Differentiation of somatic cells in the ovariuteri of the apoikogenic scorpion Euscorpius italicus (Chelicerata, Scorpiones, Euscorpiidae)

Arthropod Structure & Development xxx (2013) 1e10

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Differentiation of somatic cells in the ovariuteri of the apoikogenic scorpion Euscorpius italicus (Chelicerata, Scorpiones, Euscorpiidae) Izabela Je˛ drzejowska*, Kamil Szymusiak, Marta Mazurkiewicz-Kania, Arnold Garbiec Institute of Experimental Biology, University of Wrocław, Sienkiewicza 21, 50-335 Wrocław, Poland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 July 2013 Received in revised form 22 November 2013 Accepted 23 November 2013

In apoikogenic scorpions, growing oocytes protrude from the gonad (ovariuterus) and develop in follicles exposed to the mesosomal (i.e. hemocoelic) cavity. During subsequent stages of oogenesis (previtellogenesis and vitellogenesis), the follicles are connected to the gonad surface by prominent somatic stalks. The aim of our study was to analyze the origin, structure and functioning of somatic cells accompanying protruding oocytes. We show that these cells differentiate into two morphologically distinct subpopulations: the follicular cells and stalk cells. The follicular cells gather on the hemocoelic (i.e. facing the hemocoel) surface of the oocyte, where they constitute a cuboidal epithelium. The arrangement of the follicular cells on the oocyte surface is not uniform; moreover, the actin cytoskeleton of these cells undergoes significant modifications during oocyte growth. During initial stages of the stalk formation the stalk cells elongate and form F-actin rich cytoplasmic processes by which the stalk cells are tightly connected to each other. Additionally, the stalk cells develop microvilli directed towards the growing oocyte. Our findings indicate that the follicular cells covering hemocoelic surfaces of the oocyte and the stalk cells represent two distinct subpopulations of epithelial cells, which differ in morphology, behavior and function. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Follicular cells Stalk cells Chelicerate type ovary Epithelial morphogenesis Cellecell adhesion

1. Introduction Animal eggs are produced in the course of the process called oogenesis. During oogenesis oocytes undergo remarkable growth which proceeds in two consecutive stages: previtellogenesis and vitellogenesis. Previtellogenic growth results from the accumulation of organelles and macromolecules, whereas vitellogenic growth relies on storage of various reserve materials such as glycoproteins, lipids and glycogen in the oocyte cytoplasm (ooplasm). The oocytes growth is very often supported by additional cells of germline and/or somatic origin. The germline cells supporting the oocyte growth are called trophocytes or nurse cells. Comparative studies of the nurse cells functioning in insects have revealed that the main function of the nurse cells is the synthesis of macromolecules and their transport, together with organelles, to the oocyte cytoplasm (Büning, 1994; de Cuevas et al., 1997; Matova and Cooley, 2001; Mazurkiewicz and Kubrakiewicz, 2001; Tworzyd1o and

* Corresponding author. Department of Animal Developmental Biology, Institute of Experimental Biology, University of Wroc1aw, Sienkiewicza 21, 50-335 Wroc1aw, Poland. Tel.: þ48 71 3754027; fax: þ 48 71 3754118. E-mail addresses: [email protected] (I. Je˛ drzejowska), kamil. [email protected] (K. Szymusiak), [email protected] (M. Mazurkiewicz-Kania), [email protected] (A. Garbiec).

Kisiel, 2010). The somatic cells accompanying growing oocytes are usually referred to as the follicular cells. Numerous comparative studies of different animal groups have shown that the follicular cells may exhibit different organization and play diverse functions. In most cases, the follicular cells constitute a simple epithelium overlying either the germline clusters or individual oocytes. It has been found that among many functions, in invertebrates, the follicular cells may contribute to vitellogenesis and formation of the eggshell (reviewed in Dobens and Raftery, 2000). Moreover, based on studies of insect ovaries, it has been shown that the follicular cells are able to diversify into several distinct subpopulations that differ in morphology, behavior, function and position in relation to the germline cells (Zawadzka et al., 1997; Deng and Bownes, 1998; Dobens and Raftery, 2000; Kubrakiewicz et al., 2003; Mazurkiewicz _ and Kubrakiewicz, 2005, 2008; Tworzyd1o et al., 2005; Zelazowska, 2005; Ogorza1ek, 2007; Jaglarz et al., 2008, 2009, 2010; Garbiec and Kubrakiewicz, 2012). Among chelicerates, nutrimentary, i.e. supported with nurse cells, egg development is unique for some acarine groups (Alberti  ski et al., and Hänel, 1986; Alberti and Zeck-Kapp, 1986; Witalin 1990; Alberti and Coons, 1999; Di Palma and Alberti, 2002). In scorpions, like in most chelicerates, all germline cells differentiate into the oocytes and the oocytes growth is supported solely by the somatic cells.

1467-8039/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.asd.2013.11.004

Please cite this article in press as: Je˛ drzejowska, I., et al., Differentiation of somatic cells in the ovariuteri of the apoikogenic scorpion Euscorpius italicus (Chelicerata, Scorpiones, Euscorpiidae), Arthropod Structure & Development (2013), http://dx.doi.org/10.1016/j.asd.2013.11.004

