INTRODUCTION & BACKGROUND

F. Dugast & I. Gajda The second season for the Archaeological and Epigraphic Investigations in Tigrai Region (Pre-Aksumite & Askumite Period) took place from 15 November to 15 December 2012 in Saʿesiʿe Tsʿada Emba woreda, north-eastern Tigrai. It is part of a four year research programme, headed by Dr Iwona Gajda and Dr Fabienne Dugast from the CNRS (Umr 8167 "Orient & Méditerranée" / Paris), and supported by the French Ministry of Foreign Affairs (MAEE), the French Centre for Ethiopian Studies (CFEE / Addis Ababa), and the Bureau of Culture and Tourism of the Regional State of Tigrai (BCTRST / Wukro).

It comes as a result of a preliminary survey which took place in March 2010 in agreement with Ato Kebede Amare Belay, General Manager of the Tigrai Culture and Tourism Agency (TCTRST) at Makale, and which has been concentrated on documenting several sites first surveyed by the Ethiopian team in the region of Atsbi-Dera, east Makale (see [START_REF] Dugast | a Report on Fieldwork. First Preliminary Survey, presented to the Federal Democratic Republic of Ethiopia, Ministry of Youth, Sport and Culture[END_REF]. The archaeological site of Wakarida, in the tabia / district of Sawena, and Saʿesiʿe Tsʿada Emba woreda / region, eastern Tigrai (fig. 1), was found out in 2004 by Ato Habtamu Mekonnen from the TCTRST (Makale) and Ato Tekle Hagos from Addis Ababa University (see MEKONNEN 2008). It has been chosen to start systematic archaeological investigations which begun in March 2011 (see DUGAST & GAJDA 2011).

Position

Heart of our project is the kingdom of Aksum: that is when it rose in the early 1 st millennium AD, and mainly in what circumstances. In fact, we do not know anything really about what occurred in the 1 st millennium BC: did any polity exist before or did the local population just live in a kind of autarky, farmers ordered in several familial nuclei connected with each another? Such a question would have been less critical since archaeological remains would not have given any evidence, on several sites, of a same feature artwork, such as monumental inscriptions (see RIE), huge temples (Yeha, Haoulti, Kaskase), sculpture of high quality (Haoulti, Mäqabər Gaʾəwa)... Are they the evidence of a kind of well characterised cultural facies, of Sabaean influence? Or just the evidence of the setting up of merchant groups coming from South Arabia? Correlation is but not clear between the development -whatever short it was -of a community associated to the South-Arabian culture in the 1 st millennium BC and the Aksumite civilisation which came after. Though, both "cultures" seem to have connections, if any, with South Arabia. On the other hand, the main object brings out on cultural transfers, as soon as the 1 st millennium BC, between different civilisations which increased next to the great commercial axes linking India to the Mediterranean world, and their possible conversion or alteration in the 1 st millennium AD. In this very context, every contact that might have existed between the neighbouring communities of Soudan, Egypt, Eritrea, Somalia, and of course the whole of Ethiopia is to be taken into account.

Periodisation of northern Ethiopia's history is from now directly connected to the part this region took in the political and economical, and even cultural development of the civilisations settled either on both sides of the Red Sea or in the whole area stretched out from the Mediterranean world to India. In fact, according to ancient sources, among them the Periplus Maris Erythraei in the very 1 st c. AD (see also DIODORUS OF SICILY, STRABO, PLINY THE ELDER), the highlands of Tigrai seems to have been for long ago one of the committed countries next to the great commercial axes. Localisation: UTM 38 = 14°16'56 ''4 N / 39°43'31''9 E (altitude 2,343 m). Aksumite period (1 st millenium AD) (GoogleEarth / F. Dugast 2012 ©).

Wakarida

In this very context, it looks interesting to focus on the relations between South Arabia and the kingdom of Aksum. That is first of all considering human contacts between two countries which seem to be unconnected because of an impassable zone: the Red Sea. The latter is in fact a great extend of water, about 30 km wide at its southern point (the Gulf of Aden), but more than 350 km between the main Aksumite port of Adoulis / Zula (on the Eritrean side) and the actual ones of Jizān or Maydī (on the Arabian side). It is known to be shaky and unsafe, because of the strong currents and winds which run through from east to west. Though, it seems to be an important communication route, especially in the 1 st century AD, either from South to North, the Mediterranean world to India, or from East to West, the Arabian world to African lands.

On the Ethiopian side, the most important routes were obviously the ones which ran on the western edge of the highlands, from Aksum to the region of Adigrat, and then to Asmara and Adoulis, along on 200 km. On their course there are in fact scattered archaeological sites dated back to the Aksumite period, about 15 in number between Aksum, Hawsien and Matara, continuing from Matara to May Malatse / Asmara in Eritrea (fig. 1). Few earlier ones also existed on the axe Aksum / Matara (ANFRAY 1990; see also [START_REF] Godet E | Répertoire de sites pré-axoumites et axoumites d'Éthiopie du nord : l'Érythrée[END_REF]more recently D'ANDREA et al. 2008).

On the other hand, it seems that several settlements took all the same advantage of the eastern foothills of the highlands. Passes were for sure used to join the Danakil's depression -in the Afar region -, which gave various minerals, and above all salt in abundance, thanks to its lakes, the nearest one Assale, about 60 km to the south; they may have been used also to join the Red Sea, and along its western edge, the main port of Adoulis, some 100-150 km north. One may easily think of specific contacts between the populations living on both sides of the deep: moreover, one may think of commercial and even cultural links coming beyond the ones developed in the surroundings of the metropolis; besides, the settlement of the so called "pre-Aksumite" populations may have overstep such a usual development.

The archaeological site of Wakarida -less than 30 km east of Edaga Hamus and close to Mengela, on the actual eastern border -, may give an example of such a case, because of its remote situation, deeply enclosed in the mountains, though it looks like a crossroads which might have open towards the East, the Afar region and the Red Sea. The architectural remains tell in fact about a kind of built-up area, which appears to be coherent, as a more or less planned town can be. Furthermore, the artefacts are very similar to those we know on other sites, either in Tigrai or in its vicinity. This assemblage gives hence an opening set of evidence of a political and economic activity: was Wakarida an "advanced" economic area -a kind of "check point" -, or was it just a small urban settlement of local economy? And mostly when did it first occur?

Running & team

Our project is based on the collaboration of specialists of several domains. In 2011, geophysicists and archaeologists as well as a surveyor were part of the field work. In 2012, geographers joined the team.

It involved Ethiopian scholars: Yohannes Gebre Sellassie is part of the mission since the very beginning, and Tekle Hagos since 2011, even though they could not participate to fieldwork this year. And of course, we were helped by the French Centre for Ethiopian Studies and ARCCH (Addis Ababa), and BCTRST (Wukro), as well as the Tabia's administrator and officials from Sawena, and the local people who were very cooperative and concerned with the investigations.

The investigations were directed by Dr Iwona Gajda and Dr Fabienne Dugast, from the Centre national de la recherche scientifique (CNRS) in Paris, and team members included this year:

• Dr Xavier Peixoto, France (archaeology),

• Dr Julien Charbonnier, France / London (archaeology),

• Dr Sabina Antonini, Italy (archaeology),

• Dr Anne Benoist, France (ceramics),

• Cécile Verdellet, France (graphic design),

• Dr Olivier Barge, France (SIG & survey),

• Emmanuelle Régagnon, France (cartography & survey),

• Dr Bruno Marcolongo, Italy (geomorphology & survey),

• Xavier Crapery, France (mapping),

• Haylay Teklay, Wukro (interpreter, survey),

• Mulugeta Abegaz, Addis Ababa (official representative ARCCH),

• Tamachache Fitur & Simeneh Bacha, Addis Ababa, CFEE (drivers),

• Sisay Getachew & Godana Yohannes, Addis Ababa, CFEE (cookers),

• Hailu Abera & Woldu Hagos, Wakarida (guardians),

• 30 workmen, Wakarida (excavation),

• Aregash Woldu & Selam Hagos, Wakarida (washing).

I. FIELD OBJECTIVES

F. Dugast & I. Gajda
The archaeological site of Wakarida (formerly known as Aribara) is situated in the eastern region, about 70 km north Wukro, 2 hours trail from Edaga Hamus to Mengela, east and close to Sawena, 14°16'56"4 N / 39°43'31"9 E (altitude 2,343 m). It is dependent on Saʿesiʿe Tsʿada Emba administrative woreda, in the tabia of Sawena.

The site has been established on a rocky outcrop, which overlooks a large valley surrounded by chain of mountains, respectively from north to east and south to west: Daima, Afedadae, Dagaraebe, and Arebata (fig. 2). From now on, the place is made up of cultivated lands and farm houses, which belong to several heads of family.

Implementation

The site is completely new: none of the sources know it, and, inevitably scientific literature ignores it. It was discovered by chance by one of the local head family, Ato Hagos, who reported to the TCTRST when surveying the region of Adigrat to identify the regional State of Tigrai's patrimony (see MEKONNEN 2008). The architectural remains were then cleared out by the local people to be reused in building their own houses and walls for terrace cultivation.

The site is concerned with two main objectives, as far as its remains are related to: its remote location, while open in the direction of the Red Sea, may help in considering the relations, whatever they were, between North Ethiopia and South Arabia in the early 1 st century; on the other hand, according to the chronological sequences, it may give us sufficient elements to make out chronological markers -especially in pottery typology -which are still lacking.

 The first season objectives & supply (March 2011)

A preliminary investigation took place in 2011 (see DUGAST & GAJDA 2011): the main tasks were then to initiate archaeological excavations and research at Wakarida to be proceeded in the following years. In this purpose, this first season made a geophysical survey on the site, as well as test excavations, and initiated a provisional typology of the pottery.

Before any extensive excavation, our priority was in fact to estimate and evaluate the archaeological potential of the site, its area and organization. Specific fieldwork objectives were to implement a sampling design which employed geomantic techniques including GPS (Global Positioning System) and GIS (Geographic Information System) (see DUGAST & GAJDA 2011: II), to initiate a more systematic survey which employed specific techniques including geophysical methods. This point has been achieved thanks to an electromagnetic survey which consisted on electrodes resistivity and reflectometry measurements -taking into account the nature of sediment (see DUGAST & GAJDA 2011: III).

