The yolk cell of the zebrafish blastula harbors functional apoptosis machinery

Abstract

We recently described the implication of the Bcl-2 related anti-apoptotic Nrz protein during early zebrafish development. Nrz knock-down induces calcium-dependent cytoskeleton remodeling leading to margin constriction and premature embryo lethality. In the YSL, nrz knock-down embryos exhibit some typical features of apoptosis such as mitochondrial transmembrane potential loss and cytochrome c release. However, downstream caspase-3 activation has not been detected so far. Here, we report that the YSL contains fully functional apoptotic machinery that can activate caspase-3 following zBax ectopic expression. Furthermore, we present evidence that caspase-3 activation is actually detectable in nrz knock-down embryos when premature margin constriction is prevented.

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Figures and Tables

Figure 1 The zebrafish YSL contains inducible apoptotic machinery. (A) Western blotting detection of cytochrome C from purified YSL mitochondria treated with tBid (20 nM) for increasing time durations (0–60 min). After treatment, mitochondria were pelleted by centrifugation; at 60 min, released cytochrome C due to outer membrane permeabilization was eventually found in the supernatant. The same western blot was probed with anti-F0/F1 ATPase antibody for calibration purpose (top panels). (B) Images of Mitotracker Red-stained embryos injected with control (left panel) or zBax mRNA (right panel). White arrowheads show YSL mitochondria location; zBax-injected embryos exhibit dramatic decrease of the labeling of the YSL mitochondria belt. (C) Immunostaining of activated caspase-3 in zBax-injected embryos (top panels). Embryos were counterstained with nuclear Hoescht33342. Nrz knock-down embryos coinjected with dextran-FITC were treated with ML-7 for 3 h prior staining (bottom panels). Active caspase-3 is mainly found in the YSL (white arrowheads).

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