ZFIN ID: ZDB-FIG-160308-7
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:
Genes:
Fish:
Condition:
Anatomical Term:
Stage: Day 5
PHENOTYPE:
Fish:
Condition:
Observed In:
Stage Range: Day 5 to Days 7-13

Fig. 6

Distinct Phenotypes of mtorxu015/xu015 and rragast77/st77 Mutants

(A and B) Images show living neutral red-stained WT (A) and mtor mutant (B) larvae at 5 dpf. Dorsal views, anterior to the top.

(C and D) There was no significant reduction of the number of microglia at 5 dpf in mtor mutants or WT animals (C) treated with the mTOR inhibitor Torin1 (D).

(E) Immunoblots of 5-dpf whole-animal protein lysates showing reduction of phospho-S6p levels after Torin1 treatment compared to control DMSO treatment. The level of phospho-S6p was similar in rragast77/st77 mutants and their WT siblings. Actin is shown as a loading control.

(F–I) Lateral views of living larvae at 7 dpf show normal morphology of rragast77/st77 mutant (H) and abnormal morphology of mtorxu015/xu015 mutant (I), compared to their WT siblings (F and G, respectively).

(J and K) Analysis of survival of rragast77/st77 (J) and mtorxu015/xu015 (K) mutants. Progeny of intercross of heterozygotes were raised, and 48 animals were genotyped by PCR for the mutant lesions at the indicated time points. Very few mtorxu015/xu015 mutants survived to 10 dpf, but most rragast77/st77 mutants survived to 12 dpf. For a homozygous viable mutation, 12 mutants would be expected at each time point on average (dotted lines).

(L) qRT-PCR of whole-animal RNA samples at 5 dpf showed an increase in expression of some target genes of the lysosomal transcription factor TFEB in rragast77/st77 mutants relative to their WT siblings.

(M) Similar qRT-PCR did not detect any increased expression of the genes analyzed in mtorxu015/xu015 mutants, and many were significantly reduced. Error bars show SEM of samples analyzed in triplicate. Statistical significance determined by a two-tailed t test (*p < 0.05, **p < 0.01, and ***p < 0.005).

All scale bars, 50 µm. All larvae analyzed in (A)–(C) and (F)–(I) were genotyped by PCR. rragast77/st77 mutants analyzed in the immunoblot (E) were genotyped as described in the Experimental Procedures. rraga mutants analyzed by RT-PCR were identified by neutral red staining prior to RNA isolation.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
arhgdia WT chemical treatment by environment: torin 1 Day 5 whole organism RTPCR
asah1b WT chemical treatment by environment: torin 1 Day 5 whole organism RTPCR
rragast77/st77 standard conditions Day 5 whole organism RTPCR
bloc1s6 rragast77/st77 standard conditions Day 5 whole organism RTPCR
ctsc WT chemical treatment by environment: torin 1 Day 5 whole organism RTPCR
rragast77/st77 standard conditions Day 5 whole organism RTPCR
gabarapa WT chemical treatment by environment: torin 1 Day 5 whole organism RTPCR
rragast77/st77 standard conditions Day 5 whole organism RTPCR
hexb rragast77/st77 standard conditions Day 5 whole organism RTPCR
mapk1 rragast77/st77 standard conditions Day 5 whole organism RTPCR
mapk3 WT chemical treatment by environment: torin 1 Day 5 whole organism RTPCR
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
WT chemical treatment by environment: torin 1 Day 5 brain has normal numbers of parts of type microglial cell, normal
WT chemical treatment by environment: torin 1 Day 5 whole organism arhgdia expression decreased amount, abnormal
WT chemical treatment by environment: torin 1 Day 5 whole organism asah1b expression decreased amount, abnormal
WT chemical treatment by environment: torin 1 Day 5 whole organism ctsc expression decreased amount, abnormal
WT chemical treatment by environment: torin 1 Day 5 whole organism mapk3 expression decreased amount, abnormal
WT chemical treatment by environment: torin 1 Day 5 whole organism gabarapa expression decreased amount, abnormal
mtorxu015Gt/xu015Gt standard conditions Day 5 brain has normal numbers of parts of type microglial cell, normal
Days 7-13 trunk curved ventral, abnormal
rragast77/st77 standard conditions Day 5 whole organism hexb expression increased amount, abnormal
Day 5 whole organism bloc1s6 expression increased amount, abnormal
Day 5 whole organism gabarapa expression increased amount, abnormal
Day 5 whole organism asah1b expression increased amount, abnormal
Day 5 whole organism ctsc expression increased amount, abnormal
Day 5 whole organism mapk1 expression increased amount, abnormal
Days 7-13 trunk curvature, normal
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.