Journal of Assisted Reproduction and Genetics

, Volume 27, Issue 6, pp 265–276

Genome-wide microarray evidence that 8-cell human blastomeres over-express cell cycle drivers and under-express checkpoints

  • Ann A. Kiessling
  • Ritsa Bletsa
  • Bryan Desmarais
  • Christina Mara
  • Kostas Kallianidis
  • Dimitris Loutradis
Open AccessEmbryo biology

DOI: 10.1007/s10815-010-9407-6

Cite this article as:
Kiessling, A.A., Bletsa, R., Desmarais, B. et al. J Assist Reprod Genet (2010) 27: 265. doi:10.1007/s10815-010-9407-6

Abstract

Purpose

To understand cell cycle controls in the 8-Cell human blastomere.

Methods

Data from whole human genome (43,377 elements) microarray analyses of RNAs from normal 8-Cell human embryos were compiled with published microarrays of RNAs from human fibroblasts, before and after induced pluripotency, and embryonic stem cells. A sub database of 3,803 genes identified by high throughput RNA knock-down studies, plus genes that oscillate in human cells, was analyzed.

Results

Thirty-five genes over-detected at least 7-fold specifically on the 8-Cell arrays were enriched for cell cycle drivers and for proteins that stabilize chromosome cohesion and spindle attachment and limit DNA and centrosome replication to once per cycle.

Conclusions

These results indicate that 8-cell human blastomere cleavage is guided by cyclic over-expression of key proteins, rather than canonical checkpoints, leading to rapidly increasing gene copy number and a susceptibility to chromosome and cytokinesis mishaps, well-noted characteristics of preimplantation human embryos.

Keywords

Aurora kinaseCell cycleCLOCKCyclinsHuman embryoRB

Introduction

The human blastomere at the 8-cell stage of embryo development is a unique, totipotent cell. Despite the fundamental importance to human reproduction, little is known about cellular controls on gene expression and chromosome duplication in 8-Cell stage human blastomeres. Understanding cell cycle regulation in the 8-Cell is urgently needed to improve outcomes of assisted reproduction and provide insights into the cellular machinery used by the egg to reprogram nuclei from differentiated to totipotent. This information may markedly improve the efficiency of reprogramming unfertilized oocytes and somatic cells to patient-specific stem cells for research and therapy.

We recently reported that RB, the key G1 cell cycle checkpoint, and WEE1, a key G2 cell cycle checkpoint, were silent on microarrays of two pools of normal appearing 8-Cell stage human embryos, whereas MYC and circadian oscillators CLOCK, CRY and PER were markedly elevated [1]. Rb maintains cell cycle arrest in G1 by binding the E2F transcription factors needed for progression through G1 and the initiation of S phase. To overcome the Rb blockade, growth factors stimulate expression of CyclinD, which binds Cdk4/6 and phosphorylates Rb in a couple of locations, causing the release of E2F transcription factors that stimulate expression of Cyclin E, which binds Cdk2 and phosphorylates Rb again, rendering it inactive. Loss of functional Rb, the first tumor suppressor gene discovered, is a characteristic of many human cancers [2], suggesting that understanding cell cycle regulation in the 8-Cell may also provide unique insights into malignant transformation. Mouse fibroblasts depleted of Rb become growth factor-independent [3], as are early cleavage stage blastomeres [4, 5], which suggests the silence of RB, also reported for early cleaving mouse embryos [6], may be characteristic of totipotent embryo cells, reflecting the lack of a need for cell growth because of their large size. Like other mammals, early human embryo cell cycles are cleavage divisions that halve the volume of daughter blastomeres. Approximately six cleavage divisions, to the 64-cell stage, are needed to reduce the size of the 120 micron diameter human egg to the 10 to 20 micron diameter of normal somatic cells and embryonic stem cells (discussed in [7]).

Consistent with the absence of Rb, CCNE was markedly elevated on the microarrays of the 8-Cells [1]. In addition to activating Cdk2, Cyclin E has Cdk2-independent functions, including regulating centrosome duplication [8]. CDK2 knock-out mice are viable, presumably because Cyclin E can also bind Cdk1, but CCNE knock-out mice die in utero [9]. Cyclin E promotes cell division, and over-expression promotes aneuploidy, as has been observed for numerous human malignancies, although the mechanisms by which Cyclin E induces tumorigenesis remain elusive [10]. The silence of Wee1, the G2 checkpoint kinase that blocks cell division by inhibiting CyclinB/Cdk1 [11], suggests that Cyclin E driven aneuploidy may go unchallenged at the G2/M transition in the 8-Cell embryo. This may help explain the heteroploidy observed in early human and non-human primate embryo blastomeres [1214]. But given the fundamental importance to development of accurate chromosome duplication and allocation to daughter blastomeres, the absence of G1 and G2 checkpoints was a surprise. The over-expression of circadian oscillators in the 8-Cells [1] suggests they may help regulate the blastomere cell cycle, as has been demonstrated for many cell types, including mouse liver and muscle [15], rat fibroblasts [16] and human bone marrow [17]. By regulating WEE1 expression through its E-Box elements, mouse liver cell oscillators ensure mitosis is coupled to their circadian cycle [18].

