Sônia Furian

Laurent Barbiéro
email: barbiero@lmtg.obs-mip.fr

René Boulet

Organisation of the soil mantle in tropical southeastern Brazil (Serra do Mar) in relation to landslides processes

Keywords: Oxisol, Gibbsite, Kaolinite, Landslides, Brazil

The Serra do Mar complex in the southeastern Brazil is subject to frequent landslides especially on the Atlantic coast. The three dimensional organisation of the soil was studied in a 56 ha representative catchment. Upslope in situ soil material has developed from the parent rock, and downslope the soil has resulted from landslide processes. The soil mantle upslope is mainly characterised by slowly permeable kaolinitic horizons within a thick gibbsitic weathering horizon.

The field and microscopic relationships between the two suggest that the kaolinitic horizon has evolved at the expense of the gibbsitic material. Resilication of gibbsite to form kaolinite is postulated in this environment. The kaolinitic horizons are overlain by a microaggregated horizon.

The difference in porosity and water movement between these two horizons was estimated by density measurements, mercury porosimetry, image processing and calculation of water retention and shrinkage curves. The results indicate that water is retained briefly within the microaggregated horizon during intensive rainfall. Because of the inclination of the kaolinitic horizon, any excess water within it flows laterally downslope and accumulates in the lowest part of the slope. This leads to landslipping, the main process of landform development in the region.

Introduction

Landslides are very frequent in southeastern Brazil. Catastrophic examples are reported almost every year: Rio de Janeiro-RJ (1805,1966,1967,1988), Santos-SP (1928), Cubatao-SP (1946), Via Anchieta-SP (1956, 1964, 1966), Guaratuba-PR (1968), Campos do Jordao (1972), Caraguatatuba-SP (1944[START_REF] Felicissimo | Wegener a a escarpa da Serra do Mar: intemperismo, deslizamento e erosão[END_REF] Ubatuba-SP (1942, 1943, 1949), Tubarao-SC (1975), on the road Rio de Janeiro-Santos (1974,1975,1976,1981,1982,1984,1985) [START_REF] Bigarella | Considerações a respeito da evolução das vertentes[END_REF][START_REF] Felicissimo | Wegener a a escarpa da Serra do Mar: intemperismo, deslizamento e erosão[END_REF][START_REF] Cruz | A Serra do Mar e o litoral na area de Caraguatatuba[END_REF][START_REF] Guidicini | Estabilidade de tabules naturais e de escavações[END_REF][START_REF] Guidicini | Eusaio de correlação entre pluviosidade e escorregamentos em meio tropical úmido[END_REF][START_REF] De Ploey | Landslides in the Serra do Mar, Brazil[END_REF]Souza Sobrinho, 1988). Also there are many small landslides that do not involve loss of life or damage to property. Their real importance is unknown in areas with sparse population. Most previous studies have involved statistical estimates of the location of landsides and the conditions of climate, bedrock geology, slope steepness, vegetation cover and human occupation leading to their occurrence. This has shown that landslides occur principally in the Serra do Mar complex, at the end of the rainy season when rainfall is most intense. [START_REF] Guidicini | Eusaio de correlação entre pluviosidade e escorregamentos em meio tropical úmido[END_REF] showed that the probability of landsliding is increased when rainfall exceeds 250mm in 24hr, regardless of antecedent rainfall. [START_REF] Cruz | A Serra do Mar e o litoral na area de Caraguatatuba[END_REF] emphasised the role of slope steepness; slopes exceeding 40% encourage landslides, regardless of vegetation cover and human occupation. Landslide processes occur mainly on the lowest third of the slope. Small landslides occur in the middle slope and readjust the slope topography after the main landslides have occurred downslope. Therefore, they contribute to landscape evolution. Although more than 99 % of the rainfall infiltrates [START_REF] Furian | Estudo geomorfológico do escoamento superficial pluvial em parcelas experimentais no parque Estadual da Serra do Mar, Nucleo de Cunha/SP: Um esboço metodológico[END_REF], the flows of water in the soil mantle are unknown. Even the structure and other profile characteristics of the soil involved in the landslides are poorly understood. De Ploey and [START_REF] De Ploey | Landslides in the Serra do Mar, Brazil[END_REF] concluded that the hydrology of the slopes should be studied in more detail.

The aim of this paper is to describe the nature and origin of the soil mantle and to relate these to landslide processes.

Site description

The studied area is located at 23°S in the Brazilian humid tropical zone, between the Serra do Mar and the Atlantic Plateau in the eastern part of Sao Paulo State. The fieldwork was concentrated on a small (56 ha) catchment of the River Paraibuna in the district of Cunha, known as the "D" catchment in the Serra do Mar State Park (Fig. 1). Cunha is part of the granite-gneiss high plateaux of the Serra do Mar. Uplifted since the Oligocene [START_REF] Almeida | The system of continental rifts bordering the Santos basin, Brazil. In: Continental margins of atlantic type[END_REF], these plateaux belong, with the Serra da Mantiqueira, to the uppermost surface of the block mountains of southeastern Brazil's Atlantic margin. The chemical weathering of the crystalline Precambrian basement has resulted in sandy loamy regoliths with a maximum thickness of 15 m [START_REF] Bigarella | Considerações a respeito da evolução das vertentes[END_REF].

Recent evolution of slopes in the Serra do Mar has been mainly controlled by mass movements, including slumping and planar slides in the regolith, rockfalls and rockslides [START_REF] De Ploey | Landslides in the Serra do Mar, Brazil[END_REF]. Steeper slopes of 70-100% are related to major faults in the crystalline bedrock. Less steep slopes are 40-60%. The climate belongs to type Cwa of Köppen [START_REF] Furian | Levantamento de reconhecimento do meio físico do nucleo de Cunha-SP[END_REF] with orographic influences. Annual precipitation ranges from 2000 to 2500 mm, with a rainy season from September to March giving 71% of the annual rainfall. The natural vegetation is rain forest of the "Atlantic forest" domain which still covers large parts of the Serra do Mar.

Methods

The soil pattern of the slope was studied in detail, emphasising the geometrical relationships between the different soil horizons and genesis of the soil types [START_REF] Boulet | Analyse structurale et cartographie en pédologie . I -Les études de toposéquences et principaux apports à la connaissance des sols. II -Une méthode d'analyse prenant en compte l'analyse tridimensionnelle des couvertures pédologiques. III -Passage de la phase analytique à une cartographie générale synthétique[END_REF]. This approach has often been used to understand the links between morphogenesis and pedogenesis [START_REF] Lucas | Systèmes pédologiques en Amazonie Brésilienne[END_REF][START_REF] Fritsch | Interpretation of soil features produced by ancient and modern processes in degraded landscapes: I. A new method for constructing conceptual soil-waterlandscape models[END_REF][START_REF] Furian | Morphogenèse/pédogenèse en milieu tropical humide de la Serra do Mar, Bresil. Contribution de l'altération et de la pédogenèse à une dynamique actuelle de glissement[END_REF][START_REF] Boulet | A contribution to an understanding of landscape development through three-dimensional morphological analysis of a pedological cover (Paulinia, State of Sau Paulo, Brazil)[END_REF]. The two-dimensional soil pattern was determined in transects, one along the crest line (1) and three down the slope (2, 3 and 5)(Fig. 2). A map of the area was drawn by projecting the limits of the different horizons and selected soil features onto a horizontal plane. Soil horizons were identified, described and sampled for laboratory analysis from 47 auger holes and 24 soil pits generally located over lateral transitions between horizons.

Undisturbed blocks were collected and impregnated with an acetone-diluted ("Crystic") polyester resin after dehydration by acetone exchange [START_REF] Delaye | Notes techniques sur la préparation des lames minces dans les matériaux meubles[END_REF], and vertical thin sections (70 x 110 mm) were made and described according to [START_REF] Brewer | Fabric and mineral analysis of soils[END_REF] and [START_REF] Bullock | Handbook for soil thin section description[END_REF]. In order to visualise the macroporosity, a fluorescent dye (Ciba-Geigy Uvitex OB) was added to the resin. Some samples were collected for SEM (XL 20 Philips at 15 kV) observations and analyses of aggregates (LINK Analytical eXL energy dispersive X-Ray system).

Particle density was measured in 5 replicates of oven dried samples using a water picnometer.

Bulk density was measured on 251cm 3 calibrated cylindrical samples by weighing soil previously dried at 105°C. Total porosity was computed from these data and is expressed as a percentage of the soil core volume.

The pore size distribution, between 0.0037 and 100µm equivalent radii, was studied with a CARLO ELBA 2000 mercury porosimeter using air-dried centimetric aggregates outgassed at room temperature.

Images from thin sections were captured by reflected UV light [START_REF] Hallaire | Image analysis of pore space morphology in soil sections in relation to water movement[END_REF], which caused pore space to appear as bright areas on the dark background. Negative images are presented here. Images were digitalised using the VISILOG system with 768 x 576 pixels, a spectral resolution of 256 grey levels and a pixel size of 10 µm. The pores were identified by a simple threshold partition and the macroporosity was calculated as a volume percentage from the proportion of pore pixels (Wiebel, 1979). Overall macropore connectivity in each thin section was classified visually as low, medium or high.

Water retention curves were determined on 5-10 cm 3 samples by sorption through membrane filters according to Tessier and Berrier (1979) for the low suction range (pF 1-3) and by pressure membrane equipment (Tessier, 1978) for greater suctions. The shrinkage of the soil samples was slight, so the shrinkage curves were estimated from the apparent volumes at pF 2 and 6, using the kerosene method developed by Monnier et al. (1973).

Catena organisation

Figure 2 shows the distribution of soil horizons in sequence 2, which can be divided into two zones, the upslope and downslope domains.

Upslope domain

The upslope domain consists of a ferrallitic soil 12m thick. It is mainly composed of a sandy gibbsitic weathered material derived from the parent rock (G Horizon). The original mineral structures and that of the rock itself are preserved in gibbsite pseudomorphs. This results in a gibbsitic horizon with a box-work structure (Figs. 3 and4). The polysynthetic twinning of plagioclases and the cleavage of amphiboles are also preserved in the gibbsite, indicating that the formation of gibbsite was the first stage in weathering of the parent rock. Only quartz and muscovite were unaltered at this stage. The structure of the parent rock is also preserved in millimetric gibbsitic veins that probably infilled fissures in the parent rock. This gibbsitic weathering level (G horizon) is overlain by a pebbly and blocky horizon composed of ferruginised gibbsitic material. The structure of the parent rock is still preserved in the blocks.

Although the soil fauna has burrowed into the superficial horizons, it is still possible to identify relics of the millimetric gibbsitic veins.

In the lower part of the upslope domain, a compact clay loam kaolinitic horizon (here called K horizon) occurs within the gibbsitic horizon (G horizon). It is tongue-shaped and is truncated by the superficial porous microaggregated horizons (mh1 and mh2). These are conformable with the slope topography. In thin sections, the mh1 horizon seems to have arisen from the destruction of the gibbsitic box-work structure, whereas the mh2 horizon is composed of biofabrics and is burrowed by the soil fauna. A second clay loam kaolinitic horizon (K horizon) appears downslope at 2-2.5 m depth, also within the sandy gibbsitic weathering horizon (G horizon) but beneath the first K horizon.

