Most metazoan species lay large eggs with provisions for the entirety of embryogenesis. These eggs begin embryogenesis with rapid cell cycles, and when cell number is adequate to begin development, the cell cycles slow, and zygotic gene expression from the newly amplified nuclei begins to direct the events of morphogenesis (O’Farrell et al., 2004
). This transition from rampant proliferation to morphogenesis is called the midblastula transition (MBT; Newport and Kirschner, 1982a
). Throughout these stages, the mechanisms timing the cell cycle and those timing development are interwoven. We are probing the basis of this temporal control.
embryogenesis begins with 13 rapid, synchronous mitotic cycles that occur without zygotic gene expression. These cycles lack gap phases and cytokinesis. Instead, they oscillate between S phase and mitosis, amplifying nuclei in a syncytial cytoplasm until the MBT, which occurs in interphase of cycle 14. Early interphases extend progressively, beginning as short as 3.4 min, lengthening to 12 min by cycle 13, and abruptly jumping to 90 min or more in cycle 14, the first asynchronous cycle (Foe and Alberts, 1983
; Edgar et al., 1986
; Shermoen et al., 2010
). This temporal course is tightly coupled with development so that cycle 14 is marked by cellularization of the syncytial nuclei and onset of gastrulation.
Usually, mitosis and progress to the next cycle are triggered by Cdc25 phosphatase’s removal of inhibitory phosphate from preformed cyclin–Cdk1 complexes (Russell and Nurse, 1986
; Edgar and O’Farrell, 1989
; O’Farrell, 2001
). Indeed, at the first post-MBT mitosis, cyclins are in excess in Drosophila
, and a pulse of transcription of the string
gene, which encodes a Cdc25 phosphatase, times mitosis (Edgar and O’Farrell, 1989
; Lehner and O’Farrell, 1989
; Edgar et al., 1994
). However, the pre-MBT cycles are independent of transcription, and so, their timing cannot be governed by Cdc25 transcription.
Although it has been suggested that accumulation of cyclin times early rapid embryonic cycles (Murray and Kirschner, 1989
), studies in Drosophila
have instead implicated S-phase duration as the interphase timer. In the earliest S phases, the genome is replicated remarkably quickly by the simultaneous firing of many origins. During cycles 11 to 13, slight but increasing delays in the onset of replication of heterochromatic satellite sequences gradually extend S phase (McCleland et al., 2009a
; Shermoen et al., 2010
). During these cycles, complete deletion of S phase, by blocking the formation of prereplication complexes, shortens interphase, indicating that S phase indirectly or directly times interphase duration (McCleland et al., 2009a
). Mutations that inactivate the S-phase checkpoint, mei-41
(dATR) or grapes
; dChk1), also shorten interphase and, by the time of the thirteenth cycle, result in catastrophe when nuclei enter mitosis with incompletely replicated DNA (Sibon et al., 1997
; Yu et al., 2000
). Thus, gradually lengthening S phase acts through the S-phase checkpoint to govern interphase length.
Some experiments that altered maternal cyclin gene dose showed a cyclin influence on the length of early cycles (Edgar et al., 1994
; Stiffler et al., 1999
). These findings were initially interpreted as an indication that cyclin levels time mitotic entry; however, we would now like to test alternative interpretations that might be more consistent with findings implicating S phase as the interphase timer. Like most organisms, Drosophila
has multiple mitotic cyclins—CycA, CycB, and CycB3—which exhibit partial redundancy (Jacobs et al., 1998
). Consistent with redundancy, RNAi knockdown of all three cyclins was required to arrest early embryos in interphase (McCleland and O’Farrell, 2008
). However, unique defects that were seen when a single cyclin promoted mitosis revealed that the different cyclin types differ in their action (McCleland et al., 2009b
). Because a change in gene dose of a cyclin will alter the relative abundance of cyclin types as well as cyclin level, the consequences might be caused by either of these changes. We have made an effort to distinguish cyclin level and cyclin-type effects on the cell cycle.
Previously, we tested the influence of cyclin level without the obfuscation of changing distributions of cyclin type; two of the three mitotic cyclins were knocked down by RNAi, and the level of expression of the one remaining cyclin was altered by changing gene dose. In this situation, change in the gene dose of the remaining cyclin did not substantially alter interphase duration (), indicating that the accumulation of the cyclin protein does not limit entry into mitosis in this experimental situation (McCleland et al., 2009b
). This finding suggested that cyclin levels are in excess of requirements for mitotic entry, a conclusion in accord with the finding that S-phase duration governs interphase length (Sibon et al., 1997
; McCleland et al., 2009a
Figure 1. Cyclin-type effect on interphase timing. (A) As illustrated for RNAi knockdown of CycA (A) and CycB3 (B3), after pairwise cyclin knockdown, the dose of the remaining cyclin (CycB, B) did not significantly affect interphase length (McCleland et al., 2009b (more ...)
Thus, the past observations might be explained if changes in the distributions of cyclin type influenced interphase duration. This could be understood if the cyclins differed in their potency to trigger mitosis. For example, it was proposed that CycA could be specialized to prime entry into mitosis (Clarke et al., 1992
; Gong et al., 2007
). Alternatively, because we recently showed that down-regulation of all three “mitotic” cyclins in the early cycles extends S phase (Farrell et al., 2012
), perhaps the cyclin types could differ in abilities to accelerate S phase. Surprisingly, however, the cyclin-type input does not involve either of these mechanisms.
We knocked down two cyclins and then evaluated the ability of the remaining one to support interphase 13 progression. Each pairwise knockdown prolonged interphase to a different degree (cyclin-type effect). In the prolonged interphase, S phase was barely extended, and instead, a gap phase was introduced after S phase. Different gap-phase durations accounted for the cyclin-type effect. Inactivation of the DNA replication checkpoint eliminated this new gap phase, restored a short interphase, and removed the cyclin-type effect on interphase duration. Thus, the G2 introduced by cyclin knockdown requires the S-phase checkpoint, indicating that the G2 represents the time of recovery from the checkpoint. Consequently, the cyclin-type effect reflects differences in the ability of the different cyclins to promote checkpoint reversal.