Hugo Bernard

Barbara Garmy-Susini

Nadera Ainaoui

Loic Van Den Berghe

Alexandre Peurichard

Sophie Javerzat

Andreas Bikfalvi

David P Lane

Jean- Christophe Bourdon

Anne-Catherine Prats
email: catherine.prats@inserm.fr.

Tradgene

The p53 isoform, 133p53, stimulates angiogenesis and tumour progression

Keywords: angiogenesis, p53, isoform, cancer, glioblastoma inserm-00714607, version 1 -

The tumour suppressor p53 involved in DNA repair, cell cycle arrest and apoptosis, also inhibits blood vessel formation, i.e. angiogenesis, a process strongly contributing to tumour development. The p53 gene expresses 12 different proteins (isoforms), including TAp53 (p53or p53), p53 and p53 and 133p53 isoforms (133p53, 133p53 and 133p53133p53 was shown to modulate p53 transcriptional activity andis overexpressed in various human tumours. However its role in tumour progression is still unexplored. In the present study, we examined the involvement of 133p53 isoforms in tumoural angiogenesis and tumour growth in the highly angiogenic human glioblastoma U87.

Our data show that conditioned media from U87 cellsdepleted for133p53 isoforms blockendothelial cell migration and tubulogenesis without affecting endothelial cell proliferation in vitro.133p53 depletion in U2OS osteosarcoma cells resulted in a similar angiogenesis blockade. Furthermore, using conditioned media from U87 cells ectopically expressing each 133p53 isoform, we determined that 133p53and133p53but not 133p53stimulate angiogenesis. Our in vivo data using the chicken chorio-allantoïc membrane and mice xenografts establish that angiogenesis and growth of glioblastoma U87 tumours are inhibited upon depletion of 133p53 isoforms. By TaqMan Low Density Array, we show that alteration of expression ratio of 133p53 and TAp53 isoformsdifferentially regulatesangiogenic gene expression with 133p53 isoforms inducing pro-angiogenic gene expression and repressing anti-angiogenic gene expression.

INTRODUCTION

The well-known tumour suppressor p53 called "the guardian of the genome" was first described as a single protein. However, we have shown that the p53 gene expresses 12 protein isoforms [START_REF] Bourdon | p53 and its isoforms in cancer[END_REF][START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF]. Alternative splicing of intron 9 generates p53 isoforms bearing different Cterminal domains ( and ) (Fig. 1A). The presence of an alternative promoter in intron 4 generates 133p53 isoforms lacking the transactivation domain and part of the DNA-binding domain. In addition, initiation of translation at alternative AUG codons, codon 40 or codon 160, leads to 40p53 and 160p53 isoforms, respectively. p53 protein isoforms can be categorized into four subclasses: TAp53, 40p53, 133p53 and 160p53 protein isoforms.

Each subclass is composed of the three isoforms  and .

p53 isoforms are conserved in Drosophila, zebrafish, mouse and human, but little is known about their functions [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF][START_REF] Chen | p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish[END_REF]. A gain of expression of 133p53 mRNAs in human breast tumours versus normal breast tissue suggests a protumoural role of these isoforms [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF]. Indeed, 113p53 the zebrafishortholog of human 133p53 is able to regulate wild-type p53 (WTp53) pro-apoptotic function [START_REF] Chen | p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish[END_REF]. This activity is conserved in human cells since 133p53 can inhibit p53-mediated apoptosis and G1 cell cycle arrest without inhibiting p53mediated G2 cell cycle arrest [START_REF] Aoubala | p53 directly transactivates Delta133p53alpha, regulating cell fate outcome in response to DNA damage[END_REF]. Furthermore, increased expression of 133p53 is associated with inhibition of replicative senescence in normal human fibroblasts and colon carcinoma [START_REF] Fujita | p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence[END_REF]. However, no data are available about a direct role of 133p53 in tumour progression.

In addition to its ability to control DNA repair, cell cycle arrest and apoptosis, p53 is involved in inhibition of angiogenesis, a critical mechanism in tumour progression and metastatic dissemination [START_REF] Teodoro | Inhibition of tumor angiogenesis by p53: a new role for the guardian of the genome[END_REF]. This process is induced in most solid tumours, whose centers become hypoxic following an increase of the diffusion distance between the nutritive blood vessels and tumour cells. Hypoxia is one of the principal angiogenic stimuli leading to synthesis of angiogenic growth factors and inducing formation of new blood vessels [START_REF] Fraisl | Regulation of angiogenesis by oxygen and metabolism[END_REF][START_REF] Pouyssegur | Hypoxia signalling in cancer and approaches to enforce tumour regression[END_REF]. Such vessels, in addition to their ability to feed tumour cells, provide a way for cells to disseminate and form metastases. p53 inhibits angiogenesis by at least three mechanisms: 1) by regulating expression and activity of the central regulator of hypoxia, the hypoxia-induced transcription factor HIF1-, 2) by inhibiting production of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), and 3) by increasing production of anti-angiogenic factors [START_REF] Teodoro | Inhibition of tumor angiogenesis by p53: a new role for the guardian of the genome[END_REF][START_REF] Dameron | Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1[END_REF][START_REF] Folkman | Tumor suppression by p53 is mediated in part by the antiangiogenic activity of endostatin and tumstatin[END_REF][START_REF] Galy | Tumour suppressor p53 inhibits human fibroblast growth factor 2 expression by a post-transcriptional mechanism[END_REF][START_REF] Pal | Central role of p53 on regulation of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) expression in mammary carcinoma[END_REF][START_REF] Ravi | Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1alpha[END_REF][START_REF] Ueba | Transcriptional regulation of basic fibroblast growth factor gene by p53 in human glioblastoma and hepatocellular carcinoma cells[END_REF].p53 is able to induce tumour dormancy and the loss of WTp53 reverses the tumourswitch from a dormant non-angiogenic state to an inserm-00714607, version 1 -5 Jul 2012 angiogenic invasive phenotype [START_REF] Giuriato | Sustained regression of tumors upon MYC inactivation requires p53 or thrombospondin-1 to reverse the angiogenic switch[END_REF][START_REF] Naumov | Role of angiogenesis in human tumor dormancy: animal models of the angiogenic switch[END_REF].Clinical data also suggest that p53 plays a crucial role in controlingtumour vascularization. However, nothing is known about the role of 133p53 isoforms (,  and ) in this process. Among all solid tumours, glioblastomamultiforme (GBM) are the most angiogenic by displaying the highest degree of vascular proliferation and endothelial cell hyperplasia (for review [START_REF] Wong | Tumour angiogenesis: its mechanism and therapeutic implications in malignant gliomas[END_REF]). Such intense vascularization plays a critical role in the pathological features of GBM, including peritumouraloedema resulting from the defective blood brain barrier (BBB) in the newly formed vasculature. Thus, human glioblastoma U87, expressing WTp53, has been chosen here to address the role of 133p53 isoforms in angiogenesis and tumour progression.

In the present study we establish by knockdown and ectopic expression experiments that angiogenesis can be regulated by manipulating the expression ratio of 133p53 and p53.

RESULTS



Characterization of p53 isoforms expression inhuman glioblastoma U87.

p53 isoforms expression was analyzed at the mRNA level by nested RT-PCR inU87 human glioblastoma cells, which express wild-type p53 gene. We determined that U87 cells express TAp53 isoforms (p53 (p53) and p53 and 133p53 isoforms (133p53 and 133p53Thep53 isoforms subclass as well asp53 and 133p53were not detected at the mRNA level (Fig. 1B) To investigate the role of endogenous p53 isoforms in angiogenesis, U87 cells were transfected with the siRNAsi133p53 which targets specifically the 133p53 isoforms subclass or with the siRNA siTAp53 which targets specifically TAp53 isoforms subclass (Fig. 1A). The specificity of the above siRNAs has previously been assessed and validated [START_REF] Aoubala | p53 directly transactivates Delta133p53alpha, regulating cell fate outcome in response to DNA damage[END_REF][START_REF] Marcel | p53 regulates the transcription of its Delta133p53 isoform through specific response elements contained within the TP53 P2 internal promoter[END_REF].Efficiency of si133p53 and siTAp53 in U87 cells was confirmed by qRT-PCR and Western blotting, as compared to the control siRNA (siNON) (Fig. 1C1, 1C2, 1D1 and 1D2).

As expected, transfection of siTAp53 inhibits p53 mRNA expression but has no effect on 133p53 mRNA expression. Transfection of si133p53 in U87 cells represses 133p53 mRNA expression. However, transfection of si133p53 induces p53 expression at both protein and mRNA levels in U87 cells (Fig. 1C2 and1D2). Similar results were obtained with another siRNA specific for 133p53 isoforms, si133p53-2 (Suppl Fig. S1) [START_REF] Aoubala | p53 directly transactivates Delta133p53alpha, regulating cell fate outcome in response to DNA damage[END_REF]. Thus induction of p53 in response to si133p53 transfection seems to be due to inhibition of p53 mRNA expression by 133p53 isoforms. Co-transfection of U87 cells with siTAp53 and inserm-00714607, version 1 -5 Jul 2012 si133p53 leads to inhibition of 133p53 expression and prevents induction of p53, maintaining p53 expression at its basal level. Therefore, the expression ratio of endogenous 133p53 and p53 isoforms can be manipulated by transfection of siTAp53 and/or si133p53.

Tumour conditioned mediumfrom U87 glioblastomacells or U2OS osteosarcoma cells transfected with si133p53 impairsangiogenesis in vitro.

Anti-angiogenic activities reported for p53 prompted us to look at the effect of 133p53 isoforms in angiogenesis. U87 cells were transfected with si133p53, siTAp53 or siNON and the corresponding conditioned medium was added onto human umbilical vein endothelial cells (HUVECs). Migration of HUVECswas then analyzed in the wound healing assay (Fig. 2A1 and2A2). The ability of cultured HUVECsto form vessel-like structures (tubulogenesis) on matrigel was also investigated (Fig. 2B1 and2B2). Conditioned medium from U87 cells transfected with si133p53 inhibited HUVEC migration as well as their ability to form vessel-like structures (Fig. 2B1 and2B2) whereas conditioned medium from U87 cells transfected with sip53 had no effect. Interestingly, conditioned medium from U87 cells cotransfected with siTAp53 and si133p53 had no effect on HUVEC migration and tube formation as well as proliferation (Fig. 2A1-2B2). Cell proliferation was also assessed: neither si133p53, nor siTAp53, nor the double siRNA treatment had any effect on U87 proliferation(Suppl. Fig. S2) and the different conditioned media had no effect on HUVEC proliferation (Fig. 2C).

Endothelial cell migration was thenstudiedin a transwell migration assay. In this experiment adult bovine aortic endothelial cells (ABAE) were seeded in Boyden Chambers and their migration quantified after treatment with conditioned media of siRNA-transfected U87 cells (Suppl. Fig. S3). Here, the knockdown of 133p53 in U87 cells again resultedin a significant decrease of endothelialcell migration whereas the knockdown of p53 generated a significant increase, suggesting that the absence of increase of HUVEC migration and tubulogenesisfollowingtreatmentwith siTAp53-conditioned mediaisprobaby due to experimental conditions. Such an increasewouldprobably have been observed as for ABAE if migration and tube formation would have been quantifiedatearlier times.

