule ISH
ppargc1asa13186/sa13186(TU) standard conditions Protruding-mouth pronephric proximal convoluted tubule ISH
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-prkcz IHC TU standard conditions 10-13 somites Kupffer's vesicle apical plasma membrane
IHC TU + MO1-ppargc1a standard conditions 10-13 somites Kupffer's vesicle apical plasma membrane
Ab1-tuba IHC TU standard conditions 10-13 somites Kupffer's vesicle cilium
IHC Prim-5 pronephric tubule cilium
IHC Day 4 lateral crista cilium
IHC TU + MO1-ppargc1a standard conditions 10-13 somites Kupffer's vesicle cilium
IHC ppargc1asa13186/sa13186(TU) standard conditions Day 4 lateral crista cilium
Phenotype Details
Fish Conditions Stage Phenotype
TU + MO1-ppargc1a standard conditions 10-13 somites Kupffer's vesicle cilium assembly decreased process quality, abnormal
10-13 somites Kupffer's vesicle cilium decreased length, abnormal
10-13 somites Kupffer's vesicle has fewer parts of type Kupffer's vesicle cilium, abnormal
Long-pec determination of heart left/right asymmetry decreased process quality, abnormal
Long-pec heart mislocalised, abnormal
ppargc1asa13186/sa13186(TU) standard conditions Protruding-mouth heart edematous, abnormal
Protruding-mouth otic vesicle has extra parts of type otolith, abnormal
Protruding-mouth pronephric proximal convoluted tubule malformed, abnormal
Protruding-mouth pronephros cystic, abnormal
Protruding-mouth pronephros renal filtration decreased process quality, abnormal
Day 4 lateral crista cilium assembly decreased process quality, abnormal
Day 4 lateral crista has fewer parts of type lateral crista cilium, abnormal
Day 4 pronephros cystic, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Cell Reports for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Cell Rep.