Fig. 2

Effect of nrz Knockdown and Nrz Subcellular Localization on Epiboly and YSL Mitochondria

(A) Embryo length (L) and width (W) used for calculating the L/W ratio; left: control embryo; right: nrz morphant.

(B) Histogram showing the L/W ratio of control embryos (WT) and nrz morphants (nrzMO) at 50% epiboly/shield stage (mean ± SD; three independent experiments; p < 0.01).

(C) Schematic drawing of recombinant proteins. Nrz, full-length protein; NrzΔTM, deletion mutant lacking the TM domain; NrzCytB5, swap mutant with ER-addressing domain; NrzMaoB, swap mutant with mitochondria-addressing domain. BH and TM domains are shown as color boxes.

(D) Complementation experiments: Embryos were injected with nrz morpholinos to dampen endogenous nrz level and coinjected with mRNAs expressing the recombinant proteins displayed in (C). Embryo mortality (%) was measured at indicated (hours postfertilization). Representative data from 2–8 independent experiments. The nrz morpholino is directed against the 5′ untranslated region of the endogenous nrz mRNA; it does not match with the foreign nrz transcripts.

(E–H) Representative confocal images of Mitotracker Red-stained embryos injected with control morpholino (E), nrzMO morpholino (F), nrzMO + nrzcytb5 mRNA (G), or nrzMO + nrzmaob mRNA (H); nrzMO-injected embryos exhibit a dramatic decrease in the labeling of the YSL mitochondria belt (white arrows).

(I) Quantitative analysis of Mitotracker labeling of zebrafish embryos. Fluorescence intensity is represented as a function of the distance from the animal pole (upper panel). Intensity peaks in the region of the YSL. Statistical data are shown (bottom panel).

(J) Immunoblot analysis of Nrz protein and cytochrome c contents of purified YSL mitochondria; F0/F1 ATPase was used as a loading control. Left: mitochondrial protein extracts from control embryos and nrz morphants; loss of Nrz protein correlates with cytochrome c release. Right: same analysis carried out on nrz morphants coinjected with mRNAs expressing NrzCytB5 or NrzMaoB; cytochrome c release appears to be prevented by both Nrz swap mutants. See also Movie S1 and Figure S2.