Cyclin E Morpholino Delays Embryogenesis in XenopusYann Audic, Brian Boyle, Michael Slevin, and Rebecca S. Hartley*Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, Iowa

Received 1 May 2001; Accepted 12 June 2001Published online 23 July 2001; DOI 10.1002/gene.1041

INTRODUCTION

Cyclin E is a cell cycle regulatory protein that partnerswith Cyclin-dependent kinase 2 (Cdk2) to phosphorylatesubstrates necessary for the initiation of DNA replicationand histone biosynthesis (Ewen, 2000). Loss-of-functionmutations in Cyclin E in Drosophila and C. elegans resultin underproliferation during embryogenesis. In Drosoph-ila, DmcycE mutant embryos retain maternal cyclin Eand progress through the first 16 mitoses normally butthen arrest in the S phase of cell cycle 17 (Knoblich etal., 1994). In C. elegans, loss-of-function mutations in thecyclin E homolog (cye-1) lead to underproliferation anddefects in fertility and gut cell endoreduplication (Fayand Han, 2000). RNAi knockdown of cye-1 abolishes bothmaternal and zygotic mRNA and results in arrest beforevisible signs of morphogenesis (Fay and Han, 2000).

There are no published reports of loss-of-function mu-tations for cyclin E in vertebrate development. In Xeno-pus laevis, the inhibition of maternal cyclin E/Cdk2complexes by injection of a Cdk inhibitor results inlengthening of the early embryonic cell cycle and de-layed development prior to the midblastula transition(MBT) (Hartley et al., 1997). The MBT marks the onset ofzygotic transcription and occurs following the 12th celldivision, about 6 h postfertilization (hpf). The currentstudy aimed to use morpholinos to knock down bothmaternal and zygotic cyclin E in a vertebrate embryo. Todate, three forms of cyclin E have been cloned in Xeno-pus (cyclin E1A, E1B, and E2) (Chevalier et al., 1996;Rempel et al., 1995). These mRNAs have nearly identicalopen reading frames but differ in the 59 and 39 untrans-lated regions. Maternal cyclin E is synthesized fromstored maternal mRNA and accumulates following fertil-ization, whereas zygotic cyclin E synthesis is thought tobegin at the MBT. It is not clear which of the threecloned Xenopus cyclin E genes represent the maternaland zygotic forms; therefore, we designed a morpholinoto knockdown all three (see footnote in Table 1, CycE-MO). This morpholino spans the translation start site forall three isoforms.

* Correspondence to: Rebecca S. Hartley, I-500 BSB Department of Anat-omy and Cell Biology, College of Medicine, University of Iowa, Iowa City,Iowa 52242.

E-mail: rebecca-hartley@uiowa.edu

TABLE 1. Dose-response distribution for the Cyclin E Morpholinoinjection at 30 and 50 hours post-fertilization. For each dose, thepercentage of embryos at the indicated stage of development (Nieu-wkoop and Faber, 1967) is shown. White columns are uninjected (n5 40), grey columns are 96 ng control-MO (n 5 44), black columnsare 96 ng CycE-MO (n 5 26), horizontal striped columns are 48 ngCycE-MO (n 5 55), and diagonal striped columns are 24 ngCycE-MO (n 5 29) injected embryos. A) Embryos staged at 30 hpf.B) embryos stages at 50 hpf.

CycE-MO: 59-AAAGCCCTGAAATGCCAGTGATAAG-39, Gene-Tools negative control-MO: 59-CCTCTTACCTCAGTTACAATT-TATA-39. Data are from one experiment performed on sibling em-bryos. The experiment was repeated 3 times with similar results, butdata were not compiled due to differences in the speed of devel-opment.

© 2001 Wiley-Liss, Inc. genesis 30:107–109 (2001)

We injected high CycE-MO doses (24–96 ng) to in-crease chances of knocking down all three isoforms.Injection of CycE-MO delayed development in a dose-dependent manner, with the highest dose causing the

most severe delay (Table 1). Developmental delay wasscored according to Nieuwkoop and Faber (1967). At 30hpf, embryos injected with the highest dose (96 ng)ranged from St 22-25, whereas embryos injected with 96

FIG. 1. Delayed development of Xenopus embryos in response to Cyclin E Morpholino injection. Embryos were photographed either at 24h (a–b), or 50 h (c–e) post-fertilization. Embryos were injected with the following amounts of morpholinos and were at these developmentalstages: (a) 96 ng control-MO [St 21/22], (b) 96 ng CycE-MO [St 18 (arrowhead) to St 21], (c) 48 ng control-MO [St 35/36], (d) 24 ng CycE-MO[St 33/34] and (e) 48 ng CycE-MO [St 32]. In (c) the black arrowhead indicates the eye, the white arrowhead the cement gland, and the blackarrow the proctodeum. For (c)–(e), note that this is a different experiment from that shown for the dose response distribution in Table 1. In(f–g) embryos were injected with 192 ng of control-MO (left side) or 192 ng of CycE-MO (right side). Embryos were photographed at 7.5h (f) and 28 h post-fertilization (g). (h) Phosphorimaging of cyclin E-associated cdk2 histone H1 kinase activity. Five embryos from theexperiment shown in (f–g) were immunoprecipitated with cyclin E antibodies at the indicated hour after fertilization (hpf) and associatedkinase activity assayed using histone H1 as a substrate. As indicated below each sample, the amount of histone-H1 incorporatedradioactivity (cpm) was quantified using a phosphorimager.

