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ZFIN ID: ZDB-FIG-160308-2
Shen et al., 2016 - The Rag-Ragulator Complex Regulates Lysosome Function and Phagocytic Flux in Microglia. Cell Reports   14(3):547-59 Full text @ Cell Rep.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Gene:
Antibodies:
Fish:
Condition:
Anatomical Term:
Stage: Day 5
PHENOTYPE:
Fish:
Condition:
Observed In:
Stage Range: Protruding-mouth to Day 5

Fig. 1

Mutational Analysis Demonstrates that rraga and lamtor4 Are Essential for Microglia Development

(A–F) Comparison of microglia numbers in rragast77/st77 mutants, lamtor4st99/st99 mutants, and WT siblings. (A, B, D, and E) Neutral red-stained larvae of the indicated genotypes at 5 dpf are shown. Dorsal views, scale bar, 50 µm. (C and F) Quantifications of neutral red-stained microglia in rraga (C) and lamtor4 (F) mutants at 3, 4, and 5 dpf are shown.

(G–J) Molecular analysis of mutations in rraga. (G) Sequence chromatograms show point mutation in rragast77 mutation. (H) Sequence deleted in rragast110 mutation shows exon-intron boundary and deleted splice donor. (I) Immunoblot showing pronounced reduction of RagA protein in rragast77/st77 mutants at 5 dpf. Actin is shown as a loading control. (J) Schematic of RagA protein shows conserved domains and positions of the mutant lesions.

(K–M) Molecular analysis of mutations in lamtor4. (K) Sequence chromatograms show point mutation in lamtor4st74 mutation, which changes the ATG initiation codon to ATA. (L) Sequence deleted in lamtor4st99 mutation shows the reading frame in WT and the alteration caused by the 11-bp deletion in the mutation. (M) Schematic of the Lamtor4 protein shows positions of the mutant lesions.

Larvae shown in (A), (B), (D), and (E) were genotyped by PCR after photography.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
rraga Ab1-rraga WT standard conditions Day 5 whole organism WB
Ab1-rraga rragast110/st110 standard conditions Day 5 whole organism WB
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab9-rps6 WB WT chemical treatment by environment: torin 1 Day 5 whole organism
WB rragast77/st77 standard conditions Day 5 whole organism
Phenotype Details
Fish Conditions Stage Phenotype
WT chemical treatment by environment: torin 1 Day 5 whole organism ab9-rps6 labeling decreased amount, abnormal
lamtor4st99/st99 standard conditions Protruding-mouth brain has fewer parts of type microglial cell, abnormal
Protruding-mouth microglia development decreased process quality, abnormal
Day 4 brain has fewer parts of type microglial cell, abnormal
Day 4 microglia development decreased process quality, abnormal
Day 5 brain has fewer parts of type microglial cell, abnormal
Day 5 microglia development decreased process quality, abnormal
rragast110/st110 standard conditions Day 5 whole organism rraga expression decreased amount, abnormal
rragast77/st77 standard conditions Protruding-mouth brain has fewer parts of type microglial cell, abnormal
Protruding-mouth microglia development decreased process quality, abnormal
Day 4 brain has fewer parts of type microglial cell, abnormal
Day 4 microglia development decreased process quality, abnormal
Day 5 brain has fewer parts of type microglial cell, abnormal
Day 5 microglia development decreased process quality, 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.