Single blastomere removal from murine embryos is associated with activation of matrix metalloproteinases and Janus kinase/signal transducers and activators of transcription pathways of placental inflammation.

Single blastomere removal from cleavage-stage embryos, a common procedure used in conjunction with preimplantation genetic diagnosis (PGD), may affect reproductive outcomes. We hypothesized that negative pregnancy outcomes associated with PGD may be due to impairment of placental signaling pathways. The goal of this study was to determine the molecular mechanisms through which placental signaling is deregulated by blastomere removal. Four-cell stage murine embryos produced by in vitro fertilization were subjected to removal of a single blastomere (biopsied) or to the same manipulations without the blastomere removal (controls). Placental tissues from term (18.5 day) pregnancies obtained after embryo transfer were tested for levels of nitrosative species, interleukin 6, signal transducers and activators of transcription (STAT) 1 and 3, suppressors of cytokine signaling (SOCS) 1, 2 and 3 and matrix metalloproteinases (MMP) 1, 2, 3 and 9. Significant increases in nitrosative stress (P < 0.05), phosphorylative activation of STAT1 (P < 0.05) but not STAT3, lower levels of the inhibitors SOCS2 (P < 0.01) and SOCS3 (P < 0.001) and activation of MMP9 (P < 0.001) were observed in placentas derived from biopsied embryos, compared with controls. Such effects could contribute to greater levels of premature membrane rupture, incorrect parturition, preterm birth and intrauterine growth restriction associated with PGD. This work has determined signaling mechanisms that may be responsible for blastomere removal effects on placental function, with the potential to become targets for improving obstetric and neonatal outcomes in assisted reproduction.

Multiple functions of FADD in apoptosis, NF-κB-related signaling, and heart development in Xenopus embryos.

FADD is an adaptor protein that transmits apoptotic signals from death receptors. Additionally, FADD has been shown to play a role in various functions including cell proliferation. However, the physiological role of FADD during embryonic development remains to be delineated. Here, we show the novel roles FADD plays in development and the molecular mechanisms of these roles in Xenopus embryos. By whole-mount in situ hybridization and RT-PCR analysis, we observed that fadd is constantly expressed in early embryos. The upregulation or downregulation of FADD proteins by embryonic manipulation resulted in induction of apoptosis or size changes in the heart during development. Expression of a truncated form of FADD, FADDdd, which lacks pro-apoptotic activity, caused growth retardation of embryos associated with dramatic expressional fluctuations of genes that are regulated by NF-κB. Moreover, we isolated a homolog of mammalian cullin-4 (Cul4), a component of the ubiquitin E3 ligase family, as a FADDdd-interacting molecule in Xenopus embryos. Thus, our study shows that FADD has multiple functions in embryos; it plays a part in the regulation of NF-κB activation and heart formation, in addition to apoptosis. Furthermore, our findings provide new insights into how Cul4-based ligase is related to FADD signaling in embryogenesis.

Treating cloned embryos, but not donor cells, with 5-aza-2'-deoxycytidine enhances the developmental competence of porcine cloned embryos.

The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.

Transcriptome fingerprint of bovine 2-cell stage blastomeres is directly correlated with the individual developmental competence of the corresponding sister blastomere.

To date, gene expression profiles of bovine preimplantation embryos have only been indirectly related to developmental potential due to the invasive nature of such procedures. This study sought to find a direct correlation between transcriptome fingerprint of blastomeres of bovine 2-cell stage embryos with developmental competence of the corresponding sister blastomeres. Isolated blastomeres were classified according to the sister blastomere's development into three groups: two groups displayed developmental incompetency, including those blastomeres whose corresponding sister blastomeres either stopped cleaving after separation (2CB) or were blocked after two additional cleavages before embryonic genome activation (8CB). In the third group were competent blastomeres, which were defined as those whose sister blastomeres developed to the blastocyst stage (BL). As a result, developmental capacity of corresponding sister blastomeres was highly similar. Microarray analysis revealed 77 genes to be commonly differentially regulated among competent and incompetent blastomeres as well as blocked blastomeres. Clustering of differentially expressed genes according to molecular functions and pathways revealed antioxidant activity, NRF2-mediated oxidative stress response, and oxidative phosphorylation to be the main ontologies affected. Expression levels of selected candidate genes were further characterized in an independent model for developmental competence based on the time of first cleavage postfertilization. Moreover, overall results of this study were confirmed by higher developmental rates and more beneficial expression of CAT and PRDX1 when cultured in an antioxidative environment. These results will help us to understand molecular mechanisms defining developmental destination of individual bovine preimplantation embryos.

