Karlla Mason

Claire Boulesteix

Vincent V Brochard

Tiphaine Aguirre-Lavin

Juliette Salvaing

Renaud R Fleurot

Pierre Adenot

Walid W Maalouf

Nathalie Beaujean
email: nathalie.beaujean@jouy.inra.fr

Nuclear dynamics of histone H3 trimethylated on lysine 9 and/or phosphorylated on serine 10 in mouse cloned embryos as new markers of reprogramming?

Keywords: preimplantation embryo, nuclear transfer, histone, phosphorylation, methylation, heterochromatin

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Nuclear transfer (or cloning) is a technique with broad applications such as the production of patient ES cells (regenerative medicine), the creation of models for the study of human diseases, improvement of animal production in agriculture, the protection of endangered species and especially the opportunity to deeply investigate how epigenetic and structural changes occur in the somatic nucleus during nuclear reprogramming [START_REF] Yang | Epigenetic marks in cloned rhesus monkey embryos: comparison with counterparts produced in vitro[END_REF]. After nuclear transfer it is known that the donor cell nucleus injected into the enucleated egg must undergo biochemical changes to reverse the established constraints on the genetic potential imposed by the process of differentiation [START_REF] Kikyo | Reprogramming nuclei: insights from cloning, nuclear transfer and heterokaryons[END_REF]. On the other hand, after fertilization the genetic material of the two specialized cells, sperm and oocyte, undergoes deep remodelling to acquire the embryonic genome configuration in order to start the process of development. Similarly, the donor cell nucleus must lose its original genome conformation and be fully reorganized to acquire the embryonic genome configuration and as a result acquires proper gene regulation essential for normal development. However, the efficiency of this reversal will determine the subsequent developmental success of the reconstructed embryo and despite the successful production of clones of different species by nuclear transfer, this technique still shows extremely low success rates with high abortion and fetal death rates as well as a variety of abnormalities including obesity, large placenta and abnormal expression of genes important for development (Kang & Roh, 2010).

It is thought that these undesirable results are due to inappropriate genome reprogramming in this type of embryo and aberrant epigenetic status' such as abnormal DNA methylation and irregular histone modification patterns (Kang & Roh, 2010). However, most of the reports focused on the presence/absence of epigenetic modifications in reconstructed embryos and few analyzed the impact of the inadequate epigenetic status on chromatin reorganization within the resulting nuclei. In previous studies we demonstrated such chromatin rearrangements abnormalities in cloned embryos after ES and cumulus cell nuclear transfer using a marker of pericentromeric heterochromatin, HP1β (Martin et al 2006a;[START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF].

Undeniably, after fertilization, both parental genomes reorganize to form pronuclei, which display a peculiar distribution of pericentromeric heterochromatin with the centromeres distributed mostly around the Nucleolar Precursor Bodies (NPBs) forming a ring-like structure, while the rest of the chromosomes most probably stretch out to the periphery of the nucleus (Martin et al., 2006b). This 'cartwheel' organization exclusive to that stage has been suggested to maintain transcriptional silencing during parental genome maturation.

In embryos obtained by nuclear transfer it has been shown that pericentromeric heterochromatin is rapidly reorganized as in naturally fertilised embryos (Martin et al., 2006a;[START_REF] Merico | Epigenomic differentiation in mouse preimplantation nuclei of biparental, parthenote and cloned embryos[END_REF][START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF]. However, reprogramming after nuclear transfer is not perfect and aberrations are quite frequent: remains of somatic-like heterochromatin clumps are often observed in late 1-cell and early 2-cell clones (Martin et al., 2006b;[START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF]. These heterochromatin clumps are very similar to the chromocenters seen in the nucleus of somatic cells. Chromocenters are formed by the clustering of various centromeres of different chromosomes being basically constituted by pericentromeric and the bordering centromeric heterochromatin [START_REF] Alcobia | Spatial associations of centromeres in the nuclei of hematopoietic cells: evidence for cell-type-specific organizational patterns[END_REF][START_REF] Alcobia | The spatial organization of centromeric heterochromatin during normal human lymphopoiesis: evidence for ontogenically determined spatial patterns[END_REF].

Interestingly, we have evidenced a link between the developmental inefficiency of cloned embryos and aberrant chromatin reprogramming. We indeed observed that ES cell nuclei, that give a higher rate of survival to term after cloning, undergo better remodelling after nuclear transfer than cumulus cell nuclei [START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF]. Importantly, incubation of cloned mouse embryos in the very early hours after transfer with a histone deacetylase (HDAC) inhibitor, improved the structural remodelling of pericentric heterochromatin at 1-cell and dramatically increased the rate of full term development (a 10 fold increase) [START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF].

Altogether it indicated that the nuclear configuration of donor nuclei, and especially pericentromeric heterochromatin clustering into chromocenters, was not completely lost after nuclear transfer and that it impaired development. To further investigate this hypothesis we decided to follow pericentromeric heterochromatin rearrangements in cumulus-derived clones using various epigenetic markers. Therefore, we performed immunostainings on the two most obvious and important stages after meiosis resumption, i.e. the 1-cell stage with formation of the pronuclei and the 2-cell stage when embryonic genome activation normally occurs in the mouse.

Firstly we analyzed phosphorylation of H3 at serine 10 (H3S10P). In mammalian cells, phosphorylation of histone H3 at serine 10 is first evident in pericentromeric heterochromatin in late G2-interphase cells, spreading throughout the chromosomes arms during prophase and only getting dephosphorylated around late anaphase (Perez-Cadahia et al., 2009). The fact that H3S10P is only detected at the end of interphase and observed in the entire chromosomes lengths led to the conclusion that this epigenetic modification could be related to chromosome condensation [START_REF] Garcia | Modifications of human histone H3 variants during mitosis[END_REF]. However, this post-translational modification has also been observed outside mitosis and it is believed that it is involved with gene activation [START_REF] Lim | Chromosomal protein HMGN1 modulates histone H3 phosphorylation[END_REF][START_REF] Drobic | Promoter chromatin remodeling of immediate-early genes is mediated through H3 phosphorylation at either serine 28 or 10 by the MSK1 multi-protein complex[END_REF]. Some research groups have investigated this epigenetic modification during early mouse embryogenesis. Their reports suggest that H3S10P is linked to pericentromeric heterochromatin (WangQ et al 2006[START_REF] Huang | Comparison of histone modifications in in vivo and in vitro fertilization mouse embryos[END_REF][START_REF] Teperek-Tkacz | Phosphorylation of histone H3 serine 10 in early mouse embryos: active phosphorylation at late S phase and differential effects of ZM447439 on first two embryonic mitoses[END_REF]. We have recently shown by immunoFISH using specific pericentromeric heterochromatin probes that H3S10P indeed marks constitutive heterochromatin during interphase until the 4-cell stage in mouse embryos (Ribeiro-Mason et al., submitted).