The silence of WEE1 on the 8-Cell microarrays indicates this is not, however, a mechanism used by blastomeres. Moreover, despite recent evidence that circadian oscillators are over-expressed in the 8 Cells, and expression of Per is stimulated at fertilization of zebra fish eggs, heralding the onset of circadian oscillations in zebrafish embryos [19], cell division occurs at intervals much shorter than 24 h in early embryo cells, including human. In vivo developed mouse blastocysts reach 250 cells, eight cell doublings, by the time of implantation 5 days after fertilization, an average cell cycle time of 15 h, not 24 [4, 20]. This is accomplished by cell cycle times of 8 to12 h for the 4- to 16-cell stages, with earlier and later stages closer to 24 h, suggesting some stages are circadian intervals, but others undergo two or three cleavage divisions in one circadian interval. Similar data are not available for human embryos developing in vivo, but in vitro development to at least the 8-cell stage is also less than 24 h per cell cycle, especially for those embryos thought to be the most likely to produce offspring [21].

If the absence of G1 and G2 checkpoints accounts for rapid cell cycles in the human blastomeres, what safeguards are in place to ensure the integrity of chromosomal duplication and allocation to daughter blastomeres? Is it possible that to allow the production of sufficient embryonic proteins to signal the mother to maintain a receptive uterus, gene copy number is more important in early embryo development than accurate chromosome allocation to daughter cells? Evidence suggests that only one or two of the first 16 cells of the mouse morula give rise to the inner cell mass (ICM), which will become the embryo; the remainder of the cells become trophoblast which gives rise to placenta. Perhaps accurate chromosome allocation first becomes essential to development in the ICM, suggesting that cell cycle checkpoints first appear in ICM cells, perhaps concomitant with the need for growth factors to increase cell size, and that the cell death commonly observed in human blastocysts developed in vivo or in vitro [22, 23] reflects the elimination of defective cells, as noted for frog embryos at the midblastula transition [24].

To better understand the cell cycle in the 8-Cell human embryo, we compiled a gene data set from published high throughput strategies not feasible with human embryos that correlate cell morphology and DNA content with expression of cell cycle and cycling genes [2527]. The compiled gene list corresponds to 3,803 gene elements in our database (8CFES) of whole human genome microarrays of four cell types: 8-Cell embryos, fibroblasts before and after induced pluripotency (iPS cells) and two lines of human embryonic stem (hES) cells (Supplemental Table 1S). We then took advantage of two recent reports of MYC transcriptome [28, 29] to assess the role of the over-expression of MYC in the 8-Cells. We report here the results of this analysis which add several gene elements to cell cycle regulation, and reveal unique gene expression characteristics of the 8-Cell human blastomere.

Methods and materials

Embryos were donated by couples undergoing infertility treatment at Alexandra Hospital in Athens Greece, as described [1, 30]. The study protocol was reviewed and approved by the ethics advisory board and human subjects research committees of Alexandra Hospital and Bedford Research Foundation. Briefly, three embryos were selected for transfer 48 h following egg collection. Supernumary embryos were cultured an additional 24 h, and normal appearing, unfragmented, 8-Cell embryos were transferred to microfuge tubes in 2 ul of culture medium and flash frozen in liquid nitrogen for RNA extraction. A total of ten 8-Cell stage embryos from nine couples, divided into two pools of five each were used for microarray analyses. Seven of the nine couples achieved a pregnancy.

RNAs were isolated and amplified for fluorescent probe labeling and hybridization to Agilent whole genome microarrays as described [1]. Briefly, RNAs were isolated with Arcturus PicoPure according to manufacturer’s instructions, examined for ratios of 18S and 28S RNAs with an Agilent Bioanalyzer to assess RNA integrity, and polyadenylated RNAs were amplified to microgram quantities according to a previously reported protocol [31] shown to be linear. Microarray hybridization was done by MoGene, St. Louis, MO, and the raw hybridization intensities normalized by Agilent Feature Extraction Software. The hybridization results were verified for standard RT-PCR housekeeping genes as described [1], and combined into a Filemaker Pro database with microarray data posted at GEO (NCBI) for fibroblast RNAs before and after induced pluripotency [32] and two human embryonic stem cell lines, H9 and HES01 [33], aligned by Agilent feature number [1]. Normalized array data for each gene element were statistically compared to develop guidelines for over- and under-detection [1]. The ratio between the two pools of 8Cell embryos of fluorescence values for each gene element was 1.0 ± 3.0 [1]. For purposes of this report, two standard deviations (±7-fold) of the mean ratios of gene element fluorescent detection among the arrays was taken as the threshold for designation of over- or under-detection, as described [1].

Gene lists were submitted to DAVID (david.abcc.ncifcrf.gov) and Reactome (www.reactome.org) for function and ontology analyses, and hand annotated for accuracy. Because of the silence of canonical checkpoint genes, such as RB and WEE1, and the over-detection of unusual cyclins, such as Cyclin A1, standard pathway analysis tools were not relied upon.

