ZFIN ID: ZDB-FIG-160707-2
Michel et al., 2016 - Leptin signaling regulates glucose homeostasis, but not adipostasis, in the zebrafish. Proceedings of the National Academy of Sciences of the United States of America   113(11):3084-9 Full text @ Proc. Natl. Acad. Sci. USA
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Term:
Stage: Day 6
PHENOTYPE:
Fish:
Condition:
Knockdown Reagents:
Observed In:
Stage Range: Day 5 to Day 6

Fig. 3

Glucose homeostasis phenotypes in normal and leptin receptor mutant larval zebrafish. (A) Whole-body free glucose in WT and leprsa1508/sa1508 mutant in-crosses collected at 6 dpf. A t test shows a significant difference between genotypes, t(58) = 5.179, P < 0.0001. (B) Expression of insa and insb mRNA at 6 dpf, determined by quantitative RT-PCR (qRTPCR). insa shows significant up-regulation t (10) = 6.058, P < 0.001, whereas insb does not t(10) = 0.016, P > 0.05, t tests. (C) Expression of gcga and gcgb mRNA levels, determined by qRTPCR. gcga t(10) = 4.973, P < 0.001 and gcgb t (10) = 4.232, P < 0.01 are up-regulated, t tests. (D) A representative image of a zebrafish carrying the β-cell marker [Tg(1.2ins:H2BmCherry)], and an enlarged image showing individual β-cells. (E) Numbers of β-cells in offspring of an in-cross of heterozygous leprsa1508/+ mutant animals at 5 dpf. Homozygous leprsa1508/sa1508 mutants exhibit a significant increase in β-cell number by one-way ANOVA, F(2, 93) = 18.69, P < 0.001, Bonferroni’s MCT. (F) Number of β-cells in animals at 6 dpf, following injection of embryos with Cas9 and guide RNA directed against lepr, lepa, or lepb genes; ANOVA F(4, 263) = 11.67, P < 0.0001, Bonferroni’s MCT. (G) Number of β-cells is significantly increased in larvae homozygous for a mutation in lepa compared with their WT siblings at 6 dpf. t test t(41) = 4.738, P < 0.001. (H) A representative picture of α-cells in fish carrying the transgene [Tg(gcg:GFP)]. (I) Numbers of α-cells observed in WT and leprsa1508/sa1508 mutant siblings from heterozygous leprsa1508/+ in-crosses at 6 dpf. There was no effect of genotype [t test t(24) = 0.96, P = 0.34]. (J) Data showing gene expression analysis of fry at 6 dpf compared with WT controls. t tests show an up-regulation of glucose 6 phosphatase (g6pca.1; t (9) = 7.059, P < 0.001), mitochondrial phosphoenolpyruvate carboxykinase (pck2; t (9) = 8.601, P < 0.001), liver glycogen phosphorylase [pygl; t(9) = 4.423, P < 0.01], glucose transporters 2 (scl2a2; t (9) = 2.808, P < 0.05) and 5 [slc2a5; t(10) = 5.160, P < 0.001], but not cytoplasmic phosphoenolpyruvate carboxykinase [pck1; t(9) = 0.4822, P > 0.05], liver pyruvate kinase (pklr; t (10) = 0.2263, P > 0.05), glucose transporters 8 [slc2a8; t(10) = 0.903 P > 0.05] or 9 [slc2a9l1; t(10) = 1.31, P > 0.05). (K) Effect of metformin on the developmental increase in β-cell number. Two-factor ANOVA shows an effect of genotype [F(1,107) = 4.785, P < 0.05] and treatment [F(1,107) = 12.56, P < 0.001]. A Bonferroni post test showed a significant elevation in the DMSO-treated leprsa1508/sa1508 mutant group. Data shown as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
g6pc1a.1 WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
gcga WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
gcgb WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
ins WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
insb WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
pck1 WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
pck2 WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
pklr WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
pygl WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
slc2a2 WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
slc2a5 WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
slc2a8 WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
slc2a9l1 WT standard conditions Day 6 whole organism RTPCR
leprsa1508/sa1508 standard conditions Day 6 whole organism RTPCR
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
leprsa1508/sa1508 standard conditions Day 6 whole organism g6pc1a.1 expression increased amount, abnormal
Day 6 whole organism gcga expression increased amount, abnormal
Day 6 whole organism gcgb expression increased amount, abnormal
Day 6 whole organism ins expression increased amount, abnormal
Day 6 whole organism pck2 expression increased amount, abnormal
Day 6 whole organism pygl expression increased amount, abnormal
Day 6 whole organism slc2a2 expression increased amount, abnormal
Day 6 whole organism slc2a5 expression increased amount, abnormal
Day 6 whole organism glucose increased amount, abnormal
leprsa1508/sa1508; ia1Tg standard conditions Day 6 pancreas has normal numbers of parts of type pancreatic A cell, normal
leprsa1508/sa1508; vu513Tg standard conditions Day 5 pancreas has extra parts of type pancreatic B cell, abnormal
Day 6 pancreas has extra parts of type pancreatic B cell, abnormal
leprsa1508/sa1508; vu513Tg chemical treatment by environment: metformin Day 5 pancreas has normal numbers of parts of type pancreatic B cell, ameliorated
vu513Tg + CRISPR1-lepb standard conditions Day 6 pancreas has normal numbers of parts of type pancreatic B cell, normal
vu513Tg + CRISPR1-tyr standard conditions Day 6 pancreas has normal numbers of parts of type pancreatic B cell, normal
vu513Tg + CRISPR2-lepa standard conditions Day 6 pancreas has extra parts of type pancreatic B cell, abnormal
vu513Tg + CRISPR2-lepr standard conditions Day 6 pancreas has extra parts of type pancreatic B cell, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Proceedings of the National Academy of Sciences of the United States of America for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Proc. Natl. Acad. Sci. USA