As it is known, the chromocenters are also characterized by trimethylation of histone H3 at Lysine9 (H3K9me3) that provides a binding site for HP1β; this complex then induces transcriptional repression and heterochromatinization in the pericentromeric heterochromatin domains [START_REF] Lachner | Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins[END_REF]. In fertilised embryos, these two markers are distributed asymmetrically between maternal and paternal pronucleus: at the pronuclear stage only the maternal pronucleus contains H3K9me3 and HP1β accumulations [START_REF] Cowell | Heterochromatin, HP1 and methylation at lysine 9 of histone H3 in animals[END_REF][START_REF] Santos | Dynamic chromatin modifications characterise the first cell cycle in mouse embryos[END_REF]. This was confirmed by immuno-FISH experiments showing that both H3K9me3 and HP1β colocalise with pericentromeric heterochromatin probes, especially around the NPBs of the maternal pronuclei [START_REF] Probst | Structural differences in centromeric heterochromatin are spatially reconciled on fertilisation in the mouse zygote[END_REF].

Likewise, H3K9me3, HP1β and H3S10P, the double modification H3K9me3S10P (trimethylation of histone H3 at Lysine 9 phosphorylated at Serine 10) is also detected in the pericentromeric heterochromatin domains in somatic cells. It was first stated that both modifications coexist on the same histone tail especially during mitosis based on the results obtained by in vitro assays and analysis of the in vivo modification pattern of H3 isolated from HeLa cells [START_REF] Fischle | Binary switches and modification cassettes in histone biology and beyond[END_REF]. However, its correlation with pericentromeric heterochromatin was later described in somatic cells by peptide competition assays and immunofluorescence experiments [START_REF] Hirota | Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin[END_REF]. In fact, H3K9me3S10P can be detected at late G2 in interphase; it was found enriched in the centric and pericentric domains with a more spotted appearance on the chromosome arms during mitosis [START_REF] Fischle | Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation[END_REF][START_REF] Monier | DNA methylation promotes Aurora-Bdriven phosphorylation of histone H3 in chromosomal subdomains[END_REF]. Only one study mentioned this epigenetic modification in fertilised mouse embryos, showing that upon the first mitosis it was preferentially associated with maternal chromosomes [START_REF] Hayashi-Takanaka | Visualizing histone modifications in living cells: spatiotemporal dynamics of H3 phosphorylation during interphase[END_REF].

In this study, we investigated the status of three epigenetic markers, related to pericentromeric heterochromatin (H3K9me3, H3S10P, and H3K9me3S10P), in cloned mouse embryos at the 1-and 2-cell stages to find out if one of these marks could be used as another tool to monitor chromatin reorganization after nuclear transfer.

MATERIAL & METHODS

Animal care and handling were carried out according to European regulations on animal welfare.

Oocytes and embryos production

C57/CBA F1 female mice, 6-8 weeks of age, were superovulated with 5 IU of PMSG (pregnant mare serum gonadotropin) followed by injection with 5 IU of hCG (human chorionic gonadotropin) 48 hours later. For in vivo embryo production, females were placed together with males (one by one) after hCG administration. Embryos were collected in M2 medium containing 1 mg/ml hyaluronidase and then cultured in M16 at 37°C in a humidified atmosphere containing 5% CO2 until fixation for immunofluorescent staining. Fertilization occurred at about 12 h after hCG injection which was used as reference point for embryonic development (hours post-hCG, i.e., hphCG).

Cumulus Cell Nuclear Transfer

Oocytes were prepared by superovulating C57/CBA mice. Superovulation was induced by injecting pregnant mare serum gonadotropin (PMSG, Intervet, 5 IU) and human chorionic gonadotropin (hCG, Intervet, 5 IU) at intervals of 48 hours. Oocytes were collected from oviducts 14 hphCG and washed in M2 medium containing 1 mg/ml hyaluronidase.

Subsequently, they were incubated in M2 containing 5 μg/ml cytochalasin B and placed in a chamber on the stage of an inverted microscope (Nikon) equipped with micromanipulators (Nikon-Narishige MO-188). The chromatin spindle (visualized under differential interference contrast) was aspirated into the pipette as previously described [START_REF] Zhou | A simplified method for the reconstruction of fully competent mouse zygotes from adult somatic donor nuclei[END_REF]. For nuclear transfer, donor chromosomes were derived from cumulus cells that previously surrounded the oocytes, gently aspirating them in and out of the injection pipette (inner diameter 7-8 μm) followed by microinjection into the cytoplasm of the enucleated oocytes.

The nuclear transfer embryos were activated by incubation for 6 h in Ca 2+ -free medium containing 10 mM Sr 2+ , 5 μg/ml cytochalasin B. Embryos with visible nuclei were then considered as activated, transferred into fresh M16 medium and cultured at 37°C in a humidified atmosphere containing 5% CO 2 . Embryos were fixed during the first cell cycle (at 4, 5, 8 and10 hours post-activation, hpa), andearly andlate 2-cell stages (21 hpa and33 hpa respectively).

Immunofluorescent staining

The following antibodies were purchased from the indicated companies: rabbit polyclonal antibody against H3S10P (Abcam #5176); mouse monoclonal antibody against HP1β (Euromoedex #MOD-1A9-AS); rabbit polyclonal antibody against H3K9me3 (Upstate #07-523); rabbit polyclonal antibody against H3K9me3S10P (Abcam #5819); FITC-conjugated secondary antibody and Cy5-conjugated secondary antibody from donkey (Immunoresearch,

Jackson laboratories).

Embryos in different developmental stages were fixed with 4% PFA (paraformaldehyde) in PBS at 4 o C overnight and permeabilized with 0.5% Triton X-100 (15 min, room temperature: RT). The fixed embryos were blocked in PBS containing 2% BSA (1hour at RT) and incubated overnight at 4 o C with the specific first antibody diluted in 2% PBS-BSA (at 1:300 for H3S10P; 1:400 for HP1β; 1:400 for H3K9me3 ; 1:300 for H3K9me3S10P). The embryos were then washed twice in PBS to remove any first antibody excess. After this step the embryos were incubated with FITC or Cy5 labelled secondary antibody for 1 hour at RT (dilution 1:200). DNA counterstaining was performed with ethidium homodimer 2 or propidium iodide (Invitrogen). Embryos were then post-fixed with 2% PFA for 15 min at RT, washed and mounted on slides with an antifading agent (Citifluor) under coverslips.

High resolution microscopy

3D-preserved embryos were observed with either a Carl Zeiss AxioObserver Zl fluorescence microscope equipped with the ApoTome slider or a Zeiss LSM 510 confocal laser scanning microscope (MIMA2 Platform, INRA). On the Apotome, embryos were observed using a 63x Plan-Neofluar oil objective (NA 1.3) and single wavelength LEDs at 470 nm, 530 nm and 625 nm (Colibri illumination). Digital optical sections were collected using a Z-series acquisition feature every 1µm. As for the confocal system, embryos were visualized with an oil-immersion objective (Plan Apochromatic 63X NA 1.4) and imaging was performed with lasers at 488-, 535-and 633-nm wavelengths. Entire embryos were scanned with a distance of 0.37 µm between light optical sections. For intensity profile measurements, line scans were obtained with Image J software (http://rsbweb.nih.gov/ij/).

RESULTS

H3S10P distribution pattern in fertilised and cumulus cloned embryos

As previously described [START_REF] Huang | Comparison of histone modifications in in vivo and in vitro fertilization mouse embryos[END_REF], histone H3 was phosphorylated at serine 10 shortly after fertilization: the decondensing sperm head was already labelled for H3S10P as well as the maternal chromosomes (n=27 at 18hphCG; We also performed immunodetection of H3S10P in in vitro fertilised embryos to identify any abnormality that could have been caused by the embryo culture conditions. Based on our observations from fertilization to late 2-cell stage, we can state that the same distribution pattern for H3S10P is observed in in vivo fertilised and in vitro fertilised embryos (data not shown).