Besides, three test excavations consisting on soundings have been opened on top of the hill as well as on the eastern and northern parts of it, one of which near a cleared out building (see DUGAST & GAJDA 2011: IV). The structures unearthed on the eastern border of the outcrop -square D2 -show a plain resemblance with Aksumite architectural techniques. The walls are of stacking work, steadily scattered with levelling courses of large stone slabs, which bring to mind the architectural techniques used either at the so called "Dongur Palace" near Aksum or at Matara (see ANFRAY 1963a & 1974).

A few pedestrian surveys have also been carried out around the site, in order to recognize the real expansion of the archaeological area -which comes to be larger than first expected -, and even other settlements nearby (see DUGAST & GAJDA 2011: IV).

As "Pre-Aksumite" and Aksumite artefacts are still not well known, the fieldwork objectives were furthermore to implement a sampling of each type of ceramics in order to produce a new typology with, as far as possible, chronological references. A systematic collection of artefacts with a systematic register and stratigraphic localization has been initiated and developed into the basis of a preliminary typological study and chronological markers (see DUGAST & GAJDA 2011: V).

 Prerequisite to the second season (2012)

Together with the technical data the first season provided (2011), several implements and analysis were started to insure a plain efficiency of the next seasons to be involved:

-the complete register of the archaeological data -stratigraphic units (SU), pottery typology -has been set up thanks to a geo-referenced cartographic medium connected to several items and using ArcGIS software (O. Barge & E. Régagnon, CNRS, Archéorient / France); it has to undertake either the archaeological data from the site itself or the ones from the different field investigations, among them the geophysical survey, which tasks are to look over the territory's organisation and the communication routes, as well as the natural environment and its agricultural exploitation;

-a preliminary analysis of the natural environment, thanks to a satellite image from Digital Globe 2011, pointing out geomorphologic elements, either on the site itself or its surrounding (B. Marcolongo, CNR, IRPI / Italy).

Working hypothesis

The collection makes sure that Wakarida was a small town, established since the Aksumite period, but probably earlier. Even if no chronological connection has been yet insured between the different areas which were investigated in 2011, we know that they materialize an assemblage of housing which one may consider as a kind of quite significant built-up area which appears to be coherent, extended on almost 10 ha. Furthermore, the architectural technique the building at the eastern edge of the outcrop shows (D2 square) put forward the evidence of a public edifice, if not a monumental one, maybe the "castle" of the leader of an organised community. Besides, pottery and coinage would confirm such a hypothesis since they show a kind of a political and at least economic activity which seems to be in connection with the rise of the kingdom of Aksum.

In this very context, the site of Wakarida may enter an argument on the common establishment of communities in this actual area in connection with the commercial and exchange network on a large scale, and hence on the involvement of this kind of setting up in the development of the kingdom of Aksum.

The main task of the second season tried hence to first identify the site of Wakarida in its connection with the development of the Aksumite civilization, possibly in an earlier time; that is the origin of settlement and the reason of it. In this very sight, it focuses on the spatial organization of the site and its area depending on the chronology (well-defined excavations, stratigraphic analysis, material collection), as well as on an environmental approach that aims at giving form to a logical model of spatial setting up depending on two combined main lines: geographic and chronologic (geomorphologic analysis, archaeological survey).

II. THE NATURAL ENVIRONMENT & ITS EXPLOITATION

B. Marcolongo, O. Barge & E. Régagnon

The outcrop of Wakarida overlooks a rich and fertile valley of more than 400 ha to the south-east. The fertility of this valley does not seem to be recent, nor does its exploitation. It is obviously part of the development of the site itself: it seems difficult in fact not to expect a straight connection between the agrarian landscapes as it actually appears, their exploitation system, and the vicinity of a built-up area.

Two main lines were hence to be considered: the archaeological one as well as physiographic and geomorphologic settings, in order to have an environmental approach of the territory in its complete viewing. The main task was to study the interactions between men and physical milieu, as well as the variations of the use of the soils -from the alluvial plain in its natural structure to the terraced hillsides as plots of land-planning to facilitate their exploitation, involving important modifications of the ground of entropic origin.

Geomorphologic study

B. Marcolongo

The main task of the geomorphologic approach was to understand the relation between the archaeological site of Wakarida and its surrounding landscape and supplies, so as to be able to figure out, on a smaller scale, the process of the ancient settlement conception. In such a mean it was first important to focus on the connection that might have existed between men and natural supplies through time, taking into account the physiographic, geomorphologic and geologic description of the whole area.

 Implementation

Before any field survey though, a preliminary analysis had to be carried on -at the Consiglio nazionale delle ricerche (CNR), Istituto di ricerche per la protezione idrologica (IRPI / Padua / Italy) -on a very high resolution satellite image, black & white PAN ortho ready standard image, covering 100 sq. km precisely fixed on Wakarida (European Space Imaging/EUSI from Digital Globe, 14°17'12,92 N / 39°43'27,45 E, scene n. 10200100130CCD00). The analysis was based on geometrical and radiometrical aspects (fig. 345).

 Preliminary interpretation on satellite image

Generally speaking, the area covered by the satellite image gives a complex drainage pattern strongly controlled by tectonics. Three different drainage basins flow respectively to the NW, the NEE and the SE. Tectonics lineaments are mainly affecting the NE portion of the area, where main trends keep to faults and fractures systems with NE / SW and NW / SE directions.

This peculiar feature explains the tortuous course of Tabena Wadi, whose tributary is Wakarida valley, called by local people May Ayni -"Water Source" -, stretching upstream SW / NE, then NE / E, and turning definitely to SE and the Danakil depression. Wakarida valley itself is formed along a clear fault line SE / NW, prolonging into the other drainage basin developed to the east of its water divide. A "derivative" fault -a parallel one -is that controlling the second floor valley elongated in the same direction about 1 km south of Wakarida valley, named Kaʾebile.

Both valley floors have no visible "talweg", and only subsurface run off and/or groundwater flow are present, collecting rainfall during the monsoon season and feeding depressed area at their bottom, especially at their confluence with May Weyni and Tabena Wadi.

The two sub-basins have a roughly rectangular shape and a strong asymmetrical transversal profile. Actually, Wakarida valley covers approximately 8 sq. km surface with a south-western slope much more extended and steeper than the north-eastern one.

Sparse patches of variable dimensions of terracing (in "leopard skin spots" manner) are recognizable on the transitional plateau of Dayma range, flanking to the north Wakarida valley, and having a more gentle and regular slope to the north-east. Other limited areas of terraces are found along the steep and narrow valley of Tabena Wadi upstream of Sewane village, especially at lateral tributaries confluences. Consistent terracing forms are not developed in the other wide portions of the three drainage basins, where "dissection" pattern is absolutely prevailing and marking a highly incised ravines landscape.

 DTM analysis & contour lines anomalies

Beside this preliminary physiographic-morphologic interpretation of the satellite image, a local study has been performed on the area of Wakarida site, by superimposing a window of the georeferenced image to the contour lines model surveyed the previous year. The objective was to identify anomalies of the contour lines, which could indicate the possible presence of underground structures and archaeological evidences.

The analysis was based on a description of geomorphometric parameters and on a spatial analysis. A restitution of the site of Wakarida in digital 3D format could be useful to find potential anomalies in the elevation trend likely to hide archaeological evidences.

The followed procedure came through the following steps (fig. It clearly emerges from this analysis an elongated dorsal feature stretching SE / NW on top of Wakarida's outcrop -where an excavation area has been opened (zone 2, below). The axe is asymmetric: the natural slope facing SW, over the road to Sawena (Sawna), is steeper than those to SE and NE.

Most of the anomalies have been controlled during the field check: around north-eastern area (zone 1) and south-south-eastern one (zone 2), confirming the presence of anthropic structures buried at shallow depth.

 Few observations on a "fossil landscape"

The local landscape looks like a "fossil" one principally subjected to erosion, ever contrasted during time by men through a patient work of slope and floor valley terracing. Such a fine environment management is so diffused in Wakarida valley and the other parallel valley floor of Kaʾebile, that only long activity over centuries and centuries, performed by numerous populations, could give reason of it. It was obviously either to prevent alluvial-colluvial cover transportation or, in the mean time, to facilitate infiltration, percolation and storage of ground water, essential element for vegetation cover growth and human purposes.

These stable and favourable environmental conditions surely attracted human communities for agricultural purpose since the very past, resulting in a "fossil landscape". It may not be surprising though more ancient sites were occupied again with new settlements.

Archaeological survey

O. Barge & E. Régagnon

The archaeological survey around Wakarida was first concentrated on its vicinity, and then gradually extended to the whole valley. Preliminary investigations allowed giving greater place to a few areas instead of others. A description of the landscape as it first appears may give some reason of such a choice.

 Wakarida today: environment, milieu, territory

The site of Wakarida stands in a valley situated on a depressed point of the western edge of the highlands of Tigrai, in an intermediate position between the latter and the great valleys which plunge down to the East and the Danakil depression. This topographic unit makes a kind of headland (fig. 8).

 General aspect of the land

Everything in this area makes the qualification of the environment as completely manmade, in real contrast with the great eastern valleys (fig. 9): manmade as almost every slope, since it is not too much sharp, is converted to agricultural purpose; manmade as well as flow of water is controlled in such a way that erosion is nearly absent.

Low walls are built at regular distance at right angles to every hillside, in order to break up the flow and to drive the alluvium behind the walls. As much as possible, this type of arrangement is converted into terrace cultivation, even if the so manmade section is of modest size. On the steepest slopes grow small shrubs and scattered trees used as pasture. Even there, low walls are built along the contour lines, up to the top of the hill (fig. 10).

Every bowl is covered by such an arrangement, which changes the natural feature of the complete landscape. The relief itself is manmade and the environment appears to be very steady. In two points only, similar in their topographic position, water comes to the surface: downhill, in May Ayni valley, and in the confluence with May Weini valley, at Demba Bales locality. The valley narrows in both places, and turn into a small canyon, the mouth of which gives way to a sill where water pours to the surface. A well has been arranged in both cases: water is then directed to a canal connected to secondary waterways controlled by a gate.

From this point, the arrangement changes again: from the central point where the canal is, the low walls are built crosswise and in staggered rows, materializing a kind of network looking uphill, in order to irrigate the terrace cultivation (fig. 11).

