Philippe Chavot
email: philippe.chavot@unistra.fr

Anne Masseran

Yeny Serrano

Information and public consultation exercises concerning geothermal projects. "The Strasbourg case"

Keywords: public inquiry, information, media studies, energy transition, deep geothermal project, residents, citizen participation

Four public inquiries on deep geothermal projects in the Eurometropolis of Strasbourg were conducted during the Spring of 2015. These consultation exercises were carried out in a context of public controversy over the risk related to deep drilling. Residents' associations have been deeply involved and the media largely covered the issue. Following these consultations, three of the four projects received a negative opinion from the investigating commissioners. Later, prefectural authority granted two of the four projects.

How to interpret this opposition to urban geothermal projects? And what can we say about the way residents, stakeholders and public authorities engage with the public inquiries? To answer such questions we have decided to work on three distinct corpuses:

• The public inquiries per se, taking into account the input from citizens during the enquiry and all the documents produced in the framework of these consultation exercises.

• Local media, these public inquiries having been widely reported in the media. It is therefore crucial to examine the way journalists feature stakeholders and geothermal issues.

• A series of interviews we have carried out with different stakeholders (scientists, industrialists, associations representatives, elected officials, inquiry commissioners, etc.).

We will analyse these three corpuses according to two main guidelines. First, we will focus on the public inquiry system. Indeed, it is important to understand how it works and how the publics, the stakeholders and the investigator commissioners tackle it. At a more global level, it is important to analyse what public inquiries have led to. Has a social dialogue been established? Or, have the public inquiries led to the strengthening of demarcation lines between opponents and supporters of geothermal projects? In the background, of course, it is the public perception of the public inquiry system that becomes apparent.

The second line of enquiry concerns the public perception of deep geothermal projects and the dynamics of the public controversy. For example, how are the different issues related to geothermal projects perceived by the different stakeholders? Are geothermal projects perceived to serve populations and/or the state energy policy? How are the economic, political and social dimensions of these projects perceived? It is also important to understand the purpose for which citizens engage in the debate on geothermal projects: is it to preserve their close environment and their quality of life? Or is this engagement related to philosophical or political convictions? In this context, it is crucial to understand how the citizens get informed about the geothermal issues and the public inquiries. And more particularly, what roles do the media and the residents' associations (among others) play in this public controversy?

INTRODUCTION

In the spring of 2015, four public inquiries (PI) were held within the Eurometropolis of Strasbourg (EMS): in Strasbourg's Robertsau district near the oil port and in the towns of Mittelhausbergen, Eckbolsheim, and Illkirch-Gaffenstaden. Local residents had to express their opinion on the implementation of projects regarding exploration drilling for deep geothermal energy in order to produce heat and/or electricity. The PI are legally binding when major projects are planned in relation to urban planning, to environmentallysensitive facilities or that may affect the quality of life of local residents. They are also needed for a variety of projects relating to mining rights, as is the case for deep geothermal energy. The process is initiated and regulated by the Prefecture (CNCE 2016).

These PI prompted a strong mobilization of French and German residents. Of the four projects under investigation, the construction of a plant near a site with a Seveso 2 classification, itself close to a residential area and the German border, has had the strongest mobilizing effect on the population.

During these PI, the lack of information was particularly highlighted by the residents and investigating commissioners (IC). This may seem paradoxical, as industrial operators, local politicians and the media have indeed been communicating about these projects. In this paper, we will try to figure out how this "misunderstanding" came to be. It is necessary to take into account the fact that deep geothermal energy is staged and takes its meaning depending on the stakeholders' intentions, professional or "social worlds" affiliations [START_REF] Garrety | Social Worlds, Actor-Networks and Controversy: The Case of Cholesterol, Dietary Fat and Heart Disease[END_REF]. Citizens are informed about this still relatively unknown technology through these different frameworks. And communication actions are crucial for the firms as well as for the opponents, each seeking to convince the public. Therefore, when the promoters claim to have given enough information, they do not understand that local residents are not being more sensitive to their arguments. However, it is unrealistic to believe that a "well" informed population necessarily supports said project (Batellier 2015, p. 113-118). It is important to perceive the public as a real actor in the information production process [START_REF] Hall | Codage/décodage[END_REF]. Information must make sense to them, it must meet their interests and concerns in order to be considered. It is only then that people will decide whether the project is acceptable or not, according to their roots in a community or territory, according to their worldview or their personal, political or economic interests. [START_REF] Wynne | Misunderstood Misunderstandings: Social Identities and the Public Uptake of Science[END_REF], when analyzing other fields, shows that once the authorities -industrial or elected -neglect or even despise the social identity of local residents, new information frames can arise, legitimized by alternative social worlds that feed the opposition to these projects.

In order to grasp these dynamics, we worked on three different corpuses. All contributions from citizens as well as impact studies, position papers and different reports made during the PI were collected. Observations were conducted on the survey areas, notably when the investigating commissioners (IC) were on site, and at public meetings held during or after the PI. An analysis of media coverage by the local media was also undertaken. In addition, we conducted twenty-two in-depth interviews with the different stakeholders: scientists, industrial operators and industry partners, representatives of pro-or opposing associations, elected officials, IC and experts requested by the prefectural authorities. This research is currently supplemented by data collected from local residents.

COMMUNICATION STRATEGIES VERSUS INFORMATION

Chronology of deep geothermal energy projects within the Eurometropolis

Which informations were disclosed and received throughout the timeline of the projects and from which social actors did they come from? The answer to this question will shed light on the apparent misunderstanding between citizens who feel poorly informed -even misinformed -and project promoters who think they appropriately communicated.

Since the early 2000s, geothermal energy has been mentioned in local energy policies, in particular through the development of national and regional climate plans. A strong potential for geothermal energy in Alsace, as seen in experiments in Soultzsous-Forêts since 1986, suggests a bright future for this technology. Given this favorable context, the city of Illkirch-Graffenstaden, as soon as 2009, solicited Electricité de Strasbourg (ES) to explore the geothermal potential on its municipal territory. In April 2011, the company filed a research permit application with the Ministry of Environment. The following year, the project is exposed during the transrhinan seminar organized by the Secrétariat Permanent pour la Prévention des Pollutions Industrielles (SPPPI). ES displays its commitment to operate in full transparency. Before an audience of elected officials, heads of associations and some "representatives" from the public, the seminar provide an update on the progress achieved in the pilot plant in Soultz-sous-Forêts. The risks associated with this type of project are discussed as well as the evolution of stimulation techniques. Consequently and in this case, upstream information was made available. Opinion leaders that were present could then pass on this information to a wider audience, including associations or local residents near these new projects. However, this is an isolated case.

During the summer of 2013, a private Aquitaine company, Fonroche, shows interest in this market and files two work requests within the EMS to the Direction Régionale de l'Environnement, de l'Aménagement et du Logement (DREAL), which add up with two other requests filed from ES. It was not until autumn 2014 that the applications were validated. During those two years, with the exception of the Illkirch-Graffenstaden project, the general public has not been consulted or "informed" on these projects. Most local residents only find out about it during the PI. Thus, from summer 2014, due to the lack of official information documents, the local residents of the oil port project are warned by their German neighbors, which are already involved in actions against geothermal energy across the Rhine. The leaders of the Association pour la Défense des Intérêts de la Robertsau (ADIR) launch their campaign a few months later. A case file is later published by the ADIR journal, L'Echo de la Robertsau, in September. It is one of the first "general public" documents: the emphasis is on the "environmental impacts". Jean-Daniel Braun, engineer and main advocate of the techno-scientific arguments against the projects, provides a list of the potential risks: seismic activity, pollution of the groundwater, radon gas release, risk of explosion, trickling water contamination. He is largely inspired by the study on the risks of geothermal drilling, which was presented by a Soultz-sous-Forêts researcher during the SPPPI seminar in 2012. Braun then deconstructs the arguments of Fonroche, the oil port project operator, by highlighting all the uncertainties plaguing the assertion that seismic risk and groundwater contamination are under control. Geothermal energy is thus presented in terms of risk. The only advantage mentioned, at the end of the article, refers to a quote from the former EMS president, which states that geothermal energy "ensures energy self-sufficiency" and cheaper heating costs.