Results and discussion

A comprehensive list of genes important to cell division was compiled from three published studies of high through-put analyses of cultured cells not possible with human embryos. U20S cells, a well studied osteosarcoma cell line, were cultured with small interfering RNA (siRNA) targeting 34,373 human genes [27]. Automated single-cell fluorescence microscopy gathered data for each siRNA: cell number; percentage of cells in G1, S and G2/M; percentage of cells with >8N hyperploidy; and percentage of cells with cytokinetic defects. Depletion of 1,152 gene transcripts strongly affected cell-cycle progression. HeLa cells were cultured with endoribonuclease-prepared short interfering RNAs (esiRNAs) targeting 17,828 genes, followed by propidium iodide staining and flow cytometry analysis of DNA content [26]. Seven parameters were extracted from the DNA content analysis: G1, S, G2/M, 8N (polyploid), 4–8N (aneuploid), percent dead cells, plus mitotic index and cell size. This approach identified 1,351 genes involved in the HeLa cell cycle. RNAs were isolated for microarray analysis at 2 h intervals following synchronization of early passage human foreskin fibroblasts [25]; 480 cycling genes were identified.

The 3,803 gene elements in 8CFES (Supplemental Table S1) that corresponded to all the genes identified by the three studies DAVID-mapped to 2,377 genes, only 256 of which were designated GO term “cell cycle.” Differential expression of the genes identified in each study is described in more detail in supplementary material (Tables S1S3). Only 122 gene elements identified by the U2OS cell knock-down studies were also identified by one of the other studies, only 186 gene elements identified by the HeLa cell studies corresponded to elements in one of the other two studies, and only 136 of the cycling fibroblast genes corresponded to genes identified in the U2OS or HeLa cells. This striking lack of agreement among the three studies undoubtedly reflects experimental differences, but may more importantly emphasize the cell-type specificity of cell cycle control and the functional redundancy of many genes.

Genes in U20S and HeLa identified by RNA knock-down and cycled in fibroblasts

Only nineteen genes were identified by all three studies (Table S3), which meant they registered a phenotype in both U2OS and HeLa [26, 27] and their expression also cycled in the primary fibroblasts [25]. Seventeen were DAVID-annotated cell cycle (Table 1), and 12 were reported to be Myc targets [28, 29], which did not predict elevated levels of detection on the 8-Cell arrays. Interestingly, none brought about a cell cycle block in G1 and only two (CDC2 and PLK1) brought about a block in G2. This supports the cell-type specificity of gene expression needed for progression through G1 and G2.
Table 1

Detection levels of genes on 8CFES arrays

Cell cycle stage

Gene name

aCC

bM

Microarray fluorescence units

Entrez gene ID

8Cell (Ave)

Fibro

iPS

hES (Ave)

Detected in U2OS and HeLa and cycle in primary fibroblasts

S

CDC6

y

x

8,735

281

3,165

6,673

990

S

CHAF1A

y

 

15,120

1,162

7,100

12,999

10036

S

RRM2

y

x

18,568

1,063

2,986

3,072

6241

S

SLBP

 

x

31,828

6,272

4,242

5,342

7884

S

WEE1

y

x

61

180

652

1,223

7465

G2

CDC2

y

 

8,742

910

3,930

5,789

983

G2

PLK1

y

 

42,907

1,973

8,069

14,937

5347

M

AURKB

y

 

10,493

136

956

882

9212

M

CDCA8

y

x

36,169

2,787

6,010

9,599

55143

M

ESPL1

y

x

5,912

476

2,562

3,227

9700

M

FBXO5

y

x

10,661

367

1,064

2,620

26271

M

SFRS3

 

x

35,161

23,423

42,175

68,725

6428

M

TPX2

y

 

21,321

1,505

3,037

5,854

22974

Detected in U2OS and HeLa

G1

CUL2

y

 

3,696

385

308

573

8453

G1

KCNH5

  

5,949

2

3

19

27133

S,G1

RKHD2

 

x

7,737

34

127

120

51320

M

CYB561D1

  

13,547

368

254

4,617

284613

M

INCENP

y

x

8,945

241

622

1,812

3619

M

PLXNA1

  

242

6,425

4,546

5,157

5361

M,G1

CRKRS

 

x

3,645

64

96

248

51755

M,G1

WIPI1

  

156

17,639

1,211

2,071

55062

Detected in U2OS and cycle in primary fibroblasts

G1

BUB1

y

x

7,848

443

3,955

5,369

699

S

GINS2

y

 

31,973

963

5,092

7,624

51659

S

KIFC1

y

x

13,939

119

700

1,772

3833

G2

CABYR

  

10,963

399

1,040

1,162

26256

G2

CDKN1B

y

 

37

773

595

680

1027

M

BARD1

y

x

8,486

515

1,836

2,453

580

M

CDC25B

y

 

28,304

4,449

538

1,061

994

M

PRKD2

  

9,218

597

859

2,278

25865

M

REEP4

  

4,258

449

281

621

80346

Detected in HeLa and cycle in primary fibroblasts

G1

CCNE1

y

x

102,960

1,596

8,037

13,034

898

G1

HJURP

  