Detailed study of the contact between the K and G horizons showed that the structure of the K horizon truncates that of the G material, indicating that the latter developed first from the bedrock and subsequently changed into a kaolinitic material (Fig. 5). At this contact, the gibbsite has lost its optical properties becoming isotropic, acquiring a pale yellow colour, decreasing in crystal size and losing its box-work structure. Evidence of dissolution of quartz and gibbsite is observed by SEM (Figs. 6,7,8 and 9). Different morphological types of gibbsite are observed, from euhedral crystals to those showing extensive surface etching by dissolution (Figs. 6 and7). Therefore, the silica and alumina necessary for kaolinite formation could have been supplied by dissolution of gibbsite and quartz. Destruction of gibbsite in ferrallitic soils was originally proposed by [START_REF] Delvigne | Pédogenèse en zone tropicale: la formation des minéraux secondaires en milieu ferrallitique[END_REF] in an Ivory Coast catena, and has been observed by [START_REF] Eswaran | A scanning electron microscopy evaluation of the fabric and mineralogy of some soil in Malaysia[END_REF] in gibbsitic nodules in the surface horizon of the Malacca series in Malaysia. [START_REF] Keller | Resilication of bauxite at the Alabama street mine, Saline County, Arkansas, illustrated by scanning electron micrographs[END_REF] observed resilication of bauxite producing kaolin in Arkansas. [START_REF] Lucas | Structure, genesis and present evolution of Amazonian bauxites developed on sediments. 6[END_REF] also described resilication in Amazonian bauxites.

Downslope domain

The downslope domain (not detailed in Fig. 2) is composed of superficial horizons overlying a clay horizon with many randomly oriented blocks. The clay horizon rests directly and abruptly on the hard bedrock. Remnants of box-work gibbsite occur, but are frequently associated with unweathered minerals such as hornblende, other amphiboles, microcline and biotite (Fig. 10).

Mid-slope transition

The upslope soil cover clearly differs from the downslope, and the contact between the two is abrupt. On the middle part of the slope, the upslope soil cover is truncated over its whole thickness by the material of the downslope cover (Fig. 2).

Genesis of the soil cover

The observations suggest the following model for genesis of the soil cover. The upslope soil cover is derived from in situ gibbsitic weathering of the bedrock, the structure of which is preserved in the soil, even up to the surface horizons where gibbsitic veins occur. In contrast, in the downslope domain the blocks do not preserve the orientation of the parent rock. Gibbsite is associated with unweathered minerals, indicating a mixture of pedogenic materials which have suffered different weathering histories. We suggest that the downslope material is colluvial and results partly from reworking by landslide processes of a material similar to that observed in the upslope domain.

One of the main features of the whole soil system is the presence of kaolinitic tongues (K horizon) into the gibbsitic weathering material (G horizon). Figure 11 shows the limits of the K horizons and of the colluvium on the slope. The distribution of the K horizons is complex; they occupy a large part of the soil mantle, but are truncated downslope by the colluvium.

Physical properties of the horizons and water circulation in the catena

The dissolution of gibbsite and quartz and the formation of kaolinite generate tongues arranged like tiles on a roof. They are likely to be slowly permeable and to influence the flow of water on the slope. Table 1 shows the physical and hydrological characteristics of the gibbsitic-weathering horizon (G horizon), the kaolinitic horizon (K horizon) and the microaggregated M horizons overlying the K horizons. Mercury injection curves, macroporosity identified by image processing and water retention and shrinkage curves are presented in Figs 12, 13 and 14.

During the fieldwork, waterlogging after rainfall was observed in the M horizons overlying the K horizons. This suggests a severe decrease in the vertical infiltration rate at this contact. Density measurements indicated that the total porosity increases from the G (40%) to the K (51%) and M (64%) horizons, and the mercury injection curves show that the pore size distributions are also significantly different. The G and M horizons exhibit a bimodal distribution of pore sizes, corresponding to intra-and inter-aggregate pores as in other ferrallitic soils [START_REF] Cambier | Etude des associations argile-oxide: organisation des constituants d'un matériau ferrallitic[END_REF][START_REF] Curmi | Degradation of structure and hydraulic properties in an oxisol under cultivation (Brazil)[END_REF], whereas the K horizon has a unimodal distribution, which is almost entirely microporosity (90% of the pores intruded by mercury). The image processing also revealed significant differences in macroporosity, corresponding to 24.5% with medium connectivity in G, 9.3% with low connectivity in K, and 31.6% with high connectivity in M. Circulation of water is possible in G, but between M and K there is a clear decrease in macroporosity. K is slowly permeable because of the weak connection between macropores. This allows lateral circulation into M, which is consistent with the field observations. The water retention and shrinkage curves for these horizons confirm the information given by the macroporosity measurements. At low suction (pF3), the microaggregated M horizon retains only 50 % water (Table 1), whereas the kaolinitic K horizon is almost saturated (87 %). This means that a large proportion of the porosity in the M horizons could be briefly occupied by water during intense rainfall.

The pore size distribution suggests that some vertical drainage is possible in the gibbsitic material, but that most of the water moves laterally over the K horizons. However, this conclusion should be confirmed by an in situ analysis of the slope hydrology [START_REF] Grimaldi | Relation entre l'espace poral et le fonctionnement hydrodynamique d'une couverture pédologique sur socle de guyane française[END_REF].

Identification of the landslide risk

All the observations and measurements emphasize the contribution of weathering and pedogenesis to landscape evolution. The development of the K horizons seems to be the key to initiation of landslide processes. A three-stage model of increasing development of K horizons (Fig. 15) is proposed. In the first stage (a), the K horizons are thin and separated and vertical drainage in the sandy gibbsitic weathering material predominates. Little lateral flow is observed and the water infiltrates homogeneously along the slope. The risk of landslide is low. In the second stage (b), the K horizons are more strongly developed and the proportion of lateral subsurface drainage increases.

The risk of landslide then also increases. In the third stage (c), the K horizons are strongly developed and the subsurface lateral drainage predominates, canalizing the water downslope during intense rainfall. The water in the macroporosity of the M horizon can create a consequent overload in the lower third of the slope. The mass of retained water, within a 0.3m thick microaggregated horizon, increases from 100 kg/m² at pF3 to 200 kg/m² at pF1, increasing the slope instability as described by Moyersons (1989) in Rwanda. The risk of landslide is then very high. As the K horizons evolve laterally and upslope at the expense of the G horizon, the mass of water and the risk of landslide increase with the development of this pedological system.

Conclusion

This work emphasises the contribution of the soil system to landslide processes, and explains the relationships between landslides and environmental conditions. Landslides occur mainly on the Atlantic margin of the Serra do Mar, generally on the lower third of the slope and at the end of the rainfall season after prolonged heavy rainfall. The soil cover consists of clay loam kaolinitic K horizons arranged like the tiles of a roof, within a thick sandy gibbsitic horizon. The K horizons are overlain by a very porous microaggregated horizon. On the studied slope, this in situ soil cover is truncated downslope by a soil cover arising from landslides, in which many remnants of the upslope soil cover are preserved.

The measurements of total porosity, pore size distribution, connectivity of macropores, water retention and shrinkage curves and the field observations suggest that the drainage is vertical into the gibbsitic weathering material (G horizon), but lateral in the microaggregated M horizons over the kaolinitic horizon (K horizon), thus precluding a homogeneous distribution of the water in the soil cover. Retention curves reveal that the M horizons can retain briefly a large quantity of water during intensive rainfall, and this overloads the lower third of the slope generating landslides.

This study indicates that a better knowledge of the three dimensional distribution of different soil horizons is necessary for good management of the environment. Understanding water flow into the soil mantle is necessary for siting a drainage system that can quickly remove the excess of water during and directly after rainfall.

Various observations in the region around Cunha indicate that this type of soil cover is widespread in the Serra do Mar. For example, it has also been identified in the neighbouring Serra da Mantiqueira, where landslide processes are again frequent (Modenesi and Motta de Toledo, 1996). A simple systematic survey by hand auger should therefore indicate the extent of areas at risk. Modenesi, M.C., Motta de Toledo, M.C., 1996. Weathering and the formation of hillslope deposits in the tropical highlands of Itatiaia, southeastern Brazil. Catena 27, 81-103. Monnier, G., Stengel, P., Fies, J.C., 1973. Une méthode de mesure de petits agglomérats terreux.

Application à l'analyse des systèmes de porosité du sol. Ann. Agron. 24, 533-545. Moyersons, 1989. Les glissements de terrain au Rwanda occidental. Leurs causes et les possibilités de leur prévention. Cah. ORSTOM,Sér. Pédol. 25,[131][132][133][134][135][136][137][138][139][140][141][142][143][144][145][146][147][148][149] Souza Sobrinho, R. R. de, 1988. Uma abordagem em direção à previsão de movimentos de massa: caso da Rio-Santos (Trecho Itaguaí -Conceição de Jacareí). Geociênc. 7, 43-76. Tessier, D., 1978. Technique d'étude de l'orientation des particules argileuses utilisable sur des secs ou humides. Ann. Agron. 29, 193-207. Tessier, D., Berrier, J., 1979. Utilisation de la microscopie électronique à balayage dans l'étude des sols. Observations de sols humides, soumis à différents pF. Sci. du Sol 1, 67-82.

Wiebel, E.R., 1979. Stereological methods. Vol. 1. Practical methods for biological morphometry.

Academic Press, London.

Table 1: Porosity and water retention at pF 2 and 3 of the gibbsitic (G), kaolinitic (K) and microaggegated (M) horizons.

Fig. 1 .

 1 Fig. 1. Location of the study site in the Serra do Mar (southeastern Brazil).

Fig. 2 .

 2 Fig. 2. Distribution of the main horizons along toposequence 2.

Fig. 3 .

 3 Fig.3 . Box-work structure resulting from gibbsite pseudomorphs (Q = quartz, G = gibbsite, V =

Fig. 6 .

 6 Fig. 6. Scanning electron micrograph of the dissolution pattern of gibbsite at the contact between K

Fig. 7 .

 7 Fig. 7. Scanning micrograph showing detail of the dissolution pattern of gibbsite at the

Fig. 8 .

 8 Fig. 8. Scanning electron micrograph of the dissolution patterns of quartz at the contact between K

Fig. 9 .

 9 Fig. 9. Scanning electron micrograph of the detailed dissolution pattern of quartz showing

Fig. 10 .

 10 Fig. 10. Remnants of the gibbsitic box-work associated with unweathered minerals such as

Fig. 12 .

 12 Fig. 12. Mercury porosimetry curves of K, G and M horizons: differential and cumulative pore

Fig. 13 .

 13 Fig. 13. Distribution, size and connectivity of the macroporosity of the K Horizon (a and b) and

Fig. 14 .

 14 Fig. 14. Water retention µ and shrinkage curves λ of K and M horizons.

Fig. 15 .

 15 Fig. 15. Model proposed for the contribution of weathering and pedogenesis to landslide processes.

Acknowledgements

This research was supported by a CAPES-COFECUB cooperation. The authors thank P. Curmi, M.

Grimaldi and to the reviewers for helpful criticism and advice.


