Stawicki et al., 2014 - The zebrafish merovingian mutant reveals a role for pH regulation in hair cell toxicity and function. Disease models & mechanisms   7:847-856 Full text @ Dis. Model. Mech.

Fig. 1

merovingian mutants have inner ear defects. (A) merovingian (mero) mutants show multiple phenotypes including a failure to inflate their swim bladders, an enlarged yolk, and impaired otolith formation. Arrowhead points to otolith. (B) Quantification of the size of the posterior otolith in wild-type siblings and merovingian mutants. Otolith size is significantly reduced in merovingian mutants. Mutants were selected randomly and included eight fish lacking a posterior otolith and therefore having an otolith size of 0 (n=10 fish). (C) Hair cells expressing the brn3c:gfp transgene in the lateral crista of both wild-type siblings and merovingian mutants. (D) Quantification of the number of hair cells/crista in wild-type siblings and merovingian mutants. All three crista were used for counting. There is a significant reduction in hair cell number in merovingian mutants (n=9 fish). ***P<0.0001 by Student’s t-test; error bars indicate s.d. Scale bars: 250 μm (A), 10 μm (C).

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Term:
Stage: Day 5
PHENOTYPE:
Fish:
Observed In:
Stage: Day 5

Fig. 2

merovingian mutants are resistant to toxicant-induced hair cell death. (A) Quantification of lateral line hair cell number in wild-type siblings and merovingian mutants; ***P<0.0001 by Student’s t-test (n=10 fish). (B) Hair cells labeled with parvalbumin in merovingian mutants and wild-type siblings without (left) and with (right) neomycin treatment. merovingian mutants show reduced initial hair cell numbers, but no hair cell loss in response to neomycin. (C) merovingian mutants show a significant resistance to neomycin-induced hair cell death; P<0.0001 by two-way ANOVA (n=10 fish). (D) merovingian mutants are partially resistant to cisplatin-induced hair cell death. Genotypes are significantly different; P<0.0001 by two-way ANOVA (n=6–10 fish). Error bars indicate s.d. Scale bar: 10 μm.

EXPRESSION / LABELING:
Antibody:
Fish:
Condition:
Anatomical Term:
Stage: Day 5
PHENOTYPE:
Fish:
Observed In:
Stage: Day 5

Fig. 3

Uptake of FM1-43 and hair cell toxicants are reduced in merovingian mutants. (A) There is reduced uptake of FM1-43 into the hair cells of merovingian mutants. Hair cells are expressing the brn3c:gfp transgene. (B) Neomycin-TR fails to enter hair cells of merovingian mutants. (C) Rho-Pt uptake is reduced in merovingian mutants. (D) Quantification of FM1-43, neomycin-TR, and Rho-Pt uptake into hair cells, normalized to background fluorescence (n=10 fish); **P=0.0046, ***P<0.0001 for neomycin-TR and P=0.0006 for Rho-Pt by Student’s t-test, Welch’s correction was used for neomycin-TR and Rho-Pt. Error bars indicate s.d. Scale bars: 10 μm.

EXPRESSION / LABELING:
Gene:
Fish:
Condition:
Anatomical Term:
Stage: Day 5

Fig. 5

merovingian mutants lack H+-ATPase-rich ionocytes, but not hair cell H+-ATPases. (A) vH-ATPase labeling on the yolk of 3-dpf zebrafish. merovingian mutants lack H+-ATPase-rich ionocytes present in wild-type siblings.(B) vH-ATPase labeling in neuromasts of 5-dpf zebrafish. Although reduced in level, staining is still present in merovingian mutants. Scale bars: 100 μm (A), 10 μm (B).

EXPRESSION / LABELING:
Antibody:
Fish:
Anatomical Terms:
Stage Range: Protruding-mouth to Day 5

Fig. 6

merovingian mutants show a decreased in extracellular pH throughout their body. (A) Images of both GPI-linked and cytoplasmic pHluorin2 expressed under the control of the β-actin promoter in muscle cells. (B) The extracellular (GPI-linked) and intracellular (cytoplasmic) environment of muscle cells is acidified in merovingian mutants, as measured by a decreased 405/488 ratio in the ratiometric fluorescent pH indicator pHluorin2 (n=20 fish). (C) Images of both GPI-linked and cytoplasmic pHluorin2 expressed under the control of the myosin6b promoter in hair cells of the inner ear. (D) The extracellular and intracellular environment of inner ear hair cells is acidified in merovingian mutants, as measured by a decreased 405/488 ratio in the ratiometric fluorescent pH indicator pHluorin2 (n=15–19 fish). (E) Images of both GPI-linked and cytoplasmic pHluorin2 expressed under the control of the myosin6b promoter in hair cells of the lateral line. (F) The extracellular, but not intracellular, environment of lateral line hair cells is acidified in merovingian mutants, as measured by a decreased 405/488 ratio in the ratiometric fluorescent pH indicator pHluorin2 (n=13–16 fish). Values were normalized to the 405/488 ratio of wild-type siblings for each construct. *P<0.05, ***P<0.001 by ANOVA and Bonferroni post-hoc test. Error bars indicate s.d. Scale bars: 10 μm.

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Terms:
Stage: Day 5
Acknowledgments:
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