[Cyto B dependent and ouabain insensitive regulatory volume decrease in bicellular mouse embryo].

Mouse single-cell embryos exhibit robust Regulatory Volume Decrease (RVD). In what manner the very early mammalian embryo following zygote stage is appreciably altered by the anisotonic extracellular solution is, as yet, totally unclear. Little attention was paid to this direction since there was no way to determine the blastomere volume. This work has served to quantitatively investigate the osmotic response of bicellular mouse embryos employing Laser Scanning Microtomography (LSM) followed with three-dimensional reconstruction (3 DR). We have shown that bicellular mouse embryos in hypotonic Dulbecco's experience RVD. Embryonic cells subjected to hyposmolar exhibit rapid osmotic swelling followed by gradual shrinking back toward their original volume. The van't Hoff law defines swelling phase with the effective hydraulic conductivity of 0.3 micron x min(-1) x atm(-1). Water release during RVD in bicellular mouse embryos is abolished by Cytochalasin B (Cyto B) and the volume recovery is insensitive to ouabain treatment.

Involvement of the PKC family in regulation of early development.

Protein kinase C (PKC) isotypes have been implicated in a number of key steps during gametogenesis, fertilization, and early development. The 11-member family of PKC isotypes, many with different cofactor requirements for activation, can provide for differential activation of the specific kinases. In addition the enrichment of particular PKC isotypes to unique locations within gametes, zygotes, and early embryos likely promotes specific substrate interactions. Evidence exists to indicate involvement of PKC isotypes during sperm capacitation and the acrosome reaction, during resumption of meiosis in the oocytes, regulating the spindle organization in meiosis I and II, at fertilization, in the pronuclei, in the mitotically dividing blastomeres of the embryo, and at the plasma membranes of blastomeres at the time of embryonic compaction. Evidence also exists for crosstalk with other signaling pathways and one or more isotypes of PKC appear to be active at each major developmental transition.

Asymmetric p38 activation in zebrafish: its possible role in symmetric and synchronous cleavage.

Cleavage is one of the initial steps of embryogenesis, and is characterized by a series of symmetric and synchronous cell divisions. We showed that p38 MAP kinase (p38) is asymmetrically activated on one side of the blastodisc during the early cleavage period in zebrafish (Danio rerio) embryos. When a dominant negative (DN) form of p38 was uniformly expressed, blastomere cleavage was impaired on one side of the blastodisc, resulting in the formation of blastomeres with a large mass of cytoplasm and an enlarged nucleus on the affected side. The area affected by the DN-p38 expression did not correlate with the initial cleavage plane, but coincided with the side where dharma/bozozok, a dorsal-specific zygotic gene, was expressed (Yamanaka et al. 1998). Furthermore, UV irradiation and removal of the vegetal yolk mass before the first cleavage, both of which inhibit the initiation of the dorsalizing signals, abolished the asymmetric p38 activation. Our findings suggest that asymmetric p38 activation is required for symmetric and synchronous cleavage, and may be regulated by the same machinery that controls the initiation of dorsalizing signals.

Parameters that specify the timing of cytokinesis.

One model for the timing of cytokinesis is based on findings that p34(cdc2) can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), and this inhibition is proposed to delay cytokinesis until p34(cdc2) activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373-1386). Among these kinases and substrates is p34(cdc2) and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.

Embryonic expression of the putative gamma subunit of the sodium pump is required for acquisition of fluid transport capacity during mouse blastocyst development.

The sodium/potassium pump, Na+,K+-ATPase, is generally understood to function as a heterodimer of two subunits, a catalytic alpha subunit and a noncatalytic, glycosylated beta subunit. Recently, a putative third subunit, the gamma subunit, was cloned. This small protein (6.5 kD) coimmunoprecipitates with the alpha and beta subunits and is closely associated with the ouabain binding site on the holoenzyme, but its function is unknown. We have investigated the expression of the gamma subunit in preimplantation mouse development, where Na+, K+-ATPase plays a critical role as the driving force for blastocoel formation (cavitation). Using reverse transcriptase-polymerase chain reaction, we demonstrated that the gamma subunit mRNA accumulates continuously from the eight-cell stage onward and that it cosediments with polyribosomes from its time of first appearance. Confocal immunofluorescence microscopy revealed that the gamma subunit itself accumulates and is localized at the blastomere surfaces up to the blastocyst stage. In contrast with the alpha and beta subunits, the gamma subunit is not concentrated in the basolateral surface of the polarized trophectoderm layer, but is strongly expressed at the apical surface as well. When embryos were treated with antisense oligodeoxynucleotide complementary to the gamma subunit mRNA, ouabain-sensitive K+ transport (as indicated by 86Rb+ uptake) was reduced and cavitation delayed. However, Na+, K+-ATPase enzymatic activity was unaffected as determined by a direct phosphorylation assay ("back door" phosphorylation) applied to plasma membrane preparations. These results indicate that the gamma subunit, although not an integral component of Na+,K+-ATPase, is an important determinant of active cation transport and that, as such, its embryonic expression is essential for blastocoel formation in the mouse.