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

Izabela Rams-Pociecha

Robert Zdanowski

Malgorzata Kloc

Jacek Z Kubiak

Desmoplakin (Dsp) conditional knockout in NR5A1 + somatic cells affects germ cell survival in developing mouse gonads

Keywords: desmoplakin, cell-cell interactions, cell adhesion, germ cells, testis, testis cords, ovary, ovigerous cords, Sertoli cells, interstitium, follicular cells, gonad development, sexual differentiation, mouse

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

Desmoplakin (Dsp) conditional knockout in NR5A1+ somatic cells affects germ cell survival in developing mouse gonads

Rafal P Piprek, Izabela Rams-Pociecha, Robert Zdanowski, Malgorzata Kloc,

Jacek Z Kubiak

To cite this version:

Rafal P Piprek, Izabela Rams-Pociecha, Robert Zdanowski, Malgorzata Kloc, Jacek Z Kubiak. Desmoplakin (Dsp) conditional knockout in NR5A1+ somatic cells affects germ cell survival in developing mouse gonads. Reproduction (Cambridge), 2022, 163 (4), pp.199-207. 10.1530/REP-21-0295. hal-

Introduction

The development of gonads is decisive for sexual development and reproduction.

Testes and ovaries differentiate from the common progenitor termed the genital ridge (Piprek et al. 2016). During sexual differentiation, the gonadal primordium acquires the sex-specific features and forms either the testis or the ovary (Ungewitter and Yao 2013). In testes, the germ cells become enclosed by differentiating Sertoli cells (pre-Sertoli cells), which adhere to the germ cells and each other. The germ cells surrounded by the Sertoli epithelium built the testis cords (Svingen and Koopman 2013). Sertoli cell epithelium is underlined by the basement membrane. The peritubular myoid cells are located below the Sertoli epithelium and form the outside border of the testis cords (Svingen and Koopman 2013). The interstitial cells, including fetal Leydig cells, which produce androgens (Svingen and Koopman 2013), fill the space between testis cords. In the differentiating ovaries, the ovigerous cords contain germ cells enclosed by somatic pre-follicular cells. In mouse foetus, around the time of birth, the ovigerous cords break down into individual ovarian follicles (Grive and Freiman 2015). The ovigerous cords are much less compact and defined than the testis cords. Differences in male and female gonad tissue structure suggest that the proper intercellular adhesion should be much more important for the development of the testis than the ovaries (Piprek et al. 2017a).

In developing testes, strong adhesion between somatic cells facilitates the formation of Sertoli epithelium and the development of solid, elongated testis cords (Piprek et al. 2017a).

We showed previously that the variety and the level of expression of the mouse cell adhesion molecules (CAMs) are greater in the male (XY) than in the female (XX) gonad (Piprek et al. 2017a). We also showed that the sex-specific cell-cell interactions facilitate the development of Xenopus laevis gonads (Piprek et al. 2017b). Although these argue for the importance of CAMs in vertebrate gonad development, our knowledge of specific roles of individual CAMs in gonad development is limited, and a very high number (220) of identified CAMs complicates the studies.

Desmoplakin, encoded by the Dsp gene, is one of the CAMs showing the sex-specific differences in expression pattern, with a higher expression in the pre-Sertoli cells than in prefollicular cells (Piprek et al. 2017a). This sex-specific expression pattern suggests desmoplakin involvement in the sexual differentiation of the bipotential gonads into testes or ovaries. However, the role of desmoplakin in gonad development remains unknown.

To fill this gap, we studied the role of desmoplakin (Dsp) in mouse gonad development. This protein is present in desmosomes and is critical for desmosome assembly, stability, and its interactions with the cytoskeleton (Gallicano et al. 1998;Bornslaeger et al. 1996;Kowalczyk and Green 2013). Mutations in desmoplakin lead, directly or indirectly, to various diseases, such as cardiomyopathies, skin conditions such as keratoderma, and oropharyngeal and breast cancers (Norgett et al. 2000;Pang et al. 2004;Papagerakis et al. 2009). In mice, conventional whole-body knockout of desmoplakin eliminates the function of desmosomes, disrupts blastomere adhesion, and leads to embryo lethality before stage E6.5 (Gallicano et al. 1998).

In this study, we used conditional knockout (cre-loxP system) of desmoplakin to overcome embryo lethality. Because we were especially interested in the role of desmoplakin in the somatic-germ cell interactions during development, we removed the exon 2 of the Dsp gene, specifically in the somatic cells of developing gonads. From our previous studies on the knockout of E-and N-cadherin adhesion molecules, we knew that the absence of these CAMs in somatic cells of the gonads results in alteration of germ cell development (Piprek et al. 2019a(Piprek et al. ,2019b)). Here we crossed a mouse strain expressing the Cre recombinase under Nr5a1

promoter with a strain containing loxP sequences flanking exon 2 in the Dsp gene. The resulting deletion of exon 2 made the desmoplakin nonfunctional (Vasioukhin et al. 2001) in NR5A1-positive (NR5A1 + ) cells. The Nr5a1 (Nuclear Receptor Subfamily 5 Group A Member 1, also known as Sf1 -Steroidogenic Factor 1) is expressed from stage E10.2 in the coelomic epithelium covering the genital ridges (Ikeda et al. 1994;Hu et al. 2013). The NR5A1 + cells of the coelomic epithelium give rise to Sertoli cells and a subpopulation of interstitial cells in the testes, and follicular cells and a subpopulation of theca cells in the ovaries (Albrecht and Eicher 2001;DeFalco et al. 2011;Liu et al. 2015). Thus, the knockout we have performed eliminated desmoplakin expression only from those somatic cells, which are known to be critical for sex determination and gonad development. Such NR5A1 + Sf1 + cell-specific knockout was important because we expected that eliminating desmoplakin from all somatic cells of gonads would completely halt gonad development. We studied the gonad phenotypes by light microscopy, immunohistochemistry and analyzed the expression of selected genes by RT-qPCR.

Materials and methods

Animals

The study was approved by the First Local Commission for Ethics in Experiments on Animals. The animals were bred and housed in the Animal Facility at the Jagiellonian University (Krakow, Poland). Two transgenic mouse lines used in this study were purchased from The Jackson Laboratory (ME, USA).

The mouse strain Tg(Nr5a1-cre)7Lowl/J (Dhillon et al. 2006) was used for the Cre recombinase expression in NR5A1 + somatic cells of the gonads. The Dsp gene coding for desmoplakin was deleted in B6.129-Dsp tm1Efu /J mouse strain (Vasioukhin et al. 2001). Cre + , loxP fl/fl , and Cre -, loxP +/+ specimens were used as a negative control. The numbers of studied individuals are presented in Table 1.

Timed mating was performed by placing a single male with two females overnight.

The following morning, females were checked for the presence of the vaginal plug, and the pregnancies were designated as E0.5 (embryonic day). Females were sacrificed by spinal cord dislocation at embryonic days: E10.5, 11.5, 12.5, 13.5, 14.5, 16.5, 18.5, and the newborns were sacrificed at 1 and 2 dpp (days postpartum).

Genotyping

The sex of all studied individuals was identified by the genotyping using primers for Sly (Y chromosome) and Xlr (X chromosome) (McFarlane et al. 2013). Primers used to genotype Cre + , and loxP + specimens are listed in Table 2. We used a standard PCR protocol for genotyping.

RNA isolation from gonads and Real-Time Quantitative PCR

Gonads from mouse fetuses and newborns were pooled according to the sex and developmental stage. Total RNA was isolated using Trizol and further purified with RNeasy Mini kit according to the manufacturer's instructions (Qiagen, Valencia, CA). Total RNA in RNase-free water was stored at -80 o C and used for multigene qPCR analysis. 50 ng RNA of each sample was reverse-transcribed into cDNA using random primers and SuperScript III Reverse Transcriptase (Invitrogen, 18080044) according to the manufacturer's instructions. A list of primers is shown in Table 2. RT-qPCR was performed in 5μl reactions using SYBR Green Master Mix (Life Technologies, 4312704) on a 7500 Fast Real-Time PCR System (Applied Biosystems) with universal cycling parameters and analyzed as previously described (Svingen et al. 2009). Actb was used as a reference gene. Statistical analysis was performed using the nonparametric ANOVA Kruskal-Wallis test followed by Tukey's test. Statistica 7.0 software was used for the analyses.

Histology and Immunohistochemistry

Dissected gonads were fixed in Bouin's solution, dehydrated, and embedded in paraffin (Paraplast, Sigma, P3683). Histological staining was performed according to Debreuill's trichromatic method as previously described (Kiernan 1990;Piprek et al. 2017b).

For immunochemistry, heat-induced epitope retrieval was conducted in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6) at 95 o C for 20 minutes. Subsequently, the sections were blocked with 3% H2O2 and 10% goat serum (Sigma, G9023). Sections were incubated with the primary antibodies (rabbit polyclonal: anti-AMH, Santa Cruz Biotechnology, sc-166752; anti-collagen I, Abcam, ab34710; anti-GCNA1, Abcam, ab82527, anti-CYP17A1, Abcam, ab125022; anti-PCNA, Abcam, ab18197; anti-cleaved caspase 3, Assay BioTech, L0104; anti-Cre recombinase, Abcam, ab190177; and mouse monoclonal anti-desmoplakin, Santa Cruz Biotechnology, sc-390975) at 4 o C overnight, and the signal was detected using UltraVision Quanto Detection System (Thermo Fisher, TL-125-QHD).

Mayer's hematoxylin was used as a counterstain. Sections were examined under the Nikon Eclipse E600 microscope.

Germ cell quantification

The sections of the gonads from the animals sacrificed at 2 dpp were stained with

Trichrome. The number of germ cells was calculated in the 10,000 µm 2 area in 5 crosssections from each gonad using the ImageJ software. The diameter of testes, testis cords, and ovaries was measured in the five widest cross-sections. Average values and standard deviation were calculated using Microsoft Excel software.

Results

The effectiveness of genetic knockout

To assess the effectiveness of the knockout, first, we checked the expression of Cre recombinase using RT-qPCR and immunohistochemistry. There was a high expression of Cre gene under the control of the Nr5a1 promoter in fetal XY and XX gonads from E11.5 (Fig. 1A,D). In both sexes, the expression of Cre mRNA declined from E16.5 onward (Fig. 1A,D). The control testes did not express the CRE protein (Fig. 1B). In the knockout testes, the CRE protein was expressed in all Sertoli cells and some, but not all interstitial cells (Fig. 1C, see the dark brown staining labeled with arrows and arrowheads). In control ovaries, the CRE was not expressed (Fig. 1E). In the knockout ovaries the CRE protein was expressed in the somatic cells (Fig. 1F, arrows).

The declining expression of Dsp from E11.5 onward in the gonads of both sexes (Fig.

The effect of Dsp knockout in NR5A1 + cells on the number of germ cells

The testes of knockout males were smaller than the control testes. The apparent difference in size became visible starting from E16.5 (Fig. 2A). Histological analysis and immunolocalization of GCNA1 (germ cell marker) showed that the decrease in the size of testes correlated with the reduction of germ cell number (Fig. 2B-D). Consistent with the reduction in germ cell number observed in ovaries and testes from around E16.5, the expression of the germ cell marker Mvh (mouse vasa homolog) was also reduced in E16.5 testes and ovaries (Fig. 2G).