Figure 1: Front page of the September 2014 ADIR newsletter

In December, 300 people attended a public meeting on geothermal energy in the Robertsau district, organized by a group comprising three residents' associations: the ADIR, which associated itself with the ASsociation pour la Sauvegarde de l'Environnement de la Robertsau (ASSER) and the Association de Défense des Intérêts des Quartiers centre-est de Strasbourg (ADIQ). In March 2015, this collective urges the major candidates in the departmental elections to take a public stance on the project.

Everything seems to happen as if a large part of the official communication was prepared in response to the mobilization of local residents. ES and Fonroche launch communication actions late in the game. They produce an information/popularization brochure a few months before the inquiries. In January 2015, geothermal energy is also the subject of a small exhibition held by Fonroche in the Esplanade district in Strasbourg, which is located remotely from the planned sites for the projects, and then in Eckbolsheim. ES published a web documentary on the subject on its website (http://www.geothermie.esgroupe.fr/), in which the Illkirch-Graffenstaden project is backed by an interview with the mayor and by researchers from the Labex G-EAU-THERMIE profonde of Strasbourg.

In November 2014, the EMS published a 4 page article on geothermal energy in issue 63 of its magazine. Six projects expected to help rebalance the energy mix are mentioned. Unlike the ADIR article, geothermal energy is presented in a positive light, as an "energy of the future". The rhetoric is meant to be reassuring, citing claims by representatives of the EMS, the EOST (École et Observatoire des Sciences de la Terre) and ES. It is stated that the unfortunate incidents of Lochwiller or Basel cannot happen in Strasbourg, and that the risks are under control.

The communication strategy of the project sponsors (EMS and promoters) is to focus on the economic and energetic benefits of geothermal energy. The EMS also demonstrates its commitment to "democratic control" by announcing the creation of a wise men committee and by displaying its willingness to create a debate on the issue. Consisting of elected officials and association representatives, the committee wanted to create a dialogue by keeping the businesses at bay. But the EMS broke this rule in April 2015 by inviting ES to present their project, which lead the ADIR to withdraw itself from the committee. The EMS and the promoters have therefore, belatedly and quite superficially, provided information on the scientific, technological, and political aspects of the projects.

Organized on already contentious grounds, the spring 2015 PI have hardened positions even more. In-depth files presenting the projects, their impacts and the precautions taken to limit the dangers are shown to the general public via the DREAL website and in relevant town halls. These were very technical files. Fonroche offered comprehensive and detailed documents, but the data was presented in a relatively crude way, so that the chapters on dangers study may have given the impression that the technology was not under control. The ES files were less complex, but only presented elements of information deemed necessary, thus going back on the transparency promised during the 2012 SPPPI seminar: for instance, there was no more any mention of radioactive pollution.

However, the technical aspect of the files did not deter the local residents. Each case was examined and the inaccuracies and gaps were used to counter the projects. Thus, certain information omitted by the firms has been put back into the spotlight: subjects like Plan for the Prevention of Technological Risks (PPRT), Local Development Plan (LDP), radioactivity, energy policy... The interpretation/refocus of these files are publicized in various ways: on blogs, in local or associative newspapers, and of course in the PI records, which are read by local citizens. In addition, meetings are organized locally to alert the people, like in Oberhausbergen where the issue of geothermal energy is addressed in neighborhood councils. Lastly, the actors were also using traditional media as a tribune.

The media: influential forums?

Local media were invested by the different stakeholders -project leaders, local communities, scientists, favorable or opposing associations -with the objective of getting citizens to support their interpretation of geothermal energy. Local residents associations, for example, are mobilized not only by publishing articles on the Internet, but also by sending their texts to the local newspapers, such as the daily Dernières Nouvelles d'Alsace (DNA). However, the media are subject to standards, format, editorial and audience constraints that determine the form and visibility of its content [START_REF] Charaudeau | Les médias et l'information : l'impossible transparence du discours[END_REF]. Thus it is clearly important for stakeholders to attempt to influence the media agenda and news framing and encourage journalists to deal with these issues in a favourable light [START_REF] Voirol | Les luttes pour la visibilité[END_REF]. If a social actor manages to become a journalistic source, he/she would be first to describe the reality, using his/her own words and narratives. For example, in the present case, the opposing associations have often used the letters to the editor section as a tribune.

Who were the social actors and what were the themes favored by the media? We established a corpus of 173 articles published over the period extending from the announcement of the public inquiries to the decisions made by the Prefecture (September 2014/December 2015). The local news media (Les DNA and Rue89), blogs from local residents and environmental associations, and local communities magazines were taken into account. Our analysis shows that the media favors local authorities (elected officials from municipalities and the EMS, whether favorable or not to the project), representing 28% of the 530 sources cited. The most often quoted sources were the Mayor of Strasbourg, Roland Ries, and his assistant in charge of energy transition, Alain Jund -who are mostly in favor of geothermal energy, while also claiming to understand the concerns of citizens -and city councilor Thierry Roos, opposed to the geothermal energy project in Robertsau. Local residents associations represent 18% of the quoted sources. The promoters are next, representing 9% of cited sources (Fonroche is more often cited than ES). Finally, experts like the EOST are not really represented: because of their obligation of scientific integrity, they rarely communicate with the media.

Two media gave more importance to deep geothermal energy projects: Les DNA and le Blog de la Robertsau. Both media have a mission, a status, and a way to operate that are very different and representative of the two types of communication strategies that marked the controversy. Les DNA is a "traditional" news media which claims a certain "objectivity" of information; it still cannot escape the constraints of audience and format [START_REF] Maigret | Sociologie de la communication et des médias[END_REF]. Le Blog de la Robertsau, meanwhile, represents an alternative media, free of the constraints of a conventional redaction committee and more interactive [START_REF] Cornu | Tous connectés ! : Internet et les nouvelles frontières de l'info[END_REF][START_REF] Grilo | La blogosphère, un cinquième pouvoir[END_REF].

Both media are prioritizing the issues related to geothermal energy differently. Les DNA rely on local institutions' statements to explain the legal and administrative aspects of the projects: PI procedure, deadlines, decision-making body, etc. Some elected officials are also quoted on their position regarding the expected location within the oil port. Les DNA newspaper is relaying statements from the ADIR, the ASSER and the ADIQ, demanding more information and transparency. Some elected officials, such as Theo Klumpp, Mayor of Oberhausbergen, are on par with the associations. Klumpp says "that before taking positions, the elected officials must have full information about a complex and sensitive subject. This is far from being the case" (DNA 10-12-2014). By reporting the comments from the associations, Les DNA are also highlighting their demands for abandoning the drilling project in the oil port. In addition, the associations are using this media forum to criticize Fonroche by emphasizing their lack of experience. The main risks mentioned by the newspaper are seismicity and pollution. In conclusion, the daily newspaper is often simply relaying some of the arguments from each party, favorable or unfavorable. This also means that the project is almost never treated as a whole, the voices are heard but not put into perspective. In the Blog de la Robertsau, local residents associations are the main source used by the blog, followed by local institutions (figure 3). The firms behind the project are less visible than in Les DNA.

Based on the local residents associations' rhetoric, the Blog de la Robertsau tries to convince that the oil port project poses serious problems. Similarly, the blog relays the voices of some elected officials when they are sharing the same views. For example, the blog published Thierry Roos' entire speech to the city council on April 20, 2015, where he said to the mayor of Strasbourg: "What measures do you intend to take to honor your executive's commitments regarding this project, namely the abandonment of geothermal drilling in the oil port?" Conversely, the blog is critical towards the EMS elected officials, who had apparently not sufficiently informed the population before the PI: " …while the EMS has never held a single information meeting for the public, it has authorized Fonroche to start taking measures, and it now wants us to believe it is as pure as a newly born lamb" (26-07-2015). The blog calls for "participatory democracy" that would take into account the local residents considerations. Thus, when the blog relays the requests for information made by the associations on the potential risks, it is mainly to argue for the rejection of the project. It points out that some information is missing from the files submitted to the PI by Fonroche and ES and that it is not appropriate to leave these geothermal projects in the hands of private companies. In summary, the blog authors are developing a dual communication strategy: giving visibility to criticism on the Robertsau project and on deep geothermal energy, and limiting the scope of the voices defending the projects, including those of ES and Fonroche. A closer examination of the corpus shows that, contrary to the articles published by Les DNA -which are rather short, descriptive, and limited to quoting the sources -, the articles published by the Blog de la Robertsau are relatively long and detailed. The blog, being an associative media, intend to be an informative partisan-expert/protoexpert to online communities. Conversely, Les DNA addressed geothermal energy by conventional journalistic standards, claiming to be "objective".