 Circumstances of development & exploitation

As it is, the actual environment tells about generations of men who transmitted one another the knowledge of the natural system and of the manmade practices to control it. On the other hand, according to their complexity, transmission of the arrangements that have completely modelled the landscape from generation to generation is an absolute necessity. As a result, the natural system, as it actually works in a net of interactions between human communities and the environment, is to be considered in the long-lasting.

As regards to our periods, two hypotheses may be committed:

-the modelling and the actual environment came later on: the environmental conditions would have then been absolutely natural, the description of which is very difficult since water flow, vegetal cover, and maybe the climate itself might have been very different;

-the milieu was exactly the same as the one we actually know: the landscape would hence be a fossil one, set up by ancient populations maybe more important in number, the successive generations just keeping an achieved equilibrium.

Even so, the actual environment, in its efficiency, has been set up some time: it may be in the same time as the remains of Wakarida, perhaps earlier or later on, its length being also unknown.

In this very context, it seems interesting to detail the actual milieu, as on one hand it is essentially used and sheltered by a rural population, maintaining obviously traditional practices; on the other hand it may be the same milieu and the same system set up in ancient time, the one in which it has been "made up".

• Preliminary issues

First of all, remains of ancient settlements were surveyed at the foothills' outcrops, near the valley bottom. Whenever ancient settlements would have been fixed in the fields, they would have been completely buried, or even destroyed. As it is, no pottery shards have been collected in the valley bottom itself. On the other hand, the actual domestic settlements built on the outcrops may also have covered ancient ones, except pottery shards tell about it. Even the ridges have been investigated, following basically the watershed's limits of May Ayni valley. Such a course allowed looking over the general aspects of the milieu and understanding the topographic feature of the valley.

Survey area has been systematically registered and mapped with a GPS receiver. Since the team included two to four individuals a day, it may be counted as a 50 m wide contour from both sides of the watershed line, in order to give an easy mapping of it (fig. 12).

Artefact scatters' areas have been outlined and numbered. Pottery samples have been generated from each area: the diversity of the potteries may in fact allow identifying in each area one or more cultural facies -according to the issues of the ceramic analysis. Each architectural structure has also been registered, outlined and named (building, grave, quarry, water tank, other); walls of visible ancient structure have been marked as a line shape and qualified (height, width, masonry).

The objectives were divided into three central themes:

 Setting & site formation process

As regards to ancient periods, no hydrological or agricultural arrangement has been noticed. According to the manmade environment, it has not to be much surprising, since the actual arrangements recover often if not always previous ones, especially as regards to the ones related to water flow. Nonetheless, it is obvious that the latter have been converted to actual arrangements such as the watering places which location has been preserved anyway: Wakarida is for instance only 250 m far from a well.

The actual condition of the archaeological survey might confirm that ancient settlements were at a very similar position to the actual ones (fig. 13). Pottery shards have been found in fact on the outcrops and the foothills' sides. Few obsidian fragments, some of them visibly hewn, have also been collected.

The whole of the archaeological evidence has been recognized all over the valley, around Wakarida, but none upstream: this location has to be confirmed, though it might reveal a concentration of ancient settlements mostly down in the valley. On the ridge, the passes seem to have a symbolic position. Graves have been found at several places, as well as a peculiar arrangement, yet difficult to identify since it looks like a podium of about 10 m square. A right angle wall has been also noticed located on a pass, where the population says to be an ancient church. A mosque has been just built up on the main pass between Kaʾebile and May Ayni valley near an important and current cemetery.

 Funerary structures

Several graves have been surveyed but not excavated, which allow few hypotheses according to their shape and situation.

A first type of graves can be defined by their circular shape, of 1 to 2 m in diameter, mostly 1,50 m, surrounded by sandstone blocks about 60 x 40 x 40 cm embedded in the ground (fig. 14).

There is no special filling up inside the circle but soil; no pottery shards either. This type seems to be scattered all over the valley, mainly located on the passes or outcrops. They are very similar to those of the Bronze Age fairly known in Arabia. A second type may be identified as overhanging slabs enclosed into the cliffs (fig. 15). Since the geologic stratum shows a few degrees slope, few of these graves look like small outcrops. The slab is used as a roof covering graves arranged underneath, where holes are dig out to put the bodies in. Front of the tomb is carefully closed with a masonry of well hewn sandstones or schist slabs. As regards to the size of the masonry, the tomb may cover one are more individuals. Two assemblages of these have been documented, the number of which in both area allows thinking about a real necropolis. Their situation, looking over important water springs near the site of Wakarida, has to be noticed.

A third type of graves can be defined by their rectangular shape (fig. 16). They seem to be recent, not to say modern, since some of them are part of actual cemeteries. They look like small platforms about 2 x 1 m in size, and 0,30 m in high, made of gray schist slabs of a very neat feature. In the middle of the platform, two or three slabs about 0,40 m in high are erected at regular distance from each another.

 Communication routes

May Ayni valley has a west/north-west direction, flowing into Tabina valley which is perpendicular and tributary of one of the great tectonic lineament moving down to the East. Its direction is hence opposite to the one of the hydrographic system. Besides, the ridges which surround the basin are mostly high, in order that May Ayni looks like a dell set high up in the mountain, and turning its back on the Danakil depression, though Kibi Daga pass open to a valley flowing down to the East (fig. 8).

In this system, commercial routes are difficult to take in for questioning. On the other hand, the theoretical itineraries -which are based on a model of a walker moving according to the topography -show that the position of the site of Wakarida is obviously not on a "natural" route which might join Aksum to the Red Sea. The shorter and more practicable ones come far away the site; and even the nearest ones on the edge of the plateau come through another way than May Ayni valley to reach the Red Sea (fig. 17).

It does not mean though Wakarida was not on a commercial route. The "natural" trails may have in fact been controlled for geo-politic reasons, the "real" ones drawn within other considerations.

• Conclusions

At first sight, since the site of Wakarida seems far from the main commercial axes, it appears to be settled in the middle of a field land type rather than of a network one. Its exploitation is the result of a long process which combined human activity and environment system, in a durable feature. Such a development might have been possible since no conflict ever occurred as no defensive remains has been noticed anywhere.

The archaeological survey will have to be completed in connection with sedimentary sequences analysis to confirm these first hypotheses.

III. ARCHAEOLOGICAL EXCAVATIONS

X. Peixoto, J. Charbonnier & S. Antonini

Following the previous season and its test excavations, the archaeological investigations focused on the general organisation of the site of Wakarida itself. Since it appears to be a small town, two sectors have been opened, their choice being based on the previous results and the geomorphologic analysis (see II.1 above). The main objective was to study these areas in their morphology and feature, and their evolution (fig. 18).

Fig. 18 -Topography of the site of Wakarida & location of the excavated areas (X. Craperi 2011-2012 ©).

Considering the great quantity of artefacts removed from the previous excavations, priority has been made on an extensive study (zone 1) as well as on a deep sounding one (zone 2), in order to put together the chronological markers (see IV below).

D2 square area (zone 1)

X. Peixoto

The previous test excavation in D2 square, located on the north-eastern slope of the outcrop (see DUGAST & GAJDA 2011: 18-19) has been followed and either extended. As regards the very first results (see ibidem: 24-25), this area appears to enable evidencing a chronological margin of the ancient and effective settlement, if not a complete sequence of chronological references, which dates might be given as the pottery study will go along (see IV below).

• D2 square's main construction

The main construction in D2 square has been examined in more details in order to establish the date of its building-up as well as its feature and possible evolution.

Levelling courses set up with slabs of schist every 6 to 10 cm in high, giving a kind of stepped batter to the wall, had yet been noticed in 2011 on the main façade to the south-west (see DUGAST & GAJDA 2011: 18). Since they are very likely to the ones known at Aksum, e.g. at the so called "Palace of Dungur", or at Matara, at the "Central Monument" [START_REF] Anfray F | a "Campagnes de fouilles à Matarā près de Sénafé[END_REF]: 93-95, & ANFRAY & ANNEQUIN 1965; see MUNRO-HAY 1989: 159-164), it makes the edifice dated back to the Aksumite period (fig. 19). (Anfray 1963, pl. LXVIIIb).

The building is made up of four rooms, three of them having being rebuilt and reused as water tank by the local population. Only the fourth one, on the north-western portion of the building, could have been cleaned up in order to study it. In the middle of the room was a stonework made-up pillar (see MEKONNEN 2008), the only south-eastern angle of which is actually preserved. Its bottom, now constituted of soil and stones mixed together, has been directly based on the bedrock and was of a square section about 2,50 m (fig. 20). Few pottery shards have been collected nearby. At the south-eastern angle of the room, a wall segment of a very different construction has been noticed, integrated in the Aksumite building: since the room has been cleared out by the local population up to the bedrock, no connection could be identified.

• Two more buildings on the west side

The excavation has been extended on the south-western slope and west to D2 square building.

An angled lane runs along the latter's south-western and north-western main façade, on the other side of which are other walls of same feature (fig. 21). On the south-western side, a second building (Bt 1), composed of three or more adjoining rooms (loci 2, 3 & 4) faces the main construction; on the north side, a third one (Bt 2) has been partly uncovered, consisting of more than two rooms (loci 5 & 6). Both of these buildings are of common stone masonry. The eastern façade of the second one (Bt 1) shows the same levelling courses set up with slabs of schist. All over the surface was the occupational debris made up of alluvium mixed with stones, which may be identified as fill layers and wall collapse that accumulated over and in-between the remains of walls. This layer has been removed in the lane up to a clay surface where potteries have been broken by the destruction of the walls.

A kind of retaining wall seems to have been first set up on the bedrock, in the north-west/southeast direction, before constructing Bt 2. The separating walls would have been built afterwards. Then, a fill layer has been spread out in order to level the rooms' surface, between the walls.

Loci 2 and 3 from Bt 1 have been partly excavated. In locus 2, a hard-packed surface with gravel covers up the levelling fill layer spread out on the bedrock; there was hardly any artefact. In locus 3, the same hard-packed surface with gravel gave abundant potteries in their initial position, and for most of them, in their complete form, only crushed by the debris.

Under this occupational surface, crushing schist and soil may constitute a prepared fill layer or maybe come from architectural elements and coating which have been destroyed. This layer contains also abundant potteries, some of them being almost in their complete form. A third layer, of much more alluvium, has not yet been removed but gives already other complete potteries. Locus 4 has not been removed either, but has an entry made up of a three steps stairway, running down along the wall, from the door to the lane (fig. 22).

