455However, when additional DNA was supplied via the addition of sperm chromatin,
the extracts became sensitive to replication stress and DNA damage and arrested
their cell cycles, indicative of restored checkpoint function.
Several models have been proposed to explain the influence of the N:C ratio on
DNA damage checkpoint acquisition. One hypothesis suggests that pre-MBT
embryos cannot efficiently amplify the DNA damage signaling response in the unusu-
ally large cytoplasm associated with low N:C ratios. Injecting embryos with varying
amounts of dsDNA to mimic DNA double-strand breaks (DSBs), together with plas-
mid DNA to increase the DNA content (and thus N:C ratio), induced a precocious
DNA damage response that activated Chk1, increased phosphorylation of Cdk1, and
caused subsequent cell cycle delay (Conn et al. 2004 ). This activation of the check-
point only occurs at a critical DNA to cytoplasm ratio, demonstrating the importance
of N:C ratio in checkpoint acquisition (Conn et al. 2004 ; Peng et al. 2008 ).
Checkpoint acquisition at the MBT likely centers around Chk1 gain of function.
In zebrafish pre-MBT embryos, checkpoint proteins are maternally supplied, and the
ATM-Chk2 axis responds robustly to DNA damage, but Chk1 is not activated (Zhang
et al. 2014 ). These results suggest that lack of Chk1 activity limits checkpoint func-
tion prior to the MBT. Given these findings and the transient Chk1 activation at the
MBT observed in Xenopus (Shimuta et al. 2002 ), we propose that Chk1 activation is
sensitive to the N:C ratio and that Chk1 is a master regulator of cell cycle remodeling
at the MBT, contributing to both cell cycle elongation and checkpoint acquisition.
9.2.5.2 SAC Acquisition
To examine SAC acquisition, “mini-embryos” with reduced cytoplasmic volume
were created using a modified version of the Spemann method. A loop of baby’s
hair was placed around the animal pole of a newly fertilized Xenopus embryo to
constrict a portion of the nucleus-containing cytoplasm, effectively increasing the
N:C ratio. These mini-embryos had a cytoplasmic volume of about 1/8–1/12 the
size of a normal embryo and continued to cycle like their unperturbed counterparts,
but with a much higher N:C ratio (Clute and Masui 1995 ). The cell cycles of these
mini-zygotes became asynchronous two cycles before the usual time of the MBT
(cleavage 10). At this point, the N:C ratio of the mini-embryos corresponds to the
N:C ratio of unperturbed embryos at MBT (cleavage 12). This finding suggests that
cell cycle remodeling is controlled by the N:C ratio. Surprisingly, however, mitotic
delay after microtubule depolymerization occurred at the same time as in control
embryos, despite the disparity in N:C ratio and precocious cell cycle elongation in
the mini-zygotes. Similar findings were demonstrated with zebrafish embryos:
when pre-MBT cell cycles were artificially lengthened with the addition of acti-
vated Chk1, thereby slowing the increase of the N:C ratio, embryos still acquired a
functional SAC at 3 h post fertilization (hpf), the normal time of the MBT, despite
not having reached the usual MBT N:C ratio (Zhang et al. 2015 ).
These studies suggest that while the onset of cell cycle asynchrony depends on
the N:C ratio, the SAC is acquired at an absolute time that is independent of N:C
ratio. However, they differ from earlier findings in egg extracts, which can activate
9 Cell Cycle Remodeling and Zygotic Gene Activation at the Midblastula Transition