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Fig. 3. Inhibition of NO causes defects in ionocytes, multi-ciliated and small secretory cells
Embryos were injected by ctrl MO 34 ng (A,E,I,M, Q), nNOS MO 17 ng (B,F,J,N, R) and eNOS MO 34 ng (C,G,K,O,S) at stage
1 and fixed in 4% PFA at stage 26 (I-S), 30 (A-C) and 36 (E-G). MCCs were labeled by anti-alpha tubulin (A-C), vesicles in
SSCs were labeled by anti-5HT (E-G) and membranes by antibody against phalloidin (A-G) and imaged (apical surface of the
embryonic epidermis) by confocal microscopy (magnification 40x, scale bar = 20 μm) (A-G). MCCs, SSCs and ionocytes were
marked by alpha-tubulin (I-K) foxa1 (M-O) and foxi1e probe (Q-S) and imaged by macroscope (magnification 11,25x, scale bar
= 50 μm) (I-S). Embryos showing frequency of individual cell types after applying ctrl (A,E,I,M,Q), nNOS (B,F,J,N,R) and eNOS
(C,G,K,O,S) MO. Injection of nNOS MOS (B,F,J,N,R) and eNOS MO (C,G,K,O,S) cause reduction in number of MCCs (D),
SSCs (H) and reduced expression of scattered cell markers alpha-tubulin (L) foxa1 (P) and foxi1e (T). Quantification of
incidence of MCCs (D,L), SSCs (H,P) and ionocytes (T), error bars indicate + s.d., the P value was < 0.005 except ctrl x eNOS
(D) where was P value > 0.005. At least 5 embryos were used for each condition (One-way ANOVA, Tukey´s multiple
comparisons test). |