PUBLIC INQUIRY: A PLACE OF DEMOCRATIC EXPRESSION?

Taking part in the public inquiry

The PI is hosted by the town hall and put under the responsibility of an investigating commissioner (IC) mandated by the administrative court. Participation is opened to all citizens: it is possible to give its opinion on a register available in the town hall, to address it to the IC by mail, email, or to present it orally (figure 4). We took into account all French written opinions submitted during the four public inquiries. Of all these, only about twenty are positive. The Illkirch-Graffenstaden project has attracted the least participation (19 written opinions) and criticism. In all three other sites, however, local residents have strongly contributed to the PI to demonstrate their opposition to the project: 86 contributions in Eckbolsheim, 138 in Mittelhausbergen and 135 French contributions in Strasbourg and Robertsau (related to the oil port project), to which must be added 756 contributions from Germany. At first glance, the geographical proximity of the project(s) with dwelling places seems to motivate participation in these inquiries (figure 5). Thus, Oberhausbergen's residents have largely contributed to the investigations regarding the projects in Mittelhausbergen and Eckobolsheim, which are close to the boundaries of their municipal territory. The form of contributions varies: a simple "no" to the project (30% of opinions) is seen alongside more developed arguments (15% of opinions) or even sometimes highly detailed ones (16% of opinions).

The most elaborate documents are mainly deliberations of the municipal councils, opinions of political groups (Europe Ecologie Les Verts) and opinions from local residents associations (ADIR, ASSER, ADIQ) or from environmental protection associations (Alsace Nature, Arbres). These documents, which often have been circulating through blogs, then served as information sources to other participants.

The opinions of collective actors, as well as many individual contributions, are fed by multiple informations, sometimes cited as references: reviews of seminars organized by the SPPPI, appraisals from the Institut National de l'Environnement Industriel et des Risques (INERIS) commissioned by the prefecture, deliberations from municipal councils and opinions of local residents associations. Local media are rarely mentioned, however, even if they have contributed to the debate. If they have an influence, it would be rather indirect, by providing information which is then discussed in interpersonal circles, as we have seen during observations of the discussions happening in the PI location.

Most citizens who have submitted their opinions to the PI are informed, some have acquired the necessary proto-expertise [START_REF] Nowotny | Socially distributed knowledge: five spaces for science to meet the public[END_REF]) to handle highly technical data. Their proto-expert identity contrasts with the unrealistic image of the naïve and common sense citizen which still prevails in the philosophy of French culture (for an example of this philosophy, see Hermitte 2013).

What are citizen contributions saying?

The opinions are listing several types of risks: induced seismicity, pollution of groundwater, fluid radioactivity, risks of explosion, surface pollution (figure 6). Local residents also mention the potential negative impacts of drilling on the quality of life, on dwellings or on all nearby activities (research structure, education, trade and hotels). The opinions are therefore echoing the hierarchy of meanings suggested by the media, especially by the Blog de la Robertsau, and other information instances such as Oberhausbergen' neighborhood councils and the ADIR newsletter.

Figure 6: Occurrences of different risks in French citizens' opinions in the four EMS public inquiries

By contrast, it is as if the "official" presentation of the risk-limiting measures did not meet the expectations of citizens: these informations are faulted by local residents-cum-proto-experts, either because they believe that they are not convincing, or because they fail to address certain issues. In addition, the documents prepared by the contracting authorities and third-party appraisals conducted by the INERIS, made available to the public at the PI, are primarily written to meet the demands of the prefectural authority, and not to inform/reassure.

Therefore, when the projects advocates are claiming that they are limiting the risks, local residents are drawing the conclusion that these risks actually do exist. Dialogue consequently becomes impossible. The same goes for impact studies as well as the 2012 SPPPI seminar, during which researchers discussed the scope of the risks and the precautions taken to limit its expression. Evaluating all these arguments, local residents believe that these explanations are in some way pleading for the application of the precautionary principle.

Finally, some risks not mentioned in the files are revealed by local residents: radon pollution -missing from the ES files -or the cumulative effects of different planned drilling projects within the EMS.

Citizens are thus raising questions about issues overlooked by industry players, and also missing from traditional news coverage. The location of drilling sites is also subject to diverging interpretations. While operators and partners are stressing the opportunities linked to industries in the oil port, Robertsau residents find it absurd that a "risky" project could be implemented in an industrial complex which is the subject of a PPTR (Plan for the Prevention of Technological Risks). Another example, the industry insists on the geothermal drilling benefits for district heating in a Strasbourg neighborhood, Hautepierre, when citizens are instead noticing that the installation site is located near shops, a high school and a research center. In addition, residents are questioning the fact that many projects are emerging a few kilometers apart from each other. Are the risks accumulating? They point out that the impact studies provide no answer to these questions.

Everything happens as if the companies and the EMS politicians had failed to take into account the human, environmental and urbanized side of these projects layout. Official documents are coded according to their interests and their "philosophy": to develop a "green" technology and to make it profitable. This "oversight" is coupled with the fact that no consultation has been made before the public inquiries: it could have defined, with the help of municipalities, local residents and associations, the conditions for the installation of geothermal power plants. Lastly, the lack of a clear political framework from the EMS adds to the confusion around the debates. Indeed, the collectivity commits itself by buying prospective land for drilling while neglecting to campaign for geothermal energy; local residents are consequently not motivated to support the projects with which they will be neighbors. Besides, they doubt that the project will be profitable, while also not trusting the reliability of Fonroche. The inconsistency of politicians' attitude, the technicalities as well as, in some cases, the complacency of a company claiming to control everything, and lastly poor communication, all are considered with suspicion. Many opinions can attest to this, calling the technology "non-mature science" and the decision-makers "sorcerers' apprentices".

INDIRECT CONFRONTATION AND MEDIATION: CAN THE PUBLIC INQUIRY SOLVE PROBLEMS?

The investigating commissioner as a gatekeeper

The first task of the IC is to take ownership of the technical and legal elements of the case via the documents provided by the operator and the environmental authority, actors that it generally seeks to meet. To do this, the IC often receives training and is designated according to their prior knowledge. The IC must then make itself available to the public: during the investigation and through its constituency offices, it provides/facilitates access to the project's files by explaining and discussing them with citizens. It helps them to understand the subject, to answer their questions in order to encourage them to express their own informed opinion. When the participating phase of the PI is completed, the IC gathers, sorts and summarizes all the comments, opinions, and proposals from the audience. It is thereby fulfilling its role as a gatekeeper [START_REF] White | The 'Gatekeeper': A Case Study In the Selection of News[END_REF], valorizing, prioritizing some of the arguments/questions that will form the basis of the report that will be given to the operator. The latter will have to answer with a memorandum.

Ideally, the PI should promote dialogue between the parties involved, be mediated by the IC, and should clarify responsibilities. Did this indirect dialogue take place in the case of the EMS' PI?

The promoter's memorendum to the IC's report reveals the gap between the companies' intentions and the expectations of citizens. On at least three levels: responsibility of risks, communication, and insertion of geothermal energy in local residents' environment.

First, the promoters provide answers about risk issues. They seek to show that risk management is possible and that they are assuming responsibility. Within this framework, the precautionary principle is discussed. However, this is a rhetorical and conceptual answer: according to them, the precautions are already integrated into the scientific method on which the project is based. Yet, citizens support their claims with concrete examples: microseisms and other hazards that may affect their living environment. For the promoters, the precautionary principle means that the risks are under control, while for the residents, it should be used to postpone the projects.