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In scorpions, characteristic of almost all chelicerates, the oocytes bulge out from the gonad into the mesosomal cavity (hemocoel), and grow connected to the wall of the gonad by means of groups of somatic cells forming the oocyte stalks. This type of the ovary organization has been defined as the chelicerate type, in a contrary to mandibulate one, found e.g. in insects, in which the oocytes lack stalks and grow among the somatic cells inside the gonad (Makioka, 1988; Ikuta and Makioka, 1999). In chelicerates fertilization has been found to take place in different sites of the female reproductive system. For instance intraovarian fertilization has been reported in acarine and scorpions (Alberti and Coons, 1999; Di Palma and Alberti, 2002; Alberti and Michalik, 2004). In contrast to other arachnids, the scorpion female gonads are organs in which not only fertilization but also embryonic development take place. For these reasons the scorpion female gonads have been termed ovariuteri (Farley, 1999). Moreover, the structure of the ovariuterus is unique for scorpions and thus dissimilar to other arachnids. In all non-scorpion arachnids the ovaries are either paired or unpaired tubular structures (Makioka, 1988; Foelix, 1996; Alberti and Coons, 1999; Coons and Alberti, 1999; Felgenhauer, 1999), whereas the scorpion ovariuterus consists of a tubular network composed of longitudinal and transverse tubules that form different numbers of “cells” (Matthiesen, 1970; Francke, 1982; Hjelle, 1990; Sissom, 1990; Farley, 1999). It has been found that the scorpion ovariuteri exhibit several structural modifications with respect to the mode of embryonic development. In scorpions, two distinct patterns of embryonic development have been distinguished: apoikogenic and katoikogenic (Laurie, 1890, 1896). In the apoikogenic type, the oocytes grow in follicles exposed to the mesosomal (hemocoelic) cavity, connected to the wall of the ovariuterus by the stalk cells. The oocytes are rich in yolk. Embryonic development occurs inside the ovariuterine tubules, and embryos are nourished by yolk deposited in the oocyte cytoplasm during the course of oogenesis. In the katoikogenic type, both oocyte growth and embryo development occur inside diverticula that originate from outpocketings of the ovariuterine tubules. In the katoikogenic type the oocytes are poor in reserve materials, and the embryos are nourished via a placentalike organ. Despite that apoikogenic and katoikogenic types of embryonic development were distinguished over a century ago, to our knowledge there are only a few contributions presenting data on morphology and ultrastructure of somatic components of scorpion female gonads. Among apoikogenic scorpions such data come from studies of scorpions from family Buthidae (Warburg and Elias, 1998), Vaejovidae (Farley, 1998; Warburg and Rosenberg, 1996), and Euscorpiidae (Soranzo et al., 2000). In this paper, we present the results of histological, histochemical and ultrastructural analyses of somatic cell differentiation in the apoikogenic ovariuterus of the euscorpiid scorpion, Euscorpius italicus. The relationship and function of the somatic cells accompanying growing oocytes are discussed. 2. Material and methods The materials for this study were commercially purchased female specimens of the scorpion, E. italicus (Herbst, 1800) (Scorpiones, Euscorpiidae) at different nymphal stages (in the third, fourth and sixth instars). Soon after the specimens were obtained they were anaesthetized with chloroform and dissected. 2.1. Light and transmission electron microscopy For histological and ultrastructural observations, the material was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer

(pH ¼ 7.4) for 24 h at þ4  C. Then the material was rinsed three times in phosphate buffer and postfixed in a mixture containing 1% osmium tetraoxide and 0.8% potassium ferrocyanide (according to McDonald, 1984) for a better tissue preservation and contrast. An acetone series was used for material dehydration. The ovariuteri were embedded in Epon 812 (Serva, Heidelberg, Germany). Semithin sections (0.6 mm) were stained with 1% methylene blue in 1% borax and observed in an Olympus BHS light microscope. Ultrathin sections were contrasted with lead citrate and uranyl acetate (Reynolds, 1963) and examined in a Zeiss EM 900 at 80 Kv. For gross morphology analysis, fixed ovariuteri were viewed with the Olympus BHS light microscope equipped with Nomarski optics. 2.2. Histochemical analyses In order to detect microfilaments (F-actin) and DNA, the ovariuteri were fixed in 4% formaldehyde in phosphate-buffered saline PBS (NaCl,137 mM; KCl, 2.7 mM; Na2HPO4, 8 mM; KH2PO4,1.5 mM) for 1 h and then thoroughly rinsed in PBS. For microfilament detection, the material was stained with 2 mg/ml rhodamine-conjugated phalloidin (Sigma Chemical Co., St. Louis, MO, USA) for 20 min in darkness. Then the material was rinsed repeatedly in PBS and for DNA detection was stained with DAPI (40 ,6-diamidino-2 phenylindole dihydrochloride) (0.2 mg/ml; Sigma Chemical Co., St. Louis, MO, USA) for 20 min in darkness. Following several rinses in PBS, whole-mounted, stained ovariuterine tubes were examined with an Olympus FluoView 1000 confocal microscope. 3. Results 3.1. Gross architecture of the ovariuterus In E. italicus, the ovariuterus shows characters typical of nonbuthid scorpion species (Volschenk et al., 2008). Namely, it consists of three longitudinal and six transverse tubules, forming a sixcelled network. The ovariuterus is of apoikogenic type. In ovariuteri the oocytes grow in follicles exposed to the mesosomal (hemocoelic) cavity and remain connected to the ovariuterine wall by stalk cells. The follicles develop asynchronously and thus contain oocytes in various stages of oogenesis. 3.2. Structure of the ovariuterus in the early stages of development In early developmental stages (the third instar), longitudinal and transverse parts of the ovariuterus consist of a cylindrical mass of somatic and germline cells which considerably differ in morphology. Germline cells (oogonia, oocytes) are bigger and contain spherical nuclei. Somatic cells are smaller and contain elongate nuclei (Figs. 1A and 2A). Among somatic cells two types can be distinguished: epithelial cells and interstitial cells. The epithelial cells occupy peripheral parts of the ovariuteri and adhere to the basal lamina. The basal lamina at this stage of development constitutes the outermost layer of the ovariuterus, whereas in consecutive stages of gonad development muscle tissue appears on the surface of the epithelium (see Results 3.5). The interstitial cells take the internal parts of the ovariuteri and do not contact with the basal lamina (Figs. 3C and 4A). The germline cells are located among somatic cells on the ventral part of the ovariuterus, whereas the dorsal part of the ovariuterus is occupied only by somatic cells (Fig. 2A). Initially, the ovariuterus lacks a lumen. The latter appears during subsequent stages of gonad development (for more details of lumen formation see Results 3.5).

