Current Biology 23, 127–132, January 21, 2013 ª2013 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.11.029
of Cdc25 Degradation Terminates
Drosophila’s Early Cell-Cycle Program
Stefano Di Talia,1Richard She,1Shelby A. Blythe,1
Xuemin Lu,1,2Qi Fan Zhang,1and Eric F. Wieschaus1,*
1Howard Hughes Medical Institute, Department of Molecular
Biology, Princeton University, Princeton, NJ 08544, USA
In most metazoans, early embryonic development is charac-
terized by rapid mitotic divisions that are controlled by
maternal mRNAs and proteins that accumulate during
oogenesis . These rapid divisions pause at the midblas-
tula transition (MBT), coinciding with a dramatic increase
in gene transcription and the degradation of a subset of
maternal mRNAs [2, 3]. In Drosophila, the cell-cycle pause
is controlled by inhibitory phosphorylation of Cdk1, which
in turn is driven by downregulation of the activating Cdc25
phosphatases [4, 5]. Here, we show that the two Drosophila
Cdc25 homologs, String and Twine, differ in their dynamics
and that, contrary to current models , their downregula-
tions are not controlled by mRNA degradation but through
different posttranslational mechanisms. The degradation
rate of String protein gradually increases during the late
syncytial cycles in a manner dependent on the nuclear-
to-cytoplasmic ratio and on the DNA replication check-
points. Twine, on the other hand, is targeted for degradation
at the onset of the MBT through a switch-like mechanism
controlled, like String, by the nuclear-to-cytoplasmic ratio,
but not requiring the DNA replication checkpoints. We
demonstrate that posttranslational control of Twine degra-
dation ensures that the proper number of mitoses precede
The MBT marks the transition from the maternally controlled
amplification of nuclear number to the zygotically directed
program of differentiation and morphogenesis. Pre-MBT
nuclear amplification is driven by an abbreviated cell cycle
until the embryo reaches a defined ratio between DNA and
cytoplasmic content (N/C ratio) [1, 6, 7]. At this critical N/C
ratio, the cell cycle is rapidly reprogrammed through switch-
like inhibition of Cdk1 —driven by downregulation of the
two Cdc25 homologs present in Drosophila (String and
Twine)—to ensure a pause.
To test whether regulation of String and Twine protein rather
than mRNA provides the switch controlling the rapid inactiva-
tion of Cdk1 at the MBT, we used live imaging of embryos
maternally expressing His2Av-RFP to follow the behavior of
nuclei and String-GFP or Twine-GFP. String-GFP and Twine-
GFP were expressed using the Gal4/UAS expression system
and lacked the endogenous 30UTR mRNA sequences, which
were replaced by the SV40 30UTR sequence (Figures 1A and
1B). Because 2- to 3-fold overexpression of Twine is reported
to alter cell-cycle behavior at the MBT , we selected UAS
lines with expression levels below 50% of wild-type (see
Figure S1A available online), thereby ensuring only a slight
increase in total Twine levels. We confirmed the results ob-
tained from live imaging experiments of the transgenic
constructs by dual immunofluorescence quantification of
String and Twine (Figure 1C).
Live imaging and immunofluorescence data show that
String and Twine proteins preferentially localize to nuclei
during interphase of cleavage cycles and during early cycle
14 (Figures 1A and 1B). Both proteins disperse in the cyto-
plasm during mitosis due to nuclear envelope breakdown
(see Movies S1 and S2). Interestingly, although both String
and Twine are absent from nuclei by 20 min into cycle 14,
the dynamics of their downregulation are significantly
different. Consistent with previous results [4, 5], downregula-
tion of String does not happen specifically at the MBT but
precedes it, following a stepwise pattern of reduction during
cycles 10–13 (Figure 1D). Twine, on the other hand, does not
show significant downregulation during cycles 10–13 (Fig-
ure 1E). High levels of Twine are still present in nuclei at the
and by 20 min into cycle 14 Twine is no longer detectable
(Figure 1E). These results indicate that Twine, but not String,
is downregulated specifically at the MBT and could provide
the required switch for the cell-cycle pause.
In contrast to other systems [8, 9], quantification of the
nuclear and cytoplasmic levels of both String and Twine
indicates that they are not controlled by regulation of the
nucleocytoplasmic transport or by the activity of the
anaphase-promoting complex (see Supplemental Information
and Figure S1).
To determine whether degradation of maternal mRNA con-
trols the protein decline, we compared the protein dynamics
with the mRNA dynamics measured by quantitative RT-PCR
of precisely staged embryos. Consistent with previous results
, we found that string mRNA decline follows protein decline
with a significant lag (at least 15 min). At the beginning of cycle
14, string mRNA levels are still 60% of the cycle 12 levels,
Similarly, the decline in Twine protein precedes changes in
15 min into cycle 14 and is rapidly degraded in the 20 min that
follow (Figure 1E). The rapid downregulation of Twine protein
is observed significantly before the degradation of the
mRNA, and Twine is essentially absent when twine mRNA
we conclude that mRNA levels do not control the dynamics
of String and Twine prior to the MBT.
pause occurs in cycle 15 rather than 14 . The 14thmitosis
occurs about 15 min into cycle 14, at a time when both String
and Twine would normally be almost absent (Figures 2A and
2C). We found that, in haploid embryos, the downregulation
of both String and Twine is delayed by about one cell cycle
(Figures 2A and 2C), but, similar to diploids, the two proteins
2Present address: Verna and Marrs McLean Department of Biochemistry
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