Altogether these data show that the secretion of angiogenic and/or anti-angiogenic factors by U87 cells into the conditioned medium is regulated by the expression ratio of p53 and 133p53 and suggest that the knockdown of 133p53 in U87 cells impairs the ability of these tumour cells to stimulate angiogenesis.

inserm-00714607, version 1 -5 Jul 2012

To determine whether this effect of 133p53 can be generalized to other tumours, HUVEC tubulogenesis was performed using conditioned media of U2OS osteosarcoma cells (p53 wild type) treated with the different siRNAs. The same data were obtained as for U87 cells, showing that the pro-angiogenic effect of133p53 is not restricted to U87 glioblastoma (Fig. 2D).

133p53 and 133p53overexpression stimulates endothelial cell tubulogenesis.

In order to determine which 133p53 isoform ( or is responsible for endothelial cell migration and tubulogenesis, lentivectors expressing each of these isoforms under the control of a tetracyclin-inducible promoter were used to transduce U87 cells (Suppl Fig. S4 andS5).

To measure tubulogenesis, HUVECs were seeded on matrigel as above and incubated in conditioned media produced by doxycycline-treated U87 cells (Fig. 3). Data clearly showed that overexpression of133p53 and 133p53, but not of133p53, are able to stimulate endothelial cell tubulogenesis, suggesting that 133p53 and 133p53 isoforms have a proangiogenic activity. Since U87 cells express 133p53 and 133p53but not 133p53(Fig. 1B), we concluded that only133p53is responsible for the secretion of factors by U87 cells that regulate endothelial cell migration and tubulogenesis of HUVECs.



Si133p53 inhibits angiogenesis and tumour growth in vivo on the chorio-allantoic membrane (CAM).

The effect of133p53 on angiogenesis and tumour growth was addressed in vivo using the experimental glioma assay developed on the chicken CAM, as previously described [START_REF] Hagedorn | Accessing key steps of human tumor progression in vivo by using an avian embryo model[END_REF]. This assay recapitulates hallmarks of human GBM and allows following the first steps of tumoural angiogenesis. siRNA-transfected U87 cells were deposited on the CAM of fertilized eggs and tumours were analyzed 4 days later (Fig. 4A). Transfection of siTAp53 resulted in an increase of tumour vascularization, whereas transfection of si133p53 led to the formation of avascular tumours, showing that angiogenesis can be regulated by change in expression ratio of p53 and 133p53 (Fig. 4B and4C, left panel). Similar results were obtained with si133p53-2 (Suppl Fig. S6). Angiogenesis inhibition upon si133p53 transfection was assessed by counting chick blood vessels in tumour sections stained with SNA-1 lectin conjugated to FITC(Fig. 4C, middle panel). The anti-angiogenic effect upon si133p53 transfection was also correlated with smaller size of tumours at day 4 (Fig. 4C, right panel).

Western blot analyses confirmed that siRNAs were still efficient in tumours 4 days after transfection: tumours treated with siTAp53 or si133p53 showed an decrease or an increase inserm-00714607, version 1 -5 Jul 2012 of p53 expression, respectively (Suppl. Fig. S7). Interestingly, tumours resulting from the cotransfection of siTAp53 and si133p53 looked similar to the ones obtained after transfection of siNON (control). This confirms in vitro dataandindicates that angiogenesis can be regulated by altering the expression ratio of p53 and 133p53 si133p53 inhibits angiogenesis and tumour growth in mouse xenografts.

To study 133p53 involvement in tumour progression, siRNA-transfected glioblastoma U87 cells were subcutaneously implanted in nude mice, and followed during 44 days (Fig. 5 and Table I). At day 7 after xenograft, U87 cells transfected with si133p53 generated significantly smaller tumours compared to U87 cells transfected with si(Fig. 5A).

However, tumours generated from U87 cells transfected with siTAp53 or cotransfected with siTAp53 and si133p53 had similar sizes compared to tumours generated from U87 cells transfected with siNON. The mice were followed until day 44, which clearly showed thattumours generated after si133p53 treatment of U87 grew significantly moreslowly than the control and were drastically smaller than the control at day 35 as well as day 44 (Fig. 5Band Table I). However, in contrast to the data obtained at day 7, the size of tumours from U87 cells co-transfected with si133p53 and siTAp53 was similar to the size of tumours from U87 cells transfected with si133p53.

Angiogenesis was quantified at day 7 by staining of the endothelial cell marker, CD31. Our data showed that tumours from si133p53-transfected U87 cells were significantly less vascularized at day 7, as compared to tumours from U87 cells transfected with siNON or siTAp53 or co-transfected with siTAp53 and si133p53 (Fig. 5C).

These results are consistent with the ones obtained in CAM and confirm that change in expression ratio of p53 and 133p53 regulates angiogenesis and tumour growth in both in vivo models. Hence an increase of 133p53 compared to p53 would stimulate angiogenesis while an increase of p53 compared to 133p53 would inhibit angiogenesis.  p53 and 133p53 regulate angiogenesis by differentially modulating expression of angiogenesis-related genes.

To decipher how p53 and 133p53 regulate angiogenesis, expression of 96 angiogenesisrelated genes was analyzed by TaqMan Low Density Array (TLDA) in siRNA-transfected U87 cells, as indicated (Fig. 6). We used a Taqman human angiogenesis array providing a panel of 96 angiogenesis-related genes(Suppl Table I). In the control, expression of 14/39 inserm-00714607, version 1 -5 Jul 2012 anti-angiogenic genes (35%) and 20/28 pro-angiogenic genes (71%) was detected (Fig. 6A).

Results with si133p53 and siTAp53 were normalized to siNON-transfected U87 cells.

Regarding the anti-angiogenic factors, transfection of si133p53 significantly induced interleukin 12A (IL12A) and matrix metallopeptidase 2 (MMP2), whereas collagen IV (COL4A2) was significantly repressed (Fig. 6B and Table II). Transfection of siTAp53 induced also MMP2 expression, while no change of expression was noted for collagens IV (COL4A1, -A2 or -A3) and XVIII (COL18A1) known as targets of p53 [START_REF] Teodoro | Inhibition of tumor angiogenesis by p53: a new role for the guardian of the genome[END_REF]. In contrast, IL12A was repressed upon TAp53 isoforms depletion. Interestingly, only IL12A expression was still significantly induced upon cotransfection of U87 cells with siTAp53 and si133p53. This indicates that anti-angiogenic factors expression is differentially regulated by the expression ratio of 133p53 and p53. Moreover, it suggests that some genes such as IL12A are regulated by 133p53independently of p53.

Regarding the pro-angiogenic factors, angiogenin (ANG), midkine (MDK, neurite growthpromoting factor 2), EGF-like repeats and discoidin I-like domains 3 (EDIL3) and hepatocyte growth factor (HGF) were significantly down-regulated upon transfection of si133p53. In contrast, transfection of siTAp53 had no effect on the level of expression of these genes (Fig. 6C). Interestingly, the level of expression of the classical angiogenic growth factors VEGF, FGF1 and FGF2 was not significantly affected by any of the siRNAs. Moreover, expression of MDK, EDIL3 and HGF genes remained unchanged in U87 cells co-transfected with siTAp53 and si133p53, while ANG expression was still induced (Fig. 6C). In addition, upon transfection of si133p53, the angiogenic marker angiopoietin-like 4 (ANGPTL4) is downregulated, a repression that is not observed in U87 cells co-transfected with siTAp53 and si133p53 isoforms. This indicates that these pro-angiogenic factors and markers are differentially regulated by the expression ratio of 133p53 and p53(Fig. 6D). Moreover, it suggests that some genes such as ANG are regulated by 133p53independently of p53.

Altogether, our results indicate that upon transfection of U87 tumour cells with si133p53, which represses 133p53 expression and induces p53 anti-angiogenic factors are induced, while expression of secreted pro-angiogenic factors is repressed.

DISCUSSION

We and others have previously shown abnormal expression of p53 isoforms including p53, p53or133p53 mRNA expression in human tumours compared to normal corresponding inserm-00714607, version 1 -5 Jul 2012 tissue [START_REF] Khoury | p53 Isoforms: An Intracellular Microprocessor?[END_REF].p53 expression is associated with good prognosis in breast cancer patients expressing mutant p53 [START_REF] Bourdon | p53 mutant breast cancer patients expressing p53gamma have as good a prognosis as wild-type p53 breast cancer patients[END_REF]. Moreover, 133p53 inhibits replicative senescence, p53mediated apoptosis and G1 cell cycle arrest without preventing p53-promoted G2 cell cycle arrest by regulating gene expression [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF][START_REF] Chen | p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish[END_REF][START_REF] Aoubala | p53 directly transactivates Delta133p53alpha, regulating cell fate outcome in response to DNA damage[END_REF][START_REF] Fujita | p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence[END_REF].Altogether, it suggests that p53 isoforms play a key role in carcinogenesis. However, the role of p53 isoforms in angiogenesis was unknown. The present study reveals that 133p53 is responsible for a strong angiogenesis stimulation that mainly contributes to acceleration of human glioblastoma progression. 133p53 angiogenic effect is due to 133p53 and , but not isoform.

To study the role of p53 isoforms on angiogenesis, we used the glioblastoma U87 model expressing WTp53. Glioblastomamultiforme (GBM) are the most angiogenic by displaying the highest degree of vascular proliferation and endothelial cell hyperplasia. We have shown that U87 cells express p53 and p53 (TAp53 isoforms) as well as 133p53 and 133p53 (133p53 isoforms) at the mRNA level. Only p53 protein could be detected at the protein level because of the low affinity of current p53 antibodies and the low expression level of p53 protein isoforms. However, it is important to note that several studies have reported that p53 protein isoforms are biologically active at low expression level (3-5, 18, 22). By a knockdown approach using siRNAs specific of TAp53 isoforms or of 133p53 isoforms, we have noticed that si133p53 concomitantly represses 133p53mRNAs and induces p53 at the mRNA and protein levels in U87 cells, while siTAp53 represses p53 expression at the mRNA and protein level without altering expression of 133p53 mRNAs.

Interestingly, co-transfection of si133p53 and siTAp53 represses 133p53 mRNAs expression without inducing p53 expression. Since the specificity of siTAp53 and si133p53 has been previously validated, it suggests that 133p53 isoforms repress p53 expression in U87 cells cultured under standard conditions. We and others have shown that p53 transactivates in response to stress the internal p53 promoter giving rise to 133p53 isoforms, thus providing a negative feedback regulation of the p53 response [START_REF] Chen | p53 isoform delta113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish[END_REF][START_REF] Aoubala | p53 directly transactivates Delta133p53alpha, regulating cell fate outcome in response to DNA damage[END_REF][START_REF] Marcel | p53 regulates the transcription of its Delta133p53 isoform through specific response elements contained within the TP53 P2 internal promoter[END_REF][START_REF] Nakagawa | Autoinhibitory regulation of p73 by Delta Np73 to modulate cell survival and death through a p73-specific target element within the Delta Np73 promoter[END_REF]. Altogether, it suggests that p53 and 133p53 isoforms regulate each other expression in U87 cells. Further experiments will be required to decipher the 133p53-mediated repression of p53 in U87 cells.