11,644

357

1,430

2,133

55355

G1

NEK2

y

x

2,792

245

595

665

4751

G1

RBBP6

y

 

22,492

72

226

260

5930

S

BUB1B

y

x

17,028

439

3,941

6,268

701

S

LMNB2

 

x

221

937

1,681

5,232

84823

S

NPAT

y

 

2,058

66

206

375

4863

S

PCNA

y

x

84,348

5,278

11,990

17,444

5111

G2

DTL

y

x

5,010

250

1,614

3,345

51514

G2

H1F0

y

 

10,711

943

344

1,098

3005

G2

TIPIN

y

x

7,815

1,060

3,315

4,746

54962

M

AURKA

y

x

46,306

1,522

3,234

5,797

6790

M

CCNA2

y

 

31,573

344

1,538

2,710

890

M

CDC20

y

x

29,105

688

4,203

5,406

991

M

CENPE

y

 

7,700

755

1,972

3,390

1062

M

CIT

y

 

356

3,871

1,749

4,732

11113

M

KIF23

y

 

22,920

1,600

4,376

5,526

9493

M

ORC1L

y

x

2,007

66

812

1,326

4998

Cycle in primary fibroblasts

S

GMNN

y

x

62,273

2,432

9,468

19,070

51053

G2

UBE2C

y

x

144,929

2,903

12,472

28,375

11065

M

CCNB1

y

x

156,398

4,004

13,791

19,417

891

M

CKS1B

y

x

87,120

5,491

20,061

33,098

1163

M

PTTG1

y

x

579,305

15,479

31,030

41,398

9232

M

SKP2

y

x

26,057

159

1,139

1,873

6502

aKnown cell cycle gene

bMyc target (27,28)

Knock-down of eight of the cycling genes blocked HeLa and U20S in S phase, including WEE1, which was a surprise because although it is known that Wee1 phosphorylation of Cdk can arrest DNA-damaged cells in both S and G2, a role for Wee1 in normal progression through S phase has not been described. Three of the eight genes were over-detected in 8-Cells and hES cells relative to fibroblasts (Table 1): CDC6, an essential DNA replication licensing factor; CHAF1A, chromatin assembly factor 1, part of a nuclear complex that assembles histones onto newly replicated DNA; and RRM2, ribonucleotide reductase 2, a well known rate-limiting enzyme for DNA replication. The two genes whose knock-down blocked G2 in both HeLa and U20S, CDC2 (CDK1, cyclin dependent kinase), and PLK1 (pololike kinase 1), were both over-detected on the 8-Cell and hES gene arrays relative to fibroblasts. Both Cdc2 and Plk1 have numerous roles in cell cycle progression [34, 35].

Six cycling genes were needed for mitosis in both cell types, one of which, AURKB, was >10-fold over-detected on the 8-Cell arrays relative to all other cell types (Table 1). Aurora kinase B is a chromosomal passenger protein whose expression for normal cytokinesis is tightly controlled: over-expression or under-expression leads to centrosome amplification, defects in spindle formation, and multinucleation [36]. Four of the six cycling mitosis genes were over-detected on both 8-Cell and hES arrays relative to fibroblasts: CDCA8 (borealin), ESPL1 (separase), FBX05 (Emi1) and TPX2. Borealin is a chromosomal passenger protein required for stability of the bipolar mitotic spindle [37] and recently reported to be essential for cleavage from the 2-cell to 4-cell stage of mouse embryos [38]; separase cleaves cohesin to allow the separation of sister chromatids [39] and cleaves the linker between mother and daughter centrioles to license centriole duplication [40]; Emi1 represses APC/Ccdh1 in G2 [36], thus blocking the degradation of Cyclin A, -B and geminin, and allowing both the formation of new DNA replication initiation sites and the prevention of re-replication of DNA before mitosis. Emi1 is essential for mouse embryo development beyond the blastocyst stage [41] and for normal development in zebrafish [42]. Failure to degrade Emi1 in prophase leads to “mitotic catastrophe”, including centrosome overduplication [41]. Tpx2 is expressed exclusively at the G1/S transition and throughout G2 and activates Aurora kinase A on centrosomes to assemble mitotic spindles [43, 44].

Cell cycle genes identified by RNA knock-down in U20S and HeLa

Fifty three gene elements were identified as inhibiting cell cycle progression in both the U2OS and HeLa cells, but were not identified as cycling by the primary fibroblast study. Only 12 DAVID-mapped to cell cycle or DNA metabolism, suggesting the discovery of many new genes important for cell cycle progression that may not have redundant functions and that should be GO designated cell cycle. Interestingly, several were not detected above background levels in 8CFES (Tables S1S3). Twenty were reported targets of Myc [39, 40]. Eleven genes arrested G1, 10 arrested S phase, 10 arrested G2, and 22 arrested mitosis.