108 AUDIC ET AL.

ng of a negative control morpholino (control-MO)ranged from St 26–28. At moderate doses of CycE-MO,the distribution was St 24–27 (48 ng) and St 24–26 (24ng). At 50 hpf, CycE-MO-injected embryos had pro-gressed but were still delayed. Embryos injected with 96ng of CycE-MO were the most delayed and developmen-tally spread (St 29/30–39), whereas those injected withlower amounts were clustered between St 33/34–37/38.Embryo microinjection slightly affects developmentaltiming, thus injection of control-MO consistentlly re-sulted in a delay (St 26–28 at 24 hpf), compared withuninjected embryos (St 27/28 at 24 hpf). The amount ofcontrol-MO injected was always equal to the highestamount of cycE-MO and the delay was minimal whencompared to cycE-MO injections. Figure 1a– e shows thedelayed development of the CycE-MO-injected embryosat 24 hpf (a, 96 ng CycE-MO) compared to controls (b,96 ng control MO) and at 50 hpf (c, 48 ng control-MO; d,24 ng CycE-MO; e, 48 ng CycE-MO). In all cases, theinjection of morpholino did not inhibit development perse as embryos developed into swimming tadpoles.

Prior to the MBT, morphological delay of the cell cyclewas variable, most likely due to the cyclin E alreadypresent in the unfertilized egg, plus a variable amount ofprotein synthesized between the time of fertilization andthe time of morpholino injection. Morpholinos wereinjected starting at 30–45 min pf to avoid disruptingcortical rotation (Keller, 1991), whereas the majority ofcyclin E in the embryos is synthesized during the first1.5–2 h following fertilization (Rempel et al., 1995). Tooptimize the chances of knocking down maternal cyclinE, we injected 192 ng of CycE-MO or control-MO andassayed them morphologically for cell cycle delay (Fig-ure 1f). The main criteria to stage blastula embryos is cellsize and number (Nieuwkoop and Faber, 1967). TheCycE-MO-injected embryos (Figure 1f, right side) havefewer and larger cells than the control-MO injected em-bryos (Figure 1f, left side). This infers a longer cell cyclelength, as the embryos undergo fewer divisions in agiven period of time, as evidenced by the larger cells. InFigure 1f, after 7.5 h of development the control-MOinjected embryos are at St 9 (n 5 72), whereas CycE-MO-injected embryos are at St 8 (n 5 34). By 28 hpf, thedevelopmental delay is more drastic as compared withembryos with no pre-MBT delay (compare Figure 1gwith Figure 1a–b), with the majority of control embryosat St 24– 26 (Figure 1g, left side) and CycE-MO embryosat St 19 (Figure 1g, right side).

A cyclin E-specific antibody that immunoprecipitatescyclin E-associated kinase activity (that of Cdk2) wasused to compare relative levels of cyclin E in control andCycE-MO–injected embryos (Figure 1h). In Xenopus em-bryos, upon its synthesis cyclin E complexes with Cdk2(which is in excess), and is rate limiting for Cdk2 activ-

ity. Cyclin E-associated Cdk2 activity was immunopre-cipitated from morpholino-injected embryos and as-sessed using histone H1 as a substrate (Hartley et al.,1997). The immunoprecipitation–kinase data shown(Figure 1h) is from the experiment presented in Figure1f. Cyclin E-associated Cdk2 activity was reduced at bothconcentrations of CycE-MO, with the most dramatic de-crease at 9 hpf with 192 ng (reduced to 20% that of thecontrol-MO– injected embryos), showing that less cyclinE is available for complex formation, and thus Cdk2activation. Cyclin E protein is stable before but not afterthe MBT (Hartley et al., 1997). Therefore, the proteinsynthesized prior to morpholino injection can still formactive complexes with Cdk2, resulting in less inhibitionat 3 and 6 hpf (Figure 1h). Nevertheless, morphologicalanalyses show that this decrease in cyclin E/Cdk2 activ-ity still affects cell cycle length, and thus developmentaltiming. These results are similar to previous work inXenopus, where injection of a Cdk-inhibitor reducedcyclin E/Cdk2 activity and resulted in lengthening of thecell cycle and delayed embryonic development (Hartleyet al., 1997). In this study, we targeted all known cyclinE isoforms. Morpholinos directed against specific iso-forms should prove useful in determining which is thezygotic form and whether there are functional differ-ences between maternal and zygotic isoforms.

ACKNOWLEDGMENTS

We thank M. Rebagliati for advice and comments. B.Boyle and Y. Audic contributed equally.

LITERATURE CITED

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Fay DS, Han M. 2000. Mutations in cye-1, a Caenorhabditis eleganscyclin E homolog, reveal coordination between cell-cycle controland vulval development. Development 127:4049– 4060.

Hartley RS, Sible JC, Lewellyn AL, Maller JL. 1997. A role for cyclinE/Cdk2 in the timing of the midblastula transition in Xenopusembryos. Dev Biol 188:312– 321.

Keller R. 1991. Early embryonic development of Xenopus laevis. In:Kay BK, Peng HB, editor. Xenopus laevis : practical uses in cell andmolecular biology. San Diego: Academic Press. p 61– 113

Knoblich JA, Sauer K, Jones L, Richardson H, Saint R, Lehner CF. 1994.Cyclin E controls S phase progression and its down-regulationduring Drosophila embryogenesis is required for the arrest of cellproliferation. Cell 77:107– 120.

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109CYCLIN E MORPHOLINO DELAYS EMBRYOGENESIS IN XENOPUS