By incubating conditioned media from siRNA-transfected U87 cells with primary human or bovine endothelial cells, we have demonstrated that the expression ratio of p53 and 133p53 isoforms regulate endothelial cell migration and tubulogenesis. Furthermore their ectopic expression has revealed that 133p53 and 133p53 but not 133p53 are able to stimulate angiogenesisSince 133p53 and 133p53 but not 133p53 are endogenously expressed inserm-00714607, version 1 -5 Jul 2012 in U87 cells, we conclude that stimulation of angiogenesis is only due to endogenous 133p53 in these cells. Using in vitro and in vivo models, we have then established that the p53 and 133p53 regulateangiogenesis by modulating expression of angiogenic-related genes. Hence, an expression ratio of p53 and 133p53 in favor of 133p53 would stimulate angiogenesis while an expression ratio of p53 and 133p53 in favour of p53 would inhibit angiogenesis.  A previous report showed that 113p53, the zebrafishortholog of human 133p53, antagonizes p53-induced apoptosis via Bcl2Lactivation, and differentially modulates p53 target gene expression (3).Moreover, 133p53prevents replicative senescence by inhibiting transactivation of miR-34a by p53 [START_REF] Fujita | p53 isoforms Delta133p53 and p53beta are endogenous regulators of replicative cellular senescence[END_REF]. We have recently established that 133p53 inhibits p53-mediated apoptosis and G1 cell cycle arrest without inhibiting p53-mediated G2 cell cycle arrest. This suggests that 133p53does not exclusively act by inhibiting p53. This has been confirmed by the fact that p21, Bcl-2 and HDM2 genes are differentially regulated by Δ133p53 in response to DNA damage. Indeed, Δ133p53 represses p53 transcriptional activity on p21 promoter, while it increases p53-dependent induction of HDM2 expression and induces Bcl-2 expression independently of p53 (4). Therefore, we concluded that 133p53modulate apoptosis and cell cycle progression in response to stress by regulating gene expression in a p53-dependent and independent manner.

Our present study of angiogenesis-related gene expression by TLDA has figured out that upon siΔ133p53 transfection, which represses 133p53 and induces p53, several anti-and proangiogenic factors are differentially regulated leading to inhibition of angiogenesis.

Reciprocally, upon co-transfection of U87 tumour cells with si133p53 and siTAp53, which represses (not abolishes) 133p53 expression and maintains p53 expression at basal level, one can expect that anti-angiogenic factors are repressed, while expression of secreted proangiogenic factors is induced, leading to stimulation of endothelial cell migration and blood vessel formation (tubulogenesis). Our data suggest that 133p53upregulation affects the angiogenic balance in favour of angiogenesis, and provides an explanation to inhibition of HUVEC tubulogenesis in vitro and tumours angiogenesis in vivo observed in our study. Therefore, angiogenesis through regulation of angiogenesis-related gene expression could be controlled by modulating the expression ratio of p53 and 133p53 using specific siRNAs.

Moreover, the fact that some pro and anti-angiogenic gene factors (such as IL12A and ANG) are still regulated upon co-transfection of siTAp53 and si133p53, suggests that such genes are regulated by 133p53independently of p53. Furthermore, it is important to note that expression of the classical angiogenic VEGF or FGF genes is not significantly altered upon inserm-00714607, version 1 -5 Jul 2012 transfection by siΔ133p53, suggesting a regulation of a distinct signaling pathway involving other angiogenic factors including ANG, HGF and ANGPTL4.

The present study demonstrates for the first time the direct role of 133p53 isoform in tumour progression. Such a feature may be critical in tumours exhibiting WTp53, which represent about 72% of grade I and 35% of grade II glioblastoma and for whichangiogenesis has been recognized as a key event in their progression to malignancy [START_REF] Wong | Tumour angiogenesis: its mechanism and therapeutic implications in malignant gliomas[END_REF][START_REF] Ohgaki | Genetic pathways to primary and secondary glioblastoma[END_REF]. Our data may find an important relevance in anti-angiogenic therapeutics of cancer, presently based on angiogenesis inhibitors targeting the VEGF signaling pathway. Such treatments have been proven to be efficacious in patient survival, associated with a chemotherapy. However, while antitumoural effects and survival benefit are often evident, relapse to progressive tumour growthhas been recently reported, reflecting multiple mechanisms of adaptation to antiangiogenic therapies [START_REF] Ebos | Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis[END_REF][START_REF] Paez-Ribes | Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis[END_REF]. In this context, 133p53 may have a critical role by stimulating angiogenesis through pathways involving several angiogenic factors distinct from VEGF. Thus, targeting 133p53 may have a strong impact in cancer therapeutics.

MATERIALS AND METHODS

Cells and embryos.

HUVECs (Promocell) were cultivated in endothelial growth medium (EGM-2) containing 2% fetal bovine serum (Cambrex/Lonza). ABAE (primary cells) were cultivated as previously [START_REF] Bossard | Antiangiogenic properties of fibstatin, an extracellular FGF-2-binding polypeptide[END_REF].HumanglioblastomaU87-MG (No ATCC : HTB-14, provided by LGCpromochem, Molsheim, France), osteosarcoma U2OS (No ATCC HTB-96) and fertilized chicken eggs (E.A.R.L. Morizeau, Dangers, France) were handled as described [START_REF] Hagedorn | Accessing key steps of human tumor progression in vivo by using an avian embryo model[END_REF].

SiRNA knockdown, cell transfection, western blotting.

Small interfering RNAs (siRNAs) were purchased from Eurogentec (Liege, Belgium) and Sigma-Aldrich (Saint-Quentin Fallavier, France). Sequences are provided in Suppl Table II.

SiNON served as a negative control. SiTAp53 targets the full transactivation domain (TA) of p53 and SiΔ133p53 1 and 2 target the 5'untranslated region of Δ133p53. U87 cells were transfected with siRNAs at 50nM using InterferIN (Polyplus transfection, New York, USA) or with pΔ133p53, using FuGENE reagent (Roche, Meylan, France).

Western blots were performed as previously described [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF]. Primary antibodies were CM-1 and Sapu(2)for p53 and Δ133p53, respectively, 2A10 (Abcam, Cambridge, UK) for mdm2, TUB2.1 (Sigma-aldrich) for β-Tubulin and AC-15 (Sigma-aldrich) for β-Actin. Horseradish inserm-00714607, version 1 -5 Jul 2012 peroxidase-conjugated goat antibodies (Jackson ImmunoResearch, Baltimore), were used as secondary antibodies in immunoblots. Quantitative analysis of the immunoblot data was performed using the ImageJ 1.40g software.

Lentivector production and U87 cell transduction.

The cDNAs coding 133P53, 133P53, or 133P53 respectively, were subcloned into the lentivector pTRIP-DU3-TRE-MCS, derived from the self-inactivating lentivectorpTRIP-DU3-EF1a-EGFP [START_REF] Sirven | Enhanced transgene expression in cord blood CD34(+)-derived hematopoietic cells, including developing T cells and NOD/SCID mouse repopulating cells, following transduction with modified trip lentiviral vectors[END_REF]. The EF1a promoter was replaced by the tetracyclin-inducible pTRE promoter coming from the vector pTRE-tight (Clontech) and a multiple cloning site was inserted in place of the GFP gene (Suppl Fig. S4). The resulting lentivector plasmids were pTRIP-TRE-133P53, -133P53, and -133P53Lentivector particles were produced by the BIVIC vector facility (L. Van den Berghe, F. Gross) from HEK293 cell co-transfection by each plasmid pTRIP with plasmids pLvPack and pLvVSVg (SigmaAldrich) coding human immunodeficiency virus 1 capsid proteins and vasicular stomatitis virus G envelope protein, respectively, as previously described.

Endothelial cell migration, tube formation and proliferation.

HUVECs migration in the "wounding assay" was performed during 8 hours after treatment with conditioned media fromsiRNA-transfected U87 or U2OS cells, as described previously [START_REF] Bossard | Antiangiogenic properties of fibstatin, an extracellular FGF-2-binding polypeptide[END_REF]. Conditioned media were harvested 72h after cell transfection and administrated to HUVEC 48h after starvation. ABAE migration in the transwell migration assay was performed in Boyden Chambers Millipore) according to the manufacturer instruction. Quantification was performed 6h after treatment with conditioned media.

To assess the ability of cultured HUVECs to form vessel-like structures in culture (tube formation), Matrigel (BD Biosciences) was added to the wells of an 24-well plate in a volume of 300 l and allowed to solidify at 37°C for 30 min. After the Matrigel solidified, HUVECs (5x10 4 cells) were plated in 100 l of media EGM-2 with 0.5% serum and 300l of siRNAtumour cells transfected supernatant containing 20 ng/ml FGF2 final. Cultures were photographed and the mean number of branch points (SEM) per microscopic field was determined. Studies were performed in triplicate.

Cell proliferation was determined by using the MTT [3-(4,5-dimethylthiaxol-2-yl)-2,5diphenyltetrazolium bromide] assay. HUVECs (2 × 10 3 /well) were incubated with EBM-2 medium with 2% FBS in 96-well plates. HUVECs were serum starved overnight and inserm-00714607, version 1 -5 Jul 2012 incubated in 50 l of media EGM-2 with 0.5% serum and 150l of siRNAtumour cells transfected supernatant containing 3 ng/ml FGF2 final. The medium then was aspirated and MTT was added to each well (0.25 mg/ml). Cells then were incubated for a further 4 h at 37°C. The medium then was aspirated and the cells were lysed with DMSO and absorbance at 540 nm was measured.

Tumour in CAM and xenografts in mouse.

3 millions of siRNA-transfected U87 cells were deposited on CAM as described [START_REF] Hagedorn | Accessing key steps of human tumor progression in vivo by using an avian embryo model[END_REF]. 18 digital photos were taken on day 4 using a stereomicroscope (Nikon SMZ800).

For xenografts, 1 million cells were injected subcutaneously into nude background (n=8).

Animals were sacrificed 10 days later. Six-week-old nude mice were purchased from Janvier.

Mice were injected with 10 6 siRNA transfected U87 cells subcutaneously into the mid-back region. The tumour size was measured in three dimensions with calipers twice weekly starting at Day 3. Mice were observed for any change in behavior, appearance or weight. At the end of the experiment at Day 10, the mice were killed and xenograft specimens were harvested for further analyses.

Tumours volumes were measured according to the international formuleV= 1/2 (length x width 2 ) (29). Length and width were estimated using a stereomicroscope and Adobephotoshop software.

Histology and immunochemistry.