Induction of metaphase arrest in cleaving Xenopus embryos by the protein kinase p90Rsk.

Before fertilization, vertebrate eggs are arrested in metaphase of meiosis II by cytostatic factor (CSF), an activity that requires activation of the mitogen-activated protein kinase (MAPK) pathway. To investigate whether CSF arrest is mediated by the protein kinase p90Rsk, which is phosphorylated and activated by MAPK, a constitutively activated (CA) form of Rsk was expressed in Xenopus embryos. Expression of CA Rsk resulted in cleavage arrest, and cytological analysis showed that arrested blastomeres were in M phase with prominent spindles characteristic of meiotic metaphase. Thus, Rsk appears to be the mediator of MAPK-dependent CSF arrest in vertebrate unfertilized eggs.

Conversion of zebrafish blastomeres to an endodermal fate by TGF-beta-related signaling.

The endoderm contributes cells to the gut, and participates in the induction and patterning of the vertebrate head and heart. The mechanisms controlling the formation of endoderm are poorly understood. Commitment of endoderm cells occurs at the onset of gastrulation and requires cell interactions; studies in vitro have implicated transforming growth factor Beta (TGF-beta)-related molecules in this process. TARAM-A is a zebrafish receptor kinase that is related to the type I subunit of the TGF-beta receptor, and is expressed in presumptive endomesodermal cells at gastrulation. We provide here evidence for its involvement in endoderm formation in vivo. Activation of TARAM-A was found to drive blastomeres towards an endodermal fate. The induced endoderm behaved ad endogenous endoderm during gastrulation: it migrated in contact with the yolk and expressed endoderm-specific markers. Loss-of-function mutations in the zebrafish one-eyed-pinhead (OEP) gene lead to defects in heart formation, defects of the ventral central nervous system (CNS) and cyclopia. Mutant embryos also lack endoderm and anterior mesoderm. Endoderm formation in oep mutant embryos was found to be restored by the activation of the TARAM-A signaling pathway. Cardiac and ocular defects, but not midline CNS structures, were rescued non-autonomously, demonstrating that endoderm may provide signals that can pattern the eye anlage, and which are distinct form those specifying the ventral midline of the CNS.

Single Cell Restriction Enzyme-Based Analysis of Methylation at Genomic Imprinted Regions in Preimplantation Mouse Embryos.

The methylation of cytosines in DNA is a fundamental epigenetic regulatory mechanism. During preimplantation development, mammalian embryos undergo extensive epigenetic reprogramming, including the global erasure of germ cell-specific DNA methylation marks, to allow for the establishment of the pluripotent state of the epiblast. However, DNA methylation marks at specific regions, such as imprinted gene regions, escape this reprogramming process, as their inheritance from germline to soma is paramount for proper development. To study the dynamics of DNA methylation marks in single blastomeres of mouse preimplantation embryos, we devised a new approach-single cell restriction enzyme analysis of methylation (SCRAM). SCRAM allows for reliable, fast, and high-throughput analysis of DNA methylation states of multiple regions of interest from single cells. In the method described below, SCRAM is specifically used to address loss of DNA methylation at genomic imprints or other highly methylated regions of interest.

Heparanase improves mouse embryo implantation.