The ovaries of the knockout females had fewer germ cells, were smaller than the control ovaries (Fig. 2A), and the difference in size was apparent from E16.5. The number of cells positive for GCNA1 was lower in the knockout than in control (Fig. 2B,E,F). The level of Mvh expression was lower than in control (Fig. 2G; XX gonads are shown in the middle).

To establish if the germ cell loss resulted from a decreased proliferation or increased apoptotic cell death, we performed immunostaining with the antibody against proliferation marker PCNA and the apoptotic marker caspase 3 (Fig. 3). We did not detect any changes in the PCNA expression in fetal testes or ovaries (compare Fig. 3A with 3B, and 3E with 3F;

Fig. 3I shows a graphical presentation of the expression in time). However, caspase 3 had higher expression higher expression (stronger immunostaining) in the fetal knockout testes (compare Fig. 3C with 3D, apoptotic cells are indicated by the arrowheads, Fig. 3J shows a graphical representation of the expression in time), and ovaries (compare Fig. 3G with 3H, graphical presentation in Fig. 3J). The location of the apoptotic cells and their large nuclei suggest that they are germ cells. These data strongly suggest that the loss of germ cells in testes with the Dsp knockout in NR5A1 + cells resulted from apoptosis triggered by the absence/diminution of desmoplakin in gonadal somatic cells.

The effect of Dsp knockout in NR5A1 + cells on testes development

Although the size of control and knockout testes was different, the testis structure remained unchanged; both contained many testis cords (ts, encircled) divided by interstitium (i) (Fig. 4A-D).

We found that the developing Dsp knockout testes had typical testis cords (Fig. 4B,D, F, H, J) like those in control (Fig. 4A, C, E, G, I). Also, the basement membrane and the peritubular myoid cells (PMCs) located around the cords had normal morphology (Fig. 4C,D;

basement membrane (arrow), peritubular myoid cells (pmc)). The AMH (anti-Müllerian hormone, which is a specific marker of Sertoli cells) immunostaining showed that in the control testis, the positively stained Sertoli cells enclosed numerous germ cells (Fig. 4E,G;

germ cells (g)). The knockout testes lacked the germ cells, and their testis cords were filled with the AMH-positive Sertoli cells (Fig. 4F,H). The diameter of testis cords was significantly lower in the knockout testes (mean diameter 41.82 m +/-SD 6.51 at E18.5) than in control (49.10 m +/-SD 4.80 at E18.5), an effect that is likely to be caused by the loss of relatively voluminous germ cells. Collagen I immunostaining of control testes (Fig. 4I) and knockout testes (Fig. 4J) showed that the sterile cords in the knockout testes were enclosed by typically looking basement membranes (indicated by arrows in Fig. 4I andJ).

This indicates that the knockout of desmoplakin under Nr5a1 promoter in testis cells has not affected the integrity of the somatic component of the gonad. In the control testes, the space between the testis cords is occupied by the interstitium (i) (Figs 4C, E, G, I). We did not observe any structural changes in the interstitium of the knockout testes (Fig. 4D,F,H,J).

The effect of Dsp knockout in NR5A1 + cells on ovarian development

The knockout ovaries contained very few germ cells located in the ovarian surface epithelium, while the control ovaries were filled with many germ cells (compare Fig. 5A,C with Fig. 5B,D). The ovigerous cords were present both in the control and knockout ovaries (compare Fig. 5A, C with Fig. 5B,D; ovigerous cords are marked by a dotted line). The ovigerous cords of the control ovaries consist of the elongated groups of germ cells surrounded by the somatic cells. The ovigerous cords of the knockout ovaries consist of somatic cells only (Fig. 5B,D). This indicates that the knockout of desmoplakin under Nr5a1

promoter in ovarian cells did not affect the somatic cell adhesion and aggregation necessary for the formation of ovigerous cords. The control ovaries contained the ovarian follicles (encircled by a solid line in Fig. 5A) which consisted of a single germ cell enclosed by a monolayer of the somatic cells. However, in the knockout ovary, the ovarian follicles were absent (Fig. 5D). As we have previously suggested (Piprek et al. 2019a(Piprek et al. , 2019b)), this may indicate that germ cells are critical for forming ovarian follicles. Alternatively, the loss of Dsp may interfere with the breakdown of ovarian cysts and/or the formation of ovarian follicles resulting in the loss of germ cells.

Dsp knockout in NR5A1 + cells does not affect sex determination

The expression of three selected Sertoli cell markers and genes involved in male sex determination (Sox9, Amh, Dhh) studied by RT-qPCR were unaltered in the knockout testes (Fig. 6A). Thus, the knockout of desmoplakin performed in this study did not affect the pathways of Sertoli cell differentiation and male sex determination. Accordingly, the analysis performed by RT-qPCR in control and NR5A1 + cells KO ovaries showed no change in the expression of three selected ovarian-specific genes: Rspo1, Wnt4, Foxl2 (Fig. 6B). All these data indicate that desmoplakin deletion in NR5A1 + cells does not affect male or female sex determination.

The expression of the steroidogenic enzyme Cyp11a1, which is a specific marker of steroidogenic Leydig cells, was comparable in the knockout and control testes (Fig. 6A).

Discussion

The role of desmoplakin in gonad development was unknown before our study. The only information available was that the desmosomes are important for spermatogenesis in adult testes (Mruk and Cheng 2011). In our study, the use of CRE recombinase under the control of Nr5a1 promoter allowed us to delete desmoplakin in the descendants of NR5A1 + coelomic epithelium, i.e., Sertoli cells and a subpopulation of interstitial cells in the testes, and the follicular cells and stromal cells in the ovaries (Ikeda et al. 1994;Albrecht and Eicher 2001;DeFalco et al. 2011;Hu et al. 2013, reviewed in Piprek et al. 2016). We provided strong evidence that desmoplakin in the somatic cells of developing gonads is critical for germ cell survival and maintenance in male and female gonads. The developing knockout testes and ovaries were smaller than the control and had fewer germ cells. In both sexes, the germ cell loss occurred at embryonic day E16.5. Previously, we showed that the deletion of N-cadherin or E-cadherin in the gonadal somatic cells caused germ cell loss (Piprek et al. We showed here that in the absence of desmoplakin, somatic components of gonads in both sexes developed normally. Similarly, the knockout of the E-cadherin did not cause changes in the somatic component of the gonad, only the loss of germ cells (Piprek et al. 2019a). However, the knockout of N-cadherin led not only to the loss of germ cells but also to the disruption of testis cords and loss of fetal Leydig cells (Piprek et al. 2019b). Thus, Ncadherin, but not E-cadherin and desmoplakin, is critical for the formation of somatic components of the gonad.

The absence of ovarian follicles in the knockout gonads was the only visible sign of the change in the somatic part of the gonad. The same effect was also observed in N-and Ecadherin knockouts (Piprek et al. 2019a, b). Hypothetically the disruption in the ovarian follicle formation can be an effect of the lack of germ cells, or alternatively, granulosa cells may require desmoplakin for cell adhesion to assemble ovarian follicles. The failure of the germ cyst break down into the ovarian follicles may possibly underlie the massive germ cells death observed under Dsp knockout conditions.

We also studied the expression of male and female sex-determining genes. We showed that the knockout of desmoplakin in NR5A1 + cells, similarly to N-and E-cadherin, did not

  1G, J) proved the effectiveness of the knockout. Because the desmoplakin knockout was induced only in a subpopulation of the gonadal cells, low desmoplakin expression was still detectable in the gonad. Staining with antibodies against the fragment encoded by the deleted exon 2 of the Dsp gene showed a clear difference in the intensity of desmoplakin immunostaining between the control and the knockout testes and ovaries (Fig.1H and 1K, and compare to Fig.1I and 1L, respectively), which indicated successful knockout of Dsp expression in the somatic cells of developing male and female gonads.

  2019a, b). It is unclear why germ cell loss occurs during this particular period of embryo/gonad development. The decreased expression of Dsp in gonadal somatic cells was apparent from stage E11.5 onward, so it is possible that it takes several days (between stage E11.5 and E16.5) before the loss of desmoplakin in the somatic cells affects the germ cells.Alternatively, the germ cells could be especially susceptible to desmoplakin loss. In testes, most germ cells enter mitotic arrest between E12.5 and E14.5, and by E15.5 they are all in the mitotic arrest(Western et al. 2008). In the ovaries, at stage E17.5 some of the germ cells enter the diplotene stage of meiosis, and the germ cysts break down into ovarian follicles(Borum 1961, Pepling and Spradling 2001, Pepling et al. 2010). The knockout of desmoplakin may possibly loosen cell adhesion, which in turn may induce pro-apoptotic signaling, as it occurs in the case of carcinogenesis. Although the molecular mechanism of the germ cell loss is unclear, the E-cadherin-mediated contacts prevent apoptosis in rat granulosa cells, and the deletion of E-cadherin activates caspase 3-dependent apoptosis by inhibiting Akt kinase (protein kinase B)(Peluso et al. 2001). N-cadherin inhibits apoptosis by mediating Akt signaling and PI3K (phosphatidylinositol-3 kinase) pathway(Tran et al. 2002). Its deletion causes germ cell loss through the down regulation of these pathways(Piprek et al. 2019a, b).A mechanism by which a cell adhesion molecule can mediate cell proliferation and apoptosis may be cell type-specific. Previous studies showed that the knockdown of desmoplakin in human HaCaT keratinocytes increased cell proliferation and elevated phospho-Akt and phospho-ERK1/2 levels(Wan et al. 2007). The upregulation of desmoplakin in non-small cell lung cancer (NSCLC) inhibited proliferation and increased sensitivity to apoptotic signals(Yang et al. 2012).

  interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly. J Cell Biol 134 985-1001. Borum K 1961 Oogenesis in the mouse: a study of the meiotic prophase. Exp Cell Res 24 495-507. DeFalco T, Takahashi S & Capel B 2011 Two distinct origins for Leydig cell progenitors in the fetal testis. Dev Biol 352 14-26. Delva E, Tucker DK & Kowalczyk AP 2009 The desmosome. CSH Perspect Biol 1 RD, Edelstein EA, et al. 2006 Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis. Neuron 49 191-203. Gallicano GI, Kouklis P, Bauer C, Yin M, Vasioukhin V, Degenstein L & Fuchs E 1998 Desmoplakin is required early in development for assembly of desmosomes and cytoskeletal linkage. J Cell Biol 143 2009-2022. Garrod D & Chidgey M 2008 Desmosome structure, composition and function. BBA-Biomembranes 1778 572-587. Grive KJ & Freiman RN 2015 The developmental origins of the mammalian ovarian reserve. Development 142 2554-2563. Hu YC, Okumura LM & Page DC 2013 Gata4 is required for formation of the genital ridge in mice. PLoS Genet 9 e1003629. Huang K, Wang Y, Zhang T, He M, Sun G, Wen J, Yan H, Cai H, Yong C & Xia G 2018 JAK signaling regulates germline cyst breakdown and primordial follicle formation in mice. Biol Open 7 bio029470. Ikeda Y, Shen WH, Ingraham HA & Parker KL 1994 Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol 8 654-662. Kiernan JA 1990 Histological and Histochemical Methods: Theory and Practice. 2nd ed. Pergamon Press, Oxford, New York, Seoul, Tokyo. Kowalczyk AP & Green KJ 2013 Structure, function, and regulation of desmosomes. Prog Mol Biol Transl Sci 116 95-118. Liu C, Peng J, Matzuk MM & Yao HHC 2015 Lineage specification of ovarian theca cells requires multicellular interactions via oocyte and granulosa cells. Nat Commun 6 6934. McFarlane L, Truong V, Palmer JS & Wilhelm D 2013 Novel PCR assay for determining the genetic sex of mice. Sex Dev 7 207-211. McLaren A 2003 Primordial germ cells in the mouse. Dev Biol 262 1-15. Mruk DD & Cheng CY 2011 Desmosomes in the testis: Moving into an unchartered territory. Spermatogenesis 1 47-51. Norgett EE, Hatsell SJ, Carvajal-Huerta L, Ruiz Cabezas JC, Common J, Purkis PE, Whittock N, Leigh IM, Stevens HP & Kelsell DP 2000 Recessive mutation in desmoplakin disrupts desmoplakin-intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma. Hum Mol Genet 9 2761-2766. Pang H, Rowan BG, Al-Dhaheri M & Faber LE 2004 Epidermal growth factor suppresses induction by progestin of the adhesion protein desmoplakin in T47D breast cancer cells. Breast Cancer Res 6 R239. Papagerakis S, Shabana AH, Pollock BH, Papagerakis P, Depondt J & Berdal A 2009 Altered desmoplakin expression at transcriptional and protein levels provides prognostic information in human oropharyngeal cancer. Hum Pathol 40 1320-1329. Peluso JJ, Pappalardo A & Fernandez G 2001 E-cadherin-mediated cell contact prevents apoptosis of spontaneously immortalized granulosa cells by regulating Akt kinase activity. Biol Reprod 64 1183-1190. Pepling ME & Spradling AC 2001 Mouse ovarian germ cell cysts undergo programmed breakdown to form primordial follicles. Dev Biol 234 339-351. Pepling ME, Sundman EA, Patterson NL, Gephardt GW, Medico L & Wilson KI 2010 Differences in oocyte development and estradiol sensitivity among mouse strains. Reproduction 139 349-357. Piprek RP 2009 Molecular mechanisms underlying female sex determination-antagonism between female and male pathway. Folia Biol-Krakow 57 105-113. Piprek RP, Kloc M & Kubiak JZ 2016 Early development of the gonads: origin and differentiation of the somatic cells of the genital ridges. Results Probl Cell Differ 58 1-22. Piprek RP, Kloc M, Tassan JP & Kubiak JZ 2017b Development of Xenopus laevis bipotential gonads into testis or ovary is driven by sex-specific cell-cell interactions, proliferation rate, cell migration and deposition of extracellular matrix. Dev Biol 432 298-310. Piprek RP, Kolasa M, Podkowa D, Kloc M & Kubiak JZ 2017a Cell adhesion molecules expression pattern indicates that somatic cells arbitrate gonadal sex of differentiating bipotential fetal mouse gonad. Mech Dev 147 17-27. Piprek RP, Kolasa M, Podkowa D, Kloc M & Kubiak JZ 2019a N-Cadherin Is Critical for the Survival of Germ Cells, the Formation of Steroidogenic Cells, and the Architecture of Developing Mouse Gonads. Cells 8 1610. Piprek RP, Kolasa M, Podkowa D, Kloc M & Kubiak JZ 2019b Tissue-specific knockout of E-cadherin (Cdh1) in developing mouse gonads causes germ cells loss. Reproduction 158 147-157. Svingen T & Koopman P 2013 Building the mammalian testis: origins, differentiation, and assembly of the component cell populations. Genes Dev 27 2409-2426. Svingen T, Spiller CM, Kashimada K, Harley VR & Koopman P 2009 Identification of suitable normalizing genes for quantitative real-time RT-PCR analysis of gene expression in fetal mouse gonads. Sex Dev 3 194-204. Tran NL, Adams DG, Vaillancourt RR & Heimark RL 2002 Signal transduction from Ncadherin increases Bcl-2. Regulation of the phosphatidylinositol 3-kinase/Akt pathway by homophilic adhesion and actin cytoskeletal organization. J Biol Chem 277 32905-32914. Ungewitter EK & Yao HH 2013 How to make a gonad: cellular mechanisms governing formation of the testes and ovaries. Sex Dev 7 7-20. Vasioukhin V, Bowers E, Bauer C, Degenstein L & Fuchs E 2001 Desmoplakin is essential in epidermal sheet formation. Nat Cell Biol 3 1076. Wan H, South AP & Hart IR 2007 Increased keratinocyte proliferation initiated through down regulation of desmoplakin by RNA interference. Exp Cell Res 313 2336-2344. Western PS, Miles DC, van den Bergen JA, Burton M & Sinclair AH 2008 Dynamic regulation of mitotic arrest in fetal male germ cells. Stem Cells 26 339-347. Yang L, Chen Y, Cui T, Knosel T, Zhang Q, Albring KF, Huber O & Petersen I 2012 Desmoplakin acts as a tumor suppressor by inhibition of the Wnt/beta-catenin signaling pathway in human lung cancer. Carcinogenesis 33 1863-1870.

Fig. 1 .

 1 Fig. 1. Cre and Dsp expression. A. The expression of Cre recombinase in XY gonads is high

Fig. 2 .

 2 Fig. 2. Gonad size and the number of germ cells. A. The knockout XY and XX gonads are

Fig. 3 .

 3 Fig. 3. Proliferation (PCNA immunostaining) and apoptosis (caspase 3 immunostaining)

Fig. 4 .

 4 Fig. 4. Histology of the testes in control and desmoplakin knockout mice. A-D.Histological staining shows a similar gross morphology of control (Nr5a1-cre + Dsp +/+ ) and knockout testes (Nr5a1-cre + Dsp fl/fl ) at stage E18.5. However, in the knockout testis, testis

Fig. 5 .

 5 Fig. 5. Histology of the testes in control and desmoplakin knockout mice. A, C) Histology of control ovary (Nr5a1-Cre + Dsp +/+ ) at E18.5 show many oocytes (arrowheads) surrounded

Fig. 6 .

 6 Fig. 6. Gene expression in control (C) and desmoplakin knockout (KO) gonads at E18.5.

affect the expression of these genes (Piprek et al. 2019a, b). Thus, it seems that cell adhesion mediated by these proteins is not crucial for sex determination processes.

Further studies are necessary to uncover the potential interactions between Akt and PI3K signaling pathways, cell adhesion, apoptosis, and the germ cells of developing gonads.

Funding

The study was conducted within the project financed by the Polish National Science Centre (NCN) assigned based on the decision number DEC-2014/15/B/NZ3/02316. References Albrecht KH & Eicher EM 2001 Evidence that Sry is expressed in pre-Sertoli cells and Sertoli and granulosa cells have a common precursor. Dev Biol 240 92-107. Bornslaeger EA, Corcoran CM, Stappenbeck TS & Green KJ 1996 Breaking the connection: displacement of the desmosomal plaque protein desmoplakin from cell-cell

Declaration of interest

The authors do not have conflict of interest.

Author contributions

Conceptualization: R.P.P.; Methodology: R.P.P.; Validation: R.P.P., I.R.; Formal analysis: R.P.P., I.R.; Investigation: R.P.P., I.R.; Resources: R.P.P.; Data curation: R.P.P.; Writingoriginal draft: R.P.P., M.K., J.Z.K.; Writingreview & editing: R.P.P., M.K., J.Z.K., RZ; Supervision: R.P.P.; Project administration: R.P.P.; Funding acquisition: R.P.P.


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K Lachin

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Dimensional analysis modeling of spraying operation -Impact of fluid properties and pressure nozzle geometric parameters on the pressure-flow rate relationship

Keywords: Spray drying, atomization, pressure nozzle, operating diagram, dimensional analysis, pressure drop

Dimensional analysis modeling of spraying operation -Impact of fluid properties and pressure nozzle geometric

parameters on the pressure-flow rate relationship

Introduction

Liquid atomization is a process of prime importance in several industrial sectors for multiple purposes involving the need of fine liquid droplets. It can basically be described as a process which aims at generating droplets from a liquid which is disintegrated in a surrounding gas [START_REF] Mandato | Liquids' atomization with two different nozzles: Modeling of the effects of some processing and formulation conditions by dimensional analysis[END_REF]. When it comes to large-scale production, atomization can be found in industries such as [START_REF] Nasr | Industrial sprays and atomization: design, analysis and applications[END_REF]:

 Pharmaceutics: wet granulation, coating…;  Paper manufacturing: pulping, moisturizing…;  Food processing: spray drying, coating, agglomeration…

The atomization stage may have a direct impact on the quality of the end product (as for example, the powder size distribution for spray drying, granulation or agglomeration) and therefore needs to be perfectly understood and controlled. When using nozzles, the parameters influencing the properties of the spray generated mainly falls into three categories:

 The physicochemical properties of the sprayed fluid, including its rheological behavior, surface tension and density;  The process parameters such as operating pressure, temperature and relative velocity between the fluid and the surrounding air at the nozzle outlet;

 The geometric parameters of the nozzle.

As the main objective of atomization is to generate a fine spray, a substantial amount of the studies on the topic naturally aimed at predicting the average size of the sprayed droplets as a function of some of these parameters [START_REF] Bayvel | Liquid atomization[END_REF][START_REF] Butler Ellis | How adjuvants influence spray formation with different hydraulic nozzles[END_REF][START_REF] Hoffmann | Air and spray mixture temperature effects on atomization of agricultural sprays[END_REF][START_REF] Mandato | Liquids' atomization with two different nozzles: Modeling of the effects of some processing and formulation conditions by dimensional analysis[END_REF][START_REF] Mccreery | Drop formation mechanisms and size distributions for spray plate nozzles[END_REF]. However, the geometric parameters of the nozzle were not systematically taken into account in the establishment of the correlations as it involves a high degree of complexity in the experiments to be performed (several nozzle geometries to be studied and characterized) and in the relations to be found (more coefficients to identify).

Among the diversity of the spray nozzle existing, pressure swirl nozzles are the most commonly used because of their high reliability and low energy requirement, ensuring a good spraying quality (straight droplet size distribution) at small operating costs [START_REF] Bayvel | Liquid atomization[END_REF]. The main purpose of these devices is to convert pressure into kinetic energy so that the liquid can reach a sufficient velocity when flowing out of the nozzle to ensure the disruption of the film under the shear stress generated. By imposing a swirl motion at some location of the nozzle, the liquid undergoes centrifugal force and leaves the apparatus as a conical sheet [START_REF] Marchione | Experimental investigation of a pressure swirl atomizer spray[END_REF]. When the liquid inlet is tangential to the outlet, the liquid swirl occurs into a chamber before the nozzle exit. In the case the liquid enters axially, the swirl motion is created by an insert with slots designed to this purpose.

