Abstract
p53 is well-known for its tumour-suppressive activity. However, in the past decade it became clear that p53 is also involved in other processes including stem cell proliferation, differentiation and animal development. To investigate the role of p53 in early embryonic development, we targeted p53 by CRISPR/Cas9 to make a p53 knock-out zebrafish (Danio rerio). Our data show developmental and behavioural effects in p53-deficient zebrafish embryos and larvae. Specifically, we found that early development of zebrafish was clearly delayed in the absence of p53. However, after 1 day (1 dpf), the p53-deficient embryos appeared to recover, as evidenced by a similar level of pigmentation at 26 hpf, similar size of the eye at 4 dpf and only a minor difference in body size at 4 dpf compared to p53 wild-type siblings. The recovery of development after 1 dpf in p53-deficient embryos could be due to a compensatory mechanism involving other p53 family members. p63 and p73 were found over-expressed with respect to wild-type siblings. However, despite this adaptation, the hatching time remained delayed in p53-/- zebrafish. In addition to differences in development, p53-null zebrafish embryos also showed differences in behaviour. We observed an overall reduced activity and a reduced travel distance under non-stressed conditions and after exposing the larvae to vibration. We also observed a longer latency until the larvae started to move after touching with a needle. Overall, these data indicate that p53 is involved in early development and locomotion activities.
Highlights
Since their establishment as a model animal, zebrafish (Danio rerio) have been used in basic research and disease modelling
P53 is a transcription factor that binds to promoters of target genes leading to their transcriptional activation. p53 primarily regulates genes that control the cell cycle or induce apoptosis [2]. p53 can induce cell death in a transcription-independent manner, by interacting with pro- and anti-apoptotic proteins in the cytoplasm [3]. p53 is a member of a larger protein family that includes p63 and p73 as well as different splice versions of these three proteins [4]. p53, p63 and p73 are derived from a common ancestor and show a high homology, in the DNA binding region [5]
For knocking-out full-length p53 in zebrafish, we applied the CRISPR/Cas9 system. This system is based on the generation of DNA double strand breaks, which are introduced by the Cas9 protein after being directed to a particular sequence in the target gene via a guide RNA [19]
Summary
Since their establishment as a model animal, zebrafish (Danio rerio) have been used in basic research and disease modelling. In response to DNA damage and some other forms of cellular stress, the p53 protein is stabilised and post-translationally modified, resulting in high levels of active p53 [1]. P53 can induce cell death in a transcription-independent manner, by interacting with pro- and anti-apoptotic proteins in the cytoplasm [3]. P53, p63 and p73 are derived from a common ancestor and show a high homology, in the DNA binding region [5]. All three genes have different activities and regulate transcription of unique target genes, they regulate some common target genes under certain conditions
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