OBJECTIVE: To improve mouse embryonic implantation by recombinant heparanase supplementation. Heparanase, an endoglycosidase-degrading heparan sulfate proteoglycan, may have a role in embryonic implantation because of its enzymatic, angiogenic, and adhesive properties. Increasing endometrial receptivity could improve one of the most difficult pathologies in human fertility. DESIGN: Comparison between mouse blastocysts obtained after 24-hour incubation of morulae with or without heparanase. SETTING: Experimental laboratory in a medical center. ANIMAL(S): Mice. INTERVENTION(S): Morulae were flushed from CB6F1 female mice and incubated for 24 hours at 37 degrees C in M16 medium supplemented with 0.1 mg/mL heparanase (n = 203), with albumin (n = 60), or with medium alone (n = 258). MAIN OUTCOME MEASURE(S): Blastocysts were evaluated by heparanase immunostaining (n = 10), activity assay (n = 283), and transfer to foster mice uterine horns (n = 228). The number of implantation sites was compared. RESULT(S): Immunostaining demonstrated that heparanase is constitutively expressed in mouse morulae and blastocyts. Heparanase supplementation resulted in increased staining and enzymatic activity in blastocyts. Implantation rates for the heparanase, M16 medium, and albumin groups, were 36.9%, 17.8%, and 20%, respectively (P<.01). CONCLUSION(S): Heparanase was found to be constitutively expressed by blastocyst-stage embryos. Moreover, the amount of heparanase was markedly increased by incubation of morulae with recombinant heparanase, evaluated by immunostaining and enzymatic activity. Heparanase supplementation resulted in approximately a twofold increase in embryo implantation rate in vivo. Taken together, these data suggest that heparanase is actively involved in embryo implantation.

Immunofluorescence techniques for determining the numbers of inner and outer blastomeres in mouse morulae.

Methods are described for distinguishing inner and outer blastomeres of compact 8- to 32-cell mouse morulae. The first involves the selective labelling by immunofluorescent reagents of the exposed surface of the compact morula, disaggregation of the morula into single blastomeres and separation of these blastomeres into partially labelled (presumptive outer) and unlabeled (presumptive inner) populations. The second involves labelling blastomeres after disaggregation and is based on the recent observation that the sera on an isolated outer blastomere which originally contributed to the exposed surface of the intact morula labels more intensely than the remaining (non-exposed) surface of the blastomere. Analysis of the labelling patterns obtained from individual disaggregated morulae indicated that inner blastomeres were absent from compact 8-cell morulae, but increased in number throughout the next cleavage division until at the 16-cell stage the mean number of these blastomeres varied from 4.6 to 6.6 (with a range of 1--8) depending on the technique used. At later stages, the numbers of inner blastomeres can probably be accounted for by division of the inner cells at the 16-cell stage.

Change in the activity of Cl-,HCO3(-)-ATPase in microsome fraction during early development of the sea urchin, Hemicentrotus pulcherrimus.

In sea urchin embryos, primary mesenchyme cells, descendants from micromeres produced at the 16-cell stage, form spicules or CaCO3 deposits in their skeletal vacuoles, at the post-gastrula stage. Micromeres isolated at the 16-cell stage also differentiate into spicule-forming cells during their culture at the same time schedule as in the embryos. The present study was planned to observe change in the activity of Cl-,HCO3(-)-ATPase, which was expected to contribute to the carbonate supply for CaCO3 deposition, during development. ATP-hydrolysis in the microsome fraction, obtained from embryos of the sea urchin, Hemicentrotus pulcherrimus, and from micromere-derived cells in culture was stimulated by Cl- and HCO3- in the presence of ouabain and EGTA. The ATP-hydrolysis was inhibited by ethacrynic acid, an inhibitor of Cl-,HCO3(-)-ATPase. The activity of Cl-,HCO3(-)-ATPase in embryos and in micromere-derived cells increased during development, keeping pace with the rate of calcium deposition in spicules. Formation of calcified spicules in the cultured micromere-derived cells was inhibited by ethacrynic acid. These results indicate that Cl-,HCO3(-)-ATPase plays an important role in the mechanism of CaCO3 deposition in the primary mesenchyme cells.

Method for quantifying expression of functionally active topoisomerase II in patients with leukaemia.

AIMS: To produce a method to measure and quantify enzymatically active topoisomerase II in normal and neoplastic human cells. METHODS: A crude cell lysate from density separated mononuclear cells from either peripherial blood or bone marrow was prepared as a source of topoisomerases. Using the lysate, minicircles from the Crithedia kinetoplast DNA complex were decatenated before being separated by agarose gel electrophoresis and visualised using ethidium bromide/ultraviolet fluorescence. RESULTS: Cell number, sample volume and drug inhibition concentration required to produce reliable and reproducible assay conditions were established. Intra- and interassay standards were included which permitted the quantification of active topoisomerase II in matched peripheral blood, bone marrow, presentation, and relapse samples from patients with acute lymphoblastic leukaemia. Active topoisomerase II has been converted to a unit scale which has been used to compare topoisomerase II activities in cells from patients with normal blood and bone marrow samples. CONCLUSIONS: There was no change in topoisomerase II activities between samples taken at presentation and those taken during a recurrence. However, topoisomerase II activity in leukaemic blast populations was increased compared with topoisomerase II activity in normal cells.