KCNH5 (Eag2), one of the knocked-down genes that arrested both cell types in G1, was markedly over-detected on the 8-Cell arrays (Table 1, and 2), but nearly silent on all other 8CFES arrays. Eag2 is an outwardly rectifying voltage-gated potassium channel recently shown to bind to alpha and beta-tubulin [45] suggesting it may play an important role in the cytoskeleton of the large 8-cell blastomeres. Knock-down of RKHD2 (MEX3C), the human homolog of the C. elegans Mex-3 gene, arrested U20S in G1 and HeLa cells in S phase. One allele of RKHD2 was also markedly over-detected on the 8Cell arrays (Table 1) which is especially interesting because in combination with CUL2 (encodes the ubiquitin ligase also over-detected on the 8-Cell arrays), it has important roles in anterior-posterior cell partitioning in C. elegans embryos [46]. Only CUL2, not RKHD2 nor KCNH5, DAVID-mapped to cell cycle.
Table 2

Functional categories of cell cycle genes

Gene name

Expression

Entrez gene ID

Known CC

Myc target

8C Over

8C,hES Over

8C Under

Cyclin, cyclin-dependent kinase (cdk), cdk-inhibitor

CCNE1

x

  

898

y

x

CDC25B

x

  

994

y

 

CCNA1

x

  

8900

y

 

CCNA2

 

x

 

890

y

 

CCNB1

x

  

891

y

x

CCNB3

x

  

85417

y

 

CCND1

  

x

595

y

x

CDC2

 

x

 

983

x

 

CDC2L1

x

  

984

y

 

CDKN1A, -B

  

x

1026

y

 

CRKRS

x

  

51755

 

x

CDK6

  

x

1021

y

x

Kinase, kinase inhibitor, phosphatase

AURKA,-B

x

  

6790, 9212

y

x

AURKC

x

  

6795

y

 

BUB1,-1B

 

x

 

699, 701

y

x

CKS1B

 

x

 

1163

y

x

MAPK4(ERK3)

x

  

5596

  

NEK2

x

  

4751

y

x

PPMID

x

  

8493

y

x

PLK1

 

x

 

5347

y

 

PRKD2

 

x

 

25865

  

SKP2

x

  

6502

y

x

WEE1

  

x

7465

y

x

Transcription, translation

E2F3

  

x

1871

y

x

MYC

x

  

4609

y

 

RB

  

x

5925

y

x

RBBP4

 

x

 

5928

y

x

RBBP6

x

  

5930

  

RBM14

 

x

 

10432

y

x

RKHD2

x

  

51320

 

x

DNA replication, chromosome duplication, cohesin

CDCA8(borealin)

 

x

 

55143

y

x

CDC6

 

x

 

990

y

x

CHAF1A

 

x

 

10036

y

x

CDC45L

 

x

 

8318

y

 

CDT1

 

x

 

81620

y

x

DTL

 

x

 

51514

y

x

ESPL1(separase)

x

 

9700

y

x

GINS2,-3

 

x

 

51659, 64785

y

 

GINS4

x

  

84296

y

 

GMNN

 

x

 

51053

y

x

H1FO

x

  

3005

y

 

HJURP

 

x

 

55355

  

NPAT

x

  

4863

y

 

ORC1L

 

x

 

4998

y

x

PCNA

 

x

 

5111

y

x

POLS

x

  

11044

y

x

PTTG1,-3

x

  

9232, 26255

y

 

PTTG2

x

  

10744

y

 

RFC3

 

x

 

5983

y

 

RRM2

x

  

6241

y

x

TERF1

  

x

7013

y

 

TIPIN

 

x

 

54962

y

x

STAG3,-3L3

x

  

10734, 442578

y

x

Centrosome, centriole, securin, spindle

CENPE

 

x

 

1062

y

 

CDC20

 

x

 

991

y

x

DLG7

x

  

9787

y

 

INCENP

x

  

3619

y

x

TPX2

 

x

 

22974

y

 

Ion channel, structural protein, other

AVPI1

x

  

60370

y

x

BARD1

 

x

 

580

y

x

CABYR

x

  

26256

  

FBXO5(EMI1)

 

x

 

26271

y

x

KCNH5

x

  

27133

  

KIF23

 

x

 

9493

y

 

KIFC1

    

y

x

LMNB2

  

x

84823

 

x

CUL2,-5

x

  

8453, 8065

y

 

CIT

  

x

11113

y

 

REEP4

x

  

80346

  

Knockdown of a cytochrome B-like gene (CYB561D1) and a CDC2-related kinase, CRKRS (CRK7), the exact functions of which are unknown, blocked mitosis in both U20S and HeLa and did not DAVID-map to cell cycle. CYB561D1 was detected at higher levels on the 8-Cell and hES cell arrays than either the fibroblast or iPS cell arrays (Table S3), whereas CRKRS was detected at higher levels on the 8-Cell arrays than all other cell types (Table 1).