Tumours from CAM xenografts were fixed and cryo-sectioned as decribed [START_REF] Saidi | Experimental anti-angiogenesis causes upregulation of genes associated with poor survival in glioblastoma[END_REF].Ten micrometer sections were stained with hematoxylin and eosin. For immunohistology studies, the following primary antibodies were used: Ab-2 (clone V9, NeoMarkersAb, Montluçon, France) for vimentin, Anti Ki-67 (AnaSpec Inc., Fremont, CA) for Ki-67, fluorescein-coupled SNA-1 (AbCys, Paris, France) for Sambucusnigralectin (SNA-lectin). Cell nuclei were visualized by DAPI dye. Corresponding secondary antibodies were from Molecular Probes (Invitrogen). SNA-lectin staining allowed quantification of the capillary network invading the tumour by counting the number of green pixels on each field with Adobe photoshop software.

The whole surface of tumours has been counted because of capillary staining heterogeneity (at least 5 tumours / group).

For mice tumourxenografts, five 5 mcryosections of tissues were prepared using a Leica cryostat CM3050. Slides were fixed in ice cold acetone for two minutes, permeabilized in 0.1% Triton X-100 in phosphate buffered saline (PBS), blocked in 5% bovine serum albumin in PBS for 1 hour at room temperature and then incubated with 5 g/ml primary antibodies inserm-00714607, version 1 -5 Jul 2012

for 1 hour at room temperature. Blood vessels were detected with 5g/ml anti-CD31 (BD Pharmingen) antibody. After extensive washing, slides were incubated with 1-2 g/ml crossabsorbed goat anti-rat DyLight 549 (Tebu-bio) secondary antibody for 1 hour at room temperature. Slides were counterstained with DAPI (Tebu-bio). Coverslips were mounted with DakoCytomation fluorescent mounting medium (Dako). For quantification, number of blood vessels in 5-10 microscopic fields per cryosection (per animal) were quantified and the mean number of vessels +/-s.e.m. for the entire treatment group determined.

RNA extraction, nested RT PCR, real time qRT-PCR and TLDA.

Total RNA was isolated from U87 cells using Nucleospin RNA II kit (Macherey Nagel, Hoerdt, France), assessed and nested RT PCR was performed as decribed previously [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF].

Quantitative PCR was performed on StepOne+ (Applied Biosystems) using 10 to 100 ng of cDNA and Power SYBER Green or TaqMan Universal PCR master mix (Applied Biosystems) for detection of FLp53, Δ133p53 and 18S.

TaqManlow density array (Applied Biosystems) was performed following manufacturer instructions with 50 ng of cDNA. The results were treated with the StepOne Software v2.0 (Applied Biosystems). Primers sequences are provided in Suppl Table II.

Statistical analysis

All statistical determination was performed with GraphPad Prism, version 4.0 (GraphPad, San Diego, CA). All data are presented as the mean ± SEM. One-way and two-way analysis of variance was used to evaluate the significance of differences between groups. All statistical analyses were performed with a two-tailed Student's t-test. A p value of less than 0.05 was considered statistically significant.

CONFLICT OF INTEREST

There is no conflict of interest in relation to the work described. Fold change expression of anti-and pro-angiogenic genes upon transfection of U87 cells with siNON, siTAp53, si133p53 or siTAp53 + si133p53 as analyzed by TLDA. (A) Expression of anti-and pro-angiogenic factors in U87 cells represented as the number of genes amplified in basal conditions (SiNON). Fold change mRNA expression of 44 genes divided into 4 categories -pro-angiogenic (B), anti-angiogenic (C), markers and target genes (D) -according to Applied Biosystem classification form(Suppl. Table I). We considered as relevant repression (-) or induction (+) variations superior to two folds. Experiments were repeated twice. Details are presented in Table II. inserm-00714607, version 1 -5 Jul 2012

Figure 1 .

 1 Figure 1. Regulation of expression of endogenous p53 isoforms in U87 cells. (A) Human p53 gene structure. Alternative splicing of intron 9 generates p53 isoforms bearing different C-terminal domains ( and ). TAp53 isoforms include p53 (p53, p53 and p53, whereas133p53 isoforms include 133p53133p53and133p53(2). The different siRNAs used (siTAp53 and si133p53) and the regions they target are indicated underneath the p53 gene structure. The different p53 domains are shown: transactivation domains (TA), proline rich domain (PR), DNA binding domain (DNA BD), nuclear localization signal (NLS) and oligomerization domain (OD). (B) Gel electrophoresis (1% agarose) of p53 isoform mRNAs amplified by nested RT-PCR in U87 cells.Total RNA was extracted from U87 cells and nested RT-PCR were performed, as previously described (2). (C) 133p53 (C1) and p53 mRNAs (C2) relative expression was determined by qRT-PCR following siRNA transfection in U87 cells.Results shown are the average of two independent experiments performed in duplicate. Relative quantification of p53 isoforms was determined by the 2 -Ct by normalizing to 18S rRNA and control (siNON-transfected U87 cells)(4).Results are presented as percentage compared to control. (D1) 133p53 protein expression was analyzed by Western blotting after transfection of U87 cells with siRNA (si133p53 or siNON) and 133p53 expression vector, as indicated. (D2) p53 expression was analyzed by Western blotting after siRNA transfection (-tubulin was used as a loading control). The histogram shows densitometry analysis of multiple scanned Western blot images to quantify the percentage increase over base line of p53 (4 independent experiments).

Figure 2 .

 2 Figure 2. Tumour conditioned mediumfrom U87 cells transfected with si133p53 impairs HUVEC endothelial cell migration and tube formation, but not their proliferation. Conditioned media from siRNA-transfectedU87 cells were used to assess HUVEC migration, tube formation and proliferation. (A1) Micrographs of scratch wound healing assay of HUVEC grown in conditioned media from U87 cells transfected with siRNAs as indicated. (A2) Quantification of HUVEC migration by scratch wound healing assay. (B1) Micrographs (100x magnification) of tube formation by HUVEC grown on Matrigel containing 10ng/mL FGF2 (HUVEC migration must be stimulated to detect a possible inhibition by siRNAs) in

Figure 3 .

 3 Figure 3. 133p53 and 133p53 stimulate endothelial cell tubulogenesis. U87 cells were transduced with lentivectors expressing 133p53, 133p53, or 133p53 under the control of a tetracyclin-inducible promoter. Conditioned media from transduced and doxycycline-treated U87 cells were used to assess HUVEC tube formation as in Figure 2B, except that FGF2 was no added to Matrigel. Left panels: bright field images(100x magnification) of HUVEC in vitro "vessel" formation grown in conditioned media from U87 cells expressing 133p53 (A), 133p53 (B), or 133p53 (C) in response to doxycycline treatment, as indicated. Right panels: Quantification of the number of branch points formed during tubulogenesis. Mean number of branchingpoints per 100X field +/-SEM (*p < 0.05, § p=0.001).

Figure 4 .

 4 Figure 4. Transfection ofsi133p53 inhibits angiogenesis and tumour growthin vivo on the chorio-allantoic membrane (CAM). U87 cells transfected with siNON, siTAp53, si133p53 or siTAp53 + si133p53 (as indicated) were deposited on CAM of fertilized eggs and tumours were analyzed 4 days later.(A) Experimental protocol. (B) Tumour growth was assessed by biomicroscopy (×3 magnification) (I, II, III and IV), fluorescent immunohistochemistry (x20 magnification) (V, VI, VII and VIII) and H&E staining (x20 magnification) (IX, X, XI and XII). Tumour sections were co-stained for the mesenchymal marker vimentin (tumour cell marker, in red)and SNA-lectin (marker of endothelial cells, in green). Numerous irregular and dilated blood vessels are visible in all tumour sections (V, VI and VIII) except for tumours transfected with si133p53 isoforms (VII). H&E staining of the corresponding tumour sections is shown and

Figure 5 .

 5 Figure 5. Transfection ofsi133p53 inhibits angiogenesis and tumour growth in mouse xenografts U87 cells transfected with siNON, siTAp53, si133p53 or siTAp53 + si133p53 at 50nM werexenografted into nude mice. Tumour volumes were quantified from day 7 to day 44 (8 tumours /condition)(Table I). (A) Quantification at days 7. * = p<0,05.(B) Kinetics of tumour growth unitl day 44(C)Immunostaining to detect blood vessels in glioblastoma at day 7. Cryosectionsof xenograftedtumour of U87 cells were immunostained using anti-CD31 antibody. Nuclei were stained with DAPI (blue),200x magnification. Right panel: quantification of number of blood vessels/field. Asterisk indicates statistical significance. *, P < 0.05 vs the SiNON-treated group.

Figure 6 .

 6 Figure 6. p53 and 133p53 regulate angiogenesis by differentially modulating expression of angiogenesis-related genes.

ACKNOWLEDGMENTS

We thank Y. Barreira, S. Legonidec (animal production and phenotyping facilities of ANEXPLO platform, Inserm US006),J.-J. Maoret (GeT TQ plateau of the Genotoul Genome-Transcriptome platform), M. Pucelle (CAM facility) and C. Touriol (ABAE and HUVEC cells).

inserm-00714607, version 1 -5 Jul 2012

This workwassupported bygrantsfrom Association pour la Recherche sur le Cancer, Cancéropole GSO, INCA, Fondation de l'Avenir, Association Française contre les Myopathies (AFM). HB had a fellowship from the LigueNationaleContre Le Cancer, then from the ARC. BGS had a postdoc fellowship from the Fondation pour la RechercheMédicale, NA had a thesis fellowship from AFM. DPL andJCB were supported by Cancer Research UK (grant number: C8/A6613).


J.-B B Tary
email: tary@ualberta.ca

L Geli

Claude Guennou

P Henry

Nabil Sultan

Namik Cagatay

Valérie Vidal

L Géli

N Çağatay

Microevents produced by gas migration and expulsion at the seabed: A study based on sea bottom recordings from the Sea of Marmara

Keywords: Time series analysis, Gas and hydrate systems, Body waves, Interface waves, Seismic attenuation, Wave propagation

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

Introductiongeneral context

The Sea of Marmara is located on the North Anatolian Fault zone in NW Turkey, a major transform-plate boundary that has produced devastating historical earthquakes along its 1600 km length. After the 1999 Izmit and Düzce earthquakes, the next large (Mw > 7) earthquake is expected close to the heavily populated (>15 million inhabitants) Istanbul Area (Fig. 1).

Hence, the Sea of Marmara has been extensively surveyed during the last decade.