Second, the memorandum allows the operators to justify themselves regarding critics about their lack of communication. Hence, Fonroche saying that they communicated about their projects as early as July 2013 and took the initiative of organizing several meetings with the different stakeholders before the public inquiries. However, these actions have apparently not been withheld by local residents. It appears that they do not expect information solely produced to convince them of the merits of a project. They insist they are entitled to be informed and that they have the right to knowingly choose the best attitude to deal with these projects. These "unrewarded" efforts show that the concept of information is different according to whether one is the initiator or the receiver. On the other hand, it also seems unrealistic to believe that public information would necessarily guarantee public acceptance of technological innovations.

Finally, regarding the insertion of a central plant in an urban environment and the political and economic dimensions of these projects, the promoters relinquish all responsibilities: these strategic choices are fall within the EMS and the municipalities' jurisdiction. Again, no discussion can take place.

Enlightened by the public's open inquiries and by the answers provided by the promoter, the IC ultimately issues its opinion on the matter: it delivers its feelings about the feasibility of the project and its human environment. A feeling that turns out to be negative for the Mittelhausbergen, oil port and Eckbolsheim's IC.

Which arguments are selected, the ones the gatekeeper "lets through"? The Mittelhausbergen and oil port IC put forward the precautionary principle. The implantation site was poorly chosen in both cases: located near homes and a water catchment area, or in a Seveso zone. The IC also note the weaknesses in the companies' case files: about the cumulative effects of several drillings, the lack of accurate information about the explored fault lines and drilling directions, for example.

Finally, the gatekeeper must settle the contradiction between the promoters' memorandum, whom believe they have given enough information, and the citizens who regret the lack of information. Rejecting the arguments from the industry, the IC confront them to their responsibilities in terms of quantity and quality of information. According to them, richer, more transparent information and a serious consultation would have allowed local residents to consider deep geothermal energy projects in a more constructive way. In this context, the IC provide three functions to the informational issue:

• According to the Mittelhausbergen investigator, "good" information would lead to the acceptability of projects.

• The oil port IC defines the information: it must both answer citizens' questions and reassure. However, he considers that the documents provided by the promoter do not allow to figure out that all risks would be avoided. The information is neither able to reassure nor to answer.

• Finally, the Eckbolsheim investigator gives a more noble mission to information: if it was honest and thorough, it would allow for social appropriation of the issues related to geothermal energy, be they technological, political or social. However, these issues were not sufficiently addressed -even omitted -by the promoters and the EMS.

Discussion

The prefectural decrees authorizing the opening of mining operations in Illkirch-Graffenstaden and Eckbolsheim were signed in autumn 2015. On October 15, Christian Riguet, Secretary General of the Prefecture, reflects back on the PI during the 12th Plenary Assembly of the SPPPI:

"Citizens now feel entitled to obtain increasingly complete information and never feel sufficiently informed about the projects that affect them. [...] The decision-making process must integrate this new reality, while making sure that the audience understands that public interest comes first. General interest is not the sum of particular interests. [...] Opinions are then often expressed vehemently, sometimes irrationally. It would therefore be advisable to make use of upstream information work."

Citizens asking for information are therefore seen as "a problem" [START_REF] Felt | Vers la construction d'un public européen?[END_REF]. All it would take, according to this interpretation, is better education -which is the responsibility of promoters and politicians -to make them go along with the projects. Thus, their concerns could be eliminated and the general interest respected. The IC's role would be limited to relaying questions from local residents and answers from promoters, a far stretch from its gatekeeper mission.

This conception of information contains three classic assumptions underlying the rhetorics from promoters of controversial technological projects (Batellier 2015): (1) the "opponents" do not understand, they are ignorant and/or uninformed; (2) citizens are requesting information;

(3) if citizens were more familiar with the project, they would display a more positive attitude.

In short, acceptance of a project would be embodied in the maxim the more you know the more you like. Yet as we have seen, citizens are often well informed. This has also been highlighted by an IC which saw a major peak in attendance following an information campaign by a municipal official. In addition, more information does not necessarily lead to a more positive attitude towards the projects. Citizens are less "suggestible" and/or "reactive" than what a quick read of the controversy in terms of acceptability may suggests.

Anyone and everyone has information resources that allows them to decode information about projects with which they will have to live: resources linked to their experience of a territory, to its history, its microculture; information relayed by opinion leaders (elected officials, neighbors, family, activists ...) and, on a more global level, websites, blogs, social networks on the Internet. "Official" information, even if it came from upstream, is only one element among all these possible references... Thus, when the public requests more information when preparing their contributions to the PI, it is in many cases about unthought aspects of the projects: their inclusion in a/their territory, the cumulative effects of individual projects, the place of geothermal energy in the energy policy of the EMS... These requests have a critical dimension: the promoters may only be handing out informations in order for their projects to be accepted, projects from which they will draw benefits. Hence, the critical questions of local residents reflect, as is highlighted by Batellier, a lack of confidence in project promoters (operators and policy-makers).

What if, rather than seeing citizens as "problems" in the development of projects, the perspective was reversed? Questions, criticism, citizens' contributions should all be taken into account to build, through real dialogue, a public policy related to energy.

Figure 2 :

 2 Figure 2: Occurrence of issues related to the stakeholders in the controversy by Les DNA (analysis via the ATLAS.ti software).

  Figure 3: Occurrence of issues related to stakeholders in the controversy by the Blog de la Robertsau.

Figure 4 :

 4 Figure 4: Amount of the different forms of contributions from French citizens in the four EMS public inquiries.

Figure 5 :

 5 Figure 5: Breakdown of participation to public inquiries by area of residence.

Figure 7 :

 7 Figure 7: Excerpt of a citizen contribution to the Mittelhausbergen public inquiry.

  Yet, a meaningful dialogue can only be established if the projects are still open for discussion...

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	UQÀM, Québec, (2015).

Breton, P. and Proulx S.: L'explosion de la communication à l'aube du XXIe siècle, Ed. La Découverte, Paris, (2002).

Acknowledgements

This work was done in the framework of the project "How to deal with a public inquiry? Views from Residents and deep geothermal projects Stakeholders in Alsace" (ORA-Géo). This research has been financially supported by the Labex G-EAU-THERMIE profonde (University of Strasbourg, CNRS, ESG).


Rafal P Piprek
email: rafal.piprek@uj.edu.pl

Milena Damulewicz

Malgorzata Kloc

Jacek Z Kubiak

Transcriptome analysis identifies genes involved in sex determination and development of Xenopus laevis gonads

Keywords: gonad, testis, ovary, sex determination, gonad development, Xenopus Amh -Anti-Müllerian hormone, Bmp4 -Bone morphogenetic protein 4, Dax1 -Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene

Development of the gonads is a complex process, which starts with a period of undifferentiated, bipotential gonads. During this period the expression of sex-determining genes is initiated. Sex determination is a process triggering differentiation of the gonads into the testis or ovary. Sex determination period is followed by sexual differentiation, i.e. appearance of the first testis-and ovary-specific features. In Xenopus laevis W-linked DMdomain gene (DM-W) had been described as a master determinant of the gonadal female sex.

However, the data on the expression and function of other genes participating in gonad development in X. laevis, and in anurans, in general, are very limited. We applied microarray technique to analyze the expression pattern of a subset of X. laevis genes previously identified to be involved in gonad development in several vertebrate species. We also analyzed the localization and the expression level of proteins encoded by these genes in developing X. laevis gonads. These analyses pointed to the set of genes differentially expressed in developing testes and ovaries. Gata4, Sox9, Dmrt1, Amh, Fgf9, Ptgds, Pdgf, Fshr, and Cyp17a1 expression was upregulated in developing testes, while DM-W, Fst, Foxl2, and Cyp19a1 were upregulated in developing ovaries. We discuss the possible roles of these genes in development of X. leavis gonads.