Please cite this article in press as: Je˛ drzejowska, I., et al., Differentiation of somatic cells in the ovariuteri of the apoikogenic scorpion Euscorpius italicus (Chelicerata, Scorpiones, Euscorpiidae), Arthropod Structure & Development (2013), http://dx.doi.org/10.1016/j.asd.2013.11.004

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3.3. Epithelial cells differentiation during the oocyte protrusion In successive stages of development, the structure of ovariuterus changes considerably. The oocytes gradually enlarge due to previtellogenic growth and change position from the interior of the ovariuterus to the mesosomal cavity. Growing oocytes bulge from the ovariuterus accompanied by epithelial cells of the ovariuterus wall. During initial stages of oocyte protrusion some of the epithelial cells are elevated by protruding oocytes and become visible on the hemocoelic surface of the oocytes. At the same time, other epithelial cells, significantly elongate and become columnar. These latter epithelial cells develop among each other fingerlike cytoplasmic processes supported with microfilaments (Figs. 2B and 4A) and will contribute to the oocyte stalk formation, whereas the cells covering the hemocoelic surface of the oocyte will form a follicular epithelium. 3.4. Epithelial cells accompanying growing oocytes 3.4.1. Follicular cells The follicular cells cover the hemocoelic surface of the oocytes. Initially, the number of follicular cells is very low and the follicular cells are very flat with thin and long cellular processes (Fig. 1B, C, 2B, C and 4A). With the progress of previtellogenic growth, the follicular cells divide by mitotic divisions (Fig. 2H) so their number gradually increases. In advanced previtellogenesis and early vitellogenesis, the follicular cells are flat and form a simple epithelium (Figs. 1E, 3B and 4B). The basal parts of the follicular cells adhere to the basal lamina, and their apical parts face the oolemma. Primarily, apical membranes of the follicular cells adhere directly to the oolemma. The membranes of follicular cells remain smooth, while the oolemma forms elaborate microvilli (Fig. 3A). Then, in advanced previtellogenesis, a space between the follicular cells and the oocyte appears. The space becomes gradually filled with a fine fibrous material, a probable precursor of the vitelline envelope (Fig. 3B). Detailed analysis of the follicular cells’ ultrastructure revealed that the follicular cells contain only a few organelles and do not contain submembrane vesicles indicating that they are not engaged in endocytotic and/or exocytotic activity (Fig. 3B). Analysis of cortical actin cytoskeleton of follicular cells showed that the arrangement of follicular cells is not uniform. In advanced previtellogenesis, the follicular cells which encompass the middle

Fig. 1. Morphology of the ovariuterus in consecutive stages of the oocyte growth. Please note that the figures (AeG) are shown in upside down dorsaleventral orientation for better visualization of the oocytes and accompanying cells exposed to the

hemocoel. A. Early stage of ovariuterus development. The ovariuterus houses germline cells surrounded by somatic cells. Note that somatic cells and germline cells differ considerably in morphology. Semithin section stained with methylene blue. Scale bar: 15 mm. B, C. Early previtellogenesis. Oocytes are attached to the ovariuterus by means of short oocyte stalks. The hemocoelic surface of the oocytes is covered by flat follicular cells. At this early stage of the stalk formation the stalks are built by a few columnar stalk cells (encircled in B) with elongate nuclei (encircled in C). Note also that the central part of the stalk (asterisk) is devoid of nuclei. B. Whole mount preparation viewed in Nomarski optics. C. Semithin section stained with methylene blue. Scale bars: 25 mm. D, E. Early vitellogenesis. The follicular cells form a low cuboidal epithelium on the hemocoelic surface of the oocyte. The oocyte stalks are longer in comparison to early previtellogenic stages and composed of rows of columnar stalk cells. The central part of the stalk is denoted by asterisk. D. Whole mount preparation viewed in Nomarki optics. E. Semithin section stained with methylene blue. Scale bars: 50 mm. FeH. Late vitellogenesis. The oocytes are significantly elongated and covered by degenerating follicular cells. The oocyte stalks are long and built of numerous stalk cells. In (G) the vitelline envelope deposited on the oocyte surface is visible. In (H) note the condensed chromatin in the nuclei and the vacuoles in the cytoplasm of degenerating follicular cells. F. Whole mount preparation viewed in Nomarski optics. G. Semithin section stained with methylene blue. Scale bars: 100 mm. H. TEM. Scale bar: 1.7 mm. Abbreviations: bl, basal lamina; dFc, degenerating follicular cells; D, dorsal site of the body; E, epithelial cells of the ovariuterus; Fc, follicular cells; gc, germline cells; L, lumen of the ovariuterus; N, germline cell/oocyte nucleus; Oo, oocytes; Ov, ovariuterus; S, oocyte stalks; Sc, stalk cell; scs, somatic cells; V, ventral site of the body; v, vacuoles; ve-vitelline envelope.

Please cite this article in press as: Je˛ drzejowska, I., et al., Differentiation of somatic cells in the ovariuteri of the apoikogenic scorpion Euscorpius italicus (Chelicerata, Scorpiones, Euscorpiidae), Arthropod Structure & Development (2013), http://dx.doi.org/10.1016/j.asd.2013.11.004

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Fig. 2. Confocal images of the ovariuterus in successive stages of oocyte growth. Dorsal and ventral parts of the body are denoted to indicate the upside down orientation of the figures used for better visualization of the oocytes and accompanying cells exposed to the hemocoel. A. Early stage of the ovariuterus development. The ovariuterus contains germline cells and somatic cells. The germline cells are located at the ventral part of the ovariuterus. Germline cell nucleus encircled with dashed line. The nucleus of the somatic

Please cite this article in press as: Je˛ drzejowska, I., et al., Differentiation of somatic cells in the ovariuteri of the apoikogenic scorpion Euscorpius italicus (Chelicerata, Scorpiones, Euscorpiidae), Arthropod Structure & Development (2013), http://dx.doi.org/10.1016/j.asd.2013.11.004