Not surprising, knock-down of INCENP, inner centromere passenger protein, blocked mitosis in both U20S and HeLa, but it was surprisingly over-detected in 8-Cells relative to the other cell types, especially fibroblasts and iPS cells. In mammalian cells, 2 broad groups of centromere-interacting proteins have been described: constitutively binding centromere proteins and ‘passenger,’ or transiently interacting, proteins. The constitutive proteins are encoded by CENPA, -B, -C1 and -PD, none of which were identified by the RNA knock-down studies (Table S1). The term ‘passenger proteins’ encompasses a broad collection of proteins that localize to the centromere during specific stages of the cell cycle [47, 48]. These include CENPE and -F, cytoplasmic dyneins (e.g. DYNC1LI2, DYNLL1, DYNLRB1, Table S1) and INCENP. The passenger proteins display a broad localization along chromosomes in the early stages of mitosis but gradually become concentrated at centromeres as the cell cycle progresses into mid-metaphase. During telophase, the proteins are located within the midbody in the intercellular bridge, where they are discarded after cytokinesis. INCENP and CENPE knockout mice die at peri-implantation stage [49].

Cell cycle genes identified by RNA knock-down in U20S cells and cycle in fibroblasts

Eighteen genes were detected by knock-down in U2OS and also cycled in primary fibroblasts (Tables S1 and S3), of which 12 had previously been designated GO term cell cycle. Four blocked G1, two blocked S phase, four arrested G2, and eight blocked mitosis. Eleven are known cell cycle genes and seven are Myc targets.

Knock-down of BUB1brought about a G1 arrest in the U20S cells, a surprise because this gene encodes a kinase involved in spindle checkpoint activation, suggesting it plays a role in M phase. Mutations in this kinase, over-detected in 8-Cells, iPS cells and hES cells relative to fibroblasts, have been associated with aneuploidy and several forms of cancer [50].

One of the cycling genes in this group whose knock-down blocked U20S cells in S phase, GINS2, part of the DNA replication initiation complex, was over-detected on 8-Cells and hES cells, and one, KIFC1, a kinesin family member central to chromosome congression [51], was markedly over-detected on the 8-Cell arrays (Table 1). Only one of the knocked-down genes in this group that blocked U20S cells in G2 was over-expressed in the 8-Cells, CABYR, a calcium-binding protein, which did not DAVID-map to cell cycle. Originally thought to be testis specific, and a marker for testis cancer, several non-testis variants of this gene have been described [52]. CDKN1B (p27) was under-detected specifically on the 8-Cell arrays, thus removing another potential cell cycle block.

Of the 8 cycling genes in this group whose knock-down blocked mitosis, two were over-detected in 8Cells and hES cells, BARD1 and PRKD2, and two were over-detected specifically in 8Cells, CDC25B and REEP4. Bard1 is important to centrosome duplication and mitotic spindle assembly [53], and essential for mouse embryo development [54]; PRKD2 is a serine/threonine protein kinase about which little is known. CDC25B activates the cyclin dependent kinase CDC2 (CDK1) and is required for entry into mitosis; REEP4 is a membrane receptor accessory protein about which little is known.

Cell cycle genes identified by RNA knock-down in HeLa cells and cycle in fibroblasts

Fifty six genes were identified in the HeLa cells that also cycled in the synchronized primary fibroblasts (Tables S1 and S3), 32 of which DAVID-mapped to cell cycle, and 17 of which were identified as Myc targets. Fifteen blocked G1, 16 blocked S phase, five blocked G2, and 19 blocked mitosis.

Of the 15 cycling genes that blocked G1 progression in HeLa when knocked-down, two were over-detected on the 8-Cell arrays, CCNE1 and RBBP6, and two, HJURP and NEK2 were over-detected on both 8-Cells and hES cells. RBBP6 (retinoblastoma binding protein 6) is a multifunctional protein known to have Rb-independent activities [55]. The over-expression of HJURP, Holliday junction-recognizing protein, suggests the 8-Cells and hES cells may invoke a response to DNA double-strand breaks first described for cancer cells that prevents apoptosis in response to DNA damage and continues cell cycling [56]. Recent work suggests HJURP stabilizes Cenp A, the anchor protein for centrosome formation, and is therefore important for normal cytokinesis [57, 58]. NEK2 encodes a kinetochore-associated protein kinase that stabilizes chromosome attachment to spindle microtubules [59].

Of the 16 knocked-down genes that blocked HeLa in S phase, three were over-detected on the 8-Cell and hES cell arrays relative to fibroblasts, BUB1B, NPAT and PCNA, and one was markedly under-detected, LMNB2. Bub1B (BubR1) is a critical member of the spindle assembly checkpoint [60]. NPAT is an essential member of the p220(NPAT)/HiNF-P pathway, controlled by CyclinE/Cdk2, which activates histone H4 gene expression, essential to accurate packaging of newly synthesized DNA into chromatin [61]. PCNA, proliferating cell nuclear antigen, is a member of the DNA sliding clamp family, forming a trimeric, head to tail ring around helical DNA. It is a processivity factor for DNA polymerase delta, and also has other functions in cell cycle control [62, 63].