Bt 2 has been partly excavated. In locus 6, debris layer had more than 1 m depth and covered a clay surface with potteries in their original position. The south-western angle of the room has been destroyed by recent arrangements; no entry has been found yet. Conversely, locus 5 opens to the north-west on an elongated area, maybe a second path in a north-east/south-west direction, parallel to the first one in its northern part. A second zone has been opened on top of the outcrop, where few walls came out on the surface and part of a building has been unearthed by the local population (see DUGAST & GAJDA 2011: 16-17). Besides, at this very place, contour lines show a strong anomaly which might indicate accumulating sediment if not a debris layer (see above II.1).

Fig. 21 -Zone 2, from above (O. Barge & E. Régagnon 2012 ©).

• A kind of Aksumite architecture

A quite 10 x 15 m surface has been first opened in which a stone made building has been uncovered (fig. 23). Its main façade shows the same levelling courses set up with slabs of schist, as the ones seen in zone 1, on the eastern side of the outcrop. The outer wall of the building draws alternatively a wall plane set back and in projection, in the same way Fr. Anfray describes it at Matara on the "Central Monument" as a distinctive feature known in the Aksumite architecture (ANFRAY 1963: 102). The building counts two rows of three rooms, maybe a third one on the south-eastern side, not yet excavated.

The whole of the building was covered with a thick layer of collapsed stones. In one of the rooms, the occupational level under the layer debris is being excavated, on which a complete pottery has been found.

• A second occupational level

Two test excavations have been opened in order to determine the thickness of the sediment. In the first one, on the north-eastern border of zone 2, a more ancient level has been found, based on the bed rock and recovered by the foundation fill of the main building (fig. 22).

The second one went along the south-western façade of the same building through its foundation which trench was filled with pottery shards, charcoal and bones. Two graves were set up nearby, their chronological connection with the building being undetermined (fig. 23). A pearl of molten glass and Millefiori type has been found next to one of the graves (fig. 24).

Remains of a urban settlement

Since the excavations just start, no complete description of the remains is actually possible. Though, the latter are to be definitely identified as domestic settlements where part of Wakarida's population might have lived in the Aksumite period. Next to Bt 1 (zone 1), other walls come out on the surface, on the same north-west/south-east axe, along with the contour lines. Other lanes as well, parallel and at right angles to the uncovered ones, seem to draw a regular and orthogonal system.

On the outcrop too (zone 2), other walls come out on the surface in a fairly identical building line, though located 100 m to the west. Both settlements appear hence to be strictly organised, maybe arranged according to orthogonal circulation axes, and related to two different and dense districts.

Besides, the architectural elements -stone masonry of a neat feature, regular levelling courses set up with slabs of schist, wall planes alternatively set back and in projection -allow thinking about an architectural unit all over the outcrop, which comes through the Aksumite period, and which is almost related, since now, to a wealthy population (Matara, Aksum). Each building will have to be examined to understand its position and meaning within the site itself.

Charcoal samples have been taken from both areas to be analysed in order to have a 14 C dating (in hand). Besides, few copper coins have been found on the top surface, two of them from an Anonymous king reigning in the end of the 4 th century, another one from King Ioel reigning in the mid-6 th century (see MUNRO-HAY 1995: respectively 76 & 131), the latter giving hence the date of the latest antique occupation (fig. 24) (see IV below). Anonymous king, late 4 th c.

King Ioel, mid-6 th c.

IV. POTTERY ANALYSIS

A. Benoist & C. Verdellet
The identification of the ceramics of the highlands of Tigrai is still imperfectly defined. No real differentiation is made about productions, either at a local scale, or at the whole region of the highlands', even not at the importation one. Moreover, no real question has ever been asked about neither method of manufacture nor fabric, though it seems to proceed from a similar way even today [START_REF] Roux | Poteries éthiopiennes," Éthiopie aujourd'hui[END_REF]. The main task of a pottery analysis is hence to start a typology based on stratigraphic and technical elements, which the site of Wakarida seems to unable giving the opportunity. Since any analysis is hindered by a lack of previous systematic research on the ceramics of this region, the classification was made first blindly, without any comparison with published typologies and collections (see below).

Method of analysis

Pottery shards were observed with the naked eye, in the day light. Samples consist of shards gathered either in 2011 field season, from surface collection and test excavations, or in 2012 field season from the two excavated areas. The former ones, which comprise 6 types according to their fabric (see BUFFA, in DUGAST & GAJDA 2011: 20-24), have been hence completely re-examined according to the new classification.

A close selection was made among surface collection pottery, and typical forms only were drawn and photographed. All the ceramics from the two excavated areas and controlled stratigraphy were sorted out and posted in order to start a layout study according to the categories. Forms have been systematically registered and listed, some of them drawn and photographed.

Classification process followed two stages: first the clay has been examined, according to its texture, its temper and its forming. Secondly, surface finish and decoration of the different pots have been distinguished and classified according to their shape.

Final typology proceeds consequently in only three successive levels. Several types of ware may hence show equivalence in surface finish, decoration and form. Few special types were then selected according to a same kind of clay and decoration and making an assemblage of different forms. Since they are easily identified, these types will have to be dated back and hence will be used as chronological markers.

Classification

• Cat. 1 -Fine red ware, thinly tempered

Fabric of fine red ware is an orange, red, or light red paste, but often grey in section, with either small to tiny mineral inclusions (less than 1 mm thick) of a red or grey colour, or white inclusions, mostly sharp and matt. It sometimes has a thin and bright sandy temper, but in short supply. Clay is fairly hard with little blunt cracks, sometimes irregular with thin grooves visible inside the section. Pots are handmade, sometimes completed on a slow wheel.

• Cat. 2 -Fine red ware, with invisible temper

Another fine red ware has an orange, red paste, occasionally grey core, but thinner and harder than the former one, without any visible temper. It can have now and then very thin and slightly bright sand inclusions, in very short supply.

 Finish surface and decoration

Fine red ware (cat. 1 & 2) shows three different types of coating: without any slip (surface A); with a red or brown slip, a matt or corroded finishing in such a way that no finish surface is visible (surface B); with a red or brown slip, either burnished, polished or dry smoothed (surface C).

The main forms of the fine red ware are cups with slim everted rim, small pots with ovoid body and slim everted rim, bowls with convex wall, rounded base and simple slim rim.

• Cat. 3 -Common red ware

Fabric of common red ware is a pinkish, orange to light yellow paste, often grey in section, with many white mineral inclusions mostly faulted, as well as quartz inclusions, sometimes associated with red or grey sharp and matt inclusions. Its temper is usually made of thin and bright sand.

Thickness and size of the temper change from fine (0,5 cm or more) to thick and sometimes coarse paste. Inclusions are 1 to 2 mm thick, scarcely more. Few ceramics are partly burned by using them for cooking, and change in colour from red to dark brown. Pots are handmade, sometimes completed on a slow wheel, since the walls are uniform. Surface is mostly smoothed, either with the hand or a pebble.

The main forms of the common red ware are big necked jars with rounded or smoothed rim, a handle on the shoulder, and huge dishes with slim everted rim. Few containers have incised decorations on top of the rim. The inside wall of low plates, a ladle and a rectangular plate set up on three feet (footwasher?) coming from zone 1 have incised and impressed decorations made up with a triangular stamp (fig. 25). Common red ware has been broadly collected on the surface and zone 1, but more occasionally in zone 2, and at the deepest level.

• Cat. 4 -Dark polished ware

Fabric of dark polished ware is a thin grey or brown paste, with a mineral temper made up of grey or dark matt inclusions, white inclusions of quartz, and micaceous speck. Surface has a dark polished or burnished slip.

Collection of dark polished ware is limited, for the most part on top surface and upper level in zone 1. A complete pot has been collected in 2011; its main forms are small jars with globular body and narrow neck, bowls with convex wall, rounded base, and simple slim rim, and cups with slim everted rim (fig. 26).

• Cat. 5 -Common red ware, with bright temper

Common red ware with bright temper is an alternative common red ware (cat. 3), having abundant and bright inclusions, and a soapy surface.

The main forms are huge dishes with slim everted rim. Most of them have been collected in zone 1, in top levels.

• Cat. 6 -Dark-grey ware, tempered with steatite

Fabric of dark grey ware is a very compact clay, dark-grey paste, sometimes brown, with lots of mineral inclusions grey in colour, probably steatite or soft volcanic rock of a similar type (tacl, chlorite). Pots are handmade, the walls of which are mostly irregular, showing marks of finger pressure.

The main forms are cooking pots, with vertical handle under the rim, and sometimes a coarse incised decoration. It has been collected on the top surface in 2011, but is more frequent in zone 2, especially near the building on its southeastern side, and in the superficial levels (fig. 27).

• Cat. 7 -Fine ware, with painted and incised decoration

Fabric of fine ware with decoration is a very pure orange paste, hard and compact, without any apparent temper but a polished surface, and fine incised bands and arcs on the external wall. Some of the incised patterns are enhanced with red or black painting (fig. 28). Fine ware with painted and incised decoration has been occasionaly collected in zone 1, but is wholly absent in zone 2.

• Cat. 8 -Fine and soft red ware, with incised decoration Fabric of fine and soft red ware is an orange to brown paste, often grey in section, mostly thin, with a fine mineral temper of white inclusions and bright sand, very similar to the fine red ware one (cat. 1), and to few of the common one, but the clay seems to be more crumbly. Its surface is mostly coarse, without any slip, and partly worn.

The main forms are small jars with vertical neck, few cooking pots with rounded body and two small knobs on the shoulder. A decoration consisting of incised bands and triangles filled by simple or crossing oblique incisions are present on the top surface (fig. 29).

This type has been collected for the most part in zone 1, in the upper levels, corresponding to 10% of the assemblage. It occurs also on the top surface, in zone 2.

• Amphorae

Few shards of ridged amphorae have been collected all over the site on the top surface and in zone 2. Its fabric is a very thick paste, green in colour, set up on a wheel, with small white and crumbly inclusions and a fine vegetal temper. Slip on the surface is white and thick (fig. 30).