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part of the oocyte are aligned circumferentially in relation to the oocyte stalk. In contrast, the follicular cells which cover the distant most surface of the oocyte display a radial arrangement (Fig. 2I). With the progress of oogenesis, the oocytes continue to enlarge and change shape from spherical to ovoid (Figs. 1F, and 4C). At the beginning of vitellogenesis, the follicular cells cease mitotic divisions and become significantly stretched. Moreover, in early vitellogenesis, the actin cytoskeleton of follicular cells reorganizes. The microfilaments of the cell cortex are not longer visible, while in the basal cytoplasm of the follicular cells actin stress fibers appear. In the prevailing majority the stress fibers are aligned along the long axes of follicular cells, i.e. circumferentially in relation to the oocyte stalk (Fig. 2J). In advanced stages of vitellogenesis, the follicular cells start to degenerate. The first morphological signs of cell degenerations are observed in follicular cells neighboring the stalk cells (not shown). During successive stages of vitellogenesis the degeneration of follicular cells is massive. The regular arrangement of the follicular cells is lost and the nuclei of follicular cells are irregularly arranged in comparison to earlier stages (Fig. 1G). The chromatin in nuclei of degenerating follicular cells becomes very condensed, while in the cytoplasm numerous vacuoles appear (Fig. 1H).

3.4.2. Stalk cells The stalk cells connect the oocytes to the ovariuterus wall. Analysis of consecutive stages of the stalk formation revealed that the oocyte stalks develop due to the ongoing differentiation of epithelial cells into the stalk cells. In very early stages of the stalk formation, the stalks are composed of a few stalk cells. From the very beginning of the stalk formation the stalk cells are elongated and form among each other fingerlike cytoplasmic processes internally supported by actin filaments (Figs. 2B and 4A). With the progress of oocytes growth the stalks enlarge due to the increasing number of the stalk cells which progressively differentiate from the epithelial cells of the ovariuterus wall. The arrangement of the stalk cells is regular. At the longitudinal sections through the stalks, two layers of the columnar stalk cells joined to each other in the stalk midline are visible (Figs. 1B and 2C, D). At the cross sections through the stalks, the stalk cells exhibit a rosettelike pattern of arrangement (Fig. 2E). Observations of longitudinal and cross sections of the oocyte stalks stained with rhodamineconjugated phalloidin have showed that the central parts of the stalks are rich in actin filaments (Fig. 2D, E).

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In advanced stages of stalk development, when the lumen of the ovariuterus is clearly visible, the stalks are much longer and built of many columnar stalk cells (Figs. 1D, 2E and 4B). Analysis of developing stalk structure has shown that basal parts of the stalk cells adhere to the externally located basal lamina (Fig. 1H), while apical parts of the stalk cells occupy the stalk center (Figs. 3F and 4B). Ultrastructural analysis has revealed that the apical membranes of the stalk cells form elaborate fingerlike cytoplasmic processes, that embed in neighboring cells and by which the stalk cells are tightly connected to each other (Fig. 3F and insert, 4C). The fingerlike cytoplasmic processes are structurally supported by submembrane bundles of actin filaments (Figs. 2F and 4C). In some stalk cells, the apical membranes adhere directly to the oocytes. At the contact sites the stalk cells and the oocyte form microvilli, which interdigitate among each other (Fig. 3E, F). Analysis of confocal images of whole mount preparations stained with rhodamine-conjugated phalloidin showed that the apical parts of the stalk cells which adhere to the oocyte are rich in microfilaments (Figs. 2F and 4B, C). As mentioned above the stalk development relies on ongoing differentiation of epithelial cells into the stalk cells. Observations of the ovariuterus structure during the stalk elongation revealed that the epithelial cells of the ovariuterus which neighbor the forming stalk show resemblance to the stalk cells with respect to their morphology and organization of the actin cytoskeleton. The epithelial cells, not yet involved in the stalk structure, are columnar and their apical parts facing the lumen of the ovariuterus contain considerable amount of actin filaments (Figs. 2F and 4B). These epithelial cells become connected to each other by fingerlike cytoplasmic projections in a zip-like manner leading to the stalk elongation. The fully developed oocyte stalks, in comparison to earlier stages, consist of numerous stalk cells. Both the central part of the stalk and the contact zone between the stalk and the oocyte show distinct fluorescence after staining with rhodamine-conjugated phalloidin indicating the presence of great amounts of submembrane actin filaments. The central actin “rod” of the stalk reaches the lumen of the ovariuterus (Figs. 2G and 4C). From early stages of previtellogenesis till late vitellogenesis the stalk cells do not exhibit any morphological signs of synthetic and secretory activity. 3.5. Somatic cells of the ovariuterus wall The structure of somatic cell components of the ovariuterus wall changes with successive stages of development. In the early stages of gonad development, the ovariuterus contains the epithelial cells

