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Fig. 4 Expression of nkcc1 transcript and protein immunoreactivity are lost in lte mutants. (A-J) Whole-mount in situ hybridisation for nkcc1 reveals a reduction or loss of expression in lte mutants at all stages examined. (A,F) Four-somite stage (4S). Expression is reduced in the notochord (arrows) and somites (asterisks) in putative mutants. (B-E,G-J) Expression is reduced or lacking in the lte mutant ear from 24 hpf to 5 dpf. (K-V) Immunostaining with the T4 (Nkcc1/2) antibody. Immunoreactivity is lost at 6 dpf in the lte mutant ear (K,L). Expression of Nkcc2 in the pronephric ducts is unaffected in mutants (transverse sections M,N). (O-R) Confocal transverse section through the wild-type ear at 6 dpf; O is a brightfield image and R is an overlay of P and Q. Nkcc1 expression is restricted to the cells lining the medial face of the ventral pillar (P,R, red). TO-PRO-3 staining of nuclei (Q,R, blue) confirms that expression is restricted to the cell membrane and is highest basolaterally (P′). (S-V) Confocal transverse section through the pronephric ducts at 6 dpf showing that Nkcc2 expression is restricted to the apical cell membrane. h, semicircular canal hub; p, pronephric duct; v, ventral pillar; usc, utriculosaccular chamber; lsc, lateral semicircular canal lumen. Scale bars: 200 μm in A,F; 50 μm in B-E,G-L,O-R; 25 μm in M,N; 12.5 μm in S-V.