No shape has been yet collected: it is hence difficult to find any information relating to its origin, but the shards are very similar to the ones collected at Matara and may be identified as the well known amphores cotelées used in Mediterranean trade.

• Other ceramics

Two more shards have been collected on the top surface, the decoration of which consists of thin straight lines under the rim, and narrow and vertical red bands painted near the base. Another shard has a black and waving painted decoration associated to an incised oblique one. These shards have been related by Vittoria Buffa to Aksum painted ware (see BUFFA, in DUGAST & GAJDA 2011: 65, type 6).

A grey shard has also been found on the top surface the decoration of which consists of vertical striations, flat on top (decoration G), evoking the vertical striations of Aksum red ware (see MUNRO-HAY 1989: fig. 16-36, 37, 73, 74, 79, 88-90).

Chronology & corresponding

Ware from Wakarida consists of an original assemblage, which seems not to have similar features with other assemblages already published.

Among the fine red ware forms (cat. 1), cups with slim and spread lips, sometimes decorated with two incised lines, as well as small pots with everted rim, have been occasionally collected on other sites as Aksum (see MUNRO-HAY 1989: fig. 16-355, 360, 361, 363) or Matara (see ANFRAY 1963a: pl. LXXXVII / 12). Bowls with spread lips and geometric patterns from Matara refer to few rims from Wakarida (see ANFRAY 1963a: pl. XCII / 6, 8).

Among the common red ware (cat. 3) and the dark polished ware (cat. 4), bowls with convex body have been collected also at Aksum (see MUNRO-HAY 1989: fig. 16-399, 401).

Common red ware (cat. 3) give few parallels with red Aksumite ware: dishes with everted rim, and jars with short neck and handle on the shoulder are to be related with two items from Aksum (see MUNRO-HAY 1989: fig. 16-232 & 16-227).

The incised plate set on three feet refers exactly to items found at Aksum in 3 rd and 4 th centuries' levels (ibidem: fig. 16- 197, 198-200), and are identified as footwasher by Chittick and Munro-Hay, since a complete item is set inside a basin (ibid.: fig. 16-197). No complete items but shards have been found at Matara (see ANFRAY 1963a: pl. XCII / 5, 7): in spite of its specific use, it might have been kept on for a long time.

Red Aksumite ware has usually lots of Christian marks on the surface: mostly cross (see MUNRO-HAY 1989: fig. 16-394), and also fish (ibidem: fig. 16-213). None of these have been collected in the excavated levels; only one shard with red polished slip and crosses under the rim has been found on the top surface, near an ancient wall at SAZ 14 point, 500 m north-east from the site, on a foothills' outcrop (fig. 31). A kind of "brasero" in common red ware with red slip has been found as well near another wall part of a building, 1,5 km north-west from the site by local population (see Anfray 1966: 8, pl. XV) (fig. 32). Its form refers to incense burner coming from Mäqabər Gaʾəwa's temple near Wukro Dark-grey ware tempered with steatite (cat. 6) might refer to late dark-grey ware from Aksum as it is described by Chittick and Munro-Hay as the result of "an undisciplined and technical inferior way" and "the loss of the craft skills" in late Aksumite period, after the removal of Aksum (see MUNRO-HAY 1989: 301-303 & fig. 16-409). It only occurs in the later and top levels and on the surface.

Fine and soft red ware (cat. 8) refers to few potteries from Aksum in their shape and decoration (see MUNRO-HAY 1989: 301-303)

Conclusion

Wakarida's pottery shows actually a quite homogeneous assemblage, which partly refers to other assemblages collected on the great Aksumite sites, as they are known by currently published studies. Though, some differences occur, whether the chronology is slightly different, or the local cultural context is slightly affected by great cultural movements which go on all over the highlands and around.

Two well-defined collections have been set up: an ancient one with most of the common red ware, and a later one which only occurs in zone 2 in the upper levels. The confirmation of this analysis may allow recognizing two successive settlements, the dating of which cannot be definitively fixed at the moment. A comparison between Wakarida's dark-grey ware tempered with steatite and the one from the latest levels at Aksum gives though a possible terminus ante quem related to the removal of Aksum which is commonly placed in the 8 th , maybe 10 th century.

Unless the coins give an earlier date of the latest antique occupation, 14 C dating will enable to mark out a more precise chronology (see III above).

PRELIMINARY ISSUES

F. Dugast & I. Gajda At this stage of the 2012 field season, few preliminary issues may be put forward. First of all, the previous hypotheses seem to be easily reinforced:

-geomorphologic analysis supports the idea of a controlled development of the region, for a long time ago;

-archaeological survey reinforces this idea, since ancient remains have been observed in both May Ayni and Kaʾebile valley, next to the site of Wakarida, part of these remains being obviously earlier than the ones actually excavated at Wakarida; -excavations reinforce the idea of a build-up area, dated back to the 4 th -6 th century, the planning of which has to be better understood, but already indicates a kind of important place.

These issues allow taking out the reason why this kind of remote territory could have been developed. Abundant resources, mainly of the soil, may be sufficient to explain such human settlements. Though, even if the region seems to have been first arranged for agricultural purpose, the site of Wakarida shows the setting up of a town, as small as it may be, which indicates the presence of if not wealthy and more or less numerous people, at least a hierarchically structured population. As it is, the development of such a remote town may obviously be in connection with the kingdom of Aksum. Architecture refers in fact to what occurred at that time in the kingdom and its cities, Aksum and Matara, even if commercial trails are not plainly identifiable; even if potteries do not seem to indicate any commercial trade as one may usually understand it -but few Aksumite coins have been collected. On the other hand, the evidence of earlier settlements allows thinking about a previous organisation to consider as a "first round".

Few more issues may come forward next to a careful examination of these new elements. They will have to be completed with other investigations to be undertaken in the following years: archaeological survey has to take a more precise course according to the first issues; excavations have to be continued in order to make clear the occupation levels; buildings' mapping will be essential in order to identify them; pottery classification has to be continued and supported by 14 C analysis in order to fix the chronology.

Fig. 1 -

 1 Fig. 1 -Wakarida, in Saʿesiʿe Tsʿada Emba woreda, Tigrai region (Ethiopia). Localisation: UTM 38 = 14°16'56''4 N / 39°43'31''9 E (altitude 2,343 m). Aksumite period (1 st millenium AD) (GoogleEarth / F. Dugast 2012 ©).

Fig. 2 -

 2 Fig. 2 -The archaeological site of Wakarida and its surrounding (F. Dugast 2011 ©).

Fig. 3 -

 3 Fig. 3 -The archaeological site of Wakarida and its surrounding (satellite image, Digital Globe 2011 ©).

Fig. 4 -

 4 Fig. 4 -Selection of the portion of EUSI high resolution imagecovering a 100 sq. km area around Wakarida.

Fig. 5 -

 5 Fig. 5 -Fifth title R2C2 window centred on Wakarida site (Digital Globe 2011 ©).

  up and modelling DTM (Digital Terrain Model) under ESRI ArcGIS software, and based on benchmarks, contour lines and topographic parameters, already gathered on 2011 field season; 2/ identification of potential anomalies based on comparison criteria of contour lines trend: SA CA1 CA2 simple anomaly complex anomaly, type 1 complex anomaly, type 2 3/ DEM modelling (Digital Elevation Model) in successive phases -colour, contour lines, grey shadow -based on 3D analysis; 4/ DTM creation of DTM-derivative like slope and aspect map.

Fig. 6 -

 6 Fig. 6 -Successive phases of the process leading to DTM creation and its derivative (B. Marcolongo 2012 ©).

Fig. 7 -

 7 Fig. 7 -Aspect and slope map derived from the DTM, showing respectively the azimuth orientation of the different terrain surfaces and their slope values in percentage (B. Marcolongo 2012 ©).

Fig. 8 -

 8 Fig. 8 -The archaeological site of Wakarida: localisation in May Ayni valley (O. Barge & E. Régagnon 2012 ©).

Fig. 9 -

 9 Fig. 9-10 -May Ayni valley and its landscape (O. Barge & E. Régagnon 2012 ©). arranged with terrace cultivation and low walls in order to break up the flow.

Fig. 11 -

 11 Fig. 11 -Localisation of a well, (Digital Globe ©; O. Barge & E. Régagnon 2012 ©) 250 m far from Wakarida, and new arrangement of the terrace cultivation.

Fig. 12 -

 12 Fig. 12 -General survey map (O. Barge & E. Régagnon 2012 ©).

Fig. 13 -

 13 Fig. 13 -Arrangements layout near the site of Wakarida; insert, the necropolis (O. Barge & E. Régagnon 2012 ©).

  Fig. 14-16 -Three types of tomb: circular, overhanging slabs, and rectangular (E. Régagnon 2012 ©).

Fig-

  Fig. 17 -Theoretical itineraries (O. Barge & E. Régagnon 2012 ©). -Trails from Aksum to different points located on the Red Sea border; -Trails from the Red Sea to few localities settled in the highlands.

Fig. 19 -

 19 Fig. 19 -The main construction in D2 square & the "Central Monument" at Matara (F. Dugast 2011 ©)(Anfray 1963, pl. LXVIIIb).

Fig. 20 -

 20 Fig. 20 -The stonework made-up pillar in 2008 & after the clearing up by the local population (H. Mekonnen 2008 ©) (X. Peixoto 2012 ©).

Fig. 21 -

 21 Fig. 21 -Zone 1, from south-east, being excavated. Bt 1 consisting of three adjoining rooms (at left hand) faces the main building (at right hand), the lane in the middle (X. Peixoto 2012 ©).

Fig. 22 -

 22 Fig. 22 -Zone 1. The stairway running up from the lane to locus 4 (X. Peixoto 2012 ©).

Fig. 22 -

 22 Fig. 22 -Zone 2, test excavation Fig. 23 -Zone 2, test excavation along the southon north-east border uncovering western façade of the main building, a more ancient wall based on the and one of the two graves beside bed rock (J. Charbonnier 2012 ©). (J. Charbonnier 2012 ©).

