ZFIN ID: ZDB-FIG-190813-9
Princely Abudu et al., 2019 - NIPSNAP1 and NIPSNAP2 Act as "Eat Me" Signals for Mitophagy. Developmental Cell   49(4):509-525.e12 Full text @ Dev. Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibody:
Fish:
Knockdown Reagents:
Anatomical Terms:
Stage Range: 30%-epiboly to Day 5
PHENOTYPE:
Fish:
Condition:
Knockdown Reagents:
Observed In:
Stage Range: Protruding-mouth to Days 7-13

Fig. 7

Nipsnap1-Deficient Zebrafish Larvae Display Parkinsonism

(A) Spatial expression pattern of nipsnap1 across different development stages of zebrafish as demonstrated by whole-mount in situhybridizations. All embryos are in lateral view. Scale bar is 200 μm.

(B) Representative images of control (WT), nipsnap1 mutant, and rescue (nipsnap1 mutant + zebrafish nipsnap1 mRNA) transgenic tandem-tagged mitofish larvae at 3 dpf. Images are from head region of zebrafish larvae. Scale bars are 20 μm.

(C) Quantification of ratio of red puncta to yellow puncta (representative of mitophagy) per cell. Average of 15 cells from each of 25 larvae for control (WT), nipsnap1 mutant, and nipsnap1 mutant + rescue; tandem-tagged mitofish larvae from three independent experiments were used for quantification.

(D) Control (WT), nipsnap1−/− and nipsnap1 mutant embryos at 3 dpf incubated with CellROX and ROS levels quantified from six different experiments. Error bars represent ± SEM.

(E) Spatial expression pattern of tyrosine hydroxylase 1 (th1) gene in control (WT), nipsnap1−/− and nipsnap1 mutant embryos at 3 dpf determined by whole-mount in situ hybridization. Orientation dorsal. Scale bar is 200 μm.

(F) Quantification of the number of Th1 positive neurons from images in (D). 10 control (WT), 20 mutant and 20 knockout larvae were used for quantification. Values were normalized to control (WT) values.

(G) Representative immunoblots of Nipsnap1, Th1, and β-tubulin on whole embryo lysates of WT, nipsnap1−/− and nipsnap1 mutant embryos at 3 dpf. β-tubulin served as loading control.

(H) Representative images of TUNEL assay on control (WT), nipsnap1mutant and DNase-treated WT larvae (positive control) at 3dpf. Orientation lateral. Scale bar is 200 μm.

(I) Quantification of mean fluorescent intensity from demarcated region from images in (H). 10 control (WT), 15 nipsnap1 mutant, and 10 positive control larvae were used for quantification, respectively. Values were normalized to control (WT) values.

(J) Motility analysis of WT and nipsnap1 mutant embryos at 7 dpf using the “Zebrabox” automated videotracker (Viewpoint, Lyon). Assay was carried out during daytime, one cycle of 20 min exposure to light (white horizontal bar) followed by 20 min of darkness (black horizontal bar). Vertical bars indicate average distance moved during 5 min intervals. Controls and nipsnap1 mutants were treated or not with 5 mM of L-DOPA for 1 h, after which they were analyzed for motility. Each group consisted of 20–24 larvae.

All error bars indicate SEM. p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.0005, ∗∗∗∗ p < 0.00005, unpaired Student’s t test

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
nipsnap1 WT standard conditions 30%-epiboly whole organism ISH
75%-epiboly whole organism ISH
90%-epiboly whole organism ISH
5-9 somites endoderm ISH
5-9 somites whole organism ISH
Prim-5 endoderm ISH
Prim-5 head ISH
Long-pec head ISH
Long-pec intestine ISH
Long-pec liver ISH
Long-pec pectoral fin ISH
Protruding-mouth head ISH
Protruding-mouth intestine ISH
Protruding-mouth liver ISH
Protruding-mouth pectoral fin ISH
Day 4 head ISH
Day 4 intestine ISH
Day 4 liver ISH
Day 4 pectoral fin ISH
Day 5 head ISH
th WT standard conditions Protruding-mouth brain ISH
WT + CRISPR1-nipsnap1 + CRISPR2-nipsnap1 + CRISPR3-nipsnap1 standard conditions Protruding-mouth brain ISH
Ab1-th Protruding-mouth whole organism WB
nipsnap1sa14357/sa14357 standard conditions Protruding-mouth brain ISH
Ab1-th Protruding-mouth whole organism WB
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
WT + CRISPR1-nipsnap1 + CRISPR2-nipsnap1 + CRISPR3-nipsnap1 standard conditions Protruding-mouth brain th expression decreased amount, abnormal
Protruding-mouth brain apoptotic process increased occurrence, abnormal
Protruding-mouth dopaminergic neuron decreased amount, abnormal
Protruding-mouth whole organism th expression decreased amount, abnormal
Protruding-mouth whole organism reactive oxygen species increased amount, abnormal
Day 5 whole organism dead, abnormal
Day 5 whole organism decreased life span, abnormal
Days 7-13 swimming behavior decreased occurrence, abnormal
WT + CRISPR1-nipsnap1 + CRISPR2-nipsnap1 + CRISPR3-nipsnap1 chemical treatment by environment: L-dopa Days 7-13 swimming behavior process quality, ameliorated
nipsnap1sa14357/sa14357 standard conditions Protruding-mouth brain th expression decreased amount, abnormal
Protruding-mouth dopaminergic neuron decreased amount, abnormal
Protruding-mouth whole organism th expression decreased amount, abnormal
Protruding-mouth whole organism reactive oxygen species increased amount, abnormal
uio1Tg + CRISPR1-nipsnap1 + CRISPR2-nipsnap1 + CRISPR3-nipsnap1 standard conditions Protruding-mouth head mitophagy decreased occurrence, 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, 49(4), Princely Abudu, Y., Pankiv, S., Mathai, B.J., Håkon Lystad, A., Bindesbøll, C., Brenne, H.B., Yoke Wui Ng, M., Thiede, B., Yamamoto, A., Mutugi Nthiga, T., Lamark, T., Esguerra, C.V., Johansen, T., Simonsen, A., NIPSNAP1 and NIPSNAP2 Act as "Eat Me" Signals for Mitophagy, 509-525.e12, Copyright (2019) with permission from Elsevier. Full text @ Dev. Cell