When the sizing of a continuous atomization process is required, it may be necessary to know the relationship between the atomized liquid flowrate and the spraying pressure (or pressure drop) ΔP inside the nozzle. This type of relation can practically be of great use for the experimentalist when it comes to estimate the spraying pressure required to proceed a defined flowrate, and determine the minimal flowrate and resulting pressure drop to ensure atomization regime. The relation may be obtained establishing a dimensionless number taking into account the influence of the main parameters. In the process engineering field, pressure-flowrate studies often involve the determination of a discharge coefficient C D : ratio of the real flowrate (either volumetric or mass) over the theoretical flowrate (ideal conditions) [START_REF] Lee | Measuring air core characteristics of a pressure-swirl atomizer via a transparent acrylic nozzle at various Reynolds numbers[END_REF]. This coefficient depends on multiple factors: the shape of the vessel, the liquid properties, the flow regime…. C D is of prime importance when characterizing the continuous operation of an atomizer [START_REF] Bayvel | Liquid atomization[END_REF] as it quantifies the pressure losses due to the passage of the flow through the apparatus. This coefficient can however sometimes be difficult to estimate due to the presence of an air core in the spray: the cross-section area filled by the liquid at the nozzle exit, necessary to estimate C D , is then different from the orifice area, and this air core has to be somehow predicted to ensure reliable calculations. Due to the wide range of applications of swirl nozzle atomization, a significant amount of experimental studies were performed to investigate the influence of the product and process parameters on the spray angle or air-core diameter [START_REF] Lee | Measuring air core characteristics of a pressure-swirl atomizer via a transparent acrylic nozzle at various Reynolds numbers[END_REF][START_REF] Halder | Initiation of air core in a simplex nozzle and the effects of operating and geometrical parameters on its shape and size[END_REF][START_REF] Chu | An experimental investigation of swirl atomizer sprays[END_REF][START_REF] Moon | Air flow and pressure inside a pressure-swirl spray and their effects on spray development[END_REF][START_REF] Alidoost Dafsari | An experimental study on flow and spray characteristics of pressure swirl nozzle with variation of swirl chamber length[END_REF][START_REF] Moon | Liquid film thickness inside the high pressure swirl injectors: Real scale measurement and evaluation of analytical equations[END_REF]. However few of them focused on the study of axial inlet swirl nozzles with insert and, to the best of the author's knowledge, no extensive study on swirl nozzles with throttle insert was led. Furthermore, solely few correlations taking into account the geometric parameters of the nozzle can be found in the literature for the prediction of the discharge coefficient C D [START_REF] Bayvel | Liquid atomization[END_REF][START_REF] Jones | Factors affecting the performance of large swirl pressure jet atomizers[END_REF][START_REF] Rizk | Internal flow characteristics of simplex swirl atomizers[END_REF][START_REF] Ballester | Discharge coefficient and spray angle measurements for small pressure-swirl nozzles[END_REF][START_REF] Jain | Characterization of the full cone pressure swirl spray nozzles for the nuclear reactor containment spray system[END_REF]. And, mainly established for tangential inlet nozzles, these correlations cannot be used for accurate predictions in the case of axial swirl nozzle with throttle inserts, where the geometry can be substantially more complicated especially knowing that the influence of hydrodynamics was not systematically taken into account in these correlations.

The aim of this paper is therefore to fill in this void and to propose a pressure-flowrate relation for pressure nozzles with throttle inserts. To be as comprehensive as possible in both the product and process parameters and the geometric parameters and hydrodynamics, this relation will be established by the means of a detailed dimensional analysis approach, following the methodology described by Delaplace et al. [START_REF] Delaplace | Dimensional analysis of food processes[END_REF]. The experimental observations combined with the relations obtained will then be discussed and explored to shed some light on the mechanisms at play in this type of device and to propose some guidelines for optimal choice of atomization conditions.

Materials and methods

Sprayed liquids

Aqueous solutions of maltodextrin (MD) of dextrose equivalent (DE) 15 (C.Dry01915, Cargill) were chosen as a model of the liquid feeds usually spray dried in the food industry. Four homogeneous solutions, with a concentration ranging from 0 (pure water) to 47% w/w, were prepared by 5 min mixing at a shear rate of = 100 s -1 .

The dynamic viscosity of the solutions at T = 20°C was measured using a Physica MCR 301 rheometer (Anton Paar, Austria). Their Newtonian behavior was confirmed through measurements over a range of shear rate comprised between 100 and 1000 s -1 , representative of the shear rates that can be encountered during atomization.

The properties of the four model solutions used in this study are summarized in Table 1a.

Nozzles

Four WDA axial inlet pressure nozzles (Delavan, USA) including a 416 stainless steel distributor and an orifice disc inserted into a brass body were used for the study (Fig. 1a). Thanks to rectangular inclined slots carved into the head of the distributor, the liquid (injected axially from the top) exhibits a swirl motion just before going out of the orifice disc, allowing the generation of a hollow-cone spray at the nozzle outlet. As the distributor and orifice disc of each nozzle can be removed, it has been chosen to combine these parts to increase the number of practical nozzle geometries. Over the 16 possible combinations, 11 were selected for the experiments. In order to avoid any confusion, the following nomenclature will be adopted for the designation of the nozzles and recombined nozzles:  The distributors (inserts) are designated with numbers ranging from 1 to 4;  The orifice discs are named with letters from a to d. 1a, 2b, 3c and 4d thus represent the nozzles as commercialized by the company. For each combination, the main geometric parameters subjected to have an influence on the flow behavior had to be estimated (Fig. 1b):

 Distributor: the slot width w s , the slot depth d s , the distributor base diameter d b , the slot length l s (not represented) and the number of slots n s (not represented);  Orifice disc: the orifice minimal diameter d m , the orifice outlet diameter d o and the orifice length l o . A precise characterization of these dimensions was performed through the use of a profile projector (TESA-Visio 300, TESA, Switzerland). Motorized arms, able to move the geometry along the xyz axes with a precision of 0.5 µm, were used to perform the surface discretization. Through the measurements on two distinct nozzles for each one, it has been established that the measurement accuracy was 5*10-7 m. All the values measured for the different inserts and orifice discs used are gathered in respectively Table 1b andTable 1c.

Spraying set-up

In order to perform this study, a spraying pilot set-up (TechniProcess, France) was specifically designed (Fig. 2). It is composed by a thermo-regulated tank equipped with an adequate stirrer (A) (VDA, Milton Roy Mixing, UK), a volumetric tri-piston pump (B) (DOXA B10, Fluidcontrol, Germany), the studied spray nozzle (C) and a tank placed on a scale (D) to collect and weight the sprayed solution.

Figure 2: Pilot scale spraying set-up

The temperature of the liquid is measured through the use of thermocouples in the feeding tank and upstream the nozzle. The sprayed liquid mass measurement, performed with a scale (TSI, Timber Productions, France) associated to a timer, allows the determination of the sprayed liquid mass flow rate. For each spraying condition, the liquid flow rate is estimated from the weight measured after a period of 10 minutes. The spraying pressure is measured through a pressure sensor (Midas, JUMO, Germany) located upstream the nozzle. When using tri-piston pumps, the flowrate can be adjusted by either changing the pistons stroke or modifying the operating frequency. It has been chosen for these experiments to maintain the frequency at 50 Hz and to adjust the pistons stroke. For each of the four solutions tested and each of the eleven nozzle geometries considered, between 5 and 10 flow rates in the range 10-50 L.h -1 were chosen, corresponding to spraying pressures between 0.5 and 100 bar. Each experiment was repeated: the pressure and flowrate considered in this study are average of values obtained from both experiments. All the 264 experiments performed were supervised through the use of Labview software (Labview, National Instruments, USA).

Statistical analysis

In the same operating conditions, the difference between the measured flowrate was found to be less than 2%. It was also checked that the pulsating motion of the pump pistons did not have a significant impact on the measured pressure: the variations observed were below 2%.

Coefficients of the dimensional analysis model were determined from the experimental data set using a multiple regression based on the singular value decomposition method. The significance of the obtained coefficients value was successfully checked through a Student test. The variance analysis was also shown to follow a F test. These analyses were performed using the software MATLAB (MATLAB 7.9.0.529, MathWorks, USA).

Dimensional analysis

The dimensional analysis performed in this study was established following the guidelines proposed by Delaplace et al. [START_REF] Delaplace | Dimensional analysis of food processes[END_REF]:

 Choice of the target variable and list of the independent parameters/variables related;  Determination of the dimension of these parameters;  Use of the Vashy-Buckingham theorem;  Construction of dimensionless numbers;  Eventually, reorganization of these numbers.

Target variable and related variables/parameters

For pressure nozzles, it is well established that the atomization pressure will depend on a lot of parameters, including the liquid flowrate, the nozzle geometrical parameters and the solution physico-chemical properties. As the liquid flowrate is often imposed by the production to be achieved, the target parameter chosen here was ΔP, the difference between the pressure at the nozzle upstream and the one at the nozzle downstream (e.g. atmospheric pressure). The other variables to be considered can fall into 3 categories: Preliminary experiments showed no influence of the fluid surface tension over ΔP. This variable was thus not considered in this study. Knowing all the parameters susceptible to have a significant impact on ΔP, the general relation to identify can safely be expressed as Eq. 1:

Eq. 1

This "blind" equation can be rearranged and simplified by replacing some of the variables by hydrodynamically significant parameters. The hydrodynamic diameter d h (Eq. 2) and the total section area of the slot A s (Eq. 3) related to the slot geometry were therefore introduced to replace :

Eq. 2

Eq. 3

By introducing these intermediate variables, we thus assume that only have an impact through the parameters and , which was subsequently verified. Geometry parameters related to the orifice disc, d o and d m , can also be reinterpreted in terms of minimal section A m and outlet section A o (respectively Eq. 4 and 5):

Eq. 4

Eq. 5

When replacing these parameters in Eq. 1, we obtain the following simplified equation (Eq. 6):

Eq. 6

The values calculated for these new geometry parameters for the different nozzles considered are given in Table 2.

Dimensions of the parameters

Three fundamental dimensions are involved in the parameters listed: the mass dimension M, the length dimension L and the time dimension T. Knowing this, it is possible to build a dimension matrix D containing, for each physical parameter (on the columns) the exponent of each fundamental dimension (on the lines) (Tab. 3).

Table 3: Dimension matrix D, with central matrix C (dark grey) and Residual matrix R (white) ΔP

µ d h l s d b A o A m l o ρ A s M 1 1 0 0 0 0 0 0 1 0 0 L -1 -1 1 1 1 2 2 1 -3 2 3 T -2 -1 0 0 0 0 0 0 0 0 -1

The Vashy-Buckingham theorem

The dimensional analysis procedure used involves the use of the Vashy-Buckingham theorem [START_REF] Buckingham | On physically similar systems; illustrations of the use of dimensional equations[END_REF][START_REF] Vashy | Sur les lois de similitude en physique[END_REF]. This theorem states that as Eq. 6 expresses a relation f between 11 parameters depicted by 3 fundamental dimensions, it is possible to find a correlation F which relates (11-3) = 8 dimensionless numbers. However, it has to be noted that this theorem does not provide the form of the relationship between the dimensionless numbers.