cell is encircled with a solid line. Whole mount preparation stained with rhodamine-conjugated phalloidin and DAPI viewed in confocal microscopy. Scale bar: 20 mm. B. Early stage of the oocyte protrusion to the mesosomal cavity. The hemocoelic surface of early previtellogenic oocyte is covered by the flat follicular cell. Some of the epithelial cells, i.e. the future stalk cells, are significantly elongated and join each other by F-actin rich cellular processes (asterisk). Oocyte nucleus is encircled with dashed line. Whole mount preparation stained with rhodamine-conjugated phalloidin and DAPI viewed in confocal microscopy. Scale bar: 25 mm. C, D. Previtellogenic oocyte exposed to the mesosomal cavity. The hemocoelic surface of the oocyte is covered by follicular cells and attached to the ovariuterus by a short oocyte stalk. The stalk is composed of columnar cells with elongate nuclei. The central part of the stalk, contains F-actin rich core (asterisk) of cytoplasmic processes. Arrow indicates the follicular cell nucleus. Whole mount preparation, longitudinal section stained with DAPI (in C) and rhodamine-conjugated phalloidin (in D) viewed in confocal microscopy. Scale bar: 50 mm. E. The previtellogenic oocyte stalk viewed in cross section. The stalk cells are columnar and arranged in a rosette-like pattern. The central part of the stalk is occupied by F-actin rich apical parts of the stalk cells (asterisk). Whole mount preparation stained with rhodamine-conjugated phalloidin viewed in confocal microscopy. Scale bar: 5 mm. F. An oocyte in advanced previtellogenesis attached to the ovariuterine tube by the oocyte stalk. Apical parts of the stalk cells facing the oocyte (dashed rectangular), the central part of the stalk (asterisk), and the apical parts of the epithelial cells which neighbor the stalk (dashed ovals) contain a significant amount of microfilaments. Whole mount preparation stained with rhodamine-conjugated phalloidin viewed in confocal microscopy. Scale bar: 50 mm. G. The haemocoelic surface of vitellogenic oocyte is covered by a large number of follicular cells. The stalk connects the oocyte with the ovariuterine tube. The central part of the stalk (asterisk) and the contact zone between the stalk cells and the oocyte (dashed rectangular) are rich in F-actin. The apical part of the stalk is wider than its basal part that is covered by muscle tissue. Whole mount preparation stained with rhodamine-conjugated phalloidin and DAPI viewed in confocal microscopy. Scale bar: 100 mm. H, I. Follicular cells on the hemocoelic surface of oocytes in advanced previtellogenesis. A follicular cell in the mitotic division. Note distinct organization of the follicular cells indicated by white lines. The follicular cells located on the most distant part of the oocyte follicular cells are arranged radially, whereas in the middle part of the oocyte they are oriented circumferentially in relation to the oocyte stalk. Whole mount preparation stained with rhodamine-conjugated phalloidin and DAPI viewed in confocal microscopy. H. Scale bar: 25 mm. I. Scale bar: 100 mm. J. Follicular cells on the hemocoelic surface of early vitellogenic oocyte. The follicular cells are significantly elongate with elongate nuclei. In basal cytoplasm of follicular cells actin stress fibers (arrows) are visible. The stress fibers are arranged circumferentially in relation to the oocyte stalk. Whole mount preparation stained with rhodamine-conjugated phalloidin and DAPI viewed in confocal microscopy. Scale bar: 25 mm. Abbreviations: dc, mitotic division of follicular cells; D, dorsal site of the body; Fc, follicular cells; gc, germline cells; L, lumen of the ovariuterus; Mu, muscle tissue; N, germline cell/oocyte nucleus; NFc, follicular cell nucleus; Oo, oocytes; Ov, ovariuterus; S, oocyte stalks; Sc, stalk cells; scs, somatic cells; V, ventral site of the body.

Please cite this article in press as: Je˛ drzejowska, I., et al., Differentiation of somatic cells in the ovariuteri of the apoikogenic scorpion Euscorpius italicus (Chelicerata, Scorpiones, Euscorpiidae), Arthropod Structure & Development (2013), http://dx.doi.org/10.1016/j.asd.2013.11.004

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Fig. 3. Structure of the follicular cells (A, B), wall of the ovariuterus (C, D) and the stalk (E, F) in consecutive stages of oocyte growth. A. Follicular cells on the hemocoelic surface of early previtellogenic oocyte. Basal membranes of follicular cells are externally supported by the basal lamina. Apical membranes of follicular cells adhere to the oolemma which form microvilli. TEM, Scale bar: 1.1 mm. B. Follicular cells on the hemocoelic surface of the late previtellogenic oocyte. The space between the oocyte and follicular cells is occupied by

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and interstitial cells. Initially, the epithelial cells and the interstitial cells show morphological resemblance, they are small and elongated (Figs. 1A and 2A). During oocyte growth the epithelial cells which do not protrude to the mesosomal cavity increase in size, whereas the interstitial cells start to degenerate. We have observed the interstitial cells with characteristic electron-dense dark nuclei typical of degenerating cells in the central part of the ovariuterus wall (Fig. 3C). With the progress of oocytes growth the interstitial cells are no longer visible. The ovariuterus gains the lumen and the wall of the ovariuterus thickens. The epithelial cells of the ovariuterus wall form a simple pseudostratified epithelium which lines the lumen of the ovariuterus (Fig. 3D). Externally, epithelial cells are covered by a bilayered muscle tissue (Fig. 3D). The inner layer of muscle cells shows circular arrangement, whereas the outer one is arranged longitudinally. The muscular layers cover also the lower parts of the oocyte stalks (Fig. 2G). 4. Discussion The chelicerate type of the ovary is characteristic of almost all Cheliceriformes including Pantopoda and Chelicerata (Makioka,  ski, 2006; 1988; Ikuta and Makioka, 1999; Miyazaki and Bilin  ski et al., 2008) and its distinction relies predominantly on the Bilin external position of the stalked oocytes. However, data coming from previously conducted studies indicate that the ovaries of chelicerate type show some characters which appear to be changeable. For instance, chelicerate ovaries may differ in number of somatic cell types and also in the pattern of somatic cells differentiation. In order to complete our knowledge of somatic cells in scorpion gonads, we have analyzed the structure of the apoikogenic ovariuterus in E. italicus. On the basis of our observations we have distinguished four types of ovariuterus somatic cells excluding the muscular tissue: namely the epithelial cells of the ovariuterus, the interstitial cells, the follicular cells and the stalk cells. The follicular cells and the stalk cells accompany growing oocytes exposed to the mesosomal cavity. 4.1. Interstitial cells In the early stages of development, the ovariuterus of E. italicus consists of two types of somatic cells: the epithelial cells and the interstitial cells. When the oocytes start protruding to the mesosomal cavity, the interstitial cells located in the internal part of the ovariuterus start to degenerate. In successive stages of the ovariuterus development, the interstitial cells are no longer noticeable, while the ovariuterus gains the lumen. These findings strongly suggest that in E. italicus degeneration of interstitial cells leads to the lumen formation. Removal of inner cell mass by cell death is one of well described mechanisms of tubular organs formation and defined as cavitation (see e.g. Bryant and Mostov, 2008; Qi et al., 2012). Whether the mechanism that governs the formation of the lumen of the ovariuterus described in this paper is characteristic of all other chelicerates remains uncertain and requires further investigations.