We then checked constitutive heterochromatin rearrangements by nuclear reprogramming in Remarkably, cumulus cells had a typical somatic cell pattern for H3S10P and only very few showed a positive signal, i.e. in late G2 and mitosis (Fig 1). However, 100% of the cloned embryos we observed were positive for this mark over the two first embryonic cycles suggesting that reprogramming had happened.

H3S10P and HP1β label distinct types of heterochromatin in early cloned embryos

In order to evaluate whether H3S10P reprogramming after cloning was concomitant with nuclear remodelling and especially heterochromatin rearrangements, we then analyzed the colocalization of H3S10P and HP1β. As mentioned before, HP1β has been extensively used as a marker to check pericentromeric heterochromatin distribution and it was demonstrated that some portion of pericentromeric heterochromatin was accumulating at the nuclear periphery in embryos after nuclear transfer (Martin et al., 2006;[START_REF] Merico | Epigenomic differentiation in mouse preimplantation nuclei of biparental, parthenote and cloned embryos[END_REF][START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF].

We observed a high number of HP1β perinuclear accumulations already in early 1-cell at 4hpa, as well as a uniform euchromatic staining, but none of these HP1β foci were labelled At the 2-cell stage, HP1β followed the same distribution pattern as previously described. In Based on these findings, we suspected that cloned mouse embryos have two types of heterochromatin after nuclear transfer: one that is being remodeled, showing H3S10P and HP1β, around the NPBs and another one, with only HP1β, at the nuclear periphery. In order to confirm whether this perinuclear heterochromatin indeed corresponds to non-reprogrammed somatic heterochromatin we then focused on another typical marker of pericentromeric heterochromatin: H3K9me3.

Comparison with H3K9me3

As expected, only the maternal genome stained for this marker in fertilised embryos while the paternal one showed no labelling (n=18 at 18hphCG; Fig 3 ). This staining was then distributed all over the nucleoplasm and formed partial rings around NPBs (n=21 at 20/21phCG). At 26hphCG we observed full staining of the NPBs periphery and, remarkably, we regularly noticed one clump of H3K9me3 at the nuclear periphery similar to the clumps previously observed with HP1β (n=32; Martin et al., 2006). We later confirmed that H3K9me3 and HP1β are co-localized within these perinuclear clumps (n=33, data not shown). H3K9me3 staining was very similar to the one observed for HP1β, both in 1-cell and 2-cell cloned embryos, and that it partially differed from H3S10P after nuclear transfer.

H3K9me3S10P as a new marker of nuclear reprogramming

It is well documented that H3K9me3 is a marker of heterochromatin. Conversely, some studies stated that the double modification H3K9me3S10P could also be a marker of heterochromatin staining chromocenters in G2-phase of somatic cells. As this double modification had never been carefully investigated in mouse embryos, we analyzed its redistribution after fertilization and nuclear transfer. In conclusion, H3K9me3S10P staining did not correspond to the combination of H3K9me3 and H3S10P staining demonstrating that these two epigenetic modifications are not always adjacent within the same histone H3 tail in mouse early embryos.

DISCUSSION

One of the main issues with the nuclear transfer technique is genome reorganization of the somatic donor nucleus, a process triggered by nuclear reprogramming which is thought to be controlled mainly by the enucleated oocyte. For that reason, in this study we have investigated the dynamics of genome restructuring in early mouse embryos derived from cumulus nuclear transfer with an emphasis on pericentromeric heterochromatin related markers such as H3S10P, H3K9m3, HP1β and the double modification H3K9m3S10P.

Our results show that indeed the cumulus donor nucleus is remodelled to a certain extent in cloned mouse embryos as evidenced by the chromatin configuration seen in early 1-cell stages, as early as 4hpa (~ 18hp hCG). At this time point H3S10P showed the same distribution pattern as observed in fertilised embryos. The pseudo-pronuclei showed uniform staining for H3S10P in the nucleoplasm and strong labelling in the heterochromatin rings around the NPBs from 5hpa to 10hpa. However, at 10hpa (late 1-cell) we observed that both H3K9me3 and HP1β accumulated around the NPBs but also in the nuclear periphery, an aberration known to correlate with poor development (Martin et al., 2006b;[START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF]. Differently from these two markers, H3S10P only co-localized with the heterochromatin located around the NPBs. Altogether, this indicates that H3S10P, as opposed to H3K9me3 and HP1β, only labels remodelled pericentromeric heterochromatin located around the NPBs, resembling exactly the normal embryonic heterochromatin arrangement. This also proves that heterochromatin clumps located at the nuclear periphery are unremodelled, maintaining the epigenetic signature of the cumulus cells (with H3K9me3 and

HP1β staining on chromocenters).

As for the other epigenetic modification studied, H3K9me3S10P, a strong staining was also detected in heterochromatin accumulations in the nuclear periphery but only at very early stages (4hpa). Later on (7hpa/10hpa), the spatial distribution of H3K9me3S10P shifted and the double modification only co-localized with the heterochromatin rings around the NPBs.

We can hypothesize that this double modification was being progressively reprogrammed during the first cell cycle and finally overlapped with the portion of remodelled heterochromatin. In light of these results, we hypothesize that both epigenetic modifications H3S10P and H3K9me3S10P could be used to follow up reprogrammed heterochromatin during nuclear remodelling.

The perinuclear accumulations seen in 1-cell stage cloned embryos with H3K9me3 and HP1β staining are probably due to irregular chromatin rearrangement. The reason for this preferential positioning is unknown, however we can infer that oocytes proteins responsible for nuclear and chromatin organization are involved. It is known that the somatic chromatin configuration must be reshuffled by the reprogramming factors present in the cytoplasm of the enucleated oocyte. Among them are the nuclear lamin filaments that lie on the interface of the nuclear envelope and chromatin playing a major role in nucleoskeleton support, chromatin remodeling, as well as protein recruitment to the inner nucleolus [START_REF] Hall | Nuclear lamin antigen and messenger RNA expression in bovine in vitro produced and nuclear transfer embryos[END_REF]. It has also been suggested that additional "motor proteins" are in place to assist with chromatin organization, such as nuclear actin in mouse embryos [START_REF] Nguyen | Immunofluorescent localization of actin in relation to transcription sites in mouse pronuclei[END_REF]. It is not known how and what makes chromatin move inside the nucleus and if this process happens in a coordinated or random way. However, it might be that during the nuclear transfer procedure lamins and motor proteins are disrupted, and that as a result chromatin is misplaced. It would be of great relevance to further investigate this hypothesis, in order to better understand the role of these proteins in nuclear and chromatin organization within the early stages of reprogramming.

In fertilised embryos, it unmistakably appears that H3S10P is a better marker of pericentromeric heterochromatin as compared to other epigenetic markers such as H3K9me3 and H3K9me3S10P, since it is always correlated to this type of heterochromatin in both inherited parental genomes, from the very beginning of development. Indeed, both H3K9me3 and H3K9me3S10P epigenetic modifications showed parental asymmetry over the whole first cell stage. Similarly to H3K9me3 [START_REF] Cowell | Heterochromatin, HP1 and methylation at lysine 9 of histone H3 in animals[END_REF][START_REF] Santos | Dynamic chromatin modifications characterise the first cell cycle in mouse embryos[END_REF] this study), only the maternal pronucleus is labelled for H3K9me3S10P epigenetic modification in the rings around the NPBs and a diffuse staining is observed in the nucleoplasm after fertilization.