Introduction

At the earliest phase of development, the Xenopus laevis gonads are sexually undifferentiated and bipotential, thus they can differentiate into testes or ovaries depending on the genetic/chromosomal background: ZW in female and ZZ in male. In anuran amphibians the gonads appear around the hindlimb buds stage. In Xenopus laevis tadpole the period of undifferentiated gonad falls between Nieuwoop and Faber (NF) stage 49 and 53 [START_REF] Piprek | Development of Xenopus laevis bipotential gonads into testis or ovary is driven by sex-specific cell-cell interactions, proliferation rate, cell migration and deposition of extracellular matrix[END_REF]) (Fig. 1A), and the crucial sex-determining period occurs between stage NF50 and 52, when the W-linked DM-domain (DM-W) transcription factor starts being expressed in the ZW (genetic female) gonads [START_REF] Yoshimoto | A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis[END_REF]. The DM-W, by blocking the Doublesex and mab-3 related transcription factor 1 (DMRT1), which participates in male sex determination in vertebrates, induces female pathway [START_REF] Yoshimoto | Opposite roles of DMRT1 and its W-linked paralogue, DM-W, in sexual dimorphism of Xenopus laevis: implications of a ZZ/ZW-type sex-determining system[END_REF]. Interaction between these two genes triggers a series of changes leading to the development of testis-or ovary-specific features. The sex-determining period is followed by the period of sexual differentiation of the gonads. In X. laevis, the first differences between male and female gonads are identifiable at stage NF53, when the germ cells start dispersing within the differentiating testes, while remaining at the gonad periphery in the ovaries [START_REF] Piprek | Development of Xenopus laevis bipotential gonads into testis or ovary is driven by sex-specific cell-cell interactions, proliferation rate, cell migration and deposition of extracellular matrix[END_REF]. At stage NF56 the gonadal sex becomes anatomically recognizable; the ovary consists of the central cavity surrounded by the cortex and sterile medulla, while in the developing testes the cortex and medulla fuse in a single cell mass without the cavity. Around metamorphosis (stage NF62) the gonads are visibly differentiated, the oocytes appear in the ovaries, and the sex cords (the future seminiferous tubules) form in the testes.

X. laevis is the only amphibian species in which the sex-determining gene has been discovered. However, surprisingly, the expression of only few genes such as Sox3 [START_REF] Koyano | The Xenopus SOX3 gene expressed in oocytes of early stages[END_REF], Dmrt1 [START_REF] Osawa | Molecular cloning of Dmrt1 and its expression in the gonad of Xenopus[END_REF] and DM-W [START_REF] Yoshimoto | A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis[END_REF]) had been studied in developing X. laevis gonads. The expression of Sox9 was studied in closely related Silurana tropicalis [START_REF] Jamil | Sexspecific expression of SOX9 during gonadogenesis in the amphibian Xenopus tropicalis[END_REF]. Interestingly, there are many studies on the gene expression pattern in developing gonads of another anuran species, the Glandirana rugosa (reviewed in [START_REF] Nakamura | Sex determination in amphibians[END_REF]. G. rugosa is widely studied because of its unusual pattern of sex determination; both XX/XY and ZZ/ZW system are present in different populations of this species [START_REF] Nishioka | Four kinds of sex chromosomes in Rana rugosa[END_REF].

In order to identify genes involved in gonad development and sex determination in X. laevis, we studied the expression of genes that are known to be involved in gonad development in other vertebrates. Considering a high number of genes of interest, we used a microarray to perform a global analysis of X. laevis transcriptome. We also performed the immunolocalization of proteins encoded by the chosen genes. The level of RNA and protein expression was analyzed at four time points: stage NF50 (undifferentiated gonad), NF53 (the onset of sexual differentiation), NF56 (differentiating gonads), and NF62 (sexually differentiated gonads) (Fig. 1).

Material and Methods

Animals

Larvae of the African clawed frog (Xenopus laevis Daudin, 1802; Pipidae) were raised in the laboratory. The tadpoles were kept in 10-L aquaria (30 tadpoles per 10 L) at 22 o C and fed daily with powder food Sera Micron (Sera) as previously described [START_REF] Piprek | Development of Xenopus laevis bipotential gonads into testis or ovary is driven by sex-specific cell-cell interactions, proliferation rate, cell migration and deposition of extracellular matrix[END_REF]).

The tadpoles were staged according to Nieuwkoop and Faber (1964). After staging, the tadpoles at four stages (NF50, NF53, NF56, and NF62) were anesthetized with 0.1% MS222 solution. All specimens used in the experiments were handled according to Polish legal regulations concerning the scientific procedures on animals (Dz. U. nr 33, poz. 289, 2005) and the permission from the First Local Commission for Ethics in Experiments on Animals.

Sex determination by PCR

The genetic sex of each tadpole was determined using PCR detection of femalespecific DMW gene. DNA was isolated from tadpole tails using NucleoSpin Tissue Kit (Macherey-Nagel, 740952.240C). DMW gene (W-linked female-specific marker) and Dmrt1 gene (positive control) were used to determine ZZ or ZW status of tested animals. PCR was performed as previously described [START_REF] Piprek | Development of Xenopus laevis bipotential gonads into testis or ovary is driven by sex-specific cell-cell interactions, proliferation rate, cell migration and deposition of extracellular matrix[END_REF]. The mixture of the following pairs of primers were used: for DMW: 5'-CCACACCCAGCTCATGTAAAG-3' and 5'-GGGCAGAGTCACATATACTG-3', and for Dmrt1: 5'-AACAGGAGCCCAATTCTGAG-3' and 5'-AACTGCTTGACCTCTAATGC-3'

Immunohistochemistry

Bouin's solution-fixed and paraffin-embedded samples were serially sectioned at 4μm. Sections were deparafinated, rehydrated and heat-induced epitope retrieval was performed in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6) at 95 o C for 20 min. [START_REF] Piprek | Development of Xenopus laevis bipotential gonads into testis or ovary is driven by sex-specific cell-cell interactions, proliferation rate, cell migration and deposition of extracellular matrix[END_REF]. Sections were blocked with 3% H 2 O 2 for 10 min., blocked in 6% Bovine Serum Albumin (BSA, Sigma) for 15-min and incubated with the custom primary antibodies: anti-RSPO1 (GenScript, 674196-52), anti-WNT4 (GenScript,, and anti-SOX9a (GenScript, 674196-42) at RT. UltraVision Quanto Detection System (ThermoFisher, TL-125-QHD) was used to visualize reaction and Mayer's hematoxylin was used as a counterstain. Sections were viewed under Nikon Eclipse E600 microscope.

RNA isolation

Total RNA was isolated using Trizol as described above and further purified with Direct-zol RNA kit according to manufacturer's protocol (Zymo Research, R2061). The total RNA eluted with RNase-free water was quantified with NanoDrop 2000, and RIN (RNA Integrity Number) was assessed using Bioanalyzer 2100. All specimens used in the study had RIN above 8. Total RNA in RNase-free water was frozen at -80 o C until further use.

Microarray analysis

The microarray experiment was repeated three times. In order to obtain sufficient amount of RNA, the samples from10 individuals were pooled in each experiment. Total RNA was labeled with Cy3 using Agilent One-Color Quick Amp Labeling Protocol. Fluorescently labeled RNA samples were combined with Agilent Hi-RPM Hybridization Buffer, and hybridized at 65 o C for 17 hours in HybArray12 hybridization station (Perkin Elmer), then were washed for 1 minute each in Gene Expression Wash Buffer 1 (6X SSPE, 0.005% Nlauroylsarcosine; at RT) and Gene Expression Wash Buffer (0.06X SSPE, 0.005% Nlauroylsarcosine; at RT) for 1 minute each, immersed in a solution of acetonitrile and air dried. Slides (custom-commercial Agilent-070330 X. laevis Microarray slides) were scanned in Agilent Technologies G2505C Microarray Scanner at 5μm resolution.

Data processing

TIF files from microarray scanner were processed using Agilent Feature Extraction software version 10.5.1.1. Control features and non-uniform features were removed, and remaining values for each unique probe sequence were averaged, then log (base 2) transformed. Log base 2 intensities were median centered between arrays. Differential gene expression was filtered using both a statistical significance threshold (FDR < 0.05) and a Fold Change threshold (1.5 fold). The data are available in Gene Expression Omnibus (accession number GSE105103) and in Suppl. Table 1.