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4.2. Follicular and stalk cells The stalk cells are characteristic of almost all chelicerates. On the other hand, it is believed that in most chelicerates oocytes grow without accompany of somatic cells referred to as the follicular cells. Apart from scorpions (Farley, 1998, 1999; Soranzo et al., 2000) the presence of follicular cells has been described in pseudoscorpions (Weygoldt, 1969; Makioka, 1979; Badian and Ogorza1ek, 1982; Je˛ drzejowska et al., 2013) and some acarines (Reger, 1977;  ski, 1986; Bergmann et al., 2010). In acarines with the Witalin nutrimentary type of the ovary, the somatic cells adhering to the surface of growing oocytes have been referred to as the somacells (Alberti and Hänel, 1986; Alberti and Zeck-Kapp, 1986; Alberti and Coons, 1999; Di Palma and Alberti, 2002). It is known that the somatic cells which provide the anlage of the gonad before germ cells immigration are of mesodermal origin. One of important issues worth considerating is the mode of somatic cells differentiation into the cells accompanying growing oocytes (the stalk and follicular cells). It has been evidenced that in mites (Evans, 1992), ricinuleids (Talarico et al., 2009), schizomids (Miyazaki et al., 2001), spiders (Morishita et al., 2003) and apoikogenic scorpions (Soranzo et al., 2000) the stalk cells differentiate from epithelial cells. The results of our studies confirm that in E. italicus the epithelial cells of the ovariuterus are the source of the stalk cells. On the other hand, data regarding the origin of follicular cells in chelicerate taxa are not so obvious. In pseudoscorpions the follicular cells differentiate from interstitial cells (Je˛ drzejowska et al., 2013). In scorpions, however, the follicular cells are assumed to have an epithelial origin (Farley, 1998; Soranzo et al., 2000). Our observations of the follicular cell differentiation in E. italicus are in line with the latter notion. We show that in E. italicus the follicular cells differentiate from the epithelial cells that become elevated toward the mesosomal cavity by the growing oocytes. Soranzo et al. (2000) have suggested that the follicular cells appear on the oocyte surface as a result of forward migration of the stalk cells. In this paper we evidence that the follicular cells are mitotically active. We show also that the stalk cells tightly adhere to each other by means of fingerlike cytoplasmic projections. It suggests that the stalk cells do not change position in relation to the oocyte surface. Thus, the increasing number of follicular cells on a surface of growing oocytes apparently results from mitotic divisions rather from forward migratory activity of the stalk cells. Another relevant question worth considerating is the function of somatic cells that accompany growing oocytes. In chelicerates, the role of the follicular cells remains elusive. It has been suggested that in mites the follicular cells participate either in the formation of the  ski,1986) or perivitelline space (Witalin  ski, vitelline envelope (Witalin 1987). In pseudoscorpions, it is believed that follicular cells fulfill a mechanical role in preventing untimely oocyte migration through the lumen of the oocyte stalk (Je˛ drzejowska et al., 2013). Soranzo et al. (2000) have suggested that in Euscorpius carpaticus the follicular cells might be involved in the synthesis of the oocyte reserves. However, this is inconsistent with our findings. We did not observe

the vitelline envelope penetrated by microvilli (arrow) formed by the oolemma. Apical membranes of the follicular cells are smooth (arrowheads). TEM, Scale bar: 2.5 mm. C, D. Early (C) and advanced (D) stages of ovariuterus development. In the early stage, when the oocytes protrude to the mesosomal cavity, the wall of the ovariuterus consists of germline cells (not shown here), epithelial cells and interstitial cells. The epithelial cells are located peripherally, whereas the interstitial cells are situated internally. The nuclei of interstitial cell are noticeably electron-dense what indicates that they start to degenerate. In advanced stages of the ovariuterus development (D), the lumen of the ovariuterus is visible, while the interstitial cells are not found. Epithelial cells form a pseudostratified epithelium externally surrounded by two layers of muscle cells. C. TEM, scale bar: 0.6 mm. D. Semithin section stained with methylene blue. Scale bar: 150 mm. E, F. Ultrastructure of the oocyte stalk. E. The stalk cells, which adhere to the basal part of early previtellogenic oocyte, form numerous microvilli, which interdigitate with microvilli formed by the oolemma. F. In advanced stages of previtellogenesis, the oocyte is surrounded by the vitelline envelope that is penetrated by oocyte microvilli (arrows) that reach cytoplasmic projections of the stalk cells (encircled) facing the oocyte. The stalk cells are columnar. In the central part of the stalk, the stalk cells are connected to each other by means of fingerlike cytoplasmic projections (denoted by dashed rectangular). The latter are formed on apical parts of the stalk cells. In insert: the higher magnification of fingerlike cellular projections (denoted by dashed rectangular). E. TEM, scale bar: 1.7 mm. F. TEM, longitudinal section. Scale bar: 2.5 mm. F. TEM, scale bar: 0.6 mm. Abbreviations: bl, basal lamina; E, epithelial cells; Fc, follicular cells; Oo, oocyte; Ic, interstitial cells; L, lumen of the ovariuterus; m, mitochondria; Mu1 and Mu2, two layers of muscle cells; mv, microvilli; NFc, nucleus of follicular cell. NS, stalk cell nucleus; S, stalk cells; ve, vitelline envelope.