However, a slight decrease in H3K9meS10P intensity is seen starting from 20h phCG. This decrease most probably corresponds to chromatin decondensation and incorporation of new histones H3 upon the first replication phase as already described for H3K9me3 [START_REF] Liu | Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos[END_REF][START_REF] Wang | Dynamic reprogramming of histone acetylation and methylation in the first cell cycle of cloned mouse embryos[END_REF].

Regarding entry in the first mitosis, we observed that all three epigenetic markers were present: 1) H3S10P covered equally both parental genomes, 2) H3K9me3 strongly labelled the maternal genome and faintly stained the paternal one only at the very end of the 1-cell stage and 3) The intensity of H3K9meS10P clearly increased covering both parental genomes.

Conversely, it is known that HP1β proteins preferentially localize within condensed inactive heterochromatin and that it dissociates from chromatin during mitosis [START_REF] Minc | Localization and phosphorylation of HP1 proteins during the cell cycle inmammalian cells[END_REF][START_REF] Hayakawa | Cell cycle behavior of human HP1 subtypes: distinct molecular domains of HP1 are required for their centromeric localization during interphase and metaphase[END_REF][START_REF] Puschendorf | PRC1 and Suv39h specify parental asymmetry at constitutive heterochromatin in early mouse embryos[END_REF]. In fact, H3K9me3 and HP1 are working together to propagate heterochromatin and cause gene silencing [START_REF] Lachner | Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins[END_REF] but the binding of HP1β to the methylated H3-tail is fully reversible and highly dynamic, thereby supporting the rapid exchange of HP1β from heterochromatin [START_REF] Fischle | Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation[END_REF]. It has been proposed in somatic cells that H3S10 phosphorylation prevents the binding of HP1 to the adjacent tri-methylated Lys 9 residue of histone H3 [START_REF] Fischle | Binary switches and modification cassettes in histone biology and beyond[END_REF][START_REF] Hirota | Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin[END_REF]. Also separate observations of H3S10P staining versus H3K9me3 staining first gave the impression that the scenario in mouse embryos was in contradiction with the hypothesis made in somatic cells, we then observed that both parental genomes present a strong signal H3K9me3S10P only upon the first mitosis, exactly when HP1 disappears [START_REF] Puschendorf | PRC1 and Suv39h specify parental asymmetry at constitutive heterochromatin in early mouse embryos[END_REF] and our own unpublished results). We, however, do not know whether additional phosphorylation at the Ser10 occurs on the already tri-methylated Lys9 histone H3 or viceversa. Experiments using ZM447439 (ZM), an inhibitor of Aurora kinases activity, which is required for phosphorylation of H3S10, showed that embryos lacking almost completely H3S10 phosphorylation did not cleave properly (Teperek-Tkacz et al., 2010 and our own unpublished results). Similarly, disruption of the two mouse Suv39h HMTases (Histone Methyl Transferase) that abolishes H3-Lys9 methylation of constitutive heterochromatin induces gestation death or postnatal growth delay [START_REF] Peters | Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability[END_REF][START_REF] Peters | Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin[END_REF].

Both phosphorylation at the Ser10 and tri-methylation on Lys9 might therefore be involved in embryonic chromosome condensation.

At 2-cell stage, heterochromatin undergoes massive distribution changes, moving from the rings surrounding the NPBs towards the nuclear periphery, to form new heterochromatin domains, the chromocenters (Martin et al., 2006a;[START_REF] Merico | Epigenomic differentiation in mouse preimplantation nuclei of biparental, parthenote and cloned embryos[END_REF]). However, in cloned embryos, it appears that the heterochromatin markers H3K9me3 and HP1β have a distinct behavior. In early 2-cell cloned embryos, the two markers were observed in rings around the NPBs, a characteristic of normal early 2-cell stage embryo, but also in numerous foci within the nucleoplasm (Martin et al., 2006a;[START_REF] Merico | Epigenomic differentiation in mouse preimplantation nuclei of biparental, parthenote and cloned embryos[END_REF]this study). These foci most probably correspond to the remains of unremodelled heterochromatin clumps inherited from the donor cells. On the other hand, we observed that H3S10P and H3K9me3S10P are both labelling only pericentromeric heterochromatin rings around the NPBs in early 2-cell, following the same nuclear movements in fertilised and cloned embryos. It therefore seems that the most important restructuring events occur during the first cell stage, with relocalization of remodelled pericentric heterochromatin towards the NPBs and that cloned embryos do not undergo further reprogramming at the 2-cell stage.

There have been few studies addressing the importance of genome reorganization after nuclear transfer. In these studies, inhibitors of specific epigenetic modifications like DNA methylation and histone acetylation were applied in an attempt to improve cloning efficiency by improving chromatin remodelling [START_REF] Yamagata | Centromeric DNA hypomethylation as an epigenetic signature discriminates between germ and somatic cell lineages[END_REF][START_REF] Wang | Dynamic reprogramming of histone acetylation and methylation in the first cell cycle of cloned mouse embryos[END_REF][START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF][START_REF] Bui | Effect of trichostatin A on chromatin remodeling, histone modifications, DNA replication, and transcriptional activity in cloned mouse embryos[END_REF]. It has been shown that the use of trichostatin A (TSA), an inhibitor of deacetylases, which is known to increase the acetylation levels in somatic cells and embryos improves cloned mouse development [START_REF] Wang | Dynamic reprogramming of histone acetylation and methylation in the first cell cycle of cloned mouse embryos[END_REF]. Moreover, culturing reconstructed embryos in the presence of this drug improved chromatin reorganization. The centromeric and pericentromeric heterochromatin pattern from the TSA-treated cloned embryos resembled more the spatial distribution seen in fertilised ones and a lower amount of embryos displayed irregular heterochromatin clusters not associated to the NPBs [START_REF] Maalouf | Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term[END_REF]. Therefore, rectifying reprogramming of these epigenetic modifications at an early stage may be a strategy to improve cloning efficiency [START_REF] Shao | Effect of trychostatin A treatment on gene expression in cloned mouse embryos[END_REF]. As H3S10P and H3K9me3S10P seem to be good markers to trace remodelled pericentromeric heterochromatin after nuclear transfer, we speculate that, by the use of specific drugs, we could increase the levels of this histone H3 phosphorylation and somehow heterochromatin remodelling could be enhanced. Caffeine, a protein phosphatase inhibitor has for example been used to treat oocytes before nuclear transfer. This treatment increased the frequency of PCC as well as the development of cloned sheep embryos [START_REF] Lee | Caffeine treatment prevents age-related changes in ovine oocytes and increases cell numbers in blastocysts produced by somatic cell nuclear transfer[END_REF][START_REF] Choi | Caffeine treatment of ovine cytoplasts regulates gene expression and foetal development of embryos produced by somatic cell nuclear transfer[END_REF]. These authors believe that this increases the removal of chromatin bound proteins, thus allowing the access of oocyte derived factors involved in the reprogramming of the somatic DNA. The same principle could also be applied in regards to histone H3 phosphorylation. Treating cloned embryos with caffeine would in all probability raise the levels of the Aurora kinase responsible for H3S10 phosphorylation, making the chromatin more accessible to remodeling factors thus facilitating even more heterochromatin remodeling. At the 1-cell stage, it appears that HP1β shows accumulation in the nuclear periphery (arrows) while a stronger signal for H3S10P is visualized around the NPBs, both at 4hpa and 10hpa. At (scale bar: 10µm). In one-cell fertilised embryos the two parental pronuclei can clearly be distinguished (m: maternal and p: paternal). To make comparisons easier we rotated the images, when required, to show the paternal one on the right hand side (PB: Polar Body). We observed that H3K9me3S10P epigenetic modification showed maternal specific labelling over the whole first cell stage. By the late 1-cell stage, H3K9me3S10P intensity decreases and 5 the staining remains only on the NPBs periphery (arrowheads). Remarkably, both parental genomes were labelled during mitosis, from the very late 1-cell stage until the formation of 2cell stage nuclei ~1hour post cleavage (note that late 1-cell and 2-cell are single z-sections whereas Prophase / Metaphase / Anaphase correspond to z-stack projections). In clones, we observed strong perinuclear blocks of heterochromatin at the beginning of the 1-cell stage 10 (arrowheads) but not in late 1-cell embryos. These blocks of heterochromatin could also be observed in some donor cumulus cells that can be seen on the Metaphase II oocyte (arrows).