Generation of the dimensionless numbers

Choice of a relevant repeated variables base

In order to generate the dimensionless relation F between all the parameters retained, the choice of a base, composed of repeated variables (opposed to the non-repeated variables, i.e. the variables not included in the base) is of prime importance. The variables of the base must be dimensionally independent, cover all the dimensions, and be of the number of fundamental dimensions (i.e. 3 here). It can be convenient to choose the base so that some of the dimensionless numbers generated (as for example those related to the target variable) have a precise physical meaning without having much recombination operations to perform afterwards. Especially, in the case of the atomization process, the discharge coefficient C D was often used. The general expression for C D is (Eq. 7): Eq. 7

However, in the case of hollow-cone nozzles, the nozzle outlet section A 0 may not be completely filled with the liquid due to the generation of an air core in the spray. C D , as described by Eq. 7, is thus not recommended if a precise air core determination is not performed to allow the determination of the actual outlet section filled by the fluid. In Fluid Mechanics, it is also frequent to use the dimensionless Euler number Eu, defined as the ratio between the pressure forces and the inertial forces [START_REF] Nonnenmacher | Design of hollow cone pressure swirl nozzles to atomize Newtonian fluids[END_REF]. Eu can be calculated in the studied geometry using Eq. 8:

Eq. 8

In order to easily make this number appear in the dimensionless relation F, was chosen here as the base. The matrix defined in this base, highlighted in dark grey in Table 3, is called the central matrix (C) while the remaining matrix composed of the non-repeated variables is denoted the residual matrix (R).

Obtaining of the dimensionless numbers

Once the base chosen, the dimensionless numbers can be built by dividing each non-repeated variable by a product combination of the base variables. The purpose is then to find the adequate exponents a, b and c related to each of the base variables to ensure that, for each non-repeated variable, a dimensionless number Π arises with Π . The modified residual matrix Rm containing a, b and c values was obtained by Gaussian elimination (Eq. 9):

Eq. 9

The modified residual and central matrices arising are given in Table 4. Consequently, the dimensionless relation F is of the form depicted by Eq. 10.

ΔP µ d h l s d b A o A m l o ρ A s a 1 1 0 0 0 0 0 0 1 0 0 b -2 -0.5 0.5 0.5 0.5 1 1 0.5 0 1 0 c 2 1 0 0 0 0 0 0 0 0 1
Eq. 10

For concision concerns, the generated dimensionless numbers in Eq. 10 will be noted Π i with the i index corresponding to their position in Eq. 9 (for example, ).

Reorganization of the dimensionless numbers and expression of the dimensionless function

The chosen base allowed the direct appearance of the Eu number as Π 1 . Π 2 and Π 3 were also combined to make appear another physically relevant dimensionless number, the Reynolds number inside the distributor slots Re s (Eq. 11). Re s was substituted to Π 2 in F.

Eq. 11

By combining Π 3 and Π 5 , a dimensionless number characterizing the nozzle geometry also appeared (Eq. 12) and was substituted to Π 5 in F: Eq. 12 Eq. 10 for F therefore turned into Eq. 13 with 8 dimensionless numbers as stated by the theorem of Vashy-Buckingham:

Eq. 13

At this stage of the dimensional analysis, it is mandatory to propose a mathematical form for the relation F. As proposed by several authors [START_REF] Lallemand | Écoulement des fluides -Analyse dimensionnelle[END_REF]Scale-Up in Chemical Engineering, 2006;[START_REF] Szirtes | Applied dimensional analysis and modeling[END_REF], it was assumed that a monomial relation is well suited to the analysis on a spraying process. According to Eq. 13, this monomial relation is presented in Eq. 14, where the coefficients a 0 to a 7 are constant. They will be identified from experimental measurements of P in a wide range of conditions.

Eq. 14

Results

Influence of the fluid properties, flow rate and nozzle geometry on P

The evolution of the pressure drop P in the nozzle was measured over the range of liquid flow rate available (up to 50 L.h -1 ) changing the sprayed liquid (pure water or maltodextrin solutions) and the nozzle geometry (Fig. 3a). As expected, P depends on the nozzle geometry and increases more As suggested by the dimensionless relation obtained (Eq. 13), experimental results can also be plotted in a dimensionless space Eu vs. Re s (Fig. 3b). In this case, experiments conducted with the lowest viscosity products and highest volumetric flowrates correspond to the highest Re s , while experiments performed with the most viscous solutions correspond to the lowest Re s . As expected when considering Eq. 14, the trend observed for a specific nozzle is similar whatever the nozzle considered is, with an offset between the experimental data sets directly and solely related to the geometric parameters. Especially, for all the trends, two ruptures are observed at similar Re s values, i.e. at Re s about 200 and Re s about 1000 respectively. This similarity allows the distinction between three different atomization zones depending on the Re s value:

 Zone 1 for Re s <200;  Zone 2 for 200 <Re s <1000;  Zone 3 for Re s > 1000.

It is noteworthy that, in Zone 3, the values obtained for Eu are independent of Re s (therefore of the hydrodynamics in the throttle) and thus only depend on the geometry of the nozzle. Knowing that, a normalized Euler number Eu* was introduced (Eq. 15): Equation 15Where is the mean value of Eu in Zone 3 for the corresponding nozzle geometry. When plotting the experimental data set in the new dimensionless space Eu* vs. Re s , they all fall into a single master curve, where the three zones previously identified still appear (Fig. 4). The use of Eu* instead of Eu, thus allows to represent the impact of both the hydrodynamics and the geometry on a single curve. This validates that the offset observed in Figure 3b was actually due to the nozzle geometry and confirms the relevance of taking a monomial form for F. These results constitute a strong basis to propose ready-to-use relations for atomization set-up sizing when using swirl nozzles with throttled inlets.

Identification of coefficients of the dimensionless function

As it is confirmed that hydrodynamics have small or no influence on the Eu values obtained in Zone 3 and that the geometric parameters should be of the same influence independently on the zone considered, it was chosen to first identify the coefficients related to the geometry on the results obtained in Zone 3, and then to identify the ones related to Re s on the measurements corresponding to the other zones. The relation to be identified in Zone 3 is thus (Eq. 16):

Eq. 16

Through the multiple regression methodology mentioned in part 2.4, the values of the seven b i coefficients in Eq. 16 were obtained with a correlation coefficient R²=0.98. However, the corresponding p-values obtained with the statistical analysis showed that the coefficients b 1 and b 4 were not significant. Eq. 16 was therefore simplified and an identification procedure on Eq. 17 was performed. Values of the c i coefficients and of the p-values obtained are presented in Table 5. The correlation coefficient R² was also equal to 0.98, confirming both the adequacy between the dimensionless relation obtained and the experimental data in Zone 3 and the small influence of the dimensionless numbers associated to b 1 and b 4 .

Eq. 17 The values of these coefficients identified in Zone 3 were then used to identify the rest of the coefficients for the two other zones. The dimensionless relations obtained for each of the three zones identified are presented in Eq. 18, 19 and 20. The correlation coefficients obtained are, and R² = 0.96 for Zone 1, and R² = 0.94 for Zone 2, indicating that the monomial relations obtained give a fairly accurate description of the atomization phenomenon over the whole range of conditions studied.

Zone 1

Res < 200

Eq. 18

Zone 2

200< Res < 1000

Eq. 19

Zone 3

Res > 1000

Eq. 20

Discussion

Validity of the relations obtained

Eq. 18, 19 and 20 obtained from the experimental data set can strictly be applied in the data range where the measurements were performed (Fig. 5). Another information useful for the experimenter is the deviation expected between the actual values and the theoretical ones estimated using these relations. When plotting the values actually measured against the ones estimated in the three Re s zones (Fig. 5), almost all the values fall between the range ± 20%.

Identification of the optimal spraying conditions

Spraying regimes

In the case of swirl nozzles, several authors observed and described the existence of different spraying regimes depending on the liquid flowrate [START_REF] Ballester | Drop size measurements in heavy oil sprays from pressure-swirl nozzles[END_REF][START_REF] Cousin | Recent developments in simulations of internal flows in high pressure swirl injectors[END_REF][START_REF] Lefebvre | Atomization and sprays[END_REF]. These regimes were observed in the case of water spraying with the nozzle 4d, when progressively increasing the liquid flow rate to the maximal possible value (Fig. 6). At the lowest flowrates, the liquid tends to form a jet that fragments into large drops. The nozzle orifice is here completely filled with the liquid. As the flowrate is increased, the jet turns into an onionshaped spray and then a tulip-spray. The droplets are getting finer and finer, the spray angle increases and the orifice section filled by the liquid is smaller and smaller. Ultimately, for the highest flowrates, a constant spray flowrate angle is obtained, and the length of the intact liquid sheet at the nozzle outlet decreases until turning invisible: this is the atomization regime. The orifice section filled by the liquid is then constant and at its lowest value. It has to be noted that a similar evolution was observed with all the nozzles considered in this study. These four regimes are consistent with the information that can be found in the literature. It is interesting here to relate these regimes, and the degree of filling of the orifice, to the three atomization zones identified through the dimensionless representation of our experimental data (Fig. 7). Actually, the evolution of the orifice filling is due to the generation of an air core when the value of the Reynolds number Re s increases.

At low Reynolds number (Zone 1), the effect of the friction forces has a significant impact on the flow and, compared to the centrifugal forces in action inside the nozzle, they are too important to allow the development of an air core. This regime is to be avoided by all means since the viscous forces, compared to the other forces at stake, are too important and will not allow the generation of a spray. As a consequence, a decrease in the Eu value while increasing the Re S was observed in Zone 1. This evolution was also reported in the literature by the means, for example, of the evolution of the discharge coefficient with increasing viscosity at constant spraying pressure [START_REF] Bayvel | Liquid atomization[END_REF].

While increasing the Reynolds number, the centrifugal forces increasingly take over the friction forces up to a point in which the increase in the Reynolds number allows the generation of an air core of an increasing size: this is the regime observed in Zone 2. The existence of a range of Re s values in which Eu is increasing before reaching a plateau was also reported in the literature for swirl nozzles, either by the through the evolution of Eu [START_REF] Nonnenmacher | Design of hollow cone pressure swirl nozzles to atomize Newtonian fluids[END_REF] or C D [START_REF] Halder | Initiation of air core in a simplex nozzle and the effects of operating and geometrical parameters on its shape and size[END_REF][START_REF] Lee | Measuring air core characteristics of a pressure-swirl atomizer via a transparent acrylic nozzle at various Reynolds numbers[END_REF]. Anyway, in Zone 2, the onion-shaped and tulip shaped sprays give rise to liquid droplets with a coarse size distribution.

In Zone 3, the increase of the swirl is almost perfectly compensated by the resistance due to the friction forces at the outlet: the air core thus keeps a constant shape independently of the Reynolds value. This behavior in Zone 3, corresponding to the atomization regime, is very much recommended for performing reliable atomization trials with minimal energy loss and narrow particle size distribution. The independency of Eu vs. Re s at high Reynolds values was also confirmed in several studies with swirl nozzles [START_REF] Halder | Initiation of air core in a simplex nozzle and the effects of operating and geometrical parameters on its shape and size[END_REF][START_REF] Chu | An experimental investigation of swirl atomizer sprays[END_REF][START_REF] Datta | Numerical prediction of air core diameter, coefficient of discharge and spray cone angle of a swirl spray pressure nozzle[END_REF].