Several marine expeditions found gas emissions sites and brackish water seeps, along or near the main active faults scarps in the Sea of Marmara [Alpar, 1999;[START_REF] Halbach | Migration of the sulphatemethane reaction zone in marine sediments of the Sea of Marmaracan this mechanism be tectonically induced ?[END_REF]Armijo et al., 2005;[START_REF] Zitter | Cold seeps along the main Marmara Fault in the Sea of Marmara (Turkey)[END_REF][START_REF] Géli | Gas emissions and active tectonics within the submerged section of the North Anatolian Fault zone in the Sea of Marmara[END_REF]. Moreover, recent sediment profiler (chirp) and multibeam echosounder data acquired during the MARMESONET cruise of R/V Le Suroit [START_REF] Géli | Marmesonet Leg1 Cruise Report[END_REF] show the widespread presence of gas in the upper sediments and water column [START_REF] Dupré | Widespread gas emissions in the Sea of Marmara in relation with the tectonic and sedimentary environments: results from shipborne multibeam echosounder water column imagery (MARMESONET expedition[END_REF][START_REF] Tary | Case studies on fluids and seismicity in submarine environments based on Ocean Bottom Seismometers (OBS) recordings from the Sea of Marmara and application to the Niger Delta[END_REF]. Geochemical analysis indicates that the gas is mainly methane, and has two different origins: 1) in basins, gas is dominantly of bacterial origin, likely resulting from the decomposition of organic material in the Pleistocene sediments; 2) on the Western High and Central High (Fig. 1), gas is dominantly thermogenic, originating from the Eocene-Oligocene Thrace Basin source rocks [START_REF] Bourry | Free gas and gas hydrates from the Sea of Marmara, Turkey. Chemical and structural characterization[END_REF].

These observations and the high geohazard potential of the area are such that the Sea of Marmara has been identified as an unique, natural laboratory to study the relationships between fluids and seismicity through the EC-funded ESONET Network of Excellence (European Seafloor Observatory Network). In order to prepare the implementation of permanent multidisciplinary seafloor observatories, two preparatory experiments were conducted in 2007 and 2009-2010. Here, we report observations of non-seismic micro-events detected by Ocean Bottom Seismometers (OBS) and show that these events are probably related to gas emissions from shallow sediment layers (<5 m). These findings may help to better understand degassing processes from the seafloor. In the perspective of future multidisciplinary seafloor observatories, our results may also help establish a method to detect and characterize episodes of gas accumulation and release in shallow sediments.

Instrument characteristics and environmental settings

Between May 14 and August 30, 2007, two different experiments were carried out in the western part of the Sea of Marmara with eight, autonomous OBSs of 5 different types:

OldOBS, MicrOBS, ARMSS, SPAN, NEEDLE. The instrument locations, technical characteristics and recording periods are summarized in Table 1. Specific details, useful for the present paper, are given hereafter:

 OldOBS (deployed at sites J, K, L and M) are large instruments (1.5 m in height, weighing 240 kg) from Ifremer, designed to be deployed offboard the operating vessel. The geophones (Geospace GS-11D, its response curve is given in Fig. S1) are contained in an outer, pressure-resistant case resting on the seafloor, while the hydrophone is fixed on the instrument frame, ~0.9 m above the seafloor (Fig. 2).

 MicrOBS (deployed at site J2) is also an Ifremer instrument, weighing less than 20 kg, packaged within a 13" glass sphere, which includes the electronics, the batteries and the geophones (Geospace GS-11D). The hydrophone is fixed on the instrument frame, ~0.3 m above the seafloor. Like OldOBSs, MicrOBSs are deployed offboard the operating vessel.  ARMSS (from CGGVeritas) consists of a 0.9 m long cylinder lying horizontally on the seafloor, with a head containing both the geophones (Geospace LT-101) and the hydrophone. The three geophones are arranged in a Galperin configuration, i.e. set orthogonally and all tilted at 54.7° to the vertical axis. A vibrating system mounted around the instrument's head optimizes the coupling with the seafloor.

 SPAN (from CGGVeritas) is a ~1 m bullet shaped instrument, with geophones (SEND "Full Tilt") integrated in a titanium container three-fourth buried in the soil, or a little more due to the soft character of sediments. The data of this instrument were too noisy to be used for quantitative signals analysis.

 NEEDLE (from CGGVeritas) consists of a 4.5 m long pipe inserted in the sediments.

After penetration, a self corroding system was activated, which divided the pipe into 2 mechanically decoupled sections, the sensors (three SEND "Full Tilt" geophones and a hydrophone) being at the end of the lower section (~3 m long).

In order to test the different coupling devices, the 5 types of OBSs were all deployed for a maximum duration of 25 days, between May 14 th to June 9 th , 2007. While the Ifremer instruments (OldOBS and MicrOBS) were launched offboard R/V L'Atalante, the instruments provided by CGGVeritas (SPAN, ARMSS and NEEDLE) were installed in-situ using Nautile, the submersible of Ifremer. The second experiment involved only the 4 OldOBSs of Ifremer, which were re-deployed on June 9 th , 2007, to record the local micro-seismicity from the Western Sea of Marmara [START_REF] Tary | Sea bottom observations from the western escarpment of the Sea of Marmara[END_REF].

Based on laboratory results obtained in comparable pressure and temperature conditions, linear corrections were applied to the instruments internal clocks. For the first experiment, the drift of the internal clock was directly estimated for each instrument, based on GPS synchronization before deployment and after recovery, 25 days later. In addition, we took Figure 2 Table 1 advantage of GPS-dated seismic shots recorded by the OBSs on May 23 and 24, 2007, which ascertained that the drift of the internal clocks were nearly linear during the first experiment.

These shots were also used to improve the accuracy of the instruments position. For the second experiment (June 9 th -August 30 th , 2007), the direct estimation of the drift was not possible, because the OldOBS recordings stopped before the instruments recovery. The drifts of the internal clocks during the 2 nd experiment were thus corrected by simply applying the clock drifts that were obtained for the first experiment.

Conversion factors, from digital (counts) into physical (µm/s or Pa) units are unknown, except for the hydrophone of the MicrOBS and for the geophones of the OldOBSs. Hence, seismic shots have been used to calibrate the instruments sensors relatively to the MicrOBS hydrophone and to the OldOBS geophones, which were used as references. Conversion factors were derived assuming that the peak-to-peak amplitude of the first P-wave train in response to one given seismic shot, fired with the surface vessel directly above the OBSs (Fig. 1c), is the same for all OBSs (Fig. S2 and Table S1). Necessary information for proper sensor calibration was only available for Ifremer instruments. Therefore the instrumental response of the different sensors was not removed and the calibration is only an approximate one. The frequency response of Geospace GS-11D geophones (Fig. S1, OldOBS and MicrOBS) is almost flat in the frequency band of the micro-events (4-30 Hz). The inter-calibrated amplitudes given hereafter will be used to compare the different OBSs.

Three OldOBSs (K, L, M) were deployed so as to define a triangular network having equal sides, 10 km long, covering the North Anatolian Fault (Fig. 1). OBS L was placed on the bottom of the Tekirdag Basin, while OBS M was positioned in the northern part of the Tekirdag Basin, near WNW-ESE oriented normal faults [START_REF] Le Pichon | The main Marmara fault[END_REF][START_REF] Rangin | Strain localization in the Sea of Marmara: propagation of the North Anatolian Fault in a now inactive pull-apart[END_REF], and OBS K was positioned on the southern side of the fault, in shallower water at a depth of 546 m. Based on sediment sounder profiler (chirp) data, gas prone sediment layers were documented immediately below OBSs K and M [START_REF] Tary | Case studies on fluids and seismicity in submarine environments based on Ocean Bottom Seismometers (OBS) recordings from the Sea of Marmara and application to the Niger Delta[END_REF].

The 5 remaining OBSs (OldOBS J, MicrOBS J2, ARMSS, NEEDLE and SPAN) were positioned near the foot of the southern flank of the Tekirdag Basin, at the center of the network, within a distance of ~100 to 400 m from a cold seep called "Jack the Smoker" sitting on the seafloor trace of the North Anatolian Fault [Armijo et al., 2005;[START_REF] Zitter | Cold seeps along the main Marmara Fault in the Sea of Marmara (Turkey)[END_REF].

These OBSs are very close from each other, the closest ones, ARMSS and SPAN, being separated by only 10 m (Fig. 1c).

Micro-events general characteristics and interpretation

The dataset was first analyzed to characterize the micro-seismicity in the area. The results were published in [START_REF] Tary | Sea bottom observations from the western escarpment of the Sea of Marmara[END_REF]. Over the whole period, 270 seismic events (recorded at least at 3 stations) were identified using a STA/LTA detection algorithm (Short Term Average/Long Term Average).

The OBSs also recorded a large number of micro-events that were not detected by the above mentioned procedure, as they are generally not recorded by more than one station although their peak amplitude is comparable to the one of local micro-earthquakes (Fig. 3 andS3).

Generally, micro-events have: (1) short durations, ranging from 0.3 to 0.8 s in average, (2) frequency spectra between 4 and 30 Hz, (3) a large amplitude range of two orders of magnitude, and (4) no clear secondary arrival. Micro-events characteristics change slightly depending on the instrumental response of each OBS type (Fig. S3). Signals recorded by OldOBSs have higher frequency contents (5-30 Hz) and shorter durations (0.1-0.6 s) than that of the other instruments (4-12 Hz, 0.5-0.8 s).

Except NEEDLE, all instruments have recorded micro-events (NEEDLE only recorded aseismic signals related to its post-penetration stabilization mainly during the first two days of the experiment), discarding the hypothesis of instrumental noise to explain their origin. The absence of micro-events in the NEEDLE records can be explained by the occurrence of gassy sediments directly in contact with the geophones. The additional impedance contrast at the surface of the buried pipe created by a thin film of fluid partially reflects the wave, and thus prevents this instrument to record the micro-events.

The micro-events are visible on the hydrophones only when these are close enough to the sediments surface. The hydrophones of ARMSS and J2, situated at the seafloor and ~30 cm above the seafloor, respectively, recorded only micro-events having amplitude on the vertical geophone exceeding ~2-3 µm/s (ARMSS) and ~15 µm/s (J2). Earthquakes and seismic shots with lower amplitudes on the vertical geophone (~1 µm/s) are clearly visible on all hydrophones. The absence or low amplitudes of the signals recorded by hydrophones appear to be specific to micro-events. This could be explained by a propagation mainly along the water-sediment interface, transmitting very little motions to the water column. Hydrophones from other OBSs (OldOBS and NEEDLE) would be too far from the water-sediment interface to be able to record any micro-events.

The micro-events are most of the time not correlated from one OBS to another, implying that the source is in very close vicinity of the OBS. Some strong micro-events are simultaneously recorded only by the closest stations, ARMSS and SPAN which are 10 m apart (Fig. S3).

These pairs of signals present very different waveforms and frequency content likely due to

Figure 3 the strong resonance of OBS SPAN geophones around their natural frequency (4.5 Hz, Fig. S3). No location was attempted because of the poor quality of OBS SPAN recordings that involves large time picks uncertainties. On the other hand J2 and J, 25 m apart, did not record any correlated micro-events. The maximum spacing between two OBSs to simultaneously record micro-events of average amplitude is less than 10 m. Thus, micro-events seem to be strongly attenuated both horizontally in the sediments and vertically in the water column.

The number and temporal distribution of micro-events recorded by each instrument are shown in Table 1 and Fig. 4. About 7300 micro-events were detected. The number of micro-events is variable from one OBS to another. For instance, OBSs J and K, two OldOBSs with approximately the same recording period, recorded 915 and 3168 micro-events, respectively (Fig. 4 and Table 1).

