ZFIN ID: ZDB-FIG-130210-15
Bercier et al., 2013 - WNK1/HSN2 Mutation in Human Peripheral Neuropathy Deregulates KCC2 Expression and Posterior Lateral Line Development in Zebrafish (Danio rerio). PLoS Genetics   9(1):e1003124 Full text @ PLoS Genet.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Gene:
Fish:
Knockdown Reagents:
Anatomical Term:
Stage Range: Prim-5 to Protruding-mouth
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage Range: Prim-5 to Protruding-mouth

Fig. 4

KCC2 is overexpressed in WNK1/HSN2 knockdown embryos.

A) RT-PCR against slc12a5 (coding for kcc2) shows higher levels of RNA in WNK1/HSN2 knockdown embryos when compared with WT at 72hpf as well as at 24hpf. We overexpressed human KCC2 mRNA in WT embryos (morphology presented in (B)) to validate this result and were able to replicate the WNK1/HSN2 knockdown phenotype as assayed by (C) the number of structural and functional hair cells in PLL neuromasts and by (D) the 4-di-2-ASP score. We also validated this result by obtaining a partial rescue of the WNK1/HSN2 knockdown phenotype by knocking down kcc2 using MO1-slc12a5 in WNK1/HSN2 embryos (double knockdown-KD experiments). The embryos lacking kcc2 have morphological defects and a lower 4-di-2-ASP score (D) due to their smaller length (B), but double knockdown embryos, which are also morphologically abnormal and smaller in size have a significantly higher 4-di-2-ASP score (D) indicative of a partial rescue. For (C) The number of neuromasts counted per condition is indicated in the boxes and the total number of embryos obtained per condition is indicated in parenthesis at the bottom of the box plots. For (D), the total number of embryos is indicated in the boxes. (E) Overexpression of inactive mutant KCC2-C568A mimics hKCC2 overexpression and WNK1/HSN2 knockdown phenotype by producing PLL defects as assayed by 4-di-2-ASP vital dye staining. This indicates that WNK1/HSN2 interacts with KCC2 and regulates its transcription independent of the cotransporter′s activation. Neuromast scores were tabulated as previously done and presented as a box plot. The number of neuromasts counted per condition is indicated in the boxes. Scale bar: 100μm.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
slc12a5b WT standard conditions Prim-5 whole organism RTPCR
Protruding-mouth whole organism RTPCR
WT + MO1-wnk1b standard conditions Protruding-mouth whole organism RTPCR
WT + MO2-wnk1b standard conditions Protruding-mouth whole organism RTPCR
WT + MO3-wnk1b standard conditions Prim-5 whole organism RTPCR
Protruding-mouth whole organism RTPCR
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
WT + MO1-slc12a5b standard conditions Protruding-mouth post-vent region decreased length, abnormal
Protruding-mouth posterior lateral line neuromast decreased amount, abnormal
Protruding-mouth posterior lateral line neuromast development decreased process quality, abnormal
Protruding-mouth posterior lateral line neuromast has fewer parts of type neuromast hair cell, abnormal
WT + MO1-slc12a5b + MO3-wnk1b standard conditions Protruding-mouth posterior lateral line neuromast development decreased process quality, abnormal
Protruding-mouth posterior lateral line neuromast has fewer parts of type neuromast hair cell, abnormal
WT + MO1-wnk1b standard conditions Protruding-mouth potassium:chloride symporter activity increased process quality, abnormal
WT + MO2-wnk1b standard conditions Protruding-mouth potassium:chloride symporter activity increased process quality, abnormal
WT + MO3-wnk1b standard conditions Prim-5 potassium:chloride symporter activity increased process quality, abnormal
Protruding-mouth posterior lateral line neuromast development decreased process quality, abnormal
Protruding-mouth posterior lateral line neuromast has fewer parts of type neuromast hair cell, abnormal
Protruding-mouth potassium:chloride symporter activity increased process quality, abnormal
Acknowledgments:
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