The under-expression of LMNB2 specifically in the 8-Cells is intriguing. The lamins, A/C, B1 and B2, are intermediate filament proteins that make up the nuclear matrix next to the inner nuclear membrane. During mitosis, the lamina matrix is reversibly disassembled as the lamin proteins are phosphorylated. Structural assessment of various combinations of lamins A/C, B1 and B2 indicated polymers with B2 were “weaker” than those with B1 [64]. LmnB1 mRNA was detected on the 8CFES arrays at approximately the same level as the iPS and hES cells, all at higher levels than the fibroblasts. This suggests that the 8Cell nuclear lamina matrix may be comprised principally of LmnB1. The LMNB2 gene sequence is also a known origin of replication site, and given the rapid cell cycles of early embryos, the gene may be bound by DNA replication initiation factors nearly without interruption [65].

Of the five cycling knocked-down genes that blocked HeLa in G2, two were over-detected on the 8Cell and hES arrays: DTL, the Cdt1-targeting component of one of the ubiquitin ligases (CUL4-DDB1) responsible for degradation of Cdt1 during S phase to prevent re-replication of DNA [66], and TIPIN, encodes a replication fork binding partner of Timeless [67]. H1F0, an oocyte-specific replacement linker histone [68], was over-detected specifically on the 8-Cell array.

Of the 19 knocked-down genes that blocked HeLa in mitosis, one was over-detected on the 8-Cell arrays, AURKA, and five were over-detected on the 8-Cell and hES arrays: CCNA2, CDC20, CENPE, KIF23, and ORC1L, and one was markedly under-detected on the 8-Cell arrays, CIT, a citron kinase essential for normal cytokinesis in neuronal progenitor cells [69]. Aurora kinase A is involved in centrosome and spindle assembly and activates Plk1 at the G2/M transition [70]. Over activation of either, or both, is linked to cancer development [70]. Ccd20 is a regulatory subunit of anaphase promoting complex, APC/C, and may play a more important role in early embryo cells [71] than in somatic cells [72]; CenpE, centrosome protein E, and KIF23, are both kinesin-like motor proteins that play a role in chromosome movement [51, 73]; and Orc1l is the largest subunit of the Orc1 complex essential for the initiation of DNA replication and recently shown to also play a key role in controlling centriole and centrosome copy number in human cells [74].

Interestingly, several well described cell cycle genes shown to cycle in the primary fibroblasts were not detected by the knock-down strategies, including GMNN and CKS1B, and UBE2C over-detected on both the 8-Cell and hES arrays, and CCNB1, PTTG1 (securin) and SKP2, over-detected specifically on the 8-Cell arrays. Geminin prevents re-replication of DNA during S phase [75]; Cks1B is a kinase that blocks the inhibition of Cdks by p27 (CDKN1B) [76]. UBE2C encodes a ubiquitin-conjugating enzyme that specializes in targeting cyclins, especially G1 cyclins, for proteolysis [77]; Cyclin B1 stimulates Cdk1 during G2, and has multiple roles in the G2 to M transition [78]; securin is best known for inhibition of separase (Espl1), the enzyme that allows the separation of sister chromatids, for most of the cell cycle [79]; Skp2 is a well-studied component of a ubiquitin ligase that targets Cyclin E and p27 for proteolysis [80].

Conclusions

Using public databases of gene ontologies, combined with a gene assembly derived from high-throughput RNA knock-down and cell synchronization studies, we have identified 35 cell cycle genes over-detected on whole human genome microarrays of 8-cell stage blastomeres, 31 genes over-detected on both 8-Cell and hES microarrays, and ten genes under-detected on 8-Cell arrays (Table 2). This is by no means a comprehensive list of genes important to the cell cycle of totipotent and pluripotent cells because of the significant number of genes with unknown function, the relatively conservative (±7-fold) criteria for classification of over- or under-detection, and the lack of verification that the microarray results reflect protein expression. It does, however, lay groundwork essential for the design of the targeted experiments possible with the small numbers of human embryos available for research.

Whether or not the genes are known targets of Myc (14 of the 35 genes over-detected on the 8-Cell arrays, 18 of the 31 over-detected on both the 8-Cell and hES arrays, and five of the ten genes under-detected on the 8-Cells) did not predict level of detection on the microarrays despite the relatively high level of detection of MYC on the 8-Cell arrays. This may relate to the observation that association with multiple transcription factors in addition to Myc correlated with high level of expression in embryonic stem cells [29].

The 8-Cell blastomere is only one or two cell cycles away from initial commitment to either outer trophoblast cells, or inner cell mass cells. That commitment carries with it a new dependence upon growth factors, such as fibroblast growth factor and insulin, for both cell lineages [8183], in keeping with the detection of RB1 on the hES cell microarrays [1] and in mouse blastocysts [6]. Most of the daughter cells of the 8-Cell blastomere will commit to trophoblast, only a few to inner cell mass. Trophoblast stem cells will in turn undergo commitment to placenta, including initiating cycles of endoreduplication of the genome without cytokinesis, characteristic of placental cells [84].