No clear cycles, as the tidal cycles (less than 10 cm in the Sea of Marmara [Alpar and Yüce, 1997]), are visible on the micro-events temporal distribution. In addition, no clear correlation between the number of micro-events and the hour of the day has been found, as it could be expected if these signals resulted from the activity of some living organisms (Fig. S4).

Comparable non-seismic micro-events of short duration are very common on OBS recordings, but seismologists have paid little attention to these signals, as no useful correlation for seismological purposes can be made between distant instruments. [START_REF] Buskirk | Evidence that biological activity affects ocean bottom seismograph recordings[END_REF], [START_REF] Diaz | Atypical seismic signals at the Galicia Margin, North Atlantic Ocean, related to the resonance of subsurface fluid-filled cracks[END_REF] and [START_REF] Sohn | Hydrothermal activity at the Megaplume Site on the Southern Juan de Fuca Ridge[END_REF] have reported very similar signals in varying environmental and geodynamical settings, i.e. the Pacific Rim borders, the Galicia passive margin, and the Southern Juan de Fuca ridge, respectively. These studies reported signals with: durations between 0.5 and 1 s in the case of [START_REF] Diaz | Atypical seismic signals at the Galicia Margin, North Atlantic Ocean, related to the resonance of subsurface fluid-filled cracks[END_REF] and [START_REF] Sohn | Hydrothermal activity at the Megaplume Site on the Southern Juan de Fuca Ridge[END_REF], and between 0.5 and 4 s in the case of [START_REF] Buskirk | Evidence that biological activity affects ocean bottom seismograph recordings[END_REF]; frequency contents constituted by narrow peaks between 3 and 30 Hz; no clear secondary arrivals; and large differences in the Figure 4 number of micro-events recorded by different OBSs. In addition, no correlation was found between micro-events recorded by 2 different OBSs. The signals reported by [START_REF] Buskirk | Evidence that biological activity affects ocean bottom seismograph recordings[END_REF] have longer durations in average, are monochromatic, and show a progressive decrease of the coda amplitude. The latter could be due to the instruments resonance, although [START_REF] Buskirk | Evidence that biological activity affects ocean bottom seismograph recordings[END_REF] also noticed that the frequency content of the recorded signals is different for the same OBS deployed at different sites.

The above observations rule out the hypothesis that the micro-events we observe could result from instrumental noise. [START_REF] Buskirk | Evidence that biological activity affects ocean bottom seismograph recordings[END_REF] claimed that the number of micro-events depends on the hour of the day for instruments at depths shallower than 1000 m. In addition, the number of events decreases with the depth of the instrument, suggesting a possible relationship with the vertical distribution of biomass in the ocean. Last but not least, no micro-events were recorded within boreholes, or other environments unfavorable for fish activity. These observations, and the recovery of living organisms (eggs of unknown origin) attached to two OBSs, lead [START_REF] Buskirk | Evidence that biological activity affects ocean bottom seismograph recordings[END_REF] to suggest a biological origin of the signals.

Another argument supporting the hypothesis of biological activity could be that the number of micro-events recorded by OldOBS K (water depth ~ 546 m) is two to three times greater than the number of micro-events recorded by OldOBSs J, L and M (water depth ~1000 m).

However, besides biologic activity, pressure effects on gas bubbles can also explain the decrease in the number of micro-events with depth. Indeed, gas solubility decreases with pressure, resulting in an increase in bubble size and gas exsolution. Hence, for a same gas source and similar sediments, more bubbles will be created at lower pressure (i.e. at shallower depths). So, a decrease of biological activity is not the only parameter that could explain the decrease of the number of micro-events with depth.

Non-biological explanations for the micro-events are also far more likely for the following reasons. First, a total of 30 dives with Nautile submersible were conducted in 2007 to explore the Marmara deep seafloor [Henry, MarNaut cruise report, 2007], showing the relative scarcity of fish activity near the seabottom. Second, the occurrence of micro-events does not show any cyclicity, nor any clear dependence on the hour of the day (Fig. S4), while fish are supposed to have an internal biological clock [START_REF] Bone | Biology of fishes[END_REF]. Third, swarm of micro-events share relatively common characteristics (e.g. similar duration, frequency content, waveform), whatever the instrument design and during extended periods of time. As pointed out by [START_REF] Diaz | Atypical seismic signals at the Galicia Margin, North Atlantic Ocean, related to the resonance of subsurface fluid-filled cracks[END_REF], "the observation of events with very different amplitudes but very similar waveforms and the existence of clearly differentiated clusters of events, seem to discard a biological origin".

As mentioned earlier, the origin of the micro-events is very close to the instruments. Therefore, regional phenomena, such as tectonics or distant sources (e.g. related to human activity) can be discarded. In addition, sources in the water column (deep currents, resonating clouds of bubbles [START_REF] Pontoise | Monochromatic infra-sound waves recorded offshore Ecuador : possible evidence of methane release[END_REF], T-waves [START_REF] Talandier | T waves from underwater volcanoes in the Pacific Ocean: ringing witnesses to geyser processes ?[END_REF] and explosions/implosions) are very unlikely because micro-events were not recorded by OldOBS hydrophones. A relationship between deep currents and micro-events is also very unlikely because deep sea currents in the Sea of Marmara are, to some extent, a quasi steady state phenomenon, and cannot explain the characteristics of the observed micro-events.

On the other hand, gas is known to be common in marine sediments, and the Sea of Marmara is not an exception. Active venting sites have been found throughout the Sea of Marmara by geophysical means [Alpar, 1999;[START_REF] Géli | Gas emissions and active tectonics within the submerged section of the North Anatolian Fault zone in the Sea of Marmara[END_REF][START_REF] Dupré | Widespread gas emissions in the Sea of Marmara in relation with the tectonic and sedimentary environments: results from shipborne multibeam echosounder water column imagery (MARMESONET expedition[END_REF] and visual observations [START_REF] Zitter | Cold seeps along the main Marmara Fault in the Sea of Marmara (Turkey)[END_REF]. A chirp profile, crossing the position of OBS J and M, was collected during the Marmesonet cruise in 2009 ( [START_REF] Géli | Marmesonet Leg1 Cruise Report[END_REF], Fig. 5). On this profile, a highamplitude reflector followed by a strong attenuation of the seismic waves is clearly visible close to the fault situated below OBS M. These signatures are characteristic of the presence of gas. In addition, experiments and modeling [START_REF] Vidal | Acoustic signal associated with the bursting of a soap film which initially closes an overpressurized cavity[END_REF][START_REF] Varas | Dynamics of crater formations in immersed granular materials[END_REF][START_REF] Diaz | Atypical seismic signals at the Galicia Margin, North Atlantic Ocean, related to the resonance of subsurface fluid-filled cracks[END_REF][START_REF] Chouet | Dynamics of a Fluid-Driven Crack in Three Dimensions by the Finite Difference Method[END_REF][START_REF] Chouet | Resonance of fluid-driven crack : radiation properties and implications for the source of long-period events and harmonic tremor[END_REF][START_REF] Chouet | Long-period volcano seismicity : its source and use in eruption forecasting[END_REF][START_REF] Ferrazzini | Quantitative analysis of long-period events recorded during hydrofracture experiments at Fenton Hill, New Mexico[END_REF] of the opening and resonance of a cavity filled by fluids generate signals with waveforms qualitatively consistent with our data.

Hence, in the following we suggest that the micro-events could result from gas migration in the seafloor, considering (i) the presence of gas and a fault near OBS M (where the swarm of micro-events was recorded); (ii) Modeling of source and wave propagation. The source of the micro-events is likely quite superficial, as no correlation is observed from one OBS to another, unless they are less than 15 m apart.

Specific cluster sequence on OBS M

Sequence chronology

Interestingly, OBS M recorded a swarm of 400 micro-events in 24 hours on June 11 and 12 (Fig. 6). Despite large amplitude differences, the micro-events recorded during this crisis have very similar waveforms (Fig. S5) and frequency contents (dominant frequency between 10 and 20 Hz). The number of micro-events increases gradually over the crisis, reaching a maximum after 6 hours with 96 micro-events in 2 hours (the background rate was ~ 5 microevents/hour).

Figure 5 About two days before the swarm of micro-events, a very strong micro-event with a peak-topeak amplitude >1000 µm/s was recorded by OBS M (Fig. 3a and6). Despite its very high amplitude, the signal is not visible on the hydrophone.

As it occurred only ~40 minutes after OBS M hits the sea bottom (09/06/2007 17:48:04), it seems likely that the impact of the instrument on the seafloor has indirectly caused this strong signal. The impact may also have significantly destabilized the gas-prone sediments close to the OBS, causing gas expulsion from the superficial sediments. A similar process could also explain the increase in the number of micro-events just after the re-deployment of OBSs J, K and M.

Then, after about 40.5 h of relative quiescence, OBS M recorded the swarm. This phase constituted by signals of relatively low amplitudes could correspond to gas related processes within the superficial sediments.

Wave polarization analysis

First, the signals were detected with an automatic algorithm based on a STA/LTA threshold and visually controlled. Secondly, for each micro-event, one temporal series of N samples was extracted in order to control the origin time and average. The three-component temporal series in the OBS frame of reference were rotated in the wave frame of reference using a method based on [START_REF] Jurkevics | Polarization analysis of three-component array data[END_REF] for wave polarization analysis. The covariance matrix was calculated over the complete signal by

     N t m n nm t f t f S 1 ) ( ) ( , (1)
with N the number of samples, f the temporal series, and f n and f m the signal components on x, y or z.

Figure 6

Then, the eigenvectors (u 1 , u 2 and u 3 ) together with their corresponding eigenvalues (λ 1 , λ 2 , and λ 3 ) were determined from this symmetric 3 x 3 covariance matrix. The three pairs eigenvector-eigenvalue correspond to the polarization ellipsoid that best fit the data. As the type of waves corresponding to micro-events is not known a priori, the temporal series were only multiplied by the three eigenvectors in order to rotate the signals in the frame of reference of the polarization ellipsoid (components x1, x2, and x3) (Fig. 3).

In this frame of reference, the polarization of the micro-events presented in Fig. 3 is very different. In one case, the particle motion is mainly in the x1-x3 plane (Fig. 3a and3c), which is consistent with surface waves (Stoneley-Scholte waves, e.g. Favretto-Anrès and Rabau

[1997], [START_REF] Zakharia | Sub-bottom variability characterization using surface acoustic waves, in Impact of Littoral Environmental Variability on Acoustic Predictions and Sonar Performance[END_REF]). In the other case, the particle motion is linear in the x1 direction (Fig. 3b), which in principle is consistent with both P-and S-waves. In addition, considering the frequency content of micro-events, the P-and S-waves velocities in superficial sediments (see section 5), and a realistic source-receiver distance of less than 15 m, near-field effects should dominate micro-events waveforms. These effects will ultimately make P-and S-waves arrivals undistinguishable [e.g. [START_REF] Lokmer | Properties of the near-field term and its effect on polarization analysis and source locations of long-period (LP) and very-long-period (VLP) seismic events at volcanoes[END_REF]. Near-field effects also affect the polarization of body waves. Close to the source, both P-and S-waves can present quasielliptical polarization due to the superposition of near-field waves to the far-field wavetrains [START_REF] Vavryčuk | Polarization properties of near-field waves in homogeneous isotropic and anisotropic media: numerical modeling[END_REF]. Thus, micro-events showing a non-linear polarization are not necessarily surface waves, but could also correspond to body waves significantly disturbed by the presence of near-field waves. This could explain the absence of the very high amplitude micro-event on hydrophone recordings if this signal is mainly composed by an S-wave (Fig. 3a). The source radiation pattern, which in principle could indicate which types of waves are expected depending on the source and receiver locations, was not used here due to the lack of information about the geometry of the source of micro-events.

