p53 deficiency rescues apoptosis and differentiation of multiple cell types in zebrafish flathead mutants deficient for zygotic DNA polymerase δ1

Abstract

Cell culture work has identified the tumor suppressor p53 as a component of the S-phase checkpoint control system, while in vivo studies of this role of p53 in whole-vertebrate systems were limited. Here, we describe zebrafish mutants in the DNA polymerase delta catalytic subunit 1, based on the positional cloning of the flathead (fla) gene. fla mutants display specific defects in late proliferative zones, such as eyes, brain and cartilaginous elements of the visceral head skeleton, where cells display compromised DNA replication, followed by apoptosis, and partial or complete loss of affected tissues. Antisense-mediated knockdown of p53 in fla mutants leads to a striking rescue of all phenotypic traits, including completion of replication, survival of cells, and normal differentiation and tissue formation. This indicates that under replicationcompromised conditions, the p53 branch of the S-phase checkpoint is responsible for eliminating stalled cells that, given more time, would have otherwise finished their normal developmental program.

Ubc9 Regulates Mitosis and Cell Survival during Zebrafish Development

“…This observation is consistent with data gathered from the phenotypic analysis of morphant embryos, which first develop defects around 48 hpf, indicating that maternal Ubc9 protein is sufficient to compensate for the loss of zygotic Ubc9 protein during earlier stages of development. The high stability of maternal Ubc9 protein is consistent with data obtained for other maternally supplied zebrafish proteins (Ryu et al, 2005;Plaster et al, 2006). In addition, it is consistent with data obtained for Ubc9 in a chick cell line (Hayashi et al, 2002), where Ubc9 protein was still detectable 2 d after gene inactivation, and sumoylated proteins could even be seen for 1 additional day (Hayashi et al, 2002).…”

“…During later development ubc9.1 expression becomes more restricted, with highest transcript levels in the ciliary marginal zone of the eye, the ventricular zones of the brain, and the pharyngeal pouches. All of these domains are zones of late cell proliferation, as indicated by the expression of various marker genes, such as pcna, various cyclins, and DNA polymerase delta1, as well as by their high BrdU-incorporating activity (Plaster et al, 2006). This ubc9.1 expression pattern is in line with a role during cell proliferation in zebrafish larvae and with functional data obtained in other systems.…”

“…These results indicate that the used ubc9.1 MO efficiently blocks translation of ubc9.1 mRNA and that morphant larvae are devoid of Ubc9 protein between 48 and 96 hpf, whereas at earlier stages, maternally supplied Ubc9 protein is present. A similar high protein stability has recently been described for other maternally supplied housekeeping proteins, which could be detected throughout the first 2 d of development, compensating for the loss of zygotic gene products (Ryu et al, 2005;Plaster et al, 2006).…”

“…5-Bromo-2Ј-deoxyuridine (BrdU) incorporation assays were performed essentially as described (Plaster et al, 2006). After labeling, embryos were grown for 2 h at 28.5°C and fixed.…”

Analysis of the retina in the zebrafish model

“…To identify the population of cells that exit the cell cycle in a particular window of time, BrdU labeling can be combined with iododeoxyuridine (IdU) (Del Bene et al, 2008). Finally, another useful technique that can be used to test for cell cycle defects in mutant strains is fluorescence activated cell sorting (FACS) of dissociated retinal cells (Plaster et al, 2006;Yamaguchi et al, 2008).…”