  Fig 1). Upon formation of the pronuclei, a diffuse nucleoplasm H3S10P labelling was present in all embryos with some accumulations appearing at NPBs periphery (Nucleolar Precursor Bodies) (n=35 early 1-cellat 18 hphCG; Fig 1 arrows). This perinucleolar staining then formed full heterochromatin rings around the NPBs (n=29 at 20hphCG) that became more intense in late 1-cell embryos (n=33 at 26hphCG; Fig 1). When entering mitosis of the first cell cycle, H3S10P staining had spread out throughout the whole mitotic chromosomes as on the metaphase II chromosomes (n=25 at 28-30hphCG; Fig 1 and data not shown). For early 2-cell stage embryos, the staining was again present uniformly in the nucleoplasm and more intensely on the heterochromatin rings surrounding the NPBs (n=18 at 36hphCG; Fig 1). As the second cell cycle progresses, the pericentromeric regions of different chromosomes are assembled together to form these unique structures called chromocenters that are strongly labelled with H3S10P (n=48 at 48hphCG; Fig1 arrows). Regarding the second cell cycle, phosphorylation of histone H3 at serine 10 was also detected on the whole chromosomes following the same pattern as seen during the first mitosis (data not shown).

  cloned mouse embryos produced by somatic cell nuclear transfer with cumulus cells (Fig 1, lower panel). In these embryos, phosphorylation of histone H3 at Ser10 was detected as early as 2hpa (post activation) on the chromatin of the mouse cumulus cell which is undergoing PCC (Premature Chromosome Condensation) inside the enucleated mouse oocytes (n=22; Fig 1). At 4hpa when pseudo-pronuclei are formed, H3S10P recapitulates exactly the same pattern as observed in early fertilised embryos with uniform staining in the nucleoplasm (n=19 early 1-cell, Fig 1). Only 3 of these embryos (~16%) showed a slight labelling of the heterochromatin rings surrounding the NPBs. One hour later, 100% of the cloned embryos analyzes had these perinucleolar rings labelled with H3S10P (n=17, 5hpa, Fig 1 arrows). At the end of the 1-cell stage, all the cloned embryos showed a strong H3S10P labelling corresponding to the one observed in fertilised embryos (n=16 at 7hpa; n=17 at 8hpa and n=29 at 10hpa, Fig 1). Regarding the 2-cell stages, all the cloned embryos had the same H3S10P pattern as seen in fertilised ones (Fig 1). At 21hpa (corresponding to early 2-cell), H3S10P was observed uniformly in the nucleoplasm and a strong signal was still seen on the heterochromatin rings at NPBs periphery. This staining was then replaced by labelling in the chromocenters forming at late 2-cell (33hpa, Fig 1 arrows).

  with H3S10P (n=11, Fig 2 arrow). Remarkably, HP1β and H3S10P showed co-localization only when heterochromatin rings appeared around the NPBs. At late 1-cell (10hpa) HP1β was still observed in numerous perinuclear foci but also around all the NPBs. However H3S10P did not show any co-localization with HP1β in the clumps of heterochromatin found at the nuclear periphery, but only in the ones surrounding the NPBs (n=21; Fig 2 line scans).

  early 2-cell (21hpa) HP1β was detected in the nucleoplasm, around the NPBs and in isolated foci. As in 1-cell cloned embryos, H3S10P showed almost no co-localization with HP1β in isolated foci but only on NPBs periphery (n=15, Fig 2 line scans). This difference disappeared by the late 2-cell stage (36hpa) when both markers mostly co-localized within the newly formed chromocenters (n=10, Fig 2 arrows).

Finally

  Fig 3). Again, this suggested that cumulus inherited heterochromatin was not fully remodeled

  Fig 4 ) and both pseudo-pronuclei showed strong staining of heterochromatin clumps just

Figure 1 .

 1 Figure 1. H3S10P detection in fertilised and cloned embryos

Figure 2 .

 2 Figure 2. Double immunostaining with H3S10P and HP1β in early cloned embryos.

  early 2-cell (21hpa) H3S10P and HP1β only show co-localization on the rings of heterochromatin; again the HP1β foci lack a signal for H3S10P. Nearly full co-localization of both markers is seen by the end of the 2-cell stage (33hpa, arrows). Scale bar: 10µm.

ACKNOWLEDGEMENTS

We are grateful to Michael Jeanblanc for his help with the experiments on H3K9me3 and image acquisitions. We would also like to thank the platform MIMA2 (Microscopie et Imagerie des Microorganismes, Animaux et Elements) for confocal microscopy and IERP for animal care. The present work was supported by INRA « Jeune Equipe » funding and the European CLONET (MRTN-CT-2006-035468) grant. KM also obtained support from the Fondation pour la Recherche Médicale (FRM).

AUTHOR DISCLOSURE STATEMENT

The authors declare that no conflicting financial interests exist.

Line scans on the right column show the local intensity distribution of H3S10P (green) and HP1b (red) labellings. In the merged images, the positions and directions of the line scans are indicated by long arrows. Asterisks on the line scans point to several positions that correspond to HP1β foci without H3S10P signal. Hash symbols underline the chromocenters labelled with both HP1β and H3S10P.