Azimuths of the principal eigenvector u 1 of the polarization ellipsoid for all micro-events of the swarm were obtained using

                  z u sign x u z u sign y u Az 1 1 1 1 1 ) ( ) ( tan .
(2)

The sign of the vertical component of the principal eigenvector is included in eq. 2 to resolve the 180° ambiguity in azimuths calculation. The azimuth of the signal presented in Fig. 3a, polarized in the x1-x3 plane, was determined by eq. ( 2) using only the horizontal components of u 1 . The 180° ambiguity in azimuths calculation was not resolved in this case. These azimuths in the OBS frame of reference are shown in Fig. 7. The orientation of the principal eigenvector is very stable over the crisis, around 285° -315° in the OBS frame of reference, suggesting a localized source. Azimuths calculated for the other two eigenvectors show no preferential orientation, as expected if very little wave energy is focused on these components.

Noticeably, the strong micro-event and the micro-events constituting the swarm recorded by OBS M have similar azimuths, 294° [180°] and 285° -315°, respectively. One possibility is that both could be related to fluid migration along the fault that is visible on the chirp profile located close to OBS M (Fig. 5).

Principal Component Analysis (PCA)

In order to find the common features of micro-events and propose some physical explanations, a PCA was applied to determine the most characteristic micro-events of the swarm. Prior to the calculation of the PCA, the signals were rotated in the frame of reference of the polarization ellipsoid following the method described in section 4.2. The covariance matrix between all signals, with a common origin and a zero-average, was calculated following this formula,

Figure 7       M j i q j p i tq tp t f t f C , 1 , ) ( ) (
where

N q p t t t t   , 1 , (3)
with f i and f j the temporal series, M the number of micro-events, and N the number of samples.

Eventually, the characteristic signals (eigenvectors, V i , i = 1, M) and their data representativeness (eigenvalues, λ i , i = 1, M) are calculated from the covariance matrix. Hereafter, the representativeness of each eigenvector will be given as a percentage of the total energy (λ i ²/Σλ i ²). The micro-events constituting the swarm recorded by OBS M on June 11 and 12 present four particularities: (i) they are very impulsive; (ii) of short duration (mainly around 0.1 s); (iii) very similar; (iv) and present higher amplitudes on the horizontal components than on the vertical one (Fig. 3b). The PCA performed with this dataset indicates that the first eigenvector has a data representativeness of ~83 % on the x1 component (Fig. 8), suggesting a common source and similar source-receiver ray paths. The relatively low data representativeness of the first eigenvectors of the x2 and x3 components, ~35 and 58 %, respectively, result likely from the presence of coherent arrivals of smaller amplitudes, such as near-field waves, modifying the otherwise linear polarization of these micro-events.

The characteristic signal is very impulsive and has a duration around 0.15 s on the x1 component. Frequency spectra of the eigenvectors of the three components show one main peak between 13 and 20 Hz. Noticeably, the frequency spectrum of the first eigenvector of the x1 component show a higher frequency content than the other components (Fig. 8). The main characteristics of the first eigenvectors are summarized in Table 2.

The PCA was also applied to the complete dataset. Most of the first eigenvectors have a frequency spectrum with one dominant frequency, between 5 and 23 Hz for Ifremer OBSs (J, Table 2 Figure 8 K, L, M, and J2) and around 10 Hz for OBS ARMSS (Table S2 and Fig. S6). Considering only the x1 component, more than 80% of the micro-events energy corresponds to the first five eigenvectors (i.e. five families). The micro-events could then be grouped in families as in [START_REF] Diaz | Atypical seismic signals at the Galicia Margin, North Atlantic Ocean, related to the resonance of subsurface fluid-filled cracks[END_REF], the characteristics of these families being slightly different from one OBS to another (Fig. S6).

Source and wave propagation modeling

The numerical simulations were carried out using the SKB code [START_REF] Dietrich | Modeling of marine seismic profiles in the t-x and tau-p domains[END_REF]. This code computes, as a function of frequency and wave number, the response in terms of stress and displacement of a 3D, horizontally stratified, half-space subjected to a source positioned anywhere in the stratification. Fluid layers within or bounding the stratification can be taken into account. The attenuation is included by working with complex wave velocities [Toksöz and Johnston, 1981]. The computation is based on a recursive algorithm using reflectiontransmission coefficients as wave vector propagators [START_REF] Kennet | Seismic waves in a stratified half-space[END_REF]. The last steps of the computation are to integrate in discrete wave numbers [START_REF] Bouchon | A simple method to calculate Green's functions for elastic layered media[END_REF], and to make a convolution with a spectrum of a signal source followed by an inverse Fourier transform in time, in order to recover stresses and displacements in space and time.

According to Biot [1956], water saturation induces an attenuation that can be accounted for by a complex formulation of wave velocities, as in visco-elastic media (see also [START_REF] Géli | Seismic wave propagation in a very permeable water-saturated surface layer[END_REF]). Therefore, our computation method is adapted to the modeling of wave propagation in strongly attenuating, marine sub-surface sediments, [e.g. [START_REF] Meunier | Computation of shear waves by integral methods in stratified media[END_REF]. This computation method takes into account the complete wave field (direct, transmitted, and reflected waves), including both far-field and near-field terms [START_REF] Dietrich | Synthetic vertical seismic profiles in elastic media[END_REF].

The representation of the source in terms of forces or moment tensor appears within the SKB code in terms of equivalent stress or displacement discontinuity, allowing the calculation of stress and displacement at the receivers by using reflection-transmission coefficients. The source signal used is the zero-phase Ricker signal.

The model consists of a 1110 m thick water layer, with typical density of 1000 kg/m 3 and Pwave velocity of 1500 m/s, overlying a homogeneous half-space. Sediments P-wave velocity and density measured on cores (using a Geotek Multi-Sensor Core Logger -MSCL) collected near OBS J have been used for the numerical seismograms calculation (Fig. S7). The acoustical properties of the homogeneous half-space correspond to those of a soft and very attenuating sediment, i.e. a density of 1500 kg/m 3 , P-wave velocity and quality factor of 1550 m/s and 10, S-wave velocity and quality factor of 100 m/s [START_REF] Sultan | Detection of free gas and gas hydrate based on 3D seismic data and cone penetration testing: An example from the Nigerian Continental Slope[END_REF] and 10 [START_REF] Wang | Qs estimation for unconsolidated sediments using first-arrival SH wave critical refractions[END_REF][START_REF] Campbell | Estimates of shear-wave Q and κ0 for unconsolidated and semiconsolidated sediments in Eastern North America[END_REF]. Three types of point source were tested: an isotropic explosive source generating P-waves with the same energy in all directions, and two unidirectional forces generating P-waves with the maximum of energy in the horizontal and vertical directions. A two-component source (isotropic and single force) in an elastic half-space was studied by [START_REF] Kanamori | Analysis of Seismic Body Waves Excited by the Mount St. Helens Eruption of May 18, 1980[END_REF] for the case of the rupture of a "lid" on top of a cylinder. In the near-field, they show that the isotropic part of the source can be neglected when the radius of the cylinder is small compared to the source-receiver distance. In our case, the conduits radius, inferred from the bubble size at the seafloor, is less than 1 cm, while the sourcereceiver distance ranges between 1 and 10 m, resulting in a ratio of ~0.001-0.01. Hereafter, the waveform modeling using an isotropic source is given for comparison.

In all simulations, the source signal has a constant frequency content of 15 Hz, consistent with the observed micro-event frequency content. Numerical seismograms were calculated for 100 horizontally aligned receivers (spaced by 1 m) at four different depths (0.01 m below and 0.1, 0.3, 0.9 m above the water-sediment interface), and two source depths, 2 and 5 m below the water-sediment interface (Fig. 9).

Whatever the type and depth of the source, the computed signals are attenuated very quickly in the sediments (Fig. 10). Surface waves (Stoneley-Scholte waves) are produced by both unidirectional forces and the explosive source in superficial sediments. These waves, propagating at ~77 m/s in our configuration, are attenuated both horizontally in sediments and vertically in the water column (Fig. 10 and11). However, in the case of an explosive source, P-waves with significant amplitudes are clearly visible on the horizontal component.

The micro-event shown in Fig. 3c has about the same amplitude on the x1 component than on the x3 component, which could be compatible with Stoneley-Scholte waves produced by a shallow source.

The impulsive micro-events of the crisis have most of their energy on the x1 component and thus cannot be interpreted as Stoneley-Scholte waves (Fig. 3b). They could in principle be better explained by a P or an S-wave hitting the sediments-water interface with a high incidence angle, transmitting little energy to the water column. The corresponding wavelength would be of the order of 100 m and 6.7 m, respectively, which is probably much more than, or in the order of, the distance to the source. Most likely, these impulsive signals are generated by a weak but very close source causing horizontal displacement, which could be a pulsing conduit.

Physical hypotheses and interpretation

Physical hypotheses

Bubbles in sands are spherical, grow and migrate by displacing grains, whereas bubbles in clay are presumably oblate spheroid, and migrate by fracturing the sediments [START_REF] Johnson | Mechanical response of sediments to bubble growth[END_REF][START_REF] Boudreau | Bubble growth and rise in soft sediments[END_REF]. Hence, gas migration in fine-grained sediments is expected to depend on fracture propagation, which is a function of the mechanical properties of the medium through its Young's modulus E, shear strength, and fracture toughness K 1c [START_REF] Van Kessel | Gas production and transport in artificial sludge depots[END_REF]Algar et al., 2009]. However, it is often observed at the seafloor that bubbles escape continuously through tubular conduits, which are sometime recovered by cemented chimneys in the sediments. Such open conduits may be maintained to a few meters depth in the sediment and enable bubble-induced pore water mixing [START_REF] Haeckel | Bubble-induced porewater mixing: A 3-D model for deep porewater irrigation[END_REF].

