ZFIN ID: ZDB-FIG-080604-71
Peri et al., 2008 - Live Imaging of Neuronal Degradation by Microglia Reveals a Role for v0-ATPase a1 in Phagosomal Fusion In Vivo. Cell   133(5):916-927 Full text @ Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibody:
Fish:
Knockdown Reagents:
Anatomical Terms:
Stage: Protruding-mouth
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage: Protruding-mouth

Fig. 4 Accumulation of Apoptotic Neuronal Material in v0-ATPase a1 Knocked-Down Microglia

(A and B) Apo-E-expressing cells have a myeloid origin. Dorsal view of the wild-type fish head at 3 dpf (days post fertilization). (A) apo-E mRNA expression in the wild-type embryo. (B) apo-E mRNA is not detectable in embryos injected with a morpholino directed against the pU1 transcription factor, whose activity is required for myeloid cells specification.

(C and D) Dorsal view of the wild-type fish head at 3 dpf (days post fertilization). Acridine orange stainings detect apoptosis in the CNS. Levels of apoptosis within the CNS are comparable in wild-type (C) and atp6v0a1-MO embryos (D).

(E) Quantification of the number of apoptotic clusters per embryo. The values are comparable between wild-type, atp6v0a1-MO and Bafilomycin-A1 treated microglia. The data represent the average ± SD of ten independent experiments.

(F) Quantification of the apoptotic cluster size. Apoptotic foci in atp6v0a1-MO embryos are three times larger then in the wild-type. These values were obtained by counting the number of red vesicles per cluster in different experiments. The data represent the average ± SD of ten independent experiments.

(G) Western blotting analysis using a polyclonal antibody directed against v0-ATpase a1. This shows that the expected 116kDa band, presents in extracts of 3 dpf uninjected embryos, is absent in extracts obtained from atp6v0a1 3 dpf knockdown embryos. This confirms the efficacy of the morpholino mediated knockdown.

(H–K) Dorsal view of the wild-type fish head at 3 dpf (days post fertilization). Foci of apoptotic neurons are found inside wild-type microglia. (H) Apo-E-GFP expression. (I) NBT-dSRed expression. (J) AnnexinV-Cy5 labeling of apoptotic clusters. (K) Merge. White arrowhead points at one single apoptotic neuronal cluster.

(L–O) Dorsal view of the fish head at 3 dpf (days post fertilization) in atpv0a1 morpholino injected embryos. Foci of apoptotic neurons are larger then in wild-type. (L) Apo-E-GFP expression. (M) NBTdSRed expression. (N) AnnexinV-Cy5 labeling of apoptotic clusters. (O) Merge. White arrowhead points at one single apoptotic neuronal cluster. The size of the cluster is larger when compared to the wild-type.

Gene Expression Details
Gene Antibody Fish Conditions Stage Qualifier Anatomy Assay
apoeb WT standard conditions Protruding-mouth microglial cell ISH
WT + MO1-spi1b standard conditions Protruding-mouth Not Detected microglial cell ISH
atp6v0a1a Ab1-atp6v0a1 WT standard conditions Protruding-mouth whole organism WB
Ab1-atp6v0a1 WT + MO1-atp6v0a1a standard conditions Protruding-mouth Not Detected whole organism WB
DsRed zf147Tg; zf148Tg standard conditions Protruding-mouth CNS neuron (sensu Vertebrata) IFL
zf147Tg; zf148Tg + MO1-atp6v0a1a standard conditions Protruding-mouth CNS neuron (sensu Vertebrata) IFL
EGFP zf147Tg; zf148Tg standard conditions Protruding-mouth microglial cell IFL
zf147Tg; zf148Tg + MO1-atp6v0a1a standard conditions Protruding-mouth microglial cell IFL
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
WT + MO1-atp6v0a1a standard conditions Protruding-mouth neuron apoptotic process occurrence, normal
Protruding-mouth phagolysosome assembly arrested, abnormal
WT + MO1-spi1b standard conditions Protruding-mouth microglial cell absent, abnormal
zf147Tg; zf148Tg + MO1-atp6v0a1a standard conditions Protruding-mouth phagolysosome assembly arrested, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Cell, 133(5), Peri, F., and Nüsslein-Volhard, C., Live Imaging of Neuronal Degradation by Microglia Reveals a Role for v0-ATPase a1 in Phagosomal Fusion In Vivo, 916-927, Copyright (2008) with permission from Elsevier. Full text @ Cell