Ausrine Bartasyte
email: ausrine.bartasyte@gmail.fr

Adulfas Abrutis

Carmen Jiménez

François Weiss

Odette Chaix-Pluchery

Z Saltyte

C Jimenez

Ferroelectric PbTiO3

Keywords: liquid injection MOCVD, PbTiO 3 films, twinning

Introduction

Lead-based ferroelectric oxides, such as PbTiO 3 (PTO), PbTi 1-x Zr x O 3 (PZT) and etc., have been widely investigated as materials for applications in microelectronics [START_REF] Scott | Ferroelectric memories[END_REF], piezoelectric devices [START_REF] Ohtani | PbTiO 3 thin film ultrasonic micro-sensor fabricated on Si wafer[END_REF] and infrared sensors [START_REF] Takayama | Pyroelectric linear array infrared sensors made of c-axis oriented La-modified PbTiO 3 thin films[END_REF]. PbTiO 3 thin films attracted much attention due to high Curie temperature, low dielectric constant and high pyroelectric coefficient. Ferroelectric properties originate from the structural anisotropy, so high quality epitaxial films are required to take full advantage of the material properties and for applications in devices. Epitaxial PTO films have been grown by metalorganic chemical vapour deposition (MOCVD) [START_REF] De Keijser | The structure of heteroepitaxial lead titanate layers grown by organometallic chemical vapour deposition[END_REF][START_REF] Chen | PbTiO 3 films prepared by metalorganic chemical vapour deposition on LaAlO 3[END_REF][START_REF] Schmidt | MOCVD of ferroelectric thin films[END_REF][START_REF] Funakubo | Comparison of deposition behavior of Pb(Zr,Ti)O 3 films and its end-member oxide films prepared by MOCVD[END_REF][START_REF] Korsakov | Deposition of PbTiO 3 thin films from thd-complexes by an aerosol source MOCVD method[END_REF], pulsed laser deposition (PLD) [START_REF] Kim | Thickness effect of ferroelectric domain switching in epitaxial PbTiO 3 thin films on Pt(001)/MgO(001)[END_REF][START_REF] Kang | Preparation of epitaxial PbTiO 3 thin films by pulsed laser deposition[END_REF] and rf-magnetron sputtering [START_REF] Wasa | Crystal growth of epitaxially grown PbTiO 3 thin films on miscut SrTiO 3 substrate[END_REF]. Among various preparation methods, MOCVD has been recognized as the most promising technique due to the possibility to grow films on large area with high growth rate and good conformal step coverage.

In the literature, mainly liquid PbEt 4 and Ti(O i Pr) 4 compounds were used as precursors for MOCVD growth of PTO films by bubbling of carrier gas through containers of liquid precursors [START_REF] Chen | PbTiO 3 films prepared by metalorganic chemical vapour deposition on LaAlO 3[END_REF][START_REF] Schmidt | MOCVD of ferroelectric thin films[END_REF][START_REF] Pan | Abnormal growth of lead titanate thin film in chemical vapour deposition of Pb(C 2 H 5 ) 4 /Ti(O i Pr) 4 /O 2[END_REF][START_REF] Hong | Effect of oxygen pressure upon composition variation during chemical vapour deposition growth of lead titanate films from tetraethyl lead and titanium tetraisopropoxide[END_REF][START_REF] Byon | Corrrelation between domain structure and ferroelectricity in PbTiO 3 thin films[END_REF][START_REF] Dormans | Composition-controlled growth of PbTiO 3 on SrTiO 3 by organometallic chemical vapour deposition[END_REF][START_REF] Ramana Murty | In situ x-ray scattering study of PbTiO 3 chemical vapour deposition[END_REF]. However, some other pairs of Pb and Ti precursors have been also studied: Pb(thd) 2 (thd = 2,2,6,6-tetramethyl-3,5-heptanedionate) and Ti(O i Pr) 4 [START_REF] Kim | Microstructural, electrical and transmittance properties of PbTiO 3 films grown on p-InP (100) substrates at low temperature[END_REF][START_REF] Chung | Crystalline structures of the PbTiO 3 films prepared using the ECR PECVD method[END_REF][START_REF] Nishida | Effect of the substrate on properties of PTO thin film[END_REF][START_REF] Gao | Effects of substrate orientation and cooling rate in microstructure of PbTiO 3 thin films grown by metal-organic chemical vapour deposition[END_REF], (C 2 H 5 ) 3 PbOCH 2 C(CH 3 ) 3 and Ti(O i Pr) 4 [START_REF] Tokita | Fabrication of PbTiO 3 thin films by laser metalorganic chemical vapour deposition[END_REF], Pb(OAc) 2 and Ti(OnBu) 4 [START_REF] Malic | Effect of Precursor type on the microstructure of PbTiO 3 thin films[END_REF], PbEt 4 and Ti-npropoxyde [START_REF] Erbil | Oxide ferroelectric materials grown by metalorganic chemical vapour deposition[END_REF] or PbEt 4 and TiCl 4 [START_REF] Tong | Plasma-enhanced chemical vapour deposition of PbTiO 3[END_REF]. Only several works [START_REF] Rebane | CVD synthesis and SNMS characterization of thin films of ABO 3 perovskite-type materials (PbTiO 3 , La(Sr)CoO 3 , La(Sr)MnO 3 , LaNiO 3 )[END_REF][START_REF] Lu | Novel single-solid-source metalorganic chemical vapour processing of PT and PZT thin films[END_REF] can be found on the use of solid Pb(thd) 2 and Ti(O i Pr) 2 (thd) 2 or TiO(thd) 2 precursors for PTO depositions. In these works, the vapour phase was generated by flash evaporation of a mixture of solid Pb and Ti precursors supplied into an evaporator. Solid metalorganic precursors are often preferred because they are non-toxic and more stable at room temperature as compared with liquid and extremely toxic tetraethyl lead precursor. In the last years, single source liquid injection MOCVD techniques have been more and more used for the growth of multi-component oxide layers. Moreover, these techniques allow an easier control of the composition of multi-component films and a better reproducibility of the process than conventional CVD.

In the present paper, we report on the role of oxygen partial pressure, deposition temperature and solution composition in the growth of PbTiO 3 films by pulsed liquid injection MOCVD using Pb(thd) 2 and Ti(O i Pr) 2 (thd) 2 precursors dissolved in an organic solvent. The influence of such parameters on films microstucture, composition and morphology was studied.

Experimental details

Depositions were carried out by pulsed liquid injection MOCVD technique [START_REF] Sénateur | Procédé et dispositif d'introduction de précurseurs dans une enceinte de dépôt chimique en phase vapeur[END_REF][START_REF] Sénateur | Advances in Inorganic Films and Coatings[END_REF][START_REF] Sénateur | Innovative processing of films and nanocrystalline powders[END_REF] in a vertical hot wall reactor. Microdoses (a few microliters) of an organic solution containing a mixture of metalorganic precursors were repeatedly injected into a hot evaporator using a special computer controlled injector. The vapour mixture (precursor + solvent) formed after flash evaporation of micro-doses was transported by an Ar+O 2 gas flow into a reaction chamber to a hot substrate. The main advantages of this technique are an easy control of precursor feeding rate, vapour and film composition. Moreover, precise and reproducible pulsed microdosing of precursor solution ensures growth like "digital", and consequently a simple control of film thickness by pulse number.

Main deposition conditions are summarised in Table 1.

Pb(thd) 2 and Ti(thd) 2 (OiPr) 2 precursors were synthesized and purified in our laboratory at Vilnius university. It is worth noting that the quality of precursors, especially of Pb(thd) 2 , is crucial for obtaining good quality PTO films. For example, we tried to test some commercial Pb(thd) 2 precursors and we always had problems to prepare clear solutions without turbidity.