Laboratory experiments, where gas (air) is injected in a granular media (sodosilica grains with diameters of 100 and 400 µm), have shown that gas escapes through numerous conduits that look like tree branches [START_REF] Varas | Dynamics of crater formations in immersed granular materials[END_REF][START_REF] Varas | Venting dynamics of an immersed granular layer[END_REF]. Gas conduits seem to be intrinsically unstable, because, even without variations of the gas injection rate, conduits are created or closed continuously. The superficial sediments of the Sea of Marmara are mainly clay-rich cohesive sediments with particles smaller than 5 µm. Nevertheless, open conduits in compacting sediments are expected to be mechanically unstable except very close to the seafloor. We suspect that the opening and collapse of conduits or gas filled fractures could explain the characteristics of micro-events (Fig. 3). Considering the low cohesion of superficial sediments, the vertical force needed by the fluids to move up toward the seafloor is supposedly small. The main displacements are then produced by the side walls of the subvertical conduit. In addition, the conduits observed during laboratory experiments of gas injection in granular media [START_REF] Varas | Dynamics of crater formations in immersed granular materials[END_REF][START_REF] Varas | Venting dynamics of an immersed granular layer[END_REF] are generally nearly verticals. Explosion or vertical force types of source are still possible if a layer of higher cohesion is present on the pathway of the gas.

Then, our preferred mechanism consists of a sub-vertical fracture, pre-existent or not, gradually filled by gas within a porous and saturated medium (Fig. 12). The fracture is located close to the sediment-water interface. Gas pressure increases as the crack fills and progressively opens (step 0). When the fracture toughness threshold is reached, the gas will rise up in a moving crack, reaching or not the sediment-water interface (step 1), where it can escape to the seawater (step 2) without major deformation of the sedimentary matrix [START_REF] Johnson | Mechanical response of sediments to bubble growth[END_REF][START_REF] Boudreau | Bubble growth and rise in soft sediments[END_REF]Algar et al., 2009]. After the gas migration, the confining pressure will close the fracture. Fluid-filled crack models don't require venting, but focus mainly on the resonance characteristics of the cracks [e.g. Aki et al., 1977;[START_REF] Chouet | Dynamics of a Fluid-Driven Crack in Three Dimensions by the Finite Difference Method[END_REF][START_REF] Chouet | Resonance of fluid-driven crack : radiation properties and implications for the source of long-period events and harmonic tremor[END_REF][START_REF] Jousset | Modelling the time-dependent frequency content of low-frequency volcanic earthquakes[END_REF]. Hence, micro-events can be generated as the gas migrates in the conduit, or by its expulsion at the seafloor. The partitioning of the signal recorded by the OBSs in horizontal and vertical motions will depend on fractures tilt and depth, and on wave propagation processes.

Interpretation of the micro-events crisis

We identified two types of micro-events: small impulsive, clustered events and larger amplitude oscillatory signals, which appear less frequently and display more variability and could correspond to Stoneley-Scholte waves. [START_REF] Varas | Dynamics of crater formations in immersed granular materials[END_REF] described two regimes of bubbles emission in granular media depending on the gas injection rate. At low flow rate, large and independent bubbles are formed ("bubbling regime"), whereas at high flow rate, small bubbles supplied by a continuous channel are produced ("open-channel regime"). Except the swarm recorded by OBS M, all OBSs displayed a low daily rate of micro-events. We propose that the larger micro-events correspond to the collapse of a cavity or fracture that trapped gas at a relatively shallow depth Figure 12 below the sediments. The crisis recorded by OBS M followed such an event and could, hypothetically, result from a nearby continuous flow of bubbles in an open-channel regime which followed the expulsion of the gas from the main cavity.

Conclusions

Numerous non-seismic micro-events of short durations were recorded on different types of OBSs. These signals, of peak amplitudes comparable to those of earthquakes, were predominantly recorded by geophones. They were also recorded by those hydrophones situated less than a few tens of centimeters from the seafloor. Because the signals have similar characteristics (terms of duration, frequency content), whatever the instrument and whatever the environment, it is very unlikely -if not impossible-that these micro-events be related to instrumental artifacts, or "fish bumps".

The micro-events are generally not correlated from one OBS to another, with one noticeable exception for the closest OBSs (~10 m apart). Moreover, micro-events present a specific attenuation pattern, i.e. both horizontally in sediments and vertically in the water column.

Specific focus has been given to the micro-events recorded by OBS M during the crisis that occurred on June 11-12, 2007. The presence of gas in the superficial layers and the source modeling we performed suggest that these micro-events are likely related to gas related processes from the seafloor, such as the opening and closure of a conduit induced by degassing near the subsurface. The present work shows that OBSs can detect episodes of gas accumulation and release in shallow sediment layers. In combination with piezometers and bubble recorders, OBS could be used in the Sea of Marmara to monitor the evolution of such episodes, hence to better understand the relationships between deformation and non-seismic transients related to degassing from the sub-seafloor layers near the fault zone.

Our work also confirm recent results obtained with multibeam acoustic systems operating in the water column mode, suggesting that free gas emissions from the seafloor are likely to be more widespread than previously thought. Aki, K., Fehler, M., & Das, S., 1977 [START_REF] Rangin | Marine Atlas of the Sea of Marmara (Turkey)[END_REF][START_REF] Rangin | Strain localization in the Sea of Marmara: propagation of the North Anatolian Fault in a now inactive pull-apart[END_REF][START_REF] Imren | The North Anatolian Fault within the Sea of Marmara: a new interpretation based on multichannel seismic and multi-beam bathymetry data[END_REF]Armijo et al., 2005] Victor on the seafloor shows the outflow of fresh water as well as a fish on top of the main chimney. The white part of the chimney corresponds to bacterial mats. The black diamond shows the location of the seismic shot used for OBSs amplitude inter-calibration (see Fig. S2). Figure 10. Numerical simulations using the SKB code [START_REF] Dietrich | Modeling of marine seismic profiles in the t-x and tau-p domains[END_REF] for a horizontal, a vertical and an explosive source located at 2 m bsf (below sea floor), and sensors 1 cm within the sediments. The original numerical signals in displacement were differentiated once (velocity) and then normalized by the first numerical signal (distance: 10 m). H: horizontal motion; Z: vertical motion. Note the strong attenuation of P-and surface waves. Figure 12. Schematic explanation in 3 steps of the mechanism proposed for the micro-events source: gas migration and escape through a sub-vertical conduit. Table 2. First eigenvector characteristics of the PCA performed on the micro-events of the swarm recorded by OBS M. The number of micro-events used in the PCA is also indicated (ME: micro-events). %: data representativeness (energy); Freq.: dominant frequency; Dur.: duration.

Tables

Stations

Figures

Figure 1

Figure 1

 1 Figure 1

Figure 1 .

 1 Figure 1. (a) Map of the Sea of Marmara with active fault traces[START_REF] Rangin | Marine Atlas of the Sea of Marmara (Turkey)[END_REF][START_REF] Rangin | Strain localization in the Sea of Marmara: propagation of the North Anatolian Fault in a now inactive pull-apart[END_REF]

Fig. 5 .

 5 The faults are indicated by the other black lines. (c) Zoom in the OBS network center. Microbathymetric data were acquired during the MARMARASCARPS cruise in 2002 by the R.O.V. Victor [Armijo et al., 2005]. OBSs provided by Ifremer and CGGVeritas are indicated by black triangles and black dots, respectively. The black star shows the location of "Jack the Smoker" site where fresh water escapes from the seafloor through carbonate chimneys. The photograph taken by the R.O.V.

Figure 2 .

 2 Figure 2. Schematic structure of the OBSs deployed during the MarNaut cruise. For each

Figure 3 .

 3 Figure3. Three examples of micro-events recorded by OBS M and OBS K. The three-

Figure 4 .

 4 Figure 4. Temporal evolution of the number of micro-events recorded by OBSs J, K, L, M,

Figure 5 .

 5 Figure 5. Chirp profile acquired during the MARMESONET cruise in Nov.-Dec. 2009 ([Géli

Figure 6 .

 6 Figure 6. (a) Recordings of OBS M during the crisis of micro-events, 11/06 12:00 -12/06

Figure 7 .

 7 Figure 7. Azimuths of the first eigenvector of the polarization ellipsoid in the OBS frame of

Figure 8 .

 8 Figure 8. PCA of the micro-events constituting the crisis identified on OBS M for the

Figure 9 .

 9 Figure 9. Configuration of the model used for micro-event modeling. A point source

Figure 11

 11 Figure 11. a) Micro-events numerical simulations for a horizontal source at 2 m bsf and

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  . Abbreviations: NAFZ, North Anatolian Fault Zone; TB, Tekirdag Basin; WH, Western High; CB, Central Basin; KB, Kumburgaz Basin; CH, Central High; ÇB, Çinarcik Basin; IB: Imrali Basin; P., Peninsula; Is., Island. (b) Bathymetric map of the Tekirdag Basin. OldOBSs deployed during the MarNaut cruise in 2007 are indicated by black triangles. The black line with numbers corresponds to the ship track and trace numbers of the chirp profile in

Table 1 .

 1 Position, technical characteristics, recording period, number of micro-events recorded (N ME ) and N ME per day for each OBS. F 0 : geophones natural frequency; Fs : sampling frequency; comp.: component.

		Long. (deg)	Lat. (deg)	Depth (m)	Recording period	F0 (Hz) Fs (Hz)	Observations	NME NME/day
	J	E 27.62921 N 40.80372	1112	14 May -30 Aug. 2007	4.5	250		915	8
	J2	E 27.62902 N 40.80390	1112	22 May -28 May 2007	4.5	250	X comp. resonance	43	7
	K	E 27.6608	N 40.7613	546	14 May -19 Aug. 2007	4.5	250		3168	33
	L	E 27.5645	N 40.8044	1132	14 May -09 June 2007	4.5	250		461	18
	M	E 27.6637	N 40.8466	1110	14 May -26 Aug. 2007	4.5	250		1534	15
	ARMSS	E 27.62774 N 40.80382	1115	14 May -09 June 2007	14	500		1079	42
	SPAN	E 27.62782 N 40.80376	1117	14 May -28 May 2007	4.5	500	Low quality	85	6
	NEEDLE	E 27.62714 N 40.80337	1115	14 May -28 May 2007	4.5	500		0	0
			Eigenvector 1
	Components	%	Freq. (Hz)	Dur. (s)
		OBS M swarm (368 ME)
		x1	83	13 -20	0.14
		x2	35		14.2	0.21
		x3	58	14.5 -17	0.23

Acknowledgements

This work is part of PhD Thesis of J. B. Tary, co-funded by Total, Magnitude/BakerHughes and Ifremer. CGGVeritas kindly provided the data from the ARMSS, NEEDLE and SPAN.

The work was conducted within the Marmara Demonstration Mission of the European

Seafloor Observatory Network (ESONET) and benefited from financial support from both ESONET and Agence Nationale de la Recherche (project ANR/ISIS). Special acknowledgments to: Captain and crew of Ifremer and ITU vessels (R/V L'Atalante and R/V Yunuz) ; Francis Cerda (Total), Thomas Bardainne (Magnitude, BakerHughes) ; Renaud Daures, Emmanuel Auger (CGGVeritas) ; Bach-Lien Hua, Pascal Pelleau, Ronan Apprioual and Yves Auffret (Ifremer) ; Jacques Talandier, and Michel Dietrich.

Figure 12