Results and Discussion

Our transcriptional analysis allowed identification of genes that are sex-dimorphically expressed in developing gonads and thus are potentially involved in the control of gonad development. At stage NF50 none of the 46 analyzed genes showed sexual dimorphism in the expression pattern. This is not surprising because at this stage the gonads are still bipotential.

In the mouse Wt1, Sf1, Gata4 and Emx2 genes control the earliest phase of bipotential gonad development (reviewed by [START_REF] Piprek | Early Development of the Gonads: Origin and Differentiation of the Somatic Cells of the Genital Ridges[END_REF]). These four transcription factors are expressed in the mouse genital ridges, they regulate the expression of mouse sex-determining genes, and their mutations lead to gonadal dysgenesis [START_REF] Hammes | Two splice variants of the Wilms' tumor 1 gene have distinct functions during sex determination and nephron formation[END_REF][START_REF] Luo | A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation[END_REF][START_REF] Miyamoto | Defects of urogenital development in mice lacking Emx2[END_REF][START_REF] Miyamoto | A GATA4/WT1 cooperation regulates transcription of genes required for mammalian sex determination and differentiation[END_REF]. We chose X. laevis orthologs of these genes for our analysis. Because X. laevis is allotetraploid, some of its genes have two paralogs, a and b [START_REF] Hughes | Evolution of duplicate genes in a tetraploid animal, Xenopus laevis[END_REF]. For the Wt1 gene (Wilms tumor 1) we studied the expression of two paralogs: Wt1a and Wt1b. Both showed only moderate difference in the expression between sexes, with slight tendency to increase from stage NF53 onward in the developing testes (Fig. 2). At stage NF62 the level of Wt1a and Wt1b expression slightly increased in the testes and decreased in the ovaries. A very similar pattern of expression was revealed for Nr5a1 (Sf1) and Gata4 with the highest level of expression in the testes (Fig. 2). The expression of Emx2 (empty spiracles homeobox 2) was slightly higher in the ovaries (Fig. 2).

The review of literature on the expression of above genes in other anurans showed that the expression of Wt1 was studied only in the frog Glandirana rugosa during early sexual differentiation of the gonads where no sex dimorphism in Wt1 expression was detected [START_REF] Yamamura | Molecular cloning and expression in gonad of Rana rugosa WT1 and Fgf9[END_REF]. The Nr5a1 was found to be upregulated in differentiating ovaries of Lithobates catesbeianus [START_REF] Mayer | Sexually dimorphic expression of steroidogenic factor 1 (SF-1) in developing gonads of the American bullfrog, Rana catesbeiana[END_REF], and was expressed in the adult testes but not in the ovaries of G. rugosa [START_REF] Kawano | Expression of steroidogenic factor-1 in frog embryo and developing gonad[END_REF]. A participation of these genes in early gonad development may be conserved among vertebrates, for example Nr5a1 and Wt1 are expressed at early stages of gonad development in medaka, zebrafish, slider Trachemys scripta, American alligator and chicken [START_REF] Spotila | Expression of a new RNA-splice isoform of WT1 in developing kidney-gonadal complexes of the turtle, Trachemys scripta[END_REF][START_REF] Western | Temperature-dependent sex determination in the American alligator: expression of SF1, WT1 and DAX1 during gonadogenesis[END_REF][START_REF] Jorgensen | Expression profiles for six zebrafish genes during gonadal sex differentiation[END_REF][START_REF] Kluver | Regulatory back-up circuit of medaka Wt1 co-orthologs ensures PGC maintenance[END_REF].

Genes involved in female sex determination

DM-W (DM domain gene on W chromosome) transcription factor was identified as a master determiner of the female sex in X. laevis [START_REF] Yoshimoto | A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis[END_REF]). The DM-W probably inhibits DMRT1 action, and thus blocks male sex determination [START_REF] Yoshimoto | Opposite roles of DMRT1 and its W-linked paralogue, DM-W, in sexual dimorphism of Xenopus laevis: implications of a ZZ/ZW-type sex-determining system[END_REF]). These researchers used RT-PCR to show that DM-W gene was expressed only at the very early stage (NF48-50) of gonad development, and its expression ceased at stage NF52 [START_REF] Yoshimoto | A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis[END_REF]. We detected strong expression of DM-W throughout the ovarian development with a gradual increase from stage NF50 onward, and peaking at stageNF56 (Fig. 3). At stage NF62 the expression level of DM-W decreased to the level comparable to NF53. The difference between our and [START_REF] Yoshimoto | A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis[END_REF] data may result from different method of analysis (microarray versus PCR) or different strains of X. laevis used.

In the mouse and human, the Rspo1 and Wnt4 gene products acting through β-catenin stabilization are pivotal for the female sex determination [START_REF] Vainio | Female development in mammals is regulated by Wnt-4 signalling[END_REF][START_REF] Parma | R-spondin1 is essential in sex determination, skin differentiation and malignancy[END_REF]; reviewed by Piprek 2010). We studied the expression of Rspo1, Wnt4 and Ctnnb1 (βcatenin) in X. laevis gonads. The expression of Rspo1 was slightly higher in the female than in male gonad at stage NF56 (Fig. 3). Wnt4 and Ctnnb1 expression did not reveal significant sex difference (Fig. 3). The expression of Wnt4 gradually increased and peaked at stage NF56. To verify the potential role of these genes in gonad development we performed immunolocalization of proteins encoded by these three genes. Immunolocalization showed that WNT4 protein had strongest expression at stage NF56, which agreed with the results of microarray analysis (Fig. 4). No significant sex dimorphism was detectable in the expression of WNT4 protein. The RSPO1 protein, similar to RNA, had slightly stronger expression in the ovaries (Fig. 5). The expression level of Ctnnb1 gene was similar in both sexes.

Immunolocalization of β-catenin showed sex dimorphism with the higher expression in the ovaries (Fig. 6), The higher level of β-catenin protein in developing ovaries in X. laevis might result from the β-catenin stabilization rather than the upregulation of its expression. This mechanism may be a conserved pathway of female sex determination among vertebrates because upregulation of Rspo1 expression at early stage of ovary development is characteristic not only for mouse and X. laevis but also for zebrafish, chicken and slider T. scripta (Smith et al. 2008;[START_REF] Zhang | Molecular analysis shows differential expression of R-spondin1 in zebrafish (Danio rerio) gonads[END_REF].

Foxl2 (forkhead box L2) encodes a transcription factor, which in developing mouse ovaries plays a role in female sex determination; its deletion leads to the expression of the male sex-determining genes [START_REF] Ottolenghi | Foxl2 is required for commitment to ovary differentiation[END_REF]. This gene is upregulated also in the developing ovaries in rainbow trout, slider T. scripta, chicken, and goat [START_REF] Pailhoux | Time course of female-tomale sex reversal in 38,XX fetal and postnatal pigs[END_REF][START_REF] Loffler | Etiology of ovarian failure in blepharophimosis ptosis epicanthus inversus syndrome: FOXL2 is a conserved, early-acting gene in vertebrate ovarian development[END_REF][START_REF] Baron | An evolutionary and functional analysis of FoxL2 in rainbow trout gonad differentiation[END_REF]Smith et al. 2008). In X. laevis, there was gradual upregulation of Foxl2 in the ovaries between stage NF50 and NF62 (Fig. 7). Foxl2 was also expressed in the testes but at lower level. Several studies showed that Foxl2 was upregualted in developing ovaries of Lithobates silvaticus, Silurana tropicalis and G. rugosa (Navaro-Martin et al. 2012;[START_REF] Oshima | Molecular cloning and gene expression of Foxl2 in the frog Rana rugosa[END_REF]). Thus, Foxl2 may play a similar role in ovary development in all anurans.