Fig. 23 -

 23 Fig. 23 -The molten glass pearl (A. Benoist 2012 ©).

Fig. 24 -

 24 Fig. 24 -Coins from the top surface (C. Verdellet ©).

Fig. 25 -

 25 Fig. 25 -Pot with everted rim and plate on three feet in common red ware (C. Verdellet ©).

Fig. 26 -

 26 Fig. 26 -Bowl and small jar with straight neck in dark polished ware (C. Verdellet ©).

Fig. 27 -

 27 Fig. 27 -Dark grey ware, tempered with inclusions of steatite (C. Verdellet ©).

Fig. 28 -

 28 Fig. 28 -Bowl in fine ware, with painted and incised decoration (C. Verdellet ©).

Fig. 29 -

 29 Fig. 29 -Small jar with knobs in fine and soft red ware (C. Verdellet ©).

Fig. 30 -

 30 Fig. 30 -"Amphores cotelées" shards (C. Verdellet ©).

(

  see WOLF & NOWOTNICK 2010: fig. 9).

  : in dark polished ware from ES V(11) (fig. 16-436), and dark-grey ware handmade from the latest levels (fig. 16-409).

Fig. 31 -

 31 Fig. 31 -Fine red ware with red burnished slip and two crosses, found at point SAZ 14 (C. Verdellet ©).

Fig. 32 -

 32 Fig. 32 -Brasero found on surface at point Cn 15 (C. Verdellet ©).

On top of the rocky outcrop (zone 2) J. Charbonnier & S. Antonini


Benoit Dehapiot

Guillaume Halet
email: guillaume.halet@univ-rennes1.fr

Ran GTPase promotes oocyte polarization by regulating ERM (Ezrin/Radixin/Moesin) inactivation

Keywords: oocyte, meiosis, polarity, GTPase, actin, chromatin GV, germinal vesicle, GVBD, germinal vesicle breakdown, MI, metaphase I, MII, metaphase II

Asymmetric meiotic divisions in mammalian oocytes are driven by the eccentric positioning of the spindle, along with a dramatic reorganization of the overlying cortex, including a loss of microvilli and formation of a thick actin cap. Actin polarization relies on a Ran-GTP gradient centered on metaphase chromosomes, however the downstream signaling cascade is not completely understood. In a recent study, we have shown that Ran promotes actin cap formation via the polarized activation of Cdc42.

The related GTPase Rac is also activated in a polarized fashion in the oocyte cortex, and co-localizes with active Cdc42. In other cells, microvilli collapse can be triggered by inactivation of the ERM (Ezrin/Radixin/Moesin) family of actin-membrane crosslinkers, under the control of Rac. Accordingly, we show here that Ran-GTP promotes a substantial loss of phosphorylated ERMs in the cortex overlying the spindle, in mouse oocytes. However, this polarized phospho-ERM exclusion zone was unaffected by Rac or Cdc42 inhibition. Therefore, we suggest that Ran activates two distinct pathways, to regulate actin cap formation and microvilli disassembly in the polarized cortex of mouse oocytes. The possibility of a crosstalk between Rho GTPase and ERM signaling, and a role for ERM inactivation in promoting cortical actin dynamics, are also discussed.

Introduction

In mammalian female meiosis, successful haploidization of the maternal genome is achieved via two successive asymmetric divisions, resulting in the formation of a large oocyte, and two small polar bodies which will eventually degenerate. The small size of the polar bodies ensures that minimal amount of maternal organelles and macromolecules (eg mRNAs, proteins, mitochondria) are lost in the process. [START_REF] Brunet | Positioning to get out of meiosis: the asymmetry of division[END_REF][START_REF] Li | The road to maturation: somatic cell interaction and self-organization of the mammalian oocyte[END_REF] Early embryo viability is thus highly dependent on the establishment of oocyte asymmetry. [START_REF] Brunet | Positioning to get out of meiosis: the asymmetry of division[END_REF][START_REF] Li | The road to maturation: somatic cell interaction and self-organization of the mammalian oocyte[END_REF] The key-mechanism underlying the asymmetry of oocyte meiotic divisions is the positioning of the meiotic spindle in the vicinity of the cortex. [START_REF] Azoury | Spindle positioning in mouse oocytes relies on a dynamic meshwork of actin filaments[END_REF][START_REF] Schuh | A new model for asymmetric spindle positioning in mouse oocytes[END_REF][START_REF] Chaigne | Spindle positioning in mammalian oocytes[END_REF] In meiosis I, the spindle forms in the center of the oocyte, then moves slowly towards the cortex. At the time of homologous chromosome segregation (metaphase I-anaphase I transition), the spindle has reached a subcortical location, resulting in an asymmetric division and the formation of a small-sized first polar body. This is promptly followed by the formation of the metaphase II spindle, which remains localized in the vicinity of the cortex (Fig. 1). At fertilization, meiosis II resumes and the activated oocyte emits a small-sized second polar body, containing one set of segregated chromatids.

In both meiosis I and II, the asymmetric positioning of the meiotic spindle is accompanied by a range of polarization events, ultimately resulting in the differentiation of a polar body-forming region around the spindle. Thus, cortical granules, which are released upon fertilization to induce the zona-block to polyspermy, redistribute away from the spindle area, resulting in the formation of the so-called cortical granule-free domain (Fig. 1). [START_REF] Connors | Involvement of the cytoskeleton in the movement of cortical granules during oocyte maturation, and cortical granule anchoring in mouse eggs[END_REF] Another emblematic feature of oocyte polarization is the formation of the "actin cap", a thick layer of actin filaments that accumulates in the cortex overlying the spindle (Fig. 1). [START_REF] Longo | Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus[END_REF][START_REF] Maro | Mechanism of polar body formation in the mouse oocyte: an interaction between the chromosomes, the cytoskeleton and the plasma membrane[END_REF][START_REF] Van Blerkom | Regulation of development in the fully grown mouse oocyte: chromosomemediated temporal and spatial differentiation of the cytoplasm and plasma membrane[END_REF] Interestingly, actin caps are maintained over the chromatin clusters that remain in close apposition with the cortex, after destruction of spindle microtubules with nocodazole. [START_REF] Longo | Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus[END_REF][START_REF] Maro | Mechanism of polar body formation in the mouse oocyte: an interaction between the chromosomes, the cytoskeleton and the plasma membrane[END_REF][START_REF] Van Blerkom | Regulation of development in the fully grown mouse oocyte: chromosomemediated temporal and spatial differentiation of the cytoplasm and plasma membrane[END_REF] These observations have led to the concept that oocyte chromatin generates a signal capable of remodeling, from a distance, the nearby cortex. This signal was identified as a gradient of active Ran GTPase (Ran-GTP), which exchange factor RCC1 (Regulator of Chromosome Condensation 1) binds chromatin. [START_REF] Deng | The Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes[END_REF][START_REF] Dumont | A centriole-and RanGTPindependent spindle assembly pathway in meiosis I of vertebrate oocytes[END_REF][START_REF] Hao | Regulation of chromatin binding by a conformational switch in the tail of the Ran exchange factor RCC1[END_REF] Ran signaling was shown to promote polarized accumulation of the N-WASP-Arp2/3 machinery in the cortex overlying the spindle, thereby driving the formation of the polarized actin cap. [START_REF] Yi | Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes[END_REF] Recent studies based on live imaging of mouse oocytes, have revealed that actin filaments flow continuously away from the cortical cap. This actin flow generates cytoplasmic streaming, which is suggested to apply a directional pushing force on the spindle, thereby maintaining its off-center positioning. [START_REF] Yi | Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes[END_REF][START_REF] Yi | Actin cytoskeleton in cell polarity and asymmetric division during mouse oocyte maturation[END_REF][START_REF] Yi | Sequential actin-based pushing forces drive meiosis I chromosome migration and symmetry breaking in oocytes[END_REF] In a recent study, we have added a new protagonist, the small GTPase Cdc42, in this signaling cascade. Using a fluorescent reporter for GTP-bound Cdc42, we have shown that Cdc42 is activated in a polarized fashion in the cortex overlying the meiotic spindle, and drives the cortical accumulation of N-WASP and the formation of the actin cap in mouse oocytes. [START_REF] Dehapiot | Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes[END_REF] Furthermore, we have shown that this polarized activation of Cdc42 requires the proximity of meiotic chromatin, and Ran-GTP signaling. [START_REF] Dehapiot | Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes[END_REF] We therefore proposed the following signaling cascade for actin polarization in oocytes (Fig. 1):

Chromatin → Ran-GTP → Cdc42-GTP → N-WASP-Arp2/3 → Actin cap
Our data also suggested that the Ran/Cdc42/N-WASP signaling cascade is conserved during anaphase, promoting the formation of a thick actin layer in the cortex of the polar bodies. [START_REF] Dehapiot | Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes[END_REF] Inhibition of Cdc42 signaling resulted in a complete failure of second polar body protrusion in activated MII oocytes, associated with a relocalization of N-WASP to the cytosol. [START_REF] Dehapiot | Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes[END_REF] Our current view on these findings is that actin filaments generated via the polarized Cdc42/N-WASP pathway may provide the necessary pushing force to deform the cortex and drive polar body protrusion, in a manner similar to actin-driven leading edge extension in motile cells. [START_REF] Condeelis | Life at the leading edge: the formation of cell protrusions[END_REF][START_REF] Pollard | Cellular motility driven by assembly and disassembly of actin filaments[END_REF] In a previous study, we presented evidence for a similar polarized activation of the small GTPase Rac in the cortex of mouse oocytes, [START_REF] Halet | Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes[END_REF] also under the control of the Ran-GTP gradient (G.Halet, unpublished observations). At present, it is unclear whether Rac and Cdc42 play complementary or redundant roles in oocyte polarization. Our recent investigations suggest however that Cdc42 is the major determinant of N-WASP localization in the mouse oocyte cortex. [START_REF] Dehapiot | Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes[END_REF] Another major polarization feature is the loss of membrane microvilli in the polarized cortex (Fig. 1). [START_REF] Longo | Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus[END_REF][START_REF] Maro | Mechanism of polar body formation in the mouse oocyte: an interaction between the chromosomes, the cytoskeleton and the plasma membrane[END_REF] Microvilli provide binding and fusion sites for the fertilizing sperm, [START_REF] Yanagimachi | Sperm-egg association in mammals[END_REF][START_REF] Runge | Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution[END_REF][START_REF] Jégou | CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization[END_REF] and the rationale for their localized disassembly may be to avoid sperm fusion in the vicinity of the spindle, which could otherwise interfere with spindle function and/or lead to the untimely extrusion of paternal chromatids into the second polar body. [START_REF] Luo | Fyn kinase activity is required for normal organization and functional polarity of the mouse oocyte cortex[END_REF] The assembly and maintenance of membrane microvilli require proteins of the ERM (Ezrin, Radixin, Moesin) family, which act as membrane-cytoskeleton crosslinkers. [START_REF] Yonemura | Direct involvement of Ezrin/Radixin/Moesin (ERM)-binding membrane proteins in the organization of microvilli in collaboration with activated ERM proteins[END_REF][START_REF] Fehon | Organizing the cell cortex: the role of ERM proteins[END_REF] ERMs interact with integral membrane proteins via their N-terminal FERM (4.1-Ezrin-Radixin-Moesin) domain, and with underlying actin filaments via their C-terminus. In their inactive state, ERMs exhibit a "closed" conformation, where the N-and C-termini establish an intramolecular or intermolecular association, resulting in mutual inhibition and retention of the protein in the cytosol, as a dormant pool. According to the current model, ERM activation requires the initial binding to the phosphoinositide PIP2 via the FERM domain, facilitating the subsequent phosphorylation of a conserved threonine residue at the C-terminus (T567 in ezrin, T564 in radixin, T558 in moesin). [START_REF] Fehon | Organizing the cell cortex: the role of ERM proteins[END_REF][START_REF] Fievet | Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin[END_REF] This phosphorylation stabilizes the active conformation of ERMs (hereafter collectively referred to as phospho-ERMs) and promotes their crosslinking activity.