The morphology of the film surface was examined by using scanning electron microscopy (SEM) (Philips XL30) and atomic force microscopy (AFM) (Digital instruments Multimode Scanning Probe Microscope, tapping mode). Films on Si were etched with concentrated HF in order to form a step for thickness determination by profilometry. The film thickness on other substrates was measured from the film cross-section images obtained by SEM. Energy dispersive X-ray spectroscopy (EDS) or wavelength dispersive X-ray spectroscopy (WDS) analysis was used for the evaluation of the film composition. Crystallization, phase purity, inplane and out-of plane orientation of the films were studied by X-ray diffraction (XRD) in Bragg-Brentano and Schulz geometries using a SIEMENS D5000 diffractometer with monochromatic CuK a1 radiation (l = 0.15406 nm). The percentage of a-axis oriented domains (A, %) in the films was calculated by peak integration of the (102) reflection in j-scans corresponding to the different domains: Raman spectra were collected using a Jobin-Yvon/Horiba Labram multichannel spectrometer equipped with a liquid N 2 cooled CCD detector. Experiments were conducted in the micro-Raman mode at room temperature, in a backscattering geometry. The 514.5 nm line of an Ar + laser was focused to a spot size smaller than 1 µm. The recorded spectra were calibrated using Si spectra at room temperature. The integration times were adjusted in order to have a high signal-to-noise ratio.

Results and discussion

Effects of oxygen partial pressure

In order to test the oxygen effect on Pb content of the film, PTO films were grown at 650°C at four different oxygen partial pressures. Composition analysis showed that the maximum Pb content in films was obtained using 37.5 % oxygen in the gas flow (Fig. 1a). The maximum growth rate (1.03 mm/h) of the films was obtained also at 37.5 % O 2 . L.S. Hong et al. [START_REF] Hong | Effect of oxygen pressure upon composition variation during chemical vapour deposition growth of lead titanate films from tetraethyl lead and titanium tetraisopropoxide[END_REF] established that an increase of O 2 concentration in gas promotes the PbO formation resulting in higher growth rate. However, too high oxygen concentration resulted in Pb deficient films, that was explained by stabilisation of high valency Pb 3 O 4 and PbO 2 at higher O 2 partial pressures [START_REF] Hong | Effect of oxygen pressure upon composition variation during chemical vapour deposition growth of lead titanate films from tetraethyl lead and titanium tetraisopropoxide[END_REF]. The optimum percentage of oxygen in the gas flow is highly dependent on the precursor type and its thermal stability. For example, in case of Pb(C 2 H 5 ) 4 only 1 % of O 2 in gas flow was sufficient [START_REF] Hong | Effect of oxygen pressure upon composition variation during chemical vapour deposition growth of lead titanate films from tetraethyl lead and titanium tetraisopropoxide[END_REF], while in our case it was necessary to use much more oxygen. In further depositions, we used 37.5 % oxygen in the gas flow.

Pb may be lost from PTO film during post-deposition treatment at higher temperatures or during the slow cooling after deposition at high deposition temperature. To get more information about the lead loss at high temperatures, several series of rapid thermal annealing (RTA) for 10 min at different temperatures and oxygen partial pressures were carried out. The composition of annealed films was analyzed by WDS and results are given in Fig. 1b. The films started to lose Pb at 700 °C and the Pb loss rate rapidly increased with further increase of annealing temperature. The lead content did not change after annealing at lower temperatures.

One can see in Fig. 1b that the Pb desorption rate can be reduced by increasing oxygen partial pressure. It means that high oxygen pressure during cooling stabilizes the PbTiO 3 phase in our films. In order to minimize the Pb loss, we used 0.4 atm O 2 pressure during film cooling from high deposition temperature.

Composition and morphology

Series of films were deposited at different temperatures in varying the solution composition. The composition of films deposited on LaAlO 3 (LAO) was analyzed by WDS.

Results are given in Fig. 2. It is evident that higher deposition temperatures require higher Pb precursor content in solution to achieve the right cationic proportion in the film. This may be related to higher lead oxide desorption at higher temperatures. At low T dep = 550 ˚C, the Pb content in film gradually increases with the Pb content in solution. At higher deposition temperatures, the Pb fraction in the film increases until near stoichiometric value; it remains almost stable during further increase of the Pb content in solution. The minimum Pb content in solution necessary for achieving right Pb:Ti ratio in film may be named as "stoichiometric".

Although films grown from solution with Pb precursor excess may have right cationic ratio, they consist not only of tetragonal PbTiO 3 phase but also contain some additional phases (PbTi 0.8 O 2.6 , PbO).

The morphology of PTO films is very dependent on the solution composition especially for epitaxial films on LAO and SrTiO 3 (STO) substrates (Fig. 3). Films grown from Pb deficient or "stoichiometric" solutions on both substrates have similar surface morphologies. On the other hand, the morphology of PTO films on sapphire is fully determined by island growth at all deposition temperatures resulting in rather rough film surface (Ra = 3-7 nm).

Layers grown from solution with Pb deficit are made up of two zones, which become better separated in films deposited at higher temperatures (Fig. 3). XRD showed that these films consisted of tetragonal PTO and pyrochlore phases (Fig. 4). One zone is very smooth and the second one is formed from small crystallites. As these different zones are clearly visible by optic microscope, Raman spectra in each zone have been measured (Fig. 5). One can see that the smooth zone consists only of tetragonal PTO phase. The other zone presents a phase with lower symmetry than tetragonal PTO -it can be considered as monoclinic pyrochlore phase. Such film morphology (called "rosette-shaped") was also observed by other authors [START_REF] Chen | Texture development, microstructure evolution, and crystallization of chemically derived PZT thin films[END_REF][START_REF] Kim | The effect of RuO 2 /Pt hybrid bottom electrode structure on the leakage and fatigue properties of chemical solution derived Pb(Zr x Ti 1-x )O 3 thin films[END_REF][START_REF] Yao | Effects of postdeposition annealing on the dielectric properties of antiferroelectric lanthanum-doped lead zirconate stannate thin films derived from pulsed laser deposition[END_REF] and was considered as perovskite islands in a pyrochlore matrix.

With the increase of Pb content in solution, smooth PTO zones become bigger and all the surface becomes smooth at Pb/Ti = 1 in the film. The morphology of PTO films is defined by two factors: island growth and twinning (this aspect will be discussed in detail in the next section). Twinning results in a film morphology with perpendicular line shaped zones [START_REF] Ganpule | Imaging three dimensional polarization in epitaxial thin films grown by pulsed laser deposition[END_REF]. At higher temperatures, twinning becomes the leading factor for surface morphology of PTO films.

For example, the morphology of films grown at 650 °C and higher temperature is mainly determined by twinning. Less twinned zones are observed in films grown at 600 °C. The morphology of films deposited at 550 °C is completely determined by island growth resulting in a rather rough surface compared to the twinned layers. Thus, the roughness of the films grown from "stoichiometric" solutions decreases with the increase of deposition temperature (Fig. 6), and Ra reaches a very low value, 0.6 nm for films grown at 750 °C.

PTO films grown on STO substrates from solutions with Pb excess consist of spherical grains forming the structure of lines (Fig. 3). Such structure becomes more ordered with increasing growth temperature and at 700 °C the lines are quasi parallel. In contrast, films on LAO substrates consist also of spherical grains but the line shaped structure is not observed.

