Related Experiment Video
Updated: Jun 6, 2026
Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone
Published on: May 10, 2019
Morpholino artifacts provide pitfalls and reveal a novel role for pro-apoptotic genes in hindbrain boundary
Sebastian S Gerety1, David G Wilkinson
1Division of Developmental Neurobiology, MRC National Institute for Medical Research, London, UK.
Abstract:
Morpholino antisense oligonucleotides (MOs) are widely used as a tool to achieve loss of gene function, but many have off-target effects mediated by activation of Tp53 and associated apoptosis. Here, we re-examine our previous MO-based loss-of-function studies that had suggested that Wnt1 expressed at hindbrain boundaries in zebrafish promotes neurogenesis and inhibits boundary marker gene expression in the adjacent para-boundary regions. We find that Tp53 is highly activated and apoptosis is frequently induced by the MOs used in these studies. Co-knockdown of Tp53 rescues the decrease in proneural and neuronal marker expression, which is thus an off-target effect of MOs. While loss of gene expression can be attributed to cell loss through apoptotic cell death, surprisingly we find that the ectopic expression of hindbrain boundary markers is also dependent on Tp53 activity and its downstream apoptotic effectors. We examine whether this non-specific activation of hindbrain boundary gene expression provides insight into the endogenous mechanisms underlying boundary cell specification. We find that the pro-apoptotic Bcl genes puma and bax-a are required for hindbrain boundary marker expression, and that gain of function of the Bcl-caspase pathway leads to ectopic boundary marker expression. These data reveal a non-apoptotic role for pro-apoptotic genes in the regulation of gene expression at hindbrain boundaries. In light of these findings, we discuss the precautions needed in performing morpholino knockdowns and in interpreting the data derived from their use.
Insights
Morpholino antisense oligonucleotides (MOs) can cause off-target effects by activating Tp53. This study reveals that Tp53-induced apoptosis, not Wnt1, affects neurogenesis and boundary marker genes in zebrafish.
Area of Science:
- Developmental Biology
- Genetics
- Molecular Biology
Background:
- Morpholino antisense oligonucleotides (MOs) are common tools for gene function studies.
- MOs can induce off-target effects through Tp53 activation and apoptosis.
- Previous studies suggested Wnt1 regulates neurogenesis and boundary markers in zebrafish hindbrain.
Purpose of the Study:
- To re-examine MO-based loss-of-function studies on Wnt1 in zebrafish.
- To investigate the role of Tp53 activation in MO-induced effects.
- To understand the mechanisms regulating hindbrain boundary gene expression.
Main Methods:
- Morpholino knockdown in zebrafish.
- Tp53 co-knockdown experiments.
- Analysis of proneural and neuronal marker expression.
- Examination of hindbrain boundary marker gene expression.
- Investigating the role of Bcl genes (puma, bax-a) and the Bcl-caspase pathway.
Main Results:
- MOs used in previous studies strongly activated Tp53 and induced apoptosis.
- Co-knockdown of Tp53 rescued MO-induced decreases in proneural and neuronal markers, identifying these as off-target effects.
- Ectopic expression of hindbrain boundary markers was also dependent on Tp53 activity and apoptosis.
- Pro-apoptotic Bcl genes puma and bax-a are required for normal hindbrain boundary marker expression.
- Gain of function of the Bcl-caspase pathway led to ectopic boundary marker expression.
Conclusions:
- Tp53 activation and apoptosis are significant off-target effects of MOs, impacting gene expression studies.
- Pro-apoptotic genes have non-apoptotic roles in regulating gene expression during hindbrain boundary specification.
- Caution is needed when using MOs and interpreting data from knockdown experiments.