Follistatin (encoded by Fst gene) is an autocrine glycoprotein, which binds and bioneutralizes members of the TGF-β superfamily, and as a result inhibits formation of malespecific features, such as development of the coelomic vessels, in developing mouse ovaries [START_REF] Yao | Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis[END_REF]). Fst has not been studied in the gonad development in amphibians. Here, for the first time we showed that Fst is significantly upregulated in X. laevis ovaries, between stage NF50 and NF62, while it is expressed at very low level in testes (Fig. 7). This suggests that follistatin may be also involved in the inhibition of the male pathway in developing ovaries of anurans.

Bmp4 is involved in ovary development by promoting ovarian follicles formation in the mouse [START_REF] Nilsson | Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development[END_REF]. X. laevis Bmp4 (encoding bone morphogenetic protein 4) has two paralogs: Bmp4a and Bmp4b. We found that these two paralogs were expressed in the developing X. laevis gonads, however, with no significant sex-specific difference (Fig. 7).

The Dax1 gene, which encodes nuclear receptor, is slightly upregulated in the testes at stage NF62 in X. laevis (Fig. 7). The upregulation of this gene in developing testes was also showed for another anuran, G. rugosa [START_REF] Sugita | Expression of Dax-1 during gonadal development of the frog[END_REF]. In contrast, the expression of Dax1 was downregulated during testes development in the mouse and chicken [START_REF] Smith | Cloning and expression of a DAX1 homologue in the chicken embryo[END_REF][START_REF] Swain | Mouse Dax1 expression is consistent with a role in sex determination as well as in adrenal and hypothalamus function[END_REF], which indicates functional difference of this gene among vertebrates. In the mice mutation of Dax1 leads to male-to-female sex reversal due to inhibition of Sox9 gene -a key player in male sex determination pathway [START_REF] Bouma | Gonadal sex reversal in mutant Dax1 XY mice: a failure to upregulate Sox9 in pre-Sertoli cells[END_REF].

Genes involved in male sex determination

Dmrt1 (encoding doublesex and mab-3 related transcription factor 1) is an evolutionary conserved gene playing a role in male sex determination in vertebrates [START_REF] Koopman | Sex Determination: The Fishy Tale of Dmrt1[END_REF]. Its mutation leads to male-to-female sex reversal. In X. laevis two paralogs: Dmrt1a and Dmrt1b are present. Both showed a very strong expression in the testes from the early phase of male gonad development (NF53) (Fig. 8). Dmrt1 expression in developing testes, but not ovaries was previously shown in G. rugosa and X. laevis (Osawa et al. 2005;[START_REF] Shibata | The Dmrt1 expression in sex-reversed gonads of amphibians[END_REF].

A signaling factor FGF9 (fibroblast growth factor 9) acting via FGFR2 (fibroblast growth factor receptor 2) participates in a male sex determination in the mouse and enhances cell proliferation, which is critical for testis development [START_REF] Kim | Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination[END_REF]. In developing X. laevis gonads the Fgf9 and Fgfr2 show visible sex-specific difference in expression around metamorphosis (stage NF62) with prevalence in the male gonads (Fig. 8). This suggests that FGF9/FGRF2 pathway is involved in some aspects of testis development in X. laevis. In contrast, in developing gonads of G. rugosa there is no sex-specific difference in the expression of these genes [START_REF] Yamamura | Molecular cloning and expression in gonad of Rana rugosa WT1 and Fgf9[END_REF]. We also analyzed the expression of gene coding for FGFR3 receptor, which seems to be important in chicken ovary development [START_REF] Ayers | Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq[END_REF]. In X. laevis, the expression of Fgfr3 showed highly significant sexspecific difference at stage NF62, with the higher expression in the male gonad (Fig. 8).

Prostaglandin D2 (PGD2) is a signaling molecule, which enhances expression of genes involved in male sex determination in the mouse [START_REF] Malki | Prostaglandin D2 induces nuclear import of the sexdetermining factor SOX9 via its cAMP-PKA phosphorylation[END_REF][START_REF] Wilhelm | SOX9 regulates prostaglandin D synthase gene transcription in vivo to ensure testis development[END_REF]). In X. laevis, the expression of prostaglandin synthase Ptgds-a gene was slightly higher in the testes than in the ovaries (Fig. 8). The expression of the other paralogue of prostaglandin synthase, the Ptgds-b, showed strong upregulation in X. laevis male gonads from stage NF53 onward (Fig. 8). This indicates that prostaglandin signaling is probably involved in male sex determination and/or testis development in X. laevis.

Sox genes

We studied the expression of seven Sox genes that are known to participate in the gonad development in vertebrates. Among them SOX9 transcription factor is upregulated in developing testes, and plays a main role in male sex determination in many vertebrates [START_REF] Koopman | Sry, Sox9 and mammalian sex determination[END_REF]. We found that Sox9a and Sox9b (Sry-box 9) paralogs were significantly upregulated in X. laevis testes (Fig. 9). Sox9a was gradually upregulated in testes and ovaries.

We observed a significant upregulation of Sox9a expression in the testes between stage NF53 and NF62. The expression of Sox9b was higher in the testes than in the ovaries, but in contrast to Sox9a, there was no upregulation after stage NF53. These results suggest a potential involvement of these two Sox9 paralogs in X. laevis testis development. Because of the particularly high difference in the expression levels of Sox9 between ovaries and testes we performed immunolocalization of SOX9a protein in developing X. laevis gonads. The intensity of SOX9a immunostaining gradually increased in the male gonads and was the highest at NF62 stage, which corroborated gene expression pattern (Fig. 10). This malespecific expression resembles the expression pattern of Sox9 in other vertebrates including mammals [START_REF] Koopman | Sry, Sox9 and mammalian sex determination[END_REF]. There must be however some interspecific differences because in the G. rugosa frog the expression of Sox9 was detected in both sexes, and the upregulation in the testes was observed two months after metamorphosis when the testes are already fully developed and differentiated [START_REF] Takase | Two Sox9 messenger RNA isoforms: isolation of cDNAs and their expression during gonadal development in the frog Rana rugosa[END_REF].

Sox3 was expressed at low level in the early stages of X. laevis gonad development, however, was highly upregulated in the ovaries at stage NF62 (Fig. 9). Sox12 showed upregulation in the testes at stage NF56 and NF62 (Fig. 9). Paralog Sox18a was upregulated in the testes at stage NF62, while, Sox18b became upregulated earlier, at stage NF53, NF56 and NF62 also exclusively in the testes (Fig. 9). In summary, the upregulation of Sox9a, Sox9b, Sox18a, Sox18b, and Sox12 was characteristic for developing testis, however, Sox3 expression was characteristic for developing ovary. Previously, the expression of Sox3 in adult ovaries and Sox18 in adult testes was described in X. laevis (Hasewaga et al. 2002;[START_REF] Koyano | The Xenopus SOX3 gene expressed in oocytes of early stages[END_REF]. This indicates that for some Sox genes, the pattern of expression established during gonad development persists through the adulthood.

Genes involved in testis development

There is a subpopulation of genes involved in testis development in vertebrates [START_REF] Piprek | Molecular and cellular machinery of gonadal differentiation in mammals[END_REF]. These genes become upregulated in the testes after sex-determining period. Among these genes Pdgf (encoding platelet derived growth factor) is responsible for cell proliferation, migration and testis cord formation in the mouse testis [START_REF] Brennan | Pdgfr-α mediates testis cord organization and fetal Leydig cell development in the XY gonad[END_REF]. We found that the expression of Pdgfa and Pdgfb was upregulated in the developing testis in X. laevis in comparison to the ovaries. Pdgfa was expressed from stage NF53 with upregulation at stage NF62, and Pdgfb was upregulated in the testis later, at stage NF62 (Fig. 11). The gene encoding PDGF receptor (Pdgfrα), was upregulated in the testes starting from stage NF53, and showed visible sex dimorphism especially at stage NF62. These data suggest the involvement of PDGF pathway in the formation of testis structure in anurans.