Amplification of exon 11 of the gene for the alpha-chain of beta-N-acetylhexosaminidase in single human blastomeres.

OBJECTIVE: To evaluate the possibility of using polymerase chain reaction (PCR) technology for preimplantation diagnosis for Tay-Sachs disease. DESIGN: Polymerase chain reaction on single human blastomeres. SETTING: Bad quality embryos from IVF analyzed in an academic research genetics lab. INTERVENTIONS: Patients underwent standard IVF procedures as infertility treatment. RESULTS: Amplification was seen in 89.5% of the blastomeres; only one blank was contaminated. CONCLUSION: The technique of PCR on single blastomeres is ready to be used in clinical preimplantation diagnosis for Tay-Sachs disease.

Repression of zygotic gene expression in the putative germline cells in ascidian embryos.

In germline cells of early C. elegans and Drosophila embryos, repression of zygotic gene expression appears to be essential to maintain the germ cell fate. In these animals, specific residues in the carboxy-terminal domain (CTD) of RNA polymerase II large subunit (RNAP II LS) are dephosphorylated in the germline cells, whereas they are phosphorylated in the somatic cells. We investigated, in early embryos of the ascidian Halocynthia roretzi, the expression patterns of three genes that are essentially expressed in the entire embryo after the 32-cell stage. We found that the expression of these genes was inactive in the putative germline cells during the cleavage stages. Once cells were separated from the germline lineage by cleavages, the expression of the genes was initiated in the cells. These results suggest that repression of transcription in germline cells may also be common in chordate embryos. We then examined the phosphorylation state of the CTD of RNAP II using a phosphoepitope-specific antibody. At cleavage stages after the 32-cell stage, CTD was phosphorylated in every blastomere, including the germline cells. Therefore, in the ascidian, the inactivation of zygotic transcription is not correlated with dephosphorylation of the CTD. These observations indicate that zygotic transcription is inactivated in ascidian germline cells, but the mechanism of the repression may differ from that in C. elegans and Drosophila.

Roles for focal adhesion kinase (FAK) in blastomere abscission and vesicle trafficking during cleavage in the sea urchin embryo.

Is focal adhesion kinase (FAK) needed for embryonic cleavage? We find that FAK is expressed during early cleavage divisions of sea urchin embryos as determined by polyclonal antibodies to the Lytechinus variegatus protein. FAK is absent in eggs and zygotes and then cycles in abundance during the first cleavages after fertilization. It is maximal at anaphase, similar to the destruction and synthesis of cyclin proteins. To investigate whether FAK is needed during early cleavage, we interfered with its function by microinjecting eggs with anti-FAK antibodies or with FAK antisense morpholino oligonucleotides. Both treatments led to regression of the cleavage furrow. FAK knockdown with antibodies or morpholino oligonucleotides also resulted in an over-accumulation of endocytic vesicles. Thus, FAK could be restricting endocytosis or increasing exocytosis in localized areas important for abscission. FAK appears to be necessary for successful cleavage. These results are the first to document a functional role for FAK during embryonic cleavage.

Life-giving caspases: revealing new roles during mouse embryo preimplantation development.

Caspases, cystein proteases traditionally related to programmed cell death, have recently been found to be involved in vital processes such as cell proliferation, adhesion and differentiation. Although caspases are expressed in mouse embryos before the blastocyst stage, their role is unclear, since apoptosis does not occur significantly before implantation. In this work, we have used mouse preimplantation development as a model to evaluate the existence of non-lethal caspase activities. The use of specific caspase inhibitors during in vitro embryo culture showed that caspase 8 activity, but not caspase 2 or 9, was relevant for development. The inhibition of caspase 8 affected the compaction of morulae and the progression to the blastocyst stage. In agreement with these results, caspase 8 was expressed in mouse embryos, as shown by indirect immunofluorescence and RT-PCR. An in silico approach was used to find putative caspase targets expressed in mouse preimplantation embryos. Large-scale management of sequence data from mouse embryos was used to predict caspase substrates by tools matrix-based on known cleavage sites. A total of 510 potential caspase targets expressed in mouse embryos were identified by this procedure. The functional characterization of these proteins by Gene Onthology associations showed that many of these putative caspase targets were previously related to non-apoptotic functions and only 63 had been previously reported to be actually cleaved by caspases. Interestingly, eleven knockout mice models for caspase substrates identified in our work, i.e. catenin alpha and beta, geminin, pescadillo, calpain-2, have preimplantation lethal phenotypes. This work supports the involvement of caspases in vital functions during mouse preimplantation development and proposes a model in which the regulated cleavage of caspase substrates could account for this role.