The functions of 16 of the 35 genes over-detected on the 8-Cell arrays: AURKC, AVPI1, CABYR, CCNA1, CCNB3, CDC2L1, CRKRS, CUL2, KIFC1, RBBP6, RKHD2, PTTG2, -3, REEP4, STAG3, -3L3, are poorly understood and urgently in need of additional study. The functions in the 8-Cell blastomeres of the 19 better studied genes over-detected on the 8-Cell arrays: CCNE1, CDC25B, CCNB1, AURKA, -B, MAPK4, SKP2, MYC, NPAT, POLS, PTTG1, RRM2, DLG7, INCENP, KCNH5, PPM1D, GINS4, H1F0, and KIFC1 are also not known with certainty, although some predictions are possible.

The lack of Rb and Wee1 is similar to the absence of cell cycle checkpoints in frog embryos [24], relieves the 8-Cells from growth factor dependence for the G1 to S transition, and is consistent with the over-expression of CCNE1 and CCNA1, -2 factors essential for the G1 to S transition. RB1 was also silent, and Wee1 at threshold level of detection, on the microarrays of ovulated metaphase II human oocytes [31], indicating that unlike mouse embryos [6], these two genes are not among those that undergo degradation the first few days after fertilization of human eggs [85]. If checkpoint control in early human embryos is analogous to frog embryo development, RB and WEE1 may be turned on at the blastocyst stage, as evidenced by their detection on the hES cell microarrays [1], and those cells with abnormal karyotype or errors in DNA replication may undergo programmed cell death [2224].

Three other genes over-detected on the 8-Cells that were either not detected, or detected at very low levels in the human oocytes [31] are INCENP, KCNH5 and CCNA1, indicating these genes must also be upregulated following fertilization. In contrast, CCNE1, PTTG, and STAG3 were readily detected on the oocyte arrays, suggesting maternal messages could be still detected in the 8-Cells.

Frog embryo survival depends on becoming motile as quickly as possible after fertilization. Human embryo survival depends on sending immediate, sufficient and increasing molecular signals to the mother that development is progressing, or menses ensues. One obvious mechanism for accomplishing these goals is rapid duplication of gene copies early in development, which is more easily accomplished if cell cycle checkpoints are silent, and cell cycle drivers are over-expressed. But subsequent development requires faithful duplication and allocation of chromosomes, so how does the early embryo accomplish both?

Clues from the microarray data suggest G1 and S phase progression are augmented by ample Cyclin E, Cyclin A1, -2 and key DNA replication factors, such as ribonucleotide reductase and DNA polymerase S, thought to push strand synthesis through cohesin sites, Npat, important for chromatin organization, and Hjurp, thought to block cell cycle arrest by DNA strand breaks. Robust expression of geminin and its protector, Emi1, may prevent DNA re-replication errors. G2 to M phase is rushed by ample expression of Cyclin A and Cyclin B, Cdc25B, and elements important to timely proteolysis, Ube2C and Skp2, to allow the transition to metaphase. Attempts to ensure the integrity of chromosome allocation to daughter blastomeres include over-expression of chromosome glue, including Incenp and Stag proteins, as well as Pttg proteins to block separase (Espl1) activity and prevent pre-mature chromosome separation. Especially noteworthy on the 8-cell arrays are the high levels of detection of the Aurora kinase family, A, B and C, major players in centrosome duplication and formation of the mitotic spindle, as well as kinesins, KIFC1 and KIF23.

Taken together, the lack of well described cell cycle checkpoints in the presence of robust expression of cell cycle drivers and their controls in the 8-Cell blastomeres, suggest more dependence upon accurate cycling of gene expression, perhaps regulated by circadian oscillators [1], for faithful daughter cell formation than on the checkpoints imposed by growth factor dependency of later stage cells. This would have the advantage of rapid gene duplication, but the disadvantage of cleavage mishaps resulting from even slightly out of phase or inappropriate abundance of gene expression. The phenomenon of identical twinning indicates that only half of early cleavage cells are needed for viable offspring, suggesting at least half of the blastomeres could be genetically defective without dire consequences. Studies of the relative expression in individual normal and abnormal human embryos of the genes identified by this analysis of 8-Cell blastomere gene expression will reveal which cycling elements are the most critical for development.

Acknowledgements

This work was supported by the Bedford Research Foundation, Naylor Fund for Stem Cell Research, and special account (vat#090145420) for research grants of the National and Kapodistian University of Athens.

Open Access

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Supplementary material

10815_2010_9407_MOESM1_ESM.xls (745 kb)
Supplemental Table 1S(XLS 745 kb)
10815_2010_9407_MOESM2_ESM.doc (32 kb)
Supplemental Table 2S(DOC 32 kb)
10815_2010_9407_MOESM3_ESM.xls (83 kb)
Supplemental Table 3S(XLS 83 kb)

Copyright information

© The Author(s) 2010

Authors and Affiliations

  • Ann A. Kiessling
    • 1
  • Ritsa Bletsa
    • 2
  • Bryan Desmarais
    • 1
  • Christina Mara
    • 2
  • Kostas Kallianidis
    • 2
  • Dimitris Loutradis
    • 2
  1. 1.Bedford Stem Cell Research FoundationSomervilleUSA
  2. 2.1st Department of Obstetrics and GynecologyAthens Medical School, “Alexandra” Maternity HospitalAthensGreece