Mouse oocytes express all three ERM family members, and seem to be particularly enriched in radixin. [START_REF] Louvet | Ezrin becomes restricted to outer cells following asymmetrical division in the preimplantation mouse embryo[END_REF][START_REF] Larson | Cortical mechanics and meiosis II completion in mammalian oocytes are mediated by myosin-II and Ezrin-Radixin-Moesin (ERM) proteins[END_REF] Consistent with a role in microvilli formation, phospho-ERMs are enriched in the oocyte cortex, except for the amicrovillar region overlying the MII spindle. [START_REF] Larson | Cortical mechanics and meiosis II completion in mammalian oocytes are mediated by myosin-II and Ezrin-Radixin-Moesin (ERM) proteins[END_REF] These observations suggest that ERM dephosphorylation may be the trigger for microvilli disassembly in the polarized cortex of oocytes, as seen in other cells. [START_REF] Brown | Chemokine stimulation of human peripheral blood T lymphocytes induces rapid dephosphorylation of ERM proteins, which facilitates loss of microvilli and polarization[END_REF][START_REF] Chen | Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury[END_REF][START_REF] Kondo | ERM (Ezrin-Radixin-Moesin)based molecular mechanism of microvillar breakdown at an early stage of apoptosis[END_REF][START_REF] Nijhara | Rac1 mediates collapse of microvilli on chemokine-activated T lymphocytes[END_REF] Interestingly, Rac or Cdc42 activation were shown to promote ERM dephosphorylation and collapse of microvilli in T cells. [START_REF] Nijhara | Rac1 mediates collapse of microvilli on chemokine-activated T lymphocytes[END_REF][START_REF] Faure | ERM proteins regulate cytoskeleton relaxation promoting T cell-APC conjugation[END_REF][START_REF] Rossy | Ezrin/moesin in motile Walker 256 carcinosarcoma cells: signal-dependent relocalization and role in migration[END_REF][START_REF] Cernuda-Morollón | Rac activation by the T-cell receptor inhibits T cell migration[END_REF] Thus, one attractive hypothesis is that ERM dephosphorylation in oocytes is triggered by the polarized activation of Rac and/or Cdc42, under the control of the Ran-GTP gradient.

Here, we investigated further the mechanism of mouse oocyte polarization by asking two questions: 1-Does the polarized dephosphorylation of ERM rely on the chromatin-centered Ran-GTP gradient ? and 2-Is the polarized phospho-ERM exclusion a downstream event of Rac/Cdc42 activation ?

Results and Discussion

Polarization of the mouse oocyte cortex is induced by the proximity of meiotic chromatin, resulting in a graded polarizing response as the distance between the spindle and the cortex shortens. [START_REF] Deng | The Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes[END_REF][START_REF] Dehapiot | Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes[END_REF] To test whether this is also true for ERM activation, we examined the pattern of cortical ERM activation at successive stages of oocyte meiosis, using immuno-labeling against phospho-ERMs. [START_REF] Larson | Cortical mechanics and meiosis II completion in mammalian oocytes are mediated by myosin-II and Ezrin-Radixin-Moesin (ERM) proteins[END_REF] In agreement with a previous report, [START_REF] Larson | Cortical mechanics and meiosis II completion in mammalian oocytes are mediated by myosin-II and Ezrin-Radixin-Moesin (ERM) proteins[END_REF] phospho-ERMs and actin filament staining showed a homogenous distribution over the cortex of prophase I-arrested GV oocytes (Fig. 2Ai), consistent with the presence of numerous actin-rich microvilli. Metaphase-I oocytes with a centrally-located spindle (five hours post-germinal vesicle breakdown/GVBD) exhibited a similar distribution of activated ERMs and actin filaments all over the cortex (Fig. 2Aii). In contrast, at a later stage of metaphase I (GVBD + 7h), when the spindle apparatus exhibits an eccentric position, the phospho-ERM signal was depleted in the cortical area overlying the spindle (Fig. 2Aiii, arrow), along with the appearance of a cortical actin cap. In MII oocytes, phospho-ERMs were detected in the microvillar cortex but remained strongly excluded from the actin-rich polarized cortex overlying the MII spindle (Fig. 2Aiv, arrow). These observations suggest that the meiotic apparatus generates a signal capable of inducing, from a distance, ERM dephosphorylation in the nearby cortex, coincident with the formation of the actin cap. To investigate a possible contribution of spindle microtubules in cortical ERM inactivation, MII oocytes were treated with the microtubule-poison nocodazole, which results in the formation of chromatin clusters closely apposed to the cortex. [START_REF] Longo | Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus[END_REF][START_REF] Maro | Mechanism of polar body formation in the mouse oocyte: an interaction between the chromosomes, the cytoskeleton and the plasma membrane[END_REF][START_REF] Van Blerkom | Regulation of development in the fully grown mouse oocyte: chromosomemediated temporal and spatial differentiation of the cytoplasm and plasma membrane[END_REF] As shown in Figure 2B, each of the nocodazoleinduced chromatin clusters was associated with a localized depletion of phospho-ERM labeling, and a reciprocal increase in actin filament polymerization. Thus, similar to the polarization of actin filaments, N-WASP and activated Rac and Cdc42, [START_REF] Longo | Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus[END_REF][START_REF] Maro | Mechanism of polar body formation in the mouse oocyte: an interaction between the chromosomes, the cytoskeleton and the plasma membrane[END_REF][START_REF] Van Blerkom | Regulation of development in the fully grown mouse oocyte: chromosomemediated temporal and spatial differentiation of the cytoplasm and plasma membrane[END_REF][START_REF] Deng | The Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes[END_REF][START_REF] Dehapiot | Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes[END_REF][START_REF] Halet | Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes[END_REF] ERM dephosphorylation is driven by the proximity of meiotic chromatin, in a microtubule-independent manner.

We next examined whether chromatin-induced ERM dephosphorylation was mediated by the Ran-GTP gradient emanating from meiotic chromosomes. MII oocytes treated with nocodazole were injected with cRNA encoding RanT24N, which acts as a dominant-negative version of the GTPase. [START_REF] Deng | The Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes[END_REF][START_REF] Dumont | A centriole-and RanGTPindependent spindle assembly pathway in meiosis I of vertebrate oocytes[END_REF] Previous work from our and other groups has shown that the Ran-GTP gradient promotes activation of the Cdc42/N-WASP/Arp2/3 cascade to assemble the polarized actin cap. [START_REF] Yi | Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes[END_REF][START_REF] Dehapiot | Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes[END_REF] Therefore, disappearance of the polarized actin cap can be used as a validation of effective inhibition of Ran signaling. Interestingly, in addition to the loss of the polarized actin cap, oocytes expressing RanT24N exhibited a uniform distribution of phospho-ERMs at the cortex, including the regions overlying the chromatin clusters (Fig. 2C, right panel). These findings suggest that Ran signaling is necessary for oocyte chromatin to induce ERM dephosphorylation in the nearby cortex.

There is increasing evidence linking Rac activation to ERM dephosphorylation and microvilli collapse in other cells. [START_REF] Nijhara | Rac1 mediates collapse of microvilli on chemokine-activated T lymphocytes[END_REF][START_REF] Faure | ERM proteins regulate cytoskeleton relaxation promoting T cell-APC conjugation[END_REF][START_REF] Rossy | Ezrin/moesin in motile Walker 256 carcinosarcoma cells: signal-dependent relocalization and role in migration[END_REF][START_REF] Cernuda-Morollón | Rac activation by the T-cell receptor inhibits T cell migration[END_REF] Since our previous findings indicate that Rac, and the related GTPase Cdc42, are activated in the cortex overlying oocyte chromatin, [START_REF] Dehapiot | Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes[END_REF][START_REF] Halet | Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes[END_REF] we tested if the signaling cascade culminating in ERM dephosphorylation in the polarized cortex involved Rac or Cdc42 activation. MII oocytes were injected with mRNA encoding dominant negative RacT17N [START_REF] Halet | Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes[END_REF] or Cdc42T17N, [START_REF] Dehapiot | Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes[END_REF] and examined for phospho-ERM and actin filament distribution. Fluorescence profiles were generated along the perimeter of the oocytes, to better visualize the changes in fluorescence intensity corresponding to the actin cap and the phospho-ERM exclusion zone. MII oocytes injected with water were used as a control for the polarized phospho-ERM exclusion (Fig. 3A), while oocytes expressing RanT24N were used as a control for effective inhibition of the phospho-ERM exclusion (Fig. 3D). In oocytes expressing RacT17N, the actin cap overlying the MII spindle was preserved, and the polarized inactivation of ERMs remained also unaffected (Fig. 3B). Consistent with our recently published work [START_REF] Dehapiot | Polarized Cdc42 activation promotes polar body protrusion and asymmetric division in mouse oocytes[END_REF] , Cdc42T17N prevented the assembly of the polarized actin cap in the cortex overlying the MII chromosomes; however the polarized exclusion of phospho-ERMs was still observed (Fig. 3C). In contrast, overexpression of RanT24N induced a substantial recovery of phospho-ERM staining in the cortex overlying the MII chromosomes, in conjunction with the expected loss of the actin cap (Fig. 3D). We also tested a constitutively-active Rac mutant, Rac1Q61L, which was reported to trigger substantial ERM dephosphorylation and microvilli collapse in other cells. [START_REF] Nijhara | Rac1 mediates collapse of microvilli on chemokine-activated T lymphocytes[END_REF][START_REF] Faure | ERM proteins regulate cytoskeleton relaxation promoting T cell-APC conjugation[END_REF][START_REF] Rossy | Ezrin/moesin in motile Walker 256 carcinosarcoma cells: signal-dependent relocalization and role in migration[END_REF][START_REF] Cernuda-Morollón | Rac activation by the T-cell receptor inhibits T cell migration[END_REF] However, MII oocytes expressing EGFP-Rac1Q61L exhibited a phospho-ERM exclusion zone similar to control oocytes (data not shown).