Microstructure

PTO films on sapphire are polycrystalline. XRD spectra of PTO films on LAO and STO substrates grown from "stoichiometric" solutions consist only of (00l) and (h00) reflections (Fig. 4a) corresponding to c-axis and a-axis textured crystallites, respectively (c-and adomains). The best crystallinity was obtained for films deposited at 650 °C. PTO films on both STO and LAO substrates had very good in-plane orientation, as seen from pole figure for (102) reflection given in Fig. 7. Double peaks (split in c angle) appear in pole figure of a-domains due to the domain twinning.

As it is known, a-domains and c-domains in epitaxial ferroelectric films are tilted by a small angle from the substrate plane normal due to the twinning phenomenon [START_REF] De Keijser | The structure of heteroepitaxial lead titanate layers grown by organometallic chemical vapour deposition[END_REF]. The adomains and c-domains are bounded by the (101) twin plane, and this results in a tilt of [100] axis in a-domains, the tilt angle depending on the tetragonality of the cell (for c/a = 1.065 the tilt angle is 3.7 °) [START_REF] De Keijser | The structure of heteroepitaxial lead titanate layers grown by organometallic chemical vapour deposition[END_REF]. The tilt angle also depends on the domains volume fraction [START_REF] Kang | Crystallographic characterization of tetragonal (Pb,La)TiO 3 epitaxial thin films grown by pulsed laser deposition[END_REF]. For films with a high fraction of c-domains, the c-domains tilt angle is very small [START_REF] Foster | Substrate effects on the structure of epitaxial PbTiO 3 thin films prepared on MgO, LaAlO 3 , and SrTiO 3 by metalorganic chemical vapour deposition[END_REF]. Twinning is responsible for double rocking curves of (00l) and (h00) reflections and for the split in c angle of peaks in pole figures (Fig. 7) of a-domains. Rocking curves of (001) reflection of PTO/LAO films deposited at different temperatures are given in Fig. 8. As mentioned about the surface morphology, twinning becomes a dominant factor when deposition is taken out at 650 °C or higher temperatures whereas the microstructure of layers deposited at lower temperatures is mainly determined by island growth. When layer is fully twinned (grown at temperatures ≥ 650 °C), the (001) rocking curve is double (Fig. 8). The rocking curves of films deposited at lower temperatures are different. They become triple for film grown at 600 °C and lower temperatures: the peaks on both sides correspond to twinned zones and the central peak to nontwinned zones. This is in agreement with the fact that films grown at temperatures ≤ 600 °C are less or non-twinned and less textured than films obtained at higher temperatures. The twinning angles (tilt angles) for a-and c-domains (a a and a c respectively) in PTO films grown at various temperatures were estimated from rocking curves of (001) and (100) reflections and are given in Fig. 9. PTO films on STO have a highly dominant c-axis texture, so no split of rocking curve of (001) reflection in c-domains was observed. It is possible that the tilt angle of c-domains is too small to be detected.

PTO films grown at various temperatures on STO and LAO substrates consisted of both aand c-domains (Fig. 10). However, the c-domains growth is dominant for all range of deposition temperatures because the a-parameter of PTO cell matches better the parameter of substrate cell. The quantity of a-axis (A, %) textured crystallites in PTO films is clearly related to the deposition temperature and reaches the minimum in highly crystalline films grown at 650 °C. As a-domains form only due to twinning, C.S. Ganpule et al. [START_REF] Ganpule | Imaging three dimensional polarization in epitaxial thin films grown by pulsed laser deposition[END_REF] proposed a relation between the twinning angles of a-and c-domains and the a-domains fraction. This relation perfectly works for our PTO films on STO and LAO substrates: tana c /tana a linearly depends on A/(1-A) (Fig. 11). The bigger the amount of a-domains, the bigger the tilt angle of c-domains and the smaller the twinning angle of a-domains.

Conclusions

The influence of various deposition conditions (deposition temperature, partial oxygen pressure and solution composition) was examined for the growth of PbTiO 3 (PTO) films on various substrates by pulsed liquid injection MOCVD.

The oxygen partial pressure influences the Pb loss during the film growth and post deposition thermal treatments. The maximum Pb/(Pb+Ti) ratio in the film was obtained using 37.5 % O 2 in the gas flow during the deposition. The Pb loss during the cooling from high growth temperature can be reduced using high O 2 pressure (0.4 atm).

The evolution of film composition, microstucture and morphology with solution composition at different deposition temperatures was investigated. Higher deposition temperatures requires bigger amount of Pb precursor in solution for achieving of the right cationic proportion in the film. This may be related to higher Pb desorption at higher temperatures. The solution composition and growth temperature highly influence the film morphology and microstructure. Epitaxial films on perovskite substrates consist of dominant caxis and minor a-axis textured zones (c-domains and a-domains). The surface morphology is mainly determined by island growth for films grown at temperatures ≤ 600 ˚C and by twinning phenomenon for films grown at temperatures ≥ 650 ˚C. Fully twinned or partially twinned films

  the peak integral intensity in j-scans measured at different c angles, corresponding to a-axis (c=58° or 62°) and c-axis (c=27°) oriented crystallites (i.e. a-and c-domains). c angles were determined from c-scans of (102) reflection.

Figure captions Fig. 1 :

 captions1 Figure captions

Fig. 2 :

 2 Fig. 2: Composition of PTO/LAO films in relation with solution composition.

Fig. 3 :

 3 Fig. 3: AFM images (2x2 mm 2 ) of various compositions PTO/LAO and PTO/STO films deposited at different temperatures. The average roughness Ra and Pb/(Pb+Ti) ratio (% in film) are given on photos.

Fig. 4 :

 4 Fig. 4: XRD patterns of (a) stoichiometric and (b) Pb deficient PTO/LAO films grown at 650°C.

Fig. 5 :

 5 Fig. 5: Identification of two zones with different morphology in Pb deficient PTO/LAO film: (a) Raman spectra of two zones, (b) image of optic microscope and (c) AFM image.

Fig. 6 :

 6 Fig. 6: Average roughness (Ra) of PTO films versus deposition temperature (Pb/Ti~1).

Fig. 7 :

 7 Fig. 7: Pole figure of (102) reflection of PTO/LAO film grown at 650 ˚C.

Fig. 8 :

 8 Fig. 8: Evolution of (001) rocking curves in c-domains of PTO/LAO films with deposition temperature.

Fig. 9 :

 9 Fig. 9: Evolution of twinning angles in a-and c-domains with deposition temperature.

Fig. 10 :

 10 Fig. 10: Evolution of a-domains percentage (A, %) in films with deposition temperature.

Fig. 11 :

 11 Fig. 11: Relation between twinning angle and a-domains fraction (A/(1-A)) in PTO films.

Fig. 1

 1 Fig. 1

Fig. 2 Fig. 3

 23 Fig. 2

Fig. 4

 4 Fig. 4

Fig. 5

 5 Fig. 5

Fig. 6

 6 Fig. 6

Fig. 7

 7 Fig. 7

Fig. 8

 8 Fig. 8

Fig. 9

 9 Fig. 9

Fig. 10

 10 Fig. 10

Fig. 11

 11 Fig. 11

can be obtained by varying deposition conditions. Twinning is observed in both a-and cdomains and twinning (tilt) angles depend on a-domains fraction. The best crystallinity and the lowest a-domains fraction were obtained in epitaxial films grown at 650 ˚C on LaAlO 3 (001) and SrTiO 3 (001) substrates.