Desert hedgehog (Dhh) participates in the proliferation/survival of fetal Leydig precursors in the interstitium of the mouse testis and the formation of testis cords (Yao and Capel 2002;Yao et al. 2002). We detected a relatively low level of expression of its two paralogs Dhh-a and Dhh-b in X.laevis testes and ovaries with the higher expression level in the testes. The Dhh-b had the strongest sex dimorphism of expression at stage NF56 and NF62. The Ptch1 (protein patched homolog 1), which encodes DHH receptor, was expressed in the developing X. laevis gonads with a higher level of expression in the testes (Fig. 11).

In mammals, the inhibin B, a member of TGFβ family, is responsible for the formation of proper testis structure and its vasculature [START_REF] Yao | Sexually dimorphic regulation of inhibin beta B in establishing gonadal vasculature in mice[END_REF]). We found that in X. laevis the genes encoding three inhibins were expressed in developing gonads (Fig. 11). Inhibin A gene (Inhba) was expressed both in testes and ovaries and was gradually upregulated in the testes between stage NF56 and NF62 (Fig. 11). Inhibin B gene (Inhbb) was upregulated also in the testes after NF56, while, inhibin C gene (Inhbc) was upregulated in the testes earlier, starting from stage NF53 onward. Another TGFβ member, the anti-Müllerian hormone (AMH) is a factor secreted by developing vertebrate testes, which promotes degeneration of Müllerian ducts (oviducts) in males. We studied the expression of X. laevis ortholog of Amh and found high expression of this gene in the male gonads from stage NF56 onwards (Fig. 11). So far, the upregulation of Amh gene in the developing anuran testes was shown only for S. tropicalis and G. rugosa [START_REF] Jansson | Sex-dependent expression of anti-Müllerian hormone (amh) and amh receptor 2 during sex organ differentiation and characterization of the Müllerian duct development in Xenopus tropicalis[END_REF][START_REF] Kodama | Molecular cloning and characterization of anti-Müllerian hormone (AMH) from the Japanese wrinkled frog, Rana rugosa[END_REF], and in X. laevis using immunolocalization [START_REF] Piprek | Sexual dimorphism of AMH, DMRT1 and RSPO1 localization in the developing gonads of six anuran species[END_REF]. These data suggest AMH involvement in Müllerian ducts regression in amphibian males.

Genes of steroidogenesis and other hormones signaling

Next we focused on the expression pattern of genes involved in hormonal control of gonad development. Among genes encoding receptors of gonadotropins, the folliclestimulating hormone receptor gene (Fshr) was expressed both in testes and ovaries but was significantly upregulated in the testes starting from stage NF53 (Fig. 12). The luteinizing hormone receptor gene (Lhr) did not reveal significant sex differences and was expressed at a very low level in the gonads of both sexes (Fig. 12). The expression of Fshr receptor implies a control of testis development by pituitary FSH hormone. Gene encoding steroidogenic acute regulatory protein (Star) that is responsible for the cholesterol transfer to the mitochondria, was expressed both in testes and ovaries but showed stronger expression in the testes at stage NF62 (Fig. 12). Cyp17a1 encodes cytochrome P450 17A1 that converts progestagens to androgens. We detected significant upregulation of this gene in the testes starting from stage NF53 (Fig. 12). This indicates that there is higher steroidogenic activity in developing testes than in the ovaries. The Cyp19a1 that encodes aromatase responsible for testosterone conversion to estradiol was expressed both in testes and ovaries but was strongly upregulated in ovaries starting from stage NF53. Its expression level in testes was much lower than in ovaries and slightly increased from NF53 onward (Fig. 12). This indicates that estradiol is probably synthesized in the developing ovaries from the onset of their differentiation. The Hsd3b1 encodes 3β-hydroxysteroid dehydrogenase, which is responsible for the synthesis of progesterone. The expression of this gene showed sex-specific differences only at stage NF62 with stronger expression in the testes and decreased expression in the ovaries (Fig. 12). The expression of Srd5a1, which encodes reductase that converts testosterone to dihydrotestosterone, did not show any significant sex dimorphism (Fig. 12). The androgen receptor-coding gene (Ar) was expressed in developing ovaries and testes with strong upregulation in the testes at stage NF62 (Fig. 12). Genes of steroidogenesis were studied also in gonad development in G. rugosa. In this species the upregulation of Cyp17a1 and Ar occurred in developing testes, however, Cyp19a1 was upregulated in the ovaries [START_REF] Iwade | Elevated expression of P450c17 (CYP17) during testicular formation in the frog[END_REF][START_REF] Kato | Expression of P450 aromatase protein in developing and in sex-reversed gonads of the XX/XY type of the frog Rana rugosa[END_REF]Mauro 2008;[START_REF] Yokoyama | Androgen receptor of the frog Rana rugosa: molecular cloning and its characterization[END_REF]. Similar pattern of steroidogenic enzymes expression is also characteristic for other vertebrates such as zebrafish, T. scripta, American alligator, and chicken [START_REF] Western | Temperature-dependent sex determination in the American alligator: expression of SF1, WT1 and DAX1 during gonadogenesis[END_REF][START_REF] Jorgensen | Expression profiles for six zebrafish genes during gonadal sex differentiation[END_REF][START_REF] Matsumoto | Epigenetic control of gonadal aromatase (cyp19a1) in temperature-dependent sex determination of red-eared slider turtles[END_REF][START_REF] Shaikat | Gene expression profiling in embryonic chicken ovary during asymmetric development[END_REF].

Conclusion

Our study indicates that many genes belonging to various pathways are sexspecifically expressed and thus may be involved in the control of gonad development in X. laevis. We showed a significant sexual dimorphism in the expression of several genes. Summary of genes expressed at consecutive stages of gonad development is presented in Fig. 13. The Gata4, Sox9, Dmrt1, Amh, Fgf9, Ptgds, Pdgf, Fshr, and Cyp17a1 showed upregulation of expression in testes, while DM-W, Fst, Foxl2, and Cyp19a1 were upregulated in the ovary. An exact role of most of these genes in X. laevis gonad development remains unknown and further experimental studies with genetic or functional knockouts are necessary to identify the function of these genes.

Figure legends

Fig. 1 .

 1 Fig. 1. A. Diagram of gonad development in Xenopus laevis. At stage NF50 the gonads (arrowheads) are sexually undifferentiated and are composed of few germ cells (g) enclosed by somatic cells. The phase of undifferentiated gonads (between stage NF49and NF53) has a sex determination sub-phase when sex-determining genes are expressed. Sexual differentiation follows this sub-phase. First sex differences are noticeable at NF53 when a medulla (arrow) forms in the centre of developing ovaries. At NF56 the ovaries are easily recognizable by the ovarian cavity (asterisk) present in the medulla, whereas the cavity is absent in the testes. At NF62 oocytes (o) are present in the ovaries, however, at this stage there are no meiotic cells in the testes.

Fig. 2 .

 2 Fig. 2. Graphs of expression ofWt1, Nr5a1, Gata4, Emx2.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Graphs of expression of genes of female sex determination -DM-W, Wnt4, Rspo1, Ctnnb1.

Fig. 5 .

 5 Fig. 5. Immunohistochemistry of RSPO1. Scale bar 25 µm.

Fig. 6 .

 6 Fig. 6. Immunohistochemistry of β-catenin. Scale bar 25 µm.

Fig. 7 .

 7 Fig. 7. Graphs of expression of female sex determination genes: Foxl2, Fst, Bmp4, Dax1.

Fig. 8 .

 8 Fig.8. Graphs of expression of male sex determining genes.

Fig. 9 .

 9 Fig. 9. Graphs of expression of Sox genes.

Fig. 10 .

 10 Fig. 10. Immunohistochemistry of SOX9a. Scale bar 25 µm.

Fig. 11 .

 11 Fig. 11. Graphs of expression of testis differentiation genes.

Fig. 12 .

 12 Fig. 12. Graphs of expression of steroidogenesis genes.

Fig. 13 .

 13 Fig. 13. Diagram of genes expressed at subsequent stages of X. laevis gonad development in both sexes. Sex-specifically upregulated genes are shown in bold.

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  Fig. 1.

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  Fig. 13.

Acknowledgements

The study was conducted within the project no 0372/IP1/2015/73 financed by Polish Ministry of Science and Higher Education in the program Iuventus Plus.