Please cite this article in press as: Je˛ drzejowska, I., et al., Differentiation of somatic cells in the ovariuteri of the apoikogenic scorpion Euscorpius italicus (Chelicerata, Scorpiones, Euscorpiidae), Arthropod Structure & Development (2013), http://dx.doi.org/10.1016/j.asd.2013.11.004

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any morphological signs of secretory activity of follicular cells. However, we do not exclude the possibility that the follicular cells’ degeneration during vitellogenesis may facilitate an uptake of reserve material precursors by the oocytes. The results of our observations prompted us to speculate that in E. italicus the follicular cells might be engaged in the oocyte elongation. It has been previously evidenced that in euscorpiids the oocytes change their shape from spherical to ovoid (Soranzo et al., 2000), but the mechanism of the oocyte elongation remained unexplained. We have found that the follicular cells that surround spherical previtellogenic oocytes display a characteristic pattern of arrangement. The follicular cells which encompass the middle part of the oocyte are aligned circumferentially in relation to the oocyte stalk, while the follicular cells which cover the distant most surface of the oocyte display a radial arrangement. We demonstrate also that in E. italicus the actin cytoskeleton of the follicular cells undergoes significant reorganization leading to the appearance of actin stress fibers. Similar changes in the actin cytoskeleton organization of the follicular cells have been demonstrated in the egg chamber of the fruit fly. It has been also evidenced that in Drosophila interactions between polarized basal actin cytoskeleton of follicular cells and a polarized extracellular network of basal lamina proteins are responsible for egg elongation (He et al., 2010; Gates, 2012). We hypothesize that in E. italicus both the diversified arrangement of follicular cells on the oocyte surface and reorganization of their actin cytoskeleton enable the oocyte elongation. To support our assumption on role of follicular cells in the oocyte elongation in E. italicus further studies are required. Although the oocyte stalks are characteristic features of the chelicerate type of the ovary, both the details of the stalk structure and the mechanisms that govern the stalk formation remain elusive. In this paper we demonstrate for the first time the details of the stalk structure and formation in an apoikogenic scorpion. Our studies revealed that in E. italicus the oocyte stalks develop due to gradual differentiation of epithelial cells of the ovariuterus. From the very beginning of the stalk formation, the cells that contribute to the stalk construction develop a characteristic system of fingerlike cellular projections enriched with actin filaments. With these fingerlike processes the stalk cells tightly adhere to each other. Elongation of the stalk during the advanced stages of the stalk formation is a result of connecting the epithelial cells with fingerlike cytoplasmic processes in a zip-like manner. To our knowledge such a non-tubular structure of the stalk, built by apically ‘zipped’ epithelial cells has not been evidenced so far in chelicerates. On the other hand, comparative analyses of epithelial morphogenesis carried out in different model organisms have evidenced that formation of actin based cytoplasmic protrusions is essential in establishment of celle cells adhesion during sealing of epithelial sheaths (for a review see e.g. Vasioukhin and Fuchs, 2001). Such cytoplasmic protrusions have

Fig. 4. Schematic diagram showing modifications of the ovariuterine epithelium during oocyte growth. E. italicus, not to scale. Notation of dorsal (D) and ventral (V) parts of the body indicate the upside down orientation of the figures used for better visualization of the oocytes and accompanying cells exposed to the hemocoel. A. Structure of the ovariuterus during early stage of oocyte protrusion. The ovariuterus is composed of peripherally located epithelial cells and internal interstitial cells. The epithelial cells are externally supported by the basal lamina. During previtellogenic growth the oocytes bulge to the mesosomal cavity and elevate some epithelial cells on its surface. These epithelial cells are flat and differentiate into the follicular cells. Other epithelial cells elongate and connect to each other by fingerlike cellular processes

supported by microfilaments, and contribute to the stalk formation. B. In advanced previtellogenesis the oocytes are exposed to the mesosomal cavity and connected to the ovariuterus by the oocyte stalks. Follicular cells form a low cuboidal epithelium on the hemocoelic surface of the oocyte. The stalks are not fully formed as indicated by the stalk bifurcation at the luminal part of the stalk. The stalks cells are columnar. Most of the stalk cells are connected to each other in the central part of the stalk. Some of the stalk cells adhere with their apical membranes to the oocyte. The apical parts of the epithelial cells neighboring the stalk, and apical parts of the stalk cells are rich in Factin. The basal lamina supports externally the basal membranes of the follicular cells and the stalk cells. C. Vitellogenesis. Oocytes are elongate. The hemocoelic surface of the oocytes is covered by follicular cells. The stalks are fully developed. The central parts of the stalks reach the lumen of the ovariuterus. The stalk cells tightly adhere in the center of the stalk to each other with fingerlike cellular projections supported by actin filaments. Microvilli of the stalk cells adhering to the oocyte interdigitate with microvilli of the oocyte. The basal lamina supports externally the follicular cells and the stalk cells.

Please cite this article in press as: Je˛ drzejowska, I., et al., Differentiation of somatic cells in the ovariuteri of the apoikogenic scorpion Euscorpius italicus (Chelicerata, Scorpiones, Euscorpiidae), Arthropod Structure & Development (2013), http://dx.doi.org/10.1016/j.asd.2013.11.004