From the above data, we conclude that the signaling pathway leading to chromatin-induced ERM inactivation in the oocyte cortex, is independent of Rac or Cdc42 activation, and therefore may represent a separate Ran-dependent signaling cascade (Fig. 4, scenario 1). The mechanism and signaling intermediates leading to polarized ERM inactivation remain to be identified. One possibility is that localized ERM inactivation occurs consecutively to a depletion of the phosphoinositide PIP2, as shown in chemokine-stimulated lymphocytes. [START_REF] Hao | Phospholipase C-mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane[END_REF] However, we do not favor this hypothesis, since we have shown previously that PIP2 could still be detected in the membrane region overlying the MII spindle. [START_REF] Halet | The dynamics of plasma membrane PtdIns(4,5)P 2 at fertilization of mouse eggs[END_REF] In other cells, ERM phosphorylation is typically counteracted by calyculin A-sensitive phosphatases of the PP1/PP2a families. [START_REF] Brown | Chemokine stimulation of human peripheral blood T lymphocytes induces rapid dephosphorylation of ERM proteins, which facilitates loss of microvilli and polarization[END_REF][START_REF] Kondo | ERM (Ezrin-Radixin-Moesin)based molecular mechanism of microvillar breakdown at an early stage of apoptosis[END_REF][START_REF] Nijhara | Rac1 mediates collapse of microvilli on chemokine-activated T lymphocytes[END_REF][START_REF] Faure | ERM proteins regulate cytoskeleton relaxation promoting T cell-APC conjugation[END_REF][START_REF] Roubinet | Molecular networks linked by moesin drive remodeling of the cell cortex during mitosis[END_REF] Likewise, treatment of oocytes with calyculin A (50 nM for 15 min) prevented the development of the phospho-ERM exclusion zone in the polarized cortex (unpublished observations). This finding suggests that ERM activation in oocytes is dynamically regulated by a balance of kinase and phosphatase activities. Further investigations are needed to find out whether Ran-GTP regulates the kinase activity, the phosphatase activity, or both.

As an alternative to the dual cascade hypothesis, we can also envisage that ERM dephosphorylation lies upstream of the polarized activation of Rac/Cdc42 and actin cap formation (Fig. 4, scenario 2). For instance, through their direct interaction with Rho guanine nucleotide dissociation inhibitor/RhoGDI, phospho-ERMs have been shown to promote the activation of Rho, [START_REF] Takahashi | Direct interaction of the Rho GDP dissociation inhibitor with Ezrin/Radixin/Moesin initiates the activation of the Rho small G protein[END_REF][START_REF] Hamada | Crystallization and preliminary crystallographic studies of RhoGDI in complex with the radixin FERM domain[END_REF] which is notoriously antagonistic to Rac/Cdc42. [START_REF] Sander | Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior[END_REF][START_REF] Etienne-Manneville | Rho GTPases in cell biology[END_REF][START_REF] Vaughan | Control of local Rho GTPase crosstalk by Abr[END_REF] According to this model, a localized dephosphorylation of ERMs would downregulate Rho activity at the cortex, thus promoting polarized Rac/Cdc42 activation. Consistent with this idea, a recent study has shown that radixin depletion promotes Rac activation and cell spreading in cancer cells. [START_REF] Valderrama | Radixin regulates cell migration and cell-cell adhesion through Rac1[END_REF] Beyond the formation of an amicrovillar region unfavorable for sperm fusion, ERM inactivation could play additional roles in cortical reorganisation and polar body protrusion. Thus, ERM dephosphorylation, by releasing actin filaments from their anchoring to the overlying membrane, could enable the continuous cortical actin flow that powers cytoplasmic streaming. [START_REF] Yi | Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-complex-driven cytoplasmic streaming in mouse oocytes[END_REF] In addition, the polarized inactivation of ERMs may facilitate rapid actin-driven membrane deformation during polar body emission, by locally reducing the cortical tension which would otherwise oppose protrusion. [START_REF] Raucher | Cell spreading and lamellipodial extension rate is regulated by membrane tension[END_REF] Consistent with this idea, increasing membrane tension by expression of constitutively active ERMs prevents membrane protrusion in polarizing T cells. [START_REF] Brown | Chemokine stimulation of human peripheral blood T lymphocytes induces rapid dephosphorylation of ERM proteins, which facilitates loss of microvilli and polarization[END_REF]46 In the same way, the overactivation of moesin, by increasing cortical rigidity, interferes with polar relaxation and anaphase elongation in mitotic Drosophila cells, resulting in cytokinesis defects. [START_REF] Roubinet | Molecular networks linked by moesin drive remodeling of the cell cortex during mitosis[END_REF]47 Further investigations to test these possibilities will provide a better picture of the mammalian oocyte polarization mechanism.

Materials and Methods

Oocyte recovery and treatments: All experimental procedures were approved by the local ethics committee (CREEA, Rennes, France). OF-1 mice (8-10 week-old; Charles River) were injected with 7-10 IU PMSG (Sigma, France) for priming, followed 44-48 hours later by 5-7.5 IU hCG (Sigma) to induce ovulation. Germinal vesicle (GV)-stage oocytes were collected from antral follicles and maintained in prophase arrest by supplementing the culture medium (M2 or M16, Sigma) with 250 µM dibutyryl-cAMP (Sigma). To observe metaphase I progression, oocytes were released from the GV arrest by washing off the dibutyryl-cAMP, and cultured in M16 medium in a 5% CO2 incubator. Metaphase II (MII) oocytes were recovered from the oviducts and denuded of the surrounding cumulus cells by incubation with 300 µg/ml hyaluronidase (Sigma) in M2 medium, followed by wash. For nocodazole treatment, MII oocytes were incubated in M2 medium containing 2 µM nocodazole (Calbiochem) for 1.5 h.

Expression of RacT17N, Cdc42T17N, Rac1Q61L and RanT24N: Plasmids encoding Cdc42T17N in pRK5-myc (subsequently subcloned into pcDNA3.1) and EGFP-Rac1Q61L in pcDNA3 were obtained from Gary Bokoch via Addgene (plasmids 12973, 12981). RacT17N in pcDNA3.1 was described previously. [START_REF] Halet | Rac activity is polarized and regulates meiotic spindle stability and anchoring in mammalian oocytes[END_REF] RanT24N in pcDNA3.1 was a generous gift from Ben Margolis. After plasmid linearization, cRNAs were prepared in vitro using the mMessage mMachine T7 kit (Life Technologies) and pressure-injected in MII oocytes. Oocytes were allowed to express the mutant GTPase constructs for 3 hours before fixation and processing for immunostaining. Control oocytes were injected with an equivalent amount of water.

Immuno-labeling: Oocytes were fixed in paraformaldehyde (3.7% in PBS) for 30 minutes and permeabilized with Triton X100 (0.25% in PBS) for 20 minutes. After a 3-hour incubation in a block solution consisting of 3% BSA in PBS, oocytes were incubated overnight, at 4°C, with a rabbit monoclonal antibody that recognizes ERMs phosphorylated on the critical C-terminal threonine residue (#3149, Cell Signaling Technology). After three washes, oocytes were incubated with a secondary antibody (Alexa Fluor 488 Goat anti-rabbit IgG, Life Technologies) for 1 hour at 37°C.

Staining of actin filaments and chromatin:

To label actin filaments, permeabilized oocytes were incubated for five minutes in PBS containing Alexa Fluor 546 Phalloidin (5 units/ml; Life Technologies), followed by wash. Chromatin was labeled with To Pro-3 (5 µM; Life technologies).

Confocal imaging and image processing:

  Oocytes were placed on glass-bottom dishes (MatTek, Ashland, MA, USA) and imaged using a Leica SP5 confocal microscope equipped with 488, 561 and 633-nm laser lines. Images were processed with ImageJ. All confocal images depicted are representative of at least 20 similar observations. Fluorescence intensity profiles along the oocyte perimeter were generated using the segmented line and plot profile functions in ImageJ. The thickness of the line was 3.5 µm. 46. Liu Y, Belkina NV, Park C, Nambiar R, Loughhead SM, Patino-Lopez G et al. Constitutively active ezrin increases membrane tension, slows migration, and impedes endothelial transmigration of lymphocytes in vivo in mice. Blood 2012;119:445-53. 47. Kunda P, Rodrigues NTL, Moeendarbary E, Liu T, Ivetic A, Charras G et al. PP1-mediated moesin dephosphorylation couples polar relaxation to mitotic exit. Curr Biol 2012;22:231-6.

Figure 1 .

 1 Figure 1. Chromatin-driven polarization in the mammalian oocyte. (A) Schematic of the mouse

Figure 2 .

 2 Figure 2. Ran-dependent dephosphorylation of cortical ERMs in the vicinity of oocyte chromatin. (A)

Figure 1 Figure 4

 14 Figure 1

Acknowledgments

This work was funded by a CNRS ATIP fellowship to GH. BD is the recipient of a doctoral fellowship from the french Ministry of Research. We are grateful to Ben Margolis for the RanT24N plasmid. We appreciate the technical support from the UMS 3480 BIOSIT animal and microscopy facilities.

phospho-ERM exclusion zones in the cortical regions overlying the chromatin clusters. Note also the effective inhibition of actin cap formation over the clusters. Chromatin (labeled with To Pro-3) is shown in yellow.