A similar overall behavior was already reported by [START_REF] Halder | Initiation of air core in a simplex nozzle and the effects of operating and geometrical parameters on its shape and size[END_REF] in the case of tangential inlet swirl atomizer. The authors also described the existence of two limiting Reynolds numbers: beyond the smaller one there is no air core initiation and above the larger one, the air core reaches a stable development. Despite the geometry studied by the above mentioned authors was different from the one studied in this paper, the physical underlying principles are similar and support the findings of the present study for axial inlet pressure swirl nozzles with throttle inserts.

Atomization regime and dimensionless function

As previously mentioned, Zone 3 is highly recommended when it comes to perform efficient liquid atomization. The main question arising thus concerns the conditions needed to reach this specific zone. Through the use of the dimensional analysis performed, it is possible to clearly quantify the minimal flowrate needed to reach this zone for a specified product and geometry. According to Fig. 3, the condition to reach Zone 3 entirely lies in the value of Re s , that should be higher than 1000 (Eq. 11 and Eq.20). If we call the minimal flowrate necessary to reach the atomization regime, we obtain the following relation (Eq. 21):

Eq. 21

Then, using Eq. 13 and Eq. 20, it is possible to easily access the corresponding minimal pressure loss expected in atomization regime. Table 5 shows some results obtained with solutions 1 and 2 and nozzles 1a, 2b, 3c, 4d. Similar calculations were also performed for zone 2 using Eq. 13 and Eq. 19. The geometry has an obvious influence on the minimal flowrate needed to reach Zone 3, however the main point of vigilance lies in the fluid properties to be sprayed, especially the dynamic viscosity. While the density between solution 1 and 2 is almost similar (Table 1), the dynamic viscosity is 20 times higher for solution 2. This consequently multiplies the necessary flowrate to reach zone 3 by approximately 20, and thus the resulting pressure loss which is around 400 times higher. That means it is not possible to reach the Zone 3 with a 20 mPa.s viscosity solution with the pilot plant used in this study. For a moderately viscous fluid sprayed on these nozzles, depending on the pumping device capacity, atomization regime is thus not guaranteed. This fact legitimates the identification of Eq. 19, which can be of great use. Actually, the pressure loss to enter Zone 2 (between 20 and 30 bar for solution 2) is in that case in accordance with the capacity of the pilot plant used here. The corresponding minimum flow rate is also significantly smaller. Concerning Zone 1, according to our observations, the regime related to this zone does not correspond to an acceptable atomization.

Drying experiments

Beyond the identification of ideal spray conditions, it is also important to know their impact on the size distribution of the droplets generated. Especially, in the spray drying process, it has a great impact on the dry particle size distribution and on the final powder properties.

Whilst the measurement of the size distribution of the liquid drops generated at the nozzle outlet is not simple, the measurement of the size distribution of the particles obtained after spray drying maltodextrin solutions is relatively simple (laser granulometry).

Some spray drying trials were therefore performed to observe the impact of some parameters on the evolution of the final mean Sauter diameter of the powder (d 32 ). The dryer used was a TechniProcess SDF6 (evaporation capacity of 22 kg.h -1 ). Atomization regime was visually assessed through a transparent wall. All the drying experiments were performed using the same conditions for drying air (flowrate 550 kg.h -1 with inlet and outlet temperature respectively equals to 180°C and 80°C). The mean Sauter diameter of the recovered powder was assessed through SLS measurements (MasterSizer 2000, Malvern Instruments, UK). Two nozzles (1a, 2b) and four MD DE 15 solutions with different concentration were spray dried modifying the liquid flowrate in order to cover Re s values between about 80 and 2000. The operating conditions (mass flowrate , Re s value) and the obtained d 32 values are presented in Table 6. Droplets generated at low Reynolds (Zone 1) were too big to be efficiently dried, resulting in no powder production. This behavior is in accordance with the previous description of atomization in Zone 1. For Re s number higher than 200, at constant geometry, the effect of an increase in Re s is quite clear: the higher Re s , the lower d 32 . At constant flowrate and product, a change in geometry will induce a change in Re s , and thus in powder size.

While deeper investigation has to be proceeded in order to give more insight of the influence of some parameters (particularly the surface tension) over the size distribution obtained after spraying and then after drying, these preliminary results give interesting hints of the influence of the hydrodynamics on the average size of the powder achievable with such nozzles.

Conclusion

Through the means of a comprehensive dimensional analysis methodology, this study overall provided a set of quantitative relations allowing the prediction of the spraying pressure as a function of the relevant fluid properties, process operating conditions and nozzle geometric parameters for axial inlet swirl nozzles with throttle inserts. The mathematical relations obtained describes the influence of hydrodynamics (through the Reynolds number inside the slots) and geometric parameters over the normalized Euler number, chosen to represent the target parameter (i.e. the spraying pressure).

To identify the coefficients of the model, an experimental campaign (264 trials) was led on a pilotscale plant with solutions of maltodextrin DE 15 as model fluids exhibiting a Newtonian rheological behavior. Also, a full geometric characterization of different pressure swirl nozzles was done to have a comprehensive access to the relevant geometric properties of the nozzle (initially 7 parameters).

Three distinct spraying zones were identified. They mainly depend on the relative importance of the friction forces over the centrifugal ones. The zone above Re s = 1000 corresponds to the full atomization zone, where the conversion of pressure to kinetic energy is almost total (meaning no influence of Re s ). The relations identified can be used to precisely determine the conditions needed (especially the minimal volumetric flowrate) to achieve efficient atomization (meaning entering Zone 3) and estimate the corresponding spraying pressure.

This study also shows the benefits of a rigorous dimensional analysis approach for problems where numerous parameters have to be taken into account, allowing to obtain dimensionless relations easily usable by the scientist. It is general enough to be accurate in a large range of process conditions, fluids and geometric parameters. Such an approach is also suited to predict the influence of the process, geometric and fluid parameters over other relevant atomization parameters, as for example a characteristic droplet size.

Notations a i

Constant (i from 0 to 7) (-) A o

Outlet section (m²) A m

Orifice minimal section (m²) A s

Total section of the slots (m²) b i

Constant (i from 0 to 6) (-) c i Constant (i from 0 to 4) (-)

C

  Figure 1: Scheme (a) and geometric parameters (b) of WDA hollow cone pressure nozzles with axial liquid inlet used



  Material-related variables: Fluid dynamic viscosity µ, fluid density ρ;  Process-related variables: Volumetric flow rate ;  Nozzle geometry-related variables: see Tab. 2a and 2b.

Figure 3 :

 3 Figure 3: Evolution of the pressure drop in the nozzle (P) measured changing the sprayed liquid (20°C) and the liquid flow rate for the different nozzle geometries in the dimensional space ( vs. ) (a); and in the dimensionless space (Eu vs. Re s ) (b).

Figure 4 :

 4 Figure 4: Experimental points represented in the dimensionless space Eu* vs. Re s

  Figure 5 thus gives to the experimentalist willing to use the formula extrapolated in this study an operating diagram with a fairly comprehensive cartography of the accuracy and range of validity of the equations deduced.

Figure 5 :

 5 Figure 5: Operating diagram: Experimental Eu vs. estimated Eu values using Eq.18, 19 and 20 and validity domain of the correlations

Figure 6 :Figure 7 :

 67 Figure 6: Spraying regimes observed when increasing the liquid flow rate (nozzle 4d, water at T=20°C)

Table 1

 1

	: Measured value of the maltodextrin solutions properties (20°C) (a) and of the geometric
	parameters for the different nozzle inserts (b) and orifice discs used (c)
	a.
	Solution	1	2	3	4
	MD concentration (kg/kg sol.)	0	0.35	0.42	0.47
	Dynamic viscosity µ (x10 -3 Pa.s)	1	20	40	70
	Density ρ (kg.m -3 )	1000	1100	1150	1155
	b.
	Insert	1	2	3	4
	w s (x10 -3 m)	0.369 0.239 0.228 0.468
	d s (x10 -3 m)	0.422 0.387 0.490 0.404
	l s (x10 -3 m)	1.5	1.5	0.675 0.675
	n s (-)	4	4	6	6
	d b (x10 -3 m)	3.342 3.342 3.902 3.902
	c.
	Orifice disc	a	b	c	d
	d 0 (x10 -3 m)	0.623 0.942 0.962 0.934
	d m (x10 -3 m)	0.513 0.523 0.592 0.634
	l o (x10 -3 m)	0.413 0.322 0.138 0.227

Table 2 :

 2 Calculated nozzle geometry parameters

	1a	2b	3c	4d	1d	1b	1c	2a	2c	3a	4c
	-3 m) d h (x10 -6 m²) A s (x10 -6 m²) A o (x10 A m (x10 -6 m²) 0.207 0.215 0.394 0.295 0.623 0.370 0.305 0.697	0.311 0.670 0.727 0.275	0.434 1.134 0.685 0.316	0.394 0.623 0.685 0.316	0.394 0.623 0.697 0.215	0.394 0.623 0.727 0.275	0.295 0.370 0.305 0.207	0.295 0.370 0.727 0.275	0.311 0.670 0.305 0.207	0.434 1.134 0.727 0.275

Table 4 :

 4 Modified residual Rm and central (in grey) matrices in the repeated variables base

Table 5 :

 5 Values of coefficients c i in Eq. 17 identified from experimental points in Zone 3 and corresponding p-values

		c 0	c 1	c 2	c 3	c 4
	Value (-)	120.54	-1.88	0.47	-0.56	-0.09
	p-value (-)	3.29*10 -30	6.36*10 -28	8.18*10 -22	7.44*10 -14	2.75*10 -5

Table 5 :

 5 Minimal volumetric flowrate and corresponding spraying pressure to reach Zone 2 and 3

			Zone 2		Zone 3
	Solution	Geometry	(L.h -1 )	(bar)	(L.h -1 )	(bar)
		1a	1.1	0.08	5.7	1.9
	1	2b 3c	0.9 1.6	0.07 0.07	4.5 7.8	1.7 1.8
		4d	1.9	0.06	9.4	1.6
		1a	20.7	27.6	103.5	689.7
	2	2b 3c	16.4 28.2	25.2 26.8	82.1 141.0	630.9 669.1
		4d	34.2	23.4	171.0	586.0

Table 6 :

 6 Drying experiments performed using nozzles 1a, 2b and MD DE 15 solutions

	Nozzle	MD concentration (kg/kg sol.)	μ (Pa.s)	(kg.s -1 ) Re s (-) Zone d 32 (µm)
	1a	0.47	0.07	33.0	83	1	-
	1a	0.35	0.02	24.6	216	2	69
	2b	0.35	0.02	24.6	272	2	52
	1a	0.25	0.004	21.2	931	2	52
	1a	0.1	0.002	17.5	1555	3	39
	2b	0.1	0.002	17.8	1971	3	30

Acknowledgements

The authors would like to thank TechniProcess for funding the PhD grant of V. Pistre.