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been observed e.g. in the fruit fly oogenesis during development of the basal stalk in the Drosophila ovariole (see e.g. Godt and Laski, 1995; Roth and Lynch, 2009) and also during the sealing of epithelial sheets during dorsal closure in the course of embryogenesis (Cavey and Lecuit, 2009). As mentioned above in the chelicerate type of the ovary the oocytes grow exposed to the hemocoel supported by the oocyte stalks. The external position of growing oocytes seems to be crucial  ski et al. (2008) have evifor proper oocyte development. Bilin denced the presence of the well developed contact zone between the oocyte and the stalk cells in the pycnogonid Propallene longiceps and have proposed the role of the stalk cells in fixing the position of the oocyte in relation to the ovarian tissue. The presence of microvilli between the stalk cells and the oocyte in E. italicus supports the idea that the stalk cells participate in securing the oocyte position in relation to the ovariuterus. The stalks form also a passageway for ovulating oocytes. To our knowledge, in scorpions, the mechanism that controls ovulation, i.e. the passage of fertilized eggs through the stalk to the lumen of the ovariuterus, remains unknown. Studies carried out on invertebrates show that one of the conditions required for ovulation is programmed cell death of follicular cells at the end of oogenesis (reviewed e.g. in McCall, 2004; Baum et al., 2005). Therefore we can assume also that degeneration of follicular cells observed in E. italicus in late vitellogenesis might be a sign of forthcoming fertilization and ovulation. The other condition for ovulation is the open (patent) state of the stalk. The opening of the stalk may result from the alterations of the stalk cell intercellular junctions. Morishita et al. (2003) have reported that in the sicariid spider, the stalk cells rearrange before ovulation to form the lumen of the stalk, but they have not shown the thorough structure of the stalk before the stalk cell reorganization. In this contribution we show that in E. italicus the lumen of the stalk is closed by tightly adhering fingerlike cellular processes of the stalk cells and we postulate herein that the ‘opening’ of the stalk in E. italicus apparently relies on unzipping the connection between the stalk cells in the central part of the stalk. In order to address this problem further studies are required. It has been postulated that in acarines ovulation may result from, e.g., contraction of the stalk cells or could be induced by increasing pressure on the basal lamina encompassing the growing oocytes, and/or by the reduction of adhesion between the stalk (funicular) cells (Coons and Alberti, 1999). On the other hand it is known that in invertebrates, like e.g. Drosophila, ovulation is controlled by secretory cells of the female reproductive tract (for a review see: Sun and Spradling, 2013). To address the question what mechanism of ovulation acts in scorpions further investigations are needed. The stalk cells have been proposed to play also other than mechanical functions. It has been demonstrated that in the pseudoscorpion Chelifer cancroides the stalk cells are involved in chorion production and following ovulation in the synthesis of the nutritive fluid for the embryos (Badian and Ogorza1ek, 1982; Je˛ drzejowska et al., 2013). The nutritive role of the stalk cells has been sug ski, 2006; Bilin  ski et al., gested in pycnogonids (Miyazaki and Bilin 2008) and in ticks (de Oliveira et al., 2007), but in both cases the nutritive role of the stalk cells has not been evidenced. Data presented in this contribution do not show that in E. italicus the stalk cells are synthetically or secretory active, so their role is probably limited to establishing the oocyte position. 4.3. The stalk cells share some features with follicular cells A comprehensive analysis of the follicular cells found in invertebrates and in vertebrates revealed that the follicular cells may exhibit a distinct organization (Matova and Cooley, 2001). Moreover, it has been shown that follicular cells may undergo

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diversification into a distinct number of subpopulations, which differ in position in relation to the oocyte surface, the mode of connection with the oocyte, morphology, function and behavior (Margaritis, 1985; Zawadzka et al., 1997; Deng and Bownes, 1998; Dobens and Raftery, 2000; Kubrakiewicz et al., 2003; Mazurkiewicz and Kubrakiewicz, 2005, 2008; Tworzyd1o et al., _ 2005; Zelazowska, 2005; Ogorza1ek, 2007; Jaglarz et al., 2008, 2009, 2010; Garbiec and Kubrakiewicz, 2012). In this contribution, we demonstrate that in E. italicus the growing oocytes are accompanied by the follicular cells and the stalk cells which take a distinct position in relation to the oocyte surface. Both the follicular cells and some of the stalk cells directly adhere to the oolemma. Our observations revealed that the stalk cells show similar to the follicular cells epithelial polarization. The basal parts of the stalk cells adhere to the basal lamina, whereas the apical parts of stalk cells are directed towards the oocyte surface or the opposite stalk cells. Moreover, the stalk cells, in the contact zone with the oocyte, develop microvilli which interdigitate with microvilli of the oocyte. Common with follicular cell differentiation from epithelial cells, polarization of the stalk cells, similar to the follicular cells, and the presence of contact zones between the stalk cells and the oocyte lead us to the conclusion that in E. italicus the stalk cells could be regarded as follicular cells. The fact that not all of the stalk cells adhere to the oocyte does not rule out the possibility that the stalk cells are the follicular cells’ subpopulation. It is widely accepted that in insect ovaries the follicular cells differentiate into several subpopulations and the cells of one of them, i.e. the interfollicular stalk cells, similarly to the stalk cells in scorpions, do not adhere to the oolemma (Margaritis, 1985; reviewed in Dobens and Raftery, 2000). Consequently, in E. italicus differentiation of the epithelial cells accompanying growing oocytes is pronounced by production of two cell subpopulations differing in position on the oocyte surface (hemocoelic versus towards stalk base), morphology (low cuboidal versus columnar epithelium), formation of contact zones (well developed only between the oocyte and stalk cells), function (oocyte elongation versus mechanical support for the oocyte and passageway for the embryo), and finally the “behavior” (degeneration in vitellogenesis versus persistence till embryogenesis).

Acknowledgments We would like to thank Prof. Janusz Kubrakiewicz for sharing his experience of invertebrate oogenesis and very helpful comments.  ski from Jagiellonian UniWe are grateful to Prof. Szczepan Bilin versity (Krakow) for his critical remarks concerning our manuscript. We thank Katarzyna Pajer and Sylwia Nowak for their skillful technical assistance. This work was supported by the research grant 1068/S/IBE/2012.

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Please cite this article in press as: Je˛ drzejowska, I., et al., Differentiation of somatic cells in the ovariuteri of the apoikogenic scorpion Euscorpius italicus (Chelicerata, Scorpiones, Euscorpiidae), Arthropod Structure & Development (2013), http://dx.doi.org/10.1016/j.asd.2013.11.004

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Please cite this article in press as: Je˛ drzejowska, I., et al., Differentiation of somatic cells in the ovariuteri of the apoikogenic scorpion Euscorpius italicus (Chelicerata, Scorpiones, Euscorpiidae), Arthropod Structure & Development (2013), http://dx.doi.org